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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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DETECT ION OF NEPOVIRUSES BY E L I S A IN T I S S U E - C U L T U R E D AND FIELD-GROWN GRAPEVINES by RAYMOND CAM ILLE JOSEPH JOHNSON B . S . A . , U n i v e r s i t y o f M a n i t o b a , 1975 A THES I S SUBMITTED IN PART IAL F U L F I L L M E N T OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SC IENCE i n THE FACULTY OF GRADUATE STUDIES ( D e p a r t m e n t o f P l a n t S c i e n c e ) We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d THE UNIVERS ITY OF BR IT I SH COLUMBIA O c t o b e r , 1 9 8 8 c) Raymond Cam I I Ie J o s e p h J o h n s o n , 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P l a n t S c i e n c e The University of British Columbia Vancouver, Canada Date O c t o b e r 11. 1988  DE-6 (2/88) i i ABSTRACT The d e t e c t i o n by serology of nematode-transmit ted p o l y h e d r a l v i r u s e s (nepoviruses) i n grapevines i s o f t e n u n r e l i a b l e . Nepoviruses were detected by enzyme-1 inked immunosorbent assay (ELISA) i n t i s s u e - c u l t u r e d and f i e l d - g r o w n grapevines. Nepovirus d e t e c t i o n i n in vitro p l a n t s ( p l a n t l e t s ) was a f f e c t e d by v i r u s d i s t r i b u t i o n and growth room temperature. The r e l i a b i l i t y of v i r u s d e t e c t i o n i n f i eld-grown grapevines was improved when modified g r i n d i n g b u f f e r s were used. Arabis mosaic v i r u s (AMV) was detected by ELISA, f o r the f i r s t time, i n in vitro grapevines i n i t i a t e d from f i e l d -and screenhouse-grown p l a n t s throughout the summer. The v i r u s was not r e l i a b l y and r e p e a t e d l y detected i n in vitro p l a n t l e t s grown at 25°C. AMV and grapevine f a n l e a f v i r u s (GFLV) d i s t r i b u t i o n was not uniform throughout the p l a n t l e t s . This d i s t r i b u t i o n was a f f e c t e d by the c u l t u r e room temperature. The best p l a n t p a r t s to sample f o r v i r u s d e t e c t i o n came from the zones of r a p i d l y p r o l i f e r a t i n g shoots. The v i r u s e s were sometimes not detected i n samples taken from other t i s s u e s . Growth room temperature had an important e f f e c t on v i r u s d e t e c t i o n i n p l a n t l e t s . The highest v i r u s t i t r e s were found i n p l a n t s growing at 15°C. Temperature in c r e a s e s i n 5°C steps to 30°C reduced v i r u s t i t r e . AMV became undetectable i n n e a r l y a l l p l a n t l e t s growing at 30°C f o r as l i t t l e as 30 days. Growth at 30°C reduced ELISA absorbance i i i v a l u e s b y 7 6 % a f t e r 8 d a y s a n d a f t e r 21 d a y s t h e v a l u e s w e r e a t 4% o f p r e - t r e a t m e n t l e v e l s . T h e v i r u s t i t r e d r o p p e d b e l o w d e t e c t a b l e l e v e l s i n m o s t p l a n t l e t s . AMV c o u l d n o t b e d e t e c t e d i n p l a n t l e t s o r r o o t e d e x p l a n t s a f t e r b e i n g p l a c e d i n a 3 0 ° C t r e a t m e n t f o r 2 m o n t h s . T o m a t o r i n g s p o t v i r u s was d e t e c t e d b y E L I S A , f o r t h e f i r s t t i m e , i n in vitro g r a p e v i n e p l a n t s . T h e v i r u s was r e p e a t e d l y d e t e c t e d i n in vitro p l a n t s g r o w i n g a t 2 0 ° C . U n d e r t h e t y p i c a l s ummer c o n d i t i o n s e x p e r i e n c e d a t S i d n e y , B . C . , m o d i f y i n g t h e s t a n d a r d E L I S A g r i n d i n g b u f f e r ( 0 . 0 1 M p h o s p h a t e b u f f e r e d s a l i n e , pH 7 . 4 , 0 . 0 5 % T w e e n - 2 0 , 0 . 2 % o v a l b u m i n , 2% p o l y v i n y l p y r r o l i d o n e ) was e s s e n t i a l f o r r e l i a b l e d e t e c t i o n o f AMV o r G F L V . AMV was r e l i a b l y d e t e c t e d b y E L I S A i n f o l i a r s a m p l e s f r o m f i e l d o r s c r e e n h o u s e p l a n t s t h r o u g h o u t t h e s u m m e r when t h e g r i n d i n g b u f f e r was m o d i f i e d b y i n c r e a s i n g t h e pH t o a t l e a s t 8 . 2 a n d a d d i n g 1% n i c o t i n e o r 0 . 1 5 M p h o s p h a t e b u f f e r e d s a l i n e . T h e m o s t r e l i a b l e r e s u l t s w i t h G F L V w e r e o b t a i n e d w i t h t h e n i c o t i n e e n h a n c e d b u f f e r s . I n c o m p a r i s o n , b e c a u s e o f t h e i n c r e a s e d w o r k l o a d a s s o c i a t e d w i t h g r o w i n g p l a n t s in vitro a n d t h e u n r e l i a b l e d e t e c t i o n o f v i r u s e s i n t h e s e p l a n t s , i t r e m a i n e d p r e f e r a b l e t o d e t e c t n e p o v i r u s e s i n f i e l d p l a n t s b y E L I S A . i v TABLE OF CONTENTS PAGE TITLE PAGE ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i i LIST OF FIGURES i x ACKNOWLEDGEMENTS x i INTRODUCTION 1 LITERATURE REVIEW 7 ELISA d e t e c t i o n of v i r u s e s i n d i f f e r e n t i a t e d p l a n t t i s s u e grown in vitro 7 Temperature e f f e c t on v i r u s c o n c e n t r a t i o n i n grape p l a n t l e t s growing in vitro 8 C y t o k i n i n e f f e c t o n . v i r u s c o n c e n t r a t i o n s i n p l a n t l e t s grown in vitro 10 S e r o l o g i c a l d e t e c t i o n of n e p o v i r u s e s i n f i e l d - g r o w n g r a p e v i n e s 12 MATERIALS AND METHODS 21 T i s s u e c u l t u r e 21 Media 21 P l a n t m a t e r i a l 21 I n i t i a t i o n 22 R e c u l t u r i n g 23 S e r o l o g y 23 V i r u s p u r i f i c a t i o n 23 TABLE OF CONTENTS (c o n t ' d ) A n t i s e r u m 2 5 ^ g l o b u l i n p u r i f i c a t i o n 26 C o n j u g a t i o n 27 The ELISA t e s t 27 ELISA s e n s i t i v i t y 29 V i r u s d e t e c t i o n i n t i s s u e - c u l t u r e d p l a n t l e t s i n i t i a t e d from 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 V i r u s d i s t r i b u t i o n 32 E f f e c t of temperature and BAP c o n c e n t r a t i o n 33 V i r u s r e c o v e r y 34 V i r u s i n h i b i t i o n at 30°C 35 V i r u s d e t e c t i o n i n f i e l d p l a n t s 36 RESULTS 39 ELISA s e n s i t i v i t y 39 V i r u s d e t e c t i o n i n t i s s u e - c u l t u r e d p l a n t l e t s 39 AMV and GFLV d e t e c t i o n 39 TomRV d e t e c t i o n in vitro 42 V i r u s d i s t r i b u t i o n 43 E f f e c t of temperature and BAP c o n c e n t r a t i o n 50 v i TABLE OF CONTENTS (c o n t ' d ) V i r u s r e c o v e r y 63 E f f e c t of 30°C 63 V i r u s d e t e c t i o n i n f i e l d p l a n t s 64 DISCUSSION 72 V i r u s d e t e c t i o n i n t i s s u e - c u l t u r e d p l a n t l e t s 72 V i r u s d e t e c t i o n i n f i e l d p l a n t s 79 Comparing the r e s u l t s of ELISA t e s t s on in vitro p l a n t l e t s and f i e l d p l a n t s 90 BIBLIOGRAPHY 92 APPENDIX 99 v i i LIST OF TABLES TABLE PAGE I ELISA g r i n d i n g b u f f e r s used to d e t e c t 13 n e p o v i r u s e s i n g r a p e v i n e s I I D e t e c t i o n by ELISA of a r a b i s mosaic (AMV) 40 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 at 25°C I I I D e t e c t i o n of a r a b i s mosaic v i r u s (AMV) 41 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 at v a r i o u s temperatures from January 3, 1986 to September 17, 1986. IV D e t e c t i o n of tomato r i n g s p o t v i r u s i n 43 c u l t u r e s grown at 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 V 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 45 v a r i o u s p l a n t p a r t s taken from f o u r p l a n t l e t s grown at 25°C VI D e t e c t i o n of a r a b i s mosaic v i r u s (AMV) and 48 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 of f i v e p l a n t l e t s grown at 25°C VI I D e t e c t i o n of a r a b i s mosaic v i r u s (AMV) and 49 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 of 10 p l a n t l e t s grown at 20°C V I I I Mean ELISA absorbance v a l u e s and 54 a n a l y s i s of v a r i a n c e of temperature and N-6-benzylaminopurine (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 ( f i r s t e x p e r i m e n t ) IX R e g r e s s i o n a n a l y s e s on the e f f e c t s of temperature and N-6-benzylaminopurine c o n c e n t r a t i o n s on a r a b i s mosaic v i r u s 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 ELISA i n p l a n t l e t s X Mean absorbance v a l u e s of ELISA t e s t s 58 c a r r i e d out on 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 1 and 2 months i n t o the second experiment to t e s t the e f f e c t of temperature and N-6-benzylaminopurine (BAP) c o n c e n t r a t i o n on AMV t i t r e 56 (BAP) (AMV) v i i i LIST OF TABLES (cont'd) TABLE PAGE XI Results of ELISA t e s t s on 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 exposed to 30°C fo r three weeks 64 XII A n a l y s i s of v a r i a n c e of ELISA absorbance 65 values obtained by 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 to detect grapevine f a n l e a f v i r u s (GFLV) and a r a b i s mosaic 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 XIII E f f e c t of 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 66 the ELISA d e t e c t i o n of a r a b i s mosaic v i r u s (AMV) and grapevine 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 XIV D e t e c t i o n of a r a b i s mosaic v i r u s by ELISA 68 throughout the summer in 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 XV D e t e c t i o n of grapevine f a n l e a f v i r u s by 69 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 XVI Mean ELISA absorbance values of h e a l t h y 71 c o n t r o l p l a n t s ground in v a r i o u s b u f f e r s throughout the summer XVII A comparison of f a l s e negatives c r e a t e d by 86 using three d i f f e r e n t methods of determining 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 grapevine f a n l e a f v i r u s (GFLV) d e t e c t i o n during the summer XVIII A comparison of f a l s e p o s i t i v e s c r e a t e d by 88 using three methods of determining 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 grapevine f a n l e a f v i r u s d e t e c t i o n during the summer i x LIST OF FIGURES FIGURE PAGE 1. Frequency histogram of the v i r u s d i s t r i b u t i o n 44 as determined by ELISA i n a l l samples cut 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 i n f e c t e d p l a n t l e t s 2. Frequency histogram of a r a b i s mosaic v i r u s 46 d i s t r i b u t i o n as detected by ELISA i n d i f f e r e n t p l a n t parts d i s s e c t e d from four i n f e c t e d p l a n t l e t s 3. Mean ELISA absorbance values with one 51 standard d e v i a t i o n f o r t e s t s on a r a b i s mosaic v i r u s (AMV) and grapevine 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 at d i f f e r e n t a i r temperatures and 2 mg/l N-6-benzylamino-purine 4. Mean ELISA absorbance readings with one 52 standard d e v i a t i o n of t e s t s on 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 grown at three N-6-benzylaminopurine c o n c e n t r a t i o n s f o r 2 months at 20°C 5. Frequency histogram of ELISA absorbance 53 readings from t e s t s on ar 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 grown f o r 2 months at three N-6-benzylaminopurine (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 of three temperatures (20°C, 25°C, and 30°C) 6. Mean ELISA absorbance values from the f i r s t 57 experiment to determine the e f f e c t s of growing 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 temperatures 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-benzylaminopurine c o n c e n t r a t i o n s 7. Frequency histogram of a r a b i s mosaic v i r u s 59 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 at 20°C, 25°C, and 30°C on medium c o n t a i n i n g 2 mg/l of N-6-benzylaminopurine 8. Frequency histogram of a r a b i s mosaic v i r u s 60 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 at 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-benzylaminopurine c o n c e n t r a t i o n of 2 mg/l - second experiment LIST OF FIGURES (cont'd) FIGURE 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 at 20°C, 25°C, and 30°C on a medium c o n t a i n i n g a N-6-benzylaminopurine c o n c e n t r a t i o n of 2 mg/1 ACKNOWLEDGEMENTS The a u t h o r w i s h e s t o t h a n k t h e members o f h i s c o m m i t t e e : Dr. R. S t a c e - S m i t h ( R e s e a r c h S u p e r v i s o r ) , A g r i c u l t u r e Canada R e s e a r c h S t a t i o n , V a n c o u v e r ; Dr. R . J . Copeman ( F a c u l t y A d v i s o r ) and Dr. V.C. R u n e c k l e s , D e p a r t m e n t o f P l a n t S c i e n c e , U n i v e r s i t y o f B r i t i s h C o l u m b i a ; and D r . I.E.P. T a y l o r , D e p a r t m e n t o f B o t a n y , U n i v e r s i t y o f B r i t i s h C o l u m b i a f o r t h e i r g u i d a n c e , a s s i s t a n c e , and p a t i e n c e . The a u t h o r a l s o w i s h e s t o t h a n k t h e Food P r o d u c t i o n and I n s p e c t i o n B r a n c h , A g r i c u l t u r e Canada f o r t h e f i n a n c i a l s u p p o r t and o p p o r t u n i t y t o r e t u r n t o u n i v e r s i t y and c a r r y D o u t t h e s e s t u d i e s . A l s o , t h a n k s go t o t h e d i r e c t o r , Dr. M. W e i n t r a u b , and t h e s t a f f o f t h e V a n c o u v e r R e s e a r c h S t a t i o n , A g r i c u l t u r e C a n a d a f o r t h e i r s u p p o r t . a n d p r o v i d i n g t h e f a c i l i t i e s f o r t h i s r e s e a r c h . T h e i r h e l p has b e e n i m m e a s u r a b l e . B u t most o f a l l t h e a u t h o r w o u l d l i k e t o e x p r e s s h i s a p p r e c i a t i o n t o h i s w i f e , S u s a n , and h i s c h i l d r e n , M a t t h e w and K a t h r y n Ann, w i t h o u t whose l o v e , u n d e r s t a n d i n g , and s u p p o r t t h i s t h e s i s w o u l d n o t have been p o s s i b l e . 1 INTRODUCTION The Saanichton P l a n t Quarantine S t a t i o n i s the n a t i o n a l p o s t - e n t r y quarantine f a c i l i t y f o r indexing t r e e f r u i t and grapevines i n Canada. The program operates under the a u t h o r i t y of the P l a n t Quarantine Act, 1968-69, c.35, s . l . and the i n t e r p r e t a t i o n of t h i s act i n the corresponding P l a n t Quarantine R e g u l a t i o n s . Canada, as well as many other c o u n t r i e s around the world, has e s t a b l i s h e d import r e g u l a t i o n s which p r o h i b i t the importation of Malus, Pyrus, Prunus, Cydonia, Crataegus, Chaenomeles, and Vitis without proper c e r t i f i c a t i o n against a number of d i s e a s e - c a u s i n g organisms. This has been done i n order to prevent the i n t r o d u c t i o n and spread of these diseases i n t o Canada. Many of these d i s e a s e s , some of which have a d i r e c t impact on f r u i t and p l a n t p r o d u c t i o n or l i m i t access to export markets, are e i t h e r not found i n Canada or not widespread. The S t a t i o n plays an i n t e g r a l part i n p r o t e c t i n g Canadian a g r i c u l t u r e and p r e s e r v i n g i t s envious p o s i t i o n of r e l a t i v e freedom from d i s e a s e . Grapevine p r o d u c t i o n i s r e l a t i v e l y recent i n Canada, e s p e c i a l l y i n B r i t i s h Columbia. The c l i m a t i c c o n d i t i o n s i n Canada r e s t r i c t the use of many long e s t a b l i s h e d European wine v a r i e t i e s . Pressure has been i n c r e a s i n g to allow the i m p o r t a t i o n of new, more c o l d hardy v a r i e t i e s with b e t t e r wine-making c h a r a c t e r i s t i c s . These v a r i e t i e s are o f t e n found i n c o u n t r i e s where v i r u s diseases are widespread. V i r u s - i n d e x e d sources of these v a r i e t i e s are not always 2 a v a i l a b l e , e i t h e r because of inadequate indexing programs or because the v a r i e t i e s have only r e c e n t l y been r e l e a s e d from breeding programs p r i o r to indexing. Consequently, t h i s has r e s u l t e d i n i n c r e a s e d demand f o r indexing at the Saanichton S t a t i o n . T h i s s i t u a t i o n i s compounded because of l i m i t e d resources at the S t a t i o n and problems with v i r u s d e t e c t i o n . V i r u s e s are not e a s i l y detected i n any of the seven genera which are t e s t e d at Saanichton. The S t a t i o n i s concerned with the indexing procedures a v a i l a b l e to detect v i r u s e s i n grapevine. A vine may be i n f e c t e d with one or s e v e r a l v i r u s e s . It i s more d i f f i c u l t to detec t v i r u s e s i n woody p l a n t s than i n herbaceous hosts. The indexing techniques used on these p l a n t s can be improved, i n terms of r e l i a b i l i t y , s e n s i t i v i t y , accuracy, and time r e q u i r e d to complete them. Grapevine v i r u s e s f a l l i n t o f i v e broad c a t e g o r i e s (Bovey et a l , 1980b): the nematode-borne p o l y h e d r a l v i r u s e s ( n e p o v i r u s e s ) ; v i r u s e s t r a n s m i t t e d by s o i l f u n g i ; v i r u s e s t r a n s m i t t e d by aphids; v i r u s e s without known vectors but whose p a r t i c l e s have been c h a r a c t e r i s e d ; and v i r u s e s without known v e c t o r s , with p a r t i c l e s not c h a r a c t e r i s e d . The g r e a t e s t economic losses are caused by the v i r u s e s in the f i r s t and l a s t groups. The nepoviruses are po l y h e d r a l v i r u s e s having nematodes as v e c t o r s . In many cases, these v e c t o r s are widely d i s t r i b u t e d around the world and are able to serve as a r e s e r v o i r i n the s o i l f o r many years even a f t e r the o r i g i n a l l y i n f e c t e d vines have been destroyed. It i s 3 t h e r e f o r e important to insure that these v i r u s e s are not introduced or spread w i t h i n Canada any f u r t h e r than they are at present. C u r r e n t l y , a l l grapevines are indexed f o r nepoviruses by serology, by i n o c u l a t i n g onto herbaceous i n d i c a t o r s , and by g r a f t i n g onto woody grapevine i n d i c a t o r s i n the f i e l d . The t r a d i t i o n a l methods r e q u i r e the i n o c u l a t i o n onto herbaceous p l a n t s and woody i n d i c a t o r s . These procedures are labour i n t e n s i v e and lengthy, r e q u i r i n g up to 3 years f o r woody indexing. They are more c o s t l y due to the need f o r greenhouse f a c i l i t i e s and f i e l d p l o t maintenance and the r e s u l t s are subject to m i s i n t e r p r e t a t i o n . The symptoms caused by the v i r u s e s are sometimes f a i n t , can be confused with damage caused by i n s e c t s or other d i s e a s e s , and r e q u i r e much experience and t r a i n i n g to i n t e r p r e t . These d e t e c t i o n techniques are s u b j e c t to problems with seasonal f l u c t u a t i o n i n v i r u s t i t r e , uneven d i s t r i b u t i o n i n p l a n t s , and v i r u s - i n h i b i t i n g sap components. Of the three methods, serology shows the most promise. Under i d e a l c o n d i t i o n s , serology i s a f a s t and p r e c i s e procedure. The widely used enzyme-1 inked immunosorbent assay (ELISA) technique ( C l a r k and Adams, 1976) i s r e l a t i v e l y easy to perform with r e s u l t s being r e p r o d u c i b l e , o b j e c t i v e , and q u a n t i f i a b l e . There are, however, problems a s s o c i a t e d with using serology to detect nepoviruses i n woody grapevine m a t e r i a l s . The v i r u s e s cannot be d e t e c t e d at a l l times of the year (Uyemoto et a l . , 1976; Ramsdell et a l . , 1979; Shanmuganathan and F l e t c h e r , 1982) or may be 4 unevenly d i s t r i b u t e d w i t h i n a plant (Gilmer et a l . , 1970; Uyemoto et a l . , 1976). Furthermore, the grape sap co n t a i n s substances which may i n t e r f e r e with serology ( P i e r p o n t et a l . , 1977; Vetten, 1981). Recently, nepovirus d e t e c t i o n by ELISA i n fi e l d - g r o w n grapevines has been improved by modifying the b u f f e r used to g r i n d the l e a f m a t e r i a l (Jimenez and Goheen, 1980; Tanne, 1980; Engelbrecht, 1980). T i s s u e c u l t u r e techniques have been a p p l i e d mainly to produce new pl a n t s a f t e r v i r u s e l i m i n a t i o n by c o n v e n t i o n a l heat therapy. Very l i t t l e work has been done to de t e c t v i r u s e s s e r o l o g i c a l l y by ELISA i n p l a n t s c u l t u r e d in vitro ( p l a n t l e t s ) . Before the beginning of t h i s p r o j e c t , only the potato v i r u s e s X.Y.S.M.A, and l e a f r o l l had been d e t e c t e d by ELISA i n shoot t i p c u l t u r e s of potato (McMorran and A l l e n , 1983) and prune dwarf v i r u s i n sour cherry p l a n t s in vitro (Bauman, Casper, and Kornkamhaeng, 1984). Since then, ELISA t e s t i n g has a l s o been used to detect a r a b i s mosaic v i r u s (AMV) and grapevine f a n l e a f v i r u s (GFLV) i n grapevine p l a n t l e t s growing in vitro (Monette, 1985). Grapevines have been grown in vitro mainly on the b a s i c Murashige and Skoog (1962) medium c o n t a i n i n g the c y t o k i n i n N-6-benzylaminopurine (BAP) to enhance c e l l d i v i s i o n ( B a r l a s s and Skene, 1978, 1980a, 1980b; Chee and Pool, 1982; Goussard,1982; H a r r i s and Stevenson, 1982; Chee et a l . , 1984; L i and Eaton, 1984; Corte and de Mendoca, 1985, Mo r i n i et a l . , 1985; and Zatyko and Molnar, 1985). The BAP co n c e n t r a t i o n s used with grapevines have v a r i e d from 2 to 10 mg/l, with 2-3 mg/l being the most common c o n c e n t r a t i o n . 5 Previous r e p o r t s on the e f f e c t s of c y t o k i n i n s on v i r u s r e p l i c a t i o n i n p l a n t l e t s growing in vitro have not been c l e a r , with many r e s u l t s apparently being c o n t r a d i c t o r y ( F r a s e r and Whenham, 1982). No one has yet i n v e s t i g a t e d the e f f e c t of BAP c o n c e n t r a t i o n s on v i r u s e s i n grapevines growing in vitro. The purpose of t h i s t h e s i s p r o j e c t was to i n v e s t i g a t e methods which would improve nepovirus d e t e c t i o n i n grapevines by ELISA i n both f i e l d - g r o w n grapevines at Sidney, B.C.and i n grapevines grown in vitro . The f i r s t p art of t h i s t h e s i s i a a r e p o r t on va r i o u s aspects of nepovirus d e t e c t i o n i n d i f f e r e n t i a t e d grapevine p l a n t l e t s grown in vitro: a) the r e l i a b i l i t y of ELISA to detect AMV i n p l a n t l e t s s t a r t e d from shoot t i p s obtained from the f i e l d d u r i n g one summer, b) the e f f e c t of var i o u s c o n c e n t r a t i o n s of BAP, the primary c y t o k i n i n used i n grapevine c u l t u r e , on the d e t e c t i o n of AMV i n p l a n t l e t s , c) the e f f e c t of growth room temperature on AMV and GFLV d e t e c t i o n by the ELISA technique, d) the a b i l i t y to detect tomato r i n g s p o t v i r u s (TomRV) by ELISA i n p l a n t l e t s s t a r t e d from i n f e c t e d p l a n t s , e) and, the d i s t r i b u t i o n of AMV and GFLV w i t h i n t i s s u e - c u l t u r e d p l a n t l e t s . The second aspect of t h i s t h e s i s i s a comparison of the e f f e c t s of four g r i n d i n g b u f f e r s on the d e t e c t i o n of GFLV and AMV i n f i e l d and screenhouse-grown p l a n t s under one summer's environmental and c u l t u r a l c o n d i t i o n s Saanichton. 7 LITERATURE REVIEW ELISA d e t e c t i o n of v i r u s e s i n d i f f e r e n t i a t e d p l a n t t i s s u e grown ia vitro There are few r e p o r t s of pl a n t v i r u s d e t e c t i o n i n t i s s u e taken from d i f f e r e n t i a t e d p l a n t l e t s growing in vitro. V i r u s t e s t s on p l a n t s d e r i v e d from in vitro c u l t u r e are u s u a l l y performed a f t e r t r a n s f e r to s o i l f o r s e v e r a l months. ELISA has been used to detec t potato v i r u s e s i n potato p l a n t s grown in vitro. In 1983, McMorran and A l l e n found that potato v i r u s e s X,S,Y,A,M and l e a f r o l l were more r e l i a b l y detected i n t i s s u e c u l t u r e p l a n t l e t s growing at 15-27°C than i n greenhouse-grown p l a n t s . The r e l a t i v e l e v e l s of antigen among the leaves, stems, and roots were found to vary with each v i r u s . V i r u s e s X,S,M, and l e a f r o l l were e a s i l y detected i n a l l p l a n t p a r t s , while only stems and l e a f t i s s u e gave c o n s i s t e n t d e t e c t i o n of potato v i r u s Y. Lozoyo-Saldana and Madrigal-Vargas (1985) a l s o used ELISA to detect potato v i r u s X i n whole potato p l a n t l e t s growing at e i t h e r 28°C or 35°C. They found that the v i r u s was de t e c t e d i n a l l p l a n t l e t s kept at 28°C but was missed i n some h e l d at 35°C. Bauman et a l . (1984) e s t a b l i s h e d prune dwarf v i r u s - i n f e c t e d sour ch e r r y p l a n t l e t s in vitro. Using ELISA, they detected the v i r u s i n some but not a l l p l a n t l e t s i n i t i a t e d from i n f e c t e d p l a n t s . The c u l t u r e room temperature was not given. The v i r u s was d e t e c t a b l e only 8 a f t e r the t h i r d s u b c u l t u r e . S n i r and S t e i n (1985) c o n s i s t e n t l y d e t e c t e d prunus n e c r o t i c r i n g s p o t v i r u s by ELISA in leaves and shoots of sweet cherry p l a n t l e t s growing at 25°C. Kuo et a l . (1985) used ELISA to detect sweet potato v i r u s - A and sweet potato v i r u s - N in leaves taken from sweet potato p l a n t l e t s being growing in vitro at 22°C-32°C. Monette (1985) detected GFLV, by ELISA, i n grapevine p l a n t l e t s i n i t i a t e d from shoot t i p s . The v i r u s was d e t e c t a b l e i n 27 of 29 p l a n t l e t s i n i t i a t e d throughout the summer. Monette found no c o r r e l a t i o n between the age of the p l a n t l e t s and v i r u s c o n c e n t r a t i o n . Even though short term anti g e n f l u c t u a t i o n s were observed, there was no c o r r e l a t i o n between v i r a l c o n c e n t r a t i o n and p l a n t l e t age w i t h i n a 35 day r e c u l t u r e i n t e r v a l . In 1986, Monette used ELISA to detec t both GFLV and AMV i n 9-week o l d c u l t u r e s i n i t i a t e d from greenhouse-grown vines in. which only AMV but not GFLV was d e t e c t a b l e . Temperature e f f e c t on v i r u s c o n c e n t r a t i o n i n grape p l a n t l e t s growing in vitro Hot a i r temperatures above 35°C have been used f o r many years to e l i m i n a t e v i r u s e s from conventionally-grown grapevines (Goheen et a l . , 1965; Bovey, 1972; Goheen and Luhn, 1973). S i m i l a r l y , hot a i r has been shown to a f f e c t v i r u s c o n c e n t r a t i o n s i n d i f f e r e n t i a t e d grapevines grown in vitro. 9 Galzy (1961,1963,1964) and Galzy and Compan (1968) found that G FLV-infected grapevine p l a n t l e t s s u b j e c t e d to 35°C f o r 90 days ceased to develop symptoms i n c u l t u r e , had more normal leaves, and rooted b e t t e r than p l a n t l e t s grown at normal temperatures. These p l a n t l e t s s t i l l appeared h e a l t h y a f t e r three a d d i t i o n a l months in c u l t u r e at 20°C and a f t e r being t r a n s p l a n t e d i n t o f i e l d s . F urther s t u d i e s by Galzy (1966,1969,1972) determined that a minimum p e r i o d of 21 days at 35°C was r e q u i r e d f o r permanent e l i m i n a t i o n of v i r u s - i n d u c e d symptoms. In 1969, Galzy r e p o r t e d that root i n i t i a t i o n and growth of GFLV-infected grape p l a n t l e t s i n c r e a s e d p r o p o r t i o n a t e l y as temperature was increased over a 26°C t h r e s h o l d value to 37°C, above which the growth of both healthy and v i r u s - i n f e c t e d p l a n t l e t s was adversely a f f e c t e d . S i m i l a r l y , poor shoot growth was g r e a t l y enhanced above 30°C, with 35°C being the optimum temperature. V a l a t and Mur (1976) s u c c e s s f u l l y e l i m i n a t e d grapevine l e a f r o l l v i r u s and grapevine f l e c k from grapevines grown in vitro at a c o n t i n u o u s l y high temperature. Mur (1979) e l i m i n a t e d grapevine f l e c k , grapevine l e a f r o l l , and v e i n n e c r o s i s from in vitro p l a n t l e t s f o l l o w i n g a 70 day treatment at temperatures of 35°C or 38°C. The number of v i r u s - f r e e p l a n t l e t s increased with the number of 70 day treatments. However, he a l s o found that both f l e c k and v e i n n e c r o s i s were e l i m i n a t e d from 6 of 14 p l a n t l e t s maintained at 20°C. Fu r t h e r evidence of virus-temperature i n t e r a c t i o n s a f f e c t i n g the e l i m i n a t i o n of grapevine v i r u s e s were found by B a r l a s s et a l . i n 1982. Grapevine l e a f r o l l and summer mottle were e l i m i n a t e d from p l a n t l e t s i n i t i a t e d from fragmented shoot apices and grown at e i t h e r a constant temperature of 35°C or a f l u c t u a t i n g temperature of 27°C day/ 20°C n i g h t . Grapevine yellow speckle was e l i m i n a t e d only at the lower temperature while heat therapy (35°C) a p p l i e d e i t h e r before or during the in vitro c u l t u r e was necessary to e l i m i n a t e GFLV. Monette (1983,1986) e l i m i n a t e d both GFLV and AMV from some i n f e c t e d p l a n t l e t s by taking 2 mm shoot t i p s from p l a n t l e t s p r e v i o u s l y grown f o r as l i t t l e as 17 days at d a i l y f l u c t u a t i n g temperatures of 39°C f o r 6 hours followed by 18 hours at 22°C. A l l p l a n t l e t s produced t h i s way a f t e r 28 days of treatment were v i r u s - f r e e . However, treatments f o r up to 113 days d i d not e l i m i n a t e l e a f r o l l v i r u s from a l l p l a n t l e t s i n i t i a t e d from t i p s . He found that f l u c t u a t i n g temperatures of 35°C and 22°C f o r 12 hour periods d i d not e l i m i n a t e GFLV from a l l p l a n t l e t s when a p p l i e d f o r as long as 66 days. Likewise, AMV was not e l i m i n a t e d from a l l p l a n t l e t s a f t e r 96 days at the lower f l u c t u a t i n g temperatures. C y t o k i n i n e f f e c t on v i r u s c o n c e n t r a t i o n s i n p l a n t l e t s growing in vitro The e f f e c t s of c y t o k i n i n s on p l a n t v i r u s r e p l i c a t i o n has not yet been c l e a r l y e s t a b l i s h e d because some of the s t u d i e s have y i e l d e d c o n t r o v e r s i a l r e s u l t s (Fraser and Whenham, 1982). Most of the s t u d i e s have d e a l t with the s t i m u l a t i o n or i n h i b i t i o n of v i r u s a c t i v i t y under normal growing c o n d i t i o n s through e v a l u a t i o n of the presence or absence of l o c a l l e s i o n s on i n d i c a t o r leaves. Depending on the type and time of treatments, the time of i n o c u l a t i o n post-treatment, the c o n c e n t r a t i o n of the growth r e g u l a t o r , the l o c a l l e s i o n host, and the v i r u s , r e s u l t s have been i n c o n c l u s i v e and sometimes c o n t r a d i c t o r y . Almost no work has been done using p l a n t l e t s or p l a n t t i s s u e s growing in vitro; and s t u d i e s done to date show that i t i s i n a p p r o p r i a t e to apply the r e s u l t s of work done on conventionally-grown p l a n t s to p l a n t l e t s grown in vitro. Milo and S r i v a s t a v a (1969a), found that four of f i v e c y t o k i n i n s a p p l i e d to e x c i s e d Nicotiana glutinosa leaves p r i o r to i n o c u l a t i o n with tobacco mosaic v i r u s i n h i b i t e d l o c a l l e s i o n formation but enhanced v i r u s p r o d u c t i o n . The e x c e p t i o n was BAP which d i d not reduce l o c a l l e s i o n formation but reduced v i r u s r e p l i c a t i o n as shown by i n f e c t i v i t y assays. However, Milo and S r i v a s t a v a (1969b), using i n f e c t i v i t y assays onto Phaseolus vulgaris, a l s o found that tobacco mosaic v i r u s r e p l i c a t i o n was s t r o n g l y i n h i b i t e d i n N. tabacum p i t h t i s s u e c u l t u r e s grown f o r 28 days on media c o n t a i n i n g d i f f e r e n t c o n c e n t r a t i o n s of the f i v e c y t o k i n i n s , i n c l u d i n g BAP. V i r u s i n f e c t i v i t y dropped p r o p o r t i o n a t e l y as c y t o k i n i n c o n c e n t r a t i o n s were i n c r e a s e d from 2 to 200 ug/l. Simpkins et a l . (1981) found that i n c r e a s i n g k i n e t i n c o n c e n t r a t i o n s i n the medium from 0 to 25.6 mg/L d i d not s i g n i f i c a n t l y reduce cucumber mosaic v i r u s c o n c e n t r a t i o n i n N. rustics p l a n t l e t s over 28 days. When combined w i t h r i b a v i r i n at 10 and 50 mg/L, they found t h a t the h i g h e r c o n c e n t r a t i o n of k i n e t i n a n t a g o n i s e d the a n t i v i r a l e f f e c t of the r i b a v i r i n , s l i g h t l y i n c r e a s i n g the v i r u s c o n c e n t r a t i o n i n the p l a n t l e t s . Working w i t h p o t a t o p l a n t s growing in vitro, L o z o y a - S a l d a f i a and M a d r i g a l - V a r g a s ( 1985) found t h a t i n c r e a s e s i n k i n e t i n c o n c e n t r a t i o n from 0.3 to 300 ppm d i d not e l i m i n a t e p o t a t o v i r u s X from the in vitro p l a n t l e t s . S e r o l o g i c a l d e t e c t i o n o f n e p o v i r u s e s i n f i e l d - g r o w n g r a p e v i n e s A number of ELISA g r i n d i n g b u f f e r s have been used t o enhance n e p o v i r u s d e t e c t i o n i n g r a p e v i n e s (Table 1 ) . The s t a n d a r d ELISA g r i n d i n g b u f f e r i s a phosphate b u f f e r e d 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 20, 2% p o l y v i n y l p y r r o l i d o n e (PBS-T-PVP), sometimes m o d i f i e d w i t h 0.02% ovalbumin (PBS-T-PVP-0). Al t h o u g h the r e s u l t s have g e n e r a l l y been c o n s i s t e n t between s t u d i e s some d i s c r e p a n c i e s have been r e p o r t e d . TomRV was d e t e c t e d i n 20 of 21 i n f e c t e d v i n e s i n 1979 by Gonsalves u s i n g the ELISA t e c h n i q u e . Young, r a p i d l y expanding t e r m i n a l l e a v e s were ground i n the s t a n d a r d ELISA g r i n d i n g b u f f e r . D e t e c t i o n by t h i s method was e q u a l l y 13 Table I. ELISA grinding buffers used to detect nepoviruses i n grapevines Grinding b u f f e r 1 Tissue V i r u s 2 Tested Detection Reference PBS-T-PVP-O, pH 7.4 TomRV PBS-T-PVP-O, pH 7.4 PRMV PBS-T-PVP + 1% GFLV nicotine, pH 8.2 PBS-T-PVP-O, pH 7.4 + 1% nicotine + 0.2% NaDIECA PBS-T-PVP-O, pH 7.4 + 1% nicotine PBS-T-PVP-O, pH 7.4 PBS-T-PVP-0, pH 7.4 + 1.25% nicotine PBS-T-PVP PBS-T-PVP, +2% nicotine PBS-T-PVP-0 • 2-mercaptoethanol PBS-T-PVP PBS-T-PVP 0.2M PBS-T-PVP, pH 7.5 • 0.5% TGA +0.1% NaDIECA PBS-T-PVP, pH 7.4 + 2.5% nicotine GFLV GFLV GFLV, AMV, TBRV GFLV GFLV GFLV, AMV, TRSV GFLV, AMV leaves leaves leaves, dormant buds leaves leaves leaves leaves leaves, buds leaves leaves seasonally unreliable unreliable r e l i a b l e r e l i a b l e r e l i a b l e unreliable r e l i a b l e r e l i a b l e variable r e l i a b l e seasonally unreliable unreliable r e l i a b l e r e l i a b l e Gonsalves (1979) Ramsdell et a l . (1979) Bovey et a l . (1980a) Engelbrecht (1980) Jimenez and Goheen (1980) Tanne (1980) Jankulova et a l . (1982) Shanmuganathan and Fletcher (1982) Kearns and Mossop (1984) Walter et al.(1984) PBS-T-PVP, pH 7.4 unreliable 14 Table I. (continued) Grinding buffer 1 Tissue V i r u s 2 Tested Detection Reference PBS-T-PVP, pH 8.2 + 1% nicotine PBS-T-PVP PBS-T-PVP + nicotine GCMV GFLV, AMV, RRV, TBRV PBS-T-PVP, pH 8.2 GCMV + 1% nicotine PBS-T-PVP GFLV + 1% nicotine + 1% PEG PBS-T-PVP + 1% PEG, pH increased 0.5 M THS-T-PVP, pH 8.2 + 1% PEG 2.5% carbonate, pH 7.3 AMV, + 0.5% ascorbic acid GFLV PBS-T, pH 7.4 • 1% BSA PBS-T-PVP, pH 7.4 PBS-PVP, pH 7.4 + 1% Triton X100 • 0.2% 2-mercaptoethanol 0.5 M THS-T-PVP, pH 8.2 • 1% PEG 0.1 M THS-T-PVP, pH 8.2 • 1% BSA 2.5 % nicotine " 0.2 M THS-T-PVP, pH 8.2 0.5 M THS-T-PVP, pH 8.2 leaves r e l i a b l e buds, bark, sawdust, leaves leaves wood shavings r e l i a b l e unreliable r e l i a b l e leaves r e l i a b l e " r e l i a b l e " r e l i a b l e unreliable r e l i a b l e r e l i a b l e r e l i a b l e r e l i a b l e r e l i a b l e " less r e l i a b l e leaves r e l i a b l e Lehocky et a l . (1984) Stellmach (1985a,b) Kolber et a l . (1985) Bovey et a l . (1985) Huss et a l . (1986) and Walter and Etienne (1987) r e l i a b l e 15 Table I. (continued) Grinding buffer 1 Tissue Virus* Tested Detection Reference 2.5% nicotine PBS, pH 7.4 + 4% polyclar AT + 1% Triton X100 + 0.2% 2-mercapto-ethanol, AMV, GFLV leaves less r e l i a b l e unreliable Huss et a l . (1986) and Walter and Etienne (1987) (continued) PBS, pH 7.4 + 4% polyclar AT + 1% Triton X100 + 0.2% 2-mercapto-ethanol roots r e l i a b l e 2.5% nicotine r e l i a b l e Abbreviations for common buffer constituents: PBS - 0.01 M phosphate buffer saline T - 0.05% Tween 20 PVP - 2% polyvinylpyrrolidone 0 - 0.02% ovalbumin NaDIECA - sodium diethyldithiocarbamate TGA - t h i o g l y c o l l i c acid PEG - polyethylene glycol THS - Tris HCI saline buffer BSA - bovine serum albumin 2 TomRV - tomato ringspot virus; PRMV - peach rosette mosaic virus; GFLV - grapevine fanleaf virus; AMV - arabis mosaic virus; TBRV - tomato blackring virus; TRSV - tobacco ringspot virus; GCMC - grapevine chrome mosaic virus; RRV - raspberry ringspot virus 16 s e n s i t i v e to i n o c u l a t i o n onto herbaceous p l a n t i n d i c a t o r s . Using ELISA, Ramsdell et a l . (1979) co u l d not r e l i a b l y d e t e c t peach r o s e t t e mosaic v i r u s (PRMV) i n grapevines even though the ELISA method used could d e t e c t as l i t t l e as 10 ng of p u r i f i e d v i r u s . The v i r u s was not c o n s i s t e n t l y detected in p l a n t s using the standard g r i n d i n g b u f f e r . D e t e c t i o n was a l s o reduced i n t e s t s performed from mid-May to mid-June. The ELISA r e s u l t s were l e s s s e n s i t i v e when compared to i n o c u l a t i o n onto herbaceous i n d i c a t o r s . In 1980, Jimenez and Goheen (1980) s u c c e s s f u l l y d e t e c t e d GFLV i n crude plant sap using the ELISA technique. They found i t necessary to add a f i n a l c o n c e n t r a t i o n of 1.25% n i c o t i n e to the g r i n d i n g b u f f e r to prevent n o n - s p e c i f i c r e a c t i o n s from appearing. The b u f f e r pH was a l s o increased to 7.4 a f t e r g r i n d i n g . The a d d i t i o n of 0.01% mercaptoethanol to the b u f f e r had no e f f e c t on r e s u l t s . Also i n 1980, Engelbrecht found i t e s s e n t i a l to add n i c o t i n e ( 1 % ) , e i t h e r alone or with sodium d i e t h y l -dithiocarbamate (0.02%) (NaDIECA) to the g r i n d i n g b u f f e r to get c o n s i s t e n t GFLV d e t e c t i o n throughout the growing season. Bovey et a l . (1980a) were able to d e t e c t GFLV by ELISA durin g t e s t i n g from June to October. V i r u s d e t e c t i o n was best around f l o w e r i n g time and was reduced as the summer progressed. V i r u s c o n c e n t r a t i o n was g r e a t e s t i n young leaves and decreased from upper to lower leaves. Dormant l e a f buds were a l s o a good source of v i r u s . They used the standard g r i n d i n g b u f f e r with 1% n i c o t i n e added j u s t before g r i n d i n g . The b u f f e r pH was a d j u s t e d to 8.2 before gr ind ing . Tanne ( 1980) detected GFLV, AMV, and tomato b l a c k r i n g v i r u s (TBRV) in f r e s h or f r o z e n e x t r a c t s from s p r i n g to l a t e summer. She p r e f e r r e d using the standard g r i n d i n g b u f f e r as she found that using a b u f f e r c o n t a i n i n g 2% n i c o t i n e gave v a r i a b l e r e s u l t s . F r e e z i n g samples a f t e r g r i n d i n g g e n e r a l l y reduced absorbance values by about 50% f o r a l l three v i r u s e s . Jankulova et a l . (1982) d e s c r i b e d a method f o r q u a n t i t a t i v e determination of GFLV i n p l a n t s using ELISA. GFLV was r e l i a b l y detected i n grapevines using a modified g r i n d i n g b u f f e r c o n t a i n i n g one drop of 2-mercaptoethanol i n e i t h e r 3:1 or 10:1 (v.w) d i l u t i o n s of grapevine t i s s u e . Shanmuganathan and F l e t c h e r (1982), using the standard g r i n d i n g b u f f e r , were able to r e l i a b l y detect GFLV i n p l a n t t i s s u e only in e a r l y summer. Dormant buds and young l e a f t i s s u e gave the best r e s u l t s . In 1984, Kearns and Mossop r e p o r t e d on GFLV, AMV, and tobacco r i n g s p o t (TRSV) d e t e c t i o n i n grapevines using ELISA. They were unable to r e l i a b l y detect GFLV in shoot or l e a f t i s s u e using the standard g r i n d i n g b u f f e r . D e t e c t i o n improved when the phosphate m o l a r i t y was increased to 0.2 M and e i t h e r 2% n i c o t i n e , 0.1% NaDIECA, or 0.5% t h i o g l y c o l 1 i c a c i d (TGA) were added. Best r e s u l t s were obtained using a high m o l a r i t y phosphate b u f f e r c o n t a i n i n g both NaDIECA and TGA. Lehoczky et a l . (1984) and Kolber et a l . (1985) used ELISA to d e t e c t g r a p e v i n e chrome mosaic v i r u s (GCMV) i n f i e l d - g r o w n v i n e s i n Hungary. They used a m o d i f i e d s t a n d a r d g r i n d i n g b u f f e r c o n t a i n i n g 1% n i c o t i n e and a d j u s t e d to pH 8.2 j u s t b e f o r e use. V i r u s c o n c e n t r a t i o n s peaked i n May and June, u n t i l the end of the s m a l l b e r r y s t a g e , then dropped below d e t e c t i o n i n J u l y . V i r u s d e t e c t i o n to the end of June was s i m i l a r f o r upper, m i d d l e , and lower l e a v e s on the v i n e s . The v i r u s was d e t e c t e d a g a i n i n August and September, w i t h the upper l e a v e s g i v i n g the best r e s u l t s . Comparing d i r e c t and i n d i r e c t ELISA t e c h n i q u e s to d e t e c t GFLV and AMV, W a l t e r et a l . (1984) found t h a t b oth methods d e t e c t e d p u r i f i e d v i r u s down to a c o n c e n t r a t i o n of 10 ng/ml and always d i f f e r e n t i a t e d between the two v i r u s e s . Adding 2.5% n i c o t i n e to the g r i n d i n g b u f f e r was found to be i n d i s p e n s a b l e f o r r e l i a b l e GFLV d e t e c t i o n i n g r a p e v i n e . V i r u s d i s t r i b u t i o n e x p e r i m e n t s w i t h greenhouse-grown p l a n t s showed t h a t both GFLV and.AMV were d e t e c t e d l o n g e r i n r o o t s than l e a v e s . V i r u s c o n c e n t r a t i o n s i n f i e l d - g r o w n p l a n t s were h i g h e r i n l e a f t i s s u e i n the s p r i n g than f o r o t h e r p l a n t p a r t s . D e t e c t i o n became u n s a t i s f a c t o r y i n l a t e summer. Attempts to improve v i r u s d e t e c t i o n i n l a t e summer by a r t i f i c i a l l y f o r c i n g dormant buds i n the greenhouse were u n s u c c e s s f u l . V i r u s d e t e c t i o n i n these f o r c e d buds was comparable to t h a t found i n the f i e l d p l a n t s . U s i n g the s t a n d a r d g r i n d i n g b u f f e r , S t e l l m a c h (1985a) r e l i a b l y d e t e c t e d GFLV, AMV, and r a s p b e r r y r i n g s p o t v i r u s (RRV) i n dormant buds, bark s c r a p i n g s , and sawdust taken from dormant c u t t i n g s . These v i r u s e s were d e t e c t e d i n s a w d u s t f r o m i n f e c t e d p l a n t s d i l u t e d down t o 1% i n m i x t u r e s w i t h h e a l t h y s a w d u s t . A l s o , S t e l l m a c h ( 1 9 8 5 b ) f o u n d t h a t t h e s t a n d a r d g r i n d i n g b u f f e r was a b l e t o d e t e c t GFLV, AMV, and TBRV r e l i a b l y i n young l e a v e s and d o r m a n t buds p l u s s e c o n d a r y b a r k t a k e n f r o m f i e l d - g r o w n v i n e s , e v e n i n t h e h e a t o f summer. He s t a t e d t h a t a g r i n d i n g b u f f e r c o n t a i n i n g n i c o t i n e p r o d u c e d e r r a t i c r e s u l t s . I n a s t u d y on E L I S A d e t e c t i o n o f n e p o v i r u s e s i n g r a p e v i n e s c o n t a i n i n g m i x e d v i r u s i n f e c t i o n s , S t e l l m a c h and B e r e s ( 1 9 8 5 ) had no d i f f i c u l t i e s i n d e t e c t i n g s i n g l e n e p o v i r u s i n f e c t i o n s i n p l a n t s a l s o h a v i n g v i r u s e s o t h e r t h a n n e p o v i r u s e s . However, s i m u l t a n e o u s i n f e c t i o n w i t h two n e p o v i r u s e s s o m e t i m e s r e d u c e d t h e c o n c e n t r a t i o n o f one v i r u s , d e l a y i n g d e t e c t i o n . They recommended w a i t i n g a s u f f i c i e n t p e r i o d o f t i m e t o e n s u r e t h e e s t a b l i s h m e n t o f b o t h v i r u s e s b e f o r e t e s t i n g . The g r i n d i n g b u f f e r u s e d d i d n o t c o n t a i n any a d d i t i v e s . A l s o i n 1985, B o v e y e t a l . f o u n d t h a t GFLV was b e s t d e t e c t e d i n g r a p e v i n e s u s i n g u p p e r l e a v e s b e f o r e e a r l y J u l y . The b e s t b u f f e r was t h e one c o n t a i n i n g 1% n i c o t i n e and 1% p o l y e t h y l e n e g l y c o l (PEG) (MW 6 0 0 0 ) . N i c o t i n e c o u l d be e l i m i n a t e d i f t h e g r i n d i n g b u f f e r pH was a d j u s t e d t o c o m p e n s a t e f o r t h e a c i d i t y o f t h e g r a p e v i n e s a p . Huss e t a l . ( 1 9 8 6 ) c o n f i r m e d t h a t GFLV d e t e c t i o n i n g r a p e l e a v e s , wood s h a v i n g s , and r o o t l e t s was h i g h l y d e p e n d e n t upon t h e n a t u r e o f t h e b u f f e r u s e d t o p r e p a r e t h e e x t r a c t . GFLV was r e l i a b l y d e t e c t e d i n a l l t i s s u e s when PBS o r T R I S - H C l b a s e d b u f f e r s were u s e d , p r o v i d i n g t h e y had a 20 molarity above 0.1 M, a pH around 8, and contained 2% or 5% PVP. 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 fie 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 plastic bag. A 0.2 M Tris HCl buffer was slightly 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 failed to detect the viruses in some leaves. A 0.01 M PBS buffer was unsuitable for GFLV detection in grape leaves but worked better than the nicotine solution when wood shavings were used. Both buffers were equally acceptable when roots were used as a virus source. A low molarity Tris-HCl buffer (0.01 M) reliably 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 METHODS T i s s u e c u l t u r e . Media. The media used f o r the i n i t i a t i o n , p r o l i f e r a t i o n , and r o o t i n g of grape p l a n t l e t s (Appendix 1) are based on those d e s c r i b e d by Stevenson and Monette (1983) with a few m o d i f i c a t i o n s . The p r o l i f e r a t i o n and r o o t i n g media were a l s o adjusted to pH 5.7 and 0.7% D i f c o Bacto-agar was added before they were autoclaved at 121°C f o r 15 minutes at 20 lbs (102 kPa) pressure. T i s s u e - c u l t u r e p l a n t l e t s were grown on 10 ml volumes of media, e i t h e r i n 25 mm diameter c u l t u r e tubes covered with p l a s t i c 'Kim Kaps' and sealed with p a r a f i l m , or i n 4 f l oz (130 ml) g l a s s j a r s c l o s e d t i g h t l y with white p l a s t i c screw caps. Pl a n t m a t e r i a l . GFLV and AMV-infected shoot t i p s f o r i n i t i a t i o n i n t o c u l t u r e were taken from f i e l d - g r o w n grapevines of the c u l t i v a r LN-33 (Couderc 1613 x Vitis vinifera L . ) , obtained from v i r u s indexing p l o t s at the Saanichton Plant Quarantine S t a t i o n i n Sidney, B.C. These v i r u s - i n f e c t e d p l a n t s had been i n o c u l a t e d as dormant c u t t i n g s by chip-bud g r a f t i n g i n 1983 and then planted i n the f i e l d . The AMV and GFLV i n o c u l a came from screenhouse-grown mother p l a n t s maintained from c u t t i n g s sent by Dr. Stellmach, of the Weinbau I n s t i t u t e , West Germany. 2. TomRV-infected c u t t i n g s of the v a r i e t y De Chaunac ( VJtis hybrid) were obtained from Dr. L. Stobbs, V i n e l a n d Research S t a t i o n , V i n e l a n d , Ontario. Healthy dormant c u t t i n g s of the same v a r i e t y were obtained from the r e p o s i t o r y at the Saanichton S t a t i o n i n Sidney, B.C. A f t e r r o o t i n g , the c u t t i n g s were maintained i n the greenhouse at ambient temperature with no a d d i t i o n a l l i g h t i n g before shoot t i p s were i n i t i a t e d . I n i t i a t i o n . A l l expanded leaves and unnecessary p l a n t t i s s u e s were removed from 2 to 4 cm long, t e r m i n a l s e c t i o n s of shoots s e l e c t e d f o r i n i t i a t i o n . The shoots from f i e l d p l a n t s were s u r f a c e - d i s i n f e s t e d i n a 1:5 d i l u t i o n of household bleach ( a c t i v e i n g r e d i e n t 5.25% sodium h y p o c h l o r i t e when packed) c o n t a i n i n g 0.1% detergent ( P r o t e c t o Concentrated Dishwasher Detergent) using a B u r n e l l ' w r i s t a c t i o n ' shaker. A s i m i l a r s o l u t i o n of 1:10 bleach was used f o r to d i s i n f e s t shoots from greenhouse p l a n t s . A l l the subsequent steps were c a r r i e d out under a laminar flow hood using a s e p t i c techniques. The shoots were given three, 3- to 5-minute r i n s e s i n s t e r i l e d i s t i l l e d water with s l i g h t a g i t a t i o n and kept i n the l a s t r i n s e u n t i l use. Hand instruments were d i s i n f e s t e d by immersion i n t o 70% ethanol followed by flaming . The microscope and other surfaces were wiped with a paper towel immersed i n 70% e t h a n o l . A f t e r r i n s i n g , a l l remaining excess t i s s u e was removed under a d i s s e c t i n g microscope. Both a p i c a l and a x i l l a r y shoot t i p s measuring from 1.0 to 2.0 mm were i s o l a t e d and pl a c e d l i g h t l y i n t o the surface of i n i t i a t i n g medium i n c u l t u r e tubes. R e c u l t u r i n g . P l a n t l e t s were maintained on i n i t i a t i o n medium i n 25 mm diameter tubes u n t i l they were judged to have expanded s u i t a b l y and d i f f e r e n t i a t e d to the p o i n t where they could adapt w e l l and s u r v i v e on the p r o l i f e r a t i o n medium. The e a r l y p r o l i f e r a t i o n stages were c a r r i e d out i n the 25 mm tubes u n t i l p l a n t l e t s i z e f o r c e d the t r a n s f e r to the l a r g e r 4 oz j a r s . A l l subsequent p l a n t l e t s were maintained i n these j a r s , unless otherwise noted. P l a n t l e t s i n j a r s were u s u a l l y t r a n s f e r r e d at 6 week i n t e r v a l s , compared to 3 or 4 week i n t e r v a l s f o r those i n the tubes. N e c r o t i c t i s s u e , c a l l u s , shoots longer than 2 cm, and ro o t s were removed when r e c u l t u r i n g . A f t e r becoming w e l l - e s t a b l i s h e d on p r o l i f e r a t i o n medium, p l a n t l e t s were cut down to h a l f s i z e at each subsequent t r a n s f e r i n order to c o n t a i n t h e i r s i z e w i t h i n the v e s s e l . Wherever p o s s i b l e , masses of r a p i d l y growing and d i v i d i n g a d v e n t i t i o u s buds with short stems were r e c u l t u r e d , r a t h e r than s i n g l e shoots. Serology. V i r u s p u r i f i c a t i o n . Both AMV and GFLV were p u r i f i e d from Chenopodium quinoa Wild, which had been i n o c u l a t e d 14 to 21 days p r e v i o u s l y with grapevine f o l i a r t i s s u e . The leaves had been taken from i n f e c t e d grapevine mother p l a n t s r e c e i v e d at the Plant Quarantine S t a t i o n from France and maintained i n screenhouses. AMV and GFLV i n f e c t i o n had been confirmed i n the mother plan t s by s e r o l o g y . The o r i g i n a l i n o c u l a t i o n to C. quinoa was made using a 0.05 M potassium phosphate b u f f e r , pH 8.0 c o n t a i n i n g 0.01 M c y s t e i n e h y d r o c h l o r i d e and 3% n i c o t i n e . Subsequent i n o c u l a t i o n s between herbaceous p l a n t s were c a r r i e d out u s i n g 0.1 M potassium phosphate, 1% (w/v) PVP (MW 44,000). The herbaceous t i s s u e was homogenized in a Waring blender with 2 ml/g of 0.05 M sodium phosphate, pH 8.0 c o n t a i n i n g 0.02 M a s c o r b i c a c i d and 0.02 M 2-mercaptoethanol, both of which were f r e s h l y added to the phosphate b u f f e r p r i o r to use. The macerate was squeezed through j e r s e y c l o t h then c e n t r i f u g e d f o r 20 minutes at 12,000 g (10,000 rpm) at 4°C using a S o r v a l l SS-34 r o t o r i n a S o r v a l l SS-3 c e n t r i f u g e . A l l other low speed c e n t r i f u g a t i o n s were done with t h i s r o t o r and c e n t r i f u g e . The supernatant was adjusted to pH 5.0 with HCI and l e f t o v e r n i g h t at 4°C. The mixture was c e n t r i f u g e d f o r 20 minutes at 12,000 g (10,000 rpm) at 4°C. NaCl and PEG (MW 6,000) were added at 1% and 8% (w/v) to the supernatant and s t i r r e d g e n t l y f o r 30 minutes at room temperature. A l l the f o l l o w i n g steps were done at or near 4°C . The emulsion was c e n t r i f u g e d f o r 20 minutes at 12,000 g (10,000 rpm) and the p e l l e t s resuspended g e n t l y i n 1/10 volume of 0.05 M Na c i t r a t e pH 7.0 with 1% NaCl. A f t e r 30 minutes, the s o l u t i o n was c e n t r i f u g e d at 12,000 g (10,000 rpm) f o r 20 minutes. PEG was added at 8% to the supernatant. The a l t e r n a t i n g c y c l e s of PEG p r e c i p i t a t i o n f o l l owed by resuspension of the p e l l e t i n 1/10 volume of b u f f e r were c a r r i e d out u n t i l a concentrated volume of l e s s than 1 ml remained. The v i r u s was f u r t h e r p u r i f i e d by c e n t r i f u g a t i o n at 180,000 g (38,000 rpm) f o r 90 minutes through a 10-40% sucrose d e n s i t y g r a d i e n t using a Beckman SW-41 r o t o r i n e i t h e r a Beckman L8-80 or L8-70 c e n t r i f u g e . The g r a d i e n t was passed through an ISCO UA-5 Absorbance/Fluorescence Detector at 254 nm and the u l t r a v i o l e t absorbing band was c o l l e c t e d . The v i r u s c o n c e n t r a t i o n was estimated by measuring the absorbance at 254 nm. P u r i f i e d nepoviruses, at a c o n c e n t r a t i o n of 1 mg/ml in a 1 cm l i g h t path, have an absorbance of about 10 o p t i c a l u n i t s (Stace-Smith, 1985). Antiserum. The a n t i s e r a used i n a l l the s e r o l o g i c a l t e s t s were s u p p l i e d by R. Stace-Smith, A g r i c u l t u r e Canada, Vancouver Research S t a t i o n . The AMV, GFLV, and TomRV p o l y c l o n a l a n t i s e r a were produced from r a b b i t s and had been d i l u t e d 1:1 with g l y c e r o l and kept at 4°C u n t i l used. A monoclonal antiserum against TomRV produced from mice was a l s o used. The t i t r e had been p r e v i o u s l y determined by agar ge l double d i f f u s i o n to be 1:1,280 f o r the GFLV and TomRV p o l y c l o n a l a n t i s e r a . The t i t r e f o r AMV against 100 uq/m\ of pure v i r u s d i l u t e d i n s a l i n e s o l u t i o n (0.85 % NaCl and 0.2% NaN 3 i n d i s t i l l e d water) was determined by gel double d i f f u s i o n during these s t u d i e s to be 1:1,024. The gels were made by pouring 2 ml of a melted mixture of 1.0% Bacto-agar, 0.2% NaN 3, and 0.85% NaCl i n d i s t i l l e d water onto c o l l o d i o n - c o a t e d g l a s s microscope s l i d e s . Wells were cut i n t o the s o l i d i f i e d agar by using a template made from seven 22 c a l i b r e brass c a r t r i d g e s h e l l s (Wright and Stace-Smith, 1966) and the excess agar removed by a s p i r a t i o n . S e r i a l d i l u t i o n s of antiserum were placed around a c e n t r a l w ell f i l l e d with the v i r u s . y g l o b u l i n p u r i f i c a t i o n . One ml of p o l y c l o n a l antiserum was d i l u t e d with 9 ml of d i s t i l l e d water and incubated with 10 ml of s a t u r a t e d ( N H 4 ) 2 S 0 4 f o r 30 to 60 minutes at 4°C. The p r e c i p i t a t e was c e n t r i f u g e d f o r 10 minutes at 12,000 g (10,000 rpm), then d i s s o l v e d i n 2 ml of h a l f - s t r e n g t h PBS and d i a l y s e d three times against 500 ml of 1/2 s t r e n g t h PBS f o r 1 hour. It was then added to a column c o n t a i n i n g 7 to 10 ml of DEAE-22 Sephadex c e l l u l o s e which had been p r e - e q u i 1 i b r a t e d i n 1/2 s t r e n g t h PBS and allowed to b i n d f o r 5 minutes . The column was washed with 1/2 PBS and the f i r s t peak in the e f f l u e n t absorbing at 280 nm (ISCO UA-5 Absorbance/Fluorescence Detector) was c o l l e c t e d . The immunoglobulin c o n c e n t r a t i o n was determined using a G i l f o r d model 250 spectrophotometer at 280 nm and was adjusted to approximately 1 mg/ml ( £ z a o =1.4), e i t h e r by d i l u t i o n with 1/2 PBS, or by c o n c e n t r a t i o n using e i t h e r p r e c i p i t a t i o n with s a t u r a t e d ammonium sulphate or c e n t r i f u g a t i o n through a Centricon-30 c e n t r i f u g e f i l t e r (Amicon) at 5,000 rpm i n a SS-34 r o t o r . The monoclonal antibody to TomRV, which was an immunoglobulin G of c l a s s 2a (determined by using a commercial immunoglobulin typing k i t ) , was p u r i f i e d by a f f i n i t y chromatography using a p r o t e i n - A sepharose CL-4B column. Up to 2 ml of the immunoglobulin was mixed with to 2 ml of p r o t e i n - A sepharose p r e - e q u i 1 i b r a t e d against PBS. 27 A f t e r 30 minutes, the non-bound components were e l u t e d u n t i l the absorbance at 280 nm was 0. The bound IgG was then e l u t e d with 0.2 M g l y c i n e + 0.5 M Na C l , pH 2.5. The peak absorbing at 280 nm was c o l l e c t e d , d i l u t e d to 1 mg/l and s t o r e d at -20°C i n 0.2 a l i q u o t s u n t i l used. Conjugation. The GFLV and AMV p o l y c l o n a l a n t i s e r a and the TomRV monoclonal antiserum were conjugated as f o l l o w s . One h a l f mg of a l k a l i n e phosphatase (Boehringer-Mannheim, grade 1) was d i s s o l v e d i n 0.2 mg of ^ g l o b u l i n and d i a l y s e d overnight at 4°C ag a i n s t 130 ml of 0.06% g l u t a r a l d e h y d e ( e l e c t r o n microscope grade) d i s s o l v e d i n PBS. This was fol l o w e d by e x t e n s i v e d i a l y s i s a gainst PBS. The ^ - g l o b u l i n was d i l u t e d 1:10 with PBS and stored i n s i l i c o n caoted tubes at 4°C u n t i l used. High background readings obtained i n b u f f e r c o n t r o l w e l l s on p l a t e s with some GFLV antiserum c o n j u g a t i o n s were overcome by d i a l y s i n g the conjugated antiserum against 500 ml of 0.05 M T r i s - H C l pH 8.0 f o r 2 hours at 4°C a f t e r the PBS d i a l y s i s . The conjugate was then kept at 4°C u n t i l d i l u t e d i n the conjugate b u f f e r . The ELISA t e s t . The double antibody sandwich technique as d e s c r i b e d by C l a r k and Adams (1976) was used with minor m o d i f i c a t i o n s . Immulon II (Dynotech) p o l y s t y r e n e p l a t e s with 96 w e l l s were used. The wells were f i l l e d with 200 IA volumes of the v a r i o u s s o l u t i o n s . The conjugate b u f f e r used i n a l l t e s t s was modified by adding 0.2% ovalbumin and 2% PVP to the PBS-Tween. This same b u f f e r was used to g r i n d t i s s u e - c u l t u r e samples while four d i f f e r e n t g r i n d i n g b u f f e r s were used to detect AMV and GFLV i n field-grown p l a n t s (see methods f o r t e s t i n g f i e l d p l a n t s ) . The w e l l s were g i v e n t h r e e , 10-second washes, w i t h tap water u s i n g a homemade p l e x i g l a s s p l a t e washer which shot water d i r e c t l y i n t o a l l 96 w e l l s at once. The c o a t i n g and sample s t e p s were i n c u b a t e d o v e r n i g h t at 4°C, the c o n j u g a t e f o r 4 hours at 37°C, and the s u b s t r a t e at room temperature. A b l o c k i n g s t e p was added b e f o r e the samples were put on the p l a t e s . T h i s i n v o l v e d f i l l i n g the w e l l s c o m p l e t e l y w i t h a 2% s o l u t i o n of f e t a l c a l f serum or bovine serum albumin d i s s o l v e d i n PBS-Tween, i n c u b a t i o n f o r 30 minutes at 37°C, and r i n s i n g . The samples were always ground i n 10 volumes of g r i n d i n g b u f f e r . A t e s t p l a t e was run a f t e r each c o n j u g a t i o n t o - d e t e r m i n e the c o n c e n t r a t i o n s of c o a t i n g and c o n j u g a t e d y - g l o b u l i n which would g i v e at l e a s t a 10:1 d i f f e r e n c e i n absorbance v a l u e s (405 nm) between h e a l t h y and v i r u s - i n o c u l a t e d C. quinoa a f t e r 1 hour . As a r e s u l t , the AMV and TomRV p l a t e s were, coated at a c o n c e n t r a t i o n of 1 <yg/ml w h i l e the GFLV p l a t e s were co a t e d sometimes at 1 og/ml but u s u a l l y at 2 ug/ml. The co n j u g a t e s were d i l u t e d to 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 the 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 Dual Band M u l t i s c a n p l a t e r e a d e r (Flow L a b o r a t o r i e s I n c . ) , u n l e s s o t h e r w i s e mentioned. The v a l u e s were c o r r e c t e d f o r background b u f f e r r e a d i n g s , o b t a i n e d from w e l l s t r e a t e d i d e n t i c a l l y to o t h e r w e l l s , e x c e p t f o r the a d d i t i o n of the p l a n t sample. Because of v e r y low r e a d i n g s (<0.01) i n samples from h e a l t h y t i s s u e - c u l t u r e d p l a n t l e t s , c o r r e c t e d r e a d i n g s g r e a t e r than 0.05 were co n s i d e r e d p o s i t i v e i n t e s t s c a r r i e d out on p l a n t l e t c u l t u r e s . The d e t e c t i o n l e v e l f o r ELISA t e s t s on f i e l d p l a n t s was set at the mean of the h e a l t h y samples plus three standard d e v i a t i o n s , because of higher background readings found i n he a l t h y f i e l d p l a n t s . ELISA s e n s i t i v i t y . The s e n s i t i v i t y of the ELISA technique f o r d e t e c t i n g GFLV and AMV was determined by c a r r y i n g out the ELISA t e s t on p u r i f i e d a n t i g e n d i l u t e d i n 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 and Adams ( 1976) . V i r u s d e t e c t i o n i n t i s s u e - c u l t u r e d p l a n t l e t s i n i t i a t e d from f i e l d and greenhouse p l a n t s . AMV and GFLV d e t e c t i o n . Shoots were harvested from AMV-infected and he a l t h y f i e l d p l a n t s on three dates i n 1985: June 11, J u l y 9, and'August 26. Shoots from GFLV-infected p l a n t s were harvested only on August 26, 1985. Shoots were taken from f i v e v i r u s - i n f e c t e d p l a n t s and from f i v e non-inoculated p l a n t s , on each date and kept at 4°C u n t i l the shoot t i p s were placed i n t o c u l t u r e , w i t h i n 2 days . A l l in vitro p l a n t l e t s were maintained f o r up to 23 weeks in growth chambers at 25 x 1°C and under 50 x 5 2£m~ 2 s e c " 1 l i g h t . L i g h t at a l l stages of growth and experimentation f o r a l l p l a n t l e t s was s u p p l i e d by c o o l white f l u o r e s c e n t tubes under a 16 hour photoperiod. The p l a n t l e t s then were moved, f o r 7 weeks, to a room having the same l i g h t q u a l i t y but with temperatures f l u c t u a t i n g from 25°C at night to 29°C during the day. Then, the p l a n t l e t s were moved f o r 5 weeks i n t o a chamber having a constant temperature of 25 ± 1°C and f i n a l l y i n t o a chamber held at 20 ± 1°C where they were maintained u n t i l the end of the experiments i n September, 1986. While growing at 25°C and p r i o r to being placed at the high e r , f l u c t u a t i n g temperatures, the p l a n t l e t s i n i t i a t e d from AMV-infected f i e l d p l a n t s were t e s t e d by ELISA e i t h e r twice or three times, and those from GFLV-infected p l a n t s only once. The p l a n t l e t s were again t e s t e d 7 weeks a f t e r being moved to the room with the higher temperatures. Only 29 p l a n t l e t s i n i t i a t e d August 26 from AMV-infected p l a n t s harvest date and 33 from the GFLV-infected p l a n t s were t e s t e d 5 weeks a f t e r being returned to 25°C. The p l a n t l e t s i n i t i a t e d from the AMV-infected f i e l d p l a n t s on the f i r s t two dates were used i n an.experiment to determine the e f f e c t of temperature and BAP c o n c e n t r a t i o n on the d e t e c t i o n of AMV i n p l a n t l e t s . P l a n t l e t s from a l l three i n i t i a t i o n dates were t e s t e d at 4-6 weeks and again 24-26 weeks a f t e r being moved to 20°C from 25°C. The ELISA t e s t i n g dates c o i n c i d e d with r e c u l t u r i n g dates. Not a l l p l a n t l e t s were n e c e s s a r i l y t e s t e d on every date, t h i s being determined by the c o n d i t i o n of each p l a n t l e t and the amount of m a t e r i a l a v a i l a b l e f o r t e s t i n g a f t e r r e c u l t u r i n g . Only h e a l t h y - l o o k i n g t i s s u e from v i g o r o u s l y - g r o w i n g p l a n t s was used f o r ELISA t e s t i n g . No a t t e n t i o n was given to the type of t i s s u e samples ground before November 20, 1986. Only masses of 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 were s e l e c t e d a f t e r that date. Although always ground i n 10 volumes of g r i n d i n g b u f f e r , the f r e s h weights of the samples taken from p l a n t l e t s before they were moved to the room with the higher f l u c t u a t i n g temperatures were sma l l e r than f o r l a t e r dates. Twelve of the 178 e a r l i e r samples weighed l e s s than 0.1 g, while only three samples from the l a t e r t e s t dates had f r e s h weights of l e s s than 0.4 g. G e n e r a l l y , ELISA absorbance readings taken 2 hours a f t e r adding the s u b s t r a t e were used to determine v i r u s d e t e c t i o n , the exception being the f i r s t t e s t on GFLV p l a n t l e t s , which was read a f t e r 1 hour because of r a p i d l y d e v eloping c o l o u r . The r e s u l t s from a l l t e s t i n g dates, except the one f o l l o w i n g exposure to the higher f l u c t u a t i n g temperatures, were q u a n t i f i e d using the T i t e r t e k p l a t e reader. The r e s u l t s were estimated v i s u a l l y on that date, because of equipment mal f u n c t i o n . T r i a l s had e a r l i e r shown that the yellow c o l o u r i n t e r p r e t e d v i s u a l l y as a p o s i t i v e r e a c t i o n corresponded to an absorbance of at l e a s t 0.1 u n i t s . TomRV d e t e c t i o n . Shoot t i p s from TomRV-infected and hea l t h y c o n t r o l s were i n i t i a t e d from greenhouse c u t t i n g s on two dates, May 2 and J u l y 10, 1986. The p l a n t l e t s were maintained at 20 + 1°C under a 16 hour photoperiod at 50-75 uEm-* sec" 1 . V i r u s d e t e c t i o n by ELISA was c a r r i e d out 18 and 24 weeks a f t e r the f i r s t i n i t i a t i o n date, and 15 weeks a f t e r the second. A l l samples weighed at l e a s t 0.1 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 t i s s u e . Not a l l p l a n t l e t s from the f i r s t i n i t i a t i o n date were t e s t e d on both dates because of poor growth and m o r t a l i t y . Absorbance r e a d i n g s from the f i r s t date were taken 20 minutes f o l l o w i n g s u b s t r a t e a d d i t i o n because of r a p i d c o l o u r development, w h i l e those from the o t h e r t e s t s were read a f t e r 2 hours. R e s u l t s were q u a n t i f i e d u s i n g the p l a t e r e a d e r . V i r u s d i s t r i b u t i o n . To determine i f AMV was u n i f o r m l y d i s t r i b u t e d w i t h i n i n f e c t e d p l a n t l e t s , f o u r p l a n t l e t s o r i g i n a t i n g from the f i r s t i n i t i a t i o n date were c o m p l e t e l y d i s s e c t e d i n t o segments of v a r i o u s f r e s h weights f o l l o w i n g 21 and 22 weeks of growth at 25°C. V i r u s d e t e c t i o n by ELISA was c a r r i e d out on t h r e e p l a n t p a r t s : i n d i v i d u a l e l o n g a t e d shoots w i t h l e a v e s a t t a c h e d (LS & LVS), masses of 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 shoots ( S S ) , and r o o t s ( R ) . The f r e s h w e i g h t s of the shoots and r o o t s were u s u a l l y l e s s than those of the masses of a d v e n t i t i o u s t i s s u e s . A l l a v a i l a b l e a d v e n t i t i o u s t i s s u e s from each p l a n t l e t was d i s s e c t e d i n t o s e c t i o n s of a p p r o x i m a t e l y equal s i z e , w i t h v a r y i n g f r e s h w e i g h t s . A t o t a l of 30 e l o n g a t e d s h o o t s , 39 masses of a d v e n t i t i o u s t i s s u e s , and r o o t s from two of the p l a n t l e t s were t e s t e d . ELISA absorbance v a l u e s were measured 90 minutes a f t e r s u b s t r a t e a d d i t i o n f o r the p l a n t l e t d i s s e c t e d at 21 weeks, and 2 hours and 20 minutes a f t e r s u b s t r a t e f o r the o t h e r t h r e e p l a n t l e t s . The f i r s t d i s t r i b u t i o n study was expanded u s i n g samples of p l a n t p a r t s were taken from f i v e a d d i t i o n a l A M V - i n f e c t e d and f i v e G F L V - i n f e c t e d p l a n t l e t s growing at 25°C. The elongated shoot samples, which were a l l longer than 20 mm, were t e s t e d i n two s e c t i o n s , the stem without leaves (LS) and the leaves (LVS). ELISA absorbance values were measured 2 hours f o l l o w i n g s u b s t r a t e a d d i t i o n . The same p l a n t p a r t s were a l s o t e s t e d i n 10 AMV-infected and 10 GFLV-infected p l a n t l e t s which had been growing at 20°C f o r 4 months. Some of the elongated shoots were once again t e s t e d i n two p a r t s , while others were t e s t e d as complete e n t i t i e s (LS & LVS). ELISA absorbance values were measured 2 hours f o l l o w i n g s u b s t r a t e a d d i t i o n . E f f e c t of temperature and BAP c o n c e n t r a t i o n . P l a n t l e t s i n f e c t e d with AMV and GFLV were su b j e c t e d to v a r i o u s growth c a b i n e t temperatures and c o n c e n t r a t i o n s of BAP. I n f e c t e d and healthy p l a n t l e t s were subdivided i n t o approximately e q u a l - s i z e d parts and placed i n t o 25 mm c u l t u r e tubes, with a s u b c u l t u r e from each o r i g i n a l mother p l a n t l e t being s u b j e c t e d to each experimental f a c t o r l e v e l . A l l temperatures were constant to w i t h i n + 1°C. The p l a n t s were grown under a 16 hour photoperiod s u p p l i e d by c o o l white f l u o r e s c e n t tubes at 50 + 5 <vE m~2 s e c " 1 f o r the AMV and 75 t 5 uE m - 2 s e c - 1 f o r the GFLV experiments. T i s s u e was ground i n 10 volumes of standard g r i n d i n g b u f f e r using a V i r t i s p o l y t r o n t i s s u e g r i n d e r . A l l s u b c u l t u r e s from each mother p l a n t l e t were t e s t e d on the same ELISA p l a t e . , A n a l y s i s of va r i a n c e and r e g r e s s i o n analyses were c a r r i e d out on b u f f e r - c o r r e c t e d absorbance values 2 hours a f t e r s u b s t r a t e a d d i t i o n , using the average readings from three w e l l s l o c a t e d randomly on the p l a t e . 34 Two experiments were c a r r i e d out on AMV-infected p l a n t s . In the f i r s t , three groups of 25 p l a n t l e t s were placed at 20°, 25° and 30°C, w i t h i n which BAP was added to the medium at 2, 4, and 8 mg/l. In the second experiment, groups of 25 p l a n t s were put at 15°, 20°, 25°, and 30°C with 2, 4, and 8 mg/l of BAP t e s t e d at the 15° and 20°C temperatures. Each experiment l a s t e d 2 months. Half of each p l a n t l e t was r e c u l t u r e d a f t e r one month. ELISA t e s t i n g of AMV-infected p l a n t l e t s was c a r r i e d out at the completion of the f i r s t experiment and at 1 and 2 months during the second. Tissue samples having a f r e s h weight of 25 mg were ground a f t e r 1 month while 1 gram samples were used at the completion of each experiment. Groups of twenty GFLV-infected p l a n t l e t s were su b d i v i d e d and put at 20°, 25°, and 30°C with 2 mg/l of BAP fo r 1 month. T i s s u e samples weighing 35 ± 5 mg were t e s t e d from each c u l t u r e . ELISA.colour development was measured 21 hours a f t e r s u b s t r a t e a d d i t i o n , f o l l o w i n g i n c u b a t i o n at 4°C o v e r n i g h t , because of a slowly developing r e a c t i o n . V i r u s recovery. Nine in vitro p l a n t l e t s which had been grown at 30°C ( t r e a t e d ) f o r 2 months during the f i r s t temperature-BAP experiment were d i v i d e d i n two and moved to 20°C and 25°C. They were r e c u l t u r e d r e g u l a r l y and AMV d e t e c t i o n by ELISA was c a r r i e d out 4, 10, 16, 26, and 32 weeks l a t e r . S i m i l a r l y , s i x p l a n t l e t s were t r a n s f e r r e d to 20°C and 25°C a f t e r the second temperature experiment and t e s t e d a f t e r 6, 16, and 20 weeks. Samples weighed at l e a s t 0.2 g on the f i r s t two dates while 0.5 g samples were t e s t e d on the l a s t date. V i r u s - i n f e c t e d and healthy c o n t r o l p l a n t l e t s o r i g i n a t i n g from the same mother p l a n t s but which had never been su b j e c t e d to 30°C were grown a l o n g s i d e the t r e a t e d c u l t u r e s and a l s o t e s t e d . Shoots longer than 2 cm were rooted in vitro from both the t r e a t e d and c o n t r o l c u l t u r e s 10 and 6 weeks, r e s p e c t i v e l y , a f t e r each experimental group was moved to the lower temperatures. These p l a n t s were moved in stages from the tubes to a growth room with f l u o r e s c e n t l i g h t i n g and then to the greenhouse where they were kept under ambient (approximately 25°C) c o n d i t i o n s . F i f t e e n t r e a t e d p l a n t s produced from the f i r s t l o t were t e s t e d f o r AMV, by ELISA, 10 and 17 weeks a f t e r being removed from c u l t u r e , while 13 p l a n t s from the second group were t e s t e d a f t e r 5 1/2 weeks. These dates corresponded to 6 1/2 and 8 months of elapsed time post treatment at 30°C f o r the f i r s t group and 5 months fo r the second. Young, newly-opened leaves from these p l a n t s were ground i n the standard ELISA g r i n d i n g b u f f e r c o n t a i n i n g 1% n i c o t i n e . V i r u s i n h i b i t i o n at 30°C. In an attempt to see how q u i c k l y v i r u s d e t e c t i o n was a f f e c t e d by growth at 30°C, e i g h t AMV-infected p l a n t l e t s which had been d i v i d e d i n h a l f and grown at 20°C and 25°C f o r 6 weeks, were subdivided i n t o four parts and placed at 30°C i n a growth chamber. V i r u s d e t e c t i o n by ELISA was c a r r i e d out at the onset of t h i s experiment and weekly t h e r e a f t e r . Samples t e s t e d from each p l a n t l e t weighed at l e a s t 0.2 g, with samples on the l a s t date weighing more than 0.5 g. In an attempt to s t a n d a r d i z e r e s u l t s over time, the same c o n j u g a t e was used on a l l t e s t i n g d a t e s . Pure v i r u s , d i l u t e d to a c o n c e n t r a t i o n of 100 ng/ml i n C. quinoa f o l i a g e ground i n 10 volumes of b u f f e r , was used as a c o n t r o l to measure d e t e c t i o n s e n s i t i v i t y of each t e s t d a t e . V i r u s d e t e c t i o n i n f i e l d p l a n t s Leaf samples were taken from v i r u s - i n f e c t e d and h e a l t h y f i e l d - g r o w n and screenhouse-grown g r a p e v i n e s on f i v e d a t e s d u r i n g the summer of 1986. The screenhouse samples were ta k e n from p l a n t s i n f e c t e d w i t h one o f s i x GFLV sources or t h r e e AMV s o u r c e s . The GFLV so u r c e s c o n s i s t e d of " m i l d " and " s e v e r e " s t r a i n s from O n t a r i o , i n the v a r i e t i e s R i e s l i n g ( V. vinifera L.) and P i n o t Chardonnay ( V. vinifera L . ) , r e s p e c t i v e l y ; v e i n b a n d i n g and d e f o r m i n g s t r a i n s from C a l i f o r n i a , i n the v a r i e t y ' S t . George ( V. rupestris du L o t ) ; and two sources from France, i n two unknown V. vinifera v a r i e t i e s . Two of the AMV sources came from France, i n two unknown V. vinifera v a r i e t i e s , w i t h the o t h e r r e c e i v e d from West Germany, i n the v a r i e t y R o t b e r g e r ( V. vinifera L ) . V i r u s - i n f e c t e d f i e l d samples were taken from the v a r i e t i e s LN-33 ( V. vinifera L. x Couderc 1613), S t . George, and P i n o t N o i r ( V. vinifera L . ) , which had been chip-bud i n o c u l a t e d 2 y e a r s e a r l i e r w i t h one of the f i r s t f o u r GFLV v i r u s s o u r c e s or one of the f i r s t two AMV s o u r c e s . H e a l t h y c o n t r o l samples were taken from n o n - i n o c u l a t e d LN-33 and S t . George f i e l d p l a n t s . 37 Young, f u l l y opened leaves from the growing t i p s of three to four shoots were taken from each grapevine sampled. A f t e r removal, the leaves were kept at or near 4°C u n t i l t e s t e d , w i t h i n the next 3 days. The youngest leaves were cut i n h a l f , d i c e d i n t o 2-3 mm squares with a k n i f e , mixed thoroughly, and weighed i n t o four a l i q u o t s of 0.1 g each. The a l i q u o t s were ground with a g l a s s rod on a p o r c e l a i n spot p l a t e i n 10 volumes of each of four b u f f e r s : A - 0.15 M PBS, pH 8.4 with 0.05% Tween-20, 0.2% ovalbumin and 2% PVP, B - b u f f e r A, pH 8.4 with 1% n i c o t i n e added, C - standard 0.01 M PBS g r i n d i n g b u f f e r , pH 7.4 with 0.05% Tween-20, 0.2% ovalbumin and 2% PVP, or D - standard b u f f e r C, pH 8.2 with 1% n i c o t i n e added. The n i c o t i n e was f r e s h l y added to the b u f f e r s at the s t a r t of each day and again 3 to 4 hours l a t e r . The ground samples were put i n t o three w e l l s which had been randomly s e l e c t e d using tables of random permutations (Moses and Oakford, 1963); A l l four g r i n d i n g b u f f e r s used on each plant were put on one p l a t e . As w e l l , f o r each b u f f e r , each p l a t e had three b u f f e r c o n t r o l w e l l s and three h e a l t h y c o n t r o l w e l l s . The r e a c t i o n colour was read a f t e r 1 and 2 hours. In most cases, the 2 hour reading was used unless readings exceeding 2.0 absorbance u n i t s were observed a f t e r 1 hour. The p l a t e s were stan d a r d i s e d before a n a l y s i s by s u b t r a c t i n g the absorbance values of the b u f f e r w e l l s f o r each b u f f e r . A n a l y s i s of varia n c e was performed, followed by treatment mean comparison using the p r o t e c t e d l e a s t s i g n i f i c a n t d i f f e r e n c e (LSD) procedure which was a p p l i e d 38 only if the overall F test for treatments was significant. RESULTS ELISA s e n s i t i v i t y The most d i l u t e c o n c e n t r a t i o n s of p u r i f i e d AMV and GFLV i n standard g r i n d i n g b u f f e r which gave an ELISA absorbance read i n g at l e a s t twice that of he a l t h y C. quinoa leaves ground i n the same b u f f e r was 10 ng/ml f o r AMV and 125 ng/ml f o r GFLV. V i r u s d e t e c t i o n i n t i s s u e - c u l t u r e d p l a n t l e t s AMV and GFLV d e t e c t i o n . AMV and GFLV were not d e t e c t e d in every p l a n t l e t grown at 25°C on every t e s t date even though background absorbance values i n p l a n t l e t s s t a r t e d from healthy f i e l d p l a n t s were very low 2 hours a f t e r adding the s u b s t r a t e (Table II)..'A few AMV-infected p l a n t l e t s always te s t e d p o s i t i v e . Only 14 (58%) of a l l p l a n t l e t s t e s t e d twice and 10 (21%) of those t e s t e d three times were always p o s i t i v e . The v i r u s was detected i n e i g h t other p l a n t l e t s t e s t e d only once. The cumulative number of p l a n t l e t s t e s t i n g p o s i t i v e f o r v i r u s increased with the number of times t e s t e d . AMV was e v e n t u a l l y detected i n a l l but one p l a n t l e t s t a r t e d from the f i r s t two i n i t i a t i o n dates. T h i s p l a n t l e t d i e d at 12 weeks, a f t e r being t e s t e d only once. The v i r u s was not det e c t e d i n three p l a n t l e t s from the t h i r d i n i t i a t i o n date. Each of Table I I . D e t e c t i o n by ELISA of 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 (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 at 25°C I n i t i a t i o n Date t e s t e d (weeks P o s i t i v e s per C u m u l a t i v e date post i n i t i a t i o n ) t e s t i n g d a t e 1 p o s i t i v e s A) AMV June 12 J u l y 12 August 26 B) GFLV August 26 Sept. 4 (12) Oct. 2 (16) Nov. 20 (23) Oct. 2 (12) Nov.15 (18) Oct. 21 (9) Nov.10 (12) Dec. 29 (19) 12/21 9/24 25/25 8/14 13/13 20/36 17/34 23/33 T o t a l Nov. 17 (13) 13/36 12/21 18/27 27/27 8/14 15/16 20/36 26/37 34/37 76/80 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 than 0.05. Denominator = t o t a l number of p l a n t l e t s t e s t e d . t h e s e t h r e e c u l t u r e s had been t e s t e d t h r e e t i m e s . The v i r u s was l a t e r d e t e c t e d i n two of these p l a n t l e t s 34 weeks post i n i t i a t i o n , a f t e r growing i n a c a b i n e t w i t h a c o n s t a n t a i r temperature of 20°C f o r 6 weeks. The o t h e r p l a n t l e t d i e d b e f o r e b e i n g p l a c e d at t h i s temperature. AMV was not always d e t e c t e d i n p l a n t l e t s f o l l o w i n g the i n i t i a l d e t e c t i o n ; d e t e c t i o n b e i n g c o n s i s t e n t i n o n l y 11 of 29 p l a n t l e t s t e s t e d t w i c e more f o l l o w i n g i n i t i a l d e t e c t i o n , and i n o n l y 18 of 21 t e s t e d once more. The v i r u s was not d e t e c t e d at a l l i n o n l y one p l a n t l e t t e s t e d t w i c e f o l l o w i n g i n i t i a l d e t e c t i o n , w h i l e growing at 25°C. 41 Only 13 of 36 p l a n t l e t s t e s t e d p o s i t i v e f o r GFLV a f t e r 13 weeks. Subsequent t e s t i n g a f t e r p l a n t l e t t r a n s f e r to 20°C permitted d e t e c t i o n e v e n t u a l l y i n a l l p l a n t l e t s . AMV and GFLV d e t e c t i o n was reduced d r a s t i c a l l y a f t e r the p l a n t l e t s had been placed at the higher f l u c t u a t i n g temperatures f o r 7 weeks (Table I I I ) . Based on v i s u a l e v a l u a t i o n of ELISA t e s t p l a t e s 2 hours a f t e r s u b s t r a t e a d d i t i o n , v i r u s was detected i n only 8 of 60 AMV-infected p l a n t l e t s from the three i n i t i a t i o n dates and i n none of the 32 GFLV-infected p l a n t l e t s . P o s i t i v e c o n t r o l s , c o n s i s t i n g of samples from i n f e c t e d C. quinoa, a l l r e a c t e d p o s i t i v e l y a f t e r 2 hours, while healthy p l a n t l e t s and herbaceous c o n t r o l p l a n t s and b u f f e r w e l l s d i d not develop any c o l o u r . Table I I I . D e t e c t i o n of a r a b i s mosaic v i r u s (AMV) and grapevine 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 at v a r i o u s temperatures from January 3, 1986 to September 17,1986 Growth room Duration at Tes t temperature temperature P o s i t i v e s per date (°C) (wks) V i r u s t e s t date Feb. 20 25 to 29 7 AMV 8/60 GFLV 0/32 Mar. 29 25 5 AMV 0/29 GFLV 0/33 May 10 20 6 AMV 28/29 GFLV 31/33 Sept. 16 i it 17 20 25 AMV 19/21 GFLV 30/33 Numerator = p l a n t l e t s with ELISA absorbance readings g r e a t e r than 0.05 u n i t s . Denominator = t o t a l number of p l a n t l e t s t e s t e d . The e f f e c t of the h i g h e r temperature on v i r u s d e t e c t i o n , a l t h o u g h not permanent, was not e a s i l y r e v e r s e d ( T a b l e I I I ) . V i r u s was not d e t e c t e d i n any of the 29 A M V - i n f e c t e d and 33 G F L V - i n f e c t e d p l a n t l e t s from the t h i r d i n i t i a t i o n date which were t e s t e d 5 weeks a f t e r b e i n g r e t u r n e d to 25°C. However, these v i r u s e s became d e t e c t a b l e once a g a i n a f t e r moving the p l a n t l e t s t o 20°C f o r 6 weeks; w i t h 28 of the 29 AMV-infected and 31 of the 33 G F L V - i n f e c t e d p l a n t l e t s becoming p o s i t i v e . V i r u s 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 p l a n t s b e i n g grown at 20°C as w e l l as at 25°C (Table I I I ) . When 21 A M V - i n f e c t e d and 33 G F L V - i n f e c t e d p l a n t l e t s from the t h i r d i n i t i a t i o n date were t e s t e d a g a i n 25 weeks a f t e r b e i n g moved to 20°C, o n l y 19 of the AMV and 30 of the GFLV p l a n t l e t s were p o s i t i v e 2 hours a f t e r s u b s t r a t e a d d i t i o n . A l l f i v e of these n e g a t i v e p l a n t l e t s had p r e v i o u s l y t e s t e d p o s i t i v e by ELISA 6 w e e k s . a f t e r b e i n g p l a c e d at 20°C. A l s o , the t h r e e p l a n t l e t s i n which the v i r u s e s had not been d e t e c t e d i n the p r e v i o u s assay t e s t e d p o s i t i v e by ELISA a f t e r 25 weeks. TomRV d e t e c t i o n in vitro. TomRV was d e t e c t e d i n a l l but one p l a n t l e t o r i g i n a t i n g from i n f e c t e d greenhouse p l a n t s ( T a b l e I V ) . The p l a n t l e t i n which the v i r u s was not d e t e c t e d showed poor growth so t h a t o n l y l e a v e s were sampled. The mean ELISA absorbance v a l u e f o r samples from p l a n t l e t s s t a r t e d from h e a l t h y p l a n t s was 0.00 f o r both t e s t i n g d a t e s . 43 Table IV. D e t e c t i o n of 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 at 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 i n P o s i t i v e s 1 I n i t i a t i o n c u l t u r e (wks) 1»* 18 9/9 25 6/6 2 n A 15 8/9 Numerator = p l a n t l e t s with ELISA absorbance readings g r e a t e r than 0.05 u n i t s . Denominator = t o t a l number of p l a n t l e t s t e s t e d . V i r u s d i s t r i b u t i o n . AMV was found to be non-uniformly d i s t r i b u t e d w i t h i n the f i r s t four d i s s e c t e d p l a n t l e t s on two subsequent days ( F i g u r e 1). In one p l a n t l e t , p l a n t A, the v a r i a b i l i t y i n v i r u s c o n c e n t r a t i o n , as i n d i c a t e d by ELISA absorbance values, was very pronounced with some p l a n t l e t s e c t i o n s having values 4 times g r e a t e r than than o t h e r s . V i r u s d e t e c t i o n was l e s s dependable i n samples of elongated t i s s u e s than other types (Table V). Three elongated t i s s u e samples from p l a n t l e t C and one from p l a n t l e t D had ELISA absorbance values l e s s than 0.05 o p t i c a l u n i t s . The other negative sample from p l a n t l e t D c o n s i s t e d of a piece of n e c r o t i c p r o l i f e r a t i n g t i s s u e . The lowest reading from p l a n t l e t B (absorbance = 0.08) was als o , found i n a long shoot. The mean ELISA absorbance values were higher f o r SS t i s s u e than f o r LS & LVS t i s s u e even when the low values f o r the n e c r o t i c sample was i n c l u d e d . The g r e a t e s t mean absorbance value was found i n the R t i s s u e . 44 F i g u r e 1. Frequency h i s t o g r a m of 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 cut from each of the 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 V. Arabis mosaic v i r u s d e t e c t i o n by ELISA in v a r i o u s p l a n t parts taken from four p l a n t l e t s grown at 25°C P o s i t i v e s per p l a n t p a r t 1 P l a n t l e t LS & LVS* SS R T o t a l s A B C D 7/7 6/6 8/11 5/6 14/14 6/6 4/4 15/16 1/1 3/3 21/21 12/12 13/16 23/25 T o t a l s Mean ELISA values 26/30 0 . 59 39/40 0 .73 4/4 1.11 69/74 'Numerator = p a r t s with absorbance readings g r e a t e r than 0.05. Denominator = t o t a l number of p a r t s t e s t e d . = not t e s t e d aLS & LVS = shoots 20 mm or longer with leaves attached. SS = masses of 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 with shoots 10 mm or less i n length. R = root t i s s u e . V i r u s c o n c e n t r a t i o n a l s o v a r i e d f o r samples taken from s i m i l a r p l a n t parts w i t h i n ' t h e same p l a n t l e t s (Figure 2). Samples of SS from p l a n t l e t A had both the lowest and h i g h e s t absorbance values f o r that p l a n t . For the other three p l a n t l e t s , a wider range of values was observed i n LS 6 LVS t i s s u e s than f o r samples from other p l a n t p a r t s , with both the highest and lowest values f o r p l a n t l e t s D and B being found i n these t i s s u e s . For a l l four p l a n t l e t s , 6 of 30 samples of LS & LVS and 7 of 37 samples from SS t i s s u e s had absorbance values less than 0.10. Only 5 of 30 samples of LS & LVS and 10 of 37 samples of SS t i s s u e s , a l l from p l a n t l e t A had absorbance values g r e a t e r than 1.0 u n i t s . 46 20 10 30°C 0.5 1.0 1.5 2.0 ABSORBANC E(405nm) F i g u r e 2. Frequency h i s t o g r a m of a r a b i s mosaic 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 i n f e c t e d p l a n t l e t s : LS & LVS= samples o f e l o n g a t e d shoots w i t h l e a v e s a t t a c h e d , SS = samples taken 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 r o o t t i s s u e . AMV was detected i n a l l R samples. By comparison, mean ELISA absorbance values f o r t i s s u e from healthy p l a n t l e t s was 0.015 on the f i r s t date and 0.00 on the second. When compared, no apparent r e l a t i o n s h i p was found between the f r e s h weight of i n d i v i d u a l p l a n t p a r t s of the same type and the observed ELISA absorbance v a l u e . As an example, f o r p l a n t A, w i t h i n the 0.05 to 0.06 gm weight range, there were three samples of p r o l i f e r a t i n g t i s s u e with absorbance values g r e a t e r than 2.0, three between 1.0 and 2.0, and two l e s s than 1.0, while t i s s u e weighing 0.015 g and 0.561 g gave values of 0.787 and 0.125, r e s p e c t i v e l y . When the next f i v e p l a n t l e t s growing at 25°C were sampled, the same r e l a t i o n s h i p was found between v i r u s d e t e c t i o n and the p l a n t part sampled (Table V I ) . Both AMV and GFLV were d e t e c t e d p o o r l y i n samples taken from LS shoots. V i r u s d e t e c t i o n was lower i n LVS removed from the stems and t e s t e d i n d i v i d u a l l y . The v i r u s e s were d e t e c t e d c o n s i s t e n t l y i n SS and R samples from p l a n t l e t s where LS and LVS samples t e s t e d n e g a t i v e . The highest ELISA absorbance reading obtained from h e a l t h y samples was 0.00. The d i s t r i b u t i o n of AMV and GFLV in p l a n t l e t s growing at 20°C was d i f f e r e n t than f o r p l a n t l e t s at 25°C (Table V I I ) . Both AMV and GFLV were more e a s i l y d etected i n LS, LVS, and LS & LVS samples, with only one sample of LS & LVS being negative f o r AMV. The next lowest absorbance reading f o r samples from elongated t i s s u e from AMV-infected Table V I . D e t e c t i o n of 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 (GFLV) by ELISA i n v a r i o u s p a r t s of f i v e p l a n t l e t s grown at 25°C P o s i t i v e s per p l a n t p a r t 1 V i r u s P l a n t l e t L S 2 LVS SS R T o t a l AMV 1 0/1 0/1 2/2 1/1 3/5 2 1/1 1/1 1/1 1/1 4/5 3 1/1 1/1 2/2 1/1 5/5 4 0/1 0/1 3/3 1/1 4/6 5 1/1 0/1 2/2 1/1 4/5 T o t a l s 3/5 2/5 10/10 6/6 21/26 Mean ELISA v a l u e s 0.61 0 . 48 0 . 98 1 . 54 GFLV 1 1/1 1/1 2/2 1/1 5/5 2 1/1 0/1 2/2 1/1 4/5 3 1/1 0/1 2/2 1/1 4/5 4 2/2 0/2 1/1 1/1 4/6 5 1/1 1/1 3/3 1/1 6/6 T o t a l s 6/6 2/6 10/10 5/5 23/27 Mean ELISA v a l u e s 0 . 24 0 .07 0 . 46 0 . 44 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 than 0.05. Denominator = t o t a l number of p a r t s t e s t e d . 2 L S = shoots 20 mm or l o n g e r w i t h leaves removed. LVS = le a v e s removed from the 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 shoots 10 mm or l e s s i n l e n g t h . R = r o o t t i s s u e . p l a n t l e t s was 0.15, found i n a LVS sample. The lowest r e a d i n g f o r any sample of e l o n g a t e d t i s s u e from G F L V - i n f e c t e d p l a n t l e t s was 0.21. Table VII. D e t e c t i o n of a r a b i s mosaic v i r u s (AMV) and grapevine f a n l e a f v i r u s (GFLV) by ELISA i n va r i o u s parts of 10 p l a n t l e t s grown at 20°C P o s i t i v e s per p l a n t p a r t 1 V i r u s P l a n t l e t L S 2 LVS LS & LVS SS R T o t a l ! AMV 1 3/3 3/3 _ _ _ 2/2 1/1 9/9 2 1/1 2/2 1/1 4/4 3 1/1 1/1 3/3 1/1 6/6 4 3/3 1/1 4/4 5 1/1 1/1 1/1 3/3 6/6 6 1/1 1/1 1/1 3/3 1/1 7/7 7 1/1 4/4 1/1 6/6 8 3/3 1/1 4/4 9 1/1 1/1 1/2 2/2 1/1 6/7 10 2/2 1/1 3/3 T o t a l s 7/7 7/7 7/8 26/26 8/8 55/56 Mean ELISA 0 . 70 1 . 19 0 . 83 0.93 0 . 42 GFLV 1 2/2 2/2 2/2 1/1 7/7 2 2/2 3/3 1/1 6/6 3 1/1 3/3 1/1 5/5 4 1/1 1/1 3/3 1/1 6/6 5 1/1 1/1 3/3 1/1 6/6 6 1/1 1/1 3/3 1/1 6/6 7 1/1 1/1. 1/1 1/2 1/1 5/6 8 1/1 1/1 1/1 2/3 0/1 5/7 9 2/2 2/2 2/2 1/1 7/7 10 1/1 1/1 1/1 4/4 1/1 8/8 T o t a l s 10/10 10/10 6/6 26/28 9/10 61/64 Mean ELISA 0.35 0 . 57 0 . 79 0.15 0.14 'Numerator = p a r t s with ELISA values Denominator = t o t a l number of p a r t s g r e a t e r tes ted. than 0 .05. = not t e s t e d . 2 L S = shoots 20 mm or longer with leaves removed. LVS = leaves removed from the above LS. LS & LVS = complete long shoot with leaves. SS = masses of 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 with shoots 10 mm or l e s s i n l ength. R = root t i s s u e . V i r u s d e t e c t i o n in SS and R was d i f f e r e n t f o r the two v i r u s e s i n p l a n t l e t s growing at 20°C. Whereas AMV was f a i r l y e a s i l y detected i n a l l SS and R samples (lowest ELISA absorbance value f o r SS = 0.37, lowest f o r R = 0.19), GFLV was more d i f f i c u l t to detect i n these p l a n t p a r t s . Of the SS samples taken from three GFLV-infected p l a n t l e t s (nos. 5, 7, and 8), f i v e had absorbance values lower than 0.1 with two of these being lower than 0.05. Also , the R samples from p l a n t l e t s 5, 8, and 10 had ELISA readings l e s s than 0.1, with one value l e s s than 0.05. Two of these p l a n t l e t s , nos. 5 and 8, a l s o had the low readings i n SS m a t e r i a l . E f f e c t of temperature and BAP c o n c e n t r a t i o n . I n c r e a s i n g growth room temperatures from 15°C to 30°C caused a r e d u c t i o n i n mean absorbance readings of ELISA t e s t s c a r r i e d out on AMV- and GFLV-infected p l a n t l e t s ( F i g u r e 3). This e f f e c t came about w i t h i n 1 month. Increasing the BAP c o n c e n t r a t i o n i n the p r o l i f e r a t i o n medium a f f e c t e d the mean ELISA, absorbance readings only i n p l a n t l e t s growing at 20°C ( F i g u r e 4 ) . In the f i r s t experiment, using a t h r e s h o l d value of 0.05 absorbance u n i t s , AMV was e a s i l y detected i n a l l the p l a n t l e t s growing at 20°C, r e g a r d l e s s of BAP c o n c e n t r a t i o n ( F i g u r e 5). Even though absorbance values were lower at 25°C, a l l p l a n t l e t s had s u f f i c i e n t v i r u s t i t r e s f o r d e t e c t i o n . The lowest absorbance value found at 25°C was 0.097. However, the v i r u s was undetectable i n any p l a n t l e t s at 30°C with a l l 75 p l a n t l e t s having absorbance values below 51 F i g u r e 3 . Mean ELISA absorbance values with one standard d e v i a t i o n f o r t e s t s on a r a b i s mosaic v i r u s (AMV) and grapevine f a n l e a f v i r u s ( G F L V ) - 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 at d i f f e r e n t a i r temperatures and 2 mg/1 N - 6-benzylaminopurine: A - f i r s t experiment on AMV-infected p l a n t l e t s , 2 months a f t e r s t a r t ; B - second experiment on AMV-infected p l a n t l e t s , 1 month a f t e r s t a r t ; C - second experiment, 2 months a f t e r s t a r t ; D - GFLV-infected p l a n t l e t s , 1 month a f t e r s t a r t . 52 F i g u r e 4 . Mean ELISA absorbance readings with one standard d e v i a t i o n of t e s t s on 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 grown at three N-6-benzylaminopurine (BAP) c o n c e n t r a t i o n s f o r 2 months at 20°C : A - f i r s t experiment; B - second experiment. F i g u r e 5. Frequency histogram of ELISA absorbance readings from t e s t s on 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 grown f o r 2 months at three N-6-benzylaminopurine (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 of three temperatures (20°C, 25°C, and 30°C). 54 0.02 and nine of these having values below 0.00. Samples from healthy p l a n t l e t s submitted to the same temperatures and BAP c o n c e n t r a t i o n s had a mean absorbance value of 0.005 with a maximum value of 0.02. ELISA readings f o r p l a n t l e t s at 30°C were a l l below 0.05 absorbance u n i t s . The a n a l y s i s of v a r i a n c e of the r e s u l t s f o r t h i s experiment (Table VIII) i n d i c a t e s an i n t e r a c t i o n between temperature and BAP c o n c e n t r a t i o n . However, temperature has a f a r grea t e r e f f e c t on v i r u s d e t e c t i o n than BAP. Table V I I I . Mean ELISA absorbance values and a n a l y s i s of v a r i a n c e of temperature and N-6-benzylaminopurine (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 ( f i r s t experiment) BAP (mg/1) Temperature 2 4 8 Mean 20 0 , .80 0 . 84 0 .96 0 . 86 25 0 . 40 0 . 42 0 . 37 0 . 40 30 0 . 00 0 . 00 0 . 00 0 .00 Mean 0 . 40 0 .42 0 . 44 Sources of v a r i a t ion df SS P>F P l a n t l e t 24 Temperature 2 BAP 2 Temperature x BAP 4 0. 59 27.83 0 . 07 0.31 1.95 1112.50 2.82 6.25 0 . 0071 0.0000 0.0618 0.0001 Regression a n a l y s i s on the temperature e f f e c t at a BAP c o n c e n t r a t i o n of 2 mg/l y i e l d e d a l i n e a r equation where absorbance u n i t s = 2.381 - 0.079 temperature (T) f o r a 90.2 % r e d u c t i o n i n t o t a l sums of square (Table IX). Inc r e a s i n g the BAP c o n c e n t r a t i o n r e s u l t e d i n a s t a t i s t i c a l l y s i g n i f i c a n t i n c r e a s e i n v i r u s c o n c e n t r a t i o n only at 20°C ( F i g u r e 6). Regression a n a l y s i s on the r e s u l t s of BAP e f f e c t at 20°C gave a l i n e a r equation with absorbance = 0.738 + 0.027 BAP f o r a 15.3% r e d u c t i o n i n t o t a l sums of squares (Table IX). S i m i l a r r e s u l t s on the e f f e c t of growth room temperature on ANV c o n c e n t r a t i o n s i n p l a n t l e t s were obtained with the second experiment (Table X). V i r u s c o n c e n t r a t i o n was reduced i n p l a n t l e t s as temperature was inc r e a s e d , even a f t e r only 1 month. A n a l y s i s of v a r i a n c e of the r e s u l t s a f t e r 1 month i n d i c a t e d that 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 e f f e c t on v i r u s d e t e c t i o n at 20°C but that temperature e f f e c t at 2 mg/l of BAP was s i g n i f i c a n t (P = 0.01). Regression a n a l y s i s f i t t e d the q u a d r a t i c equation ELISA absorbance = 8.866 - 0.597 T + 0.010 T 2 to the data f o r a 52.9% r e d u c t i o n i n t o t a l sums of squares (Table IX). Although s t a t i s t i c a l l y s i g n i f i c a n t f o r the group of p l a n t l e t s , temperature e f f e c t s on AMV d e t e c t i o n by ELISA i n i n d i v i d u a l p l a n t s a f t e r 1 month was not uniform f o r a l l p l a n t l e t s at a BAP c o n c e n t r a t i o n of 2 mg/l (Figure 7). While a l l p l a n t l e t s growing at 30°C had absorbance values l e s s than 0.05, u n l i k e the f i r s t experiment, AMV was not detected i n a l l p l a n t l e t s at the two lower 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 in plantlets Sources of % reduction variation d.f. SS in total SS A) AMV - experiment 1, BAP effect at 20°C Linear 1 0.34 15.3 Residual 73 1.89 Regression equation: absorbance = 0.738 + 0.027 BAP, R2 = 0.392 B) AMV - experiment 1, temperature effect, 2 months at 2 mg/1 BAP Linear 1 7.85 90.2 Residual 73 0.85 Regression equation: absorbance = 2.381 - 0.07926 T, R2 = 0.95 C) AMV - experiment 2, temperature effect, 1 month at 2 mg BAP Linear 1 11.17 52.9 Quadratic 1 1.05 57.9 Residual 72 8.88 Regression equation: absorbance = 8.866 - 0.59688 T + 0.01005 T 2, R2 = 0.761 D) AMV - experiment 2, temperature effect, 2 months at 2 mg/1 BAP Linear 1 58.41 66.0 Quadratic 1 2.15 68.4 Residual 81 28.00 Regression equation: absorbance = 7.362 - 0.437255 T + 0.006402 T 2, R2 = 0.827 E) AMV - experiment 2, BAP effect, 2 months at 20°C Linear 1 6.22 17.5 Residual 70 29.30 Regression equation: absorbance = 0.923 + 0.1179 BAP, R2 = 0.419 F) GFLV - effect of temperature after 1 month Linear 1 3.35 39.3 Quadratic 1 0.80 48.6 Residual 57 8.53 Regression equation: absorbance = 7.606 - 0.54658 T + 0.009774 T 2, R2 = 0.697 57 1.0 < 0.0- • BAP(mg/L) F i g u r e 6. Mean ELISA absorbance values from the f i r s t experiment to determine the e f f e c t s of growing a r a b i s mosaic v i r u s - i n f e c t e d at three temperatures 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-benzylaminopurine (BAP) c o n c e n t r a t i o n s (» — » = 20°C, + + = 25°C, • •= 30°C). 58 Table X. Mean absorbance values of ELISA t e s t s c a r r i e d out on 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 1 and 2 months i n t o the second experiment to t e s t the e f f e c t of temperature and N-6-benzylaminopurine (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 (mg/l) Temperature . (°C) 2 4 8 A) 1 month 20 0. ,95 1. . 00 1 . 07 25 0 . 23 . - 1 30 0 . , 00 B) 2 months 15 2 . 24 2. . 49 2.33 20 1 . . 20 1. .35 1 .90 25 0 .44 30 0 . 01 " ~ i TweIve not t e s t e d , of a l l the p l a n t l e t s growing at 20°C and 25°C gave negative ELISA r e s u l t s a f t e r 1 month and three more had absorbance values l e s s than 0.1. By comparison, the highest value found i n the p l a n t l e t s growing at 30°C was 0.01. The mean absorbance value of healthy t i s s u e a f t e r 1 month was 0.00 with a maximum value of 0.01. The i n h i b i t i n g e f f e c t of higher temperatures on AMV d e t e c t i o n by ELISA at 2 mg/l of BAP was s t i l l evident i n the second experiment a f t e r 2 months (Figure 8). AMV was undetectable i n 24 of 25 p l a n t l e t s at 30°C, with the other p l a n t l e t having an absorbance value of 0.13. The v i r u s , however, was a l s o undetectable i n some p l a n t l e t s growing at 15°C, 20°C, and 25°C. Mean v i r u s c o n c e n t r a t i o n was g r e a t e s t i n p l a n t l e t s 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 of N-6-benzylaminopurine. 60 26 Z < u. 0 ° 26 15° C I I | • I l • | • • • l | l l l T | s D Z 2 5 ° C *4 2 0 ° C I 1 f t , . , i 1 1 1 * i - p T r " 2.0 3 0 ° C i 1 1 • 1 i • • • ' i 1 • • • i 3.0 0.0 1.0 A B S O R B A N C E (405nm) 2.0 3.0 F i g u r e 8. 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 i n p l a n t l e t s grown f o r 2 months at 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-benzylaminopurine c o n c e n t r a t i o n of 2 mg/l - second exper iment. 61 growing at 15°C (Table X). The mean ELISA rea d i n g from n o n - i n f e c t e d p l a n t l e t s was 0.00 a f t e r 2 months with a maximum value of 0.02. A n a l y s i s of v a r i a n c e on the r e s u l t s a f t e r 2 months i n d i c a t e d that 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 e f f e c t at 15°C but was s i g n i f i c a n t (P = 0.01) at 20°C. The e f f e c t of BAP on v i r u s c o n c e n t r a t i o n at 20°C was best f i t t e d by a l i n e a r equation where ELISA absorbance = 0.923 + 0.118 BAP (Table IX), f o r a 17.5 % r e d u c t i o n i n t o t a l sums of squares. When temperature e f f e c t at a BAP c o n c e n t r a t i o n of 2 mg/1 BA was analysed by r e g r e s s i o n a n a l y s i s , the r e s u l t s were f i t t e d best by a q u a d r a t i c equation where absorbance =7.362 -0.437 + 0.006, reducing the t o t a l sums of squares by 68.4 %. In comparing the r e s u l t s a f t e r 1 and 2 months, AMV was s t i l l u ndetectable, at 2 months, i n s i x p l a n t l e t s which had t e s t e d negative at 1 month. However, v i r u s became d e t e c t a b l e i n two other p l a n t l e t s with o r i g i n a l values below 0.05 a f t e r 1 month. Conversely, one p l a n t l e t with an o r i g i n a l absorbance value of 0.76 dropped to 0.01. Increasing the temperature a l s o reduced GFLV d e t e c t i o n i n p l a n t l e t s ( F i g u r e 9). While the lowest absorbance value observed i n p l a n t l e t s growing at 20°C was 0.12, only two p l a n t l e t s had values above 0.1 a f t e r growing at 25°C, and a l l p l a n t l e t s but one had values below 0.02 a f t e r growth at 30°C. The mean ELISA absorbance readings f o r p l a n t l e t s t e s t e d at each temperature were 0.58, 0.05, and 0.01; with h e a l t h y t i s s u e having a mean absorbance value of 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 at 20°C, 25°C, and 30°C on a medium c o n t a i n i n g a N-6-benzylaminopurine c o n c e n t r a t i o n of 2 mg/1. maximum value of 0.02. The q u a d r a t i c equation, absorbance 7.606 - 0.547 T + 0.01 T 2, f i t t e d the r e s u l t s f o r a 48.6 X r e d u c t i o n i n t o t a l sums of squares (Table IX). V i r u s recovery. AMV was never detected i n any of p l a n t l e t s p r e v i o u s l y grown at 30°C a f t e r they were moved to the lower temperatures while i t was r e p e a t e d l y detected on each t e s t date i n the c o n t r o l p l a n t l e t s not p r e v i o u s l y exposed to 30°C. The ELISA values were higher i n the c o n t r o l p l a n t s growing at 20°C than at 25°C. AMV was not detected i n any of the greenhouse p l a n t s produced from rooted shoots of t r e a t e d p l a n t l e t s . However, AMV was de t e c t e d i n a l l p l a n t s produced from c o n t r o l p l a n t l e t s growing at both 20°C and 25°C. Samples from h e a l t h y c o n t r o l s had an average absorbance value of 0.00 with a standard d e v i a t i o n of 0.0008. E f f e c t of 30°C. AMV d e t e c t i o n was reduced r a p i d l y a f t e r p l a c i n g the p l a n t l e t s at 30°C (Table X I ) . ELISA absorbance values dropped i n a l l p l a n t l e t s except one a f t e r 8 days. The b i g g e s t drop occurred i n p l a n t l e t s p r e v i o u s l y growing at 20°C. By the t w e n t y - f i r s t day, only two of the p l a n t l e t s from the 25°C group and f i v e from 20°C had readings of 0.05 or g r e a t e r . By comparison, a c o n t r o l sample of 100 ng/ml of pure v i r u s d i l u t e d i n C. quinoa leaves ground i n 10 volumes of g r i n d i n g b u f f e r had absorbance readings of 1.2, 1.02, 1.54, and 2.04 u n i t s r e s p e c t i v e l y , f o r the four t e s t dates. The mean ELISA, aborbance readings f o r t i s s u e from healthy p l a n t l e t s were low and s t a b l e throughout the t e s t i n g p e r i o d . Table XI. Results of ELISA t e s t s on 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 exposed to 30°C f o r three weeks Mean absorbance (days from s t a r t ) O r i g i n a l temperature P l a n t l e t 0 8 14 21 1 0. 20 0 , 07 0 . 08 0 .03 2 0 . , 28 0 . , 1 5 0 . 08 0 . 02 3 0 . 43 0 . , 04 0 .03 0 . 03 4 0 . 49 0 . 27 0 . 04 0 .05 5 0 . 46 0 . 44 0 .03 0 .01 6 0 . 91 0 . 1 1 0 . 09 0 . 03 7 0 . , 38 0 . , 23 0 . 12 0 .05 8 0 , . 48 0 . 17 0 . 06 0 . 03 Healthy 0 . , 00 0 . 00 0 . 00 0 .01 1 1 . 34 0 .21 0 .07 0 . 06 2 1 . 01 0 , . 1 5 0 .05 0 . 06 3 1 . 49 0 . 29 0 . 09 0 . 02 4 1 , . 36 0 . 19 0 . 04 0 . 04 5 2 . 02 0 . 52 0 . 10 0 .05 6 2 . 62 0 .29 0 . 18 0 .05 7 0 .75 0 . 14 0 . 10 0 . 03 8 1 . 18 0 . 36 0 . 08 0 . 12 Healthy 0 . 00 0 .01 0 .01 0 .02 V i r u s d e t e c t i o n i n f i e l d p l a n t s The g r i n d i n g b u f f e r s used on l e a f samples taken from f i e l d and screenhouse grapevines had a s i g n i f i c a n t e f f e c t (P = 0.0001) on the d e t e c t i o n of AMV and GFLV at each harvest throughout the summer (Table X I I ) . When comparing the mean ELISA absorbance values on each t e s t i n g date using the p r o t e c t e d LSD procedure, the r e s u l t s were s i m i l a r f o r the two v i r u s e s (Table X I I I ) . The mean ELISA absorbance values and the mean standard e r r o r (MSE) i s given f o r each v i r u s at each t e s t i n g date. The means Table XII. A n a l y s i s of v a r i a n c e of ELISA absorbance values obtained by 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 to dete c t grapevine f a n l e a f v i r u s (GFLV) and a r a b i s mosaic 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 est date Source df SS F Pr > F GFLV June 3 P l a n t s 24 24 . 6 24 . 06 0 . 0001 B u f f e r 3 8.3 65. 27 0 . 0001 E r r o r 72 3 . 1 June 24 P l a n t s 31 3.9 7 . 96 0 . 0001 B u f f e r 3 3 . 3 70 . 51 0 . 0001 E r r o r 93 1 . 5 J u l y 15 P l a n t s 31 3.3 7 . 42 0 . 0001 B u f f e r 3 4 . 5 103 . 28 0 . 0001 E r r o r 93 1 . 4 August 5 P l a n t s 31 24 . 6 1 1 . 23 0 . 0001 B u f f e r 3 20 . 5 96. 78 0 . 0001 E r r o r 93 6.6 September 2 P l a n t s 31 24 . 3 1 1 . 03 0 . 0001 B u f f e r 3 18.5 89 . 46 0 . 0001 E r r o r 93 6.6 AMV June 3 P l a n t s 13 4.8 7 . 73 0 . 0001 B u f f e r 3 4 . 8 33 . 14 0. 0001 E r r o r 39 1.9 June 24 P l a n t s 15 2 . 7 9 . 19 0 . 0001 B u f f e r 3 3 . 5 59 . 74 0 . 0001 E r r o r 45 0.9 J u l y 15 P l a n t s 15 3 . 0 8 . 90 0 . 0001 B u f f e r 3 1 . 0 13 . 96 0 . 0001 E r r o r 45 1 . 0 August 5 P l a n t s 1 5 3 . 4 5. 07 0 . 0001 B u f f e r 3 13.1 97 . 14 0 . 0001 E r r o r 45 2.0 September 2 P l a n t s 15 8.3 9 . 10 0. 0001 B u f f e r 3 14.4 78. 94 0 . 0001 E r r o r 45 2.7 66 Table X I I I . E f f e c t of 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 of a r a b i s mosaic v i r u s (AMV) and grapevine 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 absorbance value per t e s t i n g date B u f f e r 1 June 3 2 June 24 J u l y 15 Aug. 15 Sept. 2 A) GFLV A 1 .31 a 0 . 54 a 0 . 52 b 0 .84 b 0. 58 c B 1 .28 a 0 . 54 a 0 . 60 a 1 .03 a 0 . 93 b C 0 . 59 c 0 . 17 b 0 . 11 d 0 .10 c 0 . 15 d D 1 ,12 b 0 . 56 a 0 . ,46 c 1 .11 a 1 . 13 a LSD 0 . 12 0 . 06 0 . 06 0 . 13 0 . 13 MSE 3 0 .043 0 . ,016 0 . ,015 0 .071 0 . 069 B) AMV A 1 .16 ab 0 , 70 b 0 . 40 b 0 .87 c 0 . 76 b B 1 .27 a 0 . 84 a 0 . , 53 a 1 .13 b 1 . 28 a C 0 . 52 c 0 . 32 c 0 , .23 c 0 .13 d 0 . 21 c D 1 .09 b 0 , 93 a 0 . , 52 a 1 . 33 a 1 . 41 a LSD 0 .17 0 . 10 0 . 11 0 .15 0 . 18 MSE 0 . 048 0 .019 0 . 023 0 .045 0 . 061 '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 - 0.15 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 - b u f f e r A with 1% n i c o t i n e , pH 8.4.; C - standard g r i n d i n g b u f f e r - 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. ^Absorbance means followed by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t at P = 0.05 w i t h i n each harvest u s i n g the LSD value f o r that harvest. 3MSE = mean standard e r r o r . f o l l o w e d by the same l e t t e r are not s i g n i f i c a n t l y d i f f e r e n t u s i n g the l e a s t s i g n i f i c a n t d i f f e r e n c e i n d i c a t e d f o r each harvest date (P = 0.05). The mean absorbance values obtained by g r i n d i n g f o l i a r samples i n standard g r i n d i n g b u f f e r ( b u f f e r C) was i n f e r i o r f o r both v i r u s e s on every t e s t date. In the e a r l y summer, the mean absorbance values 67 obtained f o r the two 0.15 M PBS b u f f e r s (A and B) were s u p e r i o r to the standard g r i n d i n g b u f f e r modified by adding 1% n i c o t i n e and i n c r e a s i n g the pH; with no s i g n i f i c a n t advantage being gained by adding n i c o t i n e to the 0.15 M PBS b u f f e r . However, as the summer progressed, the a d d i t i o n of n i c o t i n e to the 0.15 M PBS b u f f e r increased the absorbance valu e s s i g n i f i c a n t l y over those of the standard 0.15 M PBS b u f f e r . Also, adding n i c o t i n e and i n c r e a s i n g the pH of the standard g r i n d i n g b u f f e r , improved the r e s u l t s such that they were equal to or b e t t e r than those obtained with the 0.15 M b u f f e r with n i c o t i n e , on the l a s t two t e s t i n g dates. A l l three b u f f e r s other than the standard g r i n d i n g b u f f e r were e q u a l l y s e n s i t i v e i n d e t e c t i n g AMV i n i n f e c t e d p l a n t s ( i . e . with absorbance values g r e a t e r than the pre-determined t h r e s h o l d ) , with the exception of one p l a n t on J u l y 15 (Table XIV). Using the standard b u f f e r , AMV was not detected i n at l e a s t one p l a n t on each of the f i r s t three dates and was harder to detec t l a t e r i n the summer, e s p e c i a l l y i n the screenhouse p l a n t s . GFLV d e t e c t i o n was l e s s s e n s i t i v e when the standard g r i n d i n g b u f f e r was used (Table XV). GFLV was, however, not det e c t e d as c o n s i s t e n t l y as AMV when the other b u f f e r s were used. Of 153 t e s t s on GFLV-infected p l a n t s during the summer, the v i r u s was det e c t e d i n 134 using the 0.15 M PBS b u f f e r , i n 141 using the same b u f f e r with IX n i c o t i n e , 87 u s i n g the standard g r i n d i n g b u f f e r , and i n 139 p l a n t s using the standard b u f f e r with IX n i c o t i n e and in c r e a s e d pH. 68 Table XIV. Detection of arabis mosaic virus by ELISA throughout the summer i n f i e l d and screenhouse plants using d i f f e r e n t grinding buffers' June 3 June 24 July IS Aug. 12 Sept. 2 Variety Plant A a B C D A B C D A B C D A B C D A B C D A) F i e l d plants St. George 1 • • • • • • + • + • - • • • • « . M 2 + + + • +*• + + + + + + + + + + • + ••. M 3 • + + • + • + + + + + + + • • + M 4 + . • • + . • + + • • + . + . . • • • + " 5 + + + + + + + • + + + + + + - + + . • + M 6 + + + + •*• + + • • + + •»• + + - + • + + . M 7 + + + + - • + + + + + + - + + + + • LN-33 8 • + • + • . + + + • + • + • . + + + • . Pinot Noir 9 • • • • • • • • . . . . . . • + B) Screenhouse plants Unknown #1 10 • • • • • • • • •. • • • • + • . • . . • • H \\ + + - • • + + • • • • • + + - • • + - • M 12 . + - . . . - . Unknown »2 13 • • • • . • • • • • . . . . _ . . . . . n 14 • • • • • . + + + • + • + • - + + . - • M 15 • • • • • • + • + . . . . . . . Rotberger 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 following grinding buffers: A - 0.1S M phosphate buffered saline <PBS). pH 8.4 with 0.05% Tween-20. 0.2X ovalbunin and 2% polyvinylpyrrolidone; B - buffer A with IX nicotine. pH 8.4.; C - 0.01 M PBS, pH 7.4, with 0.05% Tween-20, 0.2% ovalbumin and 2% polyvinylpyrrolidone; D - buffer C with 1% nicotine. pH 8.2. 69 Table XV. Detection of grapevine fanleaf virus by ELISA throughout the suoaer i n f i e l d and screenhouse plants using d i f f e r e n t grinding b u f f e r s 1 June 3 June 24 July 15 Aug. 12 Sept. 2 Variety Plant A* B C D A B C D A B C D A B C D A B C D A) F i e l d plants St. George 1 + + + + + • - • - - - - • + • + - + * 2 + + + • - - - - • + - • + • - • « 3 + • • + + • + • - - - - - - - - +. + - 4 . *• 4 + • • + - 4 - 4 • + + + ** 5 • + + + • + • • + + N 5 + + + + - • _ + + + - + + LN-33 7 + • • + . • + • • + + + + 4 4 4 4 - - - -M 3 • • • + • • • • • + + + + + + + + + ** 9 + + • + + • • • + • • + + + • + + • • • " \Q 4 4 4 4 * * * . * 4 4 - 4 + 4 . + 4 4 4 4 " 11 • + • + + • - • + • - • 4 4 - 4 4 4 4 4 " 12 + + + • • + • • + + + • • + + + 4 4 4 4 . M 13 + 4 + • 4 4 + 4 4 4 - 4 4 4 4 4 4 4 4 4 n 14 • * * • 4 • *• • + + • + 4 4 4 + 4 4 4 4 H 15 + • 4 4 * * + + 4 • + • 4 • • 4 B) Screenhouse plants R i e s l i n g 16 + 4 + + + + 4 4 - • . + 4 + 4 + • • • • H 17 + + + • • • + • - - + - 4 4 4 4 H l g • • • 4 - - - - 4 4 4 4 4 4 4 4 PinOt 19 + + +•• 4 • • 4 + + • + 4 4 + * + • + 4 Chardonnay 20 • • • • 4 + 4 4 - - -** 21 • + • • 4 • - 4 4 + + + 4 + - 4 St. George 22 • + • • 4 4 4 4 4 4 - - * 4 - + 4 4 - 4 " 23 + + - • • + • • 4 4 - 4 4 4 - 4 - - - -" 24 4 4 * 4 4 + - + 4 4 - 4 4 4 - 4 St. George 25 • • • • 4 4 4 4 - 4 4 * 4 4 4 4 4 4 4 4 H 26 • + • • 4 * * * - • - + 4 + - * 4 • - 4 Unknown # 1 2 7 + 4 + + • • • • 4 + 4 4 • + - + + ** 28 + • - • 4 * - + 4 4 * 4 H 29 4 • + • • + 4 • 4 + - * 4 * * * Unknown #2 30 4 * - + 4 4 , 4 4 4 . - 4 4 . 4 4 4 . 4 " 31 4 • • + 4 * * * 4 + - * *• 32 - 4 - * 4 4 - 4 4 4 - 4 TOTALS 25 25 23 25 31 31 23 31 19 26 11 24 30 30 15 30 29 29 15 29 '(••(•positive (-)-negative. Blank spaces were not tested. 'Refers to the following grinding buffers: A - 0.15 M phosphate buffered s a l i n e (PBS), pH 8.4 with 0.05% Tween-20, 0.2% ovalbumin and 2% polyvinylpyrrolidone; B -1 buffer A with 1% nicotine, pH 8.4; C - 0.01 H PBS, pH 7.4, with 0.05% Tween-20, 0.2% ovalbuain and 2% polyvinylpyrrolidone; D - buffer C with 1% nic o t i n e , pH 8.2. 70 The three most s e n s i t i v e b u f f e r s performed e q u a l l y well on a l l dates except f o r Ju l y 15, when both n i c o t i n e b u f f e r s , and e s p e c i a l l y the 0.15 M PBS b u f f e r with n i c o t i n e , i n c r e a s e d ELISA s e n s i t i v i t y . As of J u l y 15, GFLV became und e t e c t a b l e i n one screenhouse p l a n t (no.20), a Pinot Chardonnay i n f e c t e d with a "severe" s t r a i n of GFLV. As the summer progressed, GFLV d e t e c t i o n became l e s s r e l i a b l e u sing the standard b u f f e r . U n t i l J u l y 15, there was not much d i f f e r e n c e between the mean ELISA absorbance values of samples taken from he a l t h y c o n t r o l s of the two v a r i e t i e s t e s t e d (Table XVI). On that date, the readings obtained from LN-33 samples t e s t e d f o r GFLV were much higher than those from the St. George p l a n t s . This trend continued, though l e s s pronounced, on the l a s t two t e s t i n g dates. This v a r i e t a l d i f f e r e n c e and the a s s o c i a t e d g r e a t e r v a r i a t i o n had an e f f e c t 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 l e v e l s which a f f e c t e d GFLV d e t e c t i o n i n the St. George p l a n t s on J u l y 15. When the healthy c o n t r o l means were used s e p a r a t e l y by v a r i e t y , a l l St. George f i e l d p l a n t s became p o s i t i v e f o r the three b e t t e r b u f f e r s , while 4 of 6 plant s became p o s i t i v e when the standard g r i n d i n g b u f f e r was used. By comparison, the r e s u l t s do not change f o r the LN-33 f i e l d p l a n t s . A l s o , when the St. George c o n t r o l s were used,the p o s i t i v e t h r e s h o l d l e v e l dropped s u f f i c i e n t l y f o r the absorbance value of screenhouse mother plant no.20 to become b a r e l y p o s i t i v e f o r the three b e t t e r b u f f e r s . 71 Table XVI. Mean ELISA absorbance values of healthy c o n t r o l p l a n t s ground i n va r i o u s b u f f e r s throughout the summer GFLV 1 c o n t r o l s AMV c o n t r o l s ELISA t e s t date B u f f e r 2 LN-33 St. George LN-33 St. George June 3 A 0.01 0 .01 0 . 00 0 . 00 B 0.01 0 . 00 0.00 0 . 01 C 0 . 02 0 . 00 0.01 0 . 00 D 0.01 0 • 0 0 0.01 0 . 00 June 24 A 0 . 02 0 .01 0.01 0 . 00 B 0 .02 0 .01 0.00 0. 00 C 0.01 0 . 00 0 . 00 0 . 01 D 0 . 00 0 .01 0.01 0 . 00 J u l y 15 A 0 . 20 0 .01 0.03 -0 . 01 B 0.19 0 . 02 0 . 02 0 . 00 C 0 .03 -0 . 02 0.01 0 . 00 D 0.14 0 .01 0.01 -0 . 01 August 15 A 0 . 04 0 .01 0 . 02 0 . 02 B 0 . 04 0 .01 0 . 02 0 . 00 C 0.01 0 .00 0 . 00 0 . 00 D 0.05 0 .01 0 . 03 0 . 01 September 2 A 0 . 04 0 .02 0.01 0 . 00 B 0 . 04 0 . 02 0.01 0 . 01 C 0.02 0 . 00 0.01 0 . 00 D 0 . 04 0 . 02 0.01 0 . 00 1GFLV = grapevine f a n l e a f v i r u s . AMV = arab i s mosaic v i r u s 2 R e f e r s 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 - 0.15 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 - b u f f e r A with 1% n i c o t i n e , pH 8.4; C - standard g r i n d i n g 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. 72 DISCUSSION V i r u s d e t e c t i o n i n t i s s u e - c u l t u r e d p l a n t l e t s Although the ELISA technique i s a s e n s i t i v e method widely used to de t e c t v i r u s e s i n f i e l d p l a n t s , there have been few re p o r t s of t i s s u e - c u l t u r e d p l a n t l e t s being recommended as sources of t e s t i n g m a t e r i a l f o r ELISA (McMorran and A l l e n , 1983; Monette, 1985,1986). In vitro p l a n t c u l t u r e , a labour and r e s o u r c e - i n t e n s i v e procedure, may not be a r e l i a b l e method f o r improving v i r u s d e t e c t i o n i n grapevines by ELISA. ELISA d e t e c t i o n of AMV, GFLV, and TomRV i n p l a n t l e t s , although p o s s i b l e , was not always r e l i a b l e and r e p e a t a b l e . T h i s study i s the f i r s t to document the v a r i a b i l i t y of v i r u s c o n c e n t r a t i o n w i t h i n t i s s u e - c u l t u r e d grapevines. The f a i l u r e to detect AMV and.GFLV i n a l l p l a n t l e t s t e s t e d on each date, and re p e a t e d l y i n p l a n t l e t s r e t e s t e d at d i f f e r e n t dates suggested that there was an uneven v i r u s d i s t r i b u t i o n w i t h i n p l a n t l e t s at both 20°C and 25°C. When p l a n t l e t s growing at 25°C were d i s s e c t e d and the parts t e s t e d i n d i v i d u a l l y , the v a r i a b i l i t y i n absorbance values obtained by ELISA t e s t s .supported t h i s c o n t e n t i o n ( F i g u r e 1). Further i n v e s t i g a t i o n s with AMV and GFLV- i n f e c t e d p l a n t l e t s grown at 25°C showed that these v i r u s e s were more r e l i a b l y detected i n short r a p i d l y p r o l i f e r a t i n g t i s s u e s and roo t s r a t h e r than i n elongated shoots and leaves ( F i g u r e 2). This i n d i c a t e d that the type of t i s s u e taken f o r t e s t i n g 73 from p l a n t l e t s growing at 25°C had an e f f e c t on v i r u s d e t e c t i o n by ELISA. Sampling the wrong t i s s u e f o r t e s t i n g c o u l d r e s u l t i n f a i l u r e to d e t e c t these v i r u s e s , e s p e c i a l l y i f poor q u a l i t y antiserum was used. The p a t t e r n of v i r u s d i s t r i b u t i o n was a l t e r e d by the growth temperature. C u l t u r e at 20°C improved GFLV and AMV d e t e c t i o n i n elongated plan t shoots and leaves, with the b i g g e s t improvement o c c u r r i n g with GFLV. The lower temperature reduced v i r u s t i t r e s i n the r a p i d l y p r o l i f e r a t i n g short shoots and r o o t s . There was no apparent reason fo r t h i s . Both v i r u s e s should have been able to r e p l i c a t e well at e i t h e r 20°C or 25°C. V i r u s degradation should not have been enhanced i n one p l a n t part over another f o r e i t h e r moderate temperature. V i r u s r e p l i c a t i o n may, however, have been improved i n c e r t a i n p l a n t p a r t s . Normal p l a n t biochemical processes advantageous to v i r u s r e p l i c a t i o n may have occured at higher l e v e l s i n c e r t a i n p a r t s at d i f f e r e n t temperatures. Although McMorran and A l l e n (1983) determined that root t i s s u e was u n s u i t a b l e f o r d e t e c t i n g one of f i v e potato v i r u s e s i n t i s s u e - c u l t u r e d p l a n t s using ELISA, the r e l a t i v e a n t i g e n l e v e l s were not uniform f o r the leaves, stems, and root samples of the p l a n t l e t s i n f e c t e d with the other four v i r u s e s . D i f f e r e n t v i r u s e s behaved d i f f e r e n t l y . In the case of t h i s study, a r e p l i c a t e d experiment would be r e q u i r e d to c l a r i f y the d i s t r i b u t i o n of AMV and GFLV at d i f f e r e n t temperatures. Even though the v i r u s c o n c e n t r a t i o n was increased i n the elongated t i s s u e s when the p l a n t l e t s were grown at 20°C, AMV and GFLV d e t e c t i o n was not a s s u r e d i n a l l the p l a n t l e t s ( T a b l e I I I ) . T h i s was shown when the v i r u s e s were not d e t e c t e d i n f i v e p l a n t l e t s which had p r e v i o u s l y t e s t e d p o s i t i v e w h i l e growing at 20°C. The chance of m i s d i a g n o s i n g v i r u s i n f e c t i o n due to the t i s s u e type sampled was, however, l e s s at 20°C than at 25°C. The i n a b i l i t y to d e t e c t GFLV and AMV c o n s i s t e n t l y i n p l a n t l e t s was c o n s i s t e n t w i t h the r e s u l t s o b t a i n e d by Monette (1985), who c o u l d not d e t e c t GFLV i n a l l p l a n t l e t s i n i t i a t e d from i n f e c t e d p l a n t s . Monette r e p o r t e d s h o r t term f l u c t u a t i o n s i n GFLV c o n c e n t r a t i o n over a 14 day r e c u l t u r e p e r i o d . U n l i k e the r e s u l t s of t h i s t h e s i s study w i t h AMV and GFLV, he was a b l e to d e t e c t the v i r u s c o n s i s t e n t l y i n a l l p l a n t l e t s throughout t h a t r e c u l t u r e p e r i o d . T h i s c o n s i s t e n t d e t e c t i o n may have been due to the s h o r t e r r e c u l t u r i n g i n t e r v a l s and the growth on l i q u i d medium i n a t i l t i n g d e v i c e . S t u d i e s by H a r r i s and Stevenson (1982) showed t h a t grape p l a n t l e t s grew f a s t e r and b e t t e r under the s e c o n d i t i o n s . This r a p i d r a t e of metabolism by the p l a n t s w i t h adequate n u t r i t i o n may a l s o have i n c r e a s e d v i r u s r e p l i c a t i o n . In my study, the p l a n t s were u s u a l l y t e s t e d at the end of a s i x week r e c u l t u r e i n t e r v a l . V i r a l r e p l i c a t i o n r a t e s may have been lowered as the n u t r i e n t s were d e p l e t e d . T h i s f a c t , c o u p l e d w i t h normal v i r u s d e g r a d a t i o n i n the p l a n t s may have accounted f o r reduced v i r u s d e t e c t i o n . TomRV was a l s o d e t e c t e d i n g r a p e v i n e s growing in vitro i n i t i a t e d from i n f e c t e d greenhouse-grown p l a n t s . The f a i l u r e to d e t e c t the v i r u s i n the one p o o r l y - g r o w i n g 75 c u l t u r e a l s o r e i n f o r c e s the importance of s e l e c t i n g a p p r o p r i a t e t i s s u e f o r t e s t i n g . The whole p l a n t l e t was growing p o o r l y and the 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 of TomRV i n a l l o t h e r p l a n t l e t s compared to AMV and GFLV may have been due to n a t u r a l l y h i g h e r l e v e l s o f TomRV r e p l i c a t i o n at 20°C. Thi s study i s the f i r s t to q u a n t i f y the e f f e c t s of BAP c o n c e n t r a t i o n i n the media and growth room temperature on AMV and GFLV c o n c e n t r a t i o n s i n g r a p e v i n e s grown in vitro. BAP d i d not appear to p l a y a major r o l e i n ELISA d e t e c t i o n of AMV at the c o n c e n t r a t i o n s used i n t h i s s t u d y . Any e f f e c t s on AMV c o n c e n t r a t i o n s i n the p l a n t l e t s caused by BAP c o n c e n t r a t i o n s r a n g i n g from 2 to 8 mg/l were masked by the overwhelming e f f e c t s of temperature. The growth room temperature was the most c r i t i c a l f a c t o r a f f e c t i n g v i r u s d e t e c t i o n i n p l a n t l e t s . AMV and GFLV c o n c e n t r a t i o n s i n p l a n t l e t s , as shown by ELISA d e t e c t i o n r e s u l t s , were q u i t e h i g h at a c u l t u r e room temperature of 15°C. V i r u s c o n c e n t r a t i o n s were reduced s i g n i f i c a n t l y as the temperature was r a i s e d i n 5°C increments to 30°C where the c o n c e n t r a t i o n s dropped below the d e t e c t i o n s e n s i t i v i t y of the ELISA t e s t . T h i s r e d u c t i o n i n v i r u s c o n c e n t r a t i o n was not accompanied by a s i m i l a r r e d u c t i o n i n p l a n t v i g o u r . When v i s u a l l y compared, p l a n t l e t growth a c t u a l l y i n c r e a s e d w i t h the temperature and the best growth was ob s e r v e d at 30°C. I t was e x t r e m e l y i m p o r t a n t t o grow g r a p e v i n e p l a n t l e t s c o n t i n u o u s l y at 20°C or l e s s f o r r e l i a b l e AMV and GFLV 76 d e t e c t i o n . Exposure to temperatures above 25°C reduced the v i r u s t i t r e to l e v e l s below d e t e c t i o n t h r e s h o l d s f o r at l e a s t 2 months. Exposure to 30°C temperature had a d r a s t i c e f f e c t on AMV and GFLV c o n c e n t r a t i o n s . The e f f e c t was v e r y l o n g l a s t i n g . Treatment of AMV- i n f e c t e d p l a n t l e t s at a c o n s t a n t temperature of 30°C f o r 60 days may be s u f f i c i e n t , w i t h o u t subsequent meristem or s m a l l shoot t i p e x c i s i o n , to e l i m i n a t e AMV from p l a n t l e t s and subsequent r o o t e d e x p l a n t s grown i n p o t s . Based on the response of G F L V - i n f e c t e d p l a n t l e t s t o the 30°C temperature, i t i s l i k e l y t h a t the same tr 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 e f f e c t on GFLV t i t r e s i n t i s s u e - c u l t u r e d p l a n t l e t s . The f a i l u r e 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 the second experiment was p r o b a b l y due to t h e i r h a v i n g been grown p r e v i o u s l y i n a room where te m p e r a t u r e s f l u c t u a t e d up to 29°C d u r i n g the day. The v i r u s c o n c e n t r a t i o n was p r o b a b l y reduced and uneven w i t h i n the p l a n t . Some of the p l a n t l e t s used f o r the experiment p r o b a b l y had v e r y low l e v e l s and escaped d e t e c t i o n a f t e r one month. However, the v i r u s became d e t e c t a b l e i n more p l a n t l e t s by the end of the second month. The a b i l i t y to d e t e c t AMV i n a l l p l a n t l e t s a f t e r 2 months of growth at 15°C may have been due to chance i n s e l e c t i n g i n f e c t e d t i s s u e at the s t a r t of the experiment but, more l i k e l y , was due to growth at a temperature more f a v o r a b l e to v i r u s r e p l i c a t i o n and reduced v i r u s d e g r a d a t i o n . The r a p i d e f f e c t of the 30°C temperature on AMV c o n c e n t r a t i o n i n i n f e c t e d p l a n t l e t s was unexpected and 77 d i f f i c u l t to e x p l a i n . I t c o u l d not have been due to f a u l t y ELISA t e s t s as the same c o a t i n g and c o n j u g a t e d a n t i s e r a and b u f f e r s were used f o r a l l t e s t s and the c o n t r o l s worked w e l l . In h i s d i s c u s s i o n on the e f f e c t of temperature on v i r u s i n a c t i v a t i o n in vivo, Matthews (1981) s t a t e s t h a t s m a l l s p h e r i c a l v i r u s e s are o f t e n e a s i l y i n a c t i v a t e d in vivo w i t h i n c r e a s e d h eat. He mentions t h a t t h i s may be due to a number of p o s s i b l e mechanisms: the i n a c t i v a t i o n of v i r u s a l r e a d y p r e s e n t , the b l o c k i n g of v i r a l s y n t h e s i s by r e d u c i n g v i r a l RNA polymerase a c t i v i t y , and the p r e v e n t i o n of p r o p e r v i r u s p a r t i c l e assembly because the coat p r o t e i n cannot assume the c o r r e c t p a c k i n g c o n f i g u r a t i o n at the h i g h e r t e m p e r a t u r e . Any one of these mechanisms c o u l d be a f f e c t i n g the a b i l i t y of the v i r u s to r e p l i c a t e . The above e x p l a n a t i o n by Matthews may account f o r the reduced v i r a l r e p l i c a t i o n r a t e but does not e x p l a i n the r a p i d r e d u c t i o n i n v i r u s c o n c e n t r a t i o n observed i n t h i s s t u d y (Table X I ) . I t i s u n c l e a r whether the r a p i d r e d u c t i o n i n AMV c o n c e n t r a t i o n s was due to n a t u r a l v i r u s d e g r a d a t i o n or i n d i c a t e s t h a t a n o ther f a c t o r may be i n c r e a s i n g v i r a l d e g r a d a t i o n i n the p l a n t l e t s at 30°C. I n c r e a s e s i n growth room temperature may not o n l y have reduced the r e p l i c a t i o n r a t e of t h i s v i r u s but may a l s o be have i n c r e a s e d v i r a l d e g r a d a t i o n . Experiments have shown t h a t the s t a b i l i t y i n sap of n e p o v i r u s e s i s f a i r l y s h o r t , e s p e c i a l l y as the temperature i s i n c r e a s e d . In the sap of herbaceous p l a n t s , GFLV has a thermal e n d - p o i n t of 60-65°C and r e t a i n s i n f e c t i v i t y f o r 15-30 days at 20°C ( H e w i t t et a l . , 1970). 78 AMV, i n Petunia hybrida, l o s e s i n f e c t i v i t y a f t e r 10 m i n u t e s a t 55-61°C and s t o r a g e a t room t e m p e r a t u r e f o r 1-2 weeks ( M u r a n t , 1 9 7 0 ) . K a s s a n i s (1957) b e l i e v e d t h a t i n c r e a s e d t e m p e r a t u r e a c t u a l l y d e s t r o y e d v i r u s e s w i t h i n a p l a n t . The r e s u l t s o f t h i s s t u d y may a g r e e w i t h K a s s a n i s ' t h e o r y . W h e t h e r o r n o t t h e y do i s s p e c u l a t i o n . One w o u l d have t o d e t e r m i n e t h e v i r u s c o n c e n t r a t i o n i n a p l a n t , s u p p l y c o n d i t i o n s w h i c h w o u l d s u s p e n d v i r a l r e p l i c a t i o n o n l y and t h e n m o n i t o r v i r u s l e v e l s o v e r t i m e a t d i f f e r e n t t e m p e r a t u r e s . However, i t seems s u r p r i s i n g t o me t h a t n o r m a l v i r u s d e g r a d a t i o n r a t e s i n o t h e r w i s e a c t i v e l y g r o w i n g p l a n t l e t s w o u l d a c c o u n t f o r s u c h r a p i d r e d u c t i o n i n v i r u s t i t r e . T h e r e may a l s o be i n c r e a s e d v i r u s d e g r a d a t i o n a s s o c i a t e d w i t h i n c r e a s i n g t e m p e r a t u r e , e v e n t h o u g h t h e h i g h e r t e m p e r a t u r e i s w e l l b e l o w b o t h t h e u s u a l c o n d i t i o n s f o r v i r u s e l i m i n a t i o n by h e a t t h e r a p y and t h e t h e r m a l e n d - p o i n t i n a c t i v a t i o n t e m p e r a t u r e s . The t e m p e r a t u r e e f f e c t s f o u n d i n t h i s s t u d y r e s e m b l e t h o s e r e p o r t e d by G a l z y (1961) and G a l z y and Compan ( 1 9 6 8 ) . G a l z y f o u n d t h a t v i r u s symptoms on t i s s u e - c u l t u r e d p l a n t l e t s i n f e c t e d w i t h GFLV were r e d u c e d and p l a n t g r o w t h i m p r o v e d when t h e t e m p e r a t u r e was i n c r e a s e d above 26°C. However, G a l z y and Compan (1 9 6 8 ) f o u n d t h a t a t r e a t m e n t o f a t l e a s t 35°C f o r 90 d a y s was n e c e s s a r y t o p r e v e n t symptom r e c u r r e n c e . I n t h i s t h e s i s s t u d y , t h e i n c r e a s e d s e n s i t i v i t y o f v i r u s d e t e c t i o n by E L I S A o v e r symptom e x p r e s s i o n on p l a n t l e t s in vitro p e r m i t t e d f o l l o w i n g t h e r e d u c t i o n i n v i r u s c o n c e n t r a t i o n more c l o s e l y . The t e m p e r a t u r e e f f e c t 79 was detected as i t was o c c u r r i n g . B a r l a s s et a l . (1982) found that growth at 35°C e l i m i n a t e d GFLV from V. vinifera p l a n t l e t s while growth at f l u c t u a t i n g day/night temperatures of 27°/20°C d i d not. They d i d not i n d i c a t e the d u r a t i o n of treatment nor d i d they t r y v i r u s e l i m i n a t i o n at 30°C. They d i d mention, however, that growth at temperatures above 35°C r e s u l t e d i n death. The increased p l a n t growth and s u r v i v a l at 30°C coupled with AMV and GFLV e l i m i n a t i o n obtained i n my study i n d i c a t e d that i t i s unnecessary to sub j e c t in vitro p l a n t l e t s to n e a r - l e t h a l c o n d i t i o n s i n order to e l i m i n a t e these two v i r u s e s . V i r u s d e t e c t i o n i n f i e l d p l a n t s The r e s u l t s of these s t u d i e s i n d i c a t e t hat, under the t y p i c a l summer c o n d i t i o n s experienced at Sidney, B.C., enhancing the standard ELISA g r i n d i n g b u f f e r with n i c o t i n e was e s s e n t i a l f o r r e l i a b l e d e t e c t i o n of GFLV and AMV from f i e l d and screenhouse-grown grapevines. The lowest ELISA absorbance values on a l l the te s t dates throughout the summer occurred when the standard g r i n d i n g b u f f e r was used. The ELISA values f o r AMV-infected p l a n t s ground i n the standard b u f f e r , although s t i l l s i g n i f i c a n t l y below those obtained when us i n g the other b u f f e r s , d i d not drop to the same low l e v e l s as f o r the GFLV-infected p l a n t s . The g r e a t e s t improvement i n ELISA absorbance values during the summer was obtained when n i c o t i n e was added to both b u f f e r s . As the summer progressed, i n c r e a s i n g the b u f f e r m o l a r i t y was unnecessary when n i c o t i n e was added. The standard b u f f e r amended with n i c o t i n e o f t e n gave s t a t i s t i c a l l y higher absorbance values than the b u f f e r with i n c r e a s e d m o l a r i t y . With the ex c e p t i o n of one date, J u l y 15 f o r GFLV, using the 0.15 M g r i n d i n g b u f f e r gave ELISA absorbance values s i g n i f i c a n t l y lower than those measured using the n i c o t i n e b u f f e r s . The most important b e n e f i t of b u f f e r enhancement i s the in c r e a s e d a b i l i t y to separate p o s i t i v e ELISA absorbance values from negative values. P o s i t i v e p l a n t s were those with ELISA absorbance values greater than the t h r e s h o l d values determined by the healthy c o n t r o l p l a n t s on each date . The standard g r i n d i n g b u f f e r was always i n f e r i o r f o r d e t e c t i n g p o s i t i v e p l a n t s for both v i r u s e s when compared to the other three b u f f e r s . The standard b u f f e r was not as r e l i a b l e f o r d e t e c t i n g these v i r u s e s even at the s t a r t of the summer. Assays with t h i s b u f f e r f a i l e d to detect at l e a s t one i n f e c t e d p l a n t on each date. D e t e c t i o n r e l i a b i l i t y dropped as the summer progressed. There was a l s o no i n c r e a s e i n d e t e c t i o n with c o o l e r weather towards the end of summer. These r e s u l t s were s i m i l a r to ob s e r v a t i o n s by other v i r o l o g i s t s t e s t i n g grapevines i n warmer p a r t s of the world (Table I ) . Our c o o l e r summers do not reduce the need to optimize ELISA c o n d i t i o n s i f t h i s technique i s to be used with confidence. B u f f e r a d d i t i v e s were e s p e c i a l l y important f o r GFLV d e t e c t i o n . GFLV was more p o o r l y detected than AMV when usi n g the standard b u f f e r . It i s important to note that, even though they i n c r e a s e d d e t e c t i o n , the enhanced b u f f e r s f a i l e d to detect GFLV in some f i e l d and screenhouse p l a n t s . T h i s r e s u l t may have been due not only to a lower v i r u s t i t r e in the p l a n t s but a l s o to a lower s e n s i t i v i t y of the antiserum against GFLV. It remains c r i t i c a l , even with these b u f f e r s , to use the highest t i t r e antiserum fr e e from background r e a c t i o n s . A l s o , one should not determine v i r u s i n f e c t i o n on the r e s u l t s of s i n g l e ELISA t e s t s , e s p e c i a l l y when the absorbance values are near the t h r e s h o l d v a l u e s . When compared s e p a r a t e l y f o r each date, the three enhanced b u f f e r s performed e q u a l l y w e l l i n d e t e c t i n g the i n d i v i d u a l v i r u s e s i n f i e l d and screenhouse p l a n t s , with the e x c e p t i o n of the J u l y 15 t e s t date. An adequate number of r e p r e s e n t a t i v e c o n t r o l s must be used when c a r r y i n g out ELISA d e t e c t i o n of grapevine v i r u s e s . P r e f e r a b l y , healthy c o n t r o l s should be included f o r the same v a r i e t i e s as those being t e s t e d and growing under the same c o n d i t i o n s . Otherwise, i t would be d i f f i c u l t to d e t e c t spurious p o s i t i v e and negative r e a c t i o n s , which have been found in ELISA t e s t s with grapevines. Uniform t i s s u e samples must be s e l e c t e d to minimise spurious r e a c t i o n s and allow f o r proper a n a l y s i s of the r e s u l t s . The need to c a r e f u l l y s e l e c t healthy c o n t r o l s was supported by the r e s u l t s of the J u l y 15 t e s t date. On J u l y 15, ELISA absorbance values obtained f o r the LN-33 healthy c o n t r o l s went up s i g n i f i c a n t l y compared to those f o r the St. 82 George controls, especially when tested against the GFLV antiserum (Table XVI). Combining the values for both control varieties 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 cultural practices and pesticide spray applications prior to July 15 gave no different treatments for one variety over the other. The abnormally high 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 fully 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 April. Older expanded leaves did not give the same results. They concluded that the rapidly growing leaves were producing nonviral, noninfectious antigens which 83 were r e a c t i n g with the antiserum. They co u l d not account f o r the apparent seasonal e f f e c t on the f a l s e readings. They a l s o mentioned that s t u d i e s i n progress at that time were g i v i n g s i m i l a r r e s u l t s f o r t e s t s on healthy grape and pear p l a n t s . The grape samples taken i n t h i s study c o n s i s t e d a l s o of young, f u l l y open but not f u l l y expanded l e a v e s . A l s o , the high absorbance values were observed in J u l y and a f f e c t e d the r e s u l t s l e s s i n l a t e r months. In t h i s case, the samples taken from the LN-33 p l a n t s gave high absorbance readings only when an antiserum to GFLV was used. The same t i s s u e was used as the healthy c o n t r o l f o r both a n t i s e r a . A f t e r g r i n d i n g , the sample was a p p l i e d to ELISA p l a t e s coated against one of the two v i r u s e s . A p o s s i b l e e x p l a n a t i o n i s that only the LN-33 p l a n t s produced a normal p l a n t p r o t e i n between June 24 and J u l y 15. This p r o t e i n may have reac t e d with the a n t i b o d i e s produced a g a i n s t the p u r i f i e d GFLV.virus p r e p a r a t i o n that was i n j e c t e d i n t o the r a b b i t to produce the antiserum. Although a v i r u s p r e p a r a t i o n i s p u r i f i e d as much as p o s s i b l e f o r i n j e c t i o n i n t o an animal, there o f t e n remain some normal plant p r o t e i n s a g a i n s t which the animal produces a n t i b o d i e s . In some cases, the c o n c e n t r a t i o n of these i m p u r i t i e s i n the v i r u s p r e p a r a t i o n i s great enough to render the r e s u l t i n g p o l y c l o n a l antiserum produced by the r a b b i t nonusable because of high background r e a c t i o n s to h e a l t h y p l a n t sap. In attempts to produce an antiserum a g a i n s t GFLV, which i s found in lower t i t r e s i n herbaceous p l a n t s than AMV, a l e s s pure p r e p a r a t i o n of GFLV may have been used f o r i n j e c t i o n i n t o the animal, r e s u l t i n g i n a l e s s s p e c i f i c antiserum. In f a c t , the GFLV antiserum used i n these t e s t s was cross-adsorbed a g a i n s t healthy herbaceous p l a n t m a t e r i a l before ^ - g l o b u l i n p u r i f i c a t i o n to reduce low background ELISA r e a c t i o n s against healthy p l a n t sap components. The c r o s s - a d s o r p t i o n , which was c a r r i e d out a g a i n s t h e a l t h y herbaceous p l a n t s , may not have e l i m i n a t e d a l l unwanted a n t i b o d i e s , r e s u l t i n g i n abnormally high n e g a t i v e r e a d i n g s . The reason f o r the AMV d e t e c t i o n f a i l u r e i n the P i n o t Noir vine on J u l y 15 i s not c l e a r . I suspect that the wrong p l a n t was sampled, caused by a miscount i n the p l a n t row i n the f i e l d . The most d i f f i c u l t part i n s e p a r a t i n g p o s i t i v e p l a n t s from negative p l a n t s based on ELISA r e s u l t s i s s e t t i n g the t h r e s h o l d l e v e l above which an absorbance reading i s c o n s i d e r e d p o s i t i v e ( S u t u l a et a l . , 1986). These authors concluded that there were no easy answers for s e t t i n g 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 s when using ELISA on p l a n t s . It was important to s e l e c t a most " c o r r e c t " t h r e s h o l d , that i s one which gave the fewest f a l s e negatives and or p o s i t i v e s , a f t e r a n a l y s i n g a p o p u l a t i o n of negative and p o s i t i v e samples. They s t a t e d that the use of any t h r e s h o l d was a r b i t r a r y at l e a s t and o f t e n m i s l e a d i n g . In my study on d e t e c t i n g AMV and GFLV in i n f e c t e d f i e l d and screenhouse grapevines, the t h r e s h o l d f o r each date was determined by using Chebychev's e q u i v a l e n t because I b e l i e v e d i t to be s t a t i s t i c a l l y sound. This value i s equal 85 to the mean of the h e a l t h y c o n t r o l s , a f t e r s u b t r a c t i o n of the background c o l o u r i n b u f f e r c o n t r o l wells ( c o r r e c t e d means) plus three standard d e v i a t i o n s . This method of i d e n t i f y i n g the p o s i t i v e ELISA r e a c t i o n s was used because of the a v a i l a b i l i t y of enough healthy and b u f f e r c o n t r o l w e l l s to allow the standard d e v i a t i o n to be determined. It i s more common to s e l e c t , as a t h r e s h o l d value, a value two or three times the healthy background c o l o u r . Results p u b l i s h e d i n the s c i e n t i f i c j o u r n a l s o f t e n do not i n d i c a t e whether the b u f f e r w e l l values had been s u b t r a c t e d from the means before s e t t i n g the t h r e s h o l d v a l u e s . The s e l e c t i o n of the t h r e s h o l d c r i t e r i a may have an important e f f e c t on the r e s u l t s obtained. Comparing three methods of determining t h r e s h o l d v a l u e s , fewer f a l s e negatives were detected when using the t h r e s h o l d value equal to twice the c o r r e c t e d mean (Table XVII). This was e s p e c i a l l y true f o r samples ground in the standard g r i n d i n g b u f f e r . The increased d e t e c t i o n was o f f s e t , however, by the i n c r e a s e d chance of s e l e c t i n g f a l s e p o s i t i v e s (Table X V I I I ) . A t o t a l of 43 healthy c o n t r o l s would have been c a l l e d p o s i t i v e f o r e i t h e r GFLV or AMV d u r i n g the summer. The l a r g e number of f a l s e p o s i t i v e s may make i t d i f f i c u l t , depending on the impact of these f a l s e p o s i t i v e s , to accept t h i s method f o r s e t t i n g t h r e s h o l d s . The other two methods used to determine thresholds both gave no f a l s e p o s i t i v e s but had a g r e a t e r number of f a l s e n e g a t i v e s , e s p e c i a l l y when t e s t i n g f o r GFLV. Using Chebychev's constant e l i m i n a t e d f a l s e p o s i t i v e s but s t i l l 86 T a b l e X V I I . 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 b y u s i n g t h r e e d i f f e r e n t m e t h o d s o f d e t e r m i n i n g E L I S A 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) a n d g r a p e v i n e f a n l e a f v i r u s ( G F L V ) d e t e c t i o n d u r i n g t h e s u m m e r M e t h o d F a l s e n e g a t i v e s p e r b u f f e r 1 T e s t i n g f o r s e t t i n g d a t e t h r e s h o l d A B C D T o t a l s A ) AMV J u n e 3 c o r r . m e a n + 3 s 0 0 2 0 2 2 ( c o r r . m e a n ) 0 0 1 0 1 2 ( u n c o r r . m e a n ) 0 0 4 0 4 J u n e 24 c o r r . m e a n + 3s 0 0 1 0 1 2 ( c o r r . m e a n ) 0 0 1 0 1 2 ( u n c o r r . m e a n ) 0 0 1 0 1 J u l y 15 c o r r . m e a n + 3s 1 1 2 1 5 2 ( c o r r . m e a n ) 0 1 0 0 1 2 ( u n c o r r . m e a n ) 1 1 2 1 5 A u g . 12 c o r r . m e a n + 3s 0 0 9 0 9 2 ( c o r r . m e a n ) 0 0 0 0 0 2 ( u n c o r r . m e a n ) 3 3 7 3 16 S e p t . 2 c o r r . m e a n + 3 s 0 0 5 0 5 2 ( c o r r . m e a n ) 0 0 3 0 3 2 ( u n c o r r . m e a n ) > 0 0 7 0 7 * A - 0 . 1 5 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 . 0 5 % T w e e n - 2 0 , 0 . 2 % o v a l b u m i n a n d 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 . 0 1 M P B S , pH 7 . 4 , w i t h 0 . 0 5 % T w e e n - 2 0 , 0 . 2 % o v a l b u m i n a n d 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 . 87 Table XVII. continued Method Fal s e negatives per b u f f e r 1 T e s t i n g f o r s e t t i n g date t h r e s h o l d A B C D T o t a l s B) GFLV June 3 corr.mean + 3s 0 0 2 0 2 2 ( c o r r . mean) 0 0 0 0 0 2(uncorr. mean) 0 0 2 0 2 June 24 c o r r . mean + 3s 0 0 8 0 8 2(corr.mean) 0 0 3 0 3 2(uncorr. mean) 0 0 9 0 9 J u l y 15 c o r r . mean + 3s 12 5 20 7 44 2 ( c o r r . mean) 5 0 3 0 8 2(uncorr. mean) 1 0 13 3 17 Aug . 12 c o r r . mean + 3s 1 1 16 1 19 2 ( c o r r . mean) 1 0 4 1 6 2(uncorr. mean) 1 1 20 1 23 Sept . 2 c o r r . mean + 3s 2 2 16 2 22 2 ( c o r r . mean) 3 1 10 0 14 2(uncorr. mean) 2 2 20 2 26 XA - 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 - b u f f e r A with 1% n i c o t i n e , pH 8.4; C - standard g r i n d i n g b u f f e r - 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. 88 Table XVIII. A comparison of f a l s e p o s i t i v e s c r e a t e d by using three methods of determining 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 grapevine f a n l e a f v i r u s (GFLV) d e t e c t i o n during the summer Method F a l s e p o s i t i v e s per b u f f e r 1 T e s t i n g f o r s e t t i n g date t h r e s h o l d A B C D T o t a l s A) AMV June 3 c o r r . mean + 3s 0 0 0 0 2 ( c o r r . mean) 1 1 2 0 2(uncorr. mean) 0 0 0 0 June 24 c o r r . mean + 3s 0 0 0 0 2 ( c o r r . mean) 1 0 0 1 2(uncorr. mean) 0 0 0 0 J u l y 15 c o r r . mean + 3s 0 0 0 0 2 ( c o r r . mean) 2 1 2 2 2(uncorr. mean) 0 0 0 0 0 2 0 0 7 0 Aug. 12 c o r r . mean + 3s 0 0 0 0 2 ( c o r r . mean) 1 0 0 0 2(uncorr. mean) 0 0 0 0 0 1 0 Sept. 2 c o r r . mean + 3s 0 0 0 0 2 ( c o r r . mean) 0 1 2 3 2(uncorr. mean), 0 0 0 0 0 6 0 4A - 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 - b u f f e r A with 1% n i c o t i n e , pH 8.4; C - standard g r i n d i n g b u f f e r - 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. 89 Table XVIII. continued Method Fa l s e pos i t ives per b u f f e r 1 Tes t ing for s e t t i n g date t h r e s h o l d A B c 0 T o t a l s B) GFLV June 3 c o r r . mean + 3s 0 0 0 0 0 2 ( c o r r . mean) 1 2 1 1 5 2(uncorr. mean) 0 0 0 0 0 June 24 c o r r . mean + 3s 0 0 0 0 0 2 ( c o r r . mean) 0 1 1 1 3 2(uncorr. mean) 0 0 0 0 0 J u l y 15 c o r r . mean + 3s 0 0 0 0 0 2 ( c o r r . mean) 2 2 3 2 9 2(uncorr. mean) 0 0 0 0 0 Aug. 12 c o r r . mean + 3s 0 0 0 0 0 2<corr. mean) 0 1 3 1 5 2(uncorr. mean) 0 0 0 0 0 Sept. 2 c o r r . mean + 3s 0 0 0 0 0 2 ( c o r r . mean) 0 1 2 1 4 2(uncorr. mean) 0 0 0 0 0 XA - 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 - b u f f e r A with 1% n i c o t i n e , pH 8.4; C - standard g r i n d i n g b u f f e r - 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. 90 maintained a f a i r l y high number of f a l s e n e g a t i v e s , e s p e c i a l l y when d e t e c t i n g GFLV. Because of the tendency, at the S t a t i o n , to r e t e s t negative r e s u l t s r e p e a t e d l y i n order to confirm t h e i r s t a t u s while p o s i t i v e t e s t s are o f t e n accepted at face value, I b e l i e v e that using a t h r e s h o l d of twice the u n c o r r e c t e d mean of healthy samples would give more accurate r e s u l t s f o r the Quarantine S t a t i o n purposes. T h i s choice may not be acceptable to a l a b o r a t o r y where samples are only done once and the consequences of missing a p o s i t i v e i s g r e a t e r than f a l s e l y c a l l i n g a negative sample p o s i t i v e . The parameters of the ELISA t e s t t h r e s h o l d values should be determined f o r each separate case. It i s simple to determine the t h r e s h o l d p r o v i d i n g enough healthy c o n t r o l s are i n c l u d e d in each t e s t . It i s standard p o l i c y at the s t a t i o n to r e p e a t e d l y r e t e s t p l a n t s g i v i n g b o r d e r l i n e p o s i t i v e or negative ELISA r e s u l t s . Comparing the r e s u l t s of ELISA t e s t s on in vitro p l a n t l e t s and f i e l d p l a n t s That nepoviruses may be detected w e l l by ELISA in grapevines growing e i t h e r in vitro or i n the f i e l d and screenhouses i s an important c o n c l u s i o n of t h i s study. F a c t o r s which may a f f e c t v i r u s d e t e c t i o n by ELISA were i d e n t i f i e d and recommendations made to improve AMV, GFLV, and TomRV d e t e c t i o n i n grapevines. However, the data presented have a l s o determined that n e i t h e r method, even with the improvements d e s c r i b e d i n t h i s t h e s i s i s 100% 91 a c c u r a t e . E i t h e r method may be used with reasonable c o n f i d e n c e p r o v i d i n g the optimum parameters as d e s c r i b e d i n t h i s t h e s i s are adhered to. The g r e a t e r amount of work needed to i n i t i a t e and maintain p l a n t l e t s and the i n c r e a s e d r e l i a b i l i t y of the m o d i f i e d b u f f e r s i n d e t e c t i n g these v i r u s e s by ELISA i n d i c a t e t hat, f o r r o u t i n e t e s t i n g purposes, sampling from f i e l d p l a n t s would be more e f f i c i e n t than t e s t i n g p l a n t s grown in vitro. The need f o r r o u t i n e i n i t i a t i o n and t e s t i n g of grapevine p l a n t l e t s in vitro becomes even l e s s a t t r a c t i v e when p u b l i s h e d r e s u l t s on s u c c e s s f u l v i r u s d e t e c t i o n by ELISA i n other grapevine t i s s u e s , i n c l u d i n g dormant canes, are c o n s i d e r e d (Bovey et a l . , 1980a; Stellmach, 1985a,b; Huss et a l . , 1986; Walter and E t i e n n e , 1987). T e s t i n g of in vitro p l a n t s may be p r e f e r a b l e when used i n c o n j u n c t i o n with in vitro v i r u s e l i m i n a t i o n and subsequent r a p i d bulk propagation. However, i t i s necessary to determine the l i m i t a t i o n s of t h i s t e s t before i t can be a p p l i e d r o u t i n e l y . The v i r u s t i t r e s i n in vitro p l a n t l e t s may be reduced below d e t e c t i o n l e v e l s f o r longer periods of time. 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V i t i s t i s s u e c u l t u r e media 1 Growth stage Component I n i t i a t i o n P r o l i f e r a t i o n Root ing MS 2 3/4 f u l l 1/4 s trength s trength s trength i e . s o l ' n A 3 15.0 ml/L 20.0 ml/L 5.0 ml/L B 15.0 ml/L 20.0 ml/L 5.0 ml/L C 3.75 ml/L 5 . 0 ml/L 1.25 ml/L D 3.75 ml/L 5. 0 ml/L 1.25 ml/L E 3.75 ml/L 5 . 0 ml/L 1 .25 ml/L F 3.75 ml/L 5 . 0 ml/L 1 .25 ml/L G 3.75 ml/L 5.0 ml/L 1.25 ml/L i - i n o s i t o l 75.0 mg/L 100.0 mg/L 25.0 mg/L thiamine HCI 0.3 mg/L 0 . 4 mg/L 0.1 mg/L adenine s u l f a t e 60.0 mg/L 80 . 0 mg/L NaH 2P0 4H 20 128.0 mg/L 170.0 mg/L 150.0 mg/L BAP 4 2.0 mg/L 2.0 mg/L IBA S 0.0 23 mg/L 0.03 mg/L I AA 0.1 mg/L sucrose 22.5 g/L 30 . 0 g/L 17.5 g/L PH 5 . 7 5 . 0 5 . 0 agar 7.0 g/L 1 To prepare the c u l t u r e medium, add the d e s i r e d amount s of each s o l u t i o n and i n g r e d i e n t s to about 800 ml of d i s t i l l e d water. Bring to 1000 ml and adjust pH. If needed, heat and add agar. 2 MS c o n s i s t s of a modified Murashige and Skoog p l a n t s a l t mixture with the f o l l o w i n g i n g r e d i e n t s : 100 Appendix 1. continued S o l u t i o n Cons t i tuents Cone. (g/L) A NH 4N0 3 82.5 B KN0 3 95.0 C H 3B0 3 1.24 KH 2P0 4 34.00 KI 0.166 Na2MoO.« . 2H 20 0.05 CoCl 2.6H 20 0.005 D CaCl 2.2H 20 88.0 (or C a C l 2 ) 66.43 E MgS0 4.7H 20 74.0 MnS0 4.4H 20 3.36 (or MnS0 4.H 20) 2.55 ZnS0 4.7H 20 1.72 CuS0 4.5H 20 0.005 F* Na 2.EDTA 7.45 FeS0 4.7H 3.0 5.57 G N i c o t i n i c a c i d 0.1 P y r o x i d i n e HCl 0.1 G l y c i n e 0 . 1 4 A stock s o l u t i o n of BAP ,(.5 mg/ml) may be prepared by d i s s o l v i n g 50 mg of BAP i n a very small volume of 1 M HCl to form a s l u r r y . T h i s can then be added to 100 ml of d i s t i l l e d water and kept r e f r i g e r a t e d . 5 To prepare a stock s o l u t i o n of IBA (.lmg/ml), d i s s o l v e 10 mg i n a very small volume of 1 M NaOH to form a s l u r r y . Then slowly add 100 ml of d i s t i l l e d water while s t i r r i n g . * To prepare 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 water, heat the Na 2.EDTA s o l u t i o n , then with continuous s t i r r i n g add the FeS0 4.7H 20 s o l u t i o n . When cooled d i l u t e to 1000 ml. 

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