UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Purification and properties of potato virus M (PVM) Ahmad, Ismail Bin 1977

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1977_A6_7 A34.pdf [ 6.67MB ]
Metadata
JSON: 831-1.0094014.json
JSON-LD: 831-1.0094014-ld.json
RDF/XML (Pretty): 831-1.0094014-rdf.xml
RDF/JSON: 831-1.0094014-rdf.json
Turtle: 831-1.0094014-turtle.txt
N-Triples: 831-1.0094014-rdf-ntriples.txt
Original Record: 831-1.0094014-source.json
Full Text
831-1.0094014-fulltext.txt
Citation
831-1.0094014.ris

Full Text

PURIFICATION AND PROPERTIES OF POTATO VIRUS M (PVM) by I s m a i l B i n Ahmad B.Sc. ( A g r . ) , U n i v e r s i t y o f Malaya, 1974 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE DEPARTMENT OF PLANT SCIENCE We a c c e p t t h i s t h e s i s as con f o r m i n g t o t he r e q u i r e d s t a n d a r d THE UNIVERSITY OF March, Ismail Bin BRITISH COLUMBIA 1977 Ahmad, 1977 In p r e s e n t i n g t h i s t h e s i s i n p a r t i a l f u l f i l l m e n t o f the r e q u i r e m e n t s f o r an advanced degree a t the U n i v e r s i t y o f B r i t i s h Columbia, I agree t h a t the l i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r r e f e r e n c e and s t u d y . I f u r t h e r agree t h a t p e r m i s s i o n f o r e x t e n s i v e c o p y i n g o f t h i s t h e s i s f o r s c h o l a r l y purposes may be g r a n t e d by the Chairman o f the Department o f P l a n t S c i e n c e o r by h i s r e p r e s e n t a t i v e s . I t i s u n d e r s t o o d t h a t c o p y i n g o r p u b l i c a t i o n o f t h i s t h e s i s f o r f i n a n c i a l g a i n s h a l l not be a l l o w e d w i t h o u t m y . w r i t t e n p e r m i s s i o n . Chairman, Department o f P l a n t S c i e n c e The U n i v e r s i t y o f B r i t i s h Columbia Vancouver 8, Canada Date ik^A 1 , ( ^7 y ABSTRACT S t u d i e s on p u r i f i c a t i o n and p r o p e r t i e s o f p o t a t o v i r u s M (PVM) were c a r r i e d out u s i n g an i s o l a t e found i n B r i t i s h Columbia. The narrow ho s t range o f the v i r u s was c o n f i r m e d , and no new s u s c e p t i b l e s p e c i e s was d i s c o v e r e d . P o t a t o c u l t i v a r s f a i l e d t o deve-l o p symptomps even i n p l a n t s produced by t u b e r s o f i n o c u l a t e d p l a n t s , but none was immune. An attempt t o demonstrate t r a n s m i s s i -b i l i t y o f the v i r u s by p l a n t c o n t a c t was u n s u c c e s s f u l . In u n d i l u t e d p o t a t o sap the v i r u s had a thermal i n a c t i v a t i o n p o i n t (TIP) o f 65 to 70?:C, and a l o n g e v i t y i n v i t r o ( LIVJ o f 2 t o 4 days. The d i l u t i o n end p o i n t (DEP) was 10" 4 . The LIV and DEP o f t h e v i r u s i n tomato sap were s i m i l a r t o t h o s e i n p o t a t o sap. Crude sap d i l u t e d t o l O - 1 induced more l e s i o n s on Red Kidney bean than u n d i l u t e d sap. An e f f i c i e n t p u r i f i c a t i o n p r o c e d u r e f o r PVM was d e v e l o p e d . The v i r u s was p u r i f i e d from l e a v e s o f p o t a t o (Solanum tuberosum L . ) , by e x t r a c t i o n w i t h 0.5 M b o r a t e b u f f e r , pH 7.8, c l a r i f i c a t i o n w i t h ammonium s u l f a t e ( 2 0 % ) , and c o n c e n t r a t i o n w i t h ammonium s u l f a t e ( 3 0 % ) . F u r t h e r c o n c e n t r a t i o n was c a r r i e d out by hi g h speed c e n t r i f u g a t i o n f o l l o w e d by p o l y e t h y l e n e §lycol (PEG 6000) p r e c i p i -t a t i o n and h i g h speed c e n t r i f u g a t i o n . F i n a l p u r i f i c a t i o n was by su c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n . The y i e l d o b t a i n e d from t h i s p r o c e d u r e was 3.7 t o 4.1 mg per Kg o f i n f e c t e d l e a v e s . The p u r i f i e d p r e p a r a t i o n s c o n t a i n e d rod-shaped p a r t i c l e s 651 nm normal l e n g t h and 13.4 nm average w i d t h . The p a r t i c l e s had an A 2 6 0 / A 2 8 0 r a t i o o f ] ^ 3 ' a n A m a x / A m i n r a t i o o f '-24' a i i i maximum u l t r a v i o l e t l i g h t a b s o r p t i o n a t 260 nm, a minimum a b s o r p t i o n a t 245 nm, and a buoyant d e n s i t y i n CsCl o f 1.304 ( s u g g e s t i n g an RNA c o n t e n t o f 6.2%). The m o l e c u l a r weight o f the p r o t e i n s u b u n i t was about 39,300 d a l t o n s . i v TABLE OF CONTENTS PAGE TITLE PAGE ABSTRACT i i TABLE OF CONTENTS i v LIST.OF FIGURES v i i LIST OF TABLES i x ACKNOWLEDGEMENT x INTRODUCTION 1 REVIEW OF LITERATURE 2 (a) H i s t o r y and Nomenclature 2 (b) D i s t r i b u t i o n and Economic Importance 4 (c) Host Range and Symptomatology 5 (d) S e r o l o g y 6 (e) T r a n s m i s s i o n 7 ( f ) P u r i f i c a t i o n 9 (g) P r o p e r t i e s ^ MATERIALS AND METHODS 13 (a) The PVM I s o l a t e ~ 13 (b) Host Range and Symptomatology 14 (c) S e r o l o g y 16 (d) P r o p e r t i e s i n Crude Sap 17 (e) P u r i f i c a t i o n 18 TABLE OF CONTENTS ( c o n t ' d ) PAGE 1, E x t r a c t i o n o f v i r u s from h o s t t i s s u e s 20 2, C l a r i f i c a t i o n o f crude sap 22 3, C o n c e n t r a t i o n o f v i r u s from c l a r i f i e d 24 sap 4, F u r t h e r c o n c e n t r a t i o n o f v i r u s p r e p a r a - 26 t i o n 5, D e n s i t y g r a d i e n t c e n t r i f u g a t i o n 28 6, E l e c t r o n m i c r o s c o p y 29 ( f ) D e t e r m i n a t i o n o f the B i o p h y s i c a l and B i o c h e m i c a l P r o p e r t i e s 29 1. A b s o r p t i o n s p e c t r a 30 2. E s t i m a t i o n o f s e d i m e n t a t i o n c o e f f i c i e n t 30 3. E s t i m a t i o n o f buoyant d e n s i t y 30 4. E s t i m a t i o n o f m o l e c u l a r w e i g h t o f p r o t e i n s u b u n i t 31 RESULTS 33 (a) Host Range and Symptomatology 33 (b) S e r o l o g y 36 (c) P r o p e r t i e s i n Crude Sap 37 (d) P u r i f i c a t i o n 41 1. E x t r a c t i o n o f v i r u s from l e a f t i s s u e s 41 2, C l a r i f i c a t i o n o f c r u d e sap 42 3. C o n c e n t r a t i o n o f v i r u s from c l a r i f i e d sap 50 4, F u r t h e r c o n c e n t r a t i o n and f i n a l p u r i f i c a -t i o n 54 (e) B i o p h y s i c a l and B i o c h e m i c a l P r o p e r t i e s 64 v i TABLE OF CONTENTS ( c o n t ' d ) PAGE DISCUSSION 71 LITERATURE CITATIONS 82 APPENDICES 9 4 v i i LIST OF FIGURES FIGURE PAGE 1. A n t i b o d y p r o d u c t i o n i n r a b b i t a g a i n s t PVM a n t i g e n . Arrows i n d i c a t e the time o f v i r u s a d m i n i s t r a t i o n i n t o the immunized r a b b i t . 37 2. The r e l a t i o n s h i p s between the average number o f l o c a l l e s i o n s p er h a l f ^ l e a f o f Red Kidney bean w i t h (A) d i l u t i o n o f sap, (B) days o f p r e i n c u b a t i o n o f sap a t room t e m p e r a t u r e , and (C,D) p r e i n c u b a t i o n temperature (°C) o f sap f o r 10 min. 39 3. The r e l a t i v e c o n c e n t r a t i o n o f PVM M i n , and t u r b i d i t y a t 540 nm [•] o f , (a) 1:3 sap and (b) 1:1.5 sap, a f t e r c l a r i f i c a t i o n w i t h v a r i o u s amounts(ml) o f carbon t e t r a c h l o r i d e per 10ml o f sample. 45 4. The r e l a t i v e c o n c e n t r a t i o n o f PVM M i n , and t u r b i d i t y a t 540 nm B z f l o f , (a) 1:3 sap and (b) 1.1:5 sap, a f t e r c l a r i f i c a t i o n w i t h v a r i o u s amounts(gm) o f ammonium s u l f a t e p er 10 ml o f sample 48 5. R e l a t i v e c o n c e n t r a t i o n o f PVM M i n , and t u r b i d i t y a t 540 nmOCflof, (A) 1:3 sap from p o t a t o , (B) 1:1.5 sap from p o t a t o and (C) 1:1.5 sap from tomato, a f t e r c l a r i f i c a t i o n w i t h carbon t e t r a c h l o r i d e (CT) o r ammonium s u l f a t e (AS). 49 6. R e l a t i v e c o n c e n t r a t i o n o f PVM i n p r e p a r a t i o n s p r e c i p i t a t e d by the v a r i o u s methods: High speed c e n t r i f u g a t i o n (HC), PEG p r e c i p i t a t i o n (PEG), Ammonium, s u l f a t e p r e c i p i t a t i o n (AS) and a c i d p r e c i p i t a t i o n (AC). The p e l l e t s were resuspended i n e i t h e r 0.5 M o r 0.05 M b o r a t e b u f f e r a t o n e - t e n t h ,09] , two-tenths QlO ., o r f o u r - t e n t h s M o f the o r i g i n a l volume. 51 7. E l e c t r o n m i c r o g r a p h s o f v i r u s p r e p a r a t i o n s a f t e r t h r e e s t a g e s o f c o n c e n t r a t i o n f o l l o w e d by a s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i n SW25 r o t o r ; A. ammonium s u l f a t e method, B. carbon t e t r a c h l o r i d e method. 57 LIST OF FIGURES ( c o n t ' d ) 8. E l e c t r o n m i c r o g r a p h s o f PVM p a r t i c l e s a f t e r c l a r i f i c a t i o n and c o n c e n t r a t i o n o f the v i r u s p r e p a r a t i o n s by the f o l l o w i n g methods: A, C l a r i f i c a t i o n w i t h ammonium s u l f a t e ; B, F i r s t s t a g e o f c o n c e n t r a t i o n by p r e c i p i t a t i o n w i t h ammonium s u l f a t e -C, Second s t a g e o f c o n c e n t r a t i o n by h i g h speed c e n t r i f u g a t i o n ; D, T h i r d s t a g e o f c o n c e n t r a t i o n by p r e c i p i t a t i o n w i t h PEG 6000; E, F o u r t h s t a g e o f c o n c e n t r a t i o n by h i g h speed c e n t r i f u g a t i o n . 9. E l e c t r o n m i c r o g r a p h o f PVM p a r t i c l e s a f t e r p u r i f i c a t i o n by the ammonium s u l f a t e method. 10. S u c r o s e d e n s i t y g r a d i e n t p r o f i l e s o f PVM (254nm) o b t a i n e d i n two d i f f e r e n t e x p e r i m e n t s . The v i r u s p r e p a r a t i o n s were d e r i v e d from ammonium s u l f a t e - c l a r i f i e d sap ( a ) , carbon t e t r a c h l o r i d e , c l a r i f i e d sap ( b ) , and f r e e z e - c l a r i f i e d sap ( c ) . 11. Average number o f l o c a l l e s i o n s p er s i x h a l f - l e a v e s o f Red Kidney bean p l a n t s r e c o r d e d i n i n f e c t i v i t y a s s a y o f 1 ml f r a c t i o n s from SW 41 s u c r o s e d e n s i t y g r a d i e n t ; , absorbance a t 254 nm; • - - ^ n l , number o f l o c a l l e s i o n s . 12. F l o w - c h a r t o f the ammonium s u l f a t e method f o r the p u r i f i c a t i o n o f PVM. 13. A b s o r p t i o n spectrum o f p u r i f i e d PVM s o l u t i o n : a. u n c o r r e c t e d f o r l i g h t s c a t t e r i n g , b, c o r r e c t e d f o r l i g h t s c a t t e r i n g . 14. S c h l i e r e n p a t t e r n o f s e d i m e n t i n g PVM p a r t i c l e s i n 0.05 M b o r a t e b u f f e r , pH 7.8, 16 min a f t e r r e a c h i n g speed o f 21,740 rpm, 15. S c h l i e r e n p a t t e r n o f PVM p a r t i c l e s r e s u l t i n g from e q u i l i b r i u m banding i n cesium c h l o r i d e ; PVM bands appeared to the l e f t o f the TMV band, 16. P l o t o f m o l e c u l a r w e i g h t s o f p r o t e i n s v e r s u s t h e i r r e l a t i v e m o b i l i t i e s i n 5% p o l y a c r y l a m i d e g e l s c o n t a i n i n g 0.1% SDS. The m o l e c u l a r w e i g h t o f PVM p r o t e i n s u b u n i t was g r a p h i c a l l y e s t i m a t e d from the p l o t . The p r o t e i n s t a n d a r d s used were: Bovine serum albumin (BSA), ovalbumin (OA), c a r b o n i c anhydrase (CA), a l c o h o l dehydrogenase (AD).and myoglobin (Mg), IX LIST OF TABLES TABLE," PAGE 1. B i o p h y s i c a l p r o p e r t i e s and p r o p e r t i e s i n crude sap o f PVM as r e p o r t e d i n the l i t e r a t u r e , 11 2. Combinations o f methods used f o r the second and t h i r d s t a g e o f c o n c e n t r a t i o n - o f PVM p r e p a r a t i o n o r i g i n a l l y o b t a i n e d from ammonium s u l f a t e - o r carbon, t e t r a c h l o r i d e -c l a r i f i e d sap, 27 3. L o c a l l e s i o n symptoms on v a r i o u s s p e c i e s o r c u l t i v a r s o f p l a n t s i n c i t e d by PVM 34 4. M e c h a n i c a l and g r a f t - t r a n s m i s s i b i l i t y o f PVM i n t o the v a r i o u s p o t a t o c u l t i v a r s c a r r i e d o u t a t f o u r d i f f e r e n t t i m e s . 35 5. V a r i a t i o n s i n t h e r e l a t i v e c o n c e n t r a t i o n o f PVM i n , and the f i n a l pH o f . 1:3 sap as a r e s u l t o f u s i n g d i f f e r e n t e x t r a c t i o n b u f f e r s , a t pH 7,8, f o r homogenizing p o t a t o l e a v e s , 40 6. R e l a t i v e v i r u s c o n c e n t r a t i o n and t u r b i d i t y o f 1:3 sap a f t e r c l a r i f i c a t i o n by v a r i o u s methods. 43 7. T u r b i d i t y v a l u e s o f carbon t e t r a c h l o r i d e - c l a r i f i e d sap measured a t 540 nm,after c e n t r i f u g a t i o n a t d i f f e r e n t c o m b i n a t i o n s o f speed and time. 47 8. R e l a t i v e c o n c e n t r a t i o n o f v i r u s i n p r e p a r a t i o n s o b t a i n e d by p r e c i p i t a t i o n s w i t h d i f f e r e n t c o m b i n a t i o n s o f PEG 6000 and sodium c h l o r i d e . 53 9. R e l a t i v e c o n c e n t r a t i o n o f v i r u s p r e p a r a t i o n s a f t e r a t h i r d s t a g e o f c o n c e n t r a t i o n u s i n g f o u r d i f f e r e n t p r o c e d u r e s , 55 10, Range o f c o n c e n t r a t i o n , average ^ 2 6 0 ^ 2 8 0 r a t i o s and i n f e c t i v i t y o f PVM p r e p a r a t i o n s p u r i f i e d from 40 gm o f PV M - i n f e c t e d p o t a t o l e a v e s . 61 11. U l t r a v i o l e t a b s o r p t i o n c h a r a c t e r i s t i c s o f p o t a t o v i r u s M b e f o r e and a f t e r c o r r e c t i o n f o r l i g h t s c a t t e r i n g . 66 X ACKNOWLEDGEMENT The a u t h o r wishes t o e x p r e s s h i s g r a t i t u d e t o the members o f h i s committee, Dr. R. S t a c e - S m i t h and Dr. N.S. Wri g h t , Canada Depart-ment o f A g r i c u l t u r e , R e search S t a t i o n , Vancouver, Dr. V.C. Runekles and Dr. R.J. Uopeman ( P r o f e s s o r and Chairman, and A s s i s t a n t P r o f e s s o r , r e s p e c t i v e l y ) , Department 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 Columbia, f o r t h e i r g u i d a n c e , c r i t i c i s m and s u g g e s t i o n s p e r t a i n i n g t o t h i s t h e s i s . The a u t h o r a l s o wishes t o thank the D i r e c t o r and o t h e r s t a f f members o f the Canada Department o f A g r i c u l t u r e , Research S t a t i o n , Vancouver, f o r t h e i r i n v a l u a b l e a s s i s t a n c e and f o r p r o v i d i n g f a c i l i t i e s f o r c a r r y i n g o u t the r e s e a r c h p r o j e c t . S p e c i a l thanks i s a l s o extended t o the N a t i o n a l U n i v e r s i t y o f M a l a y s i a who p r o v i d e d f i n a n c i a l s u p p o r t f o r t h i s p r o j e c t . 1 INTRODUCTION Members o f the C a r l a v i r u s Group o c c u r i n c h a r a c t e r i s t i c a l l y low c o n c e n t r a t i o n s o f 20 t o 100 mg per l i t e r o f sap i n t h e i r r e s p e c t i v e h o s t s ( H a r r i s o n e t a l , , 1 9 7 1 ) . A l a r g e amount o f s t a r t i n g m a t e r i a l i s r e q u i r e d t o o b t a i n even a s m a l l amount o f p u r i f i e d v i r u s . Hence i t i s i m p o r t a n t to m i n i m i z e the amount o f v i r u s l o s t d u r i n g the v a r i o u s s t e p s o f any p u r i f i c a t i o n p r o c e d u r e . To s t u d y the p r o p e r t i e s o f a v i r u s , e s p e c i a l l y i t s b i o -p h y s i c a l and b i o c h e m i c a l p r o p e r t i e s , an adequate amount o f p r a c t i c a l l y p u r i f i e d v i r u s s h o u l d be a v a i l a b l e . The d i f f i c u l t y o f p u r i f i c a t i o n o f a v i r u s i s o f t e n r e f l e c t e d by the p a u c i t y o f i n f o r m a t i o n on t h e s e p r o p e r t i e s . Indeed,most o f the C a r l a v i r u s e s d e s c r i b e d i n the " D e s c r i p t i o n o f P l a n t ' V i r u s e s " o f the Commonwealth M y c o l o g i c a l I n s t i t u t e and the A s s o c i a t i o n o f A p p l i e d B i o l o g i s t s , l a c k d e t a i l s o f t h e i r b i o p h y s i c a l and b i o c h e m i c a l p r o p e r t i e s (Appendix I I ) . T h i s t h e s i s d e s c r i b e s an e f f i c i e n t p u r i f i c a t i o n p r o c e d u r e f o r p o t a t o v i r u s M (PVM), by means o f which s u f f i c i e n t amount o f the v i r u s was p u r i f i e d t o p e r m i t a s t u d y o f some of i t s b i o p h y s i c a l and b i o c h e m i c a l p r o p e r t i e s . Other p r o p e r t i e s i n c l u d i n g h o s t range, symptomatology, s u s c e p t i b i l i t y o f p o t a t o c u l t i v a r s and p r o p e r t i e s i n c r u d e sap were a l s o i n v e s t i g a t e d . 2 REVIEW OF LITERATURE ( a ) . H i s t o r y and Nomenclature. The name " p o t a t o v i r u s M" (PVM) was i n t r o d u c e d i n 1956 by B a g n a l l e t a l . t o d e s i g n a t e a t h i r d v i r u s i s o l a t e d from ' I r i s h C o b b l e r ' p o t a t o e s (Solanum tuberosum L . ) , i n f e c t e d w i t h the " i n t e r v e i n a l mosaic" d i s e a s e (McKay and D y s k t r a , 1932). The o t h e r two v i r u s e s were p o t a t o v i r u s X (PVX) and p o t a t o v i r u s S (PVS). The d i s e a s e s " l e a f r o l l i n g mosaic" ( S c h u l t z and Folsom, 1923) and " p a r a c r i n k l e " (Salaman and Le Pel l e y , 1930) were a l s o shown to be caused by PVM i n c o m b i n a t i o n w i t h PVX and PVS o r w i t h PVS o n l y ( B a g n a l l e t al.»1956, 1959). The v i r u s i s o l a t e s d e s i g n a t e d as " v i r u s K" ( K o h l e r , 1942) and "D1102" ( K o h l e r , 1953; Wetter and Brandes, 1955 and 1956) were l a t e r i d e n t i f i e d as PVM ( B a g n a l l e t al.» 1956, 1959). PVM has had a unique h i s t o r y because i t evoked a c o n t r o v e r s y on the o r i g i n o f v i r u s e s . The a p p a r e n t o b s e r v a t i o n t h a t the " p a r a c r i n k l e v i r u s " was o n l y t r a n s m i t t e d by g r a f t i n g (Bawden, 1939), l e d D a r l i n g t o n (1944) t o propose the t h e o r y t h a t the " v i r u s " had a r i s e n "de novo" i n the c u l t i v a r King Edward. T h i s t h e o r y assumed t h a t the p a r t i c l e which he c a l l e d " p r o v i r u s " c o u l d behave l i k e plasmogenes t o produce the d i s e a s e on o t h e r c u l t i v a r s . T h i s propo-s a l g a i n e d t h e ; s u p p o r t o f van der Plank (1948), but was c r i t i c i z e d by Corsan e t a l . (1944) and Smith (1946). The p r o d u c t i o n o f v i r u s -f r e e s t o c k o f 'King Edward' from v i r u s - i n f e c t e d s t o c k by t i s s u e 3 c u l t u r e ( K a s s a n i s , 1957), f i n a l l y put an end to t h e s e s p e c u l a t i o n s (Bawden and K a s s a n i s , 1965). The name " p o t a t o v i r u s M" i s now commonly a c c e p t e d and o l d e r names a r e abandoned (Beemster and Rozendaal, 1972) o r l i s t e d as i t s synonyms (MacLeod, 1962; Martyn, 1968; Wetter, 1972). The f o l l o w i n g a r e c o n s i d e r e d synomyms o f PVM: L e a f r o l l i n g mosaic v i r u s I n t e r v e i n a l mosaic v i r u s P a r a c r i n k l e v i r u s P o t a t o v i r u s E P o t a t o v i r u s 7 P o t a t o v i r u s K Solanum v i r u s 11 K a r t o f f e l - R o l l m o s a i c - V i rus P o t a t o v i r u s F,C. S c h u l t z and Folsom, 1923 McKay and D y s k t r a , 1932 Salaman and Le Pel l e y , 1930 Bawden, i n Smith, 1933 Smith, 1937 K o h l e r , 1942 Smith, 1937 K o h l e r , 1935 A u g i e r de Montgremier > • Devergne, 1958 The term " s t r a i n " has been used by v a r i o u s a u t h o r s t o d e s i g n a t e PVM i s o l a t e d from d i f f e r e n t s o u r c e s (Rozendaal and Van S l o g t e r e n , 1957; K a s s a n i s , 1960, 1961; B a r t e l and Volk 1966; Bode and Weideman, 1969, 1971 ; B a g n a l l e t al',,1959). Wetter (1972) l i s t e d the f o l l o w i n g as the s t r a i n s o f PVM: L e a f r o l l i n g mosaic i s o l a t e I n t e r v e i n a l mosaic i s o l a t e P a r a c r i n k l e i s o l a t e D1102 and Fortuna i s o l a t e s , and Dutch i s o l a t e s . 4 ( b ) . D i s t r i b u t i o n and economic importance PVM has a worldwide d i s t r i b u t i o n i n many c u l t i v a r s o f p o t a t o e s (Wetter, 1972; H i r u k i , 1973). In a d d i t i o n to North America, Germany, B r i t a i n and the N e t h e r l a n d s , the v i r u s has been r e p o r t e d from o t h e r c o u n t r i e s i n Europe (Bode, 1958; P a j a n , 1967), A s i a ( H o r i o e t al., 1969) and South America (Beemster, 1969). I t has been r e p o r t e d t h a t PVM i s the most w i d e s p r e a d p o t a t o v i r u s i n L a t v i a (Duda, 1969). The economic s i g n i f i c a n c e o f PVM has not been a s s e s s e d i n g r e a t d e t a i l . However,according t o K a s s a n i s and Schwabe (1961), and Bawden and K a s s a n i s (1965), the c u l t i v a r King Edward gave a h i g h e r y i e l d when a v i r u s - f r e e s t o c k d e r i v e d from t i s s u e c u l t u r e ( K a s s a n i s , 1957) was used f o r p l a n t i n g . S t r a i n s which cause s e v e r e symptoms on c e r t a i n c u l t i v a r s o f p o t a t o w i l l s e v e r e l y d e p r e s s y i e l d s o f t h e s e c u l t i v a r s (Beemster and Rozendaal, 1972). Tuber n e c r o s i s has been found c o r r e l a t e d w i t h the f o l i a g e symptoms (L'o ug hnane, 1957), thus d i s p r o v i n g an e a r l i e r s u g g e s t i o n by Smith (1946) t h a t PVM i s a v i r u s o f no economic s i g n i f i c a n c e . No p o t a t o c u l t i v a r has been found to be immune^ t o PVM. Z a d i n a (1971) and Hunnius (1972 a) r e p o r t e d s u s c e p t i b i l i t y o f 630 and 47 p o t a t o c u l t i v a r s , r e s p e c t i v e l y . The low e f f i c i e n c y o f a p h i d t r a n s m i s s i o n o f PVM i s e v i d e n t from the f a c t t h a t o f the 92 c u l t i v a r s t e s t e d by Bode and Weideman (1971) w i t h Myzus p e r s i c a e S u l z . , more than h a l f o f them e i t h e r f a i l e d t o become i n f e c t e d o r were i n f e c t e d i n s m a l l numbers. 5 ( c ) . Host range and Symptomatology. The h o s t range o f PVM i s r e l a t i v e l y narrow. S u s c e p t i b l e s p e c i e s l i s t e d by the v a r i o u s a u t h o r s (MacLeod 1962; V u l c i c and Hunnius 1967; H o r i o e t a l « ? 1 9 6 9 ; B a g n a l l e t al,,1956) b e l o n g t o the f a m i l i e s Amaranthaceae, Chenopodiaceae, Leguminosae and S o l a n a c e a e . USDA (1966) l i s t e d 21 s u s c e p t i b l e s p e c i e s , but r e c e n t i n v e s t i g a t i o n s i n Europe and North America have s i g n i f i c a n t l y i n c r e a s e d t h i s number (Horvath,1972; Horvath and de Bokx, 1972; B a g n a l l 1,1972; Dziewonska and Ostrowska, 1975). The m a j o r i t y o f t h e s e h o s t p l a n t s b e l o n g t o the S o l a n a c e a e (Wetter,1972). Symptoms v a r y between p l a n t s p e c i e s . I n f e c t e d Datura  metel L. e x h i b i t s l o c a l c h l o r o s i s , n e c r o s i s and s y s t e m i c n e c r o s i s . N i c o t i a n a debneyi Domin. r e a c t s t o PVM i n f e c t i o n by p r o d u c i n g l o c a l brownish n e c r o t i c l e s i o n s ( B a g n a l l e t a l . ^ 1956, 1959). The v i r u s i n d u c e s v e r y s e v e r e symptoms i n L y c o p e r s i c o n  c h i l e n s e Dun. i n the form o f e p i n a s t y , d i s t o r t i o n and l e a f a b s c i s s i o n (Kowalska and Was, 1976). In P h a s e o l u s v u l g a r i s L. cv. Red Kidney, minute brown l o c a l l e s i o n s a r e o b s e r v e d 3 to 6 days a f t e r mechanical i n o c u l a t i o n s , and t h i s s p e c i e s has been employed as a t e s t p l a n t f o r a s s a y and d e t e c t i o n f o r PVM ( H i r u k i , 1970, 1972, 1973; H i r u k i e t a l . , , 1974). A s i m i l a r t y p e o f l o c a l l e s i o n symptoms has been d e s c r i b e d i,n \ | ; i i p a : < g i n e n s i s S a v i ex. Hassk ( H o r v a t h , 1972). V a r i a t i o n s i n symptoms i n c i t e d by d i f f e r e n t i s o l a t e s o f the v i r u s have been o b s e r v e d on some h o s t p l a n t s (Rozendaal and 6 van S l o g t e r e n , 1957; B a g n a l l e t a l , , 1959; Ross, 1968; Kowalska and Was, 1976). The appearance o f symptoms i s a f f e c t e d by t e m p e r a t u r e , and symptoms a r e more pronounced a t temperatures between 16°C t o 20°C ( B a g n a l l e t a!,,1956; Rozendaal and van S l o g t e r e n , 1957; Kowalska and Was, 1976). In p o t a t o e s , symptoms range from none t o s e v e r e depending on the v i r u s s t r a i n s and the p o t a t o c u l t i v a r s (Wetter, 1972; B u r t o n , 1966). C u l t i v a r s which show no symptoms upon i n f e c t i o n by PVM have a l s o been r e p o r t e d (Bawden e t a l , , 1950j K a s s a n i s , 1956; Rozendaal and van S l o g t e r e n , 1957). D i f f e r e n c e s i n r e a c t i o n s o c c u r not o n l y between c u l t i v a r s but a l s o between p l a n t s o f the same c u l t i v a r (Chrzanowska, 1976). In the c u l t i v a r Uran,the same i s o l a t e o f PVM i n c i t e d symptoms v a r y i n g from s l i g h t l e a f - r o l l i n g o r s l i g h t c u r l i n g o f the l e a f l e t s t o some r e d u c t i o n i n growth, and sometimes even to n e c r o s i s o f l e a f p e t i o l e s and dwarfed growth, Moreover, i n d i v i d u a l p l a n t s w i t h s e v e r e symptoms were o b t a i n e d from t u b e r s o f p l a n t s w i t h s l i g h t symptoms and v i c e v e r s a , ( d ) . S e r o l o g y . PVM i s s t r o n g l y a n t i g e n i c (MacLeod, 1962; Beemster and R o z e n d a a l , 1972; Wetter, 1972) and a n t i s e r a a g a i n s t the v i r u s have been p r e p a r e d u s i n g c l a r i f i e d sap ( B a g n a l l e t a l , , 1 9 5 6 ) , p a r t i a l l y p u r i f i e d v i r u s p r e p a r a t i o n s (Rozendaal and van S l o g t e r e n , 1957; B a g n a l l e t a l . , 1959; Wetter, 1960) and i t s degraded p r o t e i n ( "D-Protein") (Shepard e t a l . , 1 9 7 1 ; Shepard, 1972). 7 In a d d i t i o n t o s e r o l o g i c a l r e l a t i o n s h i p s between v i r u s e s , PVM a n t i s e r u m has been u t i l i s e d f o r t h e e s t i m a t i o n o f v i r u s c o n c e n t r a t i o n d u r i n g p u r i f i c a t i o n ( W e t t e r , 1960), s e r o d i a g n o s i s and d e t e c t i o n o f t h e v i r u s i n p o t a t o e s and i n the t e s t p l a n t s . A c c o r d i n g t o W e t t e r (1972), s e r o l o g i c a l t e s t s a r e the b e s t methods f o r d i a g n o s i n g t h e v i r u s i n p o t a t o e s . For the purpose o f d i a g -n o s i s and d e t e c t i o n o f PVM, t h e f o l l o w i n g s e r o l o g i c a l t e c h n i q u e s have been u t i l i s e d : s l i d e p r e c i p i t i n ( W e t t e r , 1960), b e n t o n i t e f l o c c u l a t i o n (Kahn e t a l . , 1967), O u c h t e r l o n y d o u b l e - d i f f u s i o n and s i n g l e r a d i a l - d i f f u s i o n (Shepard e t a l . , 1971; Shepard, 1972). ( e ) . T r a n s m i s s i o n . The g r a f t - t r a n s m i s s i b i l i t y o f t h e p a r a c r i n k l e i s o l a t e was r e p o r t e d as e a r l y as 1930 by Salaman and Le Pel l a y . I t appeared then t h a t t h e v i r u s c o u l d n o t be t r a n s m i t t e d by o t h e r means. In o r d e r t o e x p l a i n t h i s s e e m i n g l y p e c u l i a r phenomenon, Bawden (1939), s u g g e s t e d t h a t t h e normal method o f s p r e a d o f t h e v i r u s had been l o s t , and t h a t i t s u r v i v e d and m u l t i p l i e d o n l y because i t s h o s t was p r o p a g a t e d v e g e t a t i v e l y . I t was n o t u n t i l l a t e r t h a t the s a p - t r a n s m i s s i b i l i t y o f s e v e r a l i s o l a t e s was d e m o n s t r a t e d ( K o h l e r , 1942, 1953, 1955; Bawden e t a l . , 1950; B a g n a l l e t a l . , 1956). S a p - t r a n s m i s s i o n i s a c h i e v e d when sap from young l e a v e s i s used, but i s n o t a c h i e v e d w i t h i n o c u l u m from o l d e r l e a v e s ( W e t t e r , 1972). T r a n s m i s s i o n by c o n t a c t has been r e p o r t e d (Symygla e t a l . , 1973), but t e s t s f o r seed t r a n s m i s s i o n i n p o t a t o (Corsan e t a l . , 8 1944) o r i n tomato ( H o r v a t h , 1973 j were n e g a t i v e . T r a n s m i s s i o n o f PVM by a p h i d has been demonstrated. T r a n s m i s s i b i l i t y depends on the i s o l a t e s o f t h e v i r u s and the s p e c i e s o f a p h i d . T r a n s m i s s i o n by Myzus p e r s i c a e S u l z . has been shown w i t h K ( K o h l e r , 1942), B i n t j e (Rozendaal and van S l o g t e r e n , 1957), p a r a c r i n k l e ( K a s s a n i s 1961), l e a f r o l l i n g m osaic, D1102 and F o r t u n a (Wetter and V o l k , 1960) i s o l a t e s . The Rothamsted p a r a c r i n k l e i s o l a t e ( K a s s a n i s , 1960, 1961) and the i s o l a t e s from Japan ( H o r i o e t a l . , 1969) a r e not t r a n s m i t t e d by t h i s i n s e c t s p e c i e s . A p h i d t r a n s m i s s i o n i s n o n - p e r s i s t e n t , (Bode and Weideman, 1971; Kostiw, 1975a). M. p e r s i c a e i s the most e f f i c i e n t v e c t o r , f o l l o w e d by A p h i s f r a n g u l a e Koch, A p h i s n a s t u r t i i K a l t , and Macrosiphum s o l a n i f o l i i Ashmead (Bode and Weideman, 1971), In c o n t r a s t , Kostiw (1975a, 1975b) has r e p o r t e d t h a t M. p e r s i c a e i s a poor v e c t o r o f PVM. A l t h o u g h a c o n s i d e r a b l e s p r e a d might be e x p e c t e d ( Z a d i n a , 1971), n a t u r a l t r a n s m i s s i o n o f PVM i s l e s s e x t e n s i v e than t h a t . o f most of o t h e r p o t a t o v i r u s e s (Beemster and R o z e n d a a l , 1972; Rozendaal and van S l o g t e r e n , 1957; K a s s a n i s , 1961). T h i s n a t u r a l s p r e a d i s m a i n l y by a p h i d s (Beemster and R o z e n d a a l , 1972). . F a c t o r s which i n f l u e n c e the s p r e a d o f PVM i n c l u d e the d i s t a n c e o f p l a n t s from the s o u r c e o f i n f e c t i o n (Piechowiak and G l a d y s i a k , 1972), temperature (Piechowiak and G l a d y s i a k , 1972; Chrzanowska, 1973); c u l t i v a r s o f p o t a t o e s grown (Bawden and K a s s a n i s , 1965) and the age o f p l a n t s (Hunnius, 1972b). 9 ( f ) . P u r i f i c a t i o n S e v e r a l methods f o r the p u r i f i c a t i o n o f PVM have been d e s c r i b e d by v a r i o u s a u t h o r s u s i n g p o t a t o o r tomato as p r o p a g a t i o n h o s t (Rozendaal and van S l o g t e r e n , 1957; B a g n a l l e t a l - , 1 9 5 9 ; Wetter, I960; Shepard , 1972; Maat, 1972; H i r u k i , 1974). Sol anurn demissum L i n d l . has a l s o been used as s t a r t i n g m a t e r i a l . ( H o d r e j a r v e t a l . , 1971) . Leaves o f t h e s e p l a n t s c o u l d be used f r e s h ( B a g n a l l e t a l , , 1 9 5 9 ) , f r o z e n ( H o d r e j a r v e t al.,1971) o r f r e e z e - d r i e d (Rozendaal and van S l o g t e r e n , 1957). The y i e l d s o f the PVM o b t a i n e d from the v a r i o u s methods were not i n d i c a t e d by the v a r i o u s a u t h o r s . P u r i f i c a t i o n o f PVM has been c a r r i e d out i n v a r i o u s b u f f e r s w i t h pH's r a n g i n g from 7.0 t o 8.2 These b u f f e r s i n c l u d e c i t r a t e ( H o d r e j a r v e t al., 1971), phosphate (Rozendaal and van S l o g t e r e n , 1957; B a g n a l l e t a l , , 1959; H i r u k i e t a l . , 1974), b o r a t e (Shepard, 1972) and a s c o r b i c a c i d (Wetter, 1960)( buffers'. In" t h e s t e p s o f p u r i f i c a t i o n subsequent to the h o m o g e n i z a t i o n o f t i s s u e s , b u f f e r s o f the same t y p e , but o f lower m o l a r i t y a r e used. Thus, Shepard (1972) used o.5 M b o r a t e b u f f e r f o r h o m o g e n i z a t i o n , and 0.05 M b o r a t e b u f f e r f o r the subsequent s t e p s . However, i n the method o f Wetter (1960), a s c o r b i c a c i d b u f f e r was used f o r h o m o g e n i z a t i o n and phosphate b u f f e r was used f o r the subsequent s t e p s . C l a r i f i c a t i o n o f crude sap i n the p u r i f i c a t i o n o f PVM has been a c h i e v e d by s h a k i n g w i t h o r g a n i c s o l v e n t S j _ f o r example 10 c h l o r o f o r m ( H o d r e j a r v e t al.,, 1971; Shepard, 1972), b u t a n o l and carbon t e t r a c h l o r i d e ( B a g n a l l e t al,, 1959), c o l d c h l o r o f o r m , e t h a n o l and a c e t o n e (Rozendaal and van S l o g t e r e n , 1957) d i e t h y l e t h e r and carbon t e t r a c h l o r i d e (Wetter, 1960; Maat, 1972) and n-butanol ( H i r u k i e t a l „ 1974). C l a r i f i c a t i o n o f PVM-containing sap by s t i r r i n g i t w i t h c e l i t e has a l s o been d e s c r i b e d ( A l b r e c h t o v a and K l i r , 1970). P a r t i a l l y p u r i f i e d PVM p r e p a r a t i o n s have been o b t a i n e d by s u b j e c t i n g the c l a r i f i e d sap. t o one o r two c y c l e s o f d i f f e r e n t i a l c e n t r i f u g a t i o n (Wetter, 1960; Rozendaal and van S l o g t e r e n , 1957). However, c o m b i n a t i o n s o f d i f f e r e n t i a l c e n t r i f u g a t i o n and p r e c i p i t a t i o n w i t h p o l y e t h y l e n e g l y c o l (PEG) ( A l b r e c h t o v a and K l i r , 1970; Shepard, 1972; H i r u k i e t a l , .,1974) o r p r e c i p i t a t i o n w i t h ammonium s u l f a t e (Maat, 1972) a r e more commonly p r a c t i s e d . A c c o r d i n g t o A l b r e c h t o v a and K l i r (1970), PVM s o l u t i o n s p r e p a r e d by combining PEG p r e c i p i t a t i o n and d i f f e r e n t i a l c e n t r i f u g a t i o n p r o v e d t o be p u r e r than t h o s e p r e p a r e d by means o f g e T • ' • f i l t r a t i o n w i t h Sephadex G-100, The f i n a l s t e p o f p u r i f i c a t i o n has u s u a l l y been the d e n s i t y g r a d i e n t c e n t r i f u g a t i o n (Maat, 1972; Wetter, 1960) o r e l e c t r o p h o r e s i n a s u c r o s e g r a d i e n t column ( H o d r e j a r v e t a l , f 1971). ( g ) . P r o p e r t i e s Some p h y s i c a l and b i o p h y s i c a l p r o p e r t i e s o f PVM have preytous.T been r e p o r t e d by v a r i o u s a u t h o r s , and a r e summarized i n T a b l e 1. 11 T a b l e 1, B i o p h y s i c a l p r o p e r t i e s and p r o p e r t i e s i n crude sap o f PVM as -reported' i n t h e l i t e r a t u r e P r o p e r t i e s v a l u e s f o r PVM 1. Length o f p a r t i c l e (nm) 644 ( i * ) ; 650 ( f , j , n , p ) ; 651 (e) 660 (o) 2. Width o f p a r t i c l e (nm) 12-13 (e) 15 ( f ) 3. L o n g e v i t y i n v i t r o (days) 2 ( a , b , g , h , j , ) 4. Thermal i n a c t i v a t i o n p o i n t (C°) 55 ( c ) ; 60-65 ( d , q ) ; 65-70 ( a , b , j ) 68-71 ( q ) ; 75 (m); 80-85 (k) 5. D i l u t i o n end p o i n t 1 0 - 4 ( g . h . p ) ; 1 0 " 3 ( j , l ) (a) B a g n a l l e t a l , ? 1 9 5 6 (c (e (9 ( i (k (m (o Bawden e t a l . . , 1950 Brandes e t a l . , , 1959 H i r u k i , 1972 H i t c h b o r n and H i l l , 1965 K a s s a n i s , 1956 K o h l e r , 1957 Rozendaal and van S l o g t e r e n , 1957 (q) Wetter and Brandes, 1956 (b) B a g n a l l and L a r s o n , 1957a, 1957b (d) Bode, 1958 ( f ) Brandes, 1960 (h) H i r u k i , 1973 ( j ) H o r i o e t a l , ,1969 (1) K o h l e r , 1955 (n) Lee, 1971 (p) Tu and H i r u k i , 1970 12 L i t t l e i s known o f the b i o c h e m i c a l p r o p e r t i e s o f the v i r u s . The o n l y r e p o r t was made by Nurmiste (1966) who determined t h a t the n u c l e i c a c i d o f the v i r u s i s RNA. For the purpose o f v i r u s c l a s s i f i c a t i o n , p r o b a b l e s i m i l a r i t i e s o f p r o p e r t i e s o f PVM w i t h t h o s e o f the c a r n a t i o n l a t e n t v i r u s have been assumed ( H a r r i s o n e t a l . , 1970). From the f o r e g o i n g review i t i s e v i d e n t t h a t gaps e x i s t i n our knowledge o f t h i s v i r u s . S t u d i e s which have been c a r r i e d o u t a r e m a i n l y c o n c e r n e d w i t h i t s h o s t range, symptomatology, t r a n s m i s s i o n and i t s p r o p e r t i e s i n crude sap. E v a l u a t i o n f o r immunity o r s u s c e p t i b i l i t y i n European p o t a t o c u l t i v a r s t o PVM has been c a r r i e d o u t , but t h e r e i s no c o r r e s p o n d i n g e v a l u a t i o n f o r the m a j o r i t y o f the North American p o t a t o c u l t i v a r s . Moreover, r e p o r t s on the p r o p e r t i e s o f the v i r u s i n p u r i f i e d p r e p a r a t i o n a r e - s c a r c e ; T h i s p r o b a b l y r e f l e c t s the f a c t t h a t no e f f i c i e n t p u r i f i c a t i o n p r o c e d u r e has been d e s c r i b e d . T h e r e f o r e , the o b j e c t i v e s o f the p r e s e n t s t u d i e s a r e t o : 1. E v a l u a t e the hos t range and symptomatology o f a North American i s o l a t e o f PVM, 2. e v a l u a t e the North American p o t a t o c u l t i v a r s f o r immunity o r s u s c e p t i b i l i t y t o t h i s v i r u s , 3. i n v e s t i g a t e the p r o p e r t i e s i n crude sap, 4. attempt to d e v i s e a b e t t e r p u r i f i c a t i o n p r o c e d u r e , and 5. determine as many b i o p h y s i c a l and b i o c h e m i c a l p r o p e r t i e s o f the v i r u s u s i n g the p u r i f i e d p r e p a r a t i o n a s p o s s i b l e . 13 MATERIALS AND METHODS (a) The PVM I s o l a t e The PVM i s o l a t e was k i n d l y p r o v i d e d by Dr. N.S. Wri g h t , Canada Department o f A g r i c u l t u r e , Vancouver, B r i t i s h Columbia. I t was m a i n t a i n e d i n p o t a t o p l a n t s " c u l t i v a r Banana", a l o c a l l y a s s i g n e d name to d e s i g n a t e a s e l e c t i o n which has banana-shaped t u b e r s . The i s o l a t e was f r e e from c o n t a m i n a t i o n by p o t a t o v i r u s e s A, F, S, X o r Y as i n d i c a t e d by the n e g a t i v e r e s u l t s o f b i o l o g i c a l t e s t s on i n d i c a t o r p l a n t s i n c l u d i n g Capsicum annuum L., N i c o t i a n a tabacum L. 'White B u r l e y 1 , N i c o t i a n a c l e v e l a n d i i Grey, Gomphrena g l o b o s a L,, Chenopodiurn  q u i n o a L, and Chenopodiurn a m a r a n t i c o l o r Coste and Reyn. The symptoms o b s e r v e d i n jC. a m a r a n t i c o l o r , C^ . q u i n o a and -G. g l o b o s a were t y p i c a l o f PVM i n f e c t i o n s ( B a g n a l l e t a l . , 1956; Horvath and de Bokx, 1972). S e r o l o g i c a l t e s t s a g a i n s t p o t a t o v i r u s e s S, X and Y c o n f i r m e d the n e g a t i v e r e s u l t s from i n o c u l a t i o n t e s t s . The i n i t i a l P V M - i n f e c t e d p o t a t o p l a n t s , c v . Banana,were then propagated by c u t t i n g s . Cut sho o t s were p l a c e d under m i s t f o r about 10 days f o r r o o t i n g and were then r e p l a n t e d i n t o p o t s . Leaves from the newly g e n e r a t e d p l a n t s c o u l d be h a r v e s t e d i n about 5 weeks. I t was a l s o p o s s i b l e to r o o t the c u t t i n g s i n s o i l . W a t e r i n g them t w i c e d a i l y was as e f f e c t i v e as m i s t i n g i n a m i s t chamber. Throughout the s t u d i e s the temperature o f the greenhouse was kept a t 18 to 20°C, and the v i r u s c u l t u r e was p e r i o d i c a l l y checked f o r the pres e n c e o f p o t a t o v i r u s e s S, X o r Y, 14 by i n o c u l a t i o n s on t e s t p l a n t s and by s e r o l o g y , (b) Host Range and Symptomatology. Fo r t y - o n e s p e c i e s o r c u l t i v a r s o f p l a n t s b e l o n g i n g t o the f a m i l i e s Amaranthaceae, Apocynaceae, C a r o p h y l l a c e a e , Chenopodiaceae, Compositae, C u c u r b i t a c e a e , Graminae, Leguminosae, L i l i a c e a e , C r u c i f e r a e , S o l anaceae and U m b e l l i f e r a e were t e s t e d f o r t h e i r s u s c e p t i b i l i t y t o PVM. Symptoms i n d u c e d by the v i r u s i n i n f e c t e d p l a n t s were ob s e r v e d . I n o c u l a t i o n p r o c e d u r e was as f o l l o w s : Leaves from PV M - i n f e c t e d tomato p l a n t s , 5 weeks a f t e r i n o c u l a t i o n , were ground i n a mortar. The sap was f i l t e r e d through m u s l i n c l o t h , then d i l u t e d i n 5 volumes o f 0.05 M b o r a t e b u f f e r , pH 7.8, and f i n a l l y i n o c u l a t e d w i t h the a i d o f c o t t o n swabs onto l e a v e s o r c o t y l e d o n s which had been d u s t e d w i t h carborundum. E i g h t t o twenty p l a n t s were i n o c u l a t e d per s p e c i e s o r c u l t i v a r a t the most s u s c e p t i b l e s t a g e o r age. A f t e r i n o c u l a t i o n the excess inoculum was i m m e d i a t e l y washed o f f w i t h water. Symptoms were o b s e r v e d f o r 3 t o 4 weeks, a f t e r which time the p l a n t s were t e s t e d f o r PVM i n f e c t i o n . Both i n o c u l a t e d and u n i n o c u l a t e d l e a v e s were t e s t e d by r u b b i n g onto Red Kidney bean. The h o s t range and symptomatology s t u d i e s were done t h r o u g h o u t the l a t e r h a l f o f 1975 and the f i r s t h a l f o f 1976. I n o c u l a t i o n t e s t s on some s p e c i e s o r c u l t i v a r s o f p l a n t s were r e p e a t e d when the r e s u l t s o b t a i n e d were c o n t r a r y to t h o s e r e p o r t e d i n the 1 i t e r a t u r e . In a d d i t i o n , 3 3 , c u l t i v a r s o f p o t a t o were i n o c u l a t e d to y 15 determine t h e i r immunity o r s u s c e p t i b i l i t y t o t h i s i s o l a t e o f PVM. The p l a n t s were grown from v i r u s - f r e e t u b e r s i n 5 - i n p o t s . The inoculum was p r e p a r e d as d e s c r i b e d above, but the carborundum was premixed i n the inoculum b e f o r e a p p l i c a t i o n w i t h p o l y u r e t h a n e sponges onto t h e l e a v e s o f t h e p l a n t s . Three t o e i g h t p l a n t s p er c u l t i v a r were i n o c u l a t e d a t the t w o - l e a f s t a g e . The i n o c u l a t i o n s were r e p e a t e d one week l a t e r . U n i n o c u l a t e d p l a n t s were m a i n t a i n e d as c o n t r o l s . A l l p l a n t s w e r e t h i n n e d t o a maximum o f f o u r s h o o t s . I n o c u l a t i o n s were c a r r i e d o u t i n s p r i n g 1975, w i n t e r 1975 and s p r i n g 1976. In most c a s e s i n o c u l a t i o n t e s t on a p a r t i c u l a r c u l t i v a r was done o n l y once. D e t e c t i o n o f i n f e c t i o n was c a r r i e d out s e r o l o g i c a l l y from the top f o u r l e a v e s , 5 weeks a f t e r f i r s t i n o c u l a t i o n (Hunnius, 1972b). Three to f o u r s h o o t s were t e s t e d from each p l a n t . In cases where i n f e c t i o n was n o t d e t e c t e d , t he s e r o l o g i c a l t e s t i n g was r e p e a t e d 3 weeks l a t e r . O b s e r v a t i o n f o r symptoms was c a r r i e d o u t u n t i l the 8th week p o s t i n o c u l a t i o n . Tubers from the p r i m a r i l y i n f e c t e d p o t a t o p l a n t s , c u l t i v a r s Banana, Red La Soda and White La Soda were h a r v e s t e d and r e p l a n t e d i n F a l l 1976. A t the same time some c u l t i v a r s which had f a i l e d to become i n f e c t e d w i t h the v i r u s through mechanical i n o c u l a t i o n were g r a f t - i n o c u l a t e d w i t h P V M - i n f e c t e d s h o o t s from p o t a t o p l a n t s c u l t i v a r Banana. T e s t i n g f o r v i r u s i n f e c t i o n was done 6 weeks p o s t i n o c u l a t i o n . E xperiments were a l s o c a r r i e d o u t to t e s t t r a n s m i s s i b i l i t y o f the v i r u s t h r o u g h p l a n t c o n t a c t . In one experiment tomato p l a n t s , c u l t i v a r R u t g e r s , were used. Four u n i n o c u l a t e d p l a n t s 16 were p l a c e d i n c o n t a c t w i t h f o u r m e c h a n i c a l l y i n o c u l a t e d p l a n t s t h r o u g h o u t the growing p e r i o d . Four o t h e r p l a n t s p l a c e d about 2 f e e t away were m a i n t a i n e d as c o n t r o l s . S i x weeks a f t e r i n o c u l a -t i o n t h e p l a n t s w e r e s e r o l o g i c a l l y t e s t e d f o r PVM i n f e c t i o n , and when n e g a t i v e r e s u l t s were o b t a i n e d , the d e t e c t i o n was r e p e a t e d 2 weeks l a t e r . The experiment was r e p l i c a t e d t h r e e t i m e s . In a n o t h e r e x p e r i m e n t , two v i r u s - f r e e p o t a t o p l a n t s , cv. A r r a n V i c t o r y were p l a c e d i n c o n t a c t w i t h P V M - i n f e c t e d 'White La Soda 1 p l a n t s f o r 8 weeks, a f t e r which s e r o l o g i c a l d e t e c t i o n f o r v i r u s i n f e c t i o n was c a r r i e d o u t . T h i s experiment was a l s o r e p l i c a t e d t h r e e t i m e s , (c) S e r o l o g y A n t i s e r u m a g a i n s t PVM was p r e p a r e d by i n j e c t i n g ' a young r a b b i t w i t h the v i r u s p u r i f i e d by a m o d i f i c a t i o n o f t h e method o f Shepard (1972). O v e r n i g h t i n c u b a t i o n o f the crude sap w i t h T r i t o n X-100 added to 1%, and one c y c l e o f s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n were i n c o r p o r a t e d i n t o t he p u r i f i c a t i o n p r o c e d u r e . The f i r s t i n j e c t i o n was done i n t r a m u s c u l a r l y w i t h 0.5 mg per ml o f v i r u s s o l u t i o n i n 0.05 M b o r a t e b u f f e r , pH 7.8. P r i o r t o the i n t r a m u s c u l a r i n j e c t i o n , the v i r u s s o l u t i o n was f i r s t e m u l s i f i e d w i t h Freund's complete a d j u v a n t . In each o f the subsequent i n j e c t i o n s , 1 mg o f v i r u s was used. One i n t r a v e n o u s i n j e c t i o n was c a r r i e d out 1 week a f t e r t he f i r s t i n j e c t i o n , f o l l o w e d by one i n t r a v e n o u s and one i n t r a m u s c u l a r 17 i n j e c t i o n 2 weeks l a t e r . Two " b o o s t e r " i n j e c t i o n s , one i n t r a v e n o u s and the o t h e r i n t r a m u s c u l a r , were a p p l i e d 8 weeks a f t e r the f i r s t i n j e c t i o n . The r a b b i t was b l e d weekly and the t i t e r s o f the a n t i s e r u m were e s t i m a t e d by t h e tube p r e c i p i t i n t e s t a g a i n s t sap from v i r u s - i n f e c t e d o r v i r u s - f r e e p l a n t s . A n t i s e r u m a g a i n s t normal p l a n t components was o b t a i n e d from a young r a b b i t which had been g i v e n two i n t r a m u s c u l a r and t h r e e i n t r a v e n o u s i n j e c t i o n s o f normal p l a n t components from v i r u s - f r e e p l a n t s . E x t r a c t i o n o f t h e normal p l a n t components f o l l o w e d the same method as above, e x c e p t t h a t the t r e a t m e n t s w i t h c h l o r o f o r m , T r i t o n X-100 and s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n were o m i t t e d , (d) P r o p e r t i e s i n Crude Sap The f o l l o w i n g p r o p e r t i e s i n c r u d e sap were i n v e s t i g a t e d : D i l u t i o n end p o i n t (DEP), l o n g e v i t y i n v i t r o (LIV) and thermal i n a c t i v a t i o n p o i n t ( T I P ) . Sap f o r the e x p e r i m e n t to determine the DEP and LIV was o b t a i n e d from p o t a t o p l a n t s ( c v . Banana) and tomato p l a n t s (cv. R u t g e r s ) . For the TIP d e t e r m i n a t i o n , o n l y sap from p o t a t o p l a n t s was used. The tomato p l a n t s were i n o c u l a t e d w i t h P'VM 5 weeks p r i o r to the t e s t . Leaves c o l l e c t e d from t h e s e p l a n t s were macerated i n a m o r t a r , and the sap e x t r a c t e d was t h e n f i l t e r e d t h r o u g h m u s l i n c l o t h . In a l l t h r e e e x periments the t r e a t m e n t s were d u p l i c a t e d , and each r e p l i c a t e was rubbed onto t h r e e h a l f - l e a v e s o f 10 to 12 day o l d Red Kidney bean p l a n t s . 18 The number o f l o c a l l e s i o n s per h a l f - l e a f f o r each t r e a t m e n t was r e c o r d e d 6 and 12 days a f t e r i n o c u l a t i o n . D e t e r m i n a t i o n o f DEP was made w i t h sap d i l u t e d i n 0.05 M b o r a t e b u f f e r , pH 7.8, to 1 0 _ 1 , 1 0 " 2 , 1 0 " 3 , 1 0 " 4 , 1 0 - 5 o r 1 0 - 6 o f the o r i g i n a l c o n c e n t r a t i o n . The u n d i l u t e d sap was i n o c u l a t e d onto one o f the h a l f - l e a v e s o f each Red Kidney bean p l a n t . A n o t h e r h a l f - l e a f from the o p p o s i t e p r i m a r y l e a f was i n o c u l a t e d w i t h b u f f e r o n l y . The t r e a t e d sap was then i n o c u l a t e d randomly onto the r e m a i n i n g two h a l f - l e a v e s . In the d e t e r m i n a t i o n o f TIP, the sap was i n c u b a t e d i n a water bath f o r 10 min a t t emperatures o f 30°C, 40°C, 50OC, 60°C, 70°C o r 80°C, and s u b s e q u e n t l y w i t h sap i n c u b a t e d a t t e mperatures o f 60°C, 65°C, 70°C. o r 75°C. To f a c i l i t a t e h e a t i n g and c o o l i n g , the sap samples were p l a c e d i n narrow and t h i n - w a l l e d l y o p h i l i z i n g t u b e s . They were imme d i a t e l y c o o l e d i n i c e f o r 30 s e c , then i n o c u l a t e d onto Red Kidney bean h a l f -l e a v e s . The d e t e r m i n a t i o n o f LIV was done by i n c u b a t i n g sap samples a t room temperature f o r 1, 2, 3, 4 o r 5 days, a f t e r which they were i n o c u l a t e d onto Red Kidney bean p l a n t s . F r e s h l y e x t r a c t e d sap from l e a v e s o f P V M - i n f e c t e d p o t a t o p l a n t s was c o n s i d e r e d as the u n t r e a t e d c o n t r o l . (e) P u r i f i c a t i o n The p r o g r e s s o f p u r i f i c a t i o n d u r i n g the v a r i o u s s t e p s was f o l l o w e d by s e v e r a l methods. For the e a r l y s t e p s o f p u r i f i c a t i o n the amounts o f p l a n t d e b r i s i n the p r e p a r a t i o n s were e s t i m a t e d by measuring the t u r b i d i t y v a l u e s a t 540 nm. A p r e l i m i n a r y 19 i n v e s t i g a t i o n (Appendix I) showed t h a t the v a r i a t i o n i n t h i s t u r b i d i t y v a l u e was p r o p o r t i o n a l t o the c o n c e n t r a t i o n o f d e b r i s o r i g i n a t i n g from the hos t p l a n t l e a v e s . In the case o f p a r t i a l l y p u r i f i e d v i r u s p r e p a r a t i o n t he c r i t e r i a o f p u r i t y used were a va l u e o f 1.20 t o 1,35 f o r the r a t i o s o f a b s o r p t i o n s a t 260 nm t o 280 nm (^ g Q / ^ g r - j ) , the l a c k o f r e a c t i o n w i t h a n t i s e r u m a g a i n s t normal p l a n t components and the homogeneity o f p a r t i c l e s when examined by e l e c t r o n m i c r o s c o p y . The m a j o r i t y o f v i r u s e s b e l o n g i n g t o the C a r l a v i r u s Group have ^ 2 6 0 ^ 2 8 0 v a ^ u e s between the above mentioned range. (Appendix I I ) . The &260 v a ^ u e s were a l s o used f o r the e s t i m a t i o n o f c o n c e n t r a t i o n o f p a r t i a l l y p u r i f i e d and p u r i f i e d v i r u s p r e p a r a t i o n s . The tube p r e c i p i t i n s e r o l o g i c a l t e s t was used t o e s t i m a t e the r e l a t i v e v i r u s c o n c e n t r a t i o n s which were assumed to be p r o p o r t i o n a l to the r e c i p r o c a l s o f the r e s p e c t i v e s e r o l o g i c a l d i l u t i o n end p o i n t s . The p r o c e d u r e was as f o l l o w s : Double d i l u t i o n s e r i e s o f the v i r u s p r e p a r a t i o n s were made w i t h p h y s i o l o g i c a l s a l i n e (0.85% NaCl) i n t e s t - t u b e s . To 1 ml o f each o f t h e s e d i l u t e d samples was added an equal volume o f the a n t i s e r u m a g a i n s t PVM which had been d i l u t e d 200 times i n s a l i n e . I n c u b a t i o n o f the m i x t u r e s was c a r r i e d out f o r 2 hr a t 37°C, but the p r e s e n c e o r absence o f a p o s i t i v e s e r o l o g i c a l r e a c t i o n was r e c o r d e d o n l y a f t e r a f u r t h e r i n c u b a t i o n a t room temperature o v e r n i g h t . Sap from l e a v e s o f v i r u s - f r e e p l a n t s was a l s o i n c l u d e d i n the t e s t to d e t e c t any u n s p e c i f i c s e r o l o g i c a l r e a c t i o n . 20 In e x p eriments i n v o l v i n g d i f f e r e n t b u f f e r s , o r tho s e i n v o l v i b u f f e r s w i t h d i f f e r e n t m o l a r i t i e s , pH's o r a d d i t i v e s , t h e v a r i o u s samples were f i r s t d i a l y s e d o v e r n i g h t i n a common b u f f e r . In the e x t r a c t i o n s t e p , the common b u f f e r was 0.5 M b o r a t e , pH 7.8, and i n t h e subsequent s t e p s i t was 0.05 M b o r a t e . F o l l o w i n g d i a l y s i s the samples were t r a n s f e r r e d i n t o a 10 ml measuring c y l i n d e r t o make s u r e t h a t t h e r e were no volume changes. In experiments i n -v o l v i n g volumes, examples, homogenization o f l e a f t i s s u e s o r r e s u s p e n s i o n o f p r e c i p i t a t e s , the d i f f e r e n t l y t r e a t e d samples were f i r s t c e n t r i f u g e d , then s e p a r a t e d from t he p e l l e t s , and f i n a l l y brought to the same volume f o r s e r o l o g i c a l d i l u t i o n end p o i n t d e t e r m i n a t i o n and t u r b i d i t y measurement. T e c h n i q u e s which showed minimum l o s s o f v i r u s and maximum e x c l u s i o n o f m a t e r i a l s o f p l a n t o r i g i n when employed a t a p a r t i c u l a r s t e p were chosen. U s u a l l y , a compromise between r e t e n t i o n o f v i r u s and c l a r i t y o r p u r i t y o f p r e p a r a t i o n s was made. 1. E x t r a c t i o n o f v i r u s from ho s t t i s s u e s Experiments were c a r r i e d o u t to dete r m i n e a s u i t a b l e p r o c e d u r e f o r e x t r a c t i o n o f the v i r u s from p r o p a g a t i o n h o s t p l a n t t i s s u e s . For t h i s p u r p o s e , the e f f e c t s o f homogenizing l e a f t i s s u e s i n d i f f e r e n t e x t r a c t i o n b u f f e r s , i n the same b u f f e r but o f d i f f e r e n t pH's o r i n the same b u f f e r but added i n d i f f e r e n t t i s s u e t o b u f f e r r a t i o s , were s t u d i e d . The e f f e c t o f i n c o r p o r a t i n g a r e d u c i n g o r c h e l a t i n g agent i n t o the 21 e x t r a c t i o n b u f f e r was a l s o examined. The o b j e c t was t o o b t a i n c rude sap w i t h t h e maximum c o n c e n t r a t i o n o f v i r u s . P V M - i n f e c t e d p o t a t o l e a v e s were c u t i n t o s m a l l p i e c e s . F i v e gram samples were then taken and i n d i v i d u a l l y homogenized i n a b u f f e r i n a 10 ml b l e n d e r f o r 2 min. The b l e n d e r was c o o l e d under r u n n i n g water f o r a s h o r t p e r i o d , a f t e r which t i s s u e h o mogenization was c o n t i n u e d f o r a f u r t h e r 2 min. The s t a n d a r d p r o c e d u r e was to homogenize the l e a v e s i n 0.5 M b o r a t e b u f f e r , pH 7.8, a t the r a t i o o f 1:3 (w/v), t i s s u e t o b u f f e r . No r e d u c i n g o r che l a t i n g agent was used i n the system. The o t h e r t r e a t m e n t s were e s s e n t i a l l y the v a r i a t i o n s o f t h i s system w i t h r e s p e c t to the pH o f b u f f e r , t y p e o f b u f f e r s , t i s s u e t o b u f f e r r a t i o s , o r the a d d i t i o n o f a r e d u c i n g o r a c h e l a t i n g a gent i n the b u f f e r . The homogenates were f i l t e r e d t hrough m u s l i n c l o t h and the f i l t r a t e s . were then c e n t r i f u g e d a t 9,500 rpm (10,800 g) f o r 10 min i n a S e r v a l l RC-2 c e n t r i f u g e . The v i r u s c o n c e n t r a t i o n s o f the s u p e r n a t a n t s were then compared. Experiments were undertaken t o d e t e r m i n e the most s u i t a b l e p r o p a g a t i o n h o s t f o r use i n p u r i f i c a t i o n . P o t a t o p l a n t s c v s . Banana, Columbia Russet and White La Soda, and tomato p l a n t s cvs. Rutgers and Subarctic were t e s t e d . In a d d i t i o n , the e f f e c t o f age o f p l a n t s on the c o n c e n t r a t i o n o f v i r u s was i n v e s t i g a t e d by h a r v e s t i n g the l e a v e s o f P V M - i n f e c t e d p l a n t s cv. Banana 4, 5, 6, 8, 10 o r 15 weeks a f t e r t r a n s p l a n t i n g . The p l a n t s h a r v e s t e d 4, 5 o r 6 weeks a f t e r t r a n s p l a n t i n g were 22 a l l o w e d t o regrow f o r a f u r t h e r 4 weeks, a f t e r which a second h a r v e s t was c a r r i e d o u t . An e x p e r i m e n t was a l s o c a r r i e d o u t t o determine the p o s s i b i l i t y o f u s i n g f r o z e n l e a v e s o r l e a v e s which had been s t o r e d o v e r n i g h t a t 4°C. In most experiments the r e p l i c a t i o n o f t r e a t m e n t s was f o u r but i n o t h e r s the r e p l i c a t i o n was f i v e . 2. C l a r i f i c a t i o n o f Crude Sap I n v e s t i g a t i o n s were c a r r i e d o u t to determine the s u i t a -b i l i t y o f s e v e r a l p h y s i c a l and c h e m i c a l c l a r i f i c a t i o n methods which had been employed i n v i r u s p u r i f i c a t i o n s . In a p r e l i m i n a r y s t u d y , 11 methods were " s c r e e n e d " f o r ones which showed p r o m i s i n g r e s u l t s as i n d i c a t e d by t h e i r low t u r b i d i t y v a l u e s and t h e i r h i g h r e c i p r o c a l s o f s e r o l o g i c a l d i l u t i o n end p o i n t s . P V M - i n f e c t e d p o t a t o l e a v e s were homogenized i n a Waring b l e n d e r w i t h 0.5 M b o r a t e b u f f e r , pH 7.8, a t the r a t i o o f 1:3, t i s s u e t o b u f f e r . Sodium d i e t h y l d i t h i o c a r b a m a t e (DIECA) was added as c h e l a t i n g agent a t 0.5% (w/v) f i n a l c o n c e n t r a t i o n . The homogenates were squeezed through m u s l i n c l o t h and 10 ml a l i q u o t s o f the r e s u l t i n g f i l t r a t e s were d i s p e n s e d i n t o c e n t r i f u g e t u b e s . S i x o f t h e s e samples were then i n d i v i d u a l l y c l a r i f i e d w i t h one o f the f o l l o w i n g methods: (1) E m u l s i f i i c a t i o n w i t h equal volume o f c h l o r o f o r m ; (2) E m u l s i f i c a t i o n w i t h 1.5 ml c h l o r o f o r m ; (3) I n c u b a t i o n f o r 30 min w i t h 0.8 ml n - b u t a n o l ; (.4) E m u l s i f i c a t i o n w i t h equal volume o f carbon t e t r a c h l o r i d e ; 23 C5] E m u l s i f i c a t i o n w i t h equal volume o f e t h e r ; (.6 ) O v e r n i g h t i n c u b a t i o n wi/th T r i t o n X-100 made up to 1% ( v / v ) ; (7) E m u l s i f i c a t i o n w i t h 1.5 ml c h l o r o f o r m , broken by c e n t r i f u g a t i o n , f o l l o w e d by t r e a t m e n t w i t h T r i t o n X-100 as i n 6; (8) I n c u b a t i o n a t 45°C f o r 10 min; (9) C e n t r i f u g a t i o n a t 9,500 rpm f o r 10 min, then s t i r r i n g t h e s u p e r n a t a n t w i t h 0.5 gm o f c e l i t e f o r 5 min; (10) O v e r n i g h t p r e c i p i t a t i o n a t 4°C w i t h 2.5 gm ammonium s u l f a t e ; (11) O v e r n i g h t f r e e z i n g , then thawing a t room te m p e r a t u r e . The em u l s i o n s o r s u s p e n s i o n s were then c e n t r i f u g e d a t 9,500 rpm f o r 10 min. The s u p e r n a t a n t s were brought t o the same volume by a d d i n g 0.5 M b o r a t e b u f f e r , pH 7.8. T u r b i d i t y v a l u e s a t 540 nm were measured i m m e d i a t e l y , b ut the s e r o l o g i c a l d i l u t i o n end p o i n t was det e r m i n e d o n l y a f t e r an o v e r n i g h t d i a l y s i s a g a i n s t the same b u f f e r . F u r t h e r i n v e s t i g a t i o n s were c a r r i e d o u t onemethods which showed p r o m i s i n g r e s u l t s . In a d d i t i o n , methods which r e s u l t e d i n v e r y c l e a r sap, though low i n v i r u s c o n t e n t were a l s o c o n s i d e r e d f o r f u r t h e r i n v e s t i g a t i o n . Methods which caused a p p r e c i a b l e l o s s i n v i r u s and which r e s u l t e d i n h i g h t u r b i d i t y v a l u e s were not t e s t e d f u r t h e r . Crude sap o b t a i n e d by homogenizing l e a v e s i n 0.5 M b o r a t e b u f f e r a t the r a t i o o f 1:3 o r 1:1 ,5, t i s s u e to b u f f e r , were 24 used as t e s t m a t e r i a l s . For b r e v i t y t h e s e r a t i o s were a s s i g n e d the terms "1:3 sap" o r 1:1.5 sap", r e s p e c t i v e l y . When few c l a r i f i c a t i o n methods which showed p r o m i s i n g r e s u l t s were o b t a i n e d , d i r e c t comparison between them were c a r r i e d o u t , and 1:3 and 1:1.5 sap from p o t a t o o r tomato l e a v e s were t e s t e d . 3. ' C o n c e n t r a t i o n o f v i r u s from c l a r i f i e d sap From t h i s s e c t i o n onward o n l y 1:1.5 sap was t e s t e d . B o r a t e b u f f e r , pH 7.8 was used thro u g h o u t u n l e s s o t h e r w i s e s t a t e d . Four methods f o r the c o n c e n t r a t i o n o f v i r u s from c l a r i f i e d sap were compared: h i g h speed c e n t r i f u g a t i o n , ammonium s u l f a t e p r e c i p i t a t i o n , a c i d p r e c i p i t a t i o n and polyethylene g l y c o l (MW=6000) p r e c i p i t a t i o n . Treatments were c a r r i e d o u t on 25 ml samples o f crude sap. High speed c e n t r i f u g a t i o n was c a r r i e d o u t a t 28,000 rpm f o r 1.5 hr i n a Beckman Model L u l t r a c e n t r i f u g e w i t h no.30 r o t o r . P r e c i p i t a t i o n s were c a r r i e d o u t by a d d i t i o n o f p o l y e t h y l e n e g l y c o l (PEG) and sodium c h l o r i d e added to 5% (w/v) and 4% (w/v) r e s p e c t i v e l y (Shepard, 1972), ammonium s u l f a t e t o 30% (Bawden, 1950), o r by a c i d i f i c a t i o n t o pH 4 w i t h HC1 . The p r e c i p i t a t e s were p e l l e t e d by c e n t r i f u g a t i o n a t 9,500 rpm f o r 15 min i n a S e r v a l l RC-2 c e n t r i f u g e ( h e r e a f t e r c a l l e d the low speed c e n t r i f u g a t i o n ) . P r e l i m i n a r y e x p eriments were c a r r i e d o u t t o det e r m i n e t he e f f e c t s o f m o l a r i t y and volume o f b u f f e r d u r i n g r e s u s p e n s i o n o f p e l l e t s o b t a i n e d by each o f t h e s e f o u r methods. Resuspension was c a r r i e d out o v e r n i g h t i n 2.5 ml 25 (1 : 1 0 ) , 5.0 ml (1:5) o r 10 ml (1:2.5) o f 0,5 o r 0.05 M b u f f e r ; t h e f i g u r e s i n p a r e n t h e s e s r e p r e s e n t e d t h e r a t i o s o f t h e o r i g i n a l volumes t o the f i n a l volumes o f v i r u s p r e p a r a t i o n s . F o l l o w i n g r e s u s p e n s i o n , t h e u n d i s s o l v e d p e l l e t s were removed by a low speed c e n t r i f u g a t i o n , and the s u p e r n a t a n t s were brought t o 12.5 ml (1:2) by a d d i t i o n o f the a p p r o p r i a t e b u f f e r . A f t e r o v e r n i g h t d i a l y s i s , the s e r o l o g i c a l d i l u t i o n end p o i n t s were deter m i n e d . In a s e p a r a t e e x p e r i m e n t , PVM p a r t i c l e s were p r e c i p i t a t e d from c a r b o n t e t r a c h l o r i d e - c l a r i f i e d sap by v a r i o u s c o m b i n a t i o n s o f PEG and sodium c h l o r i d e . The p e l l e t s were then resuspended o v e r n i g h t i n 5 ml o f 0.05 M b o r a t e b u f f e r , and b e f o r e t h e s e r o l o g i c a l d i l u t i o n end p o i n t s were de t e r m i n e d the s u p e r n a t a n t s were b r o u g h t t o 12.5 ml. The e f f e c t o f time o f i n c u b a t i o n w i t h ammonium s u l f a t e was a l s o i n v e s t i g a t e d . T w e n t y - f i v e ml samples o f ammonium s u l f a t e - c l a r i f i e d sap were i n d i v i d u a l l y brought t o 30% c o n c e n t r a t i o n by a d d i t i o n o f more ammonium s u l f a t e . They were then i n c u b a t e d f o r 2, 6 o r 16 hr a t 4°C. The p r e c i p i t a t e s were p e l l e t e d by a low speed c e n t r i f u g a t i o n , and then r e s u s p e n d e d o v e r n i g h t i n 5 ml o f 0.05 M b o r a t e . b u f f e r . A f t e r a low speed c e n t r i f u g a t i o n , t h e s u p e r n a t a n t s were d i l u t e d t o 12.5 ml and t h e i r s e r o l o g i c a l d i l u t i o n end p o i n t s d e t e r m i n e d . F i n a l l y , d i r e c t comparisons were made between t h e v a r i o u s methods which showed p r o m i s i n g r e s u l t s . In a l l c a s e s 25 ml 26 samples o f sap were used. The r e s u l t i n g p e l l e t s were resuspended o v e r n i g h t i n 5 ml o f 0.05 M b o r a t e b u f f e r . A f t e r a low speed c e n t r i f u g a t i o n , the s u p e r n a t a n t s were d i l u t e d t o 12.5 ml and t h e i r s e r o l o g i c a l d i l u t i o n end p o i n t s d e t e r m i n e d . The p e l l e t s were a g a i n resuspended o v e r n i g h t i n 5 ml o f the same b u f f e r . A f t e r a low speed c e n t r i f u g a t i o n , the " t h i r d s u p e r n a t a n t s " were d i l u t e d to 12.5 ml, and then t h e i r s e r o l o g i c a l d i l u t i o n end p o i n t s were dete r m i n e d . C o r r e s p o n d i n g experiments were c a r r i e d o u t w i t h sap e x t r a c t e d from tomato l e a v e s . 4. F u r t h e r . c o n c e n t r a t i o n o f v i r u s p r e p a r a t i o n s The s t a r t i n g m a t e r i a l s f o r the experiments c a r r i e d o u t i n t h i s s e c t i o n were: (1) v i r u s p r e p a r a t i o n s c l a r i f i e d by ammonium s u l f a t e , then c o n c e n t r a t e d by ammonium s u l f a t e p r e c i p i t a t i o n ; (2) v i r u s p r e p a r a t i o n s c l a r i f i e d by carbon t e t r a c h l o r i d e , then c o n c e n t r a t e d by PEG p r e c i p i t a t i o n . R e s u s p e n s i o n o f the ammonium s u l f a t e o r PEG p r e c i p i t a t e s were done t w i c e , each time w i t h o n e - f i f t h volume o f 0.05 M b o r a t e b u f f e r . The two components were then added t o g e t h e r and t h e i r s e r o l o g i c a l d i l u t i o n end p o i n t s d e t e r m i n e d . A p r e c i p i t a t i o n e x p e r i m e n t w i t h PEG c a r r i e d o u t d u r i n g t he second s t a g e o f c o n c e n t r a t i o n f o l l o w e d the same p r o c e d u r e as p r e v i o u s l y d e s c r i b e d . PEG was added a t the r a t e o f 2.5% o r 5.0 % f o l l o w e d by sodium c h l o r i d e a t the r a t e o f 2% o r 4%. T w e n t y - f i v e ml samples o f the s t a r t i n g m a t e r i a l s were f u r t h e r c o n c e n t r a t e d by h i g h speed c e n t r i f u g a t i o n o r by PEG 27 p r e c i p i t a t i o n , The r e s u l t i n g p e l l e t s were then resuspended i n o n e - f i f t h volume (5 ml) o f 0,05 M b o r a t e b u f f e r . High speed c e n t r i f u g a t i o n was c a r r i e d o u t a t 28,000 rpm f o r 1.5 hr i n a Beckman Model L u l t r a c e n t r i f u g e . P r e c i p i t a t i o n was c a r r i e d o u t by a d d i t i o n o f PEG and sodium c h l o r i d e a t 5% (w/v) and 4% (w,v), r e s p e c t i v e l y . A f t e r r e s u s p e n s i o n , t he u n d i s s o l v e d p e l l e t s were removed by a low speed c e n t r i f u g a t i o n . The s u p e r n a t a n t s were d i l u t e d t o a f i n a l volume o f 12.5 ml, and t h e i r s e r o l o g i c a l d i l u t i o n end p o i n t s were then d e t e r m i n e d . S i m i l a r e x p eriments were c a r r i e d o u t w i t h v i r u s p r e p a r a t i o n s d e r i v e d from tomato p l a n t s c u l t i v a r R u t g e r s . T a b l e 2. Combinations o f methods used f o r the second and t h i r d s t a g e s o f c o n c e n t r a t i o n o f PVM p r e p a r a t i o n o r i g i n a l l y o b t a i n e d from ammonium s u l f a t e - o r carbon t e t r a c h l o r i d e -c l a r i f i e d sap. Source o f v i r u s p r e p a r a t i o n s Ammonium s u l f a t e -c l a r i f i e d sap Carbon t e t r a c h l o r i d e -c l a r i f i e d sap Combination o f methods o f c o n c e n t r a t i o n D e s i g n a t i o n I II I I I IV Second s t a g e PEG hi g h speed PEG h i g h speed T h i r d s t a g e high speed PEG hi g h speed PEG In a n o t h e r experiment the PEG p r e c i p i t a t i o n and h i g h speed c e n t r i f u g a t i o n methods o f c o n c e n t r a t i o n were combined i n d i f f e r e n t sequences I to IV ( T a b l e 2 ) . The sample s i z e was 25 ml and 10 ml f o r the second s t a g e and t h i r d s t a g e o f c o n c e n t r a t i o n , r e s p e c t i v e l y . R esuspension o f the p e l l e t s o b t a i n e d from the second s t a g e o f c o n c e n t r a t i o n was done o v e r n i g h t . 28 In the t h i r d s t a g e , h i g h speed c e n t r i f u g a t i o n was c a r r i e d o u t i n a no, 65 r o t o r a t 35,000 rpm f o r 1.5 hr. The p e l l e t s were r e s u s -pended i n 0,3 ml o r 2.0 ml o f b o r a t e b u f f e r , and a f t e r a low speed c e n t r i f u g a t i o n t h e s u p e r n a t a n t s were brought to 10 ml f o r s e r o l o g i c a l d i l u t i o n end p o i n t d e t e r m i n a t i o n . F i n a l l y when t e n t a t i v e p r o c e d u r e s f o r PVM p u r i f i c a t i o n were o b t a i n e d , 40 gm samples o f PV M - i n f e c t e d p o t a t o l e a v e s were i n d i v i d u a l l y homogenized i n 60 ml o f 0.5 b o r a t e b u f f e r , w i t h 0.5 gm o f DIECA added t o each o f the samples as a chelating agent, Three o f the samples were s u b j e c t e d to c l a r i f i c a t i o n w i t h ammonium s u l f a t e and then t o the v a r i o u s c o n c e n t r a t i o n methods a r r a n g e d i n the f o l l o w i n g sequence: Ammonium s u l f a t e p r e c i p i t a t i o n , h i g h speed c e n t r i f u g a t i o n , PEG p r e c i p i t a t i o n , and hi g h speed c e n t r i f u g a t i o n ( h e r e a f t e r c a l l e d the ammonium s u l f a t e method). The o t h e r s were s u b j e c t e d t o c l a r i f i c a t i o n w i t h carbon t e t r a c h l o r i d e and then t o the v a r i o u s methods o f c o n c e n t r a t i o n a r r a n g e d i n the f o l l o w i n g sequence: PEG p r e c i p i t a t i o n , h i g h speed c e n t r i f u g a t i o n , PEG p r e c i p i t a t i o n , and hi g h speed c e n t r i f u g a t i o n ( h e r e a f t e r c a l l e d the carbon t e t r a c h l o r i d e method). A t the end o f each s t e p the v i r u s p r e p a r a t i o n s were examined w i t h the e l e c t r o n m i c r o s c o p e by t he n e g a t i v e s t a i n i n g method. The f i n a l c o n c e n t r a t e d v i r u s p r e p a r a t i o n s were then c e n t r i f u g e d through a s u c r o s e d e n s i t y g r a d i e n t i n SW 41 r o t o r , 5. D e n s i t y g r a d i e n t c e n t r i f u g a t i o n S u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n was c a r r i e d out i n an SW 41 o r SW 25 r o t o r s a t 27,000 rpm o r 23,000 r p m , r e s p e c t i v e l y , f o r 29 1.5 hr. U s u a l l y 5 to 35% o r 10 to 40% s u c r o s e g r a d i e n t s were p r e p a r e d . The amounts o f samples l a y e r e d onto the g r a d i e n t s were 0.3 ml and 2.0 ml f o r the SW 41 and SW 2 5 ? r e s p e c t i v e l y . The d e n s i t y g r a d i e n t s were scanned w i t h an ISCO d e n s i t y g r a d i e n t s c a n n e r . The v i r u s peak was c o l l e c t e d and then d i a l y s e d o v e r n i g h t i n 0.05 M b o r a t e b u f f e r . V i r u s p r e p a r a t i o n s from t he SW 41 g r a d i e n t s were d i l u t e d t o 2.0 ml b e f o r e t h e i r a n c^ A 2 8 0 v a ^ u e s w e r e measured. V i r u s band i n the SW 25 g r a d i e n t was sometimes removed w i t h a s y r i n g e . 6. E l e c t r o n m i c r o s c o p y A drop o f p u r i f i e d PVM p r e p a r a t i o n i n 0.05 M b o r a t e b u f f e r was a l l o w e d t o s t a n d on a g r i d f o r 5 min and then d r a i n e d o f f by t o u c h i n g the edge o f the g r i d w i t h t he edge o f f i l t e r paper. The g r i d was then n e g a t i v e l y s t a i n e d w i t h 2% ph o s p h o t u n g s t a t e s o l u t i o n , pH 7.4 f o r 1 min. Excess s o l u t i o n was d r a i n e d o f f and the g r i d was then examined w i t h a P h i l i p s EM 300 e l e c t r o n m i c r o s c o p e . Measurement o f l e n g t h o f the v i r u s p a r t i c l e s was done w i t h the a i d o f a m a g n i f y i n g g l a s s from e l e c t r o n m i c r o g r a p h s a t a m a g n i f i c a t i o n o f 39,960X. Measurement o f the p a r t i c l e w i d t h was o b t a i n e d d i r e c t l y from the n e g a t i v e s a t a m a g n i f i c a t i o n o f 23,197X w i t h t h e a i d o f a measuring G a e r t n e r Comparator. TMV p a r t i c l e s were i n c l u d e d f o r comparison. ( f ) D e t e r m i n a t i o n o f the B i o p h y s i c a l and B i o c h e m i c a l p r o p e r t i e s In t h i s s e c t i o n , p u r i f i e d v i r u s p r e p a r a t i o n o f PVM o b t a i n e d from a second s u c r o s e d e n s i t y g r a d i e n t was used. 30 l l A b s o r p t i o n s p e c t r a The a b s o r p t i o n s p e c t r a o f the p u r i f i e d p r e p a r a t i o n o f PVM i n 0.05 M b o r a t e b u f f e r , pH 7.8 were measured i n a Beckman model Du-spectrophotometer between 230 and 310 nm. C o r r e c t i o n f o r l i g h t s c a t t e r i n g was a c h i e v e d by e x t r a p o l a t i o n o f the " a b s o r p t i o n " a t wavelengths between 315 and 600 nm as d e s c r i b e d by Noordam (1973). Measurements f o r c o r r e c t i o n f o r l i g h t s c a t t e r i n g were o b t a i n e d from c o n c e n t r a t e d s o l u t i o n s o f PVM. From each o f them were taken 3 samples and d i l u t e d 1 0 - f o l d i n 0.05 M b o r a t e b u f f e r f o r measurements a t wavelengths between 230 nm and 310 nm. The i n t e r v a l s o f measurement v a r i e d from 1 nm t o 10 nm u n i t s on the u l t r a v i o l e t r e g i o n , and from 15 nm t o 50 nm u n i t s i n the v i s i b l e r e g i o n . 2. E s t i m a t i o n o f s e d i m e n t a t i o n c o e f f i c i e n t P u r i f i e d v i r u s p r e p a r a t i o n s i n 0.05 M b o r a t e b u f f e r , pH 7.8, were c e n t r i f u g e d i n a S p i n c o Model E a n a l y t i c a l u l t r a c e n t r i f u g e a t 21,740 rpm and a t 20°C. Immediately a f t e r r e a c h i n g the i n t e n d e d speed, the S c h l i e r e n p a t t e r n s were photographed a t 4 min i n t e r v a l s f o r about 20 min. The c o n c e n t r a t i o n o f v i r u s used as e s t i m a t e d by assuming the e x t i n c t i o n c o e f f i c i e n t to be 3.0 (Shepard, 1972), ranged from 0.25 mg per ml t o 0.8 mg per ml. The s e d i m e n t a t i o n c o e f f i c i e n t was then e s t i m a t e d g r a p h i c a l l y from the n e g a t i v e s by t h e method o f Mar-kham ( i 9 6 0 ) . 3, E s t i m a t i o n o f buoyant d e n s i t y The buoyant d e n s i t y o f PVM was e s t i m a t e d by the e q u i l i b r i u m banding method i n a cesium c h l o r i d e s o l u t i o n as d e s c r i b e d by Chervenka (1973). C e n t r i f u g a t i o n was done i n a S p i n c o Model E 31 analytical ultracentrifuge, with a mixture of PVM and T'MV in one c e l l and PVM alone in the other. Test samples were prepared in two volumetric tubes by dissolving 436.9 mg of cesium chloride in 0,5 ml of 0.005 M borate buffer containing 30 to 50/lg of PVM. Tec one tube was then added 0,005 M borate buffer while the other was added 10 to 35 >ug of TMV diluted in the same buffer. The samples were centrifuged at 44,770 rpm and at a temperature of 25°C. Under these conditions the bands indicating the positions of the virus p a r t i c l e s were formed at about the middle of the equilibrium density gradient p r o f i l e . The distance of each band from the center of the rotor was measured by the method of Markham (1960), accurate to 0.001 cm. The true densities of the cesium chloride solutions were estimated from t h e i r respective refractive indices determined i n an Abbe Model 60 Refractometer. Calculations of the buoyant densities of PVM and TMV standard were carried out as described by Chervenka (1973). (9) Estimation of molecular weight of protein subunit The molecular weight of protein subunit was estimated by polyacrylamide gel electrophoresis in 0.1% sodium dodecyl sulfate (SDS). The protein subunit of PVM was prepared by dissociating the virus in 0.1 M phosphate buffer, pH 7.2, containing 4 M urea, 1% SDS and 1% mercaptoethanol (Dunker and Rueckert, 1969). The sample was placed in boiling water for 90 sec to aid dissociation. The protein standards were dissolved in the same buffer 32 a t a c o n c e n t r a t i o n o f 1. mg per ml and were p l a c e d i n b o i l i n g water f o r 90 s e c . F i v e p r o t e i n s o f known m o l e c u l a r w e i g h t s , as i n d i c a t e d i n t he p a r e n t h e s e s , were used as s t a n d a r d s . These were bov i n e serum albumin (62,000), ovalbumin (43,000), a l c o h o l dehydrogenese (37,000), c a r b o n i c anhydrase (29,000) and myoglobin (17,200). The g e l s were p r e p a r e d a c c o r d i n g t o Weber and Osborne (1969) and p r o t e i n s u b u n i t o f PVM was run i n c o m b i n a t i o n w i t h BSA and Mg o r a l o n e , w h i l e the s t a n d a r d s were run i n groups o f 2 o r 3. In a l l cases a t l e a s t 2 measurements were a v a i l a b l e f o r each p r o t e i n per run. To a l l t e s t samples was added a drop o f g l y c i n e c o n t a i n i n g a "marker dye" Bromophenol b l u e , a f t e r which lOyUl were i n d i v i d u a l l y l a y e r e d on top o f the g e l s . E l e c t r o p h o r e s i s was performed w i t h gel c o n c e n t r a t i o n s o f 4% through 9% a t 50V and 7.5 t o 8.3 mA/gel f o r 3 t o 4.5 hours i n 0.1 phosphate b u f f e r , pH 7.2 c o n t a i n i n g 0.1% SDS (Weber and Osborne, 1969). F i x a t i o n and s t a i n i n g were done by o v e r n i g h t s o a k i n g o f the g e l s i n 12.5% t r i c h l o r o a c e t i c a c i d s o l u t i o n (TCA) and 12.5% TCA s o l u t i o n c o n t a i n i n g 0.01% (w/v) eoomasie b r i l l i a n t b l u e dye r e s p e c t i v e l y (Chambrach e t a l . , 1967). D e s t a i n i n g was done o v e r n i g h t i n 7.5% a c e t i c a c i d . C a l c u l a t i o n o f the r e l a t i v e m o b i l i t i e s o f each p r o t e i n w i t h r e s p e c t t o the dye, was a c h i e v e d by the method o f Weber and Osborne (1969), and the m o l e c u l a r w e i g h t o f PVM-protein s u b u n i t was e s t i m a t e d g r a p h i c a l l y from t h e p l o t o f p r o t e i n s ' r e l a t i v e m o b i l i t i e s a g a i n s t t h e i r m o l e c u l a r w e i g h t s ( S h a p i r o e t a l . , 1967). 33 RESULTS (a) Host Range and Symptomatology i n o c u l a t i o n t e s t s c o n f i r m e d p r e v i o u s r e p o r t s t h a t the h o s t range o f the v i r u s i s narrow ( B a g n a l l e t a l . , 1956; MacLeod, 1962; V u l c i c and Hunnius, 1967; H o r i o e t a l . , 1969). Of the 41 p l a n t s p e c i e s and c u l t i v a r s t e s t e d , o n l y 12 r e p r e s e n t i n g f o u r f a m i l i e s , showed s u s c e p t i b i l i t y to PVM: Amaranthaceae - G^ . g l o b o s a ; Chenopodiaceae - C^ . a m a r a n t i c o l o r , C. q u i n o a ; Leguminosae - P_. v u l g a r i s 'Red Kidney' and B o u n t i f u l ' , V_. s i n e n s i s 'Black Eye'; S o l a n a c e a e - I. e s c u l e n t u m 'Rutgers', ' S u b a r c t i c ' and 'Tiny Tim', L y c o p e r s i c o n h i r s u t u m M.B. E t h . , L_. peruvianum M i l l . and I. p i m p i n e l l i f o l i u m M i l l . A l l the s u s c e p t i b l e p l a n t s b e l o n g i n g t o the genus L y c o p e r s i c o n were s y s t e m i c a l l y i n f e c t e d but w i t h o u t symptoms. Others showed non- s y s t e m i c i n f e c t i o n s w i t h symptoms o f v a r i o u s k i n d s ( T a b l e 3 ) . PVM was not r e c o v e r e d from the f o l l o w i n g d i c o t y l e d o n o u s s p e c i e s or c u l t i v a r s b e l o n g i n g t o e i g h t f a m i l i e s : Apocynaceae - Vinca- minor I.; Car.yophyl l a c e a e - Dianthus b a r b a t u s I., [J. c a r y o p h y l u s L.; Chenopodiaeceae - Beta v u l g a r i s L., Chenopodi urn c a p i t a t u m L., S p i n a c e a o l e r a c e a e L. ;Composi.tae - Z i n n i a e l e g a n s J a c q . ; C u c u r b i t a c e a e - -Cucumis s a t i v u s , L. ' S t r a i g h t 8" ;• C r u c i f e r a e - . B r a s s i c a ca;mpestris L. 'Rapa'; Leguminosae - v u l g a r i s ' P i n t o ' and ' S t r i n g l e s s Green Pod', Pi sum s a t i v u m L.; S o l a n a c e a e - jC. annum, JJ. m e t e l , D_. stramonium L., N_. d e b n e y i , N_. c l e v e l a n d i i, JN. g l u t i n o s a L., N_. r u s t i c a , N_. tabacum L. 'Havana 425', 'Samsun NN', 'White B u r l e y ' and ' X a n t h i i ' , P h y s a l i s f l o r i d a n a Rybd., P e t u n i a h y b r i d a V i l m , ; 34 T a b l e 3 L o c a l l e s i o n symptoms on v a r i o u s s p e c i e s o r c u l t i v a r s o f p l a n t s i n c i t e d by PVM P l a n t s p e c i e s o r c u l t i v a r s C. a m a r a n t i c o l o r Symptoms C. q u i n o a G. g l o b o s a P_. vul gar i s 'Red Kidney' P . v u l g a r i s ' B o u n t i f u l ' V, s i n e n s i s " ^ l a c k Eye 1 Appearance S i z e 2 to 3 mm I r r e g u l a r y e l l o w -i s h : green l e s i o n s t u r n i n g s l i g h t l y r e d d i s h i n o l d e r i n f e c t i o n I r r e g u l a r y e l l o w -i s h green l e s i o n s t u r n i n g dark' brown i n o l d e r i n f e c t i o n D i f f u s e d brownish l e s i o n s t u r n i n g d a r k brown i n o l d e r i n f e c t i o n F a i n t g r e y i s h l e s i o n s minute t u r n i n g dark brown i n c o l o u r i n o l d e r i n f e c t i o n 2 mm Same as i n 'Red Kidney' Reddish-brown l e s i o n s t u r n i n g brown i n o l d e r i n f e c t i o n minute minute Number o f days a f t e r i n o c u l a t i o n 15 to 18 2 t o 3 mm 15 to 18 18 to 20 3 to 6 3 to 6 8 to 12 35 T a b l e 4. M e c h a n i c a l a n d g r a f t - t r a n s m i s s i b i l i t y o f PVM i n t o t h e v a r i o u s p o t a r o c u l t i v a r s c a r r i e d o u t a t f o u r d i f f e r e n t t i m e s Number o f p l a n t s i n f e c t e d o v e r t h e n u m b e r o f p l a n t s i n o c u l a t e d C u l t i v a r s S p r i n g 75 W i n t e r 75 S p r i n g 76 F a l l 76 K a t a h d i n 0/4 K e n n e b e c 0/4 0/3 N e t t e d Gem 0/4 0/4 N o r c h i p 0/3 1/3 Red L a S o d a 3/3 3/3 Red P o n t i a c 0/4 2/3 4/10 S e b a g o 0/4 War ba 0/4 W h i t e R o s e 0/3 0/3 7 / 1 0 * A v o n 0/4 6/10* C o l u m b i a R u s s e t 4/8 Wh i t e L a S o d a 7/8 A b n a k i 2/3 A l m a ( T b i q u e ) 0/3 8/8* A r r a n V i c t o r y . 2/3 2 / 2 * ; 4/1 B a n a n a 3/3 B a t o c h e 1/3 C a n u s 0/3 8/8* C a r i b o o 3/3 C h i n o o k 2/3 E p i c u r e 0/3 8/8* F u n d y 1/3 G r e e n M o u n t a i n 2/3 H u r o n 3/3 K e s w i c k 0/3 La C h i p p e r 0/3 N o o k s a k 0/3 P e c o n i c 1/3 6/10* R a r i t a n 0/3 6/10* S a b l e 0/3 1/10 S n o w c h i p 0/3 8/8* W a s e c a 0/3 N o r l a n d 1/3 * R e s u l t s o f g r a f t - i n o c u l a t i o n e x p e r i m e n t 36 U m b e l l i f e r a e - Apium g r a v e o l e n s L. A l l the t h r e e monocotyledonous p l a n t s t e s t e d were not s u s c e p t i b l e to PVM: Graminae - Hordeum v u l g a r a e L., Zea mays L.; L i l i a c e a e -A l 1 i urn porrum L. PVM was r e c o v e r e d i n o n l y 17 o f the 33 p o t a t o c u l t i v a r s which had been m e c h a n i c a l l y i n o c u l a t e d w i t h the v i r u s ( T a b l e 4 ) . However, a l l the n i n e p o t a t o c u l t i v a r s used i n the g r a f t - i n o c u l a t i o n e x p e r iment were i n f e c t e d w i t h the v i r u s , and the i n f e c t i o n s u c c e s s was 60 to 100%. None o f the i n f e c t e d p l a n t s showed symptoms. L i k e w i s e , secondary p l a n t s grown from t u b e r s d e r i v e d from PVM-i n f e c t e d p l a n t s o f the c u l t i v a r s Banana, Red La Soda and White La Soda d i d not e x h i b i t any symptoms. When s e r o l o g i c a l d e t e c t i o n was c a r r i e d o u t , a l l o f them were found t o be i n f e c t e d w i t h PVM. In the experiments to determine the t r a n s m i s s i b i l i t y o f PVM by c o n t a c t , i t was found t h a t a l l the s i x 'Arran V i c t o r y ' p l a n t s p l a c e d i n c o n t a c t w i t h the P V M - i n f e c t e d 'White La Soda' p l a n t s remained f r e e from the v i r u s . A s i m i l a r o b s e r v a t i o n was o b t a i n e d w i t h the 12 tomato p l a n t s p l a c e d i n c o n t a c t w i t h t h o s e m e c h a n i c a l l y i n o c u l a t e d w i t h the v i r u s . Thus under the c o n d i t i o n o f the e x p e r i -ment PVM was not t r a n s m i s s i b l e through p l a n t c o n t a c t , (b) S e r o l o g y The h i g h e s t t i t e r o b t a i n e d f o r a n t i s e r u m a g a i n s t PVM was 2560. The t i t e r , however, was d e c r e a s e d t o 640 two weeks l a t e r and remained so f o r about f o u r weeks. The p r o g r e s s o f the a n t i b o d y p r o d u c t i o n was f o l l o w e d weekly, and i s shown i n F i g . 1. 37 The same r a b b i t was r e i n j e c t e d i n t r a v e n o u s l y 4 months a f t e r t h e -l a s t i n j e c t i o n w i t h t he v i r u s p r e p a r e d by the p u r i f i c a t i o n p r o c e d u r e d e v e l o p e d i n t h e p r e s e n t s t u d i e s . The a n t i s e r u m t i t e r r o s e t o 128'0, but 2 months l a t e r i t had d e c r e a s e d t o 640. A t the l o w e s t d i l u t i o n t e s t e d , no r e a c t i o n was d e t e c t e d a g a i n s t h e a l t h y p l a n t sap. m EH H EH a w H En < 5120~ 1280 320 80 i 10 15 20 WEEKS AFTER FIRST INJECTION F i g . 1. A n t i b o d y p r o d u c t i o n i n r a b b i t a g a i n s t PVM a n t i g e n . Arrows i n d i c a t e the time o f v i r u s a d m i n i s t r a t i o n i n t o the immunized r a b b i t . (c) P r o p e r t i e s i n Crude Sap Twelve days a f t e r i n o c u l a t i o n s , the number o f l o c a l l e s i o n s on each o f the i n o c u l a t e d h a l f - l e a v e s was r e c o r d e d . The pr e s e n c e 38 o r the absence o f l o c a l l e s i o n s d e t e r m i n e d the p h y s i c a l p r o p e r t i e s o f the v i r u s i n crude sap. 1. D i l u t i o n end p o i n t (DEP) L o c a l l e s i o n s were o b s e r v e d on h a l f - l e a v e s which had been i n o c u l a t e d w i t h sap d i l u t e d t o 1 0 " 1 , 10-2, 1 0 - 3 o r 1 0 ~ 4 but not w i t h sap d i l u t e d to 1 0 - 5 o r 1 0 - 6 , s u g g e s t i n g t h a t the DEP o f PVM was 10" 4. Sap e x t r a c t e d from p o t a t o o r tomato l e a v e s gave s i m i l a r r e s u l t s . I t was i n t e r e s t i n g t o note t h a t the average number o f l o c a l l e s i o n s p e r h a l f - l e a f o f Red Kidney bean p l a n t s was h i g h e r when the i n o c u l a t i o n was c a r r i e d out w i t h sap d i l u t e d to 10"^ than the u n d i l u t e d sap ( F i g . 2A). 2. L o n g e v i t y i n v i t r o (LIV) One o f the two r e p l i c a t e s i n c i t e d l o c a l l e s i o n s on Red Kidney bean h a l f - l e a v e s a f t e r 3 days, and the o t h e r a f t e r 2 days o f p r e i n c u b a t i o n a t room t e m p e r a t u r e , s u g g e s t i n g t h a t the LIV o f PVM was between 2 t o 4 days. Sap e x t r a c t e d from p o t a t o o r tomato l e a v e s gave s i m i l a r r e s u l t s . The average number o f l o c a l l e s i o n s p e r h a l f - l e a f d e c r e a s e d s h a r p l y from 12 on day 0 t o 2 on day 1 ( F i g . 2B). 3. Thermal i n a c t i v a t i o n p o i n t (TIP) L o c a l l e s i o n s were o b s e r v e d on Red Kidney bean h a l f - l e a v e s which had been i n o c u l a t e d w i t h sap p r e i n c u b a t e d f o r 10 min a t room temperatures o f 20°C through 65°C, but not a t temperatures o f 70°C o r h i g h e r , thus s u g g e s t i n g a TIP between 65°C and 70°C. I t was o b s e r v e d t h a t the average number o f l o c a l l e s i o n s i n c r e a s e d when the sap was heated to 30°C o r to 40°C ( F i g . 2C). 39 TEMPERATURE 60 65 70 75 T E M P E R A T U R E F i g . 2. The r e l a t i o n s h i p s between t h e - a v e r a g e number o f l o c a l l e s i o n s per h a l f - l e a f o f Red Kidney bean w i t h (A) d i l u t i o n s o f sap, (B) days o f p r e i n c u b a t i o n o f sap a t room t e m p e r a t u r e , and (C,D) p r e i n c u b a t i o n t e mperature (°C) o f sap f o r 10 min . 40 T a b l e 5 V a r i a t i o n s i n the r e l a t i v e c o n c e n t r a t i o n o f PVM i n , and the f i n a l pH o f , 1:3 sap as a r e s u l t o f u s i n g d i f f e r e n t e x t r a c t i o n b u f f e r s , a t pH 7.8, f o r homogenizing p o t a t o l e a v e s B u f f e r * R e c i p r o c a l o f s e r o l o g i c a l d i l u t i o n end p o i n t F i n a l . p H B o r a t e , 0.5 M 32 7,8 B o r a t e , 0.2 M 32 7.8 Phosphate, 0.2 M 16 - 32 7,7 D i e t h y l b a r b i t u r a t e , . - C h i M 16 7.4 T r i s - a c e t a t e , 0.2 M 16 - 32 7.3 C i t r a t e - p h o s p h a t e , 0.2 M 16 7.5 Water 8 6.1 * C o m p o s i t i o n s a s i n A p p e n d i x I I I . 41 (d) P u r i f i c a t i o n 1. E x t r a c t i o n o f v i r u s from l e a f t i s s u e s P V M - i n f e c t e d p o t a t o l e a v e s were homogenized i n a b u f f e r a t the r a t i o o f 1:3, t i s s u e to b u f f e r . When homogenizations were c a r r i e d o u t i n 0.5 M b o r a t e b u f f e r a t pH's 6.0, 7.0, 7.8, 8.0 o r 8.5, the s e r o l o g i c a l d i l u t i o n end p o i n t s were 1:8, 1:16, 1:32, 1:32, and 1:32, r e s p e c t i v e l y . Thus, b o r a t e b u f f e r w i t h a pH range o f between 7.8 and 8.5 was s u i t a b l e f o r e x t r a c t i o n o f PVM from p o t a t o l e a v e s . I t was a l s o shown t h a t , when used a t 0.5 M o r 0.2 M, b o r a t e b u f f e r performed b e t t e r than the o t h e r b u f f e r s t e s t e d ( T a b l e 5 ) . V a r y i n g the t i s s u e to b u f f e r r a t i o to 1:1.5, 1:2.0, o r 1:2.5 d i d not reduce the e f f i c i e n c y o f the 0.5 M b o r a t e b u f f e r . I n c o r p o r a t i o n o f DIECA, mercaptoethenol o r sodium s u l f i t e to f i n a l c o n c e n t r a t i o n s o f 0.5%, 1.0% o r 0.5% r e s p e c t i v e l y , d i d not improve t h e e f f i c i e n c y o f e x t r a c t i o n . However, when the sap was l e f t f o r 2 weeks, then c l a r i f i e d w i t h c h l o r o f o r m , the u n t r e a t e d ;sap e x h i b i t e d a brownish c o l o r a t i o n , whereas the o t h e r s remained as c l e a r y e l l o w i s h - g r e e n s o l u t i o n s . F o l l o w i n g a c y c l e o f d i f f e r e n t i a l c e n t r i f u g a t i o n , PVM was s e r o l o g i c a l l y d e t e c t e d i n the p e l l e t s d e r i v e d from the t r e a t e d sap but was not d e t e c t e d i n the p e l l e t s d e r i v e d from the u n t r e a t e d sap. Crude sap e x t r a c t e d from the p r i m a r y o r the s e condary shoots o f the p o t a t o p l a n t s , cv. Banana, gave a s e r o l o g i c a l d i l u t i o n end p o i n t o f 1:32. Thus, h a r v e s t i n g o f young l e a v e s f o r p u r i f i c a t i o n o f the v i r u s c o u l d be made from i p l a n t s 4 to 15 weeks a f t e r t r a n s p l a n t i n g , o r from p l a n t s 4 weeks a f t e r the f i r s t h a r v e s t . P o t a t o p l a n t s c v s . 42 Columbia Russet and White La Soda, and tomato p l a n t s c v s . Rutgers and S u b a r c t i c , were e q u a l l y s u i t a b l e p r o p a g a t i o n h o s t s f o r the p u r i f i c a t i o n o f PVM. F r e e z i n g o f l e a v e s o v e r n i g h t r e s u l t e d i n a l o s s o f v i r u s . The s e r o l o g i c a l d i l u t i o n end p o i n t was 1:16 compared t o 1:32 f o r l e a v e s s t o r e d f o r the same d u r a t i o n a t 4°C. 2. C l a r i f i c a t i o n o f crude sap The p h y s i c a l and chemical methods o f c l a r i f i c a t i o n t e s t e d s i g n i f i c a n t l y lowered the t u r b i d i t y v a l u e s o f the sap samples compared to the c o n t r o l (P<0.01) ( T a b l e 6 ) . The methods o f c l a r i f i c a t i o n employed de t e r m i n e d the e x t e n t o f v i r u s l o s t . Among th o s e c l a r i f i c a t i o n methods which lowered the s e r o l o g i c a l d i l u t i o n end p o i n t o f the sap t o o n l y 1:16, t r e a t m e n t w i t h an equal volume o f c h l o r o f o r m gave the l o w e s t t u r b i d i t y v a l u e . In Duncan's m u l t i p l e range t e s t , t h i s t u r b i d i t y v a l u e was not s i g n i f i c a n t l y d i f f e r e n t from the t u r b i d i t y o f sap which had been c l a r i f i e d w i t h an equal volume o f carbon t e t r a c h l o r i d e . Reducing the c h l o r o f o r m to sap r a t i o t o 1:6, d i d not improve the s e r o l o g i c a l d i l u t i o n end p o i n t . On the o t h e r hand, the t u r b i d i t y v a l u e was s i g n i f i c a n t l y i n c r e a s e d ( P < 0 . 0 1 ) i n comparison t o t h a t o f sap which had been c l a r i f i e d w i t h an equal volume o f c h l o r o f o r m . When 1;1.5 sap was c l a r i f i e d w i t h an equal volume o f c h l o r o f o r m or with chloroform ^dded t o o n e - s i x t h volume o f sap, the s e r o l o g i c a l d i l u t i o n end p o i n t was 1:32. Very c l e a r p r e p a r a t i o n s were o b t a i n e d when 1:3 sap samples were 43 T a b l e 6. R e l a t i v e v i r u s c o n c e n t r a t i o n and t u r b i d i t y o f 1:3 sap a f t e r c l a r i f i c a t i o n by v a r i o u s methods C l a r i f i c a t i o n methods 1. Ammonium s u l f a t e (25%) 2. F r e e z i n g 3. C h l o r o f o r m ( 1 : 6 ) ^ , then T r i t o n X - 1 0 0 (1%) 4. C h l o r o f o r m (1:1) 5. T r i t o n X - 1 0 0 (1%) 6. Carbon t e t r a c h l o r i d e (1:1) 7. n-butanol (8%) 8. C h l o r o f o r m (1:6) 9. H e a t i n g a t 45°C, TOrmin 10. C e n t r i f u g e , C e l i t e 11. E t h e r (1:1) 12. C o n t r o l V i r u s T u r b i d i t y a t C o n c e n t r a t i o n * 540 nm** 4 0.063 a 0 0.072 a 2 0.141 a 16 0.307 b . 8 0,329 be 16 0.361 bed 16 0.400 cd 16 0.424 d 8 0.930 e 8 1.075 f 16 1.155 f 32 1.340 g * A l l t he s i x r e p l i c a t e s gave s i m i l a r s e r o l o g i c a l d i l u t i o n end p o i n t s e x c e p t i n 1 and 9 where one r e p l i c a t e d i f f e r e d ; * L e v e l o f s i g n i f i c a n c e f o r Duncan's m u l t i p l e range t e s t . Treatment means ( s i x r e p l i c a t i o n s ) not f o l l o w e d by the same l e t t e r s were s i g n i f i -c a n t l y d i f f e r e n t (P < 0.01). R a t i o o f volume o f o r g a n i c s o l v e n t t o volume o f sap. 44 subjected to overnight f reez ing , but the concentration of v irus was low. With 1:1.5 sap i t was found that removal of the f reeze-coagulated plant residues by f i l t r a t i o n through No. 54 Whatman f i l t e r paper or by centr i fugat ion at low speed resulted in supers natants with a s im i la r sero log ica l d i l u t i on end point of 1:2. The fact that PVM was not se ro log ica l l y detected from the resuspended residues or p e l l e t s , suggested that the reduction in virus concentration in the supernatants was due to virus degradation, and not due to aggregation of the v i rus par t i c les or to the i r adsorption onto the plant residues. Though n-bu tano l -c la r i f ied sap had a tu rb id i t y value comparable . to that of carbon t e t r ach lo r i de^c la r i f i ed sap, i t was greenish in colour. An attempt to remove th is colorat ion by lengthening the incubation period from 30 min to overnight, was unsuccessful. Instead, the sero log ica l d i l u t i on end point of the sap was further reduced from 1:16 to 1:8 in 1:3 sap, and from 1:32 to 1:16 in 1:1.5 sap. C l a r i f i c a t i o n with an equal volume of carbon te t rachlor ide resul ted in sap with a r e l a t i ve l y high sero logica l d i l u t i on end point and a r e l a t i v e l y low tu rb id i t y value. Later experiments revealed that the optimum concentration of the organic solvent for c l a r i f i c a t i o n of 1:3 or 1:1.5 sap was 1.25 ml per 10 ml sap. Increasing the concentration to 2.5 ml or to 5.0 ml per 10 ml sap, resul ted in a s l i gh t reduction of the sero logica l d i l u t i on end point (F ig . 3). The tu rb id i t y values were, however, not s i g n i f i c a n t l y reduced (P>0.05) when carbon te t rach lor ide was added in excess of 1.25 ml. This concentration was therefore adopted for use in < u O Z o o. ££ CL yj ( A Q y_ Z O w u o O u> u 5 LU A M O U N T O F C A R B O N T E T R A C H L O R I D E F i g . 3. R e l a t i v e c o n c e n t r a t i o n o f P V M [ H ] i n , and t u r b i d i t y a t 540 n m [ n ] o f , (a) 1:3 sap and (b) 1:1.5 sap, a f t e r c l a r i f i c a t i o n w i t h v a r i o u s amounts(ml) o f carbon t e t r a c h l o r i d e per 10 ml o f sample. 46 subsequent e x p e r i m e n t s . When carbon t e t r a c h l o r i d e - e m u l s i f i e d 1:1.5 sap was c e n t r i f u g e d a t 5,000, 6,500 o r 9,500 rpm f o r 5, 10 o r 15 min, an i d e n t i c a l s e r o l o g i c a l d i l u t i o n end p o i n t o f 1:64 was o b t a i n e d from a l l t he r e s u l t i n g s u p e r n a t a n t s . T h e i r t u r b i d i t y v a l u e s a t 540 nm were s i g n i f i c a n t l y a f f e c t e d (P <0.01) by the c o m b i n a t i o n o f time and speed o f c e n t r i f u g a t i o n . C e n t r i f u g a t i o n a t 9,500 rpm f o r 15 min r e s u l t e d i n the l o w e s t t u r b i d i t y v a l u e ( T a b l e 7 ) . Hence, t h i s c o m b i n a t i o n o f speed and time was used t h r o u g h o u t the p r e s e n t s t u d i e s . When 1:3 sap was c e n t r i f u g e d a t t h i s c o m b i n a t i o n o f speed and t i m e , the s e r o l o g i c a l d i l u t i o n end p o i n t o f 1:32 was s i m i l a r t o t h a t o f sap c e n t r i f u g e d a t 5,000 rpm f o r 5 min, En c o u r a g i n g r e s u l t s were o b t a i n e d when ammonium s u l f a t e was aidded t o the crude sap a t lower c o n c e n t r a t i o n s than the 25% used i n the p r e l i m i n a r y i n v e s t i g a t i o n ( F i g . 4 ) . A l t h o u g h the t u r b i d i t y v a l u e o f the sap was s i g n i f i c a n t l y i n c r e a s e d (P < 0.01) as the c o n c e n t r a t i o n o f the ammonium s u l f a t e was r e d u c e d , t h i s was more than compensated by the 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 . There was no d e t e c t a b l e l o s s o f v i r u s from the c l a r i f i e d sap when the amounts added were 1.5 gm o r 1.75 gm per 10 ml o f 1:3 o r 1:1.5 sap, r e s p e c t i v e l y . The s e r o l o g i c a l d i l u t i o n end p o i n t o f 1:1.5 sap which had been c l a r i f i e d w i t h 2.0 gm ammonium s u l f a t e ranged from 1:32 to 1:64. When i n c u b a t i o n was c a r r i e d o u t f o r 6 hours i n s t e a d o f o v e r n i g h t , the s e r o l o g i c a l d i l u t i o n end p o i n t s were not extended, nor were the t u r b i d i t y v a l u e s i n c r e a s e d . O v e r n i g h t i n c u b a t i o n w i t h ammonium s u l f a t e a t f i n a l c o n c e n t r a t i o n s o f 15% and 20% were t h e r e f o r e adopted f o r c l a r i f i c a t i o n o f 1:3 sap and 47 T a b l e 7. T u r b i d i t y v a l u e s o f carbon t e t r a c h l o r i d e - c l a r i f i e d sap measured a t 540 nm, a f t e r c e n t r i f u g a t i o n - a t d i f f e r e n t c o m b i n a t i o n s o f speed and time Time o f c e n t r i f u g a t i o n Speed o f ( m 1 n )  C e n t r i f u g a t i o n (rpm) 5 10 15 5,,000 0.940 f * 0.658 d 0.600 cd 6,500 0.809 e 0.591 cd 0.509 be 9,500 0.629 cd 0.408 ab 0.341 a L e v e l o f s i g n i f i c a n c e i n Duncan's m u l t i p l e range t e s t . . Treatment-, means (two r e p l i c a t i o n s , and two samples per r e p l i c a t e ) when f o l l o w e d by the same l e t t e r were s i g n i f i c a n t l y d i f f e r e n t (P < 0.05). A M O U N T O F A M M O N I U M S U L F A T E F i g . 4. R e l a t i v e c o n c e n t r a t i o n o f PVM i n [H] , and t u r b i d u t y a t 540 nm [D] o f , (a) 1:3 sap and (b) 1:1.5 sap, a f t e r c l a r i f i c a t i o n w i t h v a r i o u s amounts(gm) o f ammonium s u l f a t e per 10 ml o f sample. E e o in < > t Q CO -Pi 00 49 F i g , 5. R e l a t i v e c o n c e n t r a t i o n o f PVM [ a ] i n , and t u r b i d i t y a t 540 nm [•] o f , (A) 1:3 sap from p o t a t o , (B) 1:1.5 sap from p o t a t o and (C) 1:1.5 sap from tomato, a f t e r c l a r i f i c a t i o n w i t h carbon t e t r a c h l o r i d e (CT) o r ammonium s u l f a t e (AS). 50 1:1.5 sap, r e s p e c t i v e l y . The s e r o l o g i c a l d i l u t i o n end p o i n t o f c l a r i f i e d sap a f t e r c e n t r i f u g a t i o n a t 9,500 rpm f o r 15 min was s i m i l a r t o t h a t o f c l a r i f i e d sap which had been c e n t r i f u g e d a t 5,000 rpm f o r 5 min. In d i r e c t comparison t e s t s between c l a r i f i c a t i o n methods u t i l i s i n g t h e p r e c i p i t a t i n g o r c o a g u l a t i n g p r o p e r t i e s o f ammonium s u l f a t e o r carbon t e t r a c h l o r i d e , a l a r g e r sample s i z e o f 100 ml o f sap was used. Some samples were homogenised f o r 10 s e c w i t h carbon t e t r a c h l o r i d e i n a Waring b l e n d e r . O t h e r s were t r e a t e d w i t h the a p p r o p r i a t e amount o f ammonium s u l f a t e . S t i r r i n g was c a r r i e d o u t w i t h a magnetic s t i r r e r u n t i l a l l the ammonium s u l f a t e c r y s t a l s were d i s s o l v e d , Both t h e s e methods produced c l a r i f i e d sap w i t h s i m i l a r s e r o l o g i c a l d i l u t i o n end p o i n t s ( F i g . 5 ) . I t was s i g n i f i c a n t t h a t a l l t he r e p l i c a t e s o f 1:1.5 sap which had been c l a r i f i e d w i t h ammonium s u l f a t e added to a f i n a l c o n c e n t r a t i o n o f 20% showed a s i m i l a r s e r o l o g i c a l d i l u t i o n end p o i n t o f 1:64. 3. C o n c e n t r a t i o n o f v i r u s from c l a r i f i e d sap The r a t i o o f the volume o f a v i r u s p r e p a r a t i o n b e f o r e concen-t r a t i o n t o i t s volume a f t e r c o n c e n t r a t i o n was i m p o r t a n t . I t was found t h a t d i s s o l v i n g t he p e l l e t s i n o n e - t e n t h volume o f e i t h e r 0.5 M o r 0.05 M b o r a t e b u f f e r , i n v a r i a b l y r e s u l t e d i n lower s e r o l o g i c a l d i l u t i o n end p o i n t s compared t o thos e d i s s o l v e d i n t wo-tenths volumes o f b u f f e r ( F i g . 6 ) . B o r a t e b u f f e r a t 0.05 M was as good as a t 0.5 M when used f o r d i s s o l v i n g t he p e l l e t s F i g . 6. R e l a t i v e c o n c e n t r a t i o n o f PVM i n p r e p a r a t i o n s p r e c i p i t a t e d by the v a r i o u s methods: High speed c e n t r i f u g a t i o n (HC), PEG p r e c i p i t a t i o n (PEG), Ammonium s u l f a t e p r e c i p i t a t i o n (AS) and a c i d p r e c i p i t a t i o n (AC). The p e l l e t s were resuspended i n e i t h e r 0.5 M o r 0.05 M b o r a t e b u f f e r a t o n e - t e n t h [ s ] , two-tenths [ a ] , o r f o u r - t e n t h s [ a ] o f the o r i g i n a l volume. 52 o b t a i n e d by the v a r i o u s c o n c e n t r a t i o n methods, and a t the same time r e d u c i n g t he volume o f the v i r u s p r e p a r a t i o n t o two-rtenths o f the o r i g i n a l . I t was a l s o found t h a t r e s u s p e n s i o n o f ammonium s u l f a t e - o r P E G - p r e c i p i t a t e d p e l l e t s i n two-tenths volume o f 0.05 M. phosphate o r c i t r a t e - p h o s p h a t e b u f f e r gave s i m i l a r r e s u l t s as 0.05 M b o r a t e b u f f e r . In t h e experiment to dete r m i n e the b e s t c o m b i n a t i o n o f PEG 6000 and sodium c h l o r i d e f o r p r e c i p i t a t i o n o f PVM, i t was found t h a t the a d d i t i o n o f the former f o l l o w e d by the l a t t e r a t c o n c e n t r a -t i o n s o f 5% and 4% r e s p e c t i v e l y , was s u f f i c i e n t to p r e c i p i t a t e the v i r u s from t h e sap ( T a b l e 8 ) . I n c r e a s i n g t he c o n c e n t r a t i o n s o f e i t h e r one o r both t he c h e m i c a l s d i d not improve the s e r o l o g i c a l d i l u t i o n end p o i n t o f the c o n c e n t r a t e d v i r u s beyond 1:64, P r e v i o u s l y , ammonium s u l f a t e - t r e a t e d sap had been a l l o w e d t o s t a n d o v e r n i g h t b e f o r e c e n t r i f u g a t i o n t o p e l l e t the p r e c i p i t a t e was c a r r i e d o u t . I t was l a t e r shown t h a t even when the t r e a t e d sap was a l l o w e d t o s t a n d f o r o n l y 2 o r 6 hour s , complete p r e c i p i t a t i o n o f the v i r u s was a c h i e v e d . T h i s was i n d i c a t e d by the o b s e r v a t i o n t h a t a s i m i l a r s e r o l o g i c a l d i l u t i o n end p o i n t o f 1:64 was o b t a i n e d i n a l l the c o n c e n t r a t e d p r e p a r a t i o n s . In a d d i t i o n , PVM was not s e r o l o g i c a l l y d e t e c t e d i n the " f i r s t s u p e r n a t a n t s " . D i r e c t comparison t e s t s were c a r r i e d o u t between c o n c e n t r a t i o n methods which showed p r o m i s i n g r e s u l t s . For carbon t e t r a c h l o r i d e -c l a r i f i e d sap a comparison was made between h i g h speed c e n t r i f u g a l t i o n and PEG p r e c i p i t a t i o n i n which PEG 6000 and sodium c h l o r i d e 53 T a b l e 8. R e l a t i v e c o n c e n t r a t i o n o f v i r u s i n p r e p a r a t i o n s o b t a i n e d by p r e c i p i t a t i o n s w i t h d i f f e r e n t c o m b i n a t i o n s o f PEG 6000 and sodium c h l o r i d e , . C o n c e n t r a t i o n o f C o n c e n t r a t i o n o f sodium c h l o r i d e . ( % ) PEG 6000 (%) < 2.5 8 8 8 5.0 32 - 64 64 64 7.5 64 64 64 54 were added to f i n a l c o n c e n t r a t i o n s o f 5% and 4 % , r e s p e c t i v e l y . F o r ammonium s u l f a t e - c l a r i f i e d sap, a comparison was made between h i g h speed c e n t r i f u g a t i o n and ammonium s u l f a t e p r e c i p i t a t i o n . The ammonium s u l f a t e was added t o a f i n a l c o n c e n t r a t i o n o f 30%, and the t r e a t e d sap was then a l l o w e d to st a n d f o r 2 hours b e f o r e b e i n g c e n t r i f u g e d . In a l l cases the s e r o l o g i c a l d i l u t i o n end p o i n t o f the c o n c e n t r a t e d p r e p a r a t i o n s was 1:64. L i k e w i s e , a s i m i l a r s e r o l o g i c a l d i l u t i o n end p o i n t o f 1:4 was o b t a i n e d i n the s u p e r n a t a n t s from a second r e s u s p e n s i o n . Hence the a p p a r e n t l o s s o f v i r u s c o u l d p a r t l y be a t t r i b u t e d to the d i f f i c u l t y o f r e s u s p e n d i n g the v i r u s from the p e l l e t s f o l l o w i n g p r e c i p i t a t i o n , In a l a t e r e x p e r i m e n t i t was found t h a t the v i r u s was not s e r o l o g i c a l l y d e t e c t a b l e d u r i n g t he t h i r d r e s u s p e n s i o n o f the p e l l e t s , r e g a r d l e s s o f whether the two p r e c e e d i n g r e s u s p e n s i o n s were c a r r i e d out i n two-tenth o r f o u r - t e n t h volumes o f 0.05 M b o r a t e b u f f e r . S u b s e q u e n t l y , r e s u s p e n s i o n o f p e l l e t s o b t a i n e d from t h e f i r s t s t a g e o f c o n c e n t r a t i o n was done t w i c e , each time with t w o - t e n t h volume o f b u f f e r . 4,. F u r t h e r c o n c e n t r a t i o n and f i n a l p u r i f i c a t i o n D u r i n g t he second s t a g e o f c o n c e n t r a t i o n , e i t h e r by a h i g h speed c e n t r i f u g a t i o n or'by PEG p r e c i p i t a t i o n , the v i r u s p r e p a r a t i o n s o b t a i n e d had a s i m i l a r d i l u t i o n end p o i n t o f 1:128, r e g a r d l e s s o f the type o r s o u r c e o f the s t a r t i n g m a t e r i a l s . Hence t h e r e appeared t o be no d e t e c t a b l e l o s s i n v i r u s , as opposed to the r e s u l t s o f the f i r s t s t a g e o f c o n c e n t r a t i o n . 55 T a b l e 9. R e l a t i v e c o n c e n t r a t i o n o f v i r u s p r e p a r a t i o n s a f t e r t h e t h i r d s t a g e o f c o n c e n t r t i o n u s i n g f o u r d i f f e r e n t p r o c e d u r e s V o l u m e o f b u f f e r i n w h i c h r o c e d u r e * p e l l e t s w e r e r e s u s p e n d e d (m 10 JJ3 2.0 I 3 2 - 6 4 128 I I 32 128 I I I 32 128 IV 32 64 - 128 * I - ammonium s u l f a t e , P E G , h i g h s p e e d . I I - ammonium s u l f a t e , h i g h s p e e d , PEG. I l l - P E G , PEG, h i g h s p e e d . IV - PEG, h i g h s p e e d , PEG. 56 In t h e t h i r d s t a g e o f c o n c e n t r a t i o n , a s i g n i f i c a n t r e d u c t i o n i n d i l u t i o n end p o i n t was o b s e r v e d when the p e l l e t s were r e s u s p e n -ded i n 0.3 ml o f b u f f e r ( T a b l e 9 ) . Moreover, s e p a r a t i o n o f the u n d i s s o l v e d p e l l e t s from the s u p e r n a t a n t s f o l l o w i n g a low speed c e n t r i f u g a t i o n was d i f f i c u l t . As a r e s u l t , the v i r u s p r e p a r a t i o n s were y e l l o w i s h i n c o l o u r . T h i s was more o b v i o u s i n the case o f p o l y e t h y l e n e g l y c o l p e l l e t s from p r o c e d u r e s II (ammonium s u l f a t e , h i g h speed, PEG) o r IV (PEG" -6000, h i g h s p e e d , . PEG 6 0 0 0 ) than the h i g h speed p e l l e t s from p r o c e d u r e s I (ammonium s u l f a t e , PEG, h i g h speed) o r I I I (-.PEG 6 0 0 0 , PEG 6 0 0 0 , high s p e e d ) . Thus, h i g h speed c e n t r i f u g a t i o n was more s u i t a b l e to be employed i n the f i n a l s t a g e o f c o n c e n t r a t i o n than PEG p r e c i p i -t a t i o n . However, when the p e l l e t s were resuspended i n 2 . 0 ml o f b u f f e r , t h e r e was no d e t e c t a b l e l o s s i n v i r u s . Moreover, p a r t i a l l y p u r i f i e d v i r u s p r e p a r a t i o n s c o u l d be o b t a i n e d from t h e p r e p a r a t i o n s which had been c o n c e n t r a t e d by the pro c e d u r e s I and I I I , and s u b s e q u e n t l y c e n t r i f u g e d through 5 t o 35% s u c r o s e d e n s i t y g r a d i e n t i n SW 25 r o t o r f o r 1.5 hours a t 23 , 0 00 rpm ( F i g . 7 ) . In o r d e r t o o b t a i n p u r e r p r e p a r a t i o n s o f the v i r u s , a f o u r t h s t a g e o f c o n c e n t r a t i o n was c a r r i e d o u t . A n o t h e r c y c l e o f d i f f e r e n t i a l c e n t r i f u g a t i o n was added t o the p r o c e d u r e . The s e r o l o g i c a l d i l u t i o n end p o i n t o f t h e s e p r e p a r a t i o n s was 1 : 6 4 , showing t h a t t h e l o s s o f v i r u s was l e s s than when o n l y t h r e e s t a g e s o f c l a r i f i c a t i o n were employed. . P l a n t m a t e r i a l s were g r a d u a l l y removed from t h e v i r u s p r e p a r a t i o n s as p u r i f i c a t i o n p r o g r e s s e d . The v i r u s p a r t i c l e s appeared t o be normal F i g . 7 E l e c t r o n m i c r i g r a p h s o f v i r u s p r e p a r a t i o n s a f t e r t h r e e s t a g e s o f c o n c e n t r a t i o n f o l l o w e d by a s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n i n SW 25 r o t o r ; A. ammonium s u l f a t e method, B. carbon t e t r a c h l o r i d e method. 58 ( F i g . 8 ) . A f t e r the s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , f a i r l y pure p r e p a r a t i o n s were o b t a i n e d from both the ammonium s u l f a t e and t h e carbon t e t r a c h l o r i d e methods ( F i g . 9 ) . These p r e p a r a t i o n s f a i l e d t o r e a c t w i t h a n t i s e r u m a g a i n s t h e a l t h y p l a n t components. The ^ 2 6 0 ^ 2 8 0 r a t i o s o f 1.23 and 1.25 ( T a b l e 10) s u p p o r t e d the e l e c t r o n m i c r o s c o p i c and s e r o l o g i c a l e v i d e n c e t h a t the v i r u s p r e p a r a t i o n s d e r i v e d from t h e two p u r i f i c a t i o n p r o c e d u r e s were f a i r l y pure. Data from t he s e r o l o g i c a l d i l u t i o n end p o i n t and the A^^q d e t e r m i n -a t i o n s s u g g e s t e d t h a t t h e ammonium s u l f a t e method i s s l i g h t l y s u p e r i o r t o . the carbon t e t r a c h l o r i d e method ( T a b l e 1 0 ) . In a d d i t i o n , i t c o u l d be seen t h a t t he d e n s i t y g r a d i e n t . p r o f i l e o f the former showed a s l i g h t l y h i g h e r peak than the l a t t e r ( F i g . 10). In a d d i t i o n , , t o b e i n g f a i r l y pure, the v i r u s p r e p a r a t i o n s o b t a i n e d by the two methods were a l s o i n f e c t i o u s . In a l a t e r ; e x p e r i m e n t , i t was shown t h a t the peak o f i n f e c t i v i t y d e t e r m i n e d by assay on Red Kidney bean c o i n c i d e d w i t h t he peak o f the d e n s i t y g r a d i e n t p r o f i l e ( F i g . 11). PVM p a r t i c l e s p u r i f i e d by the ammonium s u l f a t e method had a normal l e n g t h o f 651 nm. Of the 200 p a r t i c l e s measured, 39% had t h i s l e n g t h . S i x t y - s e v e n p e r c e n t o f the p a r t i c l e s had l e n g t h s between 644 to 660 nm which i s the range o f v a l u e s r e p o r t e d as the normal l e n g t h o f PVM p a r t i c l e s ( H i t c h b o r n and H i l l , 1965; Rozendaal and van S l o g t e r e n , 1957). Only 12% o f t he p a r t i c l e s had l e n g t h s l e s s than 600 nm, and o n l y 2.5% had l e n g t h s g r e a t e r than'700 nm. ,;; D i a l y s i s o f t h e v i r u s p r e p a r a t i o n s a f t e r s u c r o s e d e n s i t y g r a d i e n t c o u l d be d e l e t e d from t he p u r i f i c a t i o n p r o c e d u r e . When 5 ml samples o f these p r e p a r a t i o n s were d i l u t e d w i t h 0.05 M b o r a t e b u f f e r and t h e , To f a c e page 59 F i g . 8. E l e c t r o n micrographs o f PVM p a r t i c l e s a f t e r c l a r i f i c a t i o n and c o n c e n t r a t i o n o f the v i r u s p r e p a r a t i o n s by the f o l l o w i n g methods: A, C l a r i f i c a t i o n w i t h ammonium s u l f a t e ; B, F i r s t s t a g e o f c o n c e n t r a t i o n by p r e c i p i t a t i o n w i t h ammonium s u l f a t e ; C, Second s t a g e o f c o n c e n t r a t i o n by hi g h speed c e n t r i f u g a t i o n ; D, T h i r d s t a g e o f c o n c e n t r a t i o n by p r e c i p i t a t i o n w i t h PEG 6000; E, F o u r t h s t a g e o f c o n c e n t r a t i o n by h i g h speed c e n t r i f u g a t i o n . 59 60 F i g . 9 E l e c t r o n m i c r o g r a p h o f PVM p a r t i c l e s a f t e r p u r i f i c a t i o n by the ammonium s u l f a t e method. 61 T a b l e 10. Range o f c o n c e n t r a t i o n , average A 2 6 0 ^ A 2 8 0 r a t l 0 S a n G l i n f e c t i v i t y o f PVM p r e p a r a t i o n s p u r i f i e d from 40 gm o f PVM - i n f e c t e d p o t a t o l e a v e s R e c i p r o c a l o f s e r o l o g i c a l Method o f d i l u t i o n . A /A p u r i f i c a t i o n end p o i n t 260 260' 280 I n f e c t i v i t y Ammonium s u l f a t e 128 0.100 1.25 P o s i t i v e Carbon t e t r a c h l o r i d e 6 4 - 1 2 8 0.081 1.23 P o s i t i v e F i g , 10, Sucrose d e n s i t y g r a d i e n t p r o f i l e s o f PVM (254 nm) o b t a i n e d i n two d i f f e r e n t e x p e r i m e n t s . The v i r u s p r e p a r a t i o n s were d e r i v e d from ammonium s u l f a t e - c l a r i f i e d sap ( a ) , carbon t e t r a c h l o r i d e ^ c l a r i f i e d sap ( b ) , and f r e e z e s ! a r i f i e d sap ( c ) . ro 63 F i g . 11. Average number o f l o c a l l e s i o n s p er s i x h a l f - l e a v e s o f Red Kidney bean p l a n t s r e c o r d e d i n i n f e c t i v i t y a s s a y o f 1 ml f r a c t i o n s from SW 41 s u c r o s e d e n s i t y g r a d i e n t ; _ , absorbance a t 254 nm; .m 0-> number o f l o c a l l e s i o n s . 64 c e n t r i g u g e d i n no. 65 r o t o r a t 35,000 rpm f o r 1.5 h r , t h e r e was no l o s s i n v i r u s i n comparison to those samples which had been d i a l y s e d o v e r n i g h t . From the f o r e g o i n g e x p e r i m e n t s , a pr o c e d u r e f o r p u r i f i c a t i o n o f PVM was d e v e l o p e d . T h i s p r o c e d u r e was t e s t e d w i t h t h r e e 400 gm samples o f p o t a t o l e a v e s as d e s c r i b e d i n the f l o w - c h a r t i n F i g . 12. The A2gQ v a l u e s f o r the t h r e e 2 ml samples o b t a i n e d a t the end o f the p r o c e d u r e was 2.45, 2.20 and 2.27. The c o r r e s p o n d i n g y i e l d s , t a k i n g an e x t i n c t i o n c o e f f i c i e n t o f 3.0, were 4.1, 3.7 and 3.8 mg o f v i r u s per Kg o f l e a v e s , r e s p e c t i v e l y , (e) B i o p h y s i c a l and B i o c h e m i c a l P r o p e r t i e s The a b s o r p t i o n s p e c t r a o f PVM s o l u t i o n b e f o r e and a f t e r c o r r e c t i o n f o r l i g h t s c a t t e r i n g i s shown i n F i g . 13. U l t r a v i o l e t a b s o r p t i o n c h a r a c t e r i s t i c s o f the v i r u s b e f o r e and a f t e r c o r r e c t i o n f o r l i g h t s c a t t e r i n g d i f f e r e d s l i g h t l y w i t h the c o r r e s p o n d i n g c o r r e c t e d v a l u e s ( T a b l e 11). The i n d i v i d u a l v a l u e s o f the s e d i m e n t a t i o n c o e f f i c i e n t o f PVM o b t a i n e d from s i x a n a l y t i c a l runs ranged from 159s t o 166s w i t h a mean v a l u e o f 162 ± I s . The S c h l i e r e n p a t t e r n showed t h a t the v i r u s p a r t i c l e s sedimented as one band ( F i g . 14), The S c h l i e r e n p a t t e r n o f the e q u i l i b r i u m banding i n ces i u m c h l o r i d e showed t h a t the PVM p a r t i c l e s formed a band c l o s e r to the c e n t e r o f t h e r o t o r than d i d the TMV p a r t i c l e s ( F i g . 15), The range o f v a l u e s o b t a i n e d from f o u r i n d i v i d u a l e s t i m a t i o n s was from 1.301 t o 1.309 w i t h a mean v a l u e o f 1.304 + 0.001. T h i s 65 («oo ,i» .Use* awl l%>*v»«» eaef&e* F * l l * t Supernatant Pellet Supernatant Pellet Supernatant Pellet - i Supernatant Pellet Supernatant Pellet w Supernatant Pellet Supernatant P e l l e t Supernatant >k«HH|«nie*il Hi »U0 nl iir 0.5 M borate Uift..r. pll a'i'l then ft l le ie.1 thruinjti •mat in o inth. I'revkplteted uvernHlht with anwurilum •ulfate I J 0 » l , then oanirtfuued at «,500 i|« for 14 «iin, More aaaonlua aulfate added to a f i n a l concentration of 10», allowed to stand for 1 hr. then centrifuged at «,500 rpe Resuspended twice, each t i e * In 140 a l of 0.05 H borate bufferi f l r a t ^suspen-sion done overnight, and aecond resus-pension done after 4 hr of incubation. Centrifuged at t.SOO rp» for IS a i n . High speed centrifuged in no.JO rotor at 21.000 rp* for 1.5 hr. Reauapended overnight In »0 a l of 0.0S M borate buffer, then centrifuged at »,500 rpa for 15 Bin. Precipitated with PtG (000 (SI) and aodiua chloride, allowed to stand for 1 hr and then centrifuged at »,500 rp* for IS Bin. Resuspended overnight i n 10 a l of 0.OS M borate buffer, than centrifuged at t.500 rpm for IS Bin. Centrifuged in no.tS rotor at 15.000 rpa for l.S hr. „ overnight in 2 a l of 0.0S M buffer, then centrifuged at »,500 rpa fo IS a i n . Centrifuged through S to J5i sucrose density gradient at 21,000 rpa for l.S hr. Virus band was collected with a syringe, dilu t e d with an equal voluae of O.OS M borate buffer, then centrifuged in no. 45 rotor at 35,000 rpa for l.S hr. Pellet was resuspended in 2 a l of 0.0SM borate buffer (VIRUS sourrioMi F i g . 12. F l o w - c h a r t o f the ammonium s u l f a t e method f o r the p u r i f i c a t i o n o f PVM. 66 T a b l e 11. U l t r a v i o l e t a b s o r p t i o n c h a r a c t e r i s t i c s o f p o t a t o v i r u s M b e f o r e and a f t e r c o r r e c t i o n f o r l i g h t s c a t t e r i n g C h a r a c t e r i s t i c s o f PVM P r o p e r t i e s U n c o r r e c t e d C o r r e c t e d A m 3 v 259 nm 260 nm max A m i n 246 nm 245 nm A 2 6 0 / A 2 8 0 1.25 ± 0.04 1 . 2 3 + 0 . 0 1 l.C'v± 0,01 Am a v / A m - i « 1.08 ± 0.01 1.24 ± 0.01 max mm 67 1.2 240 260 280 300 320 WAVELENGTH (nm) F i g , 13. A b s o r p t i o n spectrum o f p u r i f i e d PVM s o l u t i o n : a. u n c o r r e c t e d f o r l i g h t s c a t t e r i n g , b. c o r r e c t e d f o r l i g h t s c a t t e r i n g . 68 F i g . 14. S c h l i e r e n p a t t e r n o f s e d i m e n t i n g PVM p a r t i c l e s i n 0.05 M b o r a t e b u f f e r , pH 7.8, 16 min a f t e r r e a c h i n g speed o f 21,740 rpm F i g . 15. S c h l i e r e n p a t t e r n o f PVM p a r t i c l e s r e s u l t i n g from e q u i l i b r i u m banding i n cesium c h l o r i d e ; PVM bands appeared t o the l e f t o f the TMV band. 69 s u g g e s t e d t h a t t he PVM p a r t i c l e s had an RNA c o n t e n t o f 6.2% (Gibbs and H a r r i s o n , 1976). The TMV s t a n d a r d had a buoyant d e n s i t y o f 1.324. PVM p r o t e i n s u b u n i t moved through t he p o l y a c r y l a m i d e g e l as one band. I t s m o l e c u l a r w e i g h t l a y between tho s e o f c a r b o n i c anhydrase and ovalbumin ( F i g . 1 6 ) , and ranged from 37,600 to 40,300 d a l t o n s . The means o f f i v e d e t e r m i n a t i o n s f o r the i n d i v i d u a l g e l c o n c e n t r a t i o n s were 38,900, 39,100, 39,200, 39,600, 39,700 and 39,800 d a l t o n s f o r 4, 5, 6, 7, 8, and 9% g e l s r e s p e c t i v e l y . The o v e r a l l mean was 39.3 + 0.6 thousand d a l t o n s . 70 0.6 0.8 1.0 RELATIVE MOBILITY F i g . 16. P l o t o f m o l e c u l a r weights o f p r o t e i n s v e r s u s t h e i r r e l a t i v e m o b i l i t i e s \r\ 5% p o l y a c r y l a m i d e g e l s c o n t a i n i n g 0.1% SDS. The m o l e c u l a r w e i g h t o f PVM p r o t e i n s u b u n i t was g r a p h i c a l l y e s t i m a t e d from t h e p l o t . The p r o t e i n s t a n d a r d s used were: Bovine serum albumin (BSA), ovalbumin (OA), c a r b o n i c anhydrase (CA), a l c o h o l dehydrogenase (AD) and myoglobin (Mg). 71 DISCUSSION The h o s t range o f PVM i s narrow. Only p l a n t s b e l o n g i n g t o the f a m i l i e s Amaranthaceae,Chenopodiaceae, Leguminosae and S o l a n a c e a e c o u l d be m e c h a n i c a l l y i n f e c t e d by the v i r u s . Symptoms ob s e r v e d on (5. g l o b o s a , C^ . a m a r a n t i c o l o r , q u i n o a , _P. v u l g a r i s c v s . Red Kidney and B o u n t i f u l and V^ . s i n e n s i s were g e n e r a l l y s i m i l a r t o the p r e v i o u s d e s c r i p t i o n s by s e v e r a l a u t h o r s ( B a g n a l l e t a l . , 1959, H i r u k i , 1970; H o r v a t h , 1972; Kowalska and Was, 1976). MacLeod (1962) and USDA (1966) l i s t e d the s p e c i e s B_. v u l g a r i s , D. m e t e l , D_. stramoniurn and H. debneyi as h o s t s o f PVM. I n o c u l a t i o n t e s t s on t h e s e s p e c i e s o f p l a n t s were u n s u c c e s s f u l . Rozendaal and van S l o g t e r e n (1957) n o t e d t h a t the " B i n t j e " i s o l a t e d i d not i n f e c t N_. debneyi which had e a r l i e r been found to r e a c t w i t h v a r i o u s t y p e s o f l o c a l l e s i o n s t o f i v e o t h e r s t r a i n s o f the v i r u s ( B a g n a l l e t a l . , 1956). The reason g i v e n t o e x p l a i n t h i s phenomenon was t h a t the t emperature o f the greenhouses used by the Dutch workers were u n s u i t a b l e f o r PVM i n f e c t i o n . In the p r e s e n t s t u d i e s however, i n o c u l a t i o n t e s t s w i t h t h e s e f o u r s p e c i e s o f p l a n t s were conduct e d d u r i n g w i n t e r , s p r i n g and summer, i n d i c a t i n g t h a t the f a i l u r e t o a c h i e v e i n f e c t i o n i n t h e s e p l a n t s was not due to the i n f l u e n c e o f s e a s o n a l temperature v a r i a t i o n s . Moreover, Kowalska and Was (1976) r e p o r t e d t h a t o n l y one o f the t w e l v e i s o l a t e s t e s t e d produced l o c a l l e s i o n s i n N_. debneyi i r r e s p e c t i v e o f whether the p l a n t s were m a i n t a i n e d a t 16, 22 o r 28°C. 72 Immunity to PVM was not found i n North American p o t a t o c u l t i v a r s . Seven o f the 16 c u l t i v a r s which were n o t i n f e c t e d through mechanical i n o c u l a t i o n showed 60 t o 100% i n f e c t i o n when g r a f t - i n o c u l a t e d w i t h the v i r u s ( T a b l e 4 ) . A s i m i l a r g r a f t - i n o c u l a t i o n e x p e riment was not c a r r i e d o u t on the o t h e r n i n e c u l t i v a r s because v i r u s - f r e e t u b e r s were not a v a i l a b l e . The o b s e r v a t i o n t h a t one o f the ten p o t a t o p l a n t s , cv. S a b l e , was not i n f e c t e d through mechanical i n o c u l a t i o n i n f a l l 1976, but none o f the t h r e e i n s p r i n g 1976, r e f l e c t e d t h e n e c e s s i t y o f i n o c u l a t i n g more p l a n t s from each c u l t i v a r . Had t h i s been f o l l o w e d , the mechanical i n o c u l a t i o n e x p e r i m e n t s c o u l d have r e v e a l e d the s u s c e p t i b i l i t y o f more than t h e 17 found i n the p r e s e n t s t u d i e s . N o n e t h e l e s s , i t was demonstrated t h a t v a r i a t i o n s d i d e x i s t between c u l t i v a r s w i t h r e g a r d s t o the r a t i o o f the number o f i n f e c t e d p l a n t s t o the number o f i n o c u l a t e d p l a n t s . These were e x e m p l i f i e d by t h e f a c t t h a t a l l o r most o f t h e m e c h a n i c a l l y i n o c u l a t e d p o t a t o p l a n t s c v s . Banana, Red La Soda and White La Soda, and t h a t o n l y one o f t h e t e n p o t a t o p l a n t s , cv. S a b l e , were i n f e c t e d . T h i s phenomenon was p r o b a b l y due t o the e x i s t e n c e o f d i f f e r e n t i a l s u s c e p t i b i l i t y among p o t a t o c u l t i v a r s to PVM, as has been s u g g e s t e d by Z a d i n a (1971). In a l l the i n f e c t e d p o t a t o p l a n t s no symptoms d e v e l o p e d . With r e g a r d s t o t h e c u l t i v a r A r r a n V i c t o r y , t h i s was n o t an e x c e p t i o n a l case because B a g n a l l e t a l . (1959) r e p o r t e d t h a t , 73 o f the f i v e i s o l a t e s t e s t e d , o n l y the p a r a c r i n k l e and the l e a f r o l l i n g mosaic i s o l a t e s i n c i t e d symptoms. S i m i l a r l y , the progeny p l a n t s c v s . Banana, Red La Soda and White La Soda d i d not e x h i b i t any symptoms. I t has been r e p o r t e d t h a t PVM-i n f e c t e d p o t a t o p l a n t s which d i d not e x h i b i t any symptoms c o u l d produce progeny p l a n t s showing v a r i o u s degrees o f s e v e r i t y o f symptoms (Chrzanowska, 1976; B a g n a l l e t a l . , 1959). I t appeared t h e r e f o r e t h a t the v i r u s i s o l a t e used i n the p r e s e n t s t u d i e s i s a m i l d one. T r a n s m i s s i o n o f the v i r u s by p l a n t c o n t a c t had been r e p o r t e d by Symygla e t a l . (1973). Attempts t o c o n f i r m t h i s o b s e r v a t i o n were u n s u c c e s s f u l even when tomato p l a n t s which were e a s i l y i n f e c t e d through mechanical i n o c u l a t i o n were used. T h i s was p r o b a b l y due t o s t r a i n d i f f e r e n c e s . The p r e s e n t s t u d i e s showed t h a t the p h y s i c a l p r o p e r t i e s i n  v i t r o o f PVM e x t r a c t e d from p o t a t o l e a v e s were s i m i l a r t o t h o s e e x t r a c t e d from tomato l e a v e s . The d i l u t i o n end p o i n t o f the v i r u s was 1 0 " 4 , and t h i s was s i m i l a r t o the v a l u e r e p o r t e d by H i r u k i (1972) and Tu and H i r u k i (1970). The thermal i n a c t i v a t i o n p o i n t o f between 65 and 70°C f o r PVM as found i n the p r e s e n t s t u d i e s had p r e v i o u s l y been r e p o r t e d by B a g n a l l e t a l . (1956), B a g n a l l and L a r s o n (1957a, 1957b) and H o r i o e t a l . (1969). The o b s e r v a t i o n t h a t the average number o f l o c a l l e s i o n s i n c r e a s e d when the crude sap was heated to 40°C ( F i g . 2C) c o n f i r m e d the f i n d i n g o f B a g n a l l e t a l . (1956), who s u g g e s t e d t h a t h e a t i n g the 74 crude sap to t h i s temperature r e s u l t e d i n the d e i n a c t i v a t i o n o f i n h i b i t o r y s u b s t a n c e s . D i l u t i o n o f the crude sap i n 0.05 M b o r a t e b u f f e r t o 10""' caused an i n c r e a s e i n the number o f l o c a l l e s i o n s i n c i t e d by the v i r u s on Red Kidney bean ( F i g . 2A). T h i s i s the f i r s t r e p o r t on t h i s phenomenon f o r PVM. A s i m i l a r o b s e r v a t i o n has been r e p o r t e d by Saksena and Mink (1969) who noted t h a t the a p p l e c h l o r o t i c l e a f s p o t v i r u s i n cr u d e sap d i l u t e d t o 2 x 10"'' o r 10"^, produced more l o c a l l e s i o n s on C_. q u i n o a o r on Red Kidney bean than the v i r u s i n u n d i l u t e d crude sap. T h i s c o u l d be due t o the i n f l u e n c e o f i n h i b i t o r y s u b s t a n c e s i n the cru d e sap, as s u g g e s t e d by the r e l a t i v e l y s h o r t l o n g e v i t y i n v i t r o o f between 2 and 4 days. Moreover, the v i r u s p a r t i c l e s remained s t a b l e f o r a t l e a s t 2 weeks i n crude sap e x t r a c t e d w i t h 0.5 M b o r a t e b u f f e r c o n t a i n i n g a r e d u c i n g o r c h e l a t i n g agent, but not so i n crude sap e x t r a c t e d w i t h the same b u f f e r t o which a r e d u c i n g o r c h e l a t i n g agent had not been added. A s u i t a b l e b u f f e r f o r t h e e x t r a c t i o n and r e s u s p e n s i o n o f the v i r u s - c o n t a i n i n g m a t e r i a l s i s a p r e r e q u i s i t e f o r an e f f i c i e n t p u r i f i c a t i o n p r o c e d u r e . In t h i s c o n n e c t i o n i t was found t h a t the b o r a t e b u f f e r w i t h a pH range between 7.8 t o 8.5, was s u i t a b l e f o r use i n the p u r i f i c a t i o n o f PVM. B o r a t e b u f f e r , pH 8.2 had p r e v i o u s l y been used by Shepard (1972) f o r the p u r i f i c a t i o n o f t h i s v i r u s . The r e l a t i v e l y lower v i r u s c o n c e n t r a t i o n i n the sap e x t r a c t e d i n d i e t h y l b a r b i t u r a t e b u f f e r , c i t r a t e - p h o s p h a t e b u f f e r o r water c o u l d be due to the poor b u f f e r i n g c a p a c i t i e s o f t h e s e b u f f e r s , as i n d i c a t e d by the change i n t h e i r pH ( T a b l e 5 ) . 75 A l b r e c h t o v a and K l i r (1970) had p r e v i o u s l y d e s c r i b e d a p u r i f i c a t i o n p r o c e d u r e f o r PVM, i n which the d i e t h y l b a r b i t u r a t e b u f f e r was used f o r the e x t r a c t i o n and r e s u s p e n s i o n o f the v i r u s . They d i d not however, c a r r y o u t a comparison t e s t between t h i s b u f f e r and o t h e r b u f f e r s , as has been done i n the p r e s e n t s t u d i e s . The i n c o r p o r a t i o n o f a r e d u c i n g o r a c h e l a t i n g agent i n t o the b u f f e r d u r i n g homogenization o f l e a v e s was not e s s e n t i a l , but appeared to s t a b i l i z e the v i r u s p a r t i c l e s p r e s e n t i n the crude sap which had been i n c u b a t e d f o r l o n g e r t i m e s . P u r i f i c a t i o n o f the v i r u s s h o u l d be c a r r i e d o u t from f r e s h l y h a r v e s t e d l e a v e s i r r e s p e c t i v e o f whether p o t a t o o r tomato p l a n t s were used as p r o p a g a t i o n h o s t s . The r a t i o o f 1:1.5, t i s s u e t o b u f f e r , s h o u l d be used f o r economic re a s o n and f o r ease o f h a n d l i n g o f volume. The aim o f c l a r i f i c a t i o n i s t o m i n i m i z e the amount o f p l a n t m a t e r i a l i n the sap b e f o r e i t i s c o n c e n t r a t e d i n t o a s m a l l e r volume. T h i s i s n e c e s s a r y because o f the d i f f i c u l t y i n r e s u s p e n d i n g the v i r u s from the r e s u l t i n g p e l l e t s . In TMV and some o t h e r v i r u s e s , r e s u s p e n s i o n can be a i d e d by the a d d i t i o n o f T r i t o n X-100 (Nozu and Yamura, 1971). In the p r e s e n t s t u d i e s i t has been shown t h a t the d e t e r g e n t was harmful t o the v i r u s . As a r e s u l t , the v i r u s c o n t e n t o f the p r e p a r a t i o n was reduced c o n s i d e r a b l y ( T a b l e 4 ) . Ammonium s u l f a t e p r e c i p i t a t i o n and carbon t e t r a c h l o r i d e e m u l s i f i c a t i o n were e q u a l l y s u i t a b l e " ' f o r the c l a r i f i c a t i o n o f P V M - c o n t a i n i n g sap. Both methods appeared t o be s u p e r i o r t o the methods o f c l a r i f i c a t i o n w i t h c h l o r o f o r m 76 (Shepard, 1972), bu t a n o l ( H i r u k i e t a l . , 1974) o r c e l i t e ( A l b r e c h t o v a and K l i r , 1970). The a d d i t i o n o f ammonium s u l f a t e s h o u l d be made s l o w l y so t h a t i t would not o c c u r i n a hi g h c o n c e n t r a t i o n a t the a r e a s u r r o u n d i n g the d i s s o l v i n g c r y s t a l s , In t h i s way p r e c i p i t a t i o n and l o s s o f v i r u s was a v o i d e d . Shepard (1972) recommended t h a t the low speed c e n t r i f u g a t i o n i n a p u r i f i c a t i o n p r o c e d u r e f o r PVM s h o u l d be c a r r i e d o u t a t 5,000 g f o r 10 min. C e n t r i f u g a t i o n a t a h i g h e r speed f o r a l o n g e r time presumably r e s u l t e d i n a s e v e r e l o s s o f v i r u s because o f t h e s e v e r e a g g r e g a t i o n o f the v i r u s p a r t i c l e s . However, the r e s u l t s o b t a i n e d from the p r e s e n t s t u d i e s r e v e a l e d t h a t no d e t e c t a b l e l o s s o f v i r u s was o b s e r v e d when c e n t r i f u g a t i o n was c a r r i e d out a t 10,800 g (9,500 rpm) f o r 15 min. At t h i s c o m b i n a t i o n o f speed and tim e , a l a r g e r q u a n t i t y o f p l a n t m a t e r i a l c o u l d be d i s c a r d e d a t each s t e p o f t h e p u r i f i c a t i o n p r o c e d u r e . T h i s was s i g n i f i c a n t because the c o n c e n t r a t i o n o f PVM i n the p r o p a g a t i o n h o s t was low and a l a r g e q u a n t i t y o f l e a v e s had to be used a t the s t a r t o f the p u r i f i c a t i o n p r o c e d u r e . The l o s s o f v i r u s d u r i n g the f i r s t c o n c e n t r a t i o n s t e p was ma i n l y due t o the d i f f i c u l t y i n r e s u s p e n d i n g the p r e c i p i t a t e d v i r u s p a r t i c l e s from t he p e l l e t s . T h i s problem was e n c o u n t e r e d i r r e s p e c t i v e o f whether the p r e c i p i t a t i o n o f the v i r u s was c a r r i e d o u t w i t h ammonium s u l f a t e , PEG 6000 o r by a h i g h speed c e n t r i f u g a t i o n . A l b r e c h t o v a and K l i r (1970) r e p o r t e d t h a t PVM c o u l d s t i l l be d e t e c t e d a f t e r the t h i r d r e s u s p e n s i o n o f PEG p e l l e t s . 77 S i m i l a r r e s u l t s were o b t a i n e d when 0.5 M o r 0.05 M b o r a t e b u f f e r were used f o r r e s u s p e n d i n g t h e s e p e l l e t s a t two-tenths o r f o u r - t e n t h s o f the o r i g i n a l volume. Resu s p e n s i o n o f p e l l e t s i n f o u r - t e n t h s o f the o r i g i n a l volume o f 0.05 M phosphate o r 0.05 M c i t r a t e - p h o s p h a t e b u f f e r y i e l d e d s i m i l a r r e s u l t s as 0.05 M b o r a t e b u f f e r . Hence, i t was d e c i d e d t o use 0.05 M b o r a t e b u f f e r t h r o u g h o u t the subsequent s t e p s o f p u r i f i c a t i o n . With t h i s b u f f e r the v i r u s was s t i l l d e t e c t e d a f t e r the second r e s u s p e n s i o n o f p e l l e t s i n e i t h e r two-tenths o r f o u r - t e n t h s o f the o r i g i n a l volume, but not so a f t e r the t h i r d r e s u s p e n s i o n . Hence, the r e s u s p e n s i o n o f the p e l l e t s o b t a i n e d from the f i r s t s t a g e o f c o n c e n t r a t i o n was c a r r i e d o u t t w i c e , each time w i t h two-tenths o f the o r i g i n a l volume. In t h e o r y , i n c o r p o r a t i o n o f more than one c o n c e n t r a t i o n method i n t o the p u r i f i c a t i o n p r o c e d u r e s h o u l d g i v e a good f i n a l p r o d u c t . S i n c e the ammonium s u l f a t e p r e c i p i t a t i o n , PEG p r e c i p i t a t i o n and the h i g h speed c e n t r i f u g a t i o n methods gave e q u a l l y good r e s u l t s , t h e i r i n c o r p o r a t i o n i n t o the f o u r s t a g e s o f c o n c e n t r a t i o n was advantageous. In the l a s t s t a g e , h i g h speed c e n t r i f u g a t i o n was p r e f e r e d t o PEG p r e c i p i t a t i o n because i n s m a l l volumes, s e p a r a t i o n o f the s u p e r n a t a n t s from the u n d i s s o l v e d p e l l e t s d u r i n g a low speed c e n t r i f u g a t i o n was e a s i e r i n t h e former than i n the l a t t e r . Hence, p r e c i p i t a t i o n w i t h PEG s h o u l d o n l y be c a r r i e d o u t d u r i n g the f i r s t , t h e second o r the t h i r d s t a g e o f c o n c e n t r a t i o n . The minimum amounts o f PEG 6000 and sodium c h l o r i d e t o be used f o r the p r e c i p i t a t i o n o f v i r u s d u r i n g the f i r s t s t a g e o r the second s t a g e o f c o n c e n t r a t i o n 78 were 5% and 4% . r e s p e c t i v e l y . I t was then assumed t h a t t h e s e f c o n c e n t r a t i o n s were a l s o the most s u i t a b l e c o m b i n a t i o n f o r the t h i r d s t a g e o f c o n c e n t r a t i o n . T h i s c o m b i n a t i o n was recommended by Shepard (1972) f o r the p u r i f i c a t i o n o f PVM. F o l l o w i n g the s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n , v i r u s p r e p a r a t i o n s d e r i v e d from the a m m o n i u m - s u l f a t e - c l a r i f i e d sap formed o n l y one .peak when scanned by the TSCo s u c r o s e d e n s i t y g r a d i e n t s c a n n e r ( F i g . 10). S i m i l a r l y ? t h e p r e p a r a t i o n s d e r i v e d from carbon t e t r a c h l o r i d e - c l a r i f i e d sap had one peak, but had " s h o u l d e r s " which were p r o b a b l y caused by p a r t i c l e breakage o r a g g r e g a t i o n . The breakage o f p a r t i c l e s c o u l d have o c c u r r e d d u r i n g e m u l s i f i c a t i o n o f crude sap w i t h the o r g a n i c s o l v e n t i n the Waring b l e n d e r . T h i s method o f t r e a t m e n t c o u l d have been too s e v e r e f o r the v i r u s . The AggQ/^gQ r a t i o o f 1.23 f o r the p u r i f i e d p r e p a r a t i o n s o b t a i n e d by the carbon t e t r a c h l o r i d e method, and the r a t i o o f 1.25 f o r the p u r i f i e d p r e p a r a t i o n s o b t a i n e d by the ammonium s u l f a t e method, i n d i c a t e d t h a t both methods c o u l d be used f o r p u r i f i c a t i o n o f PVM. The e v i d e n c e from the ^2gQ/A2gQ r a t i o s were s u b s t a n t i a t e d by the e l e c t r o n m i c r o s c o p i c appearence o f t h e s e v i r u s p r e p a r a t i o n s ( F i g . 9 ) , which were more homogenous compared to the v i r u s p r e p a r a t i o n s shown i n F i g . 7. Assuming t h a t the e x t i n c t i o n c o e f f i c i e n t o f PVM i s 3.0, then the y i e l d s o f v i r u s i n the p r e p a r a t i o n s p u r i f i e d by the. carbon t e t r a c h l o r i d e and the ammonium s u l f a t e methods were 1.4 mg.and 1.8 mg per Kg"of p o t a t o l e a r e s p e c t i v e l y . When y i e l d s o f v i r u s were e s t i m a t e d from t h e i r s e r o l o g i c a l d i l u t i o n end p o i n t s a t the b e g i n n i n g and a t the 79 end o f the p u r i f i c a t i o n p r o c e d u r e s , the p u r i f i e d v i r u s p r e p a r a t i o n s r e p r e s e n t e d about o n e - t h i r t y f i f t h o f the amount o f v i r u s i n the c r u d e sap. The c o r r e s p o n d i n g v a l u e f o r the method o f Wetter (1960) was o n e - e i g t i e t h . Hence the p r e s e n t methods a r e s u p e r i o r than the method o f Wetter mentioned above. In the ammonium s u l f a t e method, the p a r t i c l e s o f PVM . appeared to be normal t h r o u g h o u t the p u r i f i c a t i o n p r o c e d u r e , and a f t e r t h e s u c r o s e d e n s i t y g r a d i e n t c e n t r i f u g a t i o n t h e p a r t i c l e s were f a i r l y homogenous i n l e n g t h . The normal l e n g t h was 651 nm. The p a r t i c l e w i d t h o f 13.4 nm was not d i f f e r e n t from t h e v a l u e r e p o r t e d by Brandes e t a l . (1959). The TMV p a r t i c l e s i n t r o d u c e d i n t o the PVM p r e p a r a t i o n had wi d t h o f 18.4 nm, t h i s e s t i m a t i o n i s comparable t o the measurement o f between 17 to 18 nm r e p o r t e d by Brenner and Home (1959). In bulk p u r i f i c a t i o n u s i n g the ammonium s u l f a t e method, the y i e l d s o f t h r e e r e p l i c a t e s were 4.1, 3.7 and 3.8 mg v i r u s per Kg l e a v e s . These f i g u r e s r e p r e s e n t e d more than t w i c e the y i e l d s p r e v i o u s l y o b t a i n e d . T h i s a r i s e d from t he f a c t t h a t the f i n a l c o n c e n t r a t e d v i r u s p r e p a r a t i o n s were resuspended i n 2.0 ml o f b u f f e r and then p l a c e d onto s u c r o s e d e n s i t y g r a d i e n t s i n the SW 25 r o t o r , i n s t e a d o f i n 0.3 ml o f b u f f e r f o r c e n t r i f u g a t i o n i n SW 41 r o t o r . Resuspension o f p e l l e t s i n 0.3 ml o f b u f f e r had r e s u l t e d i n a r e d u c t i o n o f the s e r o l o g i c a l d i l u t i o n end p o i n t o f the v i r u s p r e p a r a t i o n s . 80 The b i o c h e m i c a l and the b i o p h y s i c a l p r o p e r t i e s o f PVM have not been r e p o r t e d p r e v i o u s l y . The p r e s e n t s t u d i e s were s u c c e s s f u l i n d e t e r m i n i n g s e v e r a l o f t h e s e p r o p e r t i e s u s i n g p u r i f i e d v i r u s p r e p a r a t i o n s . The ^ 2 6 0 ^ 2 8 0 r a t ^ ° °^ ^ * 2 3 ^ o r PVM d e t e r m i n e d i n t h e p r e s e n t s t u d i e s was w i t h i n t h e range o f 1.14 t o 1.55 r e p o r t e d f o r the C a r l a v i r u s e s (Appendix I I ) . The v a l u e o f 1.24 f o r the k /A . r a t i o was v e r y s i m i l a r t o max mm J the v a l u e o f 1.23 r e p o r t e d f o r the c a r n a t i o n l a t e n t v i r u s (Paul and W e tter, 1964), which i s the t y p e s p e c i e s o f the C a r l a v i r u s group ( H a r r i s o n e t a l . , 1971)- The s e d i m e n t a t i o n c o e f f i c i e n t o f 162 was lower than most v i r u s e s i n t h e same group, f o r which the s e d i m e n t a t i o n c o e f f i c i e n t o f between 160 t o 168 have been r e p o r t e d . One o f the main c o n t r i b u t i o n s o f the p r e s e n t s t u d i e s i s t h e d e t e r m i n a t i o n o f t h e buoyant d e n s i t y o f PVM, which i s 1.304. T h i s i s t h e f i r s t r e p o r t made, n o t o n l y i n PVM, b u t i n t h e C a r l a v i r u s group as w e l l . T h i s v a l u e i s lower than t h a t o f b a r l e y s t r i p e mosaic v i r u s (1.309), but h i g h e r than t h a t o f t h e empty c a p s i d o f t o b a c c o r i n g s p o t v i r u s (1.290) o r p r o t e i n s (1.29) (Sehgal e t a l . , 1970). The TMV s t a n d a r d used i n t h e p r e s e n t s t u d i e s had a buoyant d e n s i t y o f 1.324. S i e g e l and Hudson (1959) r e p o r t e d the v a l u e s o f 1.325 and 1.322 f o r the buoyant d e n s i t i e s o f the 0^ and s t r a i n s o f TMV, r e s p e c t i v e l y . S i m i l a r l y , Sehgal e t a l . , (1970) found t h a t the s t r a i n had a buoyant d e n s i t y o f 1.324. 81 A l t h o u g h t he m o l e c u l a r w e i g h t o f t h e p r o t e i n s u b u n i t o f PVM appeared t o i n c r e a s e w i t h t h e gel c o n c e n t r a t i o n , t he d i f f e r e n c e s were n o t s i g n i f i c a n t ( P > 0 . 0 5 ) . The average v a l u e o f 39,000 d a l t o n s was h i g h e r compared to those o f t h e p r o t e i n s u b u n i t s o f cowpea m i l d m o t t l e v i r u s (Brunt and Kenten, 1973), a l f a l f a l a t e n t v i r u s , pea s t r e a k v i r u s and r e d c l o v e r v e i n mosaic v i r u s ( V e n k a t e s w a r l u , 1976). The m o l e c u l a r w e i g h t s o f the r e s p e c t i v e p r o t e i n s u b u n i t s were 32,000 t o 33,000, 27,000, 27,000 and 33,000 t o 34,000 d a l t o n s . The e s t i m a t e d RNA c o n t e n t o f 6.2% f o r PVM p a r t i c l e s c l o s e l y a g r e e s w i t h t h e ex p e c t e d v a l u e o f 6% f o r the C a r l a v i r u s e s ( H a r r i s o n e t a l . , 1971). The p r e s e n t s t u d i e s , t h e r e f o r e , were s u c c e s s f u l i n o b t a i n i n g a p u r i f i c a t i o n method f o r PVM by which a f a i r l y pure p r e p a r a t i o n o f the v i r u s was o b t a i n e d . The y i e l d o f the v i r u s was r e l a t i v e l y h i g h c o n s i d e r i n g t h a t t he c o n c e n t r a t i o n o f t he v i r u s i n t h e h o s t t i s s u e s i s low. The p o s s i b i l i t y o f o b t a i n i n g a s u f f i c i e n t q u a n t i t y o f the p u r i f i e d v i r u s p r e p a r a t i o n was a p r e r e q u i s i t e f o r the s u c c e s s i n the d e t e r m i n a t i o n o f the v a r i o u s p h y s i c a l , b i o p h y s i c a l and b i o c h e m i c a l p r o p e r t i e s o f the v i r u s . S t u d i e s on t h i s v i r u s a l o n g t h i s l i n e have been eschewed p r o b a b l y because o f the d i f f i c u l t y o f p u r i f i c a t i o n . I t was f u r t h e r c o n c l u d e d t h a t the p r e s e n t s t u d i e s c o n f i r m e d the narrow h o s t range o f the v i r u s , as no new h o s t s were d i s c o v e r e d . In a d d i t i o n , i t was r e v e a l e d t h a t the i s o l a t e used i n the p r e s e n t s t u d i e s i s a m i l d one. Even s o , no North American p o t a t o c u l t i v a r was immune t o i t . 82 LITERATURE CITATION A l b r e c t o v a , L. and K l i r , 0. (1970). P r e c i p i t a t i o n o f S, M, X, and Y p o t a t o v i r u s e s by p o l y e t h y l e n e g l y c o l w i t h d i f f e r e n t m o l e c u l a r w e i g h t . B i o l o g i a P l a n t arum (PRAHA) 12: 31-40. A u g i e r de Montgremier, H. and Devergne, J.C. (1958). Recherches s u r l e s p r o p r i e t e s fondamentales d'un v i r u s de l a pomme de t e r r e cecemment s i g n a l e en France p e r m e t t a n t de 1 ' i d e n t i f i e r au v i r u s M de L a r s o n . [Researches on the fundamental p r o p e r t i o f a p o t a t o v i r u s r e c e n t l y r e p o r t e d i n F r a n c e which p e r m i t i t s i d e n t i f i c a t i o n w i t h L a r s o n ' s v i r u s M.] -- C R . Acad. A g r i F r . 44: 104-110. A b s t r a c t e d i n : Rev. A p p l . Mycol. 1958, 37 B a g n a l l , R.H. (1972). R e s i s t a n c e t o p o t a t o v i r u s e s M, S, X and the s p i d l e t u b e r v i r u s i n t u b e r - b e a r i n g Solanum s p e c i e s . Amer. Pot. J . 49: 342-348. B a g n a l l , R.H. and L a r s o n , R.H. (1957a). P o t a t o v i r u s M ( A b s t r . ) . Amer. P o t a t o J . 34: 57. B a g n a l l , R.H. and L a r s o n , R.H. (1957b). P o t a t o v i r u s M ( A b s t r . ) . P h y t o p a t h o l o g y 47: 3. B a g n a l l , R.H., L a r s o n , R.H. and Walker, J.C. (1956). P o t a t o v i r u s e s M, S, and X i n r e l a t i o n to i n t e r v e i n a l mosaic o f the I r i s h C o b b l e r v a r i e t y . Res. B u l l . A g r i c . Exp. S t n . Univ. Wis. no. 198. B a g n a l l , R.H., Wetter, C. and L a r s o n R.H. (1959). D i f f e r e n t i a l 83 h o s t and s e r o l o g i c a l r e l a t i o n s h i p s o f p o t a t o v i r u s M, p o t a t o v i r u s S and c a r n a t i o n l a t e n t v i r u s . P h y t o p a t h o l o g y 49: 435-442. B a r t e l s , R. and V o l k , J . (1966). Versuche z u r Ub e r t r a g u n g von K a r t o f f e l - M - V i r u s und verwandten I s o l a t e n und d e r s e r o l o g i s c h e Nachweis des M-Virus i n Tomaten. Eur. P o t a t o J . 9: 197-207. Bawden, F.C. (1939). P l a n t V i r u s e s and V i r u s D i s e a s e s ( 1 s t . e d t ) . C h r o n i c a B o t a n i c a , L e i d e n , p. 258. Bawden, F.C. and K a s s a n i s , B. (1965). The p o t a t o v a r i e t y King Edward VII and p a r a c r i n k l e v i r u s . Rothamsted Exp. Stn Rep o r t , 1964. pp 282-290. Bawden, F . C , K a s s a n i s , B. and Nixon, H.L. (1950). The me c h a n i c a l t r a n s m i s s i o n and some p r o p e r t i e s o f p o t a t o p a r a c r i n k l e v i r u s . J . Gen. M i c r o b i o l . 4: 210-219. Beemster, A.B.R. (1964). Informe a l g a b i e r n o de C h i l e s o r b e enfermedades de c i e r t o s c u l t i v o s en C h i l e . [ A d v i c e t o the government o f C h i l e on the v i r u s d i s e a s e s o f c e r t a i n c r o p s i n C h i l e . ] -- Rome, F.A.O. (Informae no. 1812). A b s t r a c t e d i n ; Rev. A p p l . M y c o l . , 1966, 45: 249. Beemster, A.B.R. and Ro z e n d a a l , A. (1972) P o t a t o v i r u s e s : P r o p e r t i e s and symptoms. Irr. V i r u s e s o f Po t a t o e s and Se e d - p o t a t o P r o d u c t i o n (J.A. deBokx e d . ) . PUDOC, Wageningen. pp 127-129. Bode, 0. (1958). Die V i r o s e n der K a r t o f f e l und des Tabak. JTK P f F a n z l i c h e V i r p l o g i e . Band 11. Die V i r o s e n des E u r o p a i c h e r Raumes (M. K l i n k o w s k i e d . ) . Akademie-V e r l a g , B e r l i n , pp 1-30. 84 Bode, 0. and Heideman, H.L. (1969). W e i t e r e Untersuchungen M- und S - V i r u s und the Ubertragung d u r c h V e k t o r e n . P r o c , 4 t h Conf. Eur. Ass. P o t a t o Res., 1969. pp 224-226. Bode, 0. and Weideman, H.L. (1971), Untersuchungen z u r B l a t t a s u b e r t r a g b a r k e i t von K a r t o f f e l M- und S - V i r u s , P o t a t o Res. 14: 119-129. Brandes, J . , W e t t e r , C , B a g n a l l , R.H. and L a r s o n , R.H. (1959). S i z e and shape o f the p a r t i c l e s o f p o t a t o v i r u s S, p o t a t o v i r u s M and c a r n a t i o n l a t e n t v i r u s . P h y t o p a t h o l o g y 49: 443-446. Br e n n e r , S.S. and Home, R.W. (1959). A n e g a t i v e s t a i n i n g method f o r h i g h r e s o l u t i o n e l e c t r o n m i c r o s c o p y o f v i r u s e s . B i o c h i m . B i o p h y s . A c t a 34: 103-110, B r u n t , A.A. and Kenten, R.H. (1973). Cowpea m i l d m o t t l e , a newly r e c o g n i s e d v i r u s i n f e c t i n g cowpeas. ( V i g n a  u n g u i c u l a t a ) i n Ghana. Ann. A p p l . B i o l . 74: 67-74. B u r t o n , W.G., (1966). The p o t a t o : A s u r v e y o f i t s h i s t o r y and f a c t o r s o f i n f l u e n c i n g i t s y i e l d , n u t r i t i v e v a l u e , q u a l i t y and s t o r a g e . Veenman, Wageningen. pp 118-119. Chambrach, A., R e i s f e i l d , R.A., Wychoff, M. and Z a c c a r i , J . (1967). A p r o c e d u r e f o r r a p i d and s e n s i t i v e s t a i n i n g o f p r o t e i n f r a c t i o n a t e d by p o l y a c r y l a m i d e gel e l e c t r o p h o r e s i s . A n a l . Biochem. 20: 150-154. Chervenka, C H . (1973). A Manual o f Methods f o r t he A n a l y t i c a l U l t r a c e n t r i f u g e . Beckman Instrument, P a l o A l t o . 85 Chrzanowska,M. (1973), Wplyw temperatury na wykrywalnosc wirusow M and S w r o s l i n a c h z i e m n i a k a , [ E f f e c t of temperature on the d e t e c t i o n o f v i r u s e s M and S on p o t a t o p l a n t s , ] -- Z e s z y t y Problemove Postepow Nauk R o l n i c z y c h 142: 81-91. A b s t r a c t e d i n : Rev. P l a n t P a t h . , 1975, 54: 357. Chrzanowska, M. (1976). V a r i a t i o n s on symptoms caused by p o t a t o v i r u s M on p o t a t o v a r i e t y Uran. P o t a t o Res. 19: 141-146. Cors a n , G.P., Howard, H.W., Markham, R, and Smith, K.M. (1944). P a r c r i n k l e v i r u s and i n h e r i t e n c e . Nature 154: 334. D a r l i n g t o n , C D . (1944). H e r e d i t y , development and i n f e c t i o n . Nature 154: 164-169. Duda, V. (1966). V i r u s n y e b o l e z n i K a r t o f e l y a . [ V i r u s d i s e a s e s o f P o t a t o ] . - - Zashch. R a s t . , Mosk., 11: 23. A b s t r a c t e d i n Rev. A p p l . M y c o l . , 1967, 46: 315. Dunker, A.K. and R u e c k e r t , R. (1969). O b s e r v a t i o n s on m o l e c u l a r weight d e t e r m i n a t i o n on p o l y a c r y l a m i d e g e l . J . B i o l . Chem, 244: 5074-5080. Dziewonska, M.A. and Ostrowska, K. (1975). N e c r o t i c r e a c t i o n t o p o t a t o v i r u s M i n S o l anurn s t o l o n i f e r u m and Solanum m e g i s t a c r o l o b u m ( A b s t r . ) . P o t a t o Res. 18: 144. G i b b s , A. and H a r r i s o n , B. (1976). P l a n t V i r o l o g y : The p r i n c i p l e s . Edward A r n o l d L t d . , London, p 121. H a r r i s o n , B.D., F i n c h , J.T., G i b b s , A . J , , H o l l i n g s , M, Shepard, R . J , ? V a l e n t a , V. and Wetter, C (1971). S i x t e e n groups o f p l a n t 86 v i r u s e s , V i r o l o g y 45; 356-373, H i r u k i , C. (1970). Red Kidney bean, a s u c c e s s f u l b i o a s s a y h o s t f o r q u a n t i t a t i v e and q u a l i t a t i v e work w i t h p o t a t o v i r u s M. P h y t o p a t h o l o g y 60: 739-740, H i r u k i , C. (1972). The use o f the p r i m a r y l e a v e s o f Red Kidney bean i n i n d e x i n g p o t a t o v i r u s M. Proc. 5th Conf. Eur, Ass. P o t a t o Res., 1972, pp 180-181. H i r u k i , C. (1973). D e t e c t i o n o f p o t a t o v i r u s M i n t u b e r s s p r o u t s , and l e a v e s o f p o t a t o by French bean t e s t . P o t a t o Res. 16: 202-212. H i r u k i , C., Poutney, E. and Saksena, K.N. (1974). F a c t o r s a f f e c t i n g b i o a s s a y o f p o t a t o v i r u s M i n Red Kidney bean, P h y t o p a t h o l o g y 64: 807-811. H i t c h b o r n , J.H. and H i l l , G.J. (1965). The use o f n e g a t i v e s t a i n i n g i n e l e c t r o m i c r o s c o p i c e x a m i n a t i o n o f p l a n t v i r u s e s i n crude e x t r a c t s . V i r o l o g y 27: 528^540. H o d r e j a r v , U., O l s p e r t , K, and T a r a s s o v a , K, (1970). A method f o r the p u r i f i c a t i o n o f p o t a t o v i r u s . E e s t i NSV Tead. Akad. Tojm, B i o l . 19: 231 - 234. A b s t r a c t e d i n : Rev. P l a n t P a t h . , 1971, 50: 7. H o r i o , H., Yano, I. and Esumi, K. (1969). P o t a t o v i r u s M found i n Japan (Eng. A b s t r . ) . Ann. Phytopath. Soc. Japan 35: 47-54. Horv a t h , J . (1971). L y c o p e r s i c o n - A r t e n a l s nue W i r t s p f l a n z e n f u r das K a r t o f f e l - M - V i r u s ( p o t a t o v i r u s M). P o t a t o Res. 14: 297-300. 87 H o r v a t h , J . (1972). R e a c t i o n s o f v a r i o u s L y c o p e r s i c o n s p e c i e s t o i n o c u l a t i o n w i t h p o t a t o v i r u s M and p o t a t o v i r u s S. Proc. 5th Conf. Eur. Ass. P o t a t o Res. 1972. pp 181-182. H o r v a t h , J . (1973). Seed t r a n s m i s s i o n experiments o f p o t a t o v i r u s M and p o t a t o v i r u s S i n L y c o p e r s i c o n s p e c i e s . A c t a Agronomica 22: 390-392„ Horvath, J . and de Bokx, J.A. (1972). Uber d i e A n f a l l i g k e i t von Chenopodiurn A r t e n und \ S r e i t a t e n das M-Virus ( P o t a t o V i r u s M). Phytopath. Z. 73: 171-177. Hunnius, W. (1972a). Zum V e r h a l t e n des M-Virus i n der K a r t o f f e l . Z. P f l K r a n k h . P f l P a t h . P f l S c h u t z . 79: 385-399. Hunnius, W. (1972b). Zum V e r h a l t e n des M-Virus i n der K a r t o f f e l p i a n z e . P roc. 5th Conf. Eur. A s s . P o t a t o Res. 1972. p 179. Kahn, R.P., S c o t t , H.A., J o z e c v i c h , J . and V i n c e n t , M.H. (1967). D e t e c t i o n o f p o t a t o v i r u s e s X, M and S i n t he dormant p o t a t o t u b e r s by the b e n t o n i t e f l o c u l a t i o n t e s t . P h y t o p a t h o l o g y 57: 61-65. K a s s a n i s , B. (1956). S e r o l o g i c a l r e l a t i o n s h i p between P o t a t o p a r a c r i n k l e v i r u s , p o t a t o v i r u s S and c a r n a t i o n l a t e n t v i r u s . J . Gen. M i c r o b i o l . 15: 620-628. K a s s a n i s , B. (1957). The use o f t i s s u e c u l t u r e s t o produce v i r u s - f r e e c l o n e s from i n f e c t e d p o t a t o v a r i e t i e s . Ann. A p p l . B i o l . 45: 422-427. K a s s a n i s , B. (1960). P o t a t o v i r u s M and p a r a c r i n k l e Nature 188: 688. 88 K a s s a n i s , B. (1961). P o t a t o p a r a c r i n k l e v i r u s . Eur. P o t a t o J . 4: 13-24. K a s s a n i s , B. and Schwabe, W.W. (1961), The e f f e c t o f v i r u s on the growth o f King Edward p o t a t o a t d i f f e r e n t t e m p e r a t u r e s and d a y l e n g t h s , Ann. A p p l . B i o l . 49: 616-620. K o h l e r , E. (1942). Untersuchungen uber das K V i r u s d er K a r t o f f e l . Agnew. Bot. 24: 118-130. K o h l e r , E. (1953). E i n unbekantes K a r t o f f e l v i r u s . N a c h r i c h b l . D t s c h . P f l S c h u t z . 7: 22-23. Kowalska, A. and Was, M. (1976). R e t e n t i o n o f p o t a t o v i r u s M and p o t a t o v i r u s S on t e s t p l a n t s . P o t a t o Res. 19: 131-139. Kostiw, M. (1975a). Some r e s u l t s on the t r a n s m i s s i o n o f p o t a t o v i r u s Y and p o t a t o v i r u s M by ap h i d s ( A b s t r . ) . P o t a t o Res. 19: 149. Kostiw, M. (1975b). I n v e s t i g a t i o n on r e t e n t i o n o f p o t a t o v i r u s e s M and Y i n two s p e c i e s o f ap h i d s (Myzus p e r s i c a e S u l z . and A p h i s n a s t u r t i i K a l t . ) . P o t a t o Res. 18: 637-640. Loughnane, J.B, (1957). A n e c r o s e o f p o t a t o t u b e r s caused by the p a r a c r i n k l e v i r u s . Proc. 3rd Conf. P o t a t o V i r u s D i s . , L isse-Wageningen, 1957. Lee, S.T. (1971). Occurance o f p o t a t o v i r u s M and i t s s t r a i n s i n Taiwan. J . A g r i c . Ass. China 76: 79-86. A b s t r a c t e d i n : Rev. P l a n t P a t h . , 1973, 52: 401. Maat, D.Z. (1972). V i r u s P u r i f i c a t i o n . In: V i r u s e s o f P o t a t o e s and S e e d - P o t a t o P r o d u c t i o n (J.A. de Bokx e d . ) . PUDOC Wageningen. p 66. 89 MacLeod, D.J. (1962). M o s a i c and S t r e a k v i r u s e s i n the p o t a t o . Can. Dept. A g r i c . Pub!. no. 1150. pp 20-26. MacLachlan, D.S., L a r s o n , R.H. and Walker, J.C. (1954). I n t e r v e i n a l mosaic o f p o t a t o , Amer. P o t a t o J . 31: 101-105. McKay, M.B. and D y k s t r a , T.P. (1932). P o t a t o d i s e a s e s , Oregon i n v e s t i g a t i o n 1924-1929. Oregon A g r i c . Exp. s t n . B u l l . no. 294. pp 1-40. Markham, R. (1960). A g r a p h i c a l method f o r r a p i d d e t e r m i n a t i o n o f s e d i m e n t a t i o n c o e f f i c i e n t . Biochem. J . 77; 516^519. Martyn , E.B. (1968). P l a n t V i r u s Names: An a n n o t a t e d l i s t o f names and synonyms o f p l a n t v i r u s e s and d i s e a s e s . P h y t o p a t h o l o g i c a l P a p e r s , no.9, Comm. My c o l . I n s t . , England, pp 79-80. Noordam, D. (1973). I d e n t i f i c a t i o n o f P l a n t V i r u s e s : Methods and E x p e r i m e n t a t i o n . PUD0C, Wageningen. pp 88-101. Nozu, Y. and Yamura, R. (1971). Use o f T r i t o n X-100 f o r i s o l a t i o n o f t o b a c c o mosaic v i r u s from some p l a n t s o t h e r t h a n t o b a c c o . V i r o l o g y 43: 514-515. Nurmiste, B. (1966). T a i m e v i i r u s t e uumaid k a s i t u s i j a n e i l e r a j a n e v a i d t o r j e p r o b l e e m e . [Some new c o n c e p t s r e g a r d i n g v i r u s e s and c o n t r o l problems a r i s i n g from them.] -- E e s t i NSV Tead. Akad. Akad. Toim., b i o ! . S e e r . 15: 3-15. A b s t r a c t e d in;Rev. A p p l . M y c o l . , 1966, 45: 509. P a j a n , M. (1967). 0 v i r u s i m a Krumpira u J u g o s l a v i j i . [On v i r u s e s o f p o t a t o i n Y o u g o s l a v i a . ] -- Z a s t . B i l j a 18: 25-35. A b s t r a c t e d i n Rev. A p p l . M y c o l . , 1968, 90 47: 300-301. P a u l , H.L. and W e t t e r , C. (1964). Untersuchungen am C a r n a t i o n l a t e n t V i r u s . I. P r a p a r a t i o n , p h y s i k a l i s c h e und chemische E i g e n s c h a f f e n . Phytopath. Z. 49: 401-406. Peichowiak, K, and G l a d y s i a k , K. (1972). E i n f l u s s e i n i g e r F a k t o r e n a u f der V e r b r e i t u n g des V i r u s M. P r o c . 5th Conf, Eur. Ass. P o t a t o Res., 1972. p 179, Ross, H. (1968). L y c o p e r s i c o n c h i l e n c e Dun., e i n e T e s t p f l a n z e f u r d i e b e i d e n K a r t o f f e l v i r e n M und S. Eur. P o t a t o J . 11: 281-286. Roz e n d a a l , A. and van S l o g t e r e n , D.H.M. (1957). A p o t a t o v i r u s i d e n t i f i e d w i t h p o t a t o v i r u s M and i t s r e l a t i o n s h i p w i t h p o t a t o v i r u s S. P r o c . 3rd C o n f . P o t a t o V i r u s D i s e a s e s . Lisse-Wageningen. pp 20-37. Saksena, K.N. and Mink, G.I. (1969). P u r i f i c a t i o n and p r o p e r t i e s o f a p p l e c h l o r o t i c l e a f s p o t v i r u s . P h y t o p a t h o l o g y 59: 84-88. Salaman, R.N. and Le P e l l e y (1930). P a r a c r i n k l e : a p o t a t o d i s e a s e o f the v i r u s group. Proc. Roy. Soc. London, S e r i e s B, 106: 140-175. S c h u l t z , E.S. and Folsom, D, (1923). T r a n s m i s s i o n , v a r i a t i o n , and c o n t r o l o f c e r t a i n d e g e n e r a t i o n d i s e a s e s o f I r i s h p o t a t o e s . J . Agr. Res. 25: 43-117. S e h g a l , O.P., Jong-ho J e a n , B h a l l a , R.B,, Meei Meei Soong, Krause, G.F. (1970). C o r e l l a t i o n between buoyant d e n s i t y and r i b o n u c l e i c a c i d c o n t e n t i n v i r u s e s , P h y t o p a t h o l o g y 60: 1778-1783. 91 S h a p i r o , A.L., V i n u l a , E. and M a i z e l , J.V. (1967). M o l e c u l a r w e i g h t e s t i m a t i o n o f p o l y p e t i d e c h a i n s by e l e c t r o p h o r e s i s i n S D S - p o l y a c r y l a m i d e g e l s . Biochem. B i o p h s . Res. Comm. 28: 815-820. Shepard, J . F . , J u t i l a , J.W., C a t l i n , J . E . , Newman, F.S., and Hawkins, W.H., (1971). I m m u n i d i f f u s i o n a s s a y f o r p o t a t o v i r u s M i n f e c t i o n . P h y t o p a t h o l o g y 61: 873-874. Shepard, J . F . (1972). G e l - d i f f u s i o n methods f o r the s e r o l o g i c a l d e t e c t i o n o f p o t a t o v i r u s e s X, S and M. Montana A g r i c . Exp. S t n . P u b l . Mont. S t a t e U n i v . , Bozeman. S i e g e ! , A. and Hudson, W. (1959). E q u a l i b r i u m c e n t r i f u g a t i o n o f two s t r a i n s o f tobacco mosaic v i r u s i n d e n s i t y g r a d i e n t s . B i o c h i m . B i o p h s . A c t a . 34: 3514-3515. Smith, K.M., (1933). The p r e s e n t s t a t u s o f p l a n t v i r u s r e s e a r c h . B i o l . Review 8: 137-179. Smith, K.M. (1937). A Textbook o f P l a n t V i r u s D i s e a s e s . J . and A. C h u r c h i l l LTD., London, pp 365-367. Smith, K.M. (1946). V i r u s D i s e a s e s o f Farm and Garden Crops. L i t t l e b u r y and Co. LTD, p 20. Symygla, V.,Ambramova, R. and Shorova, R. (1973). U n t e r -suchungen uber das M-Virus der K a r t o f f e l . [ I n v e s t i g a t i o n on p o t a t o v i r u s M.] -- P o t a t o Res. I n s t . Korenevo, U.S.S.R. A b s t r a c t e d i n : Rev. P l a n t Path., 1973. 52: 292. 92 Tu, J.C. and H i r u k i , C. (1970) U l t r a s t r u c t u r e o f p o t a t o i n f e c t e d w i t h p o t a t o v i r u s M. V i r o l o g y 42: 238-242. T u r s k a , E. (1976). S t u d i e s o f S p r e a d i n g o f p o t a t o v i r u s e s X, S, and M up our use o f d i f f e r e n t c u l t i v a t i o n methods under f i e l d c o n d i t i o n s ( P r e l i m i n a r y Communications) (Eng. A b s t r . ) , Z e s z y t y Problemowe Postepow Naule R o l n i c z y c h Warszav/a z. 174. p 79. USDA (1966) P l a n t P e s t o f Importance to North American A g r i c u l t u r e : Index o f P l a n t V i r u s D i s e a s e s . Handbook no. 307. pp 282-283. Van der P l a n k , J . E . (1948). O r i g i n o f some p l a n t v i r u s e s . Nature 162: 291-292. V e n k a t e s w a r l u , V. (1976). Apparent m o l e c u l a r w e i g h t s o f RNA and p r o t e i n s u b u n i t s o f some C a r l a v i r u s e s . A b s t r . o f 68th Annual M e e t i n g Amer. P h y t o p a t h o l o g i c a l Soc. pp 124-125. V u l c i c , V.M. and Hunnius, W.W. (1967). Die R e a k t i o n e n v e r s c h i e d e n e n P f l a n z e n a r t e n a u f B l a t t i n f e c t i o n e n m i t S- und M- V i r u s der K a r t o f f e l . P h y t o p a t h . Z. 59: 225-248. Weber, K. and Osborne, M. (1969). The r e l i a b i l i t y o f m o l e c u l a r w e i g h t d e t e r m i n a t i o n s by dodec y l s u l f a t e - p o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s J . B i o l . Chem. 224: 4406. Wetter, C. (1960). P a r t i e l l e R e i n i g u n g e i n i g e r g e s t r e c k t e r P f l a n z e n v i r e n und i h r e Verwandung a l s A n t i g e n e b e i d e r Immunisierung m i t t e l s Freundschem Adjuvans. A r c h . M i k r o . 37: 278-292. Wetter, C. (1972). P o t a t o v i r u s M. I n : CMI/AAB D e s c r i p t i o n s o f p l a n t v i r u s e s no. 87. Wetter, C. and Brandes, J . (1956). Untersuchungen uber das K a r t o f f e l - S - V i r u s . P h y t o p a t h . Z. 26: 81-92. Wetter, C. and V o l k , J . (1960). Versuche z u r Ubertragung d e r K a r t o f f e l v i r e n M und S durch Myzus p e r s i c a e S u l z . Eur. P o t a t o J . 3: 158-163. Wetter, C, and Brandes, J . (1955). E l e c t r o n m i c r o s c o p i s c h e Untersuchungen uber G e s t a l t und Grosse des K a r t o f f e l - S -V i r u s . N a t u r w i s s e n s c h a f t e n 42: 100-101. Z a d i n a , J . (1971). P o t a t o b r e e d i n g f o r r e s i s t a n c e t o v i r u s M. Proc. 7th Conf. C z e c h o s l o v a k P l a n t V i r o l o g i s t s , High T a t r a s , 1971. pp 405-413. 94 APPENDIX I The r e l a t i o n s h i p between t u r b i d i t y al 540 nm and t h e r e l a t i v e c o n c e n t r a t i o n o f crude sap from l e a v e s o f v i r u s - f r e e p o t a t o p l a n t s Crude sap from l e a v e s o f v i r u s f r e e p o t a t o p l a n t s was e x t r a c t e d w i t h 0,5 M b o r a t e b u f f e r a t the r a t i o o f 1:3, t i s s u e to b u f f e r . A f t e r a low speed c e n t r i f u g a t i o n , t he s u p e r n a t a n t was d i l u t e d w i t h t he same b u f f e r to 0,8, 0.6, 0,4 and 0.2 o f the o r i g i n a l c o n c e n t r a t i o n . The t u r b i d i t y o f the u n d i l u t e d and d i l u t e d sap was measured a t 540 nm i n a Beckman model DU-s p e c t r o p h o t o m e t e r . A t the same t i m e , 1 ml o f the u n d i l u t e d sap was added t o an equal volume o f p a r t i a l l y p u r i f i e d PVM p r e p a r a t i o n which had been d i l u t e d to 0.8, 0.6, 0.4 ;and 0.2 o f the o r i g i n a l c o n c e n t r a t i o n . The t u r b i d i t y a t 540 nm was determined as above. The r e g r e s s i o n s o f the d i l u t i o n o f sap o r d i l u t i o n o f v i r u s (x) on the t u r b i d i t y v a l u e a t 540 nm (y) was c a l c u l a t e d w i t h t he a i d o f a computer. The r e g r e s s i o n c o e f f i c i e n t s o f 0.999 and 0.00996 f o r the d u l u t i o n o f sap and d i l u t i o n o f v i r u s , r e s p e c t i v e l y , demonstrated t h a t t he 540 nm v a l u e was p r o p o r t i o n a l t o the r e l a t i v e c o n c e n t r a t i o n o f p l a n t d e b r i s , and not t o the r e l a t i v e c o n c e n t r a t i o n o f v i r u s c o n t a i n e d i n the crude sap. 95 Appendix I (c o n t ' d ) R e g r e s s i o n o f the d i l u t i o n o f crude sap from p o t a t o l e a v e s (X) on the t u r b i d i t y v a l u e a t 540 ( Y ) . NE.,ii KU.M ? t H.'^b. i Ore ni ) I OF V r t L d t l o 1 o 1 ) A • d t d) A • d i - 3 A • 2 1 • d o .".> A J A . A I . .w i A 1 . 6 ) A . ^  i • 'oo / J A t • o 9 o ) A . 6 Y • D J 5 •j > A . 6 I • b 9 l a J A • d I 1 . 1 * 1 i J A •b r 1 • 1 0 1 d ; A • o r 1 • 1 ^ 1 3 ) A i • i I • ^ t A 1 . i 1 • Ad 1 D) A 1 . t 1 . 4!j i r ' <) . 1 6oyo * J<J id .Jd£t ->J 1 o . s A tj • 1 d O . j it) '4 vj E + <J 1 b S T - » O I - » L b b -5 • 2 3 b 3A~>dA ) 1 u > ) i « = o i -Jr»t: ic/ • 1 1 6 6 6 6 6 £ i - . l 1 rt /) • 2 2') A•,.'>/<')',) ")'*•> /11 t\d O • y V o 3 3 f 5 / d . j f . t - *yu rc ' J • 9 9 9 1 fvrs + .)•-! ( v t - i n t i o i OM d . 2 3 / v b d o J J ^ t * j i : \ f e . i I l ) U a L i i - o i l - ,*\ ti o r, £ i> :J L O '-i b b U . 3 9 6 2 3 A7 3 0 7 £-v5 2 t s i VM.A I>-i:»ut.-r.£bI O ' j-tL. b b / . \ i - 2 1 ).•)/!/J / y / / i ^ b 2 t - ; ! "< F F Jiv ifc.il u=y# C r d h . s o s / J F H*tb < 1 * 1 3 ) D F (. -V* b A t-f \ 1 J .s J ) 'i 96 Appendix I ( c o n t ' d ) R e g r e s s i o n o f the d i l u t i o n o f p a r t i a l l y p u r i f i e d v i r u s p r e p a r a t i o n ( a f t e r f u r t h e r d i l u t i o n w i t h an equal volume o f 1:3 p o t a t o sap) (X) on the t u r b i d i t y v a l u e a t 540 nm ( Y ) . i'4 £ A :>i ? JF Jfc-b 1 6 1 ) A .d r . 6 7 d; A . 2 i . 6 52 3) A • a I • 6c> 4) A • M . . 6 6 6 v>) A • 4 If • bid 6) A . A If . 6 / / i ) A • 6 r • 6 J y o ) A . 6 i . 6J9 J ) A . 6 t • 6 6 J t *J ; A • b r . 6 7 3 i i i A • o r . 67 id) A • b i • 6d i 3) A 1 • T • 65 3 i 4) A i . i . 6 6 4 i J) A 1 • 1 • 6 i '3 i O P S M ) r r I = -»+• L.A O r * o • 4vj.r) J y 1 ;>£ -» - j 3 o b A ,j . i d-ov40J:-j0dt*-0 I b b t - i J i M L i>.*S i : ' • 1 3 4 ; J y 3->9 D 3 cJ - a-wif £ U • 33J33biy v>^£-»->3 H <j . 6 6v / < 3-3 <2 I K> .i -) V • VKfdtj {i.j.ici"/ :JK-<J a t< v) • y 9 6 .3 6 1 8 3 f. - •••) 2 rs t w t o o i J.'» O - i «/ • 1 J3 J / I '"..-'a 6 iv £.:> i 'Ojnl. s a ' s i i j - : \ K U R t . . j o l J M -> J > . i '< ),Jhl :•> .*•>•}•; £ J i I t--'i .•-» I to v r \ a I M «b c-'v I*". b i \J j : - , • ) / . ' . K - i H i' F J . \ I £b 1 o • -J I 3 * F M S C I * 1 .3 J Of Appendix II V i r u s e s C a r n a t i o n l a t e n t ( 6 1 ) * Chrysanthmum B (110) Cowpea m i l d m o t t l e (140) L i l y symptomless (96) Pea s t r e a k (112) Red C l o v e r v e i n mosaic (22) P o p l a r mosaic (75) P o t a t o S (60) P o t a t o M (87) P o t a t o M ( P r e s e n t s t u d y ) Some p r o p e r t i e s o f members o f the C a r l a v i r u s e s P r o p e r t i e s A** B C D E F 6 650X12 60-65 1 0 ~ 3 2-3 167s 1.37 1,23 685X12 75-80 1 0 ~ 2 2-6 168s 1.55 1.20 650X13 65-70 1 0 " 3 165s 1.14 1.21 640X17 167s 1.20-1.43 619-630 78-80 l O " 6 2.7 160s 1,33-1.35 X? 645-12 60-65 l O ' 3 2 160s 1.14 74 2 165s 675X? 10 650X12 55-60 l O " 2 3-4 650X12 65-71 1 0 ' 2 651X13 65-70 ! 0 - 4 3-4 162s 1.23 1.24 ( )*: P u b l i c a t i o n number o f CMI/AAB D e s c r i p t i o n o f P l a n t V i r u s e s . A - s i z e ( l e n g t h X w i d t h i n nm); B-TIP ( ° C ) ; C- DEP; D- LIV ( d a y s ) ; E- S G- A /A . ; H- Absorbance a t 2 6 0 A ° ; I - buoyant d e n s i t y , max' mm' H 2.1 1.304 20 ,H F" A 2 6 0 / A 2 8 0 y8 Appendix I I I Co m p o s i t i o n s o f b u f f e r s 0.5 M B o r a t e b u f f e r , pH 7.8 B o r i c a c i d Sodium b o r a t e D i s t i l l e d water 0.2 M Phosphate b u f f e r , pH 7.8 Pota s s i u m phosphate (monobasic) 2.7 gm Sodium phosphate ( d i b a s i c ) 65.4 gm D i s t i l l e d water 1000 ml 0.1 M Sodium d i e t h y l b a r b i t u r a t e b u f f e r , pH 7.8 Sodium d i e t h y l b a r b i t u r a t e 2U.6 gm C y s t e i n e h y d r o c h l o r i d e 12.1 gm Sodium e t h y l e n e d i a m i n e t e t r a a c e t a t e (EDTA) 2.6 gm D i s t i l l e d water 1000 ml 24.6 gm 20.6 gm 1000 ml 99 Appendix I I I ( c o n t ' d ) 0.2 M C i t r a t e - p h o s p h a t e b u f f e r , pH 7.8 Sodium c i t r a t e 9.8 gm Potassium phosphate ( d i b a s i c ) 27.2 gm D i s t i l l e d water 1000 ml 0.2 M T r i s - a c e t a t e b u f f e r , pH 7.8 T r i s 24.3 gm Sodium a c e t a t e 27.2 gm EDTA 1.85 gm D i s t i l l e d water 1000 ml 

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094014/manifest

Comment

Related Items