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Characterization of potato leafroll virus Rowhani, Adib 1980

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CHARACTERIZATION OF POTATO LEAFROLL VIRUS hy ADIB^ROWHANI B.Sc. .c'Pahlavi U n i v e r s i t y , Shiraz, Iran, 1970 M.Sc, M c G i l l U n i v e r s i t y , Montreal, Canada, 1977 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF THE FACULTY OF GRADUATE STUDIES (Department of Plant Science) We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA DOCTOR OF PHILOSOPHY i n January 1980 A d i t Rowhani In p r e s e n t i n g t h i s t h e s i s in p a r t i a l f u l f i 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 at the U n i v e r s i t y o f B r i t i s h C o l u m b i a , I a g r e e that 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 p u r p o s e s may be g r a n t e d by the Head o f my Department o r by h i s r e p r e s e n t a t i v e s . It 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 my w r i t t e n p e r m i s s i o n . Department o f p l - A A / T ^ 1 The U n i v e r s i t y o f B r i t i s h Co lumbia 2075 Wesbrook Place Vancouver, Canada V6T 1WS Date 3J ^/ i i ABSTRACT Potato l e a f r o l l v i r u s (PLRV) was p u r i f i e d from i n f e c t e d potato (Splanum tuberosum L.) by using a mixture of chloroform and -"butanol f o r c l a r i f i c a t i o n and polyethylene g l y c o l f o r concentration. The vi r u s sedimented as a single band i n a sucrose density gradient. Y i e l d of p u r i f i e d v i r u s varied from 0.4-0.6 mg/kg fr e s h weight of potato f o l i a g e . V i r u s y i e l d was higher from tis s u e immediately processed a f t e r harvest than from t i s s u e stored at 4 C or -20 C f o r a day or longer. The A 260/A 2 8 0 varied from 1 . 7 4 - 1 . 8 2 . Antiserum prepared against p u r i f i e d v i r u s had a maximum t i t r e of 1024 i n agar g e l double d i f f u s i o n t e s t s . PLRV i n unconcentrated potato sap could not be detected by agar g e l t e s t s , but could be detected by enzyme-linked immunosorbent assay. A pure l i n e of the mild s t r a i n of PLRV was i s o l a t e d , and an antiserum with a maximum t i t r e of 2560 by agar g e l t e s t s was produced. PLRV had a sedimentation c o e f f i c i e n t of 127 S by l i n e a r l o g sucrose density gradient c e n t r i f u g a t i o n and 117 S by a n a l y t i c a l ultracentrifugai-t i o n . A buoyant density of 1.38-1.39 g/cm was obtained from isopycnic c e n t r i f u g a t i o n i n cesium chloride and a n a l y t i c a l u l t r a c e n t r i f u g a t i o n i n cesium sulphate. The n u c l e i c a c i d content of PLRV was estimated from the p a r t i c l e d e n s i t i e s to be 2 6 - 2 8 % . The PLRV n u c l e i c a c i d had:, a moi wt of 2.0 x 1 0 ^ ; was degraded by RNase but not by DNase; reacted with o r c i n o l but not with diphenylamine; and had a broad range of melting temperatures from 35 to 85 C i n 1 x SSC buffer with hyper-chromicity of 20-21% . These properties indicated that PLRV n u c l e i c a c i d i s a single-stranded RNA. The sedimentation c o e f f i c i e n t of the RNA molecule before and a f t e r treatment with formaldehyde was 34.5 S i i i and 20.7 S, respectively. Dissociated coat protein migrated as a single band in polyacrylamide gel electrophoresis and the average subunit moi wt was 26 300- PLRV should be considered a member of the luteovirus group. TABLE OF CONTENTS PAGE TITLE PAGE ABSTRACT , i i TABLE OF CONTENTS 'iv LIST OF FIGURES ' v i ACKNOWLEDGEMENTS i x INTRODUCTION 1 LITERATURE REVIEW 5 MATERIALS AND METHODS . 19 V i rus and vector 19 S t r a i n s 20 V i r u s p u r i f i c a t i o n 21 Serology 23 Sedimentation c o e f f i c i e n t 24 Buoyant density of v i r i o n 25 N u c l e i c a c i d 26 Coat-protein subunits 29 E l e c t r o n microscopy 30 RESULTS 31 S t r a i n s 31 Virus p u r i f i c a t i o n 32 Serology 39 Sedimentation c o e f f i c i e n t ^2 Buoyant density ^ Properties of n u c l e i c a c i d 46 Coat-protein ' 52 V TABLE OF CONTENTS (cont'd) DISCUSSION 57 SUMMARY 70 LITERATURE CITED 71 v i LIST OF FIGURES FIGURE PAGE 1 . Symptoms induced "by d i f f e r e n t s t r a i n s of PLRV on P. f l o r i d a n a yz plants A. healthy plant B. s t r a i n 1 G. s t r a i n 2 D. s t r a i n 3 E. s t r a i n 4 2 . E l e c t r o n micrograph of p u r i f i e d PLRV negatively stained with 3 5 2% PTA. The bar represents 1 0 0 nm 3 . Absorbance patterns of sucrose density gradients showing the 3 5 r e l a t i v e concentration of PLRV i n d i f f e r e n t host plants 4. Absorbance patterns of sucrose density gradients showing 36 r e l a t i v e concentration of the mild s t r a i n of PLRV i n d i f f e r e n t host plants 5 . Absorbance patterns of sucrose density gradients showing the 3 7 e f f e c t of post-harvest storage on PLRV y i e l d p u r i f i e d from in f e c t e d potato t i s s u e 6. Absorbance patterns of sucrose density gradients showing the 3 7 e f f e c t s of growing conditions on PLRV y i e l d 7 . Absorbance patterns of sucrose density gradients showing the 3 8 e f f e c t s of the molarity of resuspending b u f f e r on y i e l d of PLRV using phosphate buffer at pH 7 . 4 8 . Absorbance patterns of sucrose density gradients showing the 3 8 e f f e c t s of urea i n 0 . 0 7 M phosphate buffer, pH 7 . 4 on y i e l d of PLRV 9 . Absorbance patterns of sucrose density gradients showing the 3 9 e f f e c t s of various c l a r i f y i n g agents on y i e l d of PLRV 1 0 . Immunodiffusion re a c t i o n of PLRV i s o l a t e d i n B r i t i s h Columbia 4 1 with homologous antiserum and antiserum against a German i s o l a t e , provided by Sarkar 1 1 . Relationship between ELISA absorbance values and concentra- 4 2 t i o n of p u r i f i e d PLRV 1 2 . Absorbance p r o f i l e of a centrifuged l i n e a r l o g sucrose 4 3 density gradient containing PLRV and known i n t e r n a l standards v i i LIST OF FIGURES (cont'd) 13. Logarithm of depths of sedimentation of PLRV and known 43 i n t e r n a l standards p l o t t e d against logarithm of t h e i r respective sedimentation c o e f f i c i e n t s 1 4 . Schlieren pattern of PLRV i n 0.07 M phosphate buffer, pH 7.4 45 15. Absorbance patternaof p u r i f i e d PLRV following equilibrium 45 c e n t r i f u g a t i o n i n a GsGl gradient and the r e l a t i v e density of GsGl f r a c t i o n s 16. S c h l i e r e n pattern of PLRV centrifuged to equilibrium i n 45 cesium sulphate 17. Representative gels a f t e r electrophoresis with PLRV-nucleic 47 a c i d through -2.6% polyacrylamide g e l and treatment with enzymes 1 8 . Absorbance patterns of l i n e a r l o g sucrose density gradients 47 of PLRV-nucleic a c i d showing the e f f e c t s of RNase and DNase on the nature of the n u c l e i c a c i d 19. Melting behavior of the n u c l e i c a c i d of PLRV, SBMV i n 4 8 1 x SSG b u f f e r and c a l f thymus DNA i n 0.1 x SSG 20. Representative gels a f t e r electrophoresis withePLRV-nucleic 4 9 a c i d and standard RNAs through 2.6% polyacrylamide gel 21. Determination of moi wt of PLRV-RNA by electrophoresis on 50 2.6% polyacrylamide g e l s . The l o g of moi wt of marker RNAs i s p l o t t e d against t h e i r migration distances 22. Absorbance patterns of nu c l e i c a c i d of PLRV, TMV, SBMV and 51 BMV extracted by Tris-SDS method a f t e r c e n t r i f u g a t i o n through a l i n e a r l o g sucrose density gradient 23. Logarithm of the sedimentation c o e f f i c i e n t of RNA from 51 BMV, SBMV, TMV and PLRV, plotted against the respective logarithm of depth 2 4 . Absorbance patterns of formaldehyde-treated n u c l e i c a c i d 53 of PLRV, TMV, SBMV and BMV extracted by ammonium carbonate-SDS method, a f t e r c entrifugation through a l i n e a r l o g sucrose density gradient 25. Representative gels a f t e r electrophoresis with PLRV coat- 5^ protein subunits and standard proteins i n % polyacrylamide gel LIST OF FIGURES (cont'd) 26. Determination of the moi wt of coat-protein subunits of PLRV by electrophoresis in polyacrylamide gels. The logs of the moi wts of marker proteins are plotted against their migration distance 27. Effects of different gel concentrations on the relative mobilities of the protein standards and the PLRV in SDS-polyacrylamide gel electrophoresis i x ACKNOWLEDGEMENTS The author wishes to express his sincere appreciation to the mem-bers of his committee;. Dr. R. Stace-Smith (Thesis Supervisor), Agri-culture Canada, Research Station, Vancouver; Dr. V.G. Runeckles (Chairman) and Dr. R.J. Cope-man (Faculty -Advisor), Department of Plant Science, University of B r i t i s h Columbia; Dr. W.T. Cram, Dr. R.I. Hamil-ton and Dr. N.S. Wright, Agriculture Canada, Research Station, Vancouver for their guidance, criticism and suggestions pertaining to this thesis. Thanks are also due to Dr. S. Sarkar, University of Hohenheim, West Germany and Dr. M. Kojima, Hokkaido University, Japan for kindly providing samples of their antisera to potato l e a f r o l l virus. The author also wishes to thank the Director, Dr. M. Weintraub, and the staff of Agriculture Canada, Research Station, Vancouver for providing the f a c i l i t i e s for this research, for their invaluable assis-tance during the i n i t i a l stages and for their continued encouragement throughout this study. Finally, the author wishes to express his appreciation to his wife, Kian, for her patience, and encouragement and for typing the thesis. 1 INTRODUCTION Potato l e a f r o l l , caused by potato l e a f r o l l v i r u s (PLRV), i s a worldwide, economically important v i r u s disease of potatoes. As early as the eighteenth century, the degeneration of potatoes was considered to be i n f e c t i o u s and programmes were undertaken to destroy diseased plants i n an e f f o r t to c o n t r o l f i e l d spread of the disease:-.in Europe. The use of f r e s h seed tubers was a l s o investigated i n an attempt to control the spread of the potato l e a f r o l l disease. In England and Europe the changing of seed potato stocks continued as the major con t r o l measure u n t i l the l a t t e r part of the nineteenth century. I t was not u n t i l the e a r l y twentieth century that l e a f r o l l was considered a s p e c i f i c component of the potato "degeneration" disease complex. This break-through arose out of transmission studies which showed that the l e a f r o l l e n t i t y could be transmitted only by g r a f t i n g or by aphids. Consequently, the p o t e n t i a l host range was investigated and by the 1930's, Physalis  angulata L. and P. f l o r i d a n a Rybd. had been i d e n t i f i e d as good diagnostic hosts f o r l e a f r o l l . . v i r u s . Although l e a f r o l l had been known since the eighteenth century, i t was not u n t i l the mid 1960's that v i r u s p a r t i c l e s were shown to be associated with the disease. U l t r a t h i n sections of i n f e c t e d hosts showed that PLRV p a r t i c l e s were r e s t r i c t e d to phloem and young xylem t i s s u e s . This d i s t r i b u t i o n within the host g r e a t l y hampered the many attempts at p u r i f i c a t i o n of the v i r u s . By 1972, a l l that was known about the v i r u s was i t s s i z e and shape. The f i r s t published attempts, a f t e r t h i s time, to characterize the v i r u s i n d i c a t e d that PLRV contained 2 double-stranded DNA. A contradiction evolved as other workers t r i e d to duplicate these r e s u l t s but they found instead,that the n u c l e i c a c i d i n PLRV was single-stranded RNA. PLRV i s not mechanically transmissible but i s transmitted p r i m a r i l y through seed potatoes. Rapid f i e l d spread i s a r e s u l t of indiscriminate feeding by d i f f e r e n t aphid species, e s p e c i a l l y Myzus persicae ( S u l z e r ) , on i n f e c t e d and healthy l e a f material. Symptoms of v i r u s i n f e c t i o n are l e a f r o l l i n g and the development of s t i f f , dry, leathery leaves. In several potato v a r i e t i e s , tuber phloem necrosis i s associated with the disease. Reductions i n y i e l d may be as great as one-third to one-half the expected y i e l d . E f f o r t s to breed potato l i n e s f o r resistance to l e a f r o l l have not proven suc c e s s f u l . The problem of control, e s p e c i a l l y i n the use of seed c e r t i f i c a t i o n schemes, has been hampered by lack of r e l i a b l e detection methods. Determination of l e v e l s of c a l l o s e accumulation i n sieve tubes i s routine-l y applicable, but the t e s t does not give consistent r e s u l t s . The percent i n f e c t i o n of seed potatoes by PLRV can be estimated by growing samples of tubers during the winter i n the greenhouse or i n f i e l d p l o t s i n southern areas such as F l o r i d a . This method requires at l e a s t 6 wk of growing time before l e a f r o l l symptoms can be diagnosed, but the r e s u l t s are not r e l i a b l e . Detection of many economically important viruses depends on r a p i d and e f f i c i e n t diagnosis by s e r o l o g i c a l t e s t i n g . The d i f f i c u l t y with t h i s type of t e s t i n g i s that s p e c i f i c , high t i t r e antiserum i s e s s e n t i a l , ., and t h i s implies the need f o r s i g n i f i c a n t amounts of h i g h l y p u r i f i e d v i r u s . PLRV i s d i f f i c u l t to p u r i f y because i t i s present., i n very low 3 concentration i n host t i s s u e s and i s unstable when subjected to many of the standard p u r i f i c a t i o n techniques. In 197^ > workers i n Japan reported the production of a s p e c i f i c antiserum to PLRV and f o r some time they were the only ones to be successful. With the encouragement that i t was possible to p u r i f y s u f f i c i e n t q u a n t i t i e s of PLRV f o r a n t i -serum production, t h i s t h e s i s work was developed to determine an e f f e c t i v e -purification technique with a view to producing s u f f i c i e n t antiserum f o r screening seed-potato stocks. Having an antiserum a v a i l a b l e d i d not solve the problem of detecting l e a f r o l l i n potato t i s s u e s since the v i r u s i s not detectable i n crude sap by routine s e r o l o g i c a l methods. However, i n 1977 when t h i s t h e s i s t o p i c was being proposed, a paper was published on a promising s e r o l o g i c a l technique, enzyme-linked immunosorbent assay (ELISA), f o r detecting plant viruses that u n t i l t h i s time had proven d i f f i c u l t or impossible to diagnose because of low vi r u s concentration i n sap or because of interference from host components. With the advent of ELISA i t was conceivable that PLRV could be r e l i a b l y detected i n potato t i s s u e . I t became more important at t h i s point to determine the appropriate p u r i f i -cation scheme that would produce s u f f i c i e n t p u r i f i e d PLRV f o r i n j e c t i o n i n t o a r a b b i t to obtain antiserum.. A comparative assay was needed to determine the effectiveness of various p u r i f i c a t i o n techniques and u n t i l r e c e n t l y the only methods a v a i l a b l e required aphid transmission assays or v i s u a l i z i n g v i r u s peaks i n an 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 . With the introduction of automatic systems f o r quickly and simply scanning sucrose density gradients, the c r i t i c a l comparisons could be made under c o n t r o l l e d conditions. I t was decided that a study of PLRV was a f e a s i b l e project, 4 especially since the Japanese had shown i t was possible to obtain pure virus; since sensitive teclmiques were available to evaluate the purification methods; and since ELISA promised to be a useful tool for screening seed stocks. The objectives of this thesis were: 1) To develop an efficient purification method for PLRV; 2) To prepare an antiserum against an isolate of PLRV from British Columbia; 3) To isolate pure lines of the mild and severe strains of PLRV, and prepare specific antisera; and 4) To investigate the properties of purified PLRV. By the spring of 1979> enough progress had been made on the project that a manuscript was written in collaboration with Dr. R. Stace-Smith of Agriculture Canada, Research Station, Vancouver, and was submitted for publication in Virology. In the manuscript, i t was stated that the work reported was a portion of the thesis submitted by A. Rowhani in partial fulfilment of the requirements for a Ph. D.. degree from the University of British Columbia. This manuscript was published as "Purification and characterization of potato leafroll virus" in Virology, 1979. Vol.98:45-54 and will not be referred to as a reference in this thesis as i t is an integral part of the work reported here. 5 LITERATURE REVIEW The f i r s t d e s c r i p t i o n of potato l e a f r o l l was given by Hoppe i n 1747 i n Germany (Salaman, 19^9). Four years l a t e r , Maxwell des-cribed " c u r l " i n Great B r i t a i n as a decline i n the Galloway crops severe enough to require replacement by new stocks from Scotland and Ireland (Salaman, 19 49 ). A general outbreak of " c u r l " i n Germany encouraged s c i e n t i f i c workers to devote more at t e n t i o n to t h i s and r e l a t e d potato diseases. L e a f r o l l as a s p e c i f i c component of the potato "degeneration" complex was f i r s t recognized i n Germany and Denmark by Appel ( 1 9 0 7 ) . The f i r s t c l ear-cut recognition of l e a f r o l l as a potato disease i n America was presented by Orton (1914) a f t e r an outbreak of l e a f r o l l i n northern Colorado and western Nebraska. Quanjer et a l . ( I 9 1 6 ) reported that potato l e a f r o l l disease was gra f t transmissible and t h i s was subsequently confirmed by Murphy and Wortley ( 1 9 1 8 ) . Schultz and Folsom (1921) reported transmission of potato l e a f r o l l by the aphids M. persicae and Macrosiphum s o l a n i f o l i i Ashm. E l z e (1927 and 1931) and Smith (1929) found that. M. persicae was the most e f f i c i e n t vector of l e a f r o l l v i r u s . There are two types of i n f e c t i o n i n potato plants: primary and secondary (Webb et a l . , 1 9 5 2 ) . Primary i n f e c t i o n i s i n i t i a t e d by aphid i n o c u l a t i o n and the diagnostic symptoms are an upward r o l l i n g along the margins of the a p i c a l l e a f l e t s and the development of a pale yellow.to deep red c o l o r i n g of the leaves. Plants i n f e c t e d l a t e i n the growing season often do not e x h i b i t any symptoms. Secondary i n f e c t i o n occurs when an infe c t e d potato tuber i s used f o r seed. The symptoms of l e a f r o l l i n t h i s case are s i m i l a r to those of the primary 6 i n f e c t i o n except that l e a f r o l l i n g occurs i n the lower leaves f i r s t and i s more severe. PLRV has a r e s t r i c t e d host range and i n f e c t s mainly solanaceous plants. However, t h i s v i r u s also i n f e c t s species i n Amaranthaceae and Nolanaceae ( N a t t i et a l . , 1953)' Phloem necrosis of the potato tuber has been recognized f o r many years as being a symptom of l e a f r o l l v i r u s i n f e c t i o n (Quanjer, 1913; Orton, 1914). Stem sections of l e a f r o l l - i n f e c t e d potato plants ex-h i b i t a necrosis of the primary phloem t i s s u e . Quanjer (1913) and S h e f f i e l d (194-3) reported that l i g n i f i c a t i o n of the phloem was a dependable diagnostic symptom f o r the i d e n t i f i c a t i o n of l e a f r o l l . Douglas and Pavek (1972) found that under s p e c i f i c f i e l d and storage conditions net necrosis was associated with secondary i n f e c t i o n s i n some c u l t i v a r s . Environmental f a c t o r s influence the reaction of potato plants to l e a f r o l l i n f e c t i o n . Richards et a l . (1946) and Felton (1948) showed that i n greenhouse experiments high nitrogen l e v e l s masked the symptoms, p a r t i c u l a r l y i n dry s o i l and under low l i g h t conditions. Wilson (1951) found that during e a r l y growth i n the f i e l d , high l e v e l s of a v a i l a b l e nitrogen i n the presence of phosphorous l e d to a high degree of symptom masking. Dykstra (1933) reported that only Datura stramonium L. and D. t a t u l a L. had p o t e n t i a l as i n d i c a t o r plants f o r PLRV. Hovey and Bond (1948) found P. angulata to be a suitable assay host f o r PLRV. K i r k p a t r i c k (1948) reported P. f l o r i d a n a as a preferred i n d i c a t o r plant 7 compared with P. angulata and D. stramonium. Infection rates from single aphid transmissions were 70-100% in P. floridana, 20-40% in P. angulata and 40-70% in D. stramonium. Webb et a l . (I95l)> hy studying isolates of PLRV from 56 seed potato areas of the United States and Canada, substantiated the exis-tence of distinct strains of PLRV. Four strains were distinguished by symptoms on P. floridana and these were given number designations from 1 (mild) to 4 (severe). A l l strains were thought to exist in most potato growing areas. The extent of reaction of P. floridana infected with strains 1, 2, and 3 w a s ' dependent on temperature. The degree of recovery and subsequent growth by the infected plants i n -creased at higher temperatures. Plants infected with strain 4 were defoliated and k i l l e d at temperatures of 24 and 28 C. Symptoms of each strain were masked when the plants were kept at 35 G for 20 days but symptoms characteristic of the incitingrstrain.rapidly developed when the plants were returned to 24 C. Kassanis (1952) believed that PLRV did not exist as strains, even though i t was well-known that potato plants infected in the f i e l d showed symptoms of varying severity. Kassanis concluded that the main reason for the striking differences in severity depended on the presence of various strains of PVX. A mixture of PLRV and PVX causes symptoms of varying severity on potato plants, but Kassanis did not use P. floridana as an assay host to prove that strains of PLRV were not involved in symptom expression. Harrison (1958) reported that strains of PLRV were transmissible from infected potato to P. floridana or from P. floridana to P. floridana, and that a strain of PLRV causing mild symptoms in potato and P. floridana was 8 more r e a d i l y transmitted by M. persicae than was a severe s t r a i n . Harrison concluded that perhaps the concentration of the severe s t r a i n i n plants was lower, or that the severe s t r a i n was i n t r i n s i c a l l y l e s s r a p i d l y transmissible than the mild s t r a i n . To account f o r the extreme symptom i n s t a b i l i t y that was observed following transmission of PLRV from e i t h e r potato or P. f l o r i d a n a to P. floridana,:TtocGarthy (1963) suggested that l e a f r o l l v i r u s may be a l t e r e d within i t s vector. Ghiko and Guthrie (1969) disagreed because changes i n l e a f r o l l v i r u s within the vector would presumably occur at random and thus i n s t a b i l i t y of symptoms following aphid transmission from P. f l o r i d a n a to P. f l o r i d a n a would be u n l i k e l y . The r e s u l t s obtained by' Ghiko and Guthrie ( 1 9 6 9 ) . Webb et a l . (1952) and Harrison (1958) were i n contrast with r e s u l t s obtained by MacCarthy (1963). Chiko and Guthrie proposed that natural s e l e c t i o n of v i r u s s t r a i n s by host plants was the explanation f o r the v a r i e t y of symptom expression. Several chemical and anatomical t e s t s have been developed t o detect PLRV i n infe c t e d potato plants and tubers. Quanjer (1913) reported that l i g n i f i c a t i o n of the phloem was a dependable diagnostic symptom f o r the i d e n t i f i c a t i o n of l e a f r o l l i n f e c t i o n . One of the chemical techniques used by Perret ( I 9 2 3 ) was an iodine t e s t to detect a dark brown d i s c o l o r a t i o n as a r e s u l t of the r e a c t i o n of iodine with the excess accumulation of starch. N a t t i (1955) observed that starch grains from healthy tubers a f t e r treatment with iodine developed a blue c o l o r more r a p i d l y than d i d s i m i l a r l y treated starch grains from infec t e d tubers. S h e f f i e l d (19^-3) used phloroglucinol to s t a i n the n e c r o t i c phloem 9 c e l l s red as a r e s u l t of the reaction with l i g n i n deposit i n the ne c r o t i c phloem. Wilson (1948) used t h i s t e s t to detect PLRV-infected plants with masked symptoms, while K.lostermeyer (.1950) used t h i s method to diagnose current season i n f e c t i o n by l e a f r o l l v i r u s i n the c u l t i v a r Netted Gem. N a t t i (1952) and N a t t i and Ross (1954) reported that the ph l o r o g l u c i n o l diagnostic method was not r e l i a b l e u n t i l i n f e c t i o n had progressed beyond a c e r t a i n minimum l e v e l . C allose i s a normal constituent of the sieve plate i n many plant species, but formation of excess c a l l o s e , to the extent t h a t the tube can become completely blocked i s a response to PLRV i n f e c t i o n . Excess call o s e i n stems and tubers c a n D e detected by s t a i n i n g techniques such as the I g e l Lange t e s t (Broadbent and Heathcote, i 9 6 0 ) which requires the c a l l o s e i n tuber section to be stained with 1% r e s o r c i n blue i n 0.1% ammonia. DeBokx (1967) concluded that t h i s t e s t might be a valuable diagnostic a i d i n seed potato production f o r separation of healthy and diseased tubers. Zajac (1963) showed that the r e s o r c i n t e s t revealed v a r i a t i o n i n c a l l o s e content i n stems and tubers, but that the t e s t was not always r e l i a b l e f o r diagnosing tuber i n f e c t i o n . The main disadvantage i s that small quantities of c a l l o s e may be present i n healthy tubers making i t d i f f i c u l t to decide whether the tuber i s inf e c t e d or not. Several other techniques were used f o r diagnosis of PLRV-infected plants. Brandenburg (1962) used the diphenylamine t e s t and found that PLRV was detectable i n the plant as free DNA. This conclusion was rejected by Peters and Dielman ( 1 9 6 3 ) , Koening and Muller (1963, 1964) and Govier (1963) each of whom noted that d i f f e r e n c e s i n the color 10 v a r i a t i o n of the reagent and sap from healthy and i n f e c t e d plants were due to d i f f e r e n c e s i n sugar concentration, and not to the presence of a foreign DNA i n i n f e c t e d leaves. Other workers (Sarkar, 1963; Wenzel, 19^5) noted that the diagnostic value of t h i s technique f o r the e a r l y stages of PLRV i n f e c t i o n was dubious. Chromatography and the p r o l i n e t e s t (Omar et a l . , I 9 6 8 ) were not r e l i a b l e techniques f o r diagnosing PLRV-infected plants e i t h e r . Allam et a l . (1974) compared several chemical t e s t s f o r r e l i a b i l i t y with aphid transmission assays to detect PLRV i n potato plants and tubers and reported that the r e l i -a b i l i t y of iodine, diphenylamine and chromatography t e s t s varied from one potato v a r i e t y to another, and that s t a i n i n g f o r c a l l o s e was the most r e l i a b l e t e s t . Sarkar (1975) stained a p a r t i a l l y p u r i f i e d pre-paration of PLRV with 2% phosphotungstic a c i d f o r examination by electron microscopy and found that the technique was s i g n i f i c a n t l y quicker than the eye-cutting t e s t , and that the r e s u l t s were repro-ducible enough to be recommended f o r routine diagnosis of PLRV. U l t r a t h i n sections of i n f e c t e d P. f l o r i d a n a plants f o r electron microscopy were studied by Kojima et a l . ( 1 9 6 8 ) . Virus p a r t i c l e s of u n i -form s i z e and shape were located i n degenerated phloem c e l l s of p e t i o l e s and l e a f veins. Two independent groups of researchers i n Japan r e -ported s i m i l a r experiments that showed t h i n sections of inf e c t e d plants contained uniform s p h e r i c a l p a r t i c l e s 23-24 nm i n diameter (Kojima et a l . , 1969; A r i a et a l . , 1 9 6 9 ) . Kojima et a l . (1969) working with P. f l o r i d a n a and D. stramonium, found 23 nm p a r t i c l e s i n some of the phloem c e l l s . A r i a et a l . (1969) found p a r t i c l e s of approximately 24 nm i n diameter i n the phloem t i s s u e of P. f l o r i d a n a , D. stramonium 11 and potato. P a r t i c l e s were found within the cytoplasm or vacuoles of phloem companion c e l l s , ihloem parenchyma c e l l s and young xylem vessels, hut not i n the mesophyll c e l l s . Kojima (197*0 reported that PLRV p a r t i c l e s were r e s t r i c t e d to phloem t i s s u e of i n f e c t e d plants, confirming the r e s u l t s of other workers. Golinowski and de Zoeten (1976) reported an increase i n c a l l o s e i n sieve elements and changes i n n u c l e i of phloem parenchyma within 5 days of i n o c u l a t i o n . E l e c t r o n microscopy revealed s p h e r i c a l v e s i c l e s covered with v i r u s - l i k e par-t i c l e s , both i n the perinuclear space and outside the nucleus. Attempts to p u r i f y PLRV have met with l i t t l e success because the concentration of v i r u s i n host t i s s u e i s low. Day and Z a i t l i n (1958) c l a r i f i e d an extract of inf e c t e d P. f l o r i d a n a plants by d i f f e r e n t i a l c e ntrifugation, but f a i l e d to detect any c h a r a c t e r i s t i c p a r t i c l e s . Stegwee and Peters (1961) demonstrated that homogenates of i n f e c t i v e aphids could be centrifuged through sucrose density gradients and i n -f e c t i v i t y was associated with a p a r t i c u l a r f r a c t i o n of the gradient. Murayama and Kojima (1964) c l a r i f i e d extracts of diseased P. f l o r i d a n a plants with an organic solvent emulsion and a cycle of 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 , but found that i t was d i f f i c u l t to separate v i r u s from n o n v i r a l c e l l constituents. Peters (1965) reported two types of v i r u s -l i k e p a r t i c l e s i n homogenates of both-PLRV-free and. PLRV-carrying 1 "'-•aphids. The p a r t i c l e s had a diameter of 20 or 24 nm, although some p a r t i c l e s of 29 nm were found but the l a t t e r could be recovered equally well a f t e r p u r i f i c a t i o n from PLRV-free or PLRV-carrying aphids. No p a r t i c l e of s p e c i f i c morphology could be correlated with PLRV-carry-ing aphids. He also obtained s i m i l a r v i r u s - l i k e p a r t i c l e s from plants 12 of P. f l o r i d a n a a f t e r PLRV-free. aphids had fed on them f o r 10-14 days. Later, Peters (1967a) succeeded i n p u r i f y i n g PLRV from v i r u l i f e r o u s aphids by emulsifying the aphid homogenate with chloroform, followed by 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 sucrose density gradient c e n t r i f u g a -t i o n . A pure v i r u s preparation was obtained when the material from an i n f e c t i o u s zone of the gradient was concentrated by high speed ce n t r i f u g a t i o n . The f i n a l suspension contained p a r t i c l e s characterized by a hexagonal outline and a diameter of 23 nm. Suspensions" obtained from v i r u s - f r e e aphids i n a s i m i l a r manner were devoid of i n f e c t i v i t y and d i d not contain these p a r t i c l e s . Kojima et a l . (1968) detected 24-25 nm p a r t i c l e s i n emulsified homogenates of PLRV-infected P. f l o r i d a n a concentrated by 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 . A r i a et a l . (1969) and Kojima et a l . (1969) assayed v i r u s preparations by aphid i n j e c t i o n and demonstrated that i n f e c t i v i t y was associated with these small polyhedral p a r t i c l e s . Preliminary assays by aphid i n j e c t i o n showed that p r e c i p i t a t e s obtained by the addition of•polyethylene g l y c o l (PEG, moi wt 6000) to c l a r i f i e d sap retained i n f e c t i v i t y (Kojima and Murayama, 1972b). PEG at 8% concentration was as e f f e c t i v e as higher concentrations..in p r e c i p i t a t i n g the v i r u s . Several workers have succeeded i n p u r i f y i n g small q u a n t i t i e s of v i r u s : Murayama and Kojima, 197^-; Sarkar, 1976; Matt and de Bokx, 1978; Hepp and de Zoeten,,1978; and Mehrad et a l . , 1978. In t h e i r p u r i f i c a t i o n procedure, Takanami and Kubo (1979a) used Dris e l a s e , a macerating enzyme which e x h i b i t s pectinase and c e l l u l a s e a c t i v i t i e s . D r i s e l a s e was added to the plant homogenate to 1% and the 13 mixture incubated at 2 5 - 2 8 G f o r 2 h with shaking; chloroform-butanol was added to c l a r i f y the sap; PEG 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 were used to concentrate the v i r u s ; and sucrose density gradient cen t r i f u g a -t i o n separated the v i r u s from the remaining host material. PLRV occurs i n low concentration i n host plants and i s d i f f i c u l t t o - p u r i f y i n quantity. Kojima and Murayama (1972b) reported the y i e l d of PLRV from inf e c t e d P. f l o r i d a n a to be 0.01 mg/kg. Sarkar ( 1 9 7 6 ) , using a d i f f e r e n t p u r i f i c a t i o n procedure, reported a concentration of 0.4 mg/kg from inf e c t e d P . f l o r i d a n a . Mehrad et a l . (1978) p u r i f i e d the v i r u s from i n f e c t e d potato plants and obtained 0.03-0.1 mg/kg. Hepp and de Zoeten (1978) p u r i f i e d v i r u s from i n f e c t e d D. stramonium and obtained 0.11 mg/kg. Takanami and Kubo (1979a) improved the y i e l d of PLRV by using D r i s e l a s e enzyme and reported a y i e l d of 1.3 mg/kg of i n f e c t e d P. f l o r i d a n a . The f i r s t attempt to prepare an antiserum to PLRV was by Beemster (1955) who in j e c t e d intravenously crude sap from i n f e c t e d P. f l o r i d a n a plants that the sap had been.dialyzed, against .running tap..water.: E i g h t i n -jecti o n s t o t a l l i n g 35 ™-l were made during a period of 2 wk. A f t e r the antiserum was prepared, he p r e c i p i t a t e d the antibodies against normal plant proteins by mixing one part of healthy sap with one part of antiserum. No rea c t i o n was obtained when the cross absorbed a n t i -serum was tested against sap from infected plants. The f i r s t success i n preparing a s p e c i f i c antiserum to PLRV was reported by Kojima and Murayama (1972a), who obtained a r e l a t i v e l y crude antiserum. Murayama and Kojima (197*0 used a more hig h l y p u r i -f i e d v i r u s preparation and obtained an antiserum with high s p e c i f i c 1 4 v i r u s r e a c t i v i t y and low nonspecific r e a c t i v i t y . '.The t i t r e of t h e i r antiserum was 4096 i n p r e c i p i t i n r i n g t e s t s , and as l i t t l e as 6jag of PLRV antigen per mL reacted with homologous antiserum. Matt and de Bokx (1978) prepared antiserum to PLRV with a t i t r e of 64-256 i n agar-gel double d i f f u s i o n t e s t s . Hepp and de Zoeten (1978) prepared an a n t i -serum against PLRV p u r i f i e d from D. stramonium with a t i t r e of 64 i n m i c r o p r e c i p i t i n t e s t s . The f i r s t report of a s e r o l o g i c a l t e s t f o r PLRV i n crude plant sap was by Gasper (1977) who used ELISA to detect PLRV i n potato and P. f l o r i d a n a . He investigated the v i r u s concentration i n d i f f e r e n t parts of the plants and found that the roots of i n f e c t e d potato and P. f l o r i d a n a plants had the highest v i r u s content. Matt and de Bokx (1978) reported that ELISA could be applied r e a d i l y to t e s t the f o l i a g e of secondarily-infected, glasshouse grown potato plants f o r the presence of PLRV, although the method was l e s s r e l i a b l e when infec t e d sprouts were tested. Mehrad et a l . (1978) indicated that ELISA could be used ' as a d i r e c t means to detect PLRV and to assess the concentration i n host t i s s u e s . Clarke et a l . (1980) used ELISA to detect PLRV i n n a t u r a l l y - i n f e c t e d potato tubers by using a multiple-eye sampling and a l s o to detect PLRV i n v i r u l i f e r o u s M. persicae Hepp and de Zoeten (1978) used s e r o l o g i c a l l y s p e c i f i c e l ectron microscopy (SSEM) to detect PLRV i n crude sap. They reported that most of the v i r u s p a r t i c l e s occurred s i n g l y on the g r i d surface and could be e a s i l y confused with host material or. artifacts..produced by the negative s t a i n . I t was very d i f f i c u l t to locate and i d e n t i f y v i r i o n s probably because of the low t i t r e of t h e i r antiserum. Kojima 15 et a l . (1978) used SSEM f o r a rapid diagnosis of PLRV and found that clumps of coated v i r u s p a r t i c l e s were r e a d i l y detected. They "believed that t h i s technique was more simple and r e l i a b l e than ELISA although access to an electron microscope was e s s e n t i a l . Several papers have been published on s e r o l o g i c a l r e l a t i o n s h i p s among the luteoviruses. By feeding aphids through a membrane on v i r u s -antiserum mixtures or by subjecting the mixtures to density gradient c e n t r i f u g a t i o n before feeding, virus concentrations too low f o r con-ventional s e r o l o g i c a l techniques have been detected (Gold and Duffus, I 9 6 7 ) . Such i n f e c t i v i t y n e u t r a l i z a t i o n t e s t s demonstrated that a n t i -serum against beet western yellows v i r u s (BWYV) d i d not a f f e c t i n f e c -t i v i t y of PLRV (Duffus and Gold, 1969). Murayama and Kojima (1974) showed no close r e l a t i o n s h i p between soybean dwarf v i r u s (SDV) and PLRV, although PLRV has been reported to be r e l a t e d s e r o l o g i c a l l y to tobacco n e c r o t i c dwarf v i r u s (TNDV) another member of the l u t e o v i r u s group (Kubo and Takanami, 1978). Day and Z a i t l i n (1958) investigated the s t a b i l i t y of PLRV i n crude sap and reported that plant sap from i n f e c t e d P. f l o r i d a n a plants r e -tained i n f e c t i v e t y f o r 2 4 h at 2 G. Mueller and Ross (1961) noted that the i n f e c t i v i t y in. Kaemolymph from v i r u l i f e r o u s aphids was. retained f o r 3 days at 2 G. Stegwee and Peters (1961) reported that homogenates of v i r u l i f e r o u s aphids retained i n f e c t i v i t y f o r 2 4 h at 3 G a n d f o r 3 wk at -16 G. Murayama and Kojima (1965) investigated the s t a b i l i t y of PLRV i n crude sap of in f e c t e d P. f l o r i d a n a and determined a thermal i n a c t i v a t i o n point of about 70 G; a d i l u t i o n end-point of 10 -^; longevity i n sap 16 about 4 days at 2 G and 12-24 h at 25 C. Peters (1967b) reported that homogenates of v i r u l i f e r o u s aphids i n phosphate buffer containing 0.006 M mercaptoethanol remained i n f e c t i o u s f o r 5 days at 2 G, but only f o r 12 h at 25 G and f o r 4 h at 37 G when the homogenate was made in phosphate buffer alone. Murayama and Kojima (1973) used the sap from PLRV-infected P. f l o r i d a n a and reported that the v i r u s retained i t s i n f e c t i v i t y a f t e r storage f o r 5 days at 2 G, had a d i l u t i o n end-point of 10- and a thermal i n a c t i v a t i o n point of 70-80 G. Kojima (197*0 reported that the i n f e c t i v i t y of PLRV was retained a f t e r 5 days -3 -4 at 2 G, that the d i l u t i o n end-point was 10 -10 and that i n a c t i v a t i o n i n crude sap occurred at 70-80 G. Brandenburg (1962) reported the mechanical transmission of PLRV i n phenol extracts of inf e c t e d potato plants when the extracts had been incubated with RNase, but that incubation with DNase reduced the frequency of i n f e c t i o n s . From these r e s u l t s he concluded that PLRV was present i n the plant tissues as free DNA. Brandenburg applied the diphenylamine t e s t to expressed sap and found that sap from i n -fected potato plants gave a blue c o l o r which in d i c a t e s presence of DNA whereas sap from healthy potato plants gave a pale green color. Govier (1963) and Sarkar (1963) could not transmit PLRV mechanically i n phenol-extract with or without RNase treatment but reported that sap from potato plants infected with PLRV gave a blue c o l o r when heated with diphenylamine reagent due to the high concentration of fructose and sucrose i n sap from i n f e c t e d plants. Koening and.Mueller (1964) concluded that the color d i f f e r e n c e between extracts from healthy and infected leaves a f t e r treatment with diphenylamine reagent was due to the high concentration of ketosugars i n i n f e c t e d leaves. 17 Attempts to characterize the v i r a l n u c l e i c a c i d were reported by Sarkar and Bl e s s i n g (1973) who p u r i f i e d r e l a t i v e l y large amounts, of v i r u s from P. f l o r i d a n a and on the basis of a p o s i t i v e r e a c t i o n of phenol-extracted n u c l e i c a c i d with diphenylamine, postulated that PLRV contained DNA. A f t e r f u r t h e r work, Sarkar (1976) confirmed h i s previous report. He extracted PLRV-nucleic a c i d by two d i f f e r e n t methods: f i r s t , by treatment with 0.5 N p e r c h l o r i c a c i d at 70 G; and second, by treatment with phenol-sodium dodecyl s u l f a t e (SDS). Sarkar found that the PLRV-nucleic a c i d was r e s i s t a n t to RNase, s e n s i t i v e to DNase, possessed a sharp melting p r o f i l e with a Tm of 87.4, had a buoyant density of I.698 gm/cirr and had a moi wt of O.56 x 10 . The conclusion from these r e s u l t s was that PLRV n u c l e i c a c i d was double-stranded DNA. More recently, Sarkar (1978) used a modification of the Kleinschmidt technique and found only l i n e a r molecules of n u c l e i c a c i d , the majority of which had a length of 0.65Jum corresponding to a moi wt of 1.3 x 10°. Mehrad et a l . (1979) reported that PLRV-nucleic a c i d was s i n g l e -stranded RNA with a moi wt of 1.8-1.9 x 10^ i n polyacrylamide gels. Takanami and Kubo (1979b) obtained s i m i l a r r e s u l t s : the n u c l e i c a c i d of PLRV was .single-stranded RNA with a moi wt of 2.0 x 10 in-.poly-acrylamide gels. Takanami and Kubo (1979b) d i d not give any reason f o r the discrepancy between t h e i r r e s u l t s and those obtained by Sarkar. Mehrad et a l . (1979) noted that the lack of agreement between t h e i r r e s u l t s and Sarkar's (1976) might be due to contamination of the PLRV preparation with a large amount of heterogeneous DNase-sensitive material, e s p e c i a l l y i n the case of i s o l a t e s from leaves. 18 Sarkar (1978) reported that PLRV contained only one kind of coat-protein subunit with a moi wt of 15 000 as determined by e l e c t r o -phoresis i n SDS-containing polyacrylamide gels. This i s the only report published to date on the nature of the coat-protein subunit of PLRV. Harrison et a l . (1971) reported 16 groups of plant viruses as approved by the "Plant V i r u s Subcommittee of the In t e r n a t i o n a l Committee f o r the Nomenclature of Viruses", but PLRV was not included i n any of these groups. Shepherd et a l . (1976) reported four new plant v i r u s groups from the Pl a n t Virus Subcommittee of the I n t e r -n a t i o n a l Committee of Taxonomy of Viruses. One of these was the luteo-v i r u s group with BYDV as the type member. PLRV was not included as a member, however, Fenner (1976) i n h i s l i s t i n g of the member vir u s e s i n the group, d i d include PLRV. 19 MATERIALS AND METHODS Vi r u s and vector The source of PLRV was a single tuber of Splanum tuberosum L. cv. Netted Gem infe c t e d with the v i r u s (Wright and MacCarthy, I963)• Unless ctherwise stated, the vi r u s preparations contained a mixture of the s t r a i n s . The o r i g i n a l plant from which the tuber was obtained was i n i t i a l l y f r e e from* a l l knpwn viruses befere PLRV was intreduced by v i r u l i f e r c u s aphids (M. persicae) and i t was maintained i n an insec t prcof greenhouse. The tuber was planted i n the greenhouse and when the sprouts appeared, they were propagated as rooted cuttings. The plants were r e g u l a r l y f e r t i l i z e d with a 20-20-20 chemical f e r t i l i z e r at 2 wk i n t e r v a l s . The potato plants were r o u t i n e l y indexed f o r pptatp v i r u s X contaminaticn by manual in o c u l a t i o n of extracts to Gomphrena  globosa L. P. f l o r i d a n a seedlings were used as assay hosts and were inoculated at the cotyledon stage with v i r u l i f e r o u s aphids. Assay plants were grown i n a co n t r o l l e d climate chamber with 16 h of fluorescent l i g h t and maintained around 27 G (Wright et a l . , 1967). The plants were grown i n sand i n 5 x 5 cm pots..and" . placed i n a tra y containing nutr i e n t s o l u t i o n . Nutrient so l u t i o n was prepared according to Hoagland and Arnon ( 1 9 5 0 ) . Non-virulifereus M. persicae aphids were obtained from the insect-arium of Agr i c u l t u r e Canada, Research S t a t i c n , Vancpuver. A seurce cf v i r u l i f e r o u s aphids was established by allowing n o n - v i r u l i f e r o u s aphids to colonize PLRV-infected potato plants; n o n - v i r u l i f e r o u s aphids were kept on B r a s s i c a pekinensis Rupr. var. 'Pe T s a i (Chinese 20 cabbage). The aphids were kept i n screened cages under 16 h l i g h t period supplied by fluorescent lamps at room temperature. Virus was acquired by aphids feeding on PLRV-infected potato plants and was transmitted to healthy plants by aphids a f t e r a 4 - 2 4 h inocu-l a t i o n period. The length of the ino c u l a t i o n period depended on the experiment. For membrane feeding experiments,'"a modified version of the technique of Duffus and Gold (1965) was used: feeding cages f o r membrane feeding experiment were made from a c r y l i c tubing, 2 . 5 cm i n diameter and 3-5 °m long with one end covered by stretched parafilm. A few drops of the p u r i f i e d v i r u s preparation i n sucrose were placed on the membrane and covered with another t h i n membrane of parafilm. Non-viruliferous aphids were placed i n t o these cages. Gages with aphids and v i r u s preparations were then placed on a smooth surface with the membrane side up. A f t e r a 2 4 h a c q u i s i t i o n feeding period at 10 G, 3-5 aphids were tr a n s f e r r e d to P. f l o r i d a n a seedlings. The seedlings were caged i n d i v i d u a l l y , and the aphids were l e f t f o r a 2 4 h ino c u l a t i o n feeding. S t r a i n s PLRV has been c l a s s i f i e d into four s t r a i n s , s t r a i n s 1, 2 , 3 and 4 , i n which s t r a i n 1 i s the mildest and s t r a i n 4 i s the most severe (Webb et a l . , 1951; Wright and MacCarthy, 1963). S t r a i n s are distinguished from each other by t h e i r symptoms on P. f l o r i d a n a . The a v a i l a b l e v i r u s culture was a mixture of a l l four s t r a i n s . To obtain a pure culture of s t r a i n 1, P. f l o r i d a n a seedlings were inoculated with one v i r u l i f e r o u s aphid per plant f o r an ino c u l a t i o n period of e i t h e r 4 or 2 4 h. A f t e r 2 wk, symptoms c h a r a c t e r i s t i c of the d i f f e r e n t s t r a i n s could be distinguished on the t e s t plants. The plants with the mildest symptoms were selected assa source 21 of virus for the subsequent inoculation to P. floridana and individual aphids were used as before. After the f i f t h : such passage only strain 1 was reproducible. This procedure i s being used in an attempt to obtain a pure line of strain 4. Virus purification The virus source for most of the purification experiments was foliage of PLRV-infected potato plants. The plants were grown in a glasshouse under natural light supplemented with fluorescent light to . produce a 16 h day with the temperature ranging from 20-30 C during the summer and 20-25 C during the winter. Some purification experiments were done with potato foliage obtained from tubers and cuttings grown in a f i e l d plot, south campus, University of Bri t i s h Columbia (UBC). P. floridana, Nicotiana clevelandii Grey., and D. stramonium plants inoculated by aphids at the 5 -6 leaf stage and har-vested 1 month later, were studied as alternate sources of virus for purification. A l l purification treatments were done at or near 4 C. Low speed centrifugations were done in a Sorvall SS-3 centrifuge using a GSA rotor and high speed centrifugations in Beckman L2-50, L3-40 or L Ultracentrifuges using Spinco no. 30, 40, or SW41 rotors. Various' sources of infected tissues, buffers and purification procedures were investigated in an attempt to increase the yield of purified virus. The relative virus yield from each experiment was assessed following sucrose density ^gradient centrifugation by scanning with an,ISC0 Model UA-4 absorbance monitor at 254 nm. The following procedure was adopted for purification of the virus: 22 f r e s h l y harvested potato f o l i a g e and stems infe c t e d with PLRV were ground and the sap expressed with an Erweka KU1 f r u i t press. The ex-t r a c t was immediately mixed with Q.5 M potassium phosphate "buffer, pH 7.4, containing 0.1$ 2-mercaptoethanol and 0.1% sodium d i e t h y l d i t h i o -carbamate (Na-DIEGA) at a rate of 2 mL "buffer per gram t i s s u e . .. N-butanol-chloroform (1:1) was added gradually at the rate of 1 mL per 10 mL of extract and emulsified f o r 1 h with a magnetic s t i r r e r . The emulsion was broken by low speed centrifugation at 7000 rpm f o r 10 min. Polyethylene g l y c o l (PEG) moi wt 6000 was added t o the supernatant f r a c t i o n at 7% (wt/vol) and s t i r r e d f o r 1 h. The p r e c i p i t a t e was r e -covered by low speed centrifugation at 10 000 rpm f o r 30 min. The p e l l e t s were resuspended i n l / l O t h the o r i g i n a l volume of buffered sap with 0.07 M phosphate b u f f e r containing 0.75 M urea, pH 7.4. The sus-pension was c l a r i f i e d by low speed centrifugation at 8000 rpm f o r 10 min. The v i r u s i n the supernatant was p e l l e t e d by high speed c e n t r i -fugation i n a no. 30 r o t o r a t 27 000 rpm f o r 2.5 h. The p e l l e t s were resuspended i n 0.07 M phosphate buf f e r containing 0.75 M urea pH The v i r u s was f u r t h e r p u r i f i e d through sucrose density gradients i n an SW41 r o t o r and centrifuged at 38 000 rpm f o r 90 min. The gradients were made by l a y e r i n g 3i 3, 3 and 2 mL of 35, -.25, 15 a n < i 5f° sucrose, r e s p e c t i v e l y , dissolved i n 0.07 M phosphate buf f e r pH 7.4. The UV absorbing band was c o l l e c t e d and d i l u t e d twofold with 0.07 M phosphate buffer, pH ^M, and then centrifuged i n a no. 40 r o t o r at 38 000 rpm f o r 4 h. The p u r i f i e d v i r u s p e l l e t s were resuspended i n 0.07 M phos-phate buffer, pH 7.4, or 0.01 M T r i s - H C l buffer, pH 7.2, and stored at 4 G. The i n f e c t i v i t y of the u l t r a v i o l e t absorbing zone on sucrose 23 gradients was assayed by exposing M. persicae to the extract through a membrane as described before and then t r a n s f e r r i n g the aphids to seedlings of P. f l o r i d a n a .  Serology Antiserum against a mixture of severe and mild s t r a i n s of PLRV was obtained by immunizing a young white New Zealand r a b b i t with p u r i f i e d v i r u s by three intramuscular i n j e c t i o n s at weekly i n t e r v a l s . Each i n j e c t i o n was prepared by emulsifying the v i r u s preparation with" an equal amount of Freund's complete adjuvant. The amount of vi r u s used f o r f i r s t , second, and t h i r d i n j e c t i o n s were 0.13, 1.0 and 1.2 mg re s p e c t i v e l y , assuming an e x t i n c t i o n c o e f f i c i e n t of 5-0 (Kojima and Murayama, 1972b). The ra b b i t was bled 1 wk a f t e r the l a s t in--> j e c t i o n and thereafter at weekly i n t e r v a l s . A booster i n j e c t i o n of 1.7 mg of v i r u s was given a f t e r the t h i r d bleeding and the r a b b i t was bled at weekly i n t e r v a l s f o r 16 wk. The antiserum t i t r e was. determined by agar g e l double d i f f u s i o n t e s t s . Antiserum against the mild s t r a i n of PLRV was obtained by i n -j e c t i n g a r a b b i t 3 times i n 1 wk. The amount of virus i n j e c t e d was 0.11, 0.11 and 0.08 mg which was emulsified with an equal amount of Freund's complete adjuvant. The 1 mL preparation was i n j e c t e d i n t r a -dermally as 50-100 small b l i s t e r s on the back of the r a b b i t over an 2 approximate area of 25 cm . The ra b b i t was bled 1 wk a f t e r the l a s t i n j e c t i o n and a booster i n j e c t i o n of 0.08 mg of v i r u s was given at t h i s time. A week l a t e r the r a b b i t was bled and another booster i n -j e c t i o n of 0.07 mg was given. The rabbi t was bled at weekly i n t e r -vals a f t e r the l a s t i n j e c t i o n . The antiserum t i t r e was determined 24 against the mild s t r a i n of PLRV by agar g e l double d i f f u s i o n t e s t s . The agar gels consisted of 0.9% Ionagar, 0.8% NaCl and 0.2% NaN^ i n d i s t i l l e d water. The mixture was heated f o r 20 min i n a b o i l i n g water bath and, while the mixture was s t i l l hot, dispensed i n 2 mL a l i q u o t s onto collodion-coated microscope s l i d e s . Wells were cut i n the agar g e l by using a template made from discharged 0.22 c a l i b r e brass cartridges (Wright and Stace-Smith, 1966) and the agar plugs were removed by a s p i r a t i o n p r i o r to f i l l i n g the wells with the t e s t samples. The concentration of PLRV f o r agar g e l double d i f f u s i o n t e s t s was 80-120yU/^mL i n 0.07 M phosphate buffer pH 7-4 and the a n t i s e r a were d i l u t e d i n s a l i n e s o l u t i o n ( 0 . 8 5 % NaCl and 0.02% NaN^ i n d i s t i l l e d water). For ELISA, the method of Clark and Adams (1977) was used with minor modifications. The immunoglobulin G (igG) was concentrated from crude a n t i -serum by two cycles of ammonium su l f a t e p r e c i p i t a t i o n instead of one cycle and the f i r s t major peak which eluted from the DEAE-22 Sephadex column was c o l l e c t e d ; the conjugate was stored at 4 G without the addition of bovine serum albumin and the coated m i c r o t i t r e plates (Cooke-Dynatech) were stored at 4 C overnight or longer, p r i o r to use. A moist environment was maintained through-out the assay by enclosing the plate i n a small p l a s t i c bag held shut by an e l a s t i c band. Test samples were incubated overnight at 4 C, rather than 37 C f o r 4-6 h. The color reaction was assessed v i s u a l l y and by a spectrophotometer at 405 nm. Sedimentation c o e f f i c i e n t The sedimentation c o e f f i c i e n t of PLRV was estimated by l i n e a r l o g density gradient centrufugation (Brakke and Van P e l t , 1 9 7 0 a ) . Gradients were made by p i p e t t i n g i n t o SW4l tubes 1.3, 3>3> 2.4, 1.7 f 1.4 and 1.0 mL of 30> 26, 21, 16, 10 and 0% sucrose, r e s p e c t i v e l y , d i s s o l v e d i n 0.02 M phosphate b u f f e r pH 6.5. Brome mosaic (BMV), southern bean 25 mosaic (SBMV), tomato bushy stunt (TBSV), tobacco mosaic (TMV) and tobacco ringspot (TobRSV) viruses were used as markers of.known s e d i -mentation values. The gradients were centrifuged f o r 1.5 or 2 h at 38 000 rpm at 4 G i n an SW41 r o t o r and the p o s i t i o n (depth) attained by each v i r u s was obtained by UV flow densitometry using an ISGO UV monitor. The sedimentation c o e f f i c i e n t of PLRV was estimated from a standard curve derived from the p l o t s of the logarithm of the depth attained by the marker viruses versus the logarithm of t h e i r sedimen-t a t i o n c o e f f i c i e n t s . The sedimentation c o e f f i c i e n t of PLRV p a r t i c l e s was also deter-mined using the Beckman 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 . The v i r u s was sedimented at 33 ^50 rpi"1 and photographs were taken at 4 min i n t e r -vals using S c h l i e r e n optics. The temperature was maintained at 20 G and c e n t r i f u g a t i o n was performed at pH 7-4 i n 0.07 M phosphate buffer. S c h l i e r e n images were photographed on Kodak pro f e s s i o n a l f i l m #4154, with 15-second exposure times. C a l c u l a t i o n of ^ was done by the graphical method of Markham (19&7)• Buoyant density of v i r i o n The buoyant density of PLRV was determined by equilibrium banding i n cesium chloride gradients. Cesium chloride was di s s o l v e d i n 0.01 M T r i s - H C l buffer, pH 7-2 and discontinuous gradients were made by l a y e r i n g 1 mL each of solutions of known'densities (1.50, 1.40, 1.35, 1.30 g/crn^) i n 5 ml SW39 tubes. One m i l l i l i t e r of p u r i f i e d v i r u s suspension containing 0.06 mg of virus was layered onto each gradient and the vi r u s was centrifuged to equilibrium at 33 000 rpm f o r 20 h at 4 G. The gradients were scanned at 254 nm, 0.25 mL f r a c t i o n s were c o l l e c t e d and the d e n s i t i e s were determined at 25 G i n a Bausch and 26 Lombe .Abbe 60 refractometer. The density of cesium c h l o r i d e containing the v i r u s band was estimated by p l o t t i n g the f r a c t i o n number, density of f r a c t i o n s and absorbance at 254 nm. The density of PLRV p a r t i c l e s was a l s o determined by using cesium s u l f a t e and a Beckman Model E ana-l y t i c a l u l t r a c e n t r i f u g e at 44 770 rpm, 20 G, f o r 20 h. Cesium s u l f a t e was dissolved i n 0.07 M phosphate buffer pH 7.^ and mixed with about 72 jag of PLRV:. The f i n a l density of cesium s u l f a t e was 1.3967 g/cnP as determined by the Abbe 60 refractometer. P i c t u r e s were taken from Schlieren images at equilibrium, using 15-second exposure times. The buoyant density was calculated according to Chervenka (1973)• The percent RNA was c a l c u l a t e d according to the formula: (Sehgal et a l . , 1970) i n which/ 5 i s the density of the p a r t i c l e s . Nucleic a c i d For preparation of n u c l e i c a c i d three d i f f e r e n t techniques were used: 1. For nucleic a c i d that was to be used f o r general purposes, the Tris-SDS method of Nelson and Tremaine (1975) was used. The d i s s o c i a t i o n buffer was: 0.02 M T r i s - H C l , pH 9.0, containing 0.001 M EDTA, 1% SDS and 0.15 M NaCl. One volume of p u r i f i e d v i r u s preparation was mixed with one volume of d i s s o c i a t i o n b u f f e r and incubated at 37 G f o r 30 min. 2. For n u c l e i c a c i d that was to be formalinized, the ammonium carbo-nate-SDS method of Brakke and Rochow (197*1-) was used. D i s s o c i a t i o n b u f f e r was: 0.2 M ammonium carbonate, pH 9.0, 0.002:M EDTA, 2% SDS and 2 0 0 / i g of EDTA-treated bentonite/mL. Equal volumes of p u r i f i e d v i r u s preparation and d i s s o c i a t i o n b u f f e r were mixed and incubated at 4 G f o r RNA content = 3.88462 + 0.00026 27 16 h. The bentonite used i n t h i s procedure was treated with EDTA (Fraenkel-Conrat et a l . , 1 9 6 1 ) . 3. For n u c l e i c a c i d that was to be used f o r melting point experiments, o r c i n o l and diphenylamine t e s t s , the phenol-SES method of Ralph and Bergquist (1967) was used. The f i n a l pure n u c l e i c a c i d p e l l e t was resuspended i n SSC buf f e r (0.15 M sodium chloride and 0.015 M sodium c i t r a t e , pH 7.0) and dialyzed against SSG buffer overnight. The moi wt of PLRV n u c l e i c a c i d was determined by el e c t r o p h o r e t i c m o b i l i t y i n 2.6% c y l i n d r i c a l gels r e l a t i v e to the m o b i l i t i e s of TMV, SBMV and BMV n u c l e i c acids. Tank and gel buffers were prepared accor-ding to Dodds et a l . ( 1 9 7 7 ) , and gels were prepared according to Adesnik (1971). The gels were pre-electrophoresed f o r JO min at 3 mA/gel. Virus preparations at 2 mg/mL concentration were d i s s o c i a t e d by the Tris-SDS method, and mixed with ribonuclease-free sucrose at a f i n a l concentration of 5 percent. Samples of 20-40>uL were layered onto the gels and a marker g e l was run with bromophenol blue dye. The electrophoresis continued f o r 2-2.5 b at room temperature. The gels were removed from the tubes by rimming with a 22-gauge needle attached to a water supply so that a f i n e water stream l u b r i c a t e d the gel-tube i n t e r f a c e s . The gels were stained overnight with 0.02% t o l u i d i n e blue 0 i n 4-0% ethylene g l y c o l monomethyl ether, and destained i n tap water. The distances migrated by the nuc l e i c acids were measured and recorded. To determine the type of n u c l e i c a c i d of PLRV,. s p e c i f i c enzyme treatments were investigated. P r i o r to staining, some of the gels con-t a i n i n g electrophoresed n u c l e i c acids were incubated at room temperature f o r 2 h i n tubes containing SSG buffer with 0.5Jug/mL RNase (bovine 28 pancrease, Sigma Chemical) or with 10 /ag/mL DNase (beef pancrease, Sigma Chemical) and 0.04 M MgClg- The gels were then stained according to the procedure outlined above. In addition, n u c l e i c acids were assayed f o r s e n s i t i v i t y to RNase on l i n e a r l o g sucrose density gradients. Nucleic acids (0.5 nig/mL) were prepared from PLRV, BMV and SBMV by the Tris-SDS technique. Linear l o g sucrose density gradients were prepared by p i p e t t i n g i n t o SW41 tubes 1.6, 3.3, 2.2, 1.5, 1.3 and 1.2 mL of 32.5, 27, 21, 16, 10 and 0% RNase-free sucrose d i s s o l v e d i n SSC buffer containing 200 jug/mL bentonite (Jackson et a l . , 1973)* The gradients were stored at 4 C overnight before use. The PLRV and standard nu c l e i c acids were incubated at room temperature f o r 30 min with 0.5/ig/mL RNase or 10 /ig/mL DNase. The gradients were then loaded with 0.2 mL samples, centrifuged i n an SW41 rot o r at 38 000 rpm f o r 5 h at 4 C. A f t e r c e n t r i f u g a t i o n the gradients were scanned at 254 nm. Line a r l o g sucrose density gradients were used to estimate the sedimentation c o e f f i c i e n t of PLRV n u c l e i c a c i d by comparison of i t s depth of sedimentation with those of the marker n u c l e i c acids from BMV, SBMV and TMV. The n u c l e i c acids were prepared as described above f o r enzyme treatment. The sedimentation c o e f f i c i e n t of formaldehyde-treated PLRV n u c l e i c a c i d was determined i n the same manner. PLRV and standard virus preparations containing 0.5 mg/mL of vir u s were d i s s o c i a t e d by the ammonium carbonate-SDS method. For formaldehyde treatment, 37-*$ manufactured formaldehyde was d i l u t e d to 30% i n 0.45 M Na^HPO^ and 0.05 M NaHgPO^ as a stock s o l u t i o n (Boedtker, 1968). One volume of stock solution was added to 9 volumes of n u c l e i c a c i d preparations and 29 incubated f o r 15 min at 63 C. The samples were cooled r a p i d l y i n i c e water and immediately layered onto the gradients f o r c e n t r i f u g a t i o n i n an SW41 r o t o r at 38 000 rpm f o r 5 h. The moi wt of the n u c l e i c a c i d was c a l c u l a t e d from the sedimentation c o e f f i c i e n t according to the formula S = O..O83 M 0 , 3 8 (Brakke and Van P e l t , 1970b) i n which S i s the sedimentation c o e f f i c i e n t of formaldehyde treated RNA and M i s the moi wt of RNA. The diphenylamine and o r c i n o l t e s t s which d i s t i n g u i s h r i b o n u c l e i c a c i d from deoxyribonucleic a c i d (Pederson, 1969) were used to deter-mine the type of nu c l e i c a c i d i n PLRV p a r t i c l e s following the method of Shatkin ( 1 9 6 9 ) . For e i t h e r t e s t , p u r i f i e d n u c l e i c a c i d extracted by the phenol-SDS method was used. SBMV-RNA and c a l f thymus-DNA (Sigma Chemical) were used as standards. The melting behavior of PLRV-RNA was investigated with phenol-SDS-extracted n u c l e i c a c i d i n a G i l f o r d Model 250 Spectrophotometer com-bined with a Thermo Programmer Model 2527 and an Analog Multiplexer Model 6046. The r e s u l t was recorded on an scanning chart on a Recorder Model 6051. Coat-protein subunits The moi wt of PLRV coat-protein subunits was determined by SDS-polyacrylamide gel electrophoresis. A preparation of PLRV ( l mg/mL) was mixed with an equal volume of d i s s o c i a t i o n b u f f e r containing k- M urea, 1% SDS, 1% 2-mercaptoethanol i n 0.1 M sodium phosphate buffer, pH 7-2 (Frowd and Tremaine, 1977) and bo i l e d f o r 90 s. P r o t e i n stan-dards a t a concentration of 0.5 mg/mL were: bovine serum albumin, ovalbumin, carbonic anhydrase and myoglobin with moi wt of 66 000, 43 000, 29 000 and 17 500, r e s p e c t i v e l y . These were mixed with an 30 equal volume of d i s s o c i a t i o n b u f f e r and b o i l e d f o r 90 s. Dissociated proteins were electrophoresed i n 3> 5, 7-5 a n u - 1 0 % c y l i n d r i c a l poly-acrylamide gels. P r o t e i n samples ( 2 0 - 4 0 / J L ) i n d i s s o c i a t i o n buffer were mixed with a few drops of 10% g l y c e r o l containing 1% bromophenol blue to prevent the samples from f l o a t i n g into the tank buffer and to provide an i n -t e r n a l t racker dye f o r each g e l . The samples were layered onto the g e l surface under b u f f e r with a micropipet. The gels were electropho-resed at 6-8 mA per g e l u n t i l the marker dye was close to the end of the gels. The gels were removed from the gel tubes as before, stained with 0.2% Goomassie b r i l l i a n t blue R-15 i n a s o l u t i o n of 10% a c e t i c acid and 50% methanol overnight at room temperature, and destained i n a solution of 7% a c e t i c a c i d and 10% methanol. The moi wt of PLRV coat-protein was estimated from a standard curve derived by p l o t t i n g the l o g of the moi wt of the markers against 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 the gel, as described by Weber and Osborn ( 1 9 6 9 ) . E l e c t r o n microscopy Virus preparations f o r electron microscopy were negatively stained using 2% phosphotungstic a c i d (PTA) i n water, pH 7«2. One drop of a PLRV-suspension was placed on a 200 mesh, carbon-backed, c o l l o d i o n -coated copper g r i d f o r 1 min at room temperature, washed with PTA, b l o t t e d with f i l t e r paper and examined with a P h i l l i p s EM 300 or a P h i l l i p s EM 200 electron microscope. 31 RESULTS St r a i n s The PLRV-infected potato plants, cv. Netted Gem, grown i n the greenhouse showed t y p i c a l symptoms of PLRV i n f e c t i o n : l e a f l e t s of the lower leaves r o l l e d up at the edges, "became "britt l e and leathery and the plant exhibited mild c h l o r o s i s . P. f l o r i d a n a , inoculated with PLRV by M. persicae which had fed on these potato plants, showed a range of symptom se v e r i t y ( F i g . l ) . Based on the degree of symptom severity, the i s o l a t e s were c l a s s i f i e d into four d i f f e r e n t s t r a i n s (Webb et a l . , 1951)• s t r a i n 1- mild c h l o r o s i s , moderate r o l l i n g and basal cupping of the leaves, mild o v e r a l l stunting ( F i g . IB); s t r a i n 2-• moderate .- ch l o r o s i s , moderate r o l l i n g and basal cupping of the leaves, moderate o v e r a l l stunting ( F i g . IC); s t r a i n 3~ severe;chlorosis,- severe r o l l i n g and basal cupping of the leaves, severe p e t i o l e epinasty and twisting, severe o v e r a l l stunting ( F i g . ID); s t r a i n 4- .very severe c h l o r o s i s , very severe r o l l i n g and cupping of the leaves, severe p e t i o l e epinasty and twi s t i n g and severe o v e r a l l stunting ( F i g . IE). When the in d i c a t o r plants were inoculated by v i r u l i f e r o u s aphids ( l aphid/seed-l i n g ) over a 4 h period, fewer i n f e c t e d plants were obtained with severe symptoms than when 24 h inoculation period was used (Table l ) . A . plant with the mildest symptoms was selected as the inoculum source f o r the next t r a n s f e r and the symptoms observed were t y p i c a l of mild s t r a i n s 1 and 2. When t h i s procedure was repeated f i v e times only s t r a i n s 1 and 2 were obtained, and by the s i x t h passage only the mild-est s t r a i n ( s t r a i n l ) was produced. By t h i s method, a pure l i n e of the 32 F i g . l . Symptoms induced by different strains of PLRV on P. floridana plants: A. healthy; B. strain 1; G. strain 2; D. strain 3 and E. strain k. A l l plants were inoculated at the same age. mild strain of PLRV was obtained and i t was propagated on P. floridana for purification and subsequent production of antiserum. Similar ex-periments to obtain a pure line of strain 4 are s t i l l in progress (Table 2 ) . Virus purification Various methods of clarification and concentration were compared. Infectivity of the pure virus preparation at concentrations between 0.02 and 0.1 mg/mL from sucrose density gradients, was assayed by feeding M. persicae through a membrane. The success of transmission 33 Table 1. Results of successive transmission of PLRV by M. persicae to P. floridana to develop a pure line of the mild strain of the virus. No. of trans-mission Inoculation feeding period (h) No. of test plants No. 4 of test plants by strain 3 2 infected 1 No symp-tom -\ 4 20 4 5 2 1 8 j- 24 20 14 3 11 - 2 o 4 10 2 1 1 2 4 c. 24 10 2 3 2 1 2 4 10 l 2 4 3 J 24 10 1 2 3 2 . 2 II 4 10 _ _ 1 6 3 24 25 - 1 8 12 4 c 4 18 _ _ 3 15 J 24 20 - - 1 8 11 6 24 21 - - - 14 7 7 24 15 - - - 12 3 to P. floridana was usually greater than 70%. Fractions from sucrose density gradients containing the virus were checked by electron mic-roscopy to assess the homogeneity of the preparations (Fig. 2). The virus yield was 0.4-0.6 mg/kg of fresh potato tissue, with an A260/A280 of 1.74-1.82. P. floridana, N. clevelandii, D. stramonium and potato plants grown under similar conditions were compared as the source of PLRV for purification. The virus yield from potato foliage was almost twice as much as from P. floridana and D. stramonium while the yield from N. clevelandii was the poorest (Fig. 3 ) . The yield of the mild strain 34 Table 2. Results of successive transmissions of PLRV by M. persicae to P. floridana to develop a pure line of the very severe strain of the virus. No. of trans-mission Inoculation feeding period (h) No. of test plants No. 4 of test plants by strain 3 2 infected 1 No symp-tom 1 4 30 1 1 _ _ 28 24 36 3 2 l - 30 2 4 9 1 4 _ 1 3 24 12 2 5 2 2 1 4 18 9 l 2 6 j 24 16 10 3 1 1 1 4 4 17 7 2 _ 1 7 24 18 13 1 - 1 3 4 21 5 2 1 _ 13 24 21 8 4 4 1 4 6 4 32 4 3 2 23 24 36 9 7 6 1 13 7 4 38 8 2 _ _ 28 24 35 10 3 1 - 21 of PLRV wasshigher in D. stramonium than P. floridana and yield from potato foliage was the lowest (Fig. 4). The effect of storage on virus yield was compared. Freshly harvested material processed immediately yielded more virus than tissues stored at 4 G for 3i 10 and 21 days, or frozen for 3 days or longer (Fig. 5)• A l l virus preparations used for characterization were collected from peaks similar to the one shown in Fig. 5A. Virus yield from potato foliage grown under controlled greenhouse conditions was considerably higher than the yield from comparable foliage grown in the f i e l d (Fig. 6) . Various buffering systems, clarifying agents, concentrating tech-35 *5 • Fig. 2. Electron micrograph of purified PLRV negatively stained with 2% phosphotungstic acid. The "bar represents 100 nm. R E L A T I V E D C H M — Fig. 3. Absorbance patterns of sucrose density gradients showing the relative concentration of PLRV in different host plants: A. potato; B. P. floridana; G. D. stramonium and D. N. clevelandii. Arrow indicates position of virus peak. 36 RELATIVE DEPTH F i g . 4. Absorbance patterns of sucrose density gradients showing r e l a t i v e concentration of the mild s t r a i n of PLRV i n d i f f e r e n t host plants: A. D. stramonium; B. P. f l o r i d a n a and G. potato plants. Arrow ind i c a t e s p o s i t i o n of v i r u s peak. niques, pH's and m o l a r i t i e s of the b u f f e r were examined. Acetate, borate and phosphate buffers were compared and PLRV was found to be more stable i n phosphate buffer than i n acetate or borate b u f f e r s . Phosphate buffers at d i f f e r e n t pH *s were compared and PLRV was more stable i n n e u t r a l phosphate buffer. D i f f e r e n t m o l a r i t i e s of resuspen-ding b u f f e r were compared and y i e l d was highest when v i r u s was resus-pended i n 0.0? M phosphate bu f f e r ( F i g . 7 ) . The e f f e c t of urea'at 0.25, 0.50, 0.75 and- 1.0 M concentration i n the resuspending b u f f e r was tested and i t was found that 0.75 M urea i n the resuspending bu f f e r increased the y i e l d ( F i g . 8 ) . To c l a r i f y the crude•sap containing PLRV,- various techniques were used. Treatments included addition of chloroform, n-butanol, n-butanol-chloroform, T r i t o n X-100, ammonium s u l f a t e , or pH adjustment ( F i g . 9 ) . 37 * 1 4 _ e L A 1 V k , a • r ^ A ^ R E L A T I V E D E P T H F i g . 5- Absorbance patterns of sucrose density gradients showing the e f f e c t of post-harvest storage on PLRV yield, p u r i f i e d from i n f e c t e d potato t i s s u e : A. tissue processed immediatly a f t e r harvest; B-D. t i s s u e stored at h G f o r : B. 3 days; G. 10 days; D. 21 days; E-G. t i s s u e stored at -20. "C f o r : E. 3 days; F. 10 days; G. 21 days. Arrow Indicates p o s i t i o n of v i r u s peak. < a a o in CD < \ A - 1 t u u UL RELATIVE DEPTH F i g . 6. Absorbance patterns of sucrose density gradients showing the e f f e c t s of growing conditions on PLRV y i e l d : A. f o l i a g e from potato plants grown under c o n t r o l l e d greenhouse conditions and B. f o l i a g e from potato plants grown under f i e l d conditions. Arrow indicates p o s i t i o n of v i r u s peak. 38 R E L A T I V E D E P T H • g. 7. Absorbance patterns of sucrose density gradients showing the e f f e c t s of the molarity of resuspending b u f f e r on y i e l d of PLRV using phosphate buffer at pH 7.4: A. 0.01 M; B. 0.05 M; C 0.07 M and D. 0.1 M. Arrow in d i c a t e s p o s i t i o n of virus peak. R E L A T I V E O E P T H 8. Absorbance patterns of sucrose density gradients showing the e f f e c t s of urea i n 0.07 M phosphate -buffer, pH 7.4 on y i e l d of PLRV: A. no urea; B. 0.25 M urea; G. 0.50 M urea; D. 0.75 M urea and E. 1.0 M urea. Arrow indicates p o s i t i o n of vi r u s peak. 39 I S M IM u z « IB K o • Ml > R E L A T I V E D E P T H — • » F i g . 9. Absorbance patterns of sucrose density gradients showing the e f f e c t s of various c l a r i f y i n g agents on y i e l d of PLRV: A. n-butanol-chloroform 1:1, 10% (v/v); B. n-butanol, 10% (v/v); G. chloroform, 10% (v/v); D. T r i t o n X-100, 5% (v/v); E. ammonium s u l f a t e , 10% (wt/vol) and F. pH adjustment to pH• 5«5 with g l a c i a l a c e t i c a c i d . Arrow i n d i c a t e s p o s i t i o n of v i r u s peak. Optimal c l a r i f i c a t i o n was obtained with 10% (v/v) n-butanol-chloroform (1=1). To concentrate the c l a r i f i e d sap, d i f f e r e n t amounts of PEG from 4-10% (wt/vol) were compared; 7% PEG was as e f f e c t i v e as higher con-centrations. High speed ce n t r i f u g a t i o n i n a no. 30 r o t o r at 27 000 rpm f o r 2.5 h gave the same y i e l d of v i r u s as p r e c i p i t a t i o n with 7% PEG. Serology The antigen f o r the production of the f i r s t antiserum was prepared 40 by using potato plants i n f e c t e d with a mixture of a l l four s t r a i n s . The t i t r e of the PLRV antiserum was determined by agar g e l double d i f f u s i o n t e s t s . Before the booster i n f e c t i o n , i t was 256, and a f t e r the f i n a l i n j e c t i o n , 1024, where i t remained f o r a few weeks, before dropping to 512. The antiserum reacted with p u r i f i e d preparations of PLRV i n g e l d i f f u s i o n t e s t s but not with crude sap prepared from i n -fected potato or P. f l o r i d a n a . P u r i f i e d v i r u s prepatations used f o r immunization contained some contaminating host proteins because a non-s p e c i f i c t i t r e of 4 was obtained. The p u r i f i e d PLRV i s o l a t e from B r i t i s h Columbia reacted i n g e l d i f f u s i o n t e s t s with a n t i s e r a obtained from M. Kojima, Japan and S. Sarkar, Germany ( F i g . 1 0 ) . Homologous antigens f o r these antisera were not a v a i l a b l e , so whether or not t h i s i s o l a t e was s e r o l o g i c a l l y i d e n t i c a l to the Japanese and German i s o l a t e s of PLRV could not be determined. The heterologous reactions were s u f f i c i e n t l y strong to suggest that the three i s o l a t e s involved were c o l s e l y r e l a t e d . Antiserum against the mild s t r a i n of PLRV was prepared and the t i t r e before the f i r s t booster i n j e c t i o n was 320 and a f t e r the f i n a l i n j e c t i o n i t was 2560. This antiserum was used i n g e l d i f f u s i o n t e s t s with antigens from the mild s t r a i n and from a mixture of s t r a i n s and an i d e n t i c a l r e l a t i o n s h i p was obtained. Optimal r e s u l t s with ELISA were obtained when the m i c r o t i t r e plates were coated with PLRV IgG at a d i l u t i o n of 1:1000 and with conjugated IgG at a d i l u t i o n of 1:400. Under these conditions, p u r i f i e d PLRV could be detected at concentrations as low as 32 ng/mL ( F i g . 1 1 ) . 41 Fig. 10. Immunodiffusion reaction of PLRV isolated in B r i t i s h Columbia with homologous antiserum and antiserum against a German iso-late, provided by Sarkar: G, homologous antiserum; V, pure preparation of PLRV and G, German antiserum. To be considered positive, the extinction values (E^^nm) of the test samples had to be at least twice those of the control wells containing a l l test components except the antigen. The limit of visual detection of the color reaction corresponded to an extinction value nm = 0.1 or at a virus concentration of approximately 100 ng/mL. The possible application of ELISA for detection of PLRV in potato plants and tubers was investigated. The virus was readily detected in infected foliage: extinction values varied from 6.0 to 12.8 for different samples of PLRV-infected leaves at a 1:10 dilution while values for the healthy controls were less than 1.0. Virus could be detected in wells containing sap diluted to 1:3^0. Detection of virus in tuber tissue was unreliable because of the high nonspecific reaction 42 Fig. 1 1 . Relationship bet-ween ELISA absor-bance values and concentration of purified PLRV. CONCENTRATION OF PLRV (ng/mi) CONCENTRATION OF PLRV ( n g / m l ) with healthy material. Possibly starch granules were adsorbed to the polystyrene microtiter plates and the conjugated IgG attached nonspe-c i f i c a l l y to these starch granules. Sedimentation coefficient A mixture of PLRV, BMV ( 8 6 S), SBMV ( 1 1 5 S), TBSV ( 1 3 5 S) and TMV ( 1 8 ? S) was layered onto a linear log sucrose density gradient and after centrifligation, i t resolved into five virus peaks (Fig. 1 2 ) . The identity of each peak was determined by reference to scans of the gradients containing individual virus preparations. The sedimentation coefficient of PLRV was determined as 1 2 7 ' , + 5% S when compared with the marker values (Fig. 1 3 ) . This was repeated twice and the values of 1 2 7 S and 128 S were obtained. In addition, a mixture of PLRV and TobRSV was centrifuged through a sucrose density gradient and PLRV 43 PLRV F i g . 12. Absorbance p r o f i l e of a centrifuged l i n e a r l o g sucrose density-gradient containing potato l e a f r o l l v i r u s (PLRV) and known Internal standards. Abbreviations are BMV, brome mosaic v i r u s (86 S); SBMV, southern bean mosaic virus ( 1 1 5 S); TBSV, tomato bushy stunt v i r u s ( 1 3 5 S) and TMV, tobacco mosaic v i r u s (187 S). 0.74 T i 2 . 0 . 2 . 1 2 ^ 2 2 ^ 3 L O G S F i g . 13. Logarithm of depths of sedimentation of BMV (o), SBMV (•), TBSV (A), TMV (•) and PLRV (•) pl o t t e d against logarithm of t h e i r respective sedimentation c o e f f i c i e n t s . ii4 sedimented between the middle (9*+ S) and bottom (130 S) components of TobRSV. The calculated value f o r PLRV was 127 S. This determination agrees with the preliminary report of Kojima and Murayama (1972b) who compared t h e i r sedimentation pattern with a s i m i l a r pattern obtained by Rochow and Brakke (1964) f o r barley yellow dwarf v i r u s (BYDV) and found that the sedimentation rate of PLRV was greater than that of BYDV (115-118 S ). The sedimentation c o e f f i c i e n t of PLRV (lmg/mL) was estimated.by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n i n phosphate b u f f e r ( F i g . 14). The calculated value of 117 + 2% S was l e s s than the value obtained from l i n e a r l o g sucrose gradient centrifugation. which .may .reflect, the. d i f f e r e n t , tech-niques and conditions of cent r i f u g a t i o n . Buoyant density P u r i f i e d PLRV was centrifuged to equilibrium i n discontinuous cesium chloride gradients and when scanned, revealed a single v i r u s peak. The density of the f r a c t i o n with the highest UV absorbance was 1.392 g / c i rP , which corresponded to the density of PLRV p a r t i c l e s ( F i g . 1 5 ) . For comparison, the buoyant density of SBMV was found to be 1.350 g/cm^, which agrees with the published value of 1.359 g/cm^ (Sehgal et a l . , 1970). The density of PLRV p a r t i c l e s was also estimated by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n i n cesium s u l f a t e ( F i g . 16) since PLRV p a r t i c l e s were not stable i n cesium chloride f o r periods longer than 26 h at 4 G or a few hours at room temperature. Only a single peak was observed by Schlieren optics a f t e r 20 h cen t r i f u g a t i o n i n cesium s u l f a t e at a density of I.38 g/cm . This value agrees with that obtained from isopycnic centrifugation i n cesium chloride f o r 20 h at 4 C. The 45 F i g . 14. S c h l i e r e n pattern of potato l e a f r o l l v i r u s (PLRV) i n 0.07 M phos-phate bu f f e r pH 7.4 centrifuged at 33 450 rpm f o r 14 min at 20 G. DEPTH ** F i g . 15. Absorbance pattern at 254 nm of p u r i f i e d potato l e a f r o l l v i r u s (PLRV) following equi-l i b r i u m c e n t r i f u g a t i o n i n a GsGl gradient and the r e l a t i v e density of GsGl f r a c t i o n s . Densi-t i e s were determined by refractometry and super-imposed on the gradient scan. F i g . 16. S c h l i e r e n pattern of potato l e a f r o l l v i r u s (PLRV) centrifuged to equi l i b r i u m i n cesium sulphate f o r 20 h at 44 770 rpm. 46 n u c l e i c a c i d content of PLRV was estimated from the density of the virus to be 2 6 - 2 8 % according to the equation of Sehgal et a l . (1970). Properties of nu c l e i c a c i d The three methods of nu c l e i c a c i d extraction used were s a t i s f a c t o r y f o r PLRV. PLRV-nucleic acid i n polyacrylamide gels was s e n s i t i v e to RNase but not to DNase ( F i g . 1 7 ) . Absorbance scans of PLRV-nucleic acid i n l i n e a r l o g sucrose density gradients showed no n u c l e i c a c i d peak fo l l o w i n g RNase treatment, while sharp peaks were evident a f t e r DNase treatment and i n untreated controls ( F i g . 1 8 ) . The component that sedimented deeper i n t o the gradient was thought to be p a r t i a l l y degraded v i r i o n s . A slower sedimenting component i n the gradient was observed a f t e r DNase treatment and t h i s represented some degradation of the n u c l e i c a c i d p o s s i b l y due to contamination of the DNase stock with RNase. A decrease i n height :of the n u c l e i c a c i d peak a f t e r t r e a t -ment with DNase was al s o thought to be due to the presence of conta-minating RNase i n the DNase stock. The n u c l e i c a c i d of PLRV and the RNA standards reacted p o s i t i v e l y with o r c i n o l reagent, and had much higher readings at O D ^ Q compared with c a l f thymus DNA and the blank. When the n u c l e i c a c i d preparations were reacted with diphenylamine reagent only c a l f thymus DNA was p o s i t i v e . PLRV-nucleic a c i d showed a broad thermal t r a n s i t i o n when heated i n SSG b u f f e r ( F i g . 1 9 ) , i n d i c a t i n g a single-stranded RNA. In two r e p l i c a t e s the melting point of PLRV-nucleic a c i d was. found to be 60 G and 61 G, of SBMV-RNA, 59 G and of c a l f thymus DNA, 73 C. The pre-parations of PLRV-nucleic acid exhibited hyperchromicities of 20 and Z\%, SBMV-RNA of 2Jf0 and c a l f thymus DNA of 36 percent. The r e s u l t s 4 7 B Fig. 1 7 . Representative gels after electro-phoresis with PLRV-nucleic acid through 2.6% polyacrylamide gel and treatment with enzymes: A. control; B. treated with RNase and G. treated with DNase. Fig. 18. Absorbance pattern of PLRV-nucleic acid following centrifu-gation through a linear log sucrose density gradient: A. the pattern obtained with untreated nucleic acid; B. the pattern following DNase treatment and C. the pattern follow-ing RNase treatment. The component that sedimented deeper into the gradient was thought to be partially degraded virions. Arrow indicates position of RNA peak. 48 E0.7H 4 * 0 5 0 6 0 7 0 8 0 9 0 1 < W T E M P E R A T U R E ( C ) F i g . 19. Melting behavior of the n u c l e i c a c i d of PLRV (h—* ), SBMV (•—•) i n 1 x SSG ( 0 . 1 5 M NaCl, 0.015 M sodium c i t r a t e ) and c a l f thymus DNA (•—•) i n 0.1 x SSG. A260 s t a r t i n g values were 2.42, 2.33 and 1-37 r e s p e c t i v e l y . of enzyme treatment, c o l o r i m e t r i c methods and thermal denaturation are mutually supportive: a l l t e s t s i n d i c a t e that the n u c l e i c a c i d of PLRV i s RNA. The moi wt of PLRV-nucleic a c i d was determined by coelectropho-r e s i s with BMV, SBMV and TMV RNAs. This experiment was repeated f i v e times with three d i f f e r e n t PLRV preparations (Table 3 ) • PLRV-nucleic a c i d and the marker RNAs were, observed as -well-defined, homogeneous components i n g e l electrophoresis ( F i g . 2 0 ) . The distance migrated by the standard RNAs was plotted-, against-"'the. l o g of the.respective.moi wt ( F i g . 2 1 ) . By comparison of the mobility of PLRV-nucleic a c i d with 6 the standards a value of 2.0 x 10 was obtained f o r the moi wt of PLRV-nu c l e i c a c i d . 49 Table 3- Determination of moi wt of PLRV-RNA by comparative polyacry-lamide gel electrophoresis in 2.6% gels with v i r a l RNA standards in 5 different experiments. Source of Distance migrated (cm) , , . moi wt nucleic , ^ acid 1 2 3 5 5 ( X 1 0 ) PLRV 1.5 1.5 1.6 1.6 1.6 2.00 TMV 1 . 4 1 . 4 - - - 2.05J SBMV 2.1 2.0 2.2 2.2 2.3 1 . 4 0 BMV-L 3.0 3.0 3 - 2 3-0 3-1 1.09° B M V 2 3.0 3.1 3 . 2 3-1 3.1 0.99° BMV 3.8 3.8 4 . 1 3.9 3.9 0.75° BMV£ 6 . 2 6.0 6 . 4 6.3 6.1 0 . 2 8 ° a. Knight, 1952; b. Tremaine, 1966; c. Lane and Kaesberg, 1971. A B C 1 „ Fig. 20. Representative gels after electro-phoresis with PLRV-nucleic acid and standard RNAs through 2.6% polyacrylamide gel: A. PLRV-nucleic acid; B. SBMV-RNA and G. BMV-RNA. 50 DISTANCE MIGRATED (cm) F i g . 21. Determination of moi wt of PLRV-RNA by electrophoresis on 2.6% polyacrylamide gels. The l o g of moi wt of marker RNAs i s plotted against t h e i r migration distances. Standards and t h e i r respective moi wt are: TMV, 2.05 x 10°; SBMV, 1.4 x 10°; BMV-1, 1.09 x 1 0 6 ; BMV-2, 0.99 x 1 0 6 ; BMV-3, 0.75 x 10°; BMV-4, 0.28 x 10°. The calculated moi wt f o r • PLRV-RNA i s 2.0 x 10°. The sedimentation c o e f f i c i e n t of PLRV-RNA was determined by com-paring i t s sedimentation with that of the RNA of TMV, SBMV and BMV (Brakke and Van P e l t , 1970b) where the l o g of the depth"of sedimentation, was a l i n e a r function of the l o g of the sedimentation c o e f f i c i e n t . The n u c l e i c acids were centrifuged f o r 5 h through l i n e a r l o g sucrose density gradients i n an SW41 r o t o r at 38 000 rpm. The depths of the peaks were measured on the scanning patterns ( F i g . 22) from the middle of the peaks to the meniscus and the l o g of the depth was p l o t t e d against the l o g of the sedimentation c o e f f i c i e n t ( F i g . 2 3 ) . The sedimentation value of PLRV-RNA was estimated as 3^'5~35-0 S by comparison with the sedimentation values of the standards. 51 RELATIVE DEPTH — F i g . 22. Absorbance pattern of n u c l e i c a c i d of PLRV, TMV, SBMV and BMV extracted by Tris-SDS method a f t e r centrifugation through a l i n e a r l o g sucrose density gradient i n a SW4l r o t o r f o r 5 n at 38 000 rpm: A. p r o f i l e of PLRV-RNA; B. p r o f i l e o f TMV-RNA; G. p r o f i l e of SBMV-RNA and D. p r o f i l e of BMV-RNA. Arrow ind i c a t e s p o s i t i o n of RNA peak. T i U u ft i l 16 L O G S F i g . 23. Logarithm of the sedimentation c o e f f i c i e n t of RNA- from BMV (•), SBMV ( O ) , TMV (•) and PLRV ( a ) , p l o t t e d against the respective logarithm of depth. BMV-RNA i s l a b e l l e d 1, 2, 3 and 4 f o r the respective RNAs. 52 A value of 20.7 S was obtained f o r the sedimentation c o e f f i c i e n t of PLRV-RNA a f t e r treatment with formaldehyde, which corresponds to the S 20, W of formalinized TMV-RNA (20.7 S) (Eig :. 24-). By using the formula 0 38 S = 0.083 M f o r the r e l a t i o n s h i p between sedimentation c o e f f i c i e n t (S) of formaldehyde-treated RNA and moi wt before formaldehyde treatment (M) (Brakke and Van P e l t , 1970b), a moi wt of 2.0 x 10^ was obtained f o r PLRV-RNA. With both sucrose density gradient c e n t r i f u g a t i o n and polyacrylamide gel electrophoresis, the moi wt of PLRV-RNA was the same as that of TMV-RNA. Coat-protein To determine the moi wt of the PLRV coat-protein subunits, SDS-poly-acrylamide gel electrophoresis, was used. (Shapiro et a l . , 19 67.). The PLRV coat-protein migrated as a single band between myoglobin (moi wt 17 500) and carbonic anhydrase (moi wt 29 000) ( F i g . 2 5 ) . The mo b i l i t y of the polypeptide chains through polyacrylamide gels was cal c u l a t e d according to the formula of Weber and Osborn, ( 1 9 6 9 ) : M o b i l i t - d - i s " k a n c e °^ P r°tein migration -length of g e l before s t a i n i n g length of g e l a f t e r s t a i n i n g distance of dye migration This experiment was repeated with three d i f f e r e n t v i r u s preparations and the moi wt of PLRV coat-protein subunits ranged from 25 000 to 27 000 with an average of 26 300 ( F i g . 2 6 ) . The m o b i l i t i e s of PLRV coat-protein sub-units and protein standards r e l a t i v e to the mob i l i t y of myoglobin i n 3, 5, 7.5 and 1 0 % gels were determined (Hedrick and Smith, I968). Although p l o t s of the logarithms of t h e i r r e l a t i v e m o b i l i t i e s (Rm) against concentration extrapolated to a common intercept, Rm = O.98 rather than 1.0, at zero ge l concentration, a l l p l o t s gave s t r a i g h t l i n e s ( F i g . 2 7 ) . This r e s u l t 53 F i g . 24. Absorbance pattern of formaldehyde treated n u c l e i c a c i d of PLRV, TMV, SBMV and BMV extracted by ammonium carbo-nate-SDS method, a f t e r c e n t r i f u g a t i o n through a l i n e a r l o g sucrose density gradient i n a SW4l r o t o r f o r 5 h at 38 000 rpm: A. p r o f i l e of PLRV-RNA; B. p r o f i l e of TMV-RNA; G. p r o f i l e of SBMV-RNA and D. p r o f i l e of BMV-RNA. Arrow i n d i -cates p o s i t i o n of RNA peak. 54 A B C D E Fig. 25. Representative gels after electrophoresis with PLRV coat-protein subunits and standard proteins in 5% polyacrylamide gel j A. PLRV coat-protein subunits; B. carbonic anhydrase; G. myoglobin; D. ovalbumin and E. bovine serum albumin. 55 6.0 K4.0 O s 2.0 0.4 0.6 0.8 1.0 RELATIVE MOBILITY F i g . 26. Determination of the moi wt of coat-protein subunits of PLRV by electrophoresis i n polyacrylamide gels. The logs of the moi wts of marker proteins are plotted against t h e i r migration distances. Abbreviations are: BSA, bovine serum albumin (moi wt, 66 000); OA, ovalbumin (moi wt, 43 000); CA, carbonic an-hydrase (moi wt, 29 000) and MG, myoglobin (moi wt, 17 5 0 0 ) . G E L C O N C E N T R A T I O N F i g . 27. E f f e c t of d i f f e r e n t g e l concentrations on the r e l a t i v e m o b i l i -t i e s of the protein standards and PLRV i n SDS-polyacrylamide gel electrophoresis. Legends: f ) , myoglobin; 4, PLRV; g|, carbonic anhydrase; Q, ovalbumin; ^ , bovine serum albumin. 56 demonstrated that PLRV protein "behaved normally i n g e l electrophoresis, and that moi wt determination i s independent of gel concentration (Shapiro et a l . , 1 9 6 7 ) . 57 DISCUSSION Potato l e a f r o l l v i r u s i s u s u a l l y present i n a host as a mixture of s t r a i n s and when the vi r u s i s transmitted from t h i s source to P. f l o r i d a n a a wide range of symptoms i s observed. The four s t r a i n s of PLRV can he di s t i n g u i s h e d "by the s e v e r i t y of symptoms produced i n P. f l o r i d a n a ranging from moderate i n i t i a l symptoms and subsequent rapid recovery to severe d e f o l i a t i o n and death of the plant (Webb et a l . , 1951; Harrison, 1958; MacCarthy, I963). A mixture of two s t r a i n s might produce intermediate symptoms, or i t might show symptoms t y p i c a l of a more severe s t r a i n . The s t r a i n mixture hypothesis might explain Mac-Carthy' s (1963) f a i l u r e to obtain pure l i n e s of d i f f e r e n t s t r a i n s of PLRV by four s e r i a l transmissions. To explain the v a r i a b i l i t y of symptom expression under c o n t r o l l e d i n o c u l a t i o n and growing conditions, MacCarthy (1963) suggested three p o s s i b i l i t i e s : plants may vary even under uniform conditions; the virus s t r a i n s may be mixed i n the source plants i n varying r a t i o s and thus may be unusually d i f f i c u l t to separate; or. that the v i r u s changes within the plant or the vector. I t i s d i f f i -c u l t to categorize the s t r a i n s by v i s u a l assessment of symptom expre-ssio n . Wright et a l . (1967) indicated that symptom i n s t a b i l i t y was ob-served only when these s t r a i n s were transmitted by aphids to P. f l o r i d a n a or potato. They had observed symptoms i n s i x v a r i e t i e s of potato propagated by tubers, of very mild, mild, moderate and severe s t r a i n s which had remained unchanged f o r three years. The l e a f r o l l v i r u s i s o l a t e d from i n f e c t e d potato plants consis-t e n t l y produced varying degrees of stunting i n the i n d i c a t o r host P. f l o r i d a n a . T h i s observation indicated that my v i r u s source was carry-58 i n g a mixture of d i f f e r e n t s t r a i n s of PLRV. A f t e r several successive transmissions of vi r u s from a t e s t plant with the mildest symptom of PLRV, a pure stable l i n e of the mild s t r a i n was established. My experimental r e s u l t s with a mild s t r a i n showed that even a f t e r the fourth s e r i a l transmission, a range of symptoms on i n d i c a t o r plants was s t i l l obtained. The mild s t r a i n , free from the more severe s t r a i n s , was established when the number of transmissions was increased to 6. The f a c t that a stable l i n e of a mild s t r a i n was obtained places a new perspective on our concept of PLRV s t r a i n s . This work indicates that a s u f f i c i e n t number of t r a n s f e r s are necessary to obtain a stable i s o l a t e . The p o s s i b i l i t y remains that the s t a b i l i t y may break down with time and some v a r i a b i l i t y of symptom type may appear. In an e f f o r t to obtain the very severe s t r a i n of PLRV, the procedure which was applied f o r the mild s t r a i n was followed. I t i s more d i f f i c u l t to obtain a pure l i n e of the severe s t r a i n of PLRV since contaminating mild s t r a i n s cannot be detected by symptomatology. A second reason might be that P. f l o r i d a n a i s a good host f o r the mild s t r a i n and, whenever PLRV i s transmitted from a source with severe symptoms, the mild s t r a i n , although present i n low concentration, can mul t i p l y at a s u f f i c i e n t rate to be detected i n the next generation. P u r i f i c a t i o n of the mild s t r a i n of PLRV showed that the concen-t r a t i o n of v i r u s i n D. stramonium was higher than i n P. f l o r i d a n a or potato. T h i s r e s u l t and the symptomatology study on assay plants are supportive and in d i c a t e that PLRV occurs i n d i f f e r e n t s t r a i n s i n nature, the number of which should be investigated. Previous attempts to p u r i f y and characterize PLRV showed that even 59 under optimum conditions of propagation, a low v i r u s y i e l d was obtained. Estimates of v i r u s y i e l d from i n f e c t e d P. f l o r i d a n a t i s s u e varied from 0.01 (Kojima and Murayama, 1972b) to 0.4 mg/kg (Sarkar, 1976); from i n f e c t e d potato tissue the y i e l d varied from 0.03 to 0.1 mg/kg (Mehrad et a l . , 1 97 8) . Takanami and Kubo (1979a) reported a v i r u s y i e l d of 1.3 mg/kg "by using D r i s e l a s e enzyme. The maximum y i e l d of PLRV obtained i n t h i s study was 0.4-0.6 mg/kg. To obtain the maximum y i e l d of virus, emphasis was placed on v i r u s propagation and p u r i f i c a t i o n . The f i r s t problem was the choice of a comparative method f o r the various p u r i -f i c a t i o n treatments. Of the possible techniques a v a i l a b l e f o r assaying y i e l d s from various p u r i f i c a t i o n s , only sucrose density gradients scanned i n the UV monitor showed a peak which was s p e c i f i c to infected material. T h i s peak was thought to be caused by v i r u s p a r t i c l e s and t h i s was confirmed by electron microscopy and membrane feeding. I t was evident that the scanning of sucrose density gradients would be the i d e a l monitoring system f o r comparison of various p u r i f i c a t i o n schemes, since virus from as l i t t l e as 50 g of infected t i s s u e could be detected. Both the y i e l d and the p u r i t y of the v i r u s preparation a f t e r various treatments could be d i r e c t l y compared by scanning the gradients with the UV monitor. The f i r s t variable to be investigated was the propagative host, previous workers had used inf e c t e d P. f l o r i d a n a (Kojima and Murayama, 1972b; Sarkar, 1976; Hepp and de Zoeten, 1978) or potato plants (Mehrad et a l . , 1978) as a source of v i r u s f o r p u r i f i c a t i o n . Infected potato t i s s u e has not usually been used as a source of PLRV, possibly because potato c u l t i v a r s are frequently i n f e c t e d with l a t e n t viruses. The obvious danger i s that the f i n a l product could be contaminated with 6o viruses other than PLRV. One of the most common viruses i n f e c t i n g potato, potato v i r u s X (PVX), has a ^ of 117, which i s close to that of PLRV. When PVX in f e c t e d potato i s used f o r propagation of PLRV, the UV scan of PVX would he superimposed on that of ..PLRV. and,, because of the r e l a t i v e l y high concentration of PVX, the peak a t t r i b u t e d to PLRV w i l l be completely masked. I t i s therefore important that potato clones free from PVX be u t i l i z e d f o r propagating PLRV. Heat treatment and meristem cu l t u r e have y i e l d e d potato c u l t i v a r s which a f t e r repeated indexing proved to be free from a l l known potato viruses (Mellor and Stace-Smith, 1977)• PLRV was introduced into a v i r u s - f r e e plant and the inf e c t e d mother plant was subsequently propagated by t i p cuttings to provide a uniform supply of PLRV-infected t i s s u e . The plants were indexed at i n t e r v a l s to ensure that PLRV was the only virus being pro-pagated . When i n f e c t e d potato was compared with inf e c t e d P. f l o r i d a n a , D. stramonium and N. c l e v e l a n d i i as a source of vi r u s , the y i e l d from potato was greater than that obtained from P. f l o r i d a n a and D. s t r a -monium and the y i e l d from N. c l e v e l a n d i i was the lowest. The use of potato as the propagative host f o r PLRV had two d i s t i n c t advantages: the f i r s t was that the v i r u s y i e l d i s high; and the second was that potato grows quickly, providing more f o l i a g e per u n i t area than the other hosts. Greenhouse-grown potato was preferred over field-grown potato because the l e a f t i s s u e was more uniform and the v i r u s y i e l d more pre-d i c t a b l e . The protected environment of a greenhouse also reduced the r i s k of contamination of the source plants with other pathogens. In 61 the experiments, the vi r u s y i e l d was considerably l e s s from field-grown material but the comparison was made a f t e r the f i e l d plants had been exposed to extremes of l i g h t and temperature during the summer. F i e l d -grown material harvested i n the ea r l y summer may be more comparable to greenhouse-grown material and i f the greenhouse environment i s not av a i l a b l e and a large amount of v i r u s i s required, field-grown material could probably be substituted. Grinding of the t i s s u e and extraction of the vi r u s i s the important f i r s t step of p u r i f i c a t i o n . The l o s s of vi r u s at t h i s stage i s high since i t i s d i f f i c u l t to recover a large portion of v i r u s p a r t i c l e s from the pulp. As the experiments showed, a second extraction of the pulp y i e l d e d h a l f as much virus again as was recovered by the f i r s t e xtraction alone. Th i s i s because the vi r u s i s l i m i t e d to the phloem ti s s u e and breaking down the phloem t i s s u e to release V i r i o n s i s not as easy as r e l e a s i n g v i r i o n s from mesophyll t i s s u e . Brakke and Rochow (1974) faced the same problem with the p u r i f i c a t i o n of BYDV. They showed that the f i b r e remaining a f t e r grinding BYDV-infected leaves i n a blender contained at l e a s t three-quarters of the v i r i o n s . Takanami and Kubo (1979a) used Dr i s e l a s e enzyme to digest material before extraction. D r i s e l a s e has pectinase and c e l l u l a s e a c t i v i t i e s which dis r u p t the vascular t i s s u e s and release v i r u s p a r t i c l e s more e f f i c i e n t l y . Aggregation of PLRV p a r t i c l e s at d i f f e r e n t stages throughout the p u r i f i c a t i o n procedure may be due to hydrophobic i n t e r a c t i o n s . The ro l e of urea i n the resuspending buffer i s to weaken the a f f i n i t y of nonpolar areas on the surface of the v i r u s coat, thereby decreasing hydrophobic i n t e r a c t i o n s . This action of urea may be due to increased s o l u b i l i t y of nonpolar groups on the protein rather than s o l e l y as a 62 hydrogen bond-breaking reagent (Brunlng and Holtzer, I961). Aggregation also occurs i n concentrated pure preparation of PLRV: a f t e r resus-pending, the v i r u s preparation i s milky i n appearance, but a f t e r a few hours, the v i r u s p a r t i c l e s form a granular p r e c i p i t a t e . A d i l u t e preparation of PLRV does not e x h i b i t t h i s phenomenon. The sedimentation c o e f f i c i e n t of PLRV-particles was estimated by two d i f f e r e n t methods. Estimation through l i n e a r - l o g sucrose density, gradients gave a higher value than that obtained from a n a l y t i c a l cen-t r i f u g a t i o n . Brakke and Van P e l t (1970a) noted that the estimation of sedimentation c o e f f i c i e n t s by l i n e a r l o g gradients may d i f f e r by about % compared with other methods. The value of the sedimentation co-e f f i c i e n t of PLRV determined i n t h i s t h e s i s agrees with the preliminary report of Kojima and Murayama (1972b), who used sucrose density gradients to obtain an estimated S-value higher than 115-118 S. Takanami and Kubo ( l 9 7 9 a ) reported the sedimentation c o e f f i c i e n t of PLRV by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n to be 115 S, which agrees with the r e s u l t s reported i n t h i s t h e s i s when the same method was used. The density of PLRV-particles i n cesium chloride and cesium s u l -phate was 1.38-1.39 g/cm^, i n d i c a t i n g a compact n u c l e i c a c i d i n s i d e the coat-protein of the v i r i o n . By c a l c u l a t i o n , i t was shown that 26-28% of the t o t a l weight of the v i r i o n i s occupied by the nu c l e i c a c i d . The estimation of the sedimentation c o e f f i c i e n t of PLRV by sucrose density gradients may have been affe c t e d by the density of the particles.• A l l vir u s standards used f o r estimation of S2Q> ; w of PLRV had lower d e n s i t i e s than PLRV. In such a system, PLRV p a r t i c l e s can penetrate deeper i n t o the gradient and give correspondingly higher S-values than obtained 63 by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n . The S^Q y obtained by a n a l y t i c a l u l t r a c e n t r i f u g a t i o n i s more accurate because i t i s calculated d i r e c t l y and not by comparison with other p a r t i c l e s . The cal c u l a t e d moi wt f o r 6 6 PLRV-RNA was 2.0 x 10 , which i s close to the value 1.85 x 10 obtained by Mehrad et a l . (1979) and i d e n t i c a l to the value obtained by Takanami and Kubo (1979b). The moi wt of the RNAs of some other memebers of the lu t e o v i r u s group are, 2.0 x 10 f o r barley yellow dwarf virus (Brakke and Rochow, 1 9 7 4 ) , 1.9 x 1 0 ^ f o r beet western yellows virus (Falk et a l . , 1 9 7 7 ) , 2.0 x 10 f o r tobacco n e c r o t i c dwarf virus (Takanami and Kubo, 1979b) and 2.4 x 10 f o r pea l e a f r o l l v i r u s (Ashby and Hutting, 1979). According to Boedtker ( 1 9 6 8 ) , when the secondary structure of RNA i s destroyed by formaldehyde treatment, the sedimentation c o e f f i c i e n t should depend only on moi wt. Brakke and Van P e l t (1970b) obtained a r e l a t i o n s h i p between the sedimentation c o e f f i c i e n t of formaldehyde-treated RNA and i t s moi wt before formaldehyde treatment as described previously (see RESULTS). D u p l i c a t i n g the experimental conditions of Brakke and Van P e l t , a moi wt of 2.0 x 10 daltons was obtained f o r PLRV-RNA. Formaldehyde breaks down the secondary structure of RNA molecules and the sedimentation rate i n sucrose density gradients of these modified molecules i s correspondingly slower. Therefore, the rates of sedimentation f o r BMV, SBMV, TMV. .and PLRV-RNAs a f t e r formal-dehyde treatment are much l e s s than those obtained before formaldehyde treatment. The binding of formaldehyde by RNA i s temperature-dependent. V a r i a t i o n s of...pH over a range of f i v e to eight have no marked e f f e c t on the reaction, which i s consistent with the t h e o r e t i c a l consideration that the aminogroups of the bases are not di s s o c i a t e d within t h i s range, 64 but the s a l t concentration of the medium greatly a f f e c t s the amount of formaldehyde bound (Staehlin, 1 9 5 8 ) . The melting behavior of PLRV-nucleic a c i d was s i m i l a r to RNA of the single-stranded type. A double-stranded DNA, c a l f thymus-DNA, was used as a c o n t r o l . C a l f thymus-DNA was dissolved i n 0.1 x SSC b u f f e r and a value of 73 0 was obtained f o r i t s Tm.. The. T m of c a l f thymus-DNA i n SSC b u f f e r i s about 86 C (Marmur and Doty, 1 9 5 9 ) , "but the T m of DNA changes and s h i f t s 15-16 C lower i n 0.1 x SSC buffer (Mandel and Marmur, I968). The value obtained f o r c a l f thymus-DNA ind i c a t e s the accuracy of the technique used i n the experiments reported here and agrees with the reported value. Polyacrylamide-SDS g e l electrophoresis of d i s s o c i a t e d PLRV suggests that the capsid of t h i s v i r u s i s comprised of a s i n g l e kind of protein subunit with an average moi wt of 26 300. The binding of sodium dodecyl s u l f a t e ions to proteins has been shown f o r several protein molecules and was assumed to be the b a s i s of the separation of the denatured proteins upon SDS electrophoresis on polyacrylamide gels (Shapiro et a l . , I967). The e l e c t r o p h o r e t i c m o b i l i t i e s of d i f f e r e n t proteins through polyacrylamide gels are..independent of t h e i r i s o e l e c t r i c points and t h e i r aminoacid compositions and seem to be governed s o l e l y by the moi wt of t h e i r polypeptide chains (Weber and Osborn, 1 9 6 9 ) . The good re s o l u t i o n , the small amount of protein needed and the f a c t that an estimate of the moi wt can be obtained within a day, makes SDS gel electrophoresis a preferred method f o r moi wt determination. Considering the importance of diagnosing and c o n t r o l l i n g PLRV, e f f o r t s have been made to develop a quick and r e l i a b l e diagnostic tech-65 nique. The method used at present i n Canada and the United States to deter-mine the percent i n f e c t i o n of seed-potatoes i s to check f o r l e a f r o l l symp-toms a f t e r growing a sample of tubers e i t h e r i n a greenhouse or i n a southern area such as F l o r i d a or C a l i f o r n i a . Indexing by t h i s technique usually takes about 6 wk but i t i s not r e l i a b l e since i n f e c t i o n with the mild s t r a i n s may not produce l e a f r o l l i n g symptoms, e s p e c i a l l y i f the plants are w e l l f e r t i l i z e d . ELISA seems promising f o r use i n seed-potato c e r t i f i c a -t i o n programs, since the r e s u l t s i n t h i s t h e s i s show that the v i r u s was detected by t h i s technique i n sap from i n f e c t e d plants which had been d i l u t e d 1:320. Other i n v e s t i g a t o r s a l s o suggested that ELISA could be used ro u t i n e l y f o r indexing PLRV i n seed-potato c e r t i f i c a t i o n programs (Mehrad et al . , 1 9 ? 8 ; Clarke et a l . , 1 9 8 0 ) . ELISA d i d not provide r e l i a b l e detection of PLRV i n tuber t i s s u e because of the high nonspecific reaction possibly due to interference by starch granules. Thus, the technique at i t s present stage of development apparently can not.be applied i n seed-potato c e r t i f i c a t i o n programs to detect the v i r u s i n the tubers. According to the r e s u l t s obtained by other workers, the i s o l a t e s of PLRV from Germany, Japan, the United States and Canada are s e r o l o g i c a l l y r e l a t e d (Casper, 1977; Clarke et a l . , I98O). My i s o l a t e of PLRV reacted with a n t i s e r a prepared by Sarkar and Kojima, but i t was not possible to determine i f , i n f a c t , the immunoprecipitates which were obtained were i d e n t i c a l . Consequently i t can not be concluded from my experiments that my i s o l a t e of PLRV i s r e l a t e d to the German or Japanese i s o l a t e . Some of the properties of PLRV reported i n t h i s t h e s i s c o n f l i c t with previously published reports. The subunit moi wt of the i s o l a t e from B r i t i s h Columbia was 26 300, whereas Sarkar (1978),using a German 66 i s o l a t e , estimated the subunit moi wt as 15 000. A more s i g n i f i c a n t discrepancy, from a taxonomic point of view, i s whether PLRV contains DNA or RNA as i t s n u c l e i c a c i d . Sarkar (1976) reported that the German i s o l a t e reacted with diphenylamine, was s e n s i t i v e to DNase but not to RNase and had a sharp melting temperature curve. On the basis of these r e s u l t s , he concluded that the n u c l e i c a c i d of PLRV i s double-stranded DNA. Based on the r e s u l t s obtained f o r t h i s t h e s i s , i t i s concluded that the n u c l e i c a c i d of PLRV i s single-stranded RNA. Two independent reports (Mehrad et a l . , 1979; Takanami and Kubo, 1979h) agree with the r e s u l t s reported i n t h i s t h e s i s and confirm that the n u c l e i c a c i d of PLRV i s of the RNA type. There are three hypothesis that could explain the discrepancy between my. .results and those obtained by Sarkar: 1. Potato l e a f r o l l disease may be induced by two s e r o l o g i c a l l y r e l a t e d viruses, one an.RNA virus and the other a DNA vi r u s or the PLRV-infected .plants may be;contaminated with an unknown DNA type v i r u s ; 2. Preparations of PLRV may be contaminated with host DNA and/or host protein; 3. The y i e l d of PLRV may be too low to permit accurate c h a r a c t e r i -zation. The f i r s t hypothesis was rejected*as there i s no s e r o l o g i c a l evidence at the present time to support the idea that two viruses with d i f f e r e n t types of n u c l e i c a c i d may be involved. S p e c i f i c a l l y , the r e s u l t s i n d i c a t e that the virus used by Sarkar i n h i s studies i s sero-l o g i c a l l y the same as the one used f o r t h i s study. Sarkar's material may have been infected with two viruses: one a t y p i c a l PLRV and the 67 other an unknown DNA v i r u s capable of i n f e c t i n g solanaceous plants. I t would have to "be speculated that with the p u r i f i c a t i o n procedure' Sarkar used, the DNA-virus was recovered i n l a r g e r q u a n t i t i e s than PLRV. However, PLRV was recovered i n s u f f i c i e n t q u a n t i t i e s to be detected by membrane feeding t e s t s , and to induce s p e c i f i c antibody production i n rabbits, but i n i n s u f f i c i e n t amounts f o r accurate c h a r a c t e r i z a t i o n with respect to the type of n u c l e i c a c i d . The DNA-vi r u s , which was e i t h e r more stable or present i n higher concentrations, would y i e l d s u f f i c i e n t DNA to show a trace r e a c t i o n . This speculation i s u n l i k e l y since small plant viruses of the DNA type are rare, occurring mainly i n the t r o p i c s and are vectored by white f l i e s or leafhopper, not by aphids (Harrison et a l . , 1 9 7 7 ) ' One group of viruses, the geminiviruses, have 15-20 nm isometric p a r t i c l e s which us u a l l y occur i n p a i r s , and which contain c i r c u l a r single-stranded DNA with moi wt of 0.66-0.95 x 10^. Larger plant DNA viruses are also known which belong to the caulimovirus group (Fenner, 1 9 7 6 ) . A member of t h i s group,'if: present, would be detected by i t s d i s t i n c t s i z e (50 nm) and S 2 0 W (200-250 S) compared with PLRV. I t i s possible therefore, but not l i k e l y that Sarkar's preparations were contaminated with a new DNA,virus belonging to an undefined group of plant viruses of s i m i l a r size to PLRV and capable of i n f e c t i n g solanaceous plants. In the second hypothesis, i t i s u n l i k e l y that a l a r g e r amount of host proteins and DNA would accumulate on or around the PLRV p a r t i c l e s at the same time. I t can not be assumed that proteins or DNA would be present as free e n t i t i e s i n the virus preparation because they would have been eliminated during the p u r i f i c a t i o n procedure. However, 68 contamination of the pure preparation with host DNA could explain some of the discrepancies between my r e s u l t s and those of Sarkar. Mehrad et a l . (1979) reported that PLRV preparations, e s p e c i a l l y from leaves, can he contaminated with a large amount of heterogeneous, DNase-se n s i t i v e material. Sarkar used a lengthy p u r i f i c a t i o n procedure in v o l v i n g several f r e e z i n g and thawing steps which, when repeated In my experiments, l e d to the d i s i n t e g r a t i o n of the majority of PLRV p a r t i c l e s . The RNA may have been released and l o s t through fractures i n the protein s h e l l of the damaged p a r t i c l e s . DNase-sensitive material could have become adsorbed to the surface of the damage PLRV p a r t i c l e s , leading to the impression that DNA was the n u c l e i c a c i d associated with the PLRV v i r i o n . The t h i r d hypothesis, i . e . that Sarkar was working with micro-q u a n t i t i e s of PLRV seems to be the most pla u s i b l e explanation f o r the discrepancy between h i s r e s u l t s and mine. When small q u a n t i t i e s of DNA or RNA are reacted with .diphenylamine or o r c i n o l reagents, the c o l o r reaction may be i n d i s t i n g u i s h a b l e from the c o n t r o l . Traces of sugar i n the preparation can also react with diphenylamine and i n t e r -fere with the i n t e r p r e t a t i o n of the r e s u l t s . Sarkar (1976) treated the PLRV-nucleic a c i d with. DNase and RNase and found that a f t e r t r e a t -ment with DNase, the UV-absorption of the mixture at 260 nm was greater than that f o r n u c l e i c a c i d treated with RNase. The inherent e r r o r introduced by working with microquantities could not be avoided. Sarkar estimated the coat-protein moi wt of PLRV to be 15 000 i n poly-acrylamide g e l electrophoresis, but with low concentrations of protein, i t i s not possible to d i s t i n g u i s h the v i r a l p r otein band from other 69 bands which may e x i s t on the g e l . In a l l experiments reported i n t h i s thesis where pure PLRV-preparations were needed, highly p u r i f i e d , concentrated preparations were used to avoid a l l possible e r r o r s . Most experiments were repeated at l e a s t 3 times and the r e s u l t s obtained from d i f f e r e n t r e p l i c a t e s were very close or i d e n t i c a l . As our knowledge of plant viruses increases, considerable atten-t i o n i s devoted to c l a s s i f y i n g i n d i v i d u a l viruses i n t o groups that share s i m i l a r properties, and dendrograms have been constructed which provide convenient comparison among viruses. Tremaine (1977) construct-ed a dendrogram by s e l e c t i n g a sequence of c h a r a c t e r i s t i c s of the 22 v i r u s groups and 15 ungrouped viruses, where the nature of the n u c l e i c acid was of primary importance. I f PLRV-nucleic a c i d i s considered to be double-stranded DNA, the v i r u s must be placed near the caulimo-vi r u s group. However, i f i t s n u c l e i c a c i d i s considered to be s i n g l e -stranded RNA, as the r e s u l t s obtained i n t h i s thesis and those of others (Mehrad et a l . , 1979; Takanami and Kubo, 19791>) i n d i c a t e , the v i r u s would be included i n the l u t e o v i r u s group with BYDV, BWYV, SDV and several other viruses (Shepherd et a l . , 1 9 7 6 ) . The main c h a r a c t e r i s t i c s of the l u t e o v i r u s group are; isometric v i r i o n s about 25 nm i n diameter with a sedimentation c o e f f i c i e n t of 115-118 S; coat-protein subunit moi wt ca. 24 000; a single-stranded RNA genome with moi wt 2.0 x 10 ; low v i r u s concentration i n the host; non-transmissible by mechanical means and aphid transmissible i n a p e r s i s t e n t manner. PLRV, as characterized i n t h i s t h e s i s , conforms to a l l the major c r i t e r i a f o r i n c l u s i o n i n the l u t e o v i r u s group. 70 SUMMARY Potato l e a f r o l l v irus (PLRV) causes a disease of s i g n i f i c a n t i n t e r -n a t i o n a l importance i n potato "but, u n t i l recently, l i t t l e was known about the properties of the causal agent. The main reason so l i t t l e was known about the vi r u s properties i s the great d i f f i c u l t y i n obtain-i n g pure v i r u s preparations f o r characterization studies. As a d i r e c t r e s u l t of the need f o r f u r t h e r information, both p r a c t i c a l l y and theo-r e t i c a l l y a study of PLRV was selected as a th e s i s t o p i c . This t h e s i s contains o r i g i n a l contributions, e s p e c i a l l y i n the following areas: - I s o l a t i o n of a pure l i n e of the mild s t r a i n of PLRV; - development of a technique to determine v i r u s y i e l d ; - development of an e f f e c t i v e p u r i f i c a t i o n procedure; - preparation of s p e c i f i c antisera against a mixture of the v i r u s s t r a i n s and against the mild s t r a i n ; - a p p l i c a t i o n of the enzyme-linked immunosorbent assay f o r v i r u s detection and i t s u t i l i z a t i o n i n the B r i t i s h Columbia potato cer-t i f i c a t i o n scheme; - determination of the sedimentation c o e f f i c i e n t and density of the v i r i o n ; - determination of the type, molecular weight, sedimentation c o e f f i -cient and melting-temperature of the n u c l e i c acid; - determination of the molecular weight of the coat-protein subunits and the probable capsid structure; - c l a s s i f i c a t i o n of PLRV i n the lute o v i r u s group as a consequence of the more complete char a c t e r i z a t i o n of the v i r u s . 71 LITERATURE CITED Adesnik, M. 1971- Polyacrylamide g e l electrophoresis of v i r a l RNA. 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