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Two viruses associated with blueberry scorch disease MacDonald, Stuart Gerald 1989

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TWO VIRUSES ASSOCIATED WITH BLUEBERRY SCORCH DISEASE by Stuart Gerald MacDonald B.Sc. (Agr.), The U n i v e r s i t y of B r i t i s h Columbia A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n THE FACULTY OF GRADUATE STUDIES (The Department of Plant Science) We accept t h i s t h e s i s as conforming to the r e q u i r e d standard THE UNIVERSITY OF BRITISH COLUMBIA November, 1989 ©Stuart Gerald MacDonald, 1 9 8 9 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of The University of British Columbia Vancouver, Canada Date rJcvesmiocr 2 7 / f ^ ? D E - 6 (2/88) ABSTRACT i i Blueberry bushes with scorch symptoms were found during a survey of blueberry f i e l d s i n B r i t i s h Columbia, Washington, and Oregon. Some of these bushes were i n f e c t e d with blueberry scorch v i r u s (BBScV) while others contained a second v i r u s which was sap t r a n s m i s s i b l e to Nicotiana clevelandii, N. benthamiana, and N. tabacum cv. ''Havana 425' . This v i r u s was p u r i f i e d from N. clevelandii and had iso m e t r i c p a r t i c l e s of approximately 30 nm i n diameter, a coat p r o t e i n subunit of 27,500 daltons and a t r i p a r t i t e genome. I was unable to t r a n s f e r the v i r u s from e i t h e r i n f e c t e d N. clevelandii or i n f e c t e d blueberry to healthy N. clevelandii with Myzus persicae or Fimbriaphis fimbriata. S e r o l o g i c a l t e s t s of t h i s blueberry v i r u s with a n t i s e r a against members of the i l a r - , cucumo-, bromo-, or nepovirus groups f a i l e d to i n d i c a t e any r e l a t i o n s h i p . In a subsequent survey using enzyme-linked immunosorbent assay, t h i s i s o m e t r i c v i r u s was found i n blueberry plants from northern Washington state to c e n t r a l Oregon but has not yet been found i n B.C. Of the e s t a b l i s h e d members of the c a r l a v i r u s group examined, BBScV i s most c l o s e l y r e l a t e d to potato v i r u s S (PVS) and l e s s c l o s e l y r e l a t e d to carnation l a t e n t v i r u s (CLV) and potato v i r u s M (PVM). The d i f f e r e n c e i n host range between BBScV and PVS would i n d i c a t e that the BBScV i s not a s t r a i n of PVS but i s a separate v i r u s that i s r e l a t e d to PVS. Therefore, BBScV should be renamed blueberry scorch c a r l a v i r u s (BBSCV). BBSCV was also compared to a c a r l a v i r u s i s o l a t e d from blueberry i n the Sheep Pen H i l l blueberry growing area of New Jersey ( r e f e r r e d to as SPHV). These v i r u s e s were compared s e r o l o g i c a l l y and by use of n u c l e i c a c i d h y b r i d i z a t i o n s . BBSCV and SPHV were found to be c l o s e l y r e l a t e d and were concluded to be s t r a i n s of the same v i r u s . SPHV should be named the New Jersey s t r a i n of BBSCV. i v TABLE OF CONTENTS ABSTRACT i i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i ACKNOWLEDGEMENT v i i i GENERAL INTRODUCTION 1 CHAPTER ONE - ISOLATION AND CHARACTERIZATION OF AN ISOMETRIC VIRUS ASSOCIATED WITH BLUEBERRY SCORCH DISEASE 7 INTRODUCTION 7 MATERIALS AND METHODS 9 VIRUS ISOLATES 9 MECHANICAL TRANSMISSION OF THE BLUEBERRY ISOMETRIC VIRUS 9 ELECTRON MICROSCOPY 11 VIRUS PURIFICATION 12 DETERMINATION OF THE RELATIVE MOLECULAR MASS OF THE VIRAL COAT PROTEIN 14 RNA EXTRACTION 16 ISOLATION OF DOUBLE-STRANDED RNA . . . . . . . . 18 SEROLOGY OF THE BLUEBERRY ISOMETRIC VIRUS . . . 20 SURVEY FOR .BLUEBERRY SCORCH VIRUS AND THE BLUEBERRY ISOMETRIC VIRUS 22 APHID TRANSMISSION EXPERIMENTS 25. RESULTS 2 6 MECHANICAL TRANSMISSION OF BLUEBERRY ISOMETRIC VIRUS 2 6 VIRUS PURIFICATION 2 9 DETERMINATION OF THE RELATIVE MOLECULAR MASS OF THE VIRAL COAT PROTEIN SUBUNIT 30 RNA EXTRACTION 33 DOUBLE-STRANDED RNA EXTRACTION 33 SEROLOGY OF THE BLUEBERRY ISOMETRIC VIRUS . . . 37 SURVEY FOR BLUEBERRY SCORCH VIRUS 38 APHID TRANSMISSIONS 38 V DISCUSSION 41 CHAPTER TWO- COMPARISON OF BLUEBERRY SCORCH VIRUS WITH SHEEP PEN HILL VIRUS AND OTHER CARLAVIRUSES 4 5 INTRODUCTION 4 5 MATERIALS AND METHODS 4 6 CARLAVIRUS ISOLATES 4 6 SEROLOGICAL COMPARISONS OF THE CARLAVIRUSES . . 4 7 CARLAVIRUS RNA EXTRACTION 4 9 PREPARATION OF RANDOM PRIMED cDNA PROBES . . . 50 HYBRIDIZATIONS 51 RESULTS 53 CARLAVIRUS ISOLATES 53 SEROLOGICAL COMPARISONS OF THE CARLAVIRUSES . . 53 NUCLEIC ACID HYBRIDIZATIONS OF CARLAVIRUSES . . 54 DISCUSSION 59 GENERAL DISCUSSION 65 REFERENCES 68 v i LIST OF TABLES Table I. Survey r e s u l t s of t e s t s f o r blueberry scorch v i r u s , blueberry isometric v i r u s and tomato ringspot v i r u s i n B.C., Washington, and Oregon f o r 1987 and 1989 40 Table I I . C h a r a c t e r i s t i c s of Blueberry Isometric V i r u s . 43 Table I I I . End point t i t e r s of several p o l y c l o n a l a n t i s e r a i n homologous and heterologous i n d i r e c t ELISA r e a c t i o n s 55 Table IV. End.point t i t e r s of several p o l y c l o n a l a n t i s e r a against BBScV and SPHV i n i n d i r e c t ELISA r e a c t i o n s 56 Table V. Detection of homologous and heterologous c a r l a v i r u s e s i n double antibody sandwich ELISA . . 57 Table VI. End point t i t e r s of homologous and heterologous p o l y c l o n a l a n t i s e r a i n double antibody sandwich ELISA 58 v i i LIST OF FIGURES F i g . 1. BBScV negative Bluecrop blueberry plants i n Clark County, Washington e x h i b i t i n g l e a f and blossom b l i g h t 8 F i g . 2 . BBScV negative Bluecrop blueberry plants i n Whatcom County, Washington e x h i b i t i n g blossom and l e a f b l i g h t 8 F i g . 3 . Ringspots and oakleaf patterns on N. clevelandii l e a f 7 days a f t e r mechanical i n o c u l a t i o n from blossoms of symptomatic blueberry p l a n t s . 27 F i g . 4. Systemic n e c r o t i c l e s i o n s on expanding leaves of N. clevelandii 12 days a f t e r mechanical i n o c u l a t i o n . 27 F i g . 5. Symptoms on N. tabacum 'Havana 425' mechanically i n o c u l a t e d with leaves from i n f e c t e d N. clevelandii. 28 F i g . 6. Symptoms on N. benthamiana mechanically i n o c u l a t e d with leaves from i n f e c t e d N. clevelandii. 28 F i g . 7. E l e c t r o n micrograph of a t h i n s e c t i o n of i n f e c t e d N. clevelandii showing isometric p a r t i c l e s i n the cytoplasm and i n tubules 31 F i g . 8. Isometric p a r t i c l e s trapped out of diseased blueberry on g r i d s coated with homologous antiserum (Bar represents 100 nm) 31 F i g . 9. E l e c t r o n micrograph of negatively stained i s o m e t r i c v i r u s p a r t i c l e s from a p u r i f i e d preparation (Bar represents 100 nm) 32 F i g . 10 . Polyacrylamide g e l of the blueberry i s o m e t r i c v i r u s coat p r o t e i n 34 F i g . 11 . Polyacrylamide g e l and western b l o t of the blueberry isometric v i r u s coat p r o t e i n . . . . . . . 35 F i g . 12 . Methylmercuric hydroxide denaturing g e l of blueberry isometric v i r u s RNA 36 F i g . 13 . Non-denaturing g e l of RNase and DNase digests of blueberry isometric v i r u s RNA 39 F i g . 14. Methylmercuric hydroxide denaturing g e l of s e v e r a l c a r l a v i r u s RNA 60 F i g . 15 . H y b r i d i z a t i o n s of s i x c a r l a v i r u s e s with homologous and heterologous random primed cDNA probes i n dot b l o t h y b r i d i z a t i o n s 61 v i i i ACKNOWLEDGEMENT The author wishes to thank the members of h i s committee: Dr. R.R. Martin, A g r i c u l t u r e Canada Research Station, Vancouver; Dr. R.J. Copeman and Dr. B. E. E l l i s , Department of Plant Science, U n i v e r s i t y of B r i t i s h Columbia; Dr. R.A.J. Warren, Department of Microbiology, U n i v e r s i t y of B r i t i s h Columbia; and Dr. J.B. Hudson, Department of Pathology, U n i v e r s i t y of B r i t i s h Columbia f o r t h e i r guidance, c r i t i c i s m and suggestions. The author a l s o wishes to thank Mr. F. Skelton and Dr. F. Leggett f o r assistance with e l e c t r o n microscopy, Mr. W. MacDiarmid f o r photography, and other members of the s t a f f at the A g r i c u l t u r e Canada Research Sta t i o n , Vancouver f o r t h e i r a s s i s t a n c e . The f i n a n c i a l support of t h i s research from the B.C. Blueberry Co-op, the Washington Blueberry Commission, the Oregon Blueberry Commission and ARDSA i s g r a t e f u l l y acknowledged. 1 GENERAL INTRODUCTION Blueberry belongs to the genus Vaccinium i n the heath family (Ericaceae). The most important commercial species are highbush (V. corymbosum L. and V. australe Small), lowbush (V. angustifolium A i t . ) , and rabbiteye (V. ashei Reade) blueberry. These species are native to North America and a few commercial v a r i e t i e s are s e l e c t i o n s from the wil d . Highbush and rabbiteye b l u e b e r r i e s are u s u a l l y r e f e r r e d to as c u l t i v a t e d b l u e b e r r i e s while lowbush b l u e b e r r i e s are r e f e r r e d to as w i l d b l u e b e r r i e s . The acreage devoted to c u l t i v a t e d b l u e b e r r i e s has increased d r a m a t i c a l l y over the l a s t 40 years with new production areas developing i n A u s t r a l i a , New Zealand, Europe, and Japan. There i s an estimated 19 ,088 ha of c u l t i v a t e d b l u e b e r r i e s worldwide of which 18 ,088 ha are i n North America. Of the 18 ,088 ha i n North America 14 ,470 ha are highbush and 3 ,618 ha are rabbiteye (Eck, 1 9 8 8 ) . T o t a l North American blueberry production (both w i l d and cu l t i v a t e d ) was 214 m i l l i o n lbs i n 1988 with 120 m i l l i o n lbs of c u l t i v a t e d b l u e b e r r i e s and 94 m i l l i o n lbs of wil d b l u e b e r r i e s (B.C. Blueberry Co-op, 1989 ) . 2 The major production areas are Michigan, New Jersey, B r i t i s h Columbia, North C a r o l i n a , Washington and Oregon (Eck, 1988) . The approximate 1988 production f o r B.C., Washington and Oregon was 16, 8, and 8 m i l l i o n l b r e s p e c t i v e l y . A l l the production i n B.C., Washington and Oregon i s highbush blueberry with no commercial production of lowbush or rabbiteye b l u e b e r r i e s . There are a number of v i r u s and v i r u s - l i k e diseases which are important i n blueberry c u l t u r e . The l i t e r a t u r e up to 1982 was reviewed i n the USDA Handbook of Virus Diseases of Small F r u i t s (Converse, 1987) . A b r i e f overview of these v i r u s e s and v i r u s - l i k e diseases w i l l be given here. One of the main v i r u s diseases of blueberry i s blueberry shoestring v i r u s (BBSSV). Blueberry shoestring mainly i n f e c t s highbush b l u e b e r r i e s and i s transmitted by the blueberry aphid Illinois pepperi (Macgillivary) . It has not yet been assigned to a v i r u s group although i t shares some of the p h y s i c a l and chemical p r o p e r t i e s of the sobemovirus group (Ramsdell, 1979 ) . BBSSV has been reported i n Michigan, New Jersey, Washington, and North C a r o l i n a (Ramsdell, 1987a ) . Only one f i e l d i n Washington state has been i n f e c t e d with BBSSV (Bristow and Ramsdell, 1984) and the i n f e c t e d plants 3 have been removed. There are four nepoviruses which i n f e c t blueberry: tomato ringspot, tobacco ringspot (necrotic r i n g s p o t ) , blueberry l e a f mottle and peach rose t t e (Ramsdell et al., 1987a) . The symptoms of these v i r u s e s vary i n s u s c e p t i b l e v a r i e t i e s with symptoms i n c l u d i n g stem dieback, stunting, and c h l o r o t i c , mottled, misshapen leaves. Nepoviruses are transmitted by nematodes, but a nematode vector has not yet been i d e n t i f i e d f o r blueberry mottle. Tomato ringspot has been found i n Washington (Johnson, 1972; S.G. MacDonald, unpublished data) and Oregon (Converse and Ramsdell, 1982; S.G. Macdonald, unpublished data). Necrotic ringspot has been found i n the eastern- United States and Oregon (Converse and Ramsdell, 1982) . Blueberry l e a f mottle and peach r o s e t t e have only been found i n eastern North America (Ramsdell and G i l l e t , 1981: Ramsdell, 1987b). Blueberry red ringspot v i r u s (BRRV) i s an i s o m e t r i c (42-46 nm) p a r t i c l e (Kim et al., 1981) which occurs i n New Jersey, Arkansas, Michigan, Connecticut, Massachusetts, New York, North C a r o l i n a , and Oregon (Ramsdell et al., 1987b). The symptoms of t h i s disease are red c i r c u l a r spots or blotches on the stems and on the leaves (Hutchinson and 4 Varney, 1954). A s e r o l o g i c a l r e l a t i o n s h i p between blueberry red ringspot v i r u s and c a u l i f l o w e r mosaic v i r u s has been demonstrated (Hepp and Converse, 1989). Blueberry mosaic (BBM) i s another disease of highbush b l u e b e r r i e s . The symptoms are mottling of the leaves and mosaic patterns of chrome-yellow, yellow, yellow-green and o c c a s i o n a l l y pink. The disease has been observed on older c u l t i v a r s such as Cabot, Concord, E a r l i b l u e , Pioneer, Rubel and Stanley i n the eastern United States, Michigan, Indiana, Oregon and B r i t i s h Columbia (Ramsdell and Stretch, 1987b). The causal agent of BBM i s not known, however s p h e r i c a l p a r t i c l e s (28-30 nm) (Ramsdell and Stretch, 1987b) and filamentous v i r u s - l i k e p a r t i c l e s (Kim and Gergerich, 1985) have been observed i n t h i n sections of i n f e c t e d t i s s u e but not i n t h i n sections of healthy t i s s u e . Two diseases of b l u e b e r r i e s are caused by mycoplasma-like organisms; blueberry stunt and witches'-broom. The symptoms of blueberry stunt on highbush blueberry are stunted plants with smaller, puckered c h l o r o t i c leaves (Hutchinson et al., 1960) . Every c u l t i v a r except Rancocas i s s u s c e p t i b l e and blueberry stunt has been found i n Eastern Canada, Maine, Massachusetts, New Hampshire, New York, Michigan, N. 5 Ca r o l i n a , Pennsylvania, Maryland, V i r g i n i a and Arkansas (Ramsdell and Stretch, 1 9 8 7 a ) . Witches'-broom has only been reported i n Europe (de Leeuw, 1987) and the disease i s most important i n V. myrtillus L. Symptoms include upright new growth with short internodes and m u l t i p l e branching (witches'-broom) , smaller c h l o r o t i c leaves and i n f e c t e d plants f a i l i n g to flower. Both witches'-broom and blueberry stunt are vectored i n t h e i r hosts by d i f f e r e n t species of leafhoppers (de Leeuw, 1987 ) . Blueberry scorch disease was f i r s t seen' i n 1980 near Puyallup, WA and was shown to be associated with a c a r l a v i r u s (Martin and Bristow, 1988) . This c a r l a v i r u s has been designated as blueberry scorch v i r u s (BBScV). The symptoms of blueberry scorch occur during bloom as a blossom b l i g h t with some n e c r o t i c leaves near the b l i g h t e d flower c l u s t e r s . The s e v e r i t y of the symptoms v a r i e s with c u l t i v a r . Berkeley plants are k i l l e d i n 3-4 years while Pemberton pla n t s survive much longer e x h i b i t i n g a progressive d e c l i n e . Symptoms appear on i n f e c t e d plants every year with the r e s u l t i n g l o s s of y i e l d . Spread of the disease has been quite r a p i d through three a d j o i n i n g farms i n the Puyallup V a l l e y while the spread i n an i n f e c t e d f i e l d i n Clark Co., WA has been slower. The 6 d i f f e r e n c e s i n rate of disease spread at the two s i t e s may be due to d i f f e r e n c e s i n c u l t u r a l p r a c t i c e s such as i n s e c t and weed c o n t r o l . Another v i r u s was f i r s t reported by St r e t c h (1983) i n the Sheep Pen H i l l growing region of New Jersey. This v i r u s has been shown to be a c a r l a v i r u s (Podleckis and Davis, 1987) and w i l l be r e f e r r e d to as Sheep Pen H i l l v i r u s (SPHV) . The symptoms of SPHV are very s i m i l a r to those caused by blueberry scorch disease except that there i s an autumn f o l i a r l i n e p a t t e r n . 7 CHAPTER ONE - ISOLATION AND CHARACTERIZATION OF AN ISOMETRIC VIRUS ASSOCIATED WITH BLUEBERRY SCORCH DISEASE INTRODUCTION Blueberry f i e l d s i n B r i t i s h Columbia, Washington and Oregon were surveyed f o r BBScV i n 1987. Bushes i n se v e r a l f i e l d s had the blossom and l e a f b l i g h t i n g c h a r a c t e r i s t i c of BBScV but were negative f o r BBScV by enzyme-linked immunosorbent assay (ELISA). Figures 1 and 2 show plants which have scorch symptoms but which are negative f o r BBScV. No other cause of these symptoms such as f r o s t i n j u r y , mummy berry blossom b l i g h t , b a c t e r i a l b l i g h t , or B o t r y t i s blossom b l i g h t could be found. These symptoms d i f f e r e d s l i g h t l y from those caused by BBScV i n f e c t i o n . There was l e s s regrowth of f o l i a g e during the growing season and these bushes would show symptoms f o r 1 to 4 years then recover, e v e n t u a l l y becoming symptomless. Bushes i n f e c t e d with BBScV show symptoms every year. The v a r i e t i e s which showed these symptoms but which were negative f o r the c a r l a v i r u s included Bluecrop, Bluejay, Berkeley, Pemberton, D i x i , B luetta, Rancocas, E a r l i b l u e , F i g . 1. B B S c V n e g a t i v e B l u e c r o p b l u e b e r r y p l a n t s i n C l a r k C o u n t y , W a s h i n g t o n e x h i b i t i n g l e a f a n d b l o s s o m b l i g h t . F i g . 2 . B B S c V n e g a t i v e B l u e c r o p b l u e b e r r y p l a n t s i n Whatcom C o u n t y , W a s h i n g t o n e x h i b i t i n g b l o s s o m a n d l e a f b l i g h t . 9 a n d J e r s e y . T he o b j e c t i v e o f t h i s p o r t i o n o f t h e r e s e a r c h was t o p u r i f y a n d c h a r a c t e r i z e t h e c a u s a l a g e n t o f b l u e b e r r y s c o r c h d i s e a s e i n p l a n t s n e g a t i v e f o r B B S c V . I f t h e c a u s a l a g e n t was a v i r u s t h e n a n E L I S A t e s t w o u l d b e d e v e l o p e d a n d a s u r v e y w o u l d b e c o n d u c t e d t o d e t e r m i n e t h e r a n g e o f t h i s s e c o n d s c o r c h v i r u s . M A T E R I A L S AND METHODS V I R U S I S O L A T E S Two i s o l a t e s o f t h e s e c o n d s c o r c h v i r u s ( b l u e b e r r y i s o m e t r i c v i r u s , B I V ) w e r e s t u d i e d , one f r o m a f i e l d i n C l a r k C o u n t y i n s o u t h e r n W a s h i n g t o n s t a t e ( B a k e r i s o l a t e ) a n d o n e f r o m a f i e l d i n Whatcom c o u n t y i n n o r t h e r n W a s h i n g t o n ( A n d e r s o n i s o l a t e ) . The B B S c V i s o l a t e was a s d e s c r i b e d b y M a r t i n a n d B r i s t o w ( 1 9 8 8 ) . 10 MECHANICAL TRANSMISSION OF THE BLUEBERRY ISOMETRIC VIRUS Branches were c o l l e c t e d from diseased bushes i n a f i e l d i n C l ark County, WA which had scorch symptoms but which were negative f o r BBScV by ELISA. These branches were c o l l e c t e d i n January, 1988 fumigated i n a methyl bromide chamber and then put i n jar s of water i n the greenhouse to blossom. Blossoms were c o l l e c t e d and homogenized i n 0.05 M sodium phosphate, pH. 7.0, containing 2% p o l y v i n y l p y r r o l i d o n e 44 (PVP) then mechanically in o c u l a t e d to three p l a n t s each of Brassica juncea L. Czerniak ' F l o r i d a Broadleaf', Chenopodium amaranticolor Coste & Reyn., C. quinoa W i l l d . , Gomphrena globosa L., Nicotiana clevlandii Gray, Petunia hybrida Hort. Vilm. Andr. 'Coral Satin', Phaseolus vulgaris L. ''Top Crop', and Lycopersicon esculentum M i l l . 'Rutgers'. Plants mock ino c u l a t e d with b u f f e r served as healthy c o n t r o l s . Plants were monitored f o r symptoms and any a t y p i c a l plants were assayed f o r v i r u s i n f e c t i o n by e l e c t r o n microscopy using the l e a f dip technique (see 'Electron Microscopy' s e c t i o n ) . N. clevlandii plants i n f e c t e d with BIV were also homogenized and mechanically inoculated to three plants each of Brassica juncea ' F l o r i d a B r o a d l e a f , Chenopodium 11 amaranticolor, C. quinoa, Gomphrena globosa, Cucumus sativum L. 'Straight Eight', Nicotiana clevlandii, Phaseolus vulgaris 'Top Crop', Lycopersicon esculentum M i l l . 'Rutgers', N. tabacum L. 'Samsun', N. tabacum L. 'Havana 425', N. tabacum L. 'Harrownova', N. benthamiana L., N. glauca L., N. glutinosa L., and N. sylvestris Speg. & Comes. Again, plants mock i n o c u l a t e d with b u f f e r served as healthy c o n t r o l s . ELECTRON MICROSCOPY Leaf samples to be examined by l e a f dip e l e c t r o n microscopy were homogenized i n water and then Parlodion coated g r i d s were f l o a t e d on the homogenate f o r 1 to 2 min. The g r i d s were washed by f l o a t i n g on drops of d i s t i l l e d water and then s t a i n e d with 2% uranyl acetate or 2% phosphotungstic ac i d , pH 7.0. Immuno-electron microscopy was performed by f l o a t i n g g r i d s on a n t i s e r a (at d i l u t i o n s of 1:100 to 1:1000 of crude antisera) f o r varying lengths of time before f l o a t i n g them on l e a f homogenate. Trapped v i r u s p a r t i c l e s were decorated with a n t i s e r a by f l o a t i n g the gr i d s on a n t i s e r a (at various d i l u t i o n s ) . 12 For t h i n s e c t i o n e l e c t r o n microscopy, one mm square t i s s u e samples from blueberry and N. clevelandii plants i n f e c t e d with BIV were f i x e d i n 4% glutaraldehyde (0.1 M sodium cacodylate b u f f e r , pH 7.0) f o r 1 hour. They were po s t - s t a i n e d i n 1% osmium t e t r o x i d e f o r 1 hour and dehydrated by washing q u i c k l y i n 30% ethanol and then 2 15 min washes i n each of 50%, 70%, 95% and absolute ethanol. The samples were then washed 3 times f o r 15 min each i n propylene oxide. The samples were embedded i n Epon 812 (10 g Epon 812, 0.36 ml t r i ( d i m e t h y l aminomethyl)phenol (DMP 36) , 4.4 6 g nadic methyl anhydride, and 3 .76 g dodecenyl s u c c i n i c anhydride) and sections were cut using an LKB microtome. Sections were pos t - s t a i n e d i n 4% uranyl acetate f o r 20 min, washed three times i n d i s t i l l e d water and stained f o r 20 min i n Reynolds lead c i t r a t e (Reynolds, 1963) d i l u t e d 1:4 .5 with 0 .01N NaOH. The g r i d s were washed thoroughly i n d i s t i l l e d water and d r i e d . Thin sections and l e a f dip preparations were examined i n an H i t a c h i 600 e l e c t r o n microscope. 13 VIRUS PURIFICATION A v i r u s p u r i f i c a t i o n p r o t o c o l developed f o r tomato ringspot v i r u s by R. Stace-Smith (personal communication) was used to p u r i f y BIV. Leaves of i n f e c t e d N. clevelandii were homogenized (2 ml/g of tissue) i n 0 .03 M d i b a s i c sodium phosphate, pH 8 . 0 , containing 3 .52 g / 1 a s c o r b i c a c i d and 1.56 ml / 1 2-mercaptoethanol. The homogenate was expressed through nylon c l o t h mesh and the sap was c e n t r i f u g e d at 16, 000 g f o r 20 min (low speed c e n t r i f u g a t i o n ) . The supernatant was c o l l e c t e d , adjusted to pH 5 .0 with 6 M h y d r o c h l o r i c a c i d and l e f t overnight at 4 C. Following low speed c e n t r i f u g a t i o n , the supernatant was c o l l e c t e d . The v i r u s was p r e c i p i t a t e d by adding 8% polyethylene g l y c o l - 8 0 0 0 (PEG) plus 1% sodium c h l o r i d e and s t i r r i n g f o r 1 h at 4 C. The v i r u s was p e l l e t e d by low speed c e n t r i f u g a t i o n and the p e l l e t was allowed to dry f o r 10 min. The p e l l e t was then resuspended i n at l e a s t 1/10 the o r i g i n a l volume of 0 .05 M sodium c i t r a t e , pH 7 . 5 . A f t e r low speed c e n t r i f u g a t i o n the supernatant was c e n t r i f u g e d at 207, 000 g f o r 2 h and the p e l l e t s resuspended i n 0 .05 M sodium c i t r a t e (0 .5 ml per 10 ml tube) . A f t e r another low speed c e n t r i f u g a t i o n up to 0 .5 ml of the supernatant was layered on 10 to 40% sucrose gradients made i n 0 .05 M sodium c i t r a t e b u f f e r i n a 10 ml ce n t r i f u g e tube and c e n t r i f u g e d at 175,000 g f o r 90 min. A f t e r c e n t r i f u g a t i o n , the gradients were scanned with an ISCO UA-5 absorbance/fluorescence detector (ISCO Inc., L i n c o l n , NB) with a 254 nm f i l t e r and f r a c t i o n s with peak absorbance were c o l l e c t e d . The c o l l e c t e d f r a c t i o n s were c e n t r i f u g e d at 207, 000 g f o r 2 h and the p e l l e t was resuspended i n 0 .05 M sodium c i t r a t e b u f f e r , pH 7 . 5 . P u r i f i e d BIV d i l u t e d i n 0 .05 M sodium phosphate, pH 7 . 0 , containing 2% PVP was also mechanically i n o c u l a t e d to healthy N. clevelandii p l a n t s . These plants were kept i n the greenhouse and monitored f o r symptoms. DETERMINATION OF THE RELATIVE MOLECULAR MASS OF THE VIRAL COAT PROTEIN SUBUNIT The r e l a t i v e molecular mass of the coat p r o t e i n was determined by polyacrylamide g e l e l e c t r o p h o r e s i s on a v e r t i c a l 12% g e l with a 4% stacking g e l using a Bio-Rad mini v e r t i c a l slab g e l apparatus (Bio-Rad, Richmond, CA) . P u r i f i e d v i r u s was b o i l e d f o r 5 min i n 0 .05 M T r i s - C l , pH 6 . 8 , containing 10% g l y c e r o l , 2% sodium dodecyl sulphate (SDS), 5% 2-mercaptoethanol, and 0.00125% bromophenol blue before i t was loaded on the g e l . E l e c t r o p h o r e s i s was at 10 mA/gel u n t i l the dye f r o n t moved through the stacking g e l i n t o the separating g e l . E l e c t r o p h o r e s i s was then at 20 mA/gel f o r 40 min. A f t e r e l e c t r o p h o r e s i s , the g e l was f i x e d and s t a i n e d with 0.2% Coomassie B r i l l i a n t Blue R-250 i n 25% methanol and 7% a c e t i c a c i d . It was then destained i n 25% methanol and 7% a c e t i c a c i d (Laemmli, 1 9 7 0 ) . Western b l o t s were performed by running a p r o t e i n g e l as described above and e l e c t r o - b l o t t i n g the separated proteins onto a piece of Immobilon-P membrane ( M i l l i p o r e , Bedford, MA). The p r o t e i n was t r a n s f e r r e d to the membrane by operating the e l e c t r o p h o r e t i c t r a n s f e r c e l l (Bio-Rad, Richmond, CA) at 100 V (0 .25 A) f o r 1.5 h at 4 C i n 25 mM T r i s , 192 mM g l y c i n e , 20% methanol, pH 8 . 3 . The membrane was then blocked f o r a few h at room temperature i n PBS-Tween (0 .015 M phosphate-buffered s a l i n e plus 0.05% Tween-20) containing 1% non-fat d r i e d milk powder. The membrane was washed four times by shaking f o r 15 min per wash with PBS-Tween and the b l o t was submersed i n PBS-Tween plus BIV antibody (1 ug/ml) plus healthy sap (1 g i n 10 ml of buffer) . Excess antibody 16 was washed o f f with four washes (15 min each) of PBS-Tween. Protein-A i o d i n a t e d with 1 2 5 I and d i l u t e d i n PBS-Tween to 1-5 X 10 6 counts per ml was used to probe the b l o t . The b l o t was incubated at room temperature i n the i o d i n a t e d protein-A s o l u t i o n with a g i t a t i o n f o r 1.5 h, washed three times i n PBS-Tween and then allowed to a i r dry (Towbin et a l . , 1979) . The membranes were then wrapped i n Saran wrap and placed on a piece of X-ray f i l m between two i n t e n s i f y i n g screens. The cassette was closed, put i n a black p l a s t i c bag and placed at -70 C overnight. The autoradiographs were developed f o r 3 min i n developer, 30 s i n stop and 5 min i n f i x e r and the f i l m was well r i n s e d before drying (Maniatis, 1982) . RNA EXTRACTION P u r i f i e d v i r u s was resuspended i n 0 .5 ml of 0 .2 M T r i s -C l , pH 7 . 5 , containing 0.025 M EDTA, 0.3 M NaCl, 2% SDS and 250 g/ml • of proteinase K (W.J. Jelkman, personal communication) . Incubation at 37 C f o r 30 min was followed by two e x t r a c t i o n s with 0.5 ml phenol/chloroform ( 1 : 1 , v/v) and one e x t r a c t i o n with 0 .5 ml chloroform /isoamyl a l c o h o l ( 2 4 : 1 , v/v) e x t r a c t i o n . The RNA was p r e c i p i t a t e d with 1/10 volume of 3 M sodium acetate and 2 . 5 volumes absolute ethanol at -20 C overnight. The RNA was p e l l e t e d by c e n t r i f u g a t i o n at 16, 000 g f o r 30 min and the p e l l e t was washed twice with 70 % ethanol. Samples were c e n t r i f u g e d at 16 ,000 g f o r 10 min fo l l o w i n g each wash. The p e l l e t was d r i e d i n a Speed-vac and resuspended i n s t e r i l e deionized H 20. RNA was q u a n t i f i e d spectrophotometrically using an e x t i n c t i o n c o e f f i c i e n t of 25 mg ml"1 cm-1 at 260 nm (Rochon and Si e g a l , 1984) . Approximately 50 ng/band of RNA were loaded on a 1% denaturing agarose g e l containing 5 mM methylmercuric hydroxide. An RNA ladder of 0.3 to 9 .3 kb fragments (Bethesda Research Laboratories, Bethesda, MD) was used as RNA standards at approximately 100 ng/band/lane. A f t e r e l e c t r o p h o r e s i s f o r 1 h at 80 V i n BE (40 mM borate, pH 8 . 2 , c o n t a i n i n g 1 mM EDTA) the g e l was soaked i n 100 ml of H20 containing 70 1 of 2-mercaptoethanol to reduce the methylmercuric hydroxide. The g e l was sta i n e d f o r 10 min with the a d d i t i o n of 0 .5 g/ml ethidium bromide. The g e l was destained f o r 10 min i n H20 and the bands were v i s u a l i z e d and photographed on a t r a n s i l l u m i n a t o r (Maniatis et a l . , 1982 ) . To determine whether the extracted n u c l e i c a c i d was RNA or DNA, 400 ng of the extracted n u c l e i c a c i d from two BIV 18 i s o l a t e s were given one of four treatments. The four treatments were: untreated; mock t r e a t e d (enzyme b u f f e r s only: 50 mM T r i s , pH 7 . 5 , 10 mM MgCl 2, 10 mM 2 -mercaptoethanol) ; 5 ng RNase TI; and 5 ng DNase 1 (RNase f r e e ) . Tobacco mosaic v i r u s (TMV) RNA and plasmid DNA at the same concentrations were used as c o n t r o l s . Reactions proceeded f o r 10 min at 37 C and then 1/6 volume of sample running b u f f e r (0.25% bromophenol blue, 0.25% xylene cyanol and 30% g l y c e r o l i n H20) was added to stop the r e a c t i o n s . Samples were run on a 1% agarose g e l containing 1 g of ethidium bromide per ml of g e l and made up i n TAE (40 mM T r i s , 40 mM acetate, 2 mM EDTA, pH 8.0) b u f f e r . The running b u f f e r was TAE and the g e l was run at 70 V f o r 1 h (D.M. Rochon, personal communication) The g e l was stained, destained and photographed as described above. ISOLATION OF DOUBLE-STRANDED RNA The p r o t o c o l used f o r dsRNA e x t r a c t i o n was that of Kurppa and Martin (1986) . Twenty g of N. clevelandii t i s s u e mechanically i n o c u l a t e d 6 days e a r l i e r with the Anderson i s o l a t e of BIV were powdered i n l i q u i d nitrogen. Two volumes Of 2X STE (100 mM NaCl, 50 mM T r i s - C l , pH 7 . 1 , 1 mM EDTA), 0.4 ml of 2-mercaptoethanol, 0 .5 volumes of 10% SDS, 1.5 volumes STE saturated phenol and 1.5 volumes of chloroform were added and the mixture was shaken v i g o r o u s l y f o r 30 min at room temperature. The mixture was then c e n t r i f u g e d at 16, 000 g f o r 20 min and the aqueous phase was c o l l e c t e d and adjusted to 16% ethanol. The aqueous phase was poured onto a 1.5 cm diameter Econo-Column (Bio-Rad, Richmond, CA) containing 2 g of CF-11 c e l l u l o s e powder (Whatman I n t e r n a t i o n a l Ltd, Maidstone, GB) which had been saturated with 20 ml of STE-16% ethanol. A f t e r the l i q u i d had run through the column, the powder was washed with 80 ml of STE-16% ethanol and purged with a i r . The dsRNA was e l u t e d out of the c e l l u l o s e with 15 ml of STE i n 5 ml a l i q u o t s . The column was purged with a i r between each a l i q u o t . DsRNA samples were digested with RNase T : (2 units/ml) at 37 C f o r 1 h then 1/30 volume of 1 M MgCl 2 was added and the samples digested with DNase (1 u n i t per ml) at 37 C f o r 30 min. O n e - t h i r t i e t h the volume of 0 .5 M EDTA was added and the s o l u t i o n was adjusted to 20% ethanol. Three tenths g of Cellulose-APX (Serva, Heidlelberg, FGR) was added then shaken at room temperature f o r 20 min. This suspension was poured 20 onto a 1.5 cm diameter column and washed with 20 ml of STE-16% ethanol. The column was purged with a i r and the dsRNA elu t e d with three a l i q u o t s of 0 .5 ml of STE with the column being s t i r r e d on a vortex mixer and purged a f t e r each a d d i t i o n of STE. The contaminating Cellulose-APX which came through the column was removed by c e n t r i f u g a t i o n at 16 ,000 g f o r 1 min. The supernatant was adjusted to 0 .3 M sodium acetate (pH 5 .2) and three volumes of 95% ethanol were added. The s o l u t i o n was then a l i q u o t e d i n t o 1.5 ml eppendorf tubes and stored at -20 C overnight. The dsRNA was p e l l e t e d by c e n t r i f u g i n g at 16 ,000 g f o r 10 min and the p e l l e t was d r i e d i n a speed-vac. The p e l l e t was d i s s o l v e d i n loading b u f f e r - (TAE containing 0.04% bromophenol blue, 0.04% xylene cyan.ol, 30% g l y c e r o l ) and run f o r 2 h at 40 V on a 1% agarose g e l made up i n TAE b u f f e r . The agarose g e l had 1 g of ethidium bromide per ml of g e l incorporated i n t o the g e l a f t e r the agarose had been melted but before the g e l was poured. The running b u f f e r was TAE. E i t h e r Pst I-cut lambda DNA or Hind I l l - c u t lambda DNA were used as molecular weight markers. The dsRNA bands were v i s u a l i z e d with u l t r a v i o l e t l i g h t and photographed. 21 SEROLOGY OF THE BLUEBERRY ISOMETRIC VIRUS Three i n j e c t i o n s , 1 week apart, of 0 .3 mg of p u r i f i e d v i r u s i n 0 .5 ml of b u f f e r e m u l s i f i e d with 0 .5 ml of Freund's incomplete adjuvant were administered to a New Zealand white ra b b i t i n t r a m u s c u l a r l y . Three months l a t e r a f u r t h e r three intramuscular i n j e c t i o n s of 0.4 mg of v i r u s , again e m u l s i f i e d i n incomplete adjuvant, were administered. Virus concentrations were determined using an e x t i n c t i o n c o e f f i c i e n t of 7 .0 mg ml"1 cm"1 at 2 60 nm) . Antiserum was obtained from blood c o l l e c t e d 10 days a f t e r the f i n a l i n j e c t i o n . The t i t e r of the antiserum was determined i n agar double d i f f u s i o n t e s t s ( B a l l , 1961) with the v i r u s d i l u t e d to 0.2 mg/ml i n the center well and 2 - f o l d d i l u t i o n s of crude antiserum from 1:2 to 1:2056 i n the outer w e l l s . Tests were observed a f t e r 2 4 and 4 8 hours at room temperature f o r the formation of p r e c i p i t i n l i n e s . P u r i f i e d blueberry isometric v i r u s was also t e s t e d against p o l y c l o n a l a n t i s e r a to vi r u s e s i n the cucumo-, bromo-, i l a r - , and nepovirus groups i n agar g e l double d i f f u s i o n t e s t s . In these t e s t s , 0.2 mg/ml of p u r i f i e d v i r u s was put i n a center well and e i t h e r 1:5, 1:10, 1:20 d i l u t i o n s of raw a n t i s e r a were placed i n the outer wells or 2 - f o l d d i l u t i o n s from 1:2 to 1:2056 of raw a n t i s e r a were placed i n the outer w e l l s . A n t i s e r a to the f o l l o w i n g v i r u s e s were tes t e d : i l a r v i r u s e s (asparagus v i r u s I I , prunus n e c r o t i c ringspot, tobacco streak, apple mosaic); cucumoviruses (cucumber mosaic, peanut stunt, tomato aspermy); bromoviruses (broad bean mottle, brome mosaic, cowpea c h l o r o t i c mottle); and nepoviruses (tomato ringspot, tomato b l a c k r i n g , raspberry ringspot, tobacco ringspot, peach ro s e t t e , cherry l e a f r o l l , grapevine f a n l e a f , arabis mosaic, cherry rasp l e a f , grapevine bulgarian l a t e n t and strawberry l a t e n t ) . Several a n t i s e r a to i s o l a t e s of cucumber mosaic v i r u s and peanut stunt v i r u s were te s t e d as well as a monoclonal that reacts with a l l s t r a i n s of cucumber mosaic v i r u s (supplied by H.J. Vetten, Braunschweig) . Only one antiserum f o r each of the other v i r u s e s was t e s t e d . SURVEY FOR BLUEBERRY SCORCH VIRUS AND THE BLUEBERRY ISOMETRIC VIRUS A survey f o r BBScV and tomato ringspot v i r u s (TomRSV) i n highbush blueberry (Vaccinium corymbosum) was conducted i n 23 Washington, Oregon, and i n the Fraser V a l l e y of B.C. during the summer of 1987. Another survey of the same areas f o r BBScV, the newly i d e n t i f i e d BIV and TomRSV was conducted i n the summer of 1989. Each sample consisted of three or four f u l l y mature leaves c o l l e c t e d from d i f f e r e n t branches on a bush. The leaves were c o l l e c t e d i n p l e x i g l a s s trays c o n t a i n i n g 96 s l o t s i n an 8 X 12 format. The samples were stored at 4 C u n t i l processing. The double antibody sandwich ELISA p r o t o c o l of Clark and Adams (1977) was used except that d i f f e r e n t g r i n d i n g b u f f e r s were tes t e d . The buf f e r s t e s t e d were the standard PBS-Tween-Ov-PVP (0 .015 M phosphate-buffered s a l i n e plus 0.05% Tween-20, 0.2%(w/v) ovalbumin, 2%(w/v) PVP) ; 0 .15 M PBS-Tween-Ov-PVP, pH 7 . 5 ; 0 .1 M borate-Tween-Ov-PVP, pH 8 . 0 ; and 0 .05 M borate-Tween-Ov-PVP, pH 8 . 2 , each with and without 0.5% t e c h n i c a l n i c o t i n e (95% n i c o t i n e ) . Also, 0.1% non-fat d r i e d milk was found to be a s u i t a b l e replacement f o r ovalbumin and a gr i n d i n g b u f f e r of 0 .05 M borate-Tween-PVP-milk, pH 8 . 2 , with 0.5% n i c o t i n e was used f o r the survey (MacDonald et al., 1989 ) . A Pollahne l e a f g r i n d i n g press ( E r i c Pollahne, Wennigsen, FRG) was used to g r i n d leaves using 0 .1 g of l e a f t i s s u e and 1 ml of b u f f e r . IgGs were p u r i f i e d by ammonium sulphate p r e c i p i t a t i o n and chromatography on DEAE-22 c e l l u l o s e , and then adjusted to a concentration of 1 mg/ml. A l k a l i n e phosphatase conjugates were prepared as described by Clark and Adams (1977) . One hundred 1 of coating, sample, conjugate, and substrate were used per m i c r o t i t e r p l a t e w e l l . Two hundred 1 per well was used f o r bloc k i n g . Plates were coated with IgG (1 g/ml) f o r 4 h at 37 C and then were r i n s e d with tap water and blocked fo r 0 .5 h with PBS-Tween containing 0.1% non-fat d r i e d milk (PBS-Tween-milk). Samples were loaded and l e f t overnight at 4 C. The next morning, p l a t e s were r i n s e d with tap water then a l k a l i n e phosphatase conjugate (1 g/ml) was added and l e f t f o r 4 h at 37 C. The pl a t e s were washed again and p-nitrop h e n y l phosphate substrate (0 .5 mg/ml, Sigma) was added i n 10% diethanolamine bu f f e r , pH 9 . 8 . Substrate was allowed to develop f o r 1 hour before the A 4 0 5 absorbance values were read i n an ELISA p l a t e reader ( T i t e r t e k Multiscan Plus Mark II, Flow Laboratories Inc.). Samples were considered p o s i t i v e i f the A 4 0 5 values were greater than twice the mean of the negative c o n t r o l s (mean of s i x healthy samples). The frequency d i s t r i b u t i o n s were al s o examined f o r a bimodal d i s t r i b u t i o n of A 4 0 5 values 25 representing healthy and i n f e c t e d populations. In ad d i t i o n , the mean and standard d e v i a t i o n of s i x healthy samples on each p l a t e were c a l c u l a t e d and the mean plus three times the standard d e v i a t i o n was considered a threshold (Sutula et al., 1986). Any samples with questionable r e s u l t s were r e t e s t e d . APHID TRANSMISSION EXPERIMENTS Myzus persicae (Sulz.) aphids from colonies maintained on N. clevelandii were starved f o r 1 h by p l a c i n g them i n a p e t r i dishes l i n e d with wet paper towel. They were then placed on N. clevelandii plants i n f e c t e d with the BIV. A f t e r being given a c q u i s i t i o n access times of 30 s, 1 min, 2.5 min, 5 min, 1 h, 5 h and 18 h s i x aphids per access time were t r a n s f e r r e d to healthy N. clevelandii. Fimbriaphis fimbriata (Richards) aphids o r i g i n a l l y from blueberry, but maintained on rose and strawberry, were starved f o r 1 h i n a moist f i l t e r paper l i n e d p e t r i p l a t e . They were then placed on detached blueberry leaves i n p e t r i dishes from bushes i n f e c t e d with the BIV. A f t e r a c q u i s i t i o n access times of 30 s, 1 min, 2.5 min, 5 min, 1 h, 5 h and 18 h, s i x aphids per access time were t r a n s f e r r e d to healthy N. 26 clevelandii. The Myzus persicae and Fimbriaphis fimbriata aphids were allowed to feed on the healthy N. clevelandii plants f o r 48 h before the plants were fumigated with methyl bromide and placed i n a greenhouse. These plants were monitored f o r symptoms and t e s t e d by ELISA f o r i n f e c t i o n with BIV. RESULTS: MECHANICAL TRANSMISSION OF BLUEBERRY ISOMETRIC VIRUS N. clevelandii plants began to show ringspot symptoms on the i n o c u l a t e d leaves 7 days a f t e r mechanical i n o c u l a t i o n (Fig. 3). Twelve days a f t e r i n o c u l a t i o n the i n f e c t i o n had gone systemic with n e c r o t i c areas appearing on the young expanding leaves ( Fig. 4) . These symptoms were not present on the pla n t s mock inoculated with b u f f e r . Only N. clevelandii, N. tabacum 'Havana 425' (Fig. 5) , and N. benthamiana (Fig. 6) showed symptoms when i n f e c t e d leaves were homogenized and mechanically i n o c u l a t e d to a wide range of p l a n t s . N. clevelandii plants mechanically in o c u l a t e d with p u r i f i e d BIV developed the ringspot symptoms and the F i g . 3 . R i n g s p o t s a n d o a k l e a f p a t t e r n s on N. clevelandii l e a f 7 d a y s a f t e r m e c h a n i c a l i n o c u l a t i o n f r o m b l o s s o m s o f s y m p t o m a t i c b l u e b e r r y p l a n t s . F i g . 4. S y s t e m i c n e c r o t i c l e s i o n s on e x p a n d i n g l e a v e s o f N. clevelandii 12 d a y s a f t e r m e c h a n i c a l i n o c u l a t i o n . 28 F i g . 5. Symptoms on N. tabacum 'Havana 425' m e c h a n i c a l l y i n o c u l a t e d w i t h l e a v e s from i n f e c t e d N. clevelandii. F i g . 6. Symptoms on N. benthamiana m e c h a n i c a l l y i n o c u l a t e d w i t h l e a v e s from i n f e c t e d N. clevelandii. 2 9 systemic n e c r o t i c areas s i m i l a r to the N. clevelandii plants i n o c u l a t e d from blueberry blossoms. ELECTRON MICROSCOPY Leaf dips of i n f e c t e d N. clevelandii plants revealed i s o m e t r i c p a r t i c l e s approximately 30 nm i n diameter which were not seen i n comparable healthy m a t e r i a l . Isometric p a r t i c l e s were also seen i n t h i n sections of i n f e c t e d N. clevelandii leaves. These p a r t i c l e s were i n the cytoplasm and sometimes were seen i n tubules which appeared to be attached to the plasmodesmata (Fig. 7). No v i r u s - l i k e p a r t i c l e s were seen i n l e a f dips from symptomatic blueberry p l a n t s . However, isometric p a r t i c l e s were trapped with antiserum-coated g r i d s using the a n t i s e r a produced to t h i s v i r u s ( F i g . 8 ) . VIRUS PURIFICATION F r a c t i o n s c o l l e c t e d at the peak absorbance of the sucrose gradients i n the p u r i f i c a t i o n p r o t o c o l described above (Materials and Methods), were found to contain isometric 30 p a r t i c l e s when stained with UA and examined i n the e l e c t r o n microscope ( Fig. 9) . PTA s t a i n apparently destroyed the iso m e t r i c p a r t i c l e s as no p a r t i c l e s could be seen i n the el e c t r o n microscope when i t was used. Lower y i e l d s of the v i r u s were obtained i f the p u r i f i c a t i o n was performed l a t e r than 7 days a f t e r i n o c u l a t i o n of N. c l e v e l a n d i i . High speed c e n t r i f u g a t i o n was used to remove the sucrose and the resuspended p a r t i c l e s had a maximum absorption at 260 nm and a minimum at 24 4 nm. The A 2 6 0/A 2 8 0 r a t i o was 1.5 (uncorrected f o r l i g h t s c a t t e r i n g ) . DETERMINATION OF THE RELATIVE MOLECULAR MASS OF THE VIRAL COAT PROTEIN SUBUNIT The coat p r o t e i n of BIV migrated as two components i n a 12% polyacrylamide g e l (Fig. 10) . The major component had a Mr approximately 2 7 . 5 kDa while the minor component had a r e l a t i v e molecular mass of 53 .0 kDa. A healthy component from N. clevelandii also had a r e l a t i v e molecular mass of approximately 55 .0 kDa, but the large molecular weight-component seen with the p u r i f i e d v i r u s was shown to be v i r a l 31 F i g . 7 . E l e c t r o n micrograph of a t h i n s e c t i o n of i n f e c t e d N. clevelandii showing isometric p a r t i c l e s i n the cytoplasm and i n tubules (Bar represents 3 0 0 nm). F i g . 8. Isometric p a r t i c l e s trapped out of diseased blueberry on g r i d s coated with homologous antiserum (Bar represents 100 nm). F i g . 9. E l e c t r o n micrograph of v i r u s p a r t i c l e s from a p u r i f i e d 100 nm). n e g a t i v e l y s t a i n e d i s o m e t r i c p r e p a r a t i o n (Bar r e p r e s e n t s 33 i n o r i g i n i n the western b l o t (Fig. 11) . There was a l s o a band at approximately 7 9.0 kDa which was detected i n the western b l o t . These l a r g e r components may be a dimer and a trimer of the p r o t e i n subunit. RNA EXTRACTION E a r l y attempts at e x t r a c t i n g RNA from p u r i f i e d v i r u s f a i l e d to y i e l d RNA. The proteinase K treatment before e x t r a c t i n g the RNA r e s u l t e d i n v i s u a l i z a t i o n of bands on the g e l s . The n u c l e i c a c i d of the isometric p a r t i c l e migrated as four bands of r e l a t i v e molecular weights: 1.15, 0.90, 0.77, 0 . 30 ( X 106) (Fig. 12) . The n u c l e i c a c i d was RNA as i t was degraded by RNase but was s t a b l e to DNase. In the t e s t , TMV RNA was degraded by the RNase and not DNase while the plasmid DNA was degraded by the DNase and not the RNase (Fig. 13). DOUBLE-STRANDED RNA EXTRACTION Numerous attempts were made at e x t r a c t i n g dsRNA from N. clevelandii. DsRNA was eventually extracted from symptomless 34 A B C D F i g . 10. Polyacrylamide g e l of the blueberry isometric v i r u s coat p r o t e i n . Lanes A and D are markers with r e l a t i v e molecular masses: phosphorylase B, 92,500; bovine serum albumin, 66,200; ovalbumin, 45,000; carbonic anhydrase, 31,000; soybean t r y p s i n i n h i b i t o r , 21,500; and lysozyme, 14,400 (low-molecular-weight p r o t e i n standards, Bio-Rad, Richmond, CA) . Lanes B and C are 1.0 and 2.0 g of p u r i f i e d i s o m e t r i c p a r t i c l e r e s p e c t i v e l y . 35 F i g . 11. P o l y a c r y l a m i d e g e l a n d w e s t e r n b l o t o f t h e b l u e b e r r y i s o m e t r i c v i r u s c o a t p r o t e i n . L a n e s A t h r o u g h E a r e t h e p o l y a c r y l a m i d e g e l . L a n e s A a n d E a r e m a r k e r s ( a s d e s c r i b e d f o r F i g . 10), l a n e B i s h e a l t h y N. clevelandii ( g r o u n d 1 g i n 10 m l a n d 10 1 u s e d p e r l a n e ) , l a n e C a n d D a r e p u r i f i e d b l u e b e r r y i s o m e t r i c v i r u s a t 0.5 a n d 2 .0 g / l a n e r e s p e c t i v e l y . L a n e s F t o J c o r r e s p o n d t o l a n e s A t o E f r o m t h e p o l y a c r y l a m i d e g e l i n a w e s t e r n b l o t ( p h o t o g r a p h e d s l i g h t l y s m a l l e r t h a n t h e p o l y a c r y l a m i d e g e l ) 36 F i g . 12 . M e t h y l m e r c u r i c h y d r o x i d e d e n a t u r i n g g e l o f b l u e b e r r y i s o m e t r i c v i r u s RNA. L a n e B i s i s o m e t r i c p a r t i c l e RNA w h i l e l a n e s A a n d C a r e RNA m a r k e r s ( B e t h e s d a R e s e a r c h L a b o r a t o r i e s , B e t h e s d a MD). 37 N. clevelandii that had been mechanically i n o c u l a t e d 6 days e a r l i e r . No dsRNA was extracted from plants e x h i b i t i n g symptoms. The dsRNA that was extracted c o n s i s t e d of four bands which were approximately twice the r e l a t i v e molecular weights of the four s i n g l e stranded RNA bands extr a c t e d from p u r i f i e d BIV. Double-stranded RNA e x t r a c t i o n from blueberry leaves f a i l e d to y i e l d any appreciable amounts of dsRNA. SEROLOGY OF THE BLUEBERRY ISOMETRIC VIRUS Antiserum produced against the blueberry i s o m e t r i c v i r u s had a d i l u t i o n endpoint of 1/64 i n agar g e l double d i f f u s i o n t e s t s . When t h i s antiserum was used i n d i r e c t double antibody sandwich to detect BIV i n N. clevelandii, the no n s p e c i f i c r e a c t i o n s were higher than d e s i r e d . However, d i l u t i n g the conjugate i n healthy sap (1 g healthy N. clevelandii t i s s u e ground i n 10 ml PBS-Tween-milk) eliminated these n o n s p e c i f i c r e a c t i o n s . When t e s t i n g blueberry t i s s u e there were no n o n s p e c i f i c reactions and i n f e c t e d blueberry t i s s u e had A 4 0 5 values 10 times those of healthy c o n t r o l s . BIV d i d not react i n agar g e l double d i f f u s i o n t e s t s with any of the a n t i s e r a to the viruses l i s t e d i n the 'Materials 38 and Methods'. This included the a n t i s e r a to the d i f f e r e n t s t r a i n s of cucumber mosaic v i r u s and peanut stunt v i r u s and the monoclonal antibody that reacts with a l l s t r a i n s of cucumber mosaic v i r u s . SURVEY FOR BLUEBERRY SCORCH VIRUS, BLUEBERRY ISOMETRIC VIRUS AND ToMRSV BBScV* was found i n Pierce and Clark counties i n Washington State and i n Clackamas and Benton counties i n Oregon. TomRSV was found i n a 40 yr o l d blueberry f i e l d i n Whatcom county, Washington and i n a blueberry f i e l d i n Washington county, Oregon. BIV was found i n Whatcom, Pierce and Clark counties i n Washington and i n Clackamas and Yamhill counties i n Oregon. The three v i r u s e s have not been found i n B.C. despite extensive t e s t i n g (Table I ) . APHID TRANSMISSIONS None of the healthy N. clevelandii plants to which aphids had been t r a n s f e r r e d from i n f e c t e d N. clevelandii or blueberry developed blueberry isometric v i r u s i n f e c t i o n 39 A B C D E F G H I J K L M N O P O H t * t t 41 l | I 11 F i g . 1 3 . Non-denaturing g e l of RNase and DNase digests of blueberry isometric v i r u s RNA. Lanes A to D are tobacco mosaic v i r u s RNA, lanes E to H are the Anderson i s o l a t e of the blueberry isometric v i r u s , lanes I to L are the Baker i s o l a t e of the blueberry isometric v i r u s , lanes M to P are plasmid DNA and lane Q i s RNA markers (as described f o r F i g . 12) . Lanes A, E, I, M, and Q are untreated, lanes B, F, J, and N are mock t r e a t e d (no nuclease) , lanes C, G, K, and 0 were t r e a t e d with 5 n g RNase and lane D, H, L, and P were t r e a t e d with 5 ng of DNase. High molecular weight band (arrow) i n lanes E,F,G,I,J, and K are contaminating host m a t e r i a l which have not been seen on other denaturing gels of the RNA. 40 Table I. Survey r e s u l t s of t e s t s f o r blueberry scorch v i r u s , blueberry i s o m e t r i c v i r u s and tomato ringspot v i r u s i n B.C., Washington, and Oregon f o r 1987 and 1989. Number of farms p o s i t i v e f o r each v i r u s versus farms te s t e d 1987 a 1989 k B r i t i s h Columbia Blueberry scorch v i r u s 0/27 0/25 Blueberry isometric v i r u s - ° 0/25 Tomato ringspot v i r u s 0/27 0/25 Washington Whatcom County Blueberry scorch v i r u s 0/12 0/4 Blueberry isometric v i r u s - 3/4 Tomato ringspot v i r u s 1/12 0/4 P i e r c e County Blueberry scorch v i r u s 6/7 6/8 Blueberry isometric v i r u s - 3 /8 Tomato ringspot v i r u s 0/7 0/8 Clark County Blueberry scorch v i r u s 1/3 1/2 Blueberry i s o m e t r i c v i r u s - 1/2 Tomato ringspot v i r u s 0/3 0/2 Oregon Blueberry scorch v i r u s 0 /6 2/14 Blueberry isometric v i r u s - 2/14 Tomato ringspot v i r u s 0 /6 1/14 a In t o t a l , approximately 4 ,500 bushes were tes t e d . b In t o t a l , approximately 3 ,500 bushes were tes t e d . c a n t i s e r a not a v a i l a b l e i n 1987. 41 symptoms. In a d d i t i o n , a l l these plants were negative by ELISA f o r the blueberry isometric v i r u s . DISCUSSION These r e s u l t s show that there are at l e a s t two v i r u s e s , BBScV and an is o m e t r i c v i r u s , causing scorch symptoms i n blueberry. BBScV has been described p r e v i o u s l y (Martin and Bristow, 1988), but the new blueberry i s o m e t r i c v i r u s (BIV) has not been described before i n blueberry. BBScV has been found from c e n t r a l Washington to c e n t r a l Oregon, while BIV has been found from northern Washington to c e n t r a l Oregon. Neither of these v i r u s e s has been found i n blueberry i n B.C. The four RNA bands of BIV are s i m i l a r i n s i z e to the four RNA bands of v i r u s e s i n the cucumo-, i l a r - and bromovirus groups. RNA 4, the smallest RNA species of these v i r u s e s , i s considered to be a subgenomic of RNA 3. These v i r u s e s are s a i d to have t r i p a r t i t e genomes and thus BIV probably also has a t r i p a r t i t e genome. However, no s e r o l o g i c a l r e l a t i o n s h i p with any of the v i r u s e s t e s t e d i n these groups has been found. 42 I l a r v i r u s e s have p a r t i c l e s which can be from 20-32 nm i n diameter and a coat p r o t e i n subunit from 2 5 , 0 0 0 - 3 0 , 0 0 0 daltons depending on the v i r u s (Fulton, 1983) . In contrast, cucumoviruses have 28-30 nm diameter p a r t i c l e s and a 2 4 , 0 0 0 -25 ,000 dalton p r o t e i n subunit (Kaper and Waterworth, 1981) and bromoviruses have 26 nm diameter p a r t i c l e s and a 20 ,000 dalton coat p r o t e i n subunit (Lane, 1 9 7 9 ) . I l a r v i r u s e s g e n e r a l l y have woody hosts while cucmo- and bromoviruses have herbaceous hosts. The p a r t i c l e s of i l a r - and cucmoviruses are destroyed by PTA s t a i n s used f o r e l e c t r o n microscopy. Therefore, the s i z e of BIV p a r t i c l e s (30 nm), the s i z e of the p r o t e i n subunit (27,500 d a l t o n s ) , the woody nature of the host, and the d e s t r u c t i o n of p a r t i c l e s by PTA i n d i c a t e that t h i s v i r u s best f i t s i n t o the i l a r v i r u s group. In a d d i t i o n , cucumoviruses should have been p r e c i p i t a t e d with the plant p r o t e i n i n the pH 5 .0 c l a r i f i c a t i o n step of the p u r i f i c a t i o n p r o t o c o l (Hamilton et al., 1981 ) . The rate of spread of BIV through blueberry f i e l d s i s f a s t e r than would be expected f o r most i l a r v i r u s e s . Viruses i n i l a r - and bromovirus groups have not been shown to be aphid t r a n s m i s s i b l e except f o r a report of aphid transmission of brome mosaic v i r u s i n South A f r i c a (von Wechmar and 43 Table I I . C h a r a c t e r i s t i c s of Blueberry Isometric V i r u s P a r t i c l e s i z e : 3 0 nm i n diameter Coat p r o t e i n subunit: 27 ,500 daltons N u c l e i c a c i d : 4 RNA species 1.15 X 10 6 (Mr) Host range: narrow, blueberry N. clevelandii N. benthamiana N. tabacum cv. 'Havana 425' 0.82 X 10 6 0.65 X 10 6 0.28 X 10 6 S e r o l o g i c a l r e l a t i o n s h i p s : None found so f a r Aphid Transmission: Not shown so f a r S t a b i l i t y i n EM s t a i n : Stable i n UA but unstable i n PTA 44 Rybicki, 1985). Cucumoviruses, however, are aphid transmitted (Kaper and Waterworth, 1981). The i n a b i l i t y totransmit t h i s blueberry v i r u s i n our t e s t s i s f u r t h e r evidence of an i l a r v i r u s , although these aphid transmission experiments were not exhaustive as only two aphid species were used under laboratory c o n d i t i o n s . The speed of spread of t h i s v i r u s i n blueberry f i e l d s would also i n d i c a t e that aphids are the vector. Another i n t e r e s t i n g observation about BIV i s that plants e x h i b i t symptoms one year and then recover over the f o l l o w i n g years e v e n t u a l l y e x h i b i t i n g no symptoms. I l a r v i r u s e s , such as prunus n e c r o t i c ringspot are known to produce s i m i l a r shock r e a c t i o n i n plants (Fulton, 1981) . However, there are v i r u s e s i n other v i r u s groups which have s a t e l l i t e RNAs or d e f e c t i v e i n t e r f e r i n g p a r t i c l e s that also generate t h i s attenuation of symptoms. Further research on the epidemiology of BIV as w e l l as t e s t i n g the v i r u s against d i f f e r e n t s t r a i n s of i l a r - , cucumo-, and bromoviruses w i l l help c l a s s i f y t h i s v i r u s . This information w i l l a i d i n developing a c o n t r o l program f o r BIV. 45 CHAPTER TWO- COMPARISON OF BLUEBERRY SCORCH VIRUS WITH SHEEP PEN HILL VIRUS AND OTHER CARLAVIRUSES INTRODUCTION A C a r l a v i r u s e s are s l i g h t l y flexuous filaments normally 610-700 nm long and 12-15 nm i n diameter, with a p r o t e i n coat made up of a s i n g l e polypeptide of r e l a t i v e molecular mass of 31,000 to 3 4 , 0 0 0 , and a genome of a s i n g l e molecule of p o s i t i v e sense RNA (Mol Wt 2 . 3 - 3 . 0 X 10 6 Da). Most c a r l a v i r u s e s have r e s t r i c t e d host ranges but the d i f f e r e n t v i r u s e s occur i n a wide v a r i e t y of hosts. C a r l a v i r u s e s are u s u a l l y transmitted i n a non-persistent manner by aphids (Koenig, 1 9 8 2 ) . Blueberry scorch disease was f i r s t observed i n Pierce County i n Washington state i n 1980, and has been determined to be caused by blueberry scorch v i r u s (BBScV)(Martin and Bristow, 1 9 8 8 ) . Sheep Pen H i l l Disease was f i r s t reported from blueberry i n New Jersey i n 1983 (Stretch, 1 9 8 3 ) . It has also been found to be caused by a c a r l a v i r u s ( r e f e r r e d to as Sheep Pen H i l l v i r u s , SPHV)(Podleckis and Davis, 1987 ) . These two v i r u s e s cause s i m i l a r symptoms except that with SPHV there i s an autumn f o l i a r l i n e p a t t e r n . S e r o l o g i c a l r e l a t i o n s h i p s between BBScV, SPHV and three other c a r l a v i r u s e s were examined using p o l y c l o n a l a n t i s e r a i n double antibody sandwich ELISA (DAS-ELISA) and i n d i r e c t ELISA. The homology between the genomes of these v i r u s e s was compared by n u c l e i c a c i d h y b r i d i z a t i o n s . The o b j e c t i v e s of t h i s research were to determine i f BBScV and SPHV are d i f f e r e n t v i r u s e s or s t r a i n s of the same v i r u s , and to determine whether these are new c a r l a v i r u s e s or p r e v i o u s l y reported c a r l a v i r u s e s occurring f o r the f i r s t time i n blueberry. MATERIALS AND METHODS CARLAVIRUS ISOLATES BBScV (Martin and Bristow, 1988) was p u r i f i e d from •infected blueberry t i s s u e from f i e l d s i n the Puyallup V a l l e y i n Washington State. SPHV, which was obtained from E.V. Podleckis (USDA, B e l t s v i l l e , MA) , had been propagated and p u r i f i e d from C. quinoa. Carnation l a t e n t v i r u s (CLV) was p u r i f i e d from C. quinoa that had been in o c u l a t e d mechanically from i n f e c t e d Dianthus caryophyllus L. 'Jack' c o l l e c t e d from a greenhouse i n B r i t i s h Columbia. Potato v i r u s S (PVS) was an i s o l a t e c o l l e c t e d from potato i n B r i t i s h Columbia and was p u r i f i e d out of i n f e c t e d potatoes grown i n the greenhouse. Potato v i r u s M (PVM) was a B.C. i s o l a t e maintained i n potato and p u r i f i e d out of potato. A l l c a r l a v i r u s p u r i f i c a t i o n s were as described by Martin and Bristow ( 1 9 8 8 ) except that 0 . 0 5 M borate was used instead of 0 . 1 M borate. P u r i f i e d v i r u s preparations were stored at 4 C with approximately 0 . 0 2 % sodium azide added. SEROLOGICAL COMPARISONS OF THE CARLAVIRUSES S e r o l o g i c a l r e l a t i o n s h i p s between the d i f f e r e n t c a r l a v i r u s e s were i n v e s t i g a t e d with both i n d i r e c t ELISA and d i r e c t double antibody sandwich ELISA (DAS-ELISA) using p o l y c l o n a l a n t i s e r a . The experiments were repeated twice, once without SPHV and once with SPHV. The a n t i s e r a used were those a v a i l a b l e i n the a n t i s e r a bank at the A g r i c u l t u r e Canada Research S t a t i o n i n Vancouver except f o r antiserum to 48 SPHV (E.V. P o d l e c k i s ) . IgGs were p u r i f i e d by ammonium sulphate p r e c i p i t a t i o n and chromatography on DEAE-22 c e l l u l o s e , and then adjusted to a concentration of 1 mg/ml. A l k a l i n e phosphatase conjugates were prepared as described by Clark and Adams (1977) . A l l reagents were used at 200 1/well i n Linbro flat-bottom m i c r o t i t e r p l a t e s (Flow Laboratories Inc., McLean, VA) , except f o r the b l o c k i n g step which used 300 1/well. The pl a t e s were r i n s e d with tap water a f t e r each step of the ELISA except a f t e r the blocking step. The p l a t e s f o r i n d i r e c t ELISA (Koenig, 1978) were coated f o r 1 h with 1 g/ml of p u r i f i e d v i r u s (BBScV, SPHV, CLV, PVS, and PVM) and then blocked f o r 45 min with PBS-Tween-0.2% d r i e d milk powder (PBS-Tween-milk). Homologous and heterologous a n t i s e r a i n a 5 - f o l d d i l u t i o n s e r i e s from 1:100 to 1 : 2 , 1 8 7 , 0 0 0 were incubated on the pl a t e s f o r 2 h at 37 C. Then goat a n t i - r a b b i t a l k a l i n e phosphatase conjugate (Gibco, Mississauga, Ont) was added at the manufacturer's recommended concentration and incubated f o r 2 h at 37 C. Substrate (p-nit r o p h e n y l phosphate, Sigma 104-105) at 0 .5 mg/ml i n 10% diethanolamine, pH 9 . 8 , was added and the absorbance of each w e l l at 405 nm (A 4 0 5) was read a f t e r 1 h at 37 C and a f t e r 12 h 49 at 4 C on an ELISA pl a t e reader ( T i t e r t e k Multiscan Plus Mark II , Flow Laboratories Inc.). For DAS-ELISA, m i c r o t i t e r p l a t e s were incubated f o r 4 h at 4 C with IgG d i l u t e d to 1 g/ml i n carbonate b u f f e r , pH 9 . 6 . P l a t e s were blocked with PBS-Tween-milk f o r 30 min at room temperature. P u r i f i e d v i r u s (BBScV, SPH, CLV, PVS, and PVM) at 1 g/ml was incubated f o r 4 h at 37 C. A l k a l i n e phosphatase conjugates of the IgG used to coat the wells i n two-fold d i l u t i o n s from 1:500 to 1 :64 ,000 were incubated f o r 4 h at 37 C. Substrate was added and p l a t e s were read a f t e r 1 h and a f t e r 12 h as described above. For DAS-ELISA, p u r i f i e d tobacco mosaic v i r u s (TMV, Ul st r a i n ) was used as a negative c o n t r o l and f o r the i n d i r e c t ELISA e i t h e r p u r i f i e d TMV or t u r n i p mosaic v i r u s (TuMV) was used as negative c o n t r o l s . The negative c o n t r o l s were t r e a t e d i n the same manner as the c a r l a v i r u s e s f o r each t e s t . CARLAVIRUS RNA EXTRACTION RNA was extracted from 400 1 of p u r i f i e d v i r u s (1-10 mg/ml) with 100 1 of 0 .5 M T r i s , pH 8 . 9 , 25 1 of 20% SDS, 200 1 of chloroform/isoamyl a l c o h o l ( 2 4 : 1 ) , and 200 1 of 50 r e d i s t i l l e d phenol saturated with b u f f e r . This mixture was s t i r r e d on a vortex mixer then c e n t r i f u g e d f o r 10. min at 16,000 g. The aqueous phase was removed, and the organic phase was re-extracted with 600 1 of s t e r i l e H 20. The aqueous phases were combined and extracted with 600 1 of phenol/chloroform mixture, then c e n t r i f u g e d and the r e s u l t i n g aqueous phase was extracted with 600 1 of chloroform. The RNA was p r e c i p i t a t e d from the aqueous phase, a f t e r c e n t r i f u g a t i o n , with 0 .1 volume of 2 M sodium acetate (pH 5.4) and 2 . 25 volumes of absolute ethanol. The RNA was then stored at -20 C overnight. The RNA was p e l l e t e d by c e n t r i f u g a t i o n at 16 ,000 g, and the supernatant was poured o f f . The p e l l e t s were washed twice with 70 % c o l d ethanol and then d r i e d i n a Speed-vac. The RNA was resuspended i n s t e r i l e deionized H 20, q u a n t i f i e d spectrophotometrically and run on a methylmercuric hydroxide g e l to ensure the RNA was f u l l length (Rochon and S i e g e l , 1984) . The p u r i f i e d RNA was stored at -70 C. 51 PREPARATION OF RANDOM PRIMED CDNA PROBES Random primed cDNA was prepared i n standard reverse t r a n s c r i p t i o n mixture (0 .05 M T r i s , pH 8 . 3 , 8 mM MgCl 2, 10 mM d i t h i o t h r e i t o l , 10 1 of 100 O.D./ml DNA primers (Pharmacia), 0.6 mM dCTP, dGTP, dTTP, 5 uCi alpha 3 2P dATP, 0 .3 g RNA, 18-36 u n i t s of avian myeloblastosis v i r u s reverse t r a n s c r i p t a s e or 200 units of Malone' murine leukemia reverse t r a n s c r i p t a s e , and H20 to a f i n a l volume of 100 1) , incubated at 37 C f o r 30-120 min and the r e a c t i o n progress monitored by t r i c h l o r o a c e t i c a c i d p r e c i p i t a b l e counts. The r e a c t i o n was complete when there were 1-5 X 10 6 cpm/ g RNA incorporated. DNA was p r e c i p i t a t e d by adding 100 1 of 10 mg/ml denatured salmon sperm DNA, 30 1 of 3 M sodium acetate and 700 1 of c o l d absolute ethanol. A f t e r c e n t r i f u g a t i o n f o r 10 min, the p e l l e t e d DNA was denatured by resuspending i n 100 1 of 0.2 N NaOH and heating to 90-95 C f o r 5 min (Maniatis et al., 1982) . 52 HYBRIDIZATIONS Ten ng of each RNA sample i n 50 1 of H20 were b l o t t e d per spot onto a Nytran membrane (Schleicher & S c h n e l l Inc., Keene, NH) using a Bio-Rad dot b l o t t i n g apparatus (Bio-Rad, Richmond CA) . The Nytran had been prewetted i n 10X SSC (20X SSC = 3 M NaCl and 0.3 M trisodium c i t r a t e ) . The Nytran was then baked under vacuum f o r 1 h at 8 0 C and put i n p r e h y b r i d i z a t i o n b u f f e r (50% formamide, 4X SSPE, 1% SDS, 0.05% nonfat d r i e d milk powder, and 0.5 mg/ml denatured salmon sperm DNA) f o r 1 to 2 h at 42 C. The membrane and the l a b e l l e d probe were added to the h y b r i d i z a t i o n b u f f e r (same as p r e h y b r i d i z a t i o n b u f f e r with 50% w/v dextran s u l f a t e added and 2 concentrations of formamide 30% and 50%). The h y b r i d i z a t i o n s were c a r r i e d out f o r 16 h at 42 C with a g i t a t i o n . Then, the Nytran membranes h y b r i d i z e d i n b u f f e r containing 50% formamide were washed i n a s e r i e s of 15 min washes i n 200 ml of s o l u t i o n with decreasing concentrations of SSC at 58-62 C. The SSC concentrations used were 2X, IX, 0.5X, 0.2X, and 0.1X, and a l l the concentrations had 0.1% SDS. The membranes hy b r i d i z e d i n b u f f e r containing 30% formamide were washed twice f o r 15 min i n 2X SSC and 0.1% SDS at 42 C, twice f o r 15 min i n 5X SSC, 0.1% SDS, and 30% formamide at 42 C and b r i e f l y i n 2X SSC. The membranes were then a i r d r i e d and autoradiographed as described e a r l i e r (Determination of the R e l a t i v e Molecular Mass of the V i r a l Coat P r o t e i n Subunit s e c t i o n ) . RESULTS CARLAVIRUS ISOLATES The p u r i f i c a t i o n p r o t o c o l developed by Martin and Bristow (1988) worked w e l l f o r a l l the c a r l a v i r u s e s p u r i f i e d i n t h i s study no matter what t i s s u e they were p u r i f i e d from. Often m a t e r i a l going onto the caesium c h l o r i d e gradients was very green. However, a f t e r the gradient had been run, the green m a t e r i a l formed a s o l i d band at the top of the gradient while the v i r u s band would be halfway down the gradient. SEROLOGICAL COMPARISONS OF THE CARLAVIRUSES BBScV, SPHV, and PVS reacted strongly with homologous and heterologous a n t i s e r a i n i n d i r e c t ELISA while that of CLV 54 reacted s t r o n g l y with homologous antiserum and not as str o n g l y with heterologous a n t i s e r a . PVM only reacted with homologous antiserum and not to any heterologous a n t i s e r a (Tables I I I and IV) . PVS was also detected weakly by the TuMV antiserum, but TuMV was only detected by homologous antiserum. BBScV reacted strongly only with homologous antiserum i n DAS ELISA when t e s t e d with the a n t i s e r a used i n t h i s study. However, SPHV antiserum developed at a l a t e r date reacted st r o n g l y with BBScV (E.V. Podleckis, personal communication). SPHV reacted s t r o n g l y with homologous antiserum and BBScV antiserum, to a l e s s e r extent with PVM and PVS a n t i s e r a and f a i n t l y with CLV antiserum. PVS reacted s t r o n g l y with homologous antiserum and PVM antiserum, and weakly with BBScV, CLV, and SPHV a n t i s e r a . CLV, PVM and TMV reacted with the homologous a n t i s e r a and weakly with the SPHV antiserum (Tables V and VI) . NUCLEIC ACID HYBRIDIZATIONS OF CARLAVIRUSES The RNA molecules extracted from the d i f f e r e n t c a r l a v i r u s e s were a l l approximately 8,400 bases when run on a 55 Table I I I . End point t i t e r s of sev e r a l p o l y c l o n a l a n t i s e r a i n homologous and heterologous i n d i r e c t ELISA reactions Antigens A n t i s e r a 3 BBScV CLV PVM PVS TuMV BBSCV 312,250" 12,500 c 62 ,500 -CLV 12,500 62,500 - 12,500 -PVM 312,250 2, 500 62,500 1 ,562 ,500 -PVS 12 ,500 500 - 312 ,500 -TuMV — — - 500 62,500 a A n t i s e r a (1 mg/ml) were tes t e d i n du p l i c a t e wells of 5 - f o l d d i l u t i o n s from 1:100 to 1 : 1 , 5 6 2 , 5 0 0 . b Number i n d i c a t e s the lowest d i l u t i o n of antiserum that gave a p o s i t i v e r e a c t i o n with each v i r u s . Reactions were considered p o s i t i v e i f the absorbence was twice background absorbence. Homologous reactions are underlined. c Indicates that the antiserum d i d not detect the v i r u s at any d i l u t i o n . 56 Table IV. End point t i t e r s of several p o l y c l o n a l a n t i s e r a against BBScV and SPHV i n i n d i r e c t ELISA reactions Antigens A n t i s e r a 3 BBScV SPHV BBScV 243, 000 b 243 ,000 CLV 81,000 81 ,000 PVM 243,000 729 ,000 PVS 81,000 243 ,000 SPHV 3, 000 d 243 ,000 TMV c a A n t i s e r a (1 mg/ml) was te s t e d i n d u p l i c a t e wells of three-f o l d d i l u t i o n s from 1:1000 to 1 : 2 , 1 8 7 , 0 0 0 . b Number i n d i c a t e s the lowest d i l u t i o n of antiserum that gave a p o s i t i v e r e a c t i o n with each v i r u s . Reactions were considered p o s i t i v e i f the absorbence was twice background absorbence. Homologous reactions are underlined. c Indicates that the antiserum d i d not detect the v i r u s at any d i l u t i o n . d Later studies with SPHV a n t i s e r a r e a c t i n g s t r o n g l y with BBScV (E.V. Podleckis, personal communication). 57 Table V. Detection of homologous and heterologous c a r l a v i r u s e s i n double antibody sandwich ELISA Limit of Detection f o r Antigens 3 A n t i s e r a BBScV CLV PVM PVS TMV BBScV £ 3 b - c - - -CLV - 8. - - -PVM - - 61 16 PVS - - - 63 TMV - - - - 63 a Antigens were t e s t e d i n d u p l i c a t e wells of two-fold d i l u t i o n s from 1000 ng/ml to 8 ng/ml bNumber i n d i c a t e s the lowest d i l u t i o n of v i r a l antigen that gave a p o s i t i v e r e a c t i o n with each a n t i s e r a . Reactions were considered p o s i t i v e i f the absorbence was twice background absorbence. Homologous reactions are underlined. c Indicates that the antigen was not detected at any concentration t e s t e d . 58 Table VI. End point t i t e r s of homologous and heterologous p o l y c l o n a l a n t i s e r a i n double antibody sandwich ELISA Antigens A n t i s e r a 3 BBScV CLV PVM PVS TMV SPHV BBScV 3 2 , 0 0 0 b - c - 1,000 - 32 ,000 CLV - 64,000 - 1,000 - 1,000 PVM - - 16 ,000 16,000 - 4 ,000 PVS . - - - 32 ,000 - 4 ,000 TMV - 4 ,000 SPHV - d 500 500 500 500 32,000 a A n t i s e r a (1 mg/ml) was test e d i n wells of two-fold d i l u t i o n s from 1:500 to 1 : 6 4 , 0 0 0 . b Number i n d i c a t e s the lowest d i l u t i o n of antiserum that gave a p o s i t i v e r e a c t i o n with each v i r u s . Reactions were considered p o s i t i v e i f the absorbence was twice background absorbence. Homologous reactions are underlined. c Indicates that the antiserum d i d not detect the v i r u s at any d i l u t i o n . d Later studies with SPHV a n t i s e r a r e a c t i n g s t r o n g l y with BBScV (E.V. Podleckis, personal communication). denaturing methylmercuric hydroxide g e l (Fig. 1 4 ) . At high stringency (50% formamide h y b r i d i z a t i o n s and 60 C washes), the BBScV cDNA hy b r i d i z e d to i t s homologous RNA and to a l e s s e r extent to SPHV RNA. SPHV cDNA h y b r i d i z e d to i t s homologous RNA and f a i n t l y to BBScV RNA (Fig. 15) . The r e s t of the cDNAs only h y b r i d i z e d to the homologous RNAs at high stringency. When h y b r i d i z a t i o n s were c a r r i e d out at low stringency (30% formamide h y b r i d i z a t i o n s and 42 C washes), BBScV cDNA hy b r i d i z e d with homologous RNA and to SPHV RNA. SPHV cDNA hy b r i d i z e d to homologous RNA and to BBScV RNA. In ad d i t i o n , both BBScV and SPHV cDNAs hyb r i d i z e d to PVS RNA. PVS cDNA hy b r i d i z e d to BBScV and SPHV RNA as well as i t s homologous RNA. PVM, CLV and TMV cDNA only h y b r i d i z e d to t h e i r homologous RNAs. None of the c a r l a v i r u s cDNAs h y b r i d i z e d to t o t a l RNA extracted from healthy potato and spotted at 50 ng/dot (Fig 16) . DISCUSSION The appearance of two c a r l a v i r u s e s (BBScV and SPHV) i n blueberry i n widely separated areas at approximately the same 60 A B C D E F G H Kb F i g . 1 4 . M e t h y l m e r c u r i c h y d r o x i d e d e n a t u r i n g g e l o f s e v e r a l c a r l a v i r u s RNA. L a n e C, PVS. L a n e D, PVM, L a n e E, SPHV. L a n e F, C L V . L a n e G, B B S c V . L a n e B, TMV. L a n e s A a n d H a r e RNA markers (Bethesda Research Laboratories, Bethesda MD). 61 PROBE BBScV CLV SPHV PVM PVS TMV RNA BBScV £ 0 • • CLV * # SPHV £ # PVM PVS . » TMV HEALTHY ID BBScV| CLV SPHV • • PVM PVS TMV HEALTHY F i g . 15 . H y b r i d i z a t i o n s of s i x c a r l a v i r u s e s with homologous and heterologous random primed cDNA probes i n dot b l o t h y b r i d i z a t i o n s . Ten ng of RNA was b l o t t e d per dot. H y b r i d i z a t i o n s were c a r r i e d out at (A) low stringency (30% formamide at 42C) and at (B) high stringency (50% formamide at 4 2 C ) . BBScV probe hybridi z e d with PVS RNA at low stringency but the dots have faded during photographic t r a n s f e r . 62 time would suggest that they are the same v i r u s . This i s fu r t h e r supported by two other f a c t o r s : no other c a r l a v i r u s e s have been described i n blueberry and plant m a t e r i a l has been exchanged between the two growing areas where these v i r u s e s have been found. I n d i r e c t ELISA i s a l e s s antigen s p e c i f i c t e s t than DAS-ELISA as the probing antibodies are not conjugated to an enzyme (Koenig, 1978). In the i n d i r e c t t e s t s described, with the exceptions of PVM, a l l the c a r l a v i r u s e s reacted with a l l heterologous c a r l a v i r u s a n t i s e r a . The SPHV antiserum could only detect BBScV at a low d i l u t i o n , but a subsequent SPHV antiserum reacted strongly with BBScV (E.V. Podleckis, personal communication) . BBScV, SPHV and PVS must have s i m i l a r epitopes as the d i f f e r e n t c a r l a v i r u s a n t i s e r a used i n t h i s study gave s i m i l a r t i t e r s f o r these three v i r u s e s . None of the c a r l a v i r u s a n t i s e r a reacted with TuMV (a p o t y v i r u s ) . The TuMV a n t i s e r a reacted with i t s homologous antigen, but i t als o reacted weakly with PVS. This i s probably a non-s p e c i f i c r e a c t i o n with host m a t e r i a l c o p u r i f i e d with the PVS as potyviruses and c a r l a v i r u s e s have not been found to be s e r o l o g i c a l l y r e l a t e d (Koenig, 1982). 63 DAS-ELISA i s a more s p e c i f i c t e s t than i n d i r e c t ELISA and g e n e r a l l y only a n t i s e r a of c l o s e l y r e l a t e d v i r u s e s cross react (Koenig, 1978) . In t h i s study, PVS and SPHV were detected by heterologous a n t i s e r a although t h i s was mainly at high concentration of a n t i s e r a . BBScV antiserum reacted s t r o n g l y with SPHV, and a new SPHV antiserum has a l s o reacted s t r o n g l y with BBScV (E.V. Podleckis, personal communication) which was not seen i n these t e s t s . SPHV antiserum also reacted weakly with CLV, PVM, PVS and TMV (a tobamovirus used as a control) which may be n o n - s p e c i f i c r e a c t i o n s . The non-s p e c i f i c r e a c t i o n was not observed with BBScV since BBScV was p u r i f i e d out of a woody host while the other v i r u s e s were p u r i f i e d out of herbaceous hosts. The SPHV a n t i s e r a may have an antibody to a plant component which i s common i n the herbaceous plants and not i n the woody p l a n t . The lack of a two-way r e a c t i o n between PVS and PVM may be due to antibodies i n the a n t i s e r a being to d i f f e r e n t epitopes. The PVM a n t i s e r a has antibodies to a conserved epitope that occurs on PVM and PVS while the PVS a n t i s e r a has antibodies which are s p e c i f i c to an epitope on PVS and not on PVM. Another explanation f o r the lack of a two-way r e a c t i o n i s that the PVM a n t i s e r a was made with a p u r i f i e d preparation from a 64 mixed i n f e c t i o n of PVM and PVS. H y b r i d i z a t i o n studies with these c a r l a v i r u s e s i n d i c a t e a r e l a t i o n s h i p between BBScV, SPHV and PVS. The sequence relatedness f o r these v i r u s e s was estimated using the r e l a t i o n s h i p of 1% base mismatching decreasing T m (melting temperature) by 1.5 C (Hymen et al., 1973 ) . The Tra of the homologous RNA:DNA hybrids was c a l c u l a t e d to be 102 C using the formula of Howley et al. (1979) . Also, the r e l a t i o n s h i p that 1% formamide reduces the T m of DNA:RNA hybrids by 0 .72 C (McConaughy et al., 1969) was used. This i n d i c a t e s that h y b r i d i z a t i o n s i n 30% formamide at 42 C w i l l detect sequence homologies up to 27% base mismatching. H y b r i d i z a t i o n s i n 50% formamide at 42 C w i l l i n d i c a t e a much c l o s e r sequence relatedness with only 16% base mismatch t o l e r a t e d . Therefore, between BBScV and SPHV there i s l e s s than 16% base mismatch while between PVS and each of BBScV and SPHV there i s between 16% and 27% base mismatch. The host range of BBScV i s b e l i e v e d to be r e s t r i c t e d to blueberry (Martin and Bristow, 1988) while the host range of PVS contains species i n the f a m i l i e s Solanaceae, Chenopodiaceae and Caryophyllaceae. Hamilton et al. (1981) s t a t e d i n t h e i r g u i d e l i n e s f o r e s t a b l i s h i n g the taxonomic 65 status of a new v i r u s that v i r u s e s that are s e r o l o g i c a l l y r e l a t e d but which have d i f f e r e n t host ranges or other d i f f e r e n t c h a r a c t e r i s t i c s should be considered separate v i r u s e s . This d i f f e r e n c e i n host range between BBScV and PVS would i n d i c a t e that the BBScV i s not a s t r a i n of PVS but i s a separate v i r u s that i s r e l a t e d to PVS. I suggest that BBScV be renamed blueberry scorch c a r l a v i r u s (BBSCV). The s e r o l o g i c a l and h y b r i d i z a t i o n data along with the host range and symptomology would suggest that BBSCV and SPHV are s t r a i n s of the same v i r u s . I suggest that SPHV be named the New Jersey s t r a i n of BBSCV (BBSCV-NJ). 66 GENERAL DISCUSSION Vir u s diseases of blueberry have g e n e r a l l y been considered minor problems i n B.C., Washington and Oregon. In contrast, they are of considerable importance i n the Eastern U.S. growing regions. With the appearance of BBSCV and BIV, i t appears that v i r u s e s are becoming more of a concern i n t h i s region. It i s i n t e r e s t i n g to question where these new blueberry v i r u s e s came from as they both appeared i n blueberry at approximately the same time. In f a c t , BBSCV appeared i n Washington State and i n New Jersey, 2 widely separated areas, at the same time. One could speculate that the v i r u s o r i g i n a t e d i n New Jersey because the Sheep Pen H i l l area of New Jersey has a large nursery industry producing blueberry plants that may have been shipped to Washington. I have t e s t e d w i l d Vaccinium species around i n f e c t e d f i e l d s i n Pier c e county, and weeds i n i n f e c t e d f i e l d s but have not detected the v i r u s i n any other species of p l a n t . BIV also appeared i n Whatcom and i n Clark counties at approximately the same time as the c a r l a v i r u s appeared. I have t e s t e d f o r BIV i n w i l d plants around i n f e c t e d f i e l d s i n Whatcom County 67 but have not detected t h i s v i r u s i n the w i l d p l a n t s . It i s i n t e r e s t i n g that neither v i r u s has been found i n B.C. despite extensive t e s t i n g . B.C. growers b r i n g i n p l a n t i n g stock i n from other areas, and i f these v i r u s e s have been spread with p l a n t i n g stock i t would be s u r p r i s i n g i f these v i r u s e s have not already been brought i n t o B.C. The i n c r e a s i n g acreage of blueberry i n B.C., Washington and Oregon ( e s p e c i a l l y B.C.) w i l l mean that p l a n t i n g stock continues to be brought i n from other areas. I f e e l t h i s w i l l cause v i r u s diseases to become more of a problem i n B.C., Washington and Oregon i n future years. A v i r u s c e r t i f i c a t i o n program f o r blueberry p l a n t i n g stock using the a n t i s e r a produced i n t h i s study as well as more research on epidemiology of both BBSCV and BIV may help i n preventing the spread of these v i r u s e s . E a r l y d e t e c t i o n and removal of diseased bushes should minimize the damage caused by these v i r u s e s . ELISA can be used to i d e n t i f y diseased bushes and to ensure that a l l diseased bushes are removed. V i r u s - f r e e p l a n t i n g stock and de t e c t i o n and removal of i n f e c t e d bushes should make i t p o s s i b l e to eliminate t h i s v i r u s disease i n the P a c i f i c Northwest. 68 REFERENCES B a l l , E.M. 1961 . S e r o l o g i c a l t e s t s f o r the i d e n t i f i c a t i o n of plant v i r u s e s . Am. Phytopathol. Soc. 16 pp. B.C. Blueberry Co-op 1989. B.C. Blueberry Co-op Spring Newsletter. 8 pp. Bristow, P.R. and Ramsdell, D.C. 1984. F i r s t report of blueberry shoestring v i r u s Disease i n Washington. Plant Disease 68:450 [Abstract]. Clark, M.E. and Adams, A.N. 1977. C h a r a c t e r i s t i c s of the micro-plate method of enzyme-linked immunosorbent assay f o r the d e t e c t i o n of plant v i r u s e s . J. Gen. V i r o l . 3 4 : 4 7 5 - 4 8 3 . Converse, R.H., e d i t o r . 1987. V i r u s Diseases of Small F r u i t s . 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