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A study of luteoviruses involved in potato leafroll disease Ellis, Peter John 1991

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A STUDY OF LUTEOVIRUSES INVOLVED IN POTATO LEAFROLL DISEASE by Peter John E l l i s B.Sc.(Hon.), The University of Ottawa, 1976 M.P.M., Simon Fraser University, 1983 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES PLANT SCIENCE We accept t h i s thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1991 ® Peter John E l l i s , 1991 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of P^fl'yV S»CAoaCo The University of British Columbia Vancouver, Canada Date R p n l 2 - S ? \ 9 C \ \ DE-6 (2/88) Abstract In t o t a l , 8 0 1 samples of potato l e a f r o l l disease were co l l e c t e d and tested for potato l e a f r o l l virus (PLRV) and beet western yellows virus (BWYV) i n 1 9 8 6 , 1 9 8 7 , and 1 9 8 8 using t r i p l e antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) and v i r u s - s p e c i f i c monoclonal antibodies. The samples represented 3 2 c u l t i v a r s and originated i n eight Canadian provinces and 1 2 American states. None of the samples tested p o s i t i v e for BWYV, whereas 7 7 2 ( 9 6 . 4 % ) tested p o s i t i v e for PLRV. Neither PLRV nor BWYV could be recovered, with aphid transfers to i ndicator hosts, from 2 8 of the 2 9 samples that tested negative for both viruses. PLRV was recovered from one sample that o r i g i n a l l y tested negative by TAS-ELISA; the indicator plant tested p o s i t i v e for PLRV by TAS-ELISA. Nucleic acid spot hybridization (NASH) using random primed and cloned cDNA probes was compared with double antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) and TAS-ELISA, and aphid transmission tests for detection and i d e n t i f i c a t i o n of PLRV and BWYV i n 1 6 5 potato l e a f r o l l disease samples. A l l of the samples tested negative for BWYV with each of the assay procedures. PLRV was detected i n a l l of the samples with TAS-ELISA, NASH with a cloned cDNA probe for PLRV, and with aphid transmission to ground cherry (Physalis pubescens) . Both DAS-ELISA and NASH using random primed cDNA produced one false-negative r e s u l t . Shepherd's purse (Capsella bursa-pastoris) was a host for 72% (119/165) of the PLRV i s o l a t e s . The s u s c e p t i b i l i t y of potato to BWYV was tested by inoculating Russet Burbank with three i s o l a t e s of BWYV from Canada and four from the United States. Two of the is o l a t e s were i n a mixed i n f e c t i o n with PLRV. None of the i s o l a t e s were transmitted by Myzus persicae to vir u s - f r e e potato plants, either by themselves or i n association with PLRV. Common weeds were surveyed i n the potato-producing areas of B r i t i s h Columbia for PLRV and BWYV. In t o t a l , 10,098 weed samples, representing 98 species i n 22 plant families, were co l l e c t e d and tested by TAS-ELISA from 1986 to 1989. BWYV was detected i n 1% of the samples; the hosts were: chickweed, common groundsel, heart-podded hoary cress, hedge mustard, l i t t l e western b i t t e r c r e s s , p r i c k l y lettuce, shepherd's purse, and wild radish. PLRV was detected i n three volunteer potato plants, two samples of shepherd's purse, and one black nightshade plant. The low incidence of PLRV i n plants other than potato indicates that weeds are of minor importance i n the epidemiology of potato l e a f r o l l disease i n B r i t i s h Columbia. i i i Table of Contents Abstract i i L i s t of Tables v i i L i s t of Figures v i i i L i s t of Abbreviations x Acknowledgement x v i 1 Introduction 1 1.1 Luteoviruses 1 1.2 Luteoviruses i n f e c t i n g potato 7 1.3 D e f i n i t i o n of the problem 11 1. 4 Objectives 18 2 Production of monoclonal antibodies 20 2.1 Introduction 20 2.2 Materials and methods 21 2.2.1 Virus i s o l a t e s and p u r i f i c a t i o n 21 2.2.2 Antibodies 25 2.2.3 Hybridoma production, screening, and isotyping 25 2.2.4 ELISA 28 2.3 Results 30 2.3.1 Production of hybridomas 30 2.3.2 Monoclonal antibody s p e c i f i c i t y . . 31 2.4 Discussion 34 3 Detection and i d e n t i f i c a t i o n using monoclonal antibodies. 37 3.1 Introduction 37 i v 3.2 Materials and methods 38 3.2.1 Co l l e c t i o n of samples 38 3.2.2 Virus i s o l a t e s and p u r i f i c a t i o n 41 3.2.3 Antisera and monoclonal antibodies 41 3.2.4 ELISA 42 3.2.5 Aphid transmission tests . 43 3.3 Results 44 3.4 Discussion 58 4 Comparison of ELISA and nucleic a c i d spot hybridization f o r detection and i d e n t i f i c a t i o n of PLRV and BWYV 63 4 .1 Introduction 63 4 .2 Materials and methods . 65 4.2.1 Virus i s o l a t e s , p u r i f i c a t i o n , and RNA extraction 65 4.2.2 Synthesis and cloning of cDNA 66 4.2.3 Northern blots 67 4.2.4 Co l l e c t i o n of samples 67 4.2.5 Preparation of plant extracts 69 4.2.6 Preparation of nucleic acid probes 69 4.2.7 NASH procedures 70 4.2.8 Antisera and monoclonal antibodies 72 4.2.9 Double antibody sandwich ELISA 72 4.2.10 T r i p l e antibody sandwich ELISA 73 4.2.11 Aphid transmission tests 74 4.3 Results 74 4.4 Discussion 87 v 5 S u s c e p t i b i l i t y of potato to BWYV i s o l a t e s from Canada and the United States 91 5 . 1 Introduction 9 1 5 . 2 Materials and methods 9 2 5 . 2 . 1 Virus i s o l a t e s and transmission t e s t s . . . . 9 2 5 . 2 . 2 ELISA 9 3 5 . 3 Results 9 3 5 . 4 Discussion 9 5 6 A survey of weeds as reservoirs f o r BWYV and PLRV. ...... .97 6 . 1 Introduction 97 6 . 2 Materials and methods 9 8 6 . 2 . 1 C o l l e c t i o n of samples 98 6 . 2 . 2 Antisera and monoclonal antibodies 9 8 6 . 2 . 3 ELISA . 9 9 6 . 2 . 4 Aphid transmissions 9 9 6 . 3 Results 1 0 0 6 . 4 Discussion 1 0 9 7 Summary and conclusions 115 7 . 1 Summary of results 1 1 5 7 . 2 Conclusions 1 1 8 Bibliography 121 v i L i s t of Tables Table I. D e f i n i t i v e members of the luteovirus group 3 Table I I . Comparison of the properties of beet western yellows virus (BWYV) and potato l e a f r o l l virus (PLRV) 9 Table I I I . Luteovirus i s o l a t e s used to determine s p e c i f i c i t y of monoclonal antibodies 2 3 Table IV. Monoclonal antibody subclasses 3 1 Table V. S p e c i f i c i t y of monoclonal antibodies 3 2 Table VI. Origins of potato l e a f r o l l disease samples 4 0 Table VII. Virus i s o l a t e s used i n aphid transmission t e s t s . . . 9 2 Table VIII. Mean v i r u s - s p e c i f i c TAS-ELISA (A405) r e s u l t s of attempts to transmit beet western yellows (BWYV) and potato l e a f r o l l virus (PLRV) to potato 94 Table IX. Plants surveyed for beet western yellows virus and potato l e a f r o l l virus i n the potato producing areas of B r i t i s h Columbia 1 0 1 Table X. Reservoir hosts of beet western yellows virus (BWYV) and potato l e a f r o l l virus (PLRV) i n southern B r i t i s h Columbia 1 0 8 v i i L i s t of Figures F i g . 1. Secondary symptoms of potato l e a f r o l l virus from tuber-borne infection 39 Fi g . 2. TAS-ELISA analysis of p u r i f i e d PLRV and BWYV 45 Fi g . 3A. Histogram of 1986 F l o r i d a winter test r e s u l t s for PLRV i n 55 potato l e a f r o l l disease samples .46 Fi g . 3B. Histogram of 1986 F l o r i d a winter test r e s u l t s for BWYV i n 55 potato l e a f r o l l disease samples 47 Fi g . 4A. Histogram of 1987 C a l i f o r n i a winter t e s t r e s u l t s for PLRV i n 257 potato l e a f r o l l disease samples 48 Fi g . 4B. Histogram of 1987 C a l i f o r n i a winter test r e s u l t s for BWYV i n 257 potato l e a f r o l l disease samples 49 Fi g . 5A. Histogram of 1987 F l o r i d a winter test r e s u l t s for PLRV i n 51 potato l e a f r o l l disease samples. . . ... .50 F i g . 5B. Histogram of 1987 Fl o r i d a winter test r e s u l t s for BWYV i n 51 potato l e a f r o l l disease samples 51 Fi g . 6A. Histogram of 1987 Oregon winter test r e s u l t s for PLRV i n 86 potato l e a f r o l l disease samples 52 Fi g . 6B. Histogram of 1987 Oregon winter test r e s u l t s for BWYV i n 86 potato l e a f r o l l disease samples 53 Fi g . 7A. Histogram of 1988 C a l i f o r n i a winter test r e s u l t s for PLRV i n 109 potato l e a f r o l l disease samples 54 Fi g . 7B. Histogram of 1988 C a l i f o r n i a winter test r e s u l t s for BWYV i n 109 potato l e a f r o l l disease samples 55 v i i i F i g . 8A. Histogram of 1988 F l o r i d a winter test r e s u l t s for PLRV i n 243 potato l e a f r o l l disease samples 56 Fi g . 8B. Histogram of 1988 F l o r i d a winter test r e s u l t s for BWYV i n 243 potato l e a f r o l l disease samples 57 Fi g . 9A. Detection of BWYV is o l a t e s using a cloned cDNA probe 75-76 F i g . 9B. Detection of BWYV RNA i n a d i l u t i o n series using random primed cDNA probe 75-7 6 F i g . 9C. Detection of BWYV RNA i n a d i l u t i o n series using a cloned cDNA probe 75-76 F i g . 10. DAS-ELISA f a i l e d to detect BWYV i n potato l e a f r o l l disease samples 77 Fi g . 11. TAS-ELISA f a i l e d to detect BWYV i n potato l e a f r o l l disease samples 78 Fig . 12. NASH of 20 potato l e a f r o l l disease samples using cloned cDNA probes for PLRV and BWYV 80-81 Fig . 13. NASH of potato l e a f r o l l disease samples using PLRV and BWYV random primed cDNA 82-83 Fi g . 14. Capsella bursa-pastoris infected with PLRV 84 Fi g . 15. NASH of potato l e a f r o l l disease samples on Nytran and n i t r o c e l l u l o s e membranes using a cloned cDNA probe 85-86 ix L i s t of Abbreviations A 2 6 0 - absorbance at 2 60 nm A 2 8 0 - absorbance at 280 nm A 4 0 5 - absorbance at 4 05 nm AAT - a sele c t i o n medium for growing hybridoma cultures a i - active ingredient ATP (dATP) - adenosine triphosphate (deoxy adenosine triphosphate) BALB/c - a mouse l i n e used for producing hybridomas BLRV - bean l e a f r o l l virus bp - base p a i r BMYV - beet mild yellowing virus BMYV-324 - a sugar beet i s o l a t e of BMYV from England BW/PL-5.2 - a mixed i n f e c t i o n of BWYV and PLRV i n ground cherry BWYV - beet western yellows virus BWYV-12H - a common groundsel i s o l a t e of BWYV from B r i t i s h Columbia, Canada BWYV-210E - a common groundsel i s o l a t e of BWYV from B r i t i s h Columbia, Canada BWYV-40-8003 - a Malva i s o l a t e of BWYV from Tasmania BWYV-150 - a b r o c c o l i i s o l a t e of BWYV from C a l i f o r n i a , USA BWYV-B10 - a beet i s o l a t e of BWYV from B e l t s v i l l e , USA BWYV-BC - sugar beet i s o l a t e of BWYV from B r i t i s h Columbia, Canada x BWYV-CA - a Malva i s o l a t e of BWYV from Oceanside, C a l i f o r n i a , USA BWYV-D3 - an i s o l a t e of BWYV from the state of Washington, USA BWYV-MP4BF - a c r u c i f e r i s o l a t e of BWYV from Germany BWYV-MY4 - a Malva i s o l a t e of BWYV from C a l i f o r n i a , USA BWYV-NETH - a lettuce i s o l a t e of BWYV from the Netherlands BWYV-RB2 - a b r o c c o l i i s o l a t e of BWYV from C a l i f o r n i a , USA BWYV-RY-7 - a br o c c o l i i s o l a t e of BWYV from C a l i f o r n i a , USA BWYV-RY-1-R - a br o c c o l i i s o l a t e of BWYV from C a l i f o r n i a , USA BWYV-SAL(9) - a lettuce i s o l a t e of BWYV from Germany BYDV - barley yellow dwarf virus BYDV-MAV-NY - the New York type-strain of BYDV-MAV that i s transmitted s p e c i f i c a l l y by Sitobion (=Macrosiphum) avenae BYDV-RPV-IL - a RPV i s o l a t e of BYDV that i s s p e c i f i c a l l y transmitted by Rhopalosiphum padi BYDV-RPV-NY - the New York type-strain of BYDV-RPV that i s s p e c i f i c a l l y transmitted by Rhopalosiphum padi BYDV-RPV-T - a RPV i s o l a t e of BYDV from Tasmania that i s s p e c i f i c a l l y transmitted by Rhopalosiphum padi C - Celsius cDNA - complementary DNA CLRV - carrot redleaf virus cm (mm) - centimeter (millimeter) CsCl - cesium chloride DAS-ELISA - double antibody sandwich enzyme-linked immunosorbent assay x i DNA - deoxyribonucleic acid DMEM - Dulbecco's Modified Eagle Medium ds - double-stranded ds-RNA - double stranded RNA EDTA - ethylenediaminetetraacetic acid ELISA - enzyme-linked immunosorbent assay EM - electron microscopy microscopy FCS - f e t a l c a l f serum FOX-NY - a myeloma c e l l l i n e used to produce hybridomas g (mg,fj.g,pg) - gram (milligram, microgram,picogram) g - gravity GPA - green peach aphid (Myzus persicae) GRAV - groundnut rosette a s s i s t o r virus hr - hour IgG - immunoglobulin G IgM - immunoglobulin M IPTT752 - a mixed i n f e c t i o n of BWYV and PLRV from turnip i n the state of Washington, USA ISEM - immunosorbent electron microscopy (=immuno EM) ISDV - Indonesian soybean dwarf virus kb - kilobase kDa - ki l o d a l t o n 1 (ml,[i.l) - l i t e r ( m i l l i l i t e r , m i c r o l i t e r ) LYV - legume yellows virus m (cm,mm,nm) - meter (centimeter,millimeter,nanometer) M (mM) - molar (millimolar) x i i MAb - monoclonal antibody MAV - see BYDV-MAV MiAV - Michigan a l f a l f a virus M-MLV - Moloney murine lukemia virus MYV - Malva yellows virus min - minute Mr - molecular weight NaCl - sodium c l o r i d e NaOH - sodium hydroxide NASH - nucleic acid spot hybridization nt - nucleotide ORF - open reading frame PAV - a vector nonspecific i s o l a t e of BYDV (BWYV-PAV) PBS - phosphate-buffered saline PEG - polyethylene g l y c o l PeLRV - pea l e a f r o l l virus PLRV - potato l e a f r o l l virus PLRV-BC - an i s o l a t e of PLRV from B r i t i s h Columbia PLRV-ID - an i s o l a t e of PLRV from Idaho PLRV-ORE - an i s o l a t e of PLRV from Oregon PLRV-ST4 - a severe i s o l a t e of PLRV from B r i t i s h Columbia polyA - polyadenylate; an oligonucleotide formed e n t i r e l y of adenosine residues Pst 1 - a r e s t r i c t i o n enzyme i s o l a t e d from Providencea stuartii that cuts at the recognition sequence GTGCAG pUC9 - a plasmid cloning vector x i i i PVP - polyvinylpyrrolidone RBF/Dn - a mouse l i n e used i n the production of hybridomas RNA - ribonucleic acid RMV - a vector s p e c i f i c s t r a i n of BYDV (BYDV-RMV) that i s transmitted by Rhopalosiphum madis RPV - a vector s p e c i f i c s t r a i n of BYDV (BYDV-RPV) that i s transmitted by Rhopalosiphum padi RGV - r i c e guillaume virus RY-l-R - see BWYV-RY-1-R RYV - radish yellows virus sw,2o ~ sedimentation c o e f f i c i e n t - the rate of sedimentation per unit c e n t r i f u g a l f i e l d measured i n Svedberg units (S) and corrected to sedimentation i n water at 20 C SCLRV - subterranean clover redleaf virus SDI - s e r o l o g i c a l d i f f e r e n t i a t i o n index SDS - sodium dodecyl sulfate SDV - soybean dwarf virus SDV-AP - an i s o l a t e of SDV that i s transmitted by Acyrthosiphon pisum SDV-AS - an i s o l a t e of SDV that i s transmitted by Aulacorthum solani SGV - a vector s p e c i f i c s t r a i n of BYDV (BYDV-SGV) that i s transmitted by Schizaphis graminum SMYEV - strawberry mild yellow edge virus ss - single-stranded xiv SSC - a buffer containing 0.015 M sodium chloride and 0.015 mM trisodium c i t r a t e SSPE - a buffer containing 0.15 M sodium chloride, 0.01 M sodium phosphate, and 1 mM EDTA SYV - Solanum yellows virus SYV-21 - i s o l a t e 21 of SYV from B e l t s v i l l e , USA SYV-44 - i s o l a t e 44 of SYV from B e l t s v i l l e , USA TAS-ELISA - t r i p l e antibody sandwich enzyme-linked immunosorbent assay TNDV - tobacco necrotic dwarf virus Tris-HCl — tris(hydroxymethyl)aminomethane - hydrochloric acid TuYV - turnip yellows virus TYTV - tomato yellow top virus |XEm"2s_1 - microergs per square meter per second UV - u l t r a v i o l e t vol - volumn VPg - genome-linked protein X-gal - 5-bromo-4-chloro-3-indolyl-P-D-galactoside xv Acknowledgements I wish to thank Dr. M. Weintraub and the s t a f f of the Vancouver Research Station for t h e i r help and support, and for providing the research f a c i l i t i e s for my work. Special thanks to Dr. R. I. Hamilton, my senior supervisor, for introducing me to the fascinating world of plant virology, and for his patience during my research. Thanks to the rest of my committee, Drs. R. Copeman, J. Hudson, and J. Tremaine, for t h e i r help and encouragement. I am indebted to Dr. Bud Wright for sharing his laboratory and his vast knowledge of potato viruses with me. I would also l i k e to thank Dennis Kirkham and Paul Froese for t h e i r help i n c o l l e c t i n g potato l e a f r o l l virus i s o l a t e s . I am g r a t e f u l to Mr. Wes MacDiarmid for producing a l l the plates i n t h i s t h e s i s . Thanks to Andrew Wieczorek for his help i n the monoclonal lab and his excellent technical advice. I thank my wife Coral and my children, Rein and Meghan, for t o l e r a t i n g my lack of humour during the writing of t h i s t h e s i s . I express my sincere appreciation to Dr. R. Stace-Smith for his valuable advice, comments, and discussion throughout my work on t h i s project; there i s no substitute for experience. F i n a l l y , I thank Dr. H. R. MacCarthy for his encouragement and many hours of e d i t i n g . This thesis would not have been completed without his help. xvi Chapter 1 Introduct ion 1.1 Luteoviruses The luteovirus group was f i r s t recognized by the International Committee on Taxonomy of Viruses i n 1975 (Shepherd et al., 1976) . The group name i s from the Latin "luteus", meaning yellow, because infected plants tend to show yellowing symptoms (Casper, 1988) . Other c h a r a c t e r i s t i c symptoms are reddening, leaf r o l l i n g or curling, and b r i t t l e n e s s , although some luteoviruses are latent i n many hosts (Waterhouse, Gildow, and Johnston, 1988). Luteoviruses occur worldwide i n many important crops and can cause substantial economic losses; barley yellow dwarf virus (BYDV), beet western yellows virus (BWYV), and potato l e a f r o l l virus (PLRV) are the most important members of t h i s group (Rochow and Duffus, 1981). Luteovirus taxonomy i s i n a state of f l u x . The number of d e f i n i t i v e members has changed several times, from three i n 1975 to 15 i n 1982 (Matthews, 1982) and then back to 14 i n 1985 (Francki, Milne, and Hatta, 1985) . The d e f i n i t i v e members of the group are l i s t e d i n Table I and c l o s e l y related strains and synonyms are l i s t e d under each member. The c l a s s i f i c a t i o n I have presented i n Table I l i s t s nine of the ten d e f i n i t i v e 1 members selected by Waterhouse et al. (1988). The tenth, BYDV-RPV, i s grouped as a s t r a i n of BWYV as suggested by Casper (1988) because of i t s strong s e r o l o g i c a l r e l a t i o n s h i p to BWYV (Rochow, 1984; Rochow and Duffus, 1978) . This grouping i s also supported by investigations of cytopathological e f f e c t s ( G i l l and Chong, 1979a,b) and nucleic acid hybridization studies showing homology between BWYV and the RPV i s o l a t e of BYDV (Martin and D'Arcy, 1990). The number of d e f i n i t i v e luteoviruses w i l l l i k e l y decrease again i n the future when a more complete evaluation of the group can be made using multiple t r a i t s , including host range, serology, nucleic acid homologies, and vector relationships (Martin et al., 1990; Waterhouse et al., 1988) . Casper (1988) and Waterhouse et al. (1988) have recently reviewed the properties of the luteovirus group. Luteoviruses have isometric p a r t i c l e s about 25 nm in diameter, w i l l sediment as a single component between 104 and 118 S, and have a buoyant density of about 1.40 g/cm3 i n CsCl. The coat protein subunits are composed of a single polypeptide of Mr about 24 x 103. The genome i s a single molecule of po s i t i v e sense, single-stranded RNA of Mr approximately 2.0 x 106 with a genome-linked protein (VPg) , Mr about 7.0 x 103 for PLRV and 17.0 x 103 for BYDV, and no polyA t a i l (Mayo et al., 1982; Murphy, D'Arcy, and Clark, 1989). The v i r i o n s have high A 2 6 0/A 2 8 0 r a t i o s of between 1.6 and 1.9, an RNA content of about 28-30%, and are strongly immunogenic. 2 Table I. D e f i n i t i v e members of the luteovirus group 9 Members Reference Barley yellow dwarf (BYDV) MAV BYDV isolates PAV and SGV Rochow (1984) Bean l e a f r o l l (BLRV) Pea l e a f r o l l (PeLRV) Legume yellows (LYV) Michigan a l f a l f a (MiAV) Ashby (1984) Ashby & Johnstone (1985) Ashby (1984) Thottappilly et al. (1977) Beet western yellows (BWYV) BYDV isolates RPV and RMV Beet mild yellowing (BMYV) Malva yellows (MYV) Radish yellows (RYV) Rice giallume virus (RGV) Turnip yellows (TuYV) Rochow & Duffus (1981) Rochow (1984) Govier (1985) Duffus (1972) Duffus (1960, 1972) Osier (1984) Duffus (1972) Carrot red leaf (CRLV) Waterhouse & Murant (1982) Groundnut rosette assistor (GRAV) Casper et al. (1983) Indonesian soybean dwarf (ISDV) Rajeshwari & Murant (1988) Potato l e a f r o l l (PLRV) Solanum yellows (SYV) Tomato yellow top (TYTV) Harrison (1984) Milbrath & Duffus (1978) Thomas (1984) Soybean dwarf virus (SDV) Subterranean clover red leaf (SCRLV) Strawberry mild yellow edge (SMYEV) Tamada & Kojima (1977) Ashby & Johnstone (1985) Martin & Converse (1985) Tobacco necrotic dwarf (TNDV) Kubo (1981) aWaterhouse, Gildow, & Johnstone, 1988 3 Luteoviruses are transmitted by aphids i n the persistent ( c i r c u l a t i v e and nonpropagative) manner (Eskandari, Sylvester, and Richardson, 1979). Generally, luteoviruses have a high degree of vector s p e c i f i c i t y ; each being transmitted e f f i c i e n t l y by one or a few species of aphid. Aphids acquire luteoviruses while feeding i n the phloem tissue of infected plants (Leonard and Holbrook, 1978). Using e l e c t r o n i c monitoring, Leonard and Holbrook (1978) demonstrated that the green peach aphid (GPA), Myzus persicae Sulz., could aquire PLRV in as l i t t l e as 1-2 min once the s t y l e t s had penetrated and remained i n the sieve elements. U l t r a s t r u c t u r a l studies indicate that luteovirus p a r t i c l e s pass through the hindgut by c e l l u l a r transport into the aphid's haemocoel where they then c i r c u l a t e i n the haemolymph (Gildow, 1985) . Following endocytosis into the aphid's accessory s a l i v a r y gland and transport within coated v e s i c l e s through the s a l i v a r y gland secretory c e l l s , the virus p a r t i c l e s are released by exocytosis into the s a l i v a r y duct (Gildow, 1985) . Transmission probably occurs during the egestion phase of feeding (Leonard and Holbrook, 1978) . E f f i c i e n t transmission of most luteoviruses requires a c q u i s i t i o n and inoculation feeding periods of 24 hr each. A minimum latent period (defined as the time from the st a r t of the a c q u i s i t i o n feeding u n t i l the aphid can i n f e c t plants with the virus) i s usually between 12 and 24 hr (Duffus, 1977). 4 None of the luteoviruses has been transmitted by mechanical inoculation or by seed (Rochow and Duffus, 1981) U n t i l recently, l i t t l e was known about the molecular biology of luteoviruses because the p a r t i c l e s are phloem-limited, scarce, and d i f f i c u l t to p u r i f y . M i l l e r et al. (1988a) determined the complete nucleotide sequence of a BYDV-PAV serotype and i t s genome organization was deduced from the open reading frames (ORF's). The c i s t r o n closest to the 5' end encodes the putative RNA-dependent RNA polymerase, the coat protein c i s t r o n i s near the middle of the genome, and there i s a possible t r a n s l a t i o n a l readthrough of the coat protein ORF to produce a 69 kd protein. The nucleotide sequence of the genomic RNA (5641 nt) of BWYV has recently been determined and i t s genomic organization proposed (Veidt et al., 1988). BWYV has six long ORF's and a clus t e r of three of these, including the coat protein c i s t r o n , have extensive amino acid sequence homology with the corresponding ORF's of BYDV-PAV (Veidt et al., 1988; M i l l e r et al., 1988b). The putative polymerase regions, i n contrast, are d i s t i n c t from one another; the BWYV RNA polymerase ORF resembles that of southern bean mosaic, whereas that of BYDV-PAV appears to share common ancestry with carnation mottle virus (Veidt et a l . , 1988; M i l l e r , Waterhouse and Gerlach, 1988). Kozak (1986) suggested that RNA viruses may evolve by exchanging functional 5 sequence modules with one another; t h i s mechanism could explain the juxtaposition of simi l a r and di f f e r e n t ORF's i n BWYV and BYDV-PAV (Veidt et al., 1 9 8 8 ) . It i s now clear that recombination between v i r a l RNA molecules i s not a rare event, indeed i t i s now considered to be one of the major mechanisms of RNA virus evolution (Goldbach and Wellink, 1 9 8 8 ) . Mayo et al. ( 1 9 8 9) reported the nucleotide sequence of PLRV; i t s genome organization i s very si m i l a r to that of BWYV. Si g n i f i c a n t amino acid sequence homology exists between a l l of the analogous ORF's in BWYV and PLRV except i n the f i r s t ORF. The genome organization of BWYV and PLRV i s d i s t i n c t from BYDV-PAV by having an additional ORF at the 5 ' end; there i s no corresponding ORF i n the BYDV-PAV genome (Martin et al., 1 9 9 0 ) . The sequences of four d i f f e r e n t i s o l a t e s of PLRV have been compared by Keese et al. ( 1 9 9 0 ) . Although the is o l a t e s originated from widely separated locations (Australia, Canada, Netherlands, and Scotland), they were clos e l y related with more than 9 3 % sequence homology. It appears there i s l i t t l e d i v e r s i t y i n PLRV i s o l a t e s worldwide. Subgenomic RNA's have been detected i n nucleic acid extracts from PLRV-infected potato (Mr 1 x 1 0 6 ) and from BYDV-PAV infected oats (c. 0 . 8 and 2 . 8 kb) (Barker, Mayo, and Robinson, 1 9 8 4 ; Gerlach, M i l l e r , and Waterhouse, 1 9 8 7 ) . Gildow, Ballinger, and Rochow ( 1 9 8 3 ) reported f i v e species of double-stranded RNA (ds-RNA) i n tissue infected with BYDV-RPV and four 6 from tiss u e infected with BYDV-MAV. Falk and Duffus (1984) found f i v e ds-RNA's i n nucleic acid extracts from plants infected with BWYV. Incomplete t r a n s l a t i o n of PLRV and SDV RNA in vitro and the presence of subgenomic RNA i n plants infected with these viruses suggests that luteoviruses may be translated v i a subgenomic messenger RNA species (Waterhouse, Gildow and Johnstone, 1988) . A recent review on the evolution and molecular biology of luteoviruses (Martin et al., 1990) suggests several d i f f e r e n t gene expression strategies for t h i s group including i n t e r n a l i n i t i a t i o n , t r a n s l a t i o n a l frameshifting, and subgenomic RNA for expression of ORF's i n the 3' ha l f of the genome. 1.2 Luteoviruses i n f e c t i n g potato PLRV, the causal agent of potato l e a f r o l l disease, occurs worldwide and i s economically the most important virus a f f e c t i n g potato crops (Harrison, 1984). Recently Duffus (1981a,b) has reported that BWYV i s a common and major component i n the potato l e a f r o l l disease complex i n North America. Isolates were recovered from potatoes with l e a f r o l l symptoms from widely separated locations including B r i t i s h Columbia, C a l i f o r n i a , Maine, Oregon, and Wisconsin. They reacted s e r o l o g i c a l l y i n a sim i l a r manner to BWYV is o l a t e s obtained from beets, c r u c i f e r s , and composites but they d i f f e r e d i n s p e c i f i c s e r o l o g i c a l 7 reactions. In a simi l a r study, Duffus and Johnstone (1982a) concluded that the l e a f r o l l syndrome i n Tasmania was e s s e n t i a l l y the same as i t i s i n North America. They suggested that, since s t r i c t quarantine measures had been enforced i n Tasmania since the early 1930's, BWYV had already spread with the movement of potato tubers throughout the world before the establishment of c e r t i f i c a t i o n schemes and quarantine r e s t r i c t i o n s . For example, Kyrikou, Close, and Ashby (1983) found potatoes to be infected i n the f i e l d with a New Zealand i s o l a t e of BWYV. The properties of BWYV and PLRV are compared i n Table I I . It i s clear from t h i s comparison that these viruses are s i m i l a r . The most apparent difference between them i s t h e i r host range. BWYV has the widest host range of the luteoviruses, and causes stunting and chlorosis i n a wide range of dicotyledonous species including many economic hosts such as b r o c c o l i , broad bean, cabbage, cauliflower, chick pea, clover, crambe, cucumber, flax, mustard, oil s e e d rape, pea, pepper, pumpkin, radish, spinach, soybean, sugar beet, sunflower, swede, table beet, tomato, turnip, and watermelon. Over 150 species i n 23 dicotyledonous families are susceptible to BWYV (Duffus, 1960,1964,1972,1977; Duffus and Johnstone, 1982b) as are at least a few monocotyledonous ones (Ashby and Johnstone, 1985; Duffus and Rochow, 1978). 8 Table I I . Comparison of the properties of beet western yellows virus (BWYV) and potato l e a f r o l l virus (PLRV) Main host families Main aphid vector Serological properties 8 Close s e r o l o g i c a l r e l a t i o n s h i p SDI 0-3 Moderate s e r o l o g i c a l r e l a t i o n s h i p SDI 4-6 Distant s e r o l o g i c a l r e l a t i o n s h i p SDI > 6 BWYV Amaranthaceae6 Chenopodiaceae Compositae Cruciferae Leguminosae Solanaceae Myzus persicae BMYV TuYV MYV BYDV-RPV SDV GRAV BLRV CRLV PLRV SMYEV PLRV Amaranthaceae7 Solanaceae M. persicae TYTV SYV TNDV BWYV GRAV SDV BLRV CLRV P a r t i c l e properties Sedimentation 1141 . 1152 c o e f f i c i e n t (S20,w) Diameter (nm) 251 242 Protein subunits 241 26.33 Mr (xlO 3) •^260/280 1 . 65 1 . 7 8 Density i n CsCl 1.421 1.393 (g/cm3) Genome properties SSRNA Mr (xlO 6) 1.91 2.03 size (nt) 56414 58835 1 Hewings & D'Arcy, 1983 2 Takanami & Kubo, 1979 3 Rowhani & Stace-Smith, 197 9 4 Veidt et al., 1988 5 Keese et al., 1988 Duffus, 1960,1964 7 N a t t i , Kirkpatrick & Ross, 1953 8 Waterhouse, Gildow & Johnstone, 1988 6 9 In contrast, most of the known hosts of PLRV (about 20 species) are i n the Solanaceae (Harrison, 1984) . A few species i n the Amaranthaceae, Cruciferae, Portulacaceae, and Nolanaceae are susceptible to PLRV (Natti, Kirkpatrick, and Ross, 1953; Thomas,1984; Tamada, Harrison and Roberts, 1984). The question of which viruses are responsible for causing potato l e a f r o l l disease i s of more than t h e o r e t i c a l i n t e r e s t . North American seed potato c e r t i f i c a t i o n programs are based on an extensive t e s t i n g program to ensure that the e l i t e stock i s vir u s - f r e e (Stace-Smith, 1987; Martin, 1987). Currently the reliance i s on ser o l o g i c a l t e s t i n g using antiserum prepared against PLRV. The occurrence of BWYV is o l a t e s i n potato stocks raises serious questions as to the v a l i d i t y of any currently used s e r o l o g i c a l indexing procedures (Duffus, 1981a). Further, BWYV has a very wide host range, and many common weeds i n the Cruciferae and Compositae serve as overwintering sources of BWYV (Wallis, 1967a,b). It i s possible that the extensive natural host range of BWYV could act as a reservoir of inoculum i n potato-producing and even i n is o l a t e d seed potato-producing areas (Duffus, 1981a). 10 1.3 D e f i n i t i o n of the problem Duffus (1981a) used three l i n e s of evidence to demonstrate the occurrence of BWYV i n potato plants affected with potato l e a f r o l l disease: (1) Indicator hosts for BWYV and PLRV. He used two indicator hosts, ground cherry {Physalis floridana Rydb. =P. pubescens L.) and shepherd's purse {Capsella bursa-pastoris [L.] M e d i c ) . These two were chosen because PLRV and BWYV cause si m i l a r reactions i n ground cherry, whereas shepherd's purse i s a commonly used indicator for BWYV. At the time Duffus (1981a,b) reported his work, shepherd's purse was not known to be a host for PLRV. Duffus found that f i v e potato sources with l e a f r o l l symptoms had naturally occurring i s o l a t e s that infected shepherd's purse. (2) Serological t e s t s . Virus i s o l a t e s from infected shepherd's purse were c l a r i f i e d by low-speed centrifugation and p e l l e t t e d by ul t r a c e n t r i f u g a t i o n . P e l l e t s were resuspended i n buffer to approximately 3.0% of the o r i g i n a l volume of sap. The re s u l t i n g virus samples were incubated for 0.5 hr at 37 C with equal volumes of antisera prepared against strains of BWYV, legume yellows luteovirus (LYV), and healthy 11 shepherd's purse sap as a control. The incubated mixtures were subjected to density-gradient centrifugation and analyzed photometrically. The reduction or elimination of virus antigen i n the scanning patterns was considered a p o s i t i v e test for BWYV. Duffus found that virus i s o l a t e s from potato l e a f r o l l - a f f e c t e d plants reacted s e r o l o g i c a l l y the same as other BWYV is o l a t e s and LYV, but not with antisera to healthy shepherd's purse. He also found that antiserum to PLRV reacted with a l l BWYV strains tested. Duffus maintained that the rec i p r o c a l reaction, BWYV antiserum against PLRV i s o l a t e s , was negative i n a l l tests except the distant r e l a t i o n s h i p demonstrated by Roberts, Tamada, and Harrison (1980) but he did not include t h i s control i n his experiment. (3) Host range studies. Duffus found that the host range of one i s o l a t e from l e a f r o l l - a f f e c t e d potato (P5) was simi l a r to that of other i s o l a t e s of BWYV is o l a t e d from c r u c i f e r s and composites. Duffus' conclusion that BWYV i s widely d i s t r i b u t e d i n North America i s supported by the work of Sibara and Slack (1985b) who reported that 65% of 519 potato l e a f r o l l - a f f e c t e d plants were infected with both BWYV and PLRV, 4% with BWYV alone, and 16% with PLRV alone. Neither BWYV nor PLRV was detected i n 15% of the symptomatic samples and they suggested that a s i g n i f i c a n t component of the potato l e a f r o l l complex was not detected by t h e i r antisera. These samples represented 18 c u l t i v a r s o r i g i n a t i n g from Wisconsin, Maine, Minnesota, Nebraska, and North Dakota. Gallenberg, Z i t t e r , and Jones (1987) reached s i m i l a r conclusions when they detected BWYV i n potato l e a f r o l l samples from nine states and provinces i n North America. In contrast to evidence that supports Duffus' work are several studies that question the importance or involvement of BWYV as a causal agent of potato l e a f r o l l disease. Clarke, Powelson and Beraha (1983) f a i l e d to detect BWYV i n Russet Burbank potatoes affected with net necrosis. Foliage sampled from plants with l e a f r o l l symptoms also were negative for BWYV but were p o s i t i v e for PLRV. Marco (1984) reported that i s o l a t e s of BWYV from Israel were not able to i n f e c t potato or several other members of the Solanaceae. Barker (1986) tested several potato c u l t i v a r s for s u s c e p t i b i l i t y to two B r i t i s h i s o l a t e s of BWYV. Neither i s o l a t e , either by i t s e l f or i n association with PLRV, was transmitted by GPA to vir u s - f r e e potato plants. Barker concluded that although l o c a l strains do not present any i d e n t i f i e d r i s k to the potato crop i n B r i t a i n , tests for BWYV are already included i n quarantine procedures for potatoes imported into B r i t a i n . The results of ISEM (immunosorbent electron microscopy) studies indicate that BMYV, a s t r a i n of BWYV, i s clo s e l y related s e r o l o g i c a l l y to Scottish i s o l a t e s of PLRV (Roberts and Harrison, 1979) . Richter et al. (1983) compared PLRV and BMYV s e r o l o g i c a l l y by agar gel double d i f f u s i o n , ELISA, and immuno-EM decoration. Nine antisera prepared against PLRV and three against BMYV were tested by agar gel double d i f f u s i o n against both viruses. In each case a cross-reaction was obtained, t i t e r s being lower with the homologous than the heterologous virus by a factor of 3-5, ind i c a t i n g a distant s e r o l o g i c a l r e l a t i o n . Decoration tests performed i n the same way gave equivalent r e s u l t s . Reactions were weaker for the heterologous reaction than for the homologous one. However, i n ELISA no cross-reaction was found with 1-100 p.g/ml of the heterologous virus, whereas the detection l i m i t for the homologous virus was 10 ng/ml. Marco (1985) found i n ISEM tests that PLRV and BWYV could be e a s i l y detected by coating grids with either homologous or heterologous antiserum. He also demonstrated that PLRV and BWYV could be trapped on ELISA plates with either homologous or heterologous antiserum. Both results c l e a r l y indicate s e r o l o g i c a l cross-r e a c t i v i t y between PLRV and BWYV. Tamada, Harrison, and Roberts (1984) suggest that cross-reactions between BWYV, BMYV, and PLRV in ELISA may account for at least some of the ser o l o g i c a l evidence that BWYV occurs i n potato. The results of other tests with BWYV antiserum i n which no relati o n s h i p with PLRV (Duffus and Gold, 1969) or only a weak relat i o n s h i p was found (Roberts et al. 1980) may r e f l e c t antigenic v a r i a t i o n i n BWYV or possibly the u n a v a i l a b i l i t y , at the time, of hig h - t i t e r e d antisera (Tamada et al., 1984). In reaching his conclusions, Duffus (1981a,b) has made a number of assumptions which might be questioned: (1) Duffus assumed that virus i s o l a t e s that are aphid transmitted from l e a f r o l l - a f f e c t e d potato to shepherd's purse must be BWYV and not PLRV because shepherd's purse was not known to be a host for PLRV at that time. Thomas (1981) found that an Australian i s o l a t e of tomato yellow top virus (TYTV), a s t r a i n of PLRV (Harrison, 1984; Casper, 1988), had a host range that was mainly r e s t r i c t e d to the Solanaceae, but also infected shepherd's purse asymptomatically. Further, an i s o l a t e from l e a f r o l l - a f f e c t e d potato, which caused t y p i c a l l e a f r o l l symptoms i n potato and symptomless i n f e c t i o n i n shepherd's purse, reacted i n gel d i f f u s i o n tests with PLRV antiserum (Thomas, 1981) . S y l l e r (1985) found that six PLRV i s o l a t e s , representing a range of symptom severity on both ground cherry and potato, a l l infected shepherd's purse but the frequency of successful i n f e c t i o n depended on both the i s o l a t e and the source plant. His work also showed that the concentration of PLRV i n potato i s dependent on both the virus i s o l a t e and on 15 the s u s c e p t i b i l i t y of the c u l t i v a r . S y l l e r concluded that i t i s not necessary to assume the existence of two or more viruses, even clo s e l y related ones, i n order to explain why the virus could be transmitted to shepherd's purse from some potato plants but not from others. Barker ( 1 9 8 6 ) succeeded i n transmitting a virus from l e a f r o l l - a f f e c t e d potatoes to shepherd's purse, but the virus was PLRV not BWYV. Fox et al. ( 1 9 9 0 ) reported transmission of an i s o l a t e of PLRV to, and recovery from, two wild c r u c i f e r s , shepherd's purse and Sisymbrium altissimum L. (Jim H i l l or tumble mustard). (2) The ser o l o g i c a l test Duffus used to confirm that the i s o l a t e s transmitted to shepherd's purse were BWYV was based on the assumption that BWYV antiserum does not cross-react with PLRV even though i n the reci p r o c a l test PLRV antiserum cross-reacts with BWYV. The technique he used involved incubating semi-p u r i f i e d virus and antiserum mixtures, subjecting the mixture to density gradient centrifugation, and scanning the gradient photometrically. A p o s i t i v e assay was based on the reduction or elimination of the virus peak i n the scanning patterns of density gradient columns. While t h i s test would provide evidence that a virus-antiserum i n t e r a c t i o n had 1 6 occurred, i t may not di s t i n g u i s h between a se r o l o g i c a l l y related antigen and a s e r o l o g i c a l l y s p e c i f i c antigen (Stace-Smith, 1987) . Since BWYV and PLRV are s e r o l o g i c a l l y related (Marco, 1985; Harrison, 1984; Thomas, 1984), a reduction i n the scanning pattern i n a virus-antiserum mixture where PLRV i s incubated with an antiserum prepared against BWYV would be expected. Duffus' r e s u l t s could be interpreted as showing that i s o l a t e s of PLRV, s e r o l o g i c a l l y related to BWYV, can be transmitted by aphids from potato to shepherd's purse (Stace-Smith, 1987) . This l a s t statement i s supported by some of Duffus' own work; Milbrath and Duffus (1978) demonstrated that solanum yellows virus (SYV), now known to be a s t r a i n of PLRV (Casper, 1988; Waterhouse et al., 1988), i s s e r o l o g i c a l l y related to BWYV and BYDV-RPV using 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 and ELISA t e s t s . (3) Host range study. Duffus (1981a) used shepherd's purse, infected with an i s o l a t e of BWYV (P-5) from potato as a source plant for the host range study, not the o r i g i n a l infected potato plant. Because shepherd's purse i s a good host for many stra i n s of BWYV and i s very a t t r a c t i v e to the vector GPA, i t i s possible that the shepherd's purse may have become infected with BWYV from a source other than potato. Transmission of a virus from potato d i r e c t l y to a host range t y p i c a l of BWYV would have been more convincing. Casper (1983) did not succeed i n attempts to transmit a virus from l e a f r o l l - a f f e c t e d potatoes to the hosts of BWYV and concluded that BWYV i s probably not important as a cause of potato l e a f r o l l disease i n Germany. 1.4 Objectives The role of BWYV i n potato l e a f r o l l disease has not been unequivocally determined. The effectiveness of control programs for t h i s disease w i l l be threatened i f BWYV proves to be an important component of potato l e a f r o l l disease. Consequently, a study was undertaken to determine the importance of BWYV as a cause of potato l e a f r o l l disease i n Canada and the United States. The objectives of t h i s research were: (1) to produce monoclonal antibodies (MAbs) that react with BWYV but do not cross-react with PLRV; 18 (2) to determine the incidence of BWYV and PLRV i n potato l e a f r o l l disease samples from several locations i n Canada and the United States using v i r u s - s p e c i f i c MAbs; (3) to produce nucleic acid probes (cDNA) for BWYV and PLRV and compare nucleic acid hybridization methods with ELISA for detection of BWYV and PLRV; (4) to determine the s u s c e p t i b i l i t y of potato to several i s o l a t e s of BWYV from Canada and the United States; (5) and, to determine the importance of weeds as reservoirs of BWYV and PLRV i n the potato-producing areas of B r i t i s h Columbia. 19 Chapter 2 Production of monoclonal antibodies 2.1 Introduction Potato l e a f r o l l , caused by potato l e a f r o l l virus (PLRV), i s a major disease of potato (Banttari, E l l i s , and Khurana, 1990) and i s responsible for high y i e l d losses throughout the world wherever potatoes are grown (Peters and Jones, 1981). In 1981 Duffus (1981a) reported that beet western yellows virus (BWYV) i s a common and important component of a virus complex responsible for t h i s disease. Seed potato c e r t i f i c a t i o n programs that r e l y on antisera prepared against PLRV to confirm the diagnosis of potato l e a f r o l l disease may not detect plants infected with BWYV (Marco, 1985; Stace-Smith, 1987) . In the past, polyclonal antisera prepared against luteoviruses in rabbits often have had r e l a t i v e l y high l e v e l s of nonspecific reaction (Clarke, Converse and Kojima, 1980; Martin and Stace-Smith, 1984) and those reactions can lead to f a l s e - p o s i t i v e r e s u l t s i n ELISA (Gunn and Pares, 1988) . V a r i a b i l i t y i n the q u a l i t y of polyclonal antisera can lead to disagreements among investigators working with the same antigen i n d i f f e r e n t laboratories (Halk and DeBoer, 1985). Often, high qu a l i t y reference or diagnostic polyclonal antisera are not produced i n s u f f i c i e n t quantities for general d i s t r i b u t i o n . Hybridoma 20 technology, introduced by Kohler and M i l s t e i n (1975), revolutionized antibody production and eliminated many of the problems associated with polyclonal antiserum. This technology provides a means to produce an unlimited, uniform supply of antibody of a required s p e c i f i c i t y (Goding, 1986) . The objective of t h i s research was to produce MAbs that could be used i n an ELISA procedure to detect and d i f f e r e n t i a t e BWYV and PLRV. 2.2 Materials and methods 2.2.1 Virus i s o l a t e s and p u r i f i c a t i o n The source of the PLRV i s o l a t e used for p u r i f i c a t i o n was a single tuber of the potato c u l t i v a r Russet Burbank infected with a severe s t r a i n of the virus (Wright and MacCarthy, 1963). This i s o l a t e was characterized by Rowhani and Stace-Smith (1979) and w i l l be c a l l e d PLRV-BC. PLRV-BC was propagated i n Physalis pubescens L. (=P. floridana Rybd.) inoculated by v i r u l i f e r o u s GPA i n a growth chamber (21 C, 105 (lEm^s-1 fluorescent and incandescent l i g h t ) for a 72 hr inoculation access period. The aphids were k i l l e d by spraying the plants with pirimicarb (0.25 g ai/1, Chipman Inc., Winnipeg, Manitoba) and the plants were moved to a greenhouse (15-25 C) for 4 to 6 weeks. Leaf tissue was harvested and used immediately or frozen at -80 C u n t i l used. A sugar beet i s o l a t e of BWYV from B r i t i s h Columbia (MacCarthy, 1 9 6 9 ) was used for virus p u r i f i c a t i o n and w i l l be referred to as BWYV-BC. P. pubescens was inoculated with BWYV-BC and propagated i n a separate growth chamber and greenhouse using the same procedure as for PLRV-BC. The luteovirus i s o l a t e s used to determine the s p e c i f i c i t y of MAbs, and t h e i r sources are l i s t e d i n Table I I I . Both PLRV and BWYV were p u r i f i e d using a modification of the procedure used by D'Arcy et al. ( 1 9 8 3 ) . Virus-infected tissue was frozen i n l i q u i d nitrogen and crushed into small pieces with a wooden pestle. The frozen tissue was then transferred to a stainle s s s t e e l blender jar, pre-cooled with l i q u i d nitrogen, and ground to a fine powder. The powdered tissue was added to 0 . 1 M phosphate buffer pH 6.0 (2 ml/g of tissue) containing 0 . 1 % 2-mercaptoethanol, 0 . 2 % (w/v) Ultrazym 1 0 0 (Schweizerische Ferment AG, Basel, Switzerland), and 0 . 0 2 % sodium azide. The mixture was s t i r r e d slowly overnight at 4 C. Trito n X - 1 0 0 (1% v/v) was added and the preparation was s t i r r e d for 2 hr at room temperature followed by the addition 2 2 Table I I I . Luteovirus i s o l a t e s used to determine s p e c i f i c i t y of monoclonal antibodies. Virus i s o l a t e Origin Host Source BWYV-B10 B e l t s v i l l e , USA beet J. E. Duffus 1 BWYV-RB2 C a l i f o r n i a , USA b r o c c o l i J. E. Duffus BWYV-MY4 C a l i f o r n i a , USA Malva sp. J. E. Duffus BWYV-RY-1-R C a l i f o r n i a , USA b r o c c o l i J. E. Duffus BWYV-150 C a l i f o r n i a , USA broad bean J. E. Duffus BWYV-RY-7 C a l i f o r n i a , USA b r o c c o l i J. E. Duffus BWYV-MP4BF Germany c r u c i f e r J. E. Duffus BWYV-SAL(9) Germany lettuce J. E. Duffus BWYV-NETH Netherlands lettuce J. E. Duffus BWYV-40-8003 Tasmania ground cherry G. Johnstone 2 BWYV-BC B.C., Canada sugar beet H. MacCarthy 3 BMYV-324 England sugar beet J. E. Duffus TuYV England turnip J. E. Duffus BYDV-RPV-NY New York, USA oats B. W. Falk 4 BYDV-MAV-NY New York, USA oats B. W. Falk BYDV-RPV-IL I l l i n o i s , USA oats C. D'Arcy 5 BYDV-RPV-T Tasmania oats G. Johnstone SDV-AS Tasmania clover G. Johnstone SDV-AP Tasmania clover G. Johnstone SYV-21 I l l i n o i s , USA Solanum sp. J. E. Duffus SYV-44 I l l i n o i s , USA Solarium sp. J. E. Duffus PLRV-BC B.C., Canada potato N. S. Wright 3 PLRV-ST4 B.C., Canada potato N. S. Wright PLRV-ORE Oregon, USA potato 0. Gutbrod 6 PLRV-ID Idaho, USA potato R. G. Clarke 7 1 USDA, ARS, Salinas, C a l i f o r n i a , USA. 2 Tasmania Department of Agriculture, Hobart, A u s t r a l i a . 3 Agriculture Canada, Vancouver Research Station, Vancouver, B.C., Canada. 4 University of C a l i f o r n i a , Davis, C a l i f o r n i a , USA. 5 University of I l l i n o i s , Urbana, I l l i n o i s , USA. 6 Oregon State University, C o r v a l l i s , Oregon, USA. 7 Idaho Crop Improvement Association, Idaho F a l l s , Idaho, USA. 23 of 1/10 vol of chloroform:butanol (1:1) with continued s t i r r i n g for 30 min at room temperature. The emulsion was c l a r i f i e d by centrifugation at 13,200 x g for 20 min. S o l i d NaCl and polyethylene g l y c o l 6000 (PEG) were added to the supernatant to give 0.4 M and 8% (w/v) respectively and the mixture was s t i r r e d for 1 hr at 4 C. The p r e c i p i t a t e was recovered by centrifugation at 13,200 x g for 30 min and the p e l l e t s were resuspended with a glass homogenizer i n l/10th the o r i g i n a l vol of 0.05 M phosphate buffer pH 7.0. The suspension was c l a r i f i e d by low speed centrifugation as above. The virus p a r t i c l e s were p e l l e t e d by high speed centrifugation at 184,000 x g for 90 min through a 10 ml 20% sucrose cushion. The p e l l e t s were resuspended i n 0.05 M phosphate buffer pH 7.0. Following one additional cycle of low and high speed centrifugation, the viruses were further p u r i f i e d on 10-40% l i n e a r sucrose density gradients, prepared by the freeze-thaw method of Davis and Pearson (1978), centrifuged i n a Beckman SW 41 rotor at 178,000 x g for 90 min. Gradients were scanned photometrically with an ISCO UA-5 absorbance monitor and the fractions containing the virus were c o l l e c t e d . The fractions were combined and d i l u t e d twofold with 0.05 M phosphate buffer pH 7.0 and then centrifuged at 290, 000 x g for 2 hr. The p e l l e t s were resuspended i n a small vol of 0.05 M, phosphate buffer pH 7.0 and stored at 4 C. The virus concentration was estimated from the UV-absorption spectra (220-320 nm, Hewlett-Packard Model 5451A spectrophotometer) assuming an extinction c o e f f i c i e n t of A 2 6 0 0 - 1 % = 8.6 based on the value calculated for PLRV by Takanami and Kubo (1979) . 2 . 2 . 2 Antibodies The rabbit antiserum against PLRV used i n t h i s study was prepared by Rowhani and Stace-Smith (1979). A rabbit antiserum against a C a l i f o r n i a n i s o l a t e of BWYV from b r o c c o l i (RY-l-R) was a g i f t from Dr. J. Duffus (USDA, ARS, Salinas, C a l i f o r n i a ) . MAb (371A) , s p e c i f i c for PLRV, was a g i f t from Dr. R. Martin (Vancouver Research Station, Agriculture Canada, Vancouver, B.C.) . Immunoglobulins were p u r i f i e d from antisera by ammonium sulfate p r e c i p i t a t i o n and DEAE-cellulose column chromatography as described by Clarke, L i s t e r , and Bar-Joseph, (1986). The immunoglobulins were adjusted to approximately 1 mg/ml (A 2 8 0 =1 . 4) and stored i n aliquots of 0 . 5 ml at - 2 0 C. 2 . 2 . 3 Hybridoma production, screening and isotyping Two immunization procedures were used: (1) RBF/Dn mice (Jackson Laboratories, Inc., Bar Harbour, Maine) were immunized with three inje c t i o n s of p u r i f i e d BWYV i n 0 . 0 5 M phosphate 2 5 buffer pH 7.0. The f i r s t i n j e c t i o n , 50 |ig of BWYV emulsified with Freund's incomplete adjuvant, was given subcutaneously. The second and t h i r d injections of 50 \ig each were administered i n t r a p e r i t o n e a l l y at 4 and 6 weeks following the f i r s t i n j e c t i o n . Three days following the t h i r d i n j e c t i o n , the mice were k i l l e d by C02 asphixiation and the spleens were removed a s e p t i c a l l y . (2) BALB/c mice were immunized with three in j e c t i o n s of v i r u s . The f i r s t i n j e c t i o n was 50 jig each of p u r i f i e d BWYV and PLRV emulsified with Freund's incomplete adjuvant and given subcutaneously. The second i n j e c t i o n was 50 |ig of PLRV given 4 weeks l a t e r i n t r a p e r i t o n e a l l y i n 0.05 M phosphate buffer pH 7.0. The t h i r d and f i n a l booster, 6 weeks aft e r the f i r s t , was 50 fig of BWYV given i n t r a p e r i t o n e a l l y i n phosphate buffer. Three days l a t e r the mice were s a c r i f i c e d and the spleens were harvested as above. The same fusion protocol was used for both immunization procedures. FOX-NY myeloma c e l l s (Hyclone Laboratories, Inc., Logan, Utah) were cultured i n Dulbecco's Modified Eagle Medium (DMEM, Gibco Canada Inc., Burlington, Ontario) supplemented with 1 mM pyruvate and 2 mM L-glutamine and 10% f e t a l c a l f serum (FCS). Spleen c e l l s were fused with myeloma c e l l s (spleen c e l l to myeloma c e l l r a t i o approximately 10:1) i n 50% polyethylene g l y c o l 4000 (BDH Chemicals Canada Ltd., Vancouver, B.C.) as described by Kannangara, Wieczorek and Lavender (1989). A l l myelomas and hybridomas were grown at 37 C i n an atmosphere of 10% C02. The c e l l fusion mixture was dispensed into f i v e 96-well culture plates (Nunc, Denmark) and incubated overnight i n nonselective media (DMEM containing 20% FCS) with mouse thymocytes as feeder c e l l s . The microcultures were then fed with AAT selection media [7.5 x 10~5 M adenine, 8 x 10"7 M aminopterin, and 1.6 x 10"5 M thymidine (Taggart and Samloff, 1983)] . After 10 days, culture f l u i d s from the hybridomas were screened for anti-BWYV and anti-PLRV antibody production by an in d i r e c t t r i p l e antibody sandwich ELISA [(TAS-ELISA) , Martin and Stace-Smith, 1984] described i n d e t a i l below. Culture f l u i d from the hybridomas were also screened for antibodies against healthy plant sap. Hybridoma c e l l l i n e s which produced antibodies .that tested p o s i t i v e l y with either BWYV or PLRV and negatively against healthy potato and ground cherry sap were cloned twice by l i m i t i n g d i l u t i o n , grown i n c e l l culture, retested, and the po s i t i v e cultures were stored under l i q u i d nitrogen. For production of antibodies i n a s c i t i c f l u i d , BALB/c mice were primed i n t r a p e r i t o n e a l l y w i t h 2,6,10,14-tetramethylpentadecane (Pristane; A l d r i c h Chemical Co., Milwaukee, Wisconsin) 10 days before intr a p e r i t o n e a l i n j e c t i o n of approximately 107 hybridoma c e l l s . Some BALB/c mice were also immunosuppressed by i n j e c t i o n of cyclophosphamide (0.025 g/g animal weight, Sigma Chemical Co., St. Louis, Missouri) 24 hr before i n j e c t i o n of the hybridoma c e l l s . A s c i t i c f l u i d was 27 c o l l e c t e d 10 to 20 days l a t e r by in s e r t i n g an 18-gauge needle into the peritoneal cavity of mice showing pronounced abdominal swelling. After low speed centrifugation at 8,700 x g for 20 min, to remove c e l l u l a r debris, the a s c i t i c f l u i d supernatant was mixed with an equal vol of saturated ammonium sulfate and stored at 4 C u n t i l required. The MAb isotypes were determined by double antibody sandwich ELISA (DAS-ELISA) using a mouse hybridoma sub-isotyping k i t (Calbiochem, Behring Diagnostics, La J o l l a , C a l i f o r n i a ) . Subcloned hybridoma supernatant f l u i d s were tested using the manufacturer's protocol. 2.2.4 ELISA An i n d i r e c t t r i p l e antibody sandwich ELISA (TAS-ELISA) procedure was used for screening hybridomas for antibody production and for t e s t i n g antibody s p e c i f i c i t y (Martin and Stace-Smith, 1984) . A l l reagents were used at 100 (Xl per well i n flat-bottomed Lindbro microtiter plates (Flow Laboratories, Mississauga, Ontario) except for the blocking steps, which were 300 [Ll per well. Plates were coated overnight at 4 C with polyclonal immunoglobulin homologous to the antigen to be tested, d i l u t e d to 1 p.g/ml i n phosphate-buffered saline [ (PBS) 127 mM NaCl, 2.6 mM KC1, 8.5 mM Na2HP04, 1.1 mM KH2P04] . Plates 28 were blocked with 0.2% BLOTTO [BLOTTO (Johnson et al., 1984) i s 10 g nonfat dried milk made up to 100 ml with d i s t i l l e d water and 0.02% sodium azide as a preservative] i n PBS for 30 min at room temperature. Leaf tiss u e from infected and healthy plants was ground i n a sap extractor (Erich Pollahne, F.G.R.) while adding grinding buffer (PBS/ 0.05% Tween 20/ 2% PVP/ 0.2% BLOTTO) drop-wise onto the bevelled r o l l e r s (1 ml per 0.1 g leaf tissue) . Isolates provided by Dr. J. E. Duffus (Table 3) as freeze-dried cultures were ground with a mortar and pestle (0.02 g/ml grinding b u f f e r ) . Extracts were incubated i n the wells overnight at 4 C. Plates were then washed three times for 20 sec with PBS containing 0.05% Tween 20 (PBS-T) and then blocked again as above. Homologous or heterologous MAbs, from culture f l u i d supernatants, were d i l u t e d 1:1 i n PBS-T containing 0.2% BLOTTO (PBS-T-BLOTTO) and incubated i n the wells for 3 hr at room temperature. Plates were washed as above arid conjugate, rabbit anti-mouse al k a l i n e phosphatase (Jackson Immunoresearch Laboratories, Inc., Avondale, Pennslyvania), d i l u t e d 1:5,000 i n PBS-T-BLOTTO was incubated i n the wells for 3 hr at room temperature. Afte r washing as above, substrate (p-nitrophenyl phosphate, Sigma 104-105, Sigma Chemical Co.) at 0.5 mg/ml i n 10% (v/v) diethanolamine pH 9.8 was added to the wells and incubated for 2 hr at room temperature, then overnight at room temperature. The absorbance of each well was read at 405 nm (A 4 0 5) in a Titertek Multiscan MCC plate reader (Flow Laboratories). ELISA reactions were scored +++ for readings of 1.0 and greater, ++ for readings of 0.5 to 1.0, and + for readings of less than 0.5 but greater than three times the mean of the readings of the healthy control plants. Readings below t h i s threshold were scored - for negative (Voller, Bidwell, and B a r t l e t t , 1977) . 2.3 Results 2.3.1 Production of hybridomas From the f i r s t immunization protocol and fusion two hybridoma clones secreting BWYV-specific antibodies were obtained. Clones 510H and 112E produced a s c i t i c f l u i d s i n BALB/c mice, a f t e r immunosupression with cyclophosphamide, with t i t r e s of 10 - 8 and 10 - 4 respectively i n TAS-ELISA. Clone 510H produced between 3-8 ml of a s c i t i c f l u i d per mouse whereas clone 112E tended to produce s o l i d tumors and less than 1 ml of ascites per mouse. The fusion from the second immunization protocol resulted in 18 wells of 420 with hybridomas t e s t i n g p o s i t i v e for production of antibody to either BWYV or PLRV, or both, but not 30 to healthy plant sap from potato or P. pubescens. Seven of these hybridomas were successfully cloned and tested further. The isotypes of the MAbs are presented i n Table IV. Table IV. Monoclonal antibody subclasses Monoclonal Isotype Homologous antibody antigen 510H IgG2a BWYV-BC 112E IgGl BWYV-BC 13CD IgM BWYV-BC 15CD IgM BWYV-BC 31CC IgM BWYV-BC 43GB IgM BWYV-BC 43BC IgM BWYV-BC/PLRV-BC 26BE IgGl PLRV-BC 41BC IgM PLRV-BC 2.3.2 Monoclonal antibody s p e c i f i c i t y The s p e c i f i c i t y of the MAbs was determined i n TAS-ELISA using 25 luteovirus i s o l a t e s (Table III) and the resu l t s are given i n Table V. Sixteen of the is o l a t e s are BWYV [BWYV includes i s o l a t e s i d e n t i f i e d by the synonyms (Table I) beet mild yellowing virus (BMYV), turnip yellows virus (TuYV), and the RPV is o l a t e of barley yellow dwarf (Casper, 1988)] . Two MAbs, 510H and 112E, reacted with a l l of the BWYV is o l a t e s tested and four others (13CD, 15CD, 31CC, and 43GB) detected a l l of the BWYV is o l a t e s except the RPV i s o l a t e of BYDV. MAbs 26BE and 41BC 31 Table V. S p e c i f i c i t y of monoclonal antibodies* Monoclonal antibodies Antigen 510H 112E 13CD 15CD 31CC 43GB 43BC 26BE 41BC 371A* BWYV-BC + + + + + + + + + + + + + + + -BWYV-RY-1-R + + + + + + + + + + + + + + + + + -BWYV-RY-7 + + + + + + + + + + + + + + + + + + + -BWYV-SALO) + + + + + + + + + + + + + + + + + + + + + - -BWYV-NETH + + + + + + + + + + + + + + + + + + + -BWYV-150 + + + + + + + + + + + - -BWYV-4 0-008 _|_ _j_ _|_ BWYV-B10 + + + + + ; + + + - - -BWYV—RB2 + + + + + + + + + + + + + + + + + + -BWYV—MP 4 + + + + + + + + + - - -BWYV-MY4 + + + + + + + + + -BMYV-324 + + + + + + + + + + - - -TuYV + + + + + + + + + + + + + + + + + + + -BYDV—RPV—NY _|__|_ + + _ — _ — — — — — BYDV-RPV-T _)__(_ BYDV—MAV—NY — - — BYDV-PAV-IL — — — — — — — 32 Table V. Continued A n t i g e n 510H 112E Monoclonal antibodies 13CD 15CD 31CC 43GB 43BC 26BE 41BC 371A' PLRV-BC - - " " " - + ++ + + + + + + PLRV-ST4 - - - - - - • ++ + + + + + + PLRV-ORE - - ++ + + + + + + PLRV-ID - - - " " - • +++ + + + + + + SYV-21 - - - " " " + + + + SYV-44 - - - " - " + ++ + + + + SDV-AS - - _ _ _ _ _ - - -SDV-AP - - _ _ _ _ _ - - -* Reactions (A 4 0 5) after overnight incubation of substrate +++ = 1.0 or more ++ = 0.5 - 1.0 + = less than 0.5 =. negative *MAb 371A was produced by Martin and Stace-Smith (1984) reacted with a l l of the PLRV is o l a t e s screened including two is o l a t e s of a s t r a i n of PLRV c a l l e d solanum yellows virus (SYV). One MAb (43BC) reacted with an epitope common to BWYV and PLRV. None of the MAbs reacted with the other luteoviruses nor with healthy plant sap. 33 2.4 Discussion Although the ser o l o g i c a l relationships among the luteoviruses are complex (Waterhouse et al., 1988), MAbs provide a t o o l to discriminate among them. Several clear advantages of MAbs over polyclonal antisera have been reported (Halk and DeBoer, 1985): 1. the a v a i l a b i l i t y of an unlimited supply of uniform antibody s p e c i f i c for a single epitope; 2. MAbs can be produced from a small amount of antigen, even i f the antigen i s impure; 3. hybridomas can be stored i n l i q u i d nitrogen, ensuring a continuous supply of antibody over time; 4. MAbs obviate the q u a l i t a t i v e and quantitative antibody content encountered i n the use of d i f f e r e n t batches of polyclonal antiserum; 5. and, s p e c i f i c MAbs may reveal s e r o l o g i c a l relationships between antigens that were previously unrecognizable using polyclonal antiserum. The objective of t h i s study, to produce MAbs that would be suitable for detecting and i d e n t i f y i n g BWYV and PLRV i n plant tissu e using ELISA, was achieved. The TAS-ELISA procedure has two advantages over DAS-ELISA: (1) a single anti-mouse conjugate can be used to detect a l l mouse MAbs and (2) the s p e c i f i c i t y of the MAb i s not altered by the conjugation of an enzyme (Koenig, 1978) . MAbs 510H and 112E detected a l l the BWYV strains tested including the RPV i s o l a t e of BYDV that i s now recognized as a st r a i n of BWYV (Casper, 1988; D'Arcy, Torrance, and Martin, 1989) . Four other MAbs detected a l l the BWYV strains except BYDV-RPV. Any of these six MAbs would be suitable for screening potato samples for BWYV because the host range of BYDV-RPV does not include dicotyledonous species (Rochow and Duffus, 1978). MAb 510H was selected for further large scale t e s t i n g because of i t s broad s p e c i f i c i t y for BWYV, detecting 16 i s o l a t e s from six countries on four d i f f e r e n t continents, and because of i t s high t i t e r i n ascites f l u i d . Two MAbs, 26BC and 41BC, detected a l l of the PLRV i s o l a t e s tested including the two strai n s of PLRV named solanum yellows virus (SYV) . None of the MAbs detected either s t r a i n of soybean dwarf virus (SDV) or the MAV and PAV is o l a t e s of BYDV. Because the s p e c i f i c i t y of MAbs 26BC. and 41BC appeared to be the same as MAb 371A produced by Martin and Stace-Smith (1984), 371A was chosen for further t e s t i n g because i t was already available as a s c i t i c f l u i d and i t had been widely tested against many is o l a t e s of PLRV. Clone 43BC produced antibody that detected an epitope common to both BWYV and PLRV. This epitope has also been reported by D'Arcy et a l . (1989), and i t may provide an 35 explanation for the confusion concerning the presence of BWYV in potato affected with potato l e a f r o l l disease. In Duffus' work demonstrating the presence of BWYV i n potato (Duffus, 1981a) i t had been assumed that BWYV antiserum did not cross-react with PLRV. If some of the antibodies i n his BWYV polyclonal antiserum were against an epitope common to both viruses, then the antigen scanning pattern analysis used by him, would not be s p e c i f i c for BWYV. Since a po s i t i v e test was based on the reduction or elimination of the virus peak, t h i s assay would have been evidence that an antigen-antibody i n t e r a c t i o n had occurred, but the inter a c t i o n may have involved an epitope not unique to BWYV. The res u l t s of t h i s study show that the MAbs described here can be used e f f e c t i v e l y i n a routine diagnostic TAS-ELISA for the detection and i d e n t i f i c a t i o n of BWYV and PLRV i n plant t i s s u e . 36 Chapter 3 Detection and i d e n t i f i c a t i o n using monoclonal antibodies 3.1 Introduction Potato l e a f r o l l disease i s one of the most important diseases of potato and i t occurs worldwide wherever potatoes are grown (Peters and Jones, 1981): The disease i s usually considered to be caused by PLRV (Barker, 198 6) but i n North America (Duffus, 1981a,b) and i n Tasmania (Duffus and Johnstone, 1982) BWYV has been i s o l a t e d from plants with t y p i c a l potato l e a f r o l l disease symptoms. The conclusion that BWYV occurs i n potato (Duffus, 1981a,b) was made on the basis of two assumptions: that shepherd's purse (Capsella bursa-pastoris) i s a host for BWYV but i t i s not a host for PLRV, and that BWYV antiserum does not cross-react with PLRV. Both assumptions are challenged by more recent evidence i n the s c i e n t i f i c l i t e r a t u r e (Stace-Smith, 1987) . - Several researchers have demonstrated that PLRV can indeed i n f e c t shepherd's purse (Fox et al., 1990; S y l l e r , 1985; Thomas, 1984) and others have reported that BWYV antiserum does cross-react with PLRV i n some se r o l o g i c a l tests (Marco, 1985; Richter et al., 1983). 37 Immunological techniques are among the most important tools for the detection and i d e n t i f i c a t i o n of plant viruses (Halk, 198 6). Recent advances i n methodology, including ELISA and monoclonal antibody (MAb) production, have greatly improved the s e n s i t i v i t y , s p e c i f i c i t y , and ease of luteovirus diagnosis (D'Arcy et al., 1989). BWYV i s p o t e n t i a l l y a greater threat to seed potato production than PLRV because i t has a much wider host range. Many common weeds are hosts of BWYV and they may serve as virus reservoirs i n seed production areas. The objective of t h i s study was to determine i f BWYV i s an important component of a complex causing potato l e a f r o l l disease i n Canada and the United States using virus s p e c i f i c MAbs. A preliminary report has been published ( E l l i s and Wieczorek, 1988). 3.2 Materials and methods 3.2.1 C o l l e c t i o n of samples Most of the samples were c o l l e c t e d i n the seed potato c e r t i f i c a t i o n winter test plots near Homestead, F l o r i d a and Oceanside, C a l i f o r n i a . With the assistance of seed potato o f f i c i a l s from each l i s t e d state or province (Table VI), foliage samples were c o l l e c t e d from plants with secondary l e a f r o l l symptoms such as l e a f r o l l i n g of the lower leaves, chlorosis, and stunting (Fig. 1). The samples were placed i n l a b e l l e d p l a s t i c 38 i F i g . 1. Secondary symptoms of potato l e a f r o l l v i r u s from tuber-borne i n f e c t i o n . 39 bags and shipped to Vancouver i n coolers. Some of the samples were provided by seed c e r t i f i c a t i o n o f f i c i a l s at Oregon State University, and by Food Production and Inspection Branch o f f i c i a l s of Agriculture Canada. In sum, 801 samples, representing 32 c u l t i v a r s , o r i g i n a t i n g i n 8 Canadian provinces and 12 American states were c o l l e c t e d and tested i n 1986, 1987, and 1988 (Table VI). Table VI. Origins of potato l e a f r o l l disease samples Province or state Number of samples 1986 1987 1988 Canada Alberta 2 4 B r i t i s h Columbia 20 8 Manitoba 2 New Brunswick 6 63 Nova Scotia 8 Ontario 8 3 Prince Edward Island 8 10 Quebec 9 United States C a l i f o r n i a 9 Colorado 39 6 Idaho 209 96 Maine 26 22 Michigan 7 18 Minnesota 7 Nebraska 20 North Dakota 6 16 Oregon 86 Utah 3 Wisconsin 12 64 Wyoming 4 40 3.2.2 Virus i s o l a t e s and p u r i f i c a t i o n As i n section 2.2.1 3.2.3 Antisera and monoclonal antibodies An antiserum against BWYV-BC was produced by immunizing a young, White New Zealand rabbit with 1 mg of p u r i f i e d v i r u s . The f i r s t i n j e c t i o n , 100 |ig of virus emulsified with an equal volume of Freund's complete adjuvant, was administered intramuscularly i n a hind leg. The second i n j e c t i o n of 200 \iq of virus was given intravenously 10 days l a t e r . The next three i n j e c t i o n s of approximately 300, 200, and 100 (ig were emulsified with Freund's incomplete adjuvant and injected intramuscularly in a hind leg at 2-week i n t e r v a l s . Blood was c o l l e c t e d at 2-week i n t e r v a l s following the l a s t i n j e c t i o n . A booster i n j e c t i o n of 100 (ig of virus was given aft e r the second bleeding. The antiserum t i t e r was determined by agar gel double d i f f u s i o n tests as described by Rowhani and Stace-Smith (1979). The PLRV antiserum used was a g i f t from Dr. R. Stace-Smith (Vancouver Research Station, Agriculture Canada). The immunoglobulins were p u r i f i e d from the PLRV antiserum, adjusted to 1 mg/ml, and stored i n 0.5 ml aliquots at -20 C as 41 d e s c r i b e d i n s e c t i o n 2.2.2. The BWYV antiserum was p u r i f i e d by a f f i n i t y chromatography u s i n g P r o t e i n A-Sepharose CL-4B (Pharmacia F i n e Chemicals AB, Sweden). Immunoglobulins (IgG) were adsorbed t o the column i n 0.1 M sodium phosphate b u f f e r pH 7.0. The column was then washed with 10 v o l o f the same b u f f e r t o remove any unbound m a t e r i a l . The immunoglobulins were e l u t e d with 1.0 M a c e t i c a c i d , immediately n e u t r a l i z e d with 1.0 M T r i s -HC1 pH 9.0, then p r e c i p i t a t e d by adding an equal v o l of s a t u r a t e d ammonium s u l f a t e and i n c u b a t i n g o v e r n i g h t at 4 C. The immunoglobulins were c o l l e c t e d by c e n t r i f u g a t i o n at 12,100 x g f o r 20 min. The p e l l e t was resuspended i n PBS, a d j u s t e d t o approximately 1 mg/ml (A 2 8 0 = 1 . 4) and s t o r e d i n a l i q u o t s of 0.5 ml at -20 C. MAb 371A, s p e c i f i c f o r PLRV, was p r o v i d e d by Dr. R. M a r t i n (Vancouver Research S t a t i o n , A g r i c u l t u r e Canada). BWYV-specific MAb 510H d e s c r i b e d i n s e c t i o n 2.3.2 was s e l e c t e d f o r r o u t i n e d e t e c t i o n of BWYV because of i t s broad s p e c i f i c i t y t o BWYV i s o l a t e s . 3.2.4 ELISA Each sample was t e s t e d s e p a r a t e l y f o r PLRV and BWYV u s i n g v i r u s - s p e c i f i c MAbs, 371A and 510H r e s p e c t i v e l y , u s i n g the i n d i r e c t TAS-ELISA p r o t o c o l d e s c r i b e d i n d e t a i l i n s e c t i o n 2.2.4. Each sample was tested twice. Reactions were considered p o s i t i v e when the A 4 0 5 readings were greater than three times the mean of the readings for f i v e healthy ^controls (Voller et al., 1977) . The s e n s i t i v i t y of the TAS-ELISA was determined by t e s t i n g a t e n f o l d d i l u t i o n series of p u r i f i e d PLRV-BC and BWYV-BC. Each virus was s e r i a l l y d i l u t e d i n viru s - f r e e potato sap d i l u t e d 1:9 i n grinding buffer. Duplicates of each virus d i l u t i o n , from 1 Hg/ml to 1 pg/ml, were tested as above. 3.2.5 Aphid transmission tests Mature wingless GPA were used i n attempts to transmit virus from potato to the test species Physalis pubescens and Capsella bursa-pastoris. The potato leaves were placed on moistened f i l t e r paper i n p e t r i dishes. The aphids were transferred from plant to plant with a camel-hair brush. About 50 nonviruliferous aphids, reared on Chinese cabbage (Brassica pekinensis Rupr.), were placed on the l e a f . A f t e r a 4 8 hr ac q u i s i t i o n feeding, about 20 aphids were transferred to each of the test species. The plants were infested with aphids at the 2-to 4- true leaf stage (Lutman and Tucker, 1987) and kept caged i n small p l a s t i c cylinders covered on top with f i n e wire mesh. After a 48 hr inoculation access feeding, the aphids were k i l l e d by s p r a y i n g the p l a n t s with p i r i m i c a r b at 0.25 g a i / 1 (Chipman I n c . ) . The p l a n t s were moved t o a greenhouse (15-20 G) f o r 6 weeks and then r a t e d f o r development of symptoms. The t e s t p l a n t s were a l s o screened f o r PLRV and BWYV u s i n g TAS-ELISA as d e s c r i b e d above. 3.3 R e s u l t s The t i t e r of the BWYV antiserum was 512 b e f o r e the f i n a l b o o ster, 1024 two weeks a f t e r the booster, and then i t f e l l t o 512 on the next b l e e d i n g . Antiserum from the t h i r d b l e e d i n g was used i n a l l the experiments. The TAS-ELISA c o u l d r e l i a b l y d e t e c t PLRV and BWYV at 1 ng/ml or 100 pg/well ( F i g . 2). The mean A 4 0 5 readings f o r 1 ng/ml of PLRV and BWYV were 0.111 and 0.125 r e s p e c t i v e l y , more than f i f t e e n times the mean readings f o r the v i r u s - f r e e c o n t r o l s . The ELISA r e s u l t s are pres e n t e d as histograms (Sutula et al., 1986) and each histogram r e p r e s e n t s a data s et f o r each l o c a t i o n and year i n which samples were c o l l e c t e d (Figs 3A-8B). An i n t e r v a l of 0.025 OD i s used f o r a l l of the histograms on the x a x i s . 44 s <r Ld O Z < CD Q_ O Ul CD < 3.00-2.50-2.00-1.50-1.00-0.50-0.00 '| 1000.00 i l i l l M i l l I I I r M i l l I—I T I I 100.00 10.00 1.00 0.10 0.01 CONCENTRATION (ng/ml) F i g . 2 TAS-ELISA analysis of p u r i f i e d potato l e a f r o l l v i r u s (PLRV •) and beet western yellows v i r u s (BWYV A ) s e r i a l l y d i l u t e d i n v i r u s - f r e e potato l e a f sap d i l u t e d 1:9 with grinding buffer. Each point i s the mean of two t e s t s . The mean of the ELISA readings (A 4 0 5) of f i v e v i r u s - f r e e control plants was 0.007 f o r the PLRV te s t and 0.008 f o r the BWYV t e s t . 45 6-T 5-0.0 0.5 1.0 1.5 2.0 2.5 3.0 ABSORBANCE (A**) F i g . 3A. Histogram of 1986 F l o r i d a winter t e s t ELISA r e s u l t s (mean of two tests) f o r potato l e a f r o l l v i r u s i n 55 potato l e a f r o l l disease samples. 46 >-o z Ld Z> a LU ' i i i i i i i i i I i i i i i i i i i I i i i i i i i i i I i i i i i i i i i I i i i i i i i i i I i i i i i i i i i I .0 0.5 1.0 1.5 2.0 2.5 3.0 ABSORBANCE (A405) F i g . 3B. Histogram of 1986 F l o r i d a winter t e s t ELISA r e s u l t s (mean of two tests) f o r beet western yellows virus i n 55 potato l e a f r o l l disease samples. 47 >-o a Ld D_ I I I I I I I I | f 1 1 1 II I I I | I I f I I I I I T | I I I I I I I I T | I I T 1 I f f I T p T T T T T T ' f T J T T T T f I I I I 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ABSORBANCE (A**) F i g . 4A. Histogram of 1987 C a l i f o r n i a winter t e s t ELISA r e s u l t s (mean of two tests) f o r potato l e a f r o l l v i r u s i n 257 potato l e a f r o l l disease samples. 48 >-o z LJ Z> o L U en 140-120-100-0 11 11 11 11 1 1 1 11 11 11 11 11 1 1 1 11 11 11 11 11 11 1 1 1 11 11 11 11 11 1 1 1 11 11 11 i 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ABSORBANCE (A4 0 5) F i g . 4B. Histogram of 1987 C a l i f o r n i a winter t e s t ELISA res u l t s (mean of two tests) f o r beet western yellows v i r u s i n 257 potato l e a f r o l l disease samples. 49 6 5-4-Z> 3-O i n o 2 o w V 2-1-0-0.0 T I T I I T T T I | I " 1 I I I I I I | I I I I I I I I I ' | u 0.5 1.0 • " I i I 1.5 2.0 ABSORBANGE ( A ^ ) 1 1 1 1 1 1 1 1 1 1 2.5 3.0 F i g . 5A. Histogram of 1987 F l o r i d a winter t e s t ELISA re s u l t s (mean of two tests) f o r potato l e a f r o l l v i r u s i n 51 potato l e a f r o l l disease samples. 50 >-o o 1_J c_ ABSORBANCE (A**) F i g . 5B. Histogram of 1987 F l o r i d a winter t e s t ELISA re s u l t s (mean of two tests) f o r beet western yellows v i r u s i n 51 potato l e a f r o l l disease samples. 51 20-15->-O 3 10-a Ld 5-0 I D O H ffl '"I 'I' l"l | I l " l I I l " l | I I T I I I I I I | I I I I I I I I I | I 1 1 I I I I I 0.0 0.5 1.0 1.5 2.0 2.5 3.0 ABSORBANCE ( A « ) F i g . 6A. Histogram of 1987 Oregon winter t e s t ELISA re s u l t s (mean of two tests) f o r potato l e a f r o l l v i r u s i n 86 potato l e a f r o l l disease samples. 52 >-o z 1_U ZD a L U (Z LL_ 1.0 1.5 2.0 ABSORBANCE ( A ™ ) F i g . 6B. Histogram of 1987 Oregon winter t e s t ELISA re s u l t s (mean of two tests) f o r beet western yellows virus i n 86 potato l e a f r o l l disease samples. 53 >-o a L U 20-15-10-5-0-oo o 3? o 0.0 T 0.5 1 1 1 1 1 1 H 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 i ? 1 1 1 1 P H 1.0 1.5 2.0 2.5 ABSORBANCE ( A ^ ) i i 11 3.0 3.5 F i g . 7A. Histogram of 1988 C a l i f o r n i a winter t e s t ELISA r e s u l t s (mean of two tests) f o r potato l e a f r o l l virus i n 109 potato l e a f r o l l disease samples. 54 >-o 2_ LJ ZD a Ld G_ 100 80-60-40-• 20-0— 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 • 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ABSORBANCE (A^) F i g . 7B. Histogram of 1988 C a l i f o r n i a winter t e s t ELISA re s u l t s (mean of two tests) f o r beet western yellows virus i n 109 potato l e a f r o l l disease samples. 55 20 15-ABSORBANCE (A40O F i g . 8A. Histogram of 1988 F l o r i d a winter t e s t ELISA re s u l t s (mean of two tests) f o r potato l e a f r o l l v i r u s i n 243 potato l e a f r o l l disease samples. 56 250 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 ABSORBANCE (A**) F i g . 8B. Histogram of 1988 F l o r i d a winter t e s t ELISA r e s u l t s (mean of two tests) f o r beet western yellows virus i n 243 potato l e a f r o l l disease samples. 57 None of the potato samples tested p o s i t i v e for BWYV; i n a l l cases the data were clustered near zero. BWYV-infected P. pubescens, used as a po s i t i v e control, produced a strong signal (A 4 0 5 > 1.0) i n a l l of the BWYV ELISA t e s t s . Most of the samples, 772/801 (96.4%), tested p o s i t i v e for PLRV. Neither PLRV nor BWYV could be recovered, by aphid transfers to indicator hosts, from 28 of the samples that tested negative for both viruses. One sample that was'scored negative for both viruses by TAS-ELISA tested p o s i t i v e i n the aphid transmission test to P. pubescens. The indicator plant tested p o s i t i v e for PLRV by TAS-ELISA. 3.4 Discussion The r e s u l t s indicate that BWYV i s neither a common nor important component of a complex that causes potato l e a f r o l l disease. The small number of samples (28/801 or 3.5%) that were i d e n t i f i e d i n the f i e l d as having l e a f r o l l symptoms, but that tested negative for PLRV and BWYV, were probably affected by a phys i o l o g i c a l l e a f r o l l i n g . Nonvirus l e a f r o l l (LeClerg, 1944) i s a symptom caused by the impairment of carbohydrate translocation from the f o l i a g e . When starch accumulates i n the leaves they become leathery and r o l l upwards and the symptoms are e a s i l y mistaken for potato l e a f r o l l virus disease. PLRV-like symptoms 58 may be caused by Rhizoctonia stem canker and other diseases, mechanical injury to the stems, and s o i l n u t r i t i o n a l conditions such as nitrogen t o x i c i t y or boron deficiency (Hooker, 1981) . The samples tested i n t h i s study represented a wide range of c u l t i v a r s and geographical o r i g i n s . The resu l t s are d i s t i n c t l y d i f f e r e n t from those reported by Sibara and Slack (1985b) and Gallenberg et al. (1987) ; both groups found BWYV to be common i n potato l e a f r o l l disease samples from Canada and the United States. This i s p a r t i c u l a r l y i n t e r e s t i n g because they also c o l l e c t e d many samples from the F l o r i d a winter test plots that I had sampled. The questions a r i s e ; did BWYV, common in many of t h e i r samples c o l l e c t e d i n 1983 to 1986, suddenly disappear i n the years following, or did these investigators detect something other than BWYV with t h e i r BWYV polyclonal antiserum, or did the MAb that I used i n my study f a i l to detect BWYV i s o l a t e s occuring i n the potatoes? It' i s very u n l i k e l y that BWYV was common i n potato up u n t i l 1986 and then suddenly disappeared i n the following years because both PLRV and BWYV are e f f i c i e n t l y vectored by the GPA and BWYV has a much wider host range than PLRV. The BWYV MAb used here detected a l l i s o l a t e s of BWYV against which i t was screened including many strai n s (BMYV, TuYV, and the RPV i s o l a t e of BYDV) from several locations i n six countries. Moreover, tests using BWYV polyclonal antiserum prepared against a B r i t i s h Columbia i s o l a t e of BWYV gave i d e n t i c a l results (section 4.3); no potato l e a f r o l l disease samples tested p o s i t i v e for BWYV. Some of the samples c o l l e c t e d from the F l o r i d a winter test plots i n 1988 were also tested independently by a commercial laboratory (Agdia Inc., Mishawaka, Indiana) for PLRV and BWYV. The resu l t s were the same; viz. no po s i t i v e assays for BWYV (Dr. C. Sutula, personel communication). Reconciliation of the differences between my resu l t s and those of Sibara and Slack (1985b), and of Gallenberg et al. (1987) requires some speculation. Because the actual ELISA readings from the experiments of Sibara and Slack (1985b) were not presented i n t h e i r abstract and the paper has not yet been published, I w i l l comment only on the re s u l t s of Gallenberg et al. (1987). Gallenberg et al. (1987) noted that the BWYV antiserum they used had a higher l e v e l of background interference and an ove r a l l lower l e v e l of reaction with infected samples than the PLRV antisera they used. However, they did not attempt to cross-absorb the globulin to reduce non-specific reactions and possibly increase the signal-to-noise r a t i o . Moreover, they prepared the gamma globulin by ammonium sulfate p r e c i p i t a t i o n only and further p u r i f i c a t i o n by f i l t r a t i o n through DEAE c e l l u l o s e was not attempted. Although they rated many samples as weak pos i t i v e s , they did not apparently confirm t h e i r ELISA re s u l t s with aphid transmission t e s t s . Because P. pubescens i s 60 a host for both PLRV and BWYV, i t could have been used as a transmission host and the plants could have been retested l a t e r s e r o l o g i c a l l y a f t e r BWYV had developed a reasonable t i t e r . Without an independent test, t h e i r weak ELISA re s u l t s are not unequivocal proof that BWYV was present i n the samples they tested. To obtain these ELISA results they had to use high reagents concentrations. The coating antibody and conjugate were used at 4 |lg/ml and sap was d i l u t e d 1:5 with sample buffer. Recent work by Gunn and Pares (1988) demonstrated the presence of a stress-induced antigen which co - p u r i f i e d with PLRV. The antigen also appeared to be produced i n uninfected but ph y s i o l o g i c a l l y stressed potato plants, and reacted with PLRV antisera i n ELISA tests, r e s u l t i n g i n f a l s e - p o s i t i v e r e s u l t s . It i s possible that antibodies to a stress-induced protein may be present i n the BWYV polyclonal antiserum which would be one explanation for the low signal-to-noise r a t i o i n t h e i r ELISA r e s u l t s . This may also explain why some of t h e i r samples tested p o s i t i v e for BWYV. The samples that were tested represented many d i f f e r e n t c u l t i v a r s , and c u l t i v a r s may respond d i f f e r e n t l y to p h y s i o l o g i c a l stress. In Fl o r i d a , where t h e i r samples were collected, many of the plots were on s i t e s with less than i d e a l s o i l s . In fact, i t was not uncommon to f i n d locations with less than one foot of s o i l over the coral parent material. At one s i t e I saw a gravel ridge running through the tes t p l o t s ; potatoes planted here would be growing under conditions of stress. The r e s u l t s presented here indicate that, i n Canada and the United States, potato l e a f r o l l disease i s caused by one virus, PLRV. I could not f i n d any evidence to support the e a r l i e r findings that BWYV i s a common component of potato l e a f r o l l disease. My resu l t s support the work of Tamada et al. (1984); they found no evidence of antigenic v a r i a t i o n among potato i s o l a t e s of PLRV. From a p r a c t i c a l viewpoint i t appears that BWYV does not present a r i s k to potato c u l t i v a t i o n at the present time. 62 Chapter 4 Comparison of ELISA and nucleic a c i d spot hybridization f o r detection and i d e n t i f i c a t i o n of PLRV and BWYV 4.1 Introduction The introduction of ELISA to plant virology by V o l l e r et al. (1976) and Clark and Adams (1977) was a technological breakthrough (Clark et al., 1986). ELISA has become widely accepted and used as a rapid and sensitive method of immunodetection, to study relationships among plant viruses, as a diagnostic t o o l , and as a quantitative assay to measure the concentration of virus i n d i f f e r e n t plant tissues (Eweida, Oxelfelt, and Tomenius, 1988) . The application of hybridoma technology to plant virology has further enhanced the performance of ELISA as a diagnostic t o o l by eliminating or reducing the background problems often associated with polyclonal antiserum. Rapid nucleic acid hybridization techniques using complementary DNA (cDNA) were f i r s t developed for the detection of Epstein-Barr virus i n animal c e l l s (Brandsma and M i l l e r , 1980) and shortly afterward for the detection of potato spindle tuber v i r o i d (Owens and Diener, 1981; Salazar et a l . , 1983). This technique became known as dot-blot or nucleic acid spot 63 hybridization (NASH) and has become a useful a l t e r n a t i v e to ELISA as a highly s p e c i f i c and sensitive method of i d e n t i f y i n g and comparing plant viruses (Maule, Hull, and Danson, 1983; Baulcombe, F l a v e l l and J e l l i s , 1984a,b). NASH i s p a r t i c u l a r l y suited to the study of luteoviruses because of t h e i r low concentrations i n plant tissues and because of the p o s s i b i l i t y of genomic masking (or transcapsidation). When two viruses r e p l i c a t e simultaneously i n the same plant c e l l some of the virus p a r t i c l e s may contain the genome of one virus completely encapsidated by the coat protein of the other v i r u s . This phenomenon i s c a l l e d genomic masking or transcapsidation and has been demonstrated to occur with some luteoviruses (Creamer and Falk, 1990) . It i s obvious that ELISA would only detect the antigenic properties of the coat protein whereas NASH can i d e n t i f y the v i r a l nucleic acid. Genomic masking of heterologous v i r a l RNA by PLRV" protein has never been reported to occur i n nature despite the common occurrence of PLRV i n mixed infec t i o n s with other potato viruses. However, Waterhouse and Murant (1983) have presented experimental evidence that PLRV can substitute for carrot red leaf virus (CRLV) i n packaging carrot mottle virus nucleic acid (CMotV). Although Myzus persicae does not normally transmit CMotV, i t can from plants infected with both PLRV and CMotV. The objectives of t h i s study were to produce cDNA clones of BWYV RNA which can be used i n NASH assays to detect v i r a l 64 nucleic acid; and to compare NASH with ELISA and aphid transmission tests for the detection of PLRV and BWYV i n potato l e a f r o l l disease samples from Canada and the United States. 4.2 Materials and methods 4.2.1 Virus i s o l a t e s , p u r i f i c a t i o n , and RNA extraction A sugar beet i s o l a t e of BWYV (MacCarthy, 1969) was propagated and p u r i f i e d as described i n section 2.2.1. RNA was extracted by vigorously mixing (Maxi Mix II, Thermolyne Corporation, Dubuque, Iowa) p u r i f i e d virus i n alkaline-SDS (0.25 M Tris-HCl, pH 8.9, 1.0 mM EDTA, 2% (w/v) SDS) with an equal vol of phenol:chloroform:isoamyl alcohol (25:24:1). The emulsion was broken by centrifugation at 10, 000 x g for 2 min i n an Eppendorf microcentrifuge (Brinkman Instruments, Ltd., Rexdale Ontario). The aqueous phase was pipetted into a s t e r i l e microcentrifuge tube and set on i c e . The organic phase was re-extracted with an equal vol of s t e r i l e , deionized water and the aqueous phases were combined. An equal vol of phenol:chloroform:isoamyl alcohol (25:24:1) was added to the combined aqueous phase, the phases were mixed b r i e f l y and centrifuged as above. The aqueous phase was pipetted into another s t e r i l e microcentrifuge tube and the RNA was pr e c i p i t a t e d by the addition of 0.1 vol of 2 M sodium acetate, 65 pH 5.8 and 2 vol of absolute ethanol. After mixing well and incubating overnight at -20 C, the RNA was p e l l e t t e d by centrifugation at 10,000 x g for 15 min. The p e l l e t was washed with 70% ethanol and recovered by centrifugation, dried under vacuum, and resuspended i n s t e r i l e , deionized water. The concentration of RNA was estimated 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 2 60 nm. The purit y and i n t e g r i t y of the RNA was determined by electrophoresis i n denaturing agarose gels containing methylmercuric hydroxide (Bailey and Davidson, 1976) . The RNA's were p r e c i p i t a t e d with ethanol, resuspended i n deionized s t e r i l e water, and stored at -70 C u n t i l used. Five BWYV i s o l a t e s : BWYV-BC, BWYV-Sal(9), BWYV RY-l-R, BWYV RY-7, BWYV-NETH; and four PLRV i s o l a t e s : PLRV-BC, PLRV-ST4, PLRV-ORE, and PLRV-ID (Table III) were used to determine the s p e c i f i c i t y of cloned BWYV cDNA. 4.2.2 Synthesis and cloning of cDNA BWYV RNA was polyadenylated in vitro by the method of Sippel (1973). The po l y ( A ) - t a i l e d RNA, heat-denatured at 68 C for 5 min and immediately quenched on ice, served as a template for oligo(dT)-primed, f i r s t - s t r a n d cDNA synthesis using cloned Moloney murine leukemia virus (M-MLV) reverse transcriptase (Bethesda Research Laboratories, Gaithersburg, Maryland). The second strand reaction was e s s e n t i a l l y as described by Gubler and Hoffman (1983), using Escherichia coli DNA polymerase I and RNase H (Bethesda Research Laboratories). Double-stranded cDNA was dC-tailed at the 3' termini using terminal transferase (Bethesda Research Laboratories) and annealed to Pstl-digested pUC9, dG-tailed by the same method. The recombinant DNA was used to transform competent E. coli DH5a c e l l s which were then plated on Luria-Bertani agar containing a m p i c i l l i n and X-gal. A m p i c i l l i n - r e s i s t a n t recombinants were screened by in situ colony hybridization (Gergen, Stern, and Wensink, 1979) using random primed, 3 2 P - l a b e l l e d cDNA synthesized using BWYV RNA as a template (Taylor, Illemensee, and Summers, 1976). F i f t y colonies showing the strongest signals were selected and the plasmids were i s o l a t e d using the alk a l i n e l y s i s method (Maniatis, F r i t s c h , and Sambrook, 1982) . Plasmids were digested with PstI to release the DNA inserts and the mixture was electrophoresed i n agarose gels to determine the size of the in s e r t s . 4.2.3 Northern blots Approximately 100 ng of PLRV and BWYV RNA, and 1 \ig of t o t a l RNA from healthy potato and P. pubescens, prepared by the method of Siegel et al. (1976), were analyzed by electrophoresis 67 i n denaturing methylmercury gels (Bailey and Davidson, 1976) and transferred to charge-modified nylon membranes of Nytran (Schleicher and Schuell Inc., Keene, New Hampshire) as described by V r a t i , Mann, and Reed (1987) . 4.2.4 C o l l e c t i o n of samples With the assistance of seed potato o f f i c i a l s from twenty states or provinces (Table VI), 127 tuber samples were c o l l e c t e d from plants showing symptoms of potato l e a f r o l l disease at the C a l i f o r n i a and F l o r i d a winter test p l o t s . In addition, 38 tuber samples were obtained from the winter test program at Oregon State University. The samples, representing 8 c u l t i v a r s , originated i n four provinces and seven states. The tubers were placed i n l a b e l l e d paper bags, packed i n coolers, and shipped by a i r to Vancouver. The tuber samples were stored for 4 months at 4 C, planted i n 6-inch p l a s t i c pots and the r e s u l t i n g plants were grown i n an aphid-free greenhouse. Leaf samples were c o l l e c t e d a f t e r a l l of the plants were at least 15 cm high. Healthy potato plants were grown from v i r u s - f r e e tubers i n a separate aphid-free greenhouse. Healthy seedlings of ground cherry(Physalis pubescens) and shepherd's purse (Capsella bursa-pastoris) were grown from seed and kept i n the same greenhouse as the v i r u s - f r e e potatoes. 68 4.2.5 Preparation of plant extracts Total nucleic acid extracts from both healthy and v i r u s -infected potato, and BWYV-infected P. pubescens leaves were prepared by a modification of the method of H a b i l i , Mclnnes and Symons (1987). Intact leaf tissue (0.25 g) of each sample was crushed between the r o l l e r s of a sap extractor (Erich Pollahne, F.G.R.) during the dropwise addition of 0.5 ml TS buffer (TS buffer i s 50 mM Tris-HCl, pH 7.4, 2.0% SDS). Each s l u r r y sample was c o l l e c t e d i n a 1.5 ml microfuge tube, mixed with an equal vol of water-saturated phenol:chloroformrisoamyl alcohol (25:24:1, v/v/v), mixed for 15 sec, and centrifuged at 10,000 x g at room temperature for 5 min. The aqueous phase (4 00 |il) was pipetted into a clean, s t e r i l e 1.5 ml microcentrifuge tube and the nucleic acids were pr e c i p i t a t e d by -adding 2.5 vol c h i l l e d absolute ethanol, mixing b r i e f l y , and incubating at -20 C overnight. After centrifugation at 10, 000 x g for 15 min at room temperature, the nucleic acid p e l l e t was washed with cold 70% ethanol, dried under vacuum, resuspended i n 40 (Xl of 0.1 mM EDTA, and stored at -7 0 C. 4.2.6 Preparation of nucleic acid probes Random primed, 3 2 P - l a b e l l e d cDNA probes were synthesized using PLRV and BWYV RNAs as templates by the method described by Taylor et al. (1976). A recombinant DNA clone of PLRV i n the plasmid vector Bluescript (Stratagene, San Diego, California) was obtained from L. Kawchuk and R. Martin, Agriculture Canada, Vancouver. The clone, designated pLP7 9, contained an inse r t of approximately 3.5 kb (Kawchuk et al., 1989). A recombinant DNA clone of BWYV, pBW79 described i n section 4.3, i n the plasmid vector pUC9 contained an insert of approximately 0.7 kb. Recombinant plasmid PLRV and BWYV DNA were prepared according to the modified a l k a l i n e l y s i s protocol (Maniatis et al., 1982) and l a b e l l e d with [a- 3 2P] dATP using the o l i g o - l a b e l l i n g procedure of Feinberg and Vogelstein (1983, 1984). Unincorporated 3 2 P - l a b e l l e d nucleotides were removed by p r e c i p i t a t i n g the probes twice with ammonium acetate and ethanol. The s p e c i f i c a c t i v i t i e s of the probes were greater than 1 x 109 cpm/u.g DNA. 4.2.7 NASH procedures N i t r o c e l l u l o s e f i l t e r s (Schleicher and Schuell, Inc.) were cut to 12 x 15 cm, marked l i g h t l y at 96 equally spaced points with a blunt lead p e n c i l (HB), soaked i n glass d i s t i l l e d water for 5 min and then i n 20x SSC (SSC i s 0.15 M NaCl, 0.015 M sodium c i t r a t e ) for 15 min and a i r dried. Charge-modified nylon f i l t e r s of Nytran (Schleicher and Schuell), were cut and marked with p e n c i l as above and soaked i n g l a s s - d i s t i l l e d water before a i r drying. 70 Each sample of t o t a l nucleic acid was thawed, mixed b r i e f l y , and 2 | i l was spotted on the premarked f i l t e r s . Each sample was spotted twice on both nit r o c e l l u o s e and Nytran f i l t e r s . A f t e r spotting, the membranes were dried under a lamp for 5 min and then baked under vacuum at 80 C for 2 hr. The f i l t e r s were then transferred to p l a s t i c , snap-seal containers and prehybridized at 50 C for 4 hr i n prehybridization buffer consisting of 50% (v/v) deionized formamide, 1.5x SSPE (SSPE i s 0.18 M NaCl, 0.01 M sodium phosphate, pH 7.0, 0.01 M EDTA), 0.5% BLOTTO, 1% (w/v) SDS, and 0.5 mg/ml sheared, denatured salmon sperm DNA (Reed, 1986). The hybridizations themselves were ca r r i e d out at 50 C [Tm = -15] (Martin and D'Arcy, 1990) for 16 hr i n fresh buffer containing 50% deionized formamide, 10% dextran sulfate, 1.5x SSPE, 0.5% BLOTTO, and 0.5 mg/ml sheared, denatured salmon sperm DNA (Reed, 1986) . At the completion of hybridization, the membranes were removed from the hybridization solution and washed b r i e f l y i n 2x SSC, then successively by vigorous agitation for 15 min at room temperature i n the following solutions (using approximately 50 ml/membrane) : (1) 2.Ox SSC/0.1% SDS, (2) 0.5x SSC/0.1% SDS, and (3) O.lx SSC/0.1% SDS. The membranes were given a f i n a l wash at 50 C for 20 min. i n prewarmed O.lx SSC/1% SDS, rinsed i n O.lx SSC, b l o t t e d l i g h t l y to remove excess moisture, and immediately wrapped i n p l a s t i c f i l m . The membranes were exposed to X-OMAT f i l m (Kodak) for 24 to 72 hr at -70 C i n an X-ray cassette containing Lightning Plus (Dupont) i n t e n s i f y i n g screens. 4.2.8 Antisera and monoclonal antibodies As i n section 3.2.2. 4.2.9 Double antibody sandwich ELISA A modification of the double antibody sandwich ELISA (DAS-ELISA) of Clark and Adams (1977) was used. A l l reagents were used at 100 |ll/well except for blocking steps which were 300 (i l / w e l l . Linbro flat-bottomed m i c r o t i t e r plates (Flow Laboratories Inc.) were coated with p u r i f i e d immunoglobulin d i l u t e d to 1 (lg/ml i n PBS overnight at 4 C. The plates were then blocked with 2% BLOTTO in PBS for 30 min at room temperature. The blocking agent was shaken out and plant extract added and incubated overnight at 4 C. Each plant sample (0.1 g) was extracted with a sap press (Erich Pollahne, F.G.R.) while adding 900 | l l grinding buffer (PBS: 0.5% Tween 20: 0.2% BLOTTO: 2% PVP, v:v:v:w) dropwise on the r o l l e r s . Following the incubation step, the plant extract was washed out of the wells with PBS-T, three times for 1 min each. The plates were blocked again, as before. Blocking agent was shaken out of the wells and a l k a l i n e phosphatase conjugates (cross-absorbed for 1 hr at room temperature with sap from 1 g healthy potato leaf tissue i n 10 ml grinding buffer) d i l u t e d to 1 \ig/ml i n PBS-T were added and incubated for 3 hr at room temperature. The wells were washed again as above except that the f i n a l wash was with tap water to remove any remaining phosphate. Substrate (p-nitrophenyl phosphate, Sigma 104-105, 0.5 mg/ml in 10% diethanolamine, pH 9.8) was added and incubated for 1 hr at room temperature. The absorbance at 405 nm (A405) of each well was determined i n a Titertek Multiscan MCC plate reader (Flow Laboratories Inc.). Reactions were considered to be p o s i t i v e when the A 4 0 5 readings were greater than three times the mean of the readings for f i v e healthy control samples (Voller et al., 1977) . 4.2.10 T r i p l e antibody sandwich ELISA Each sample was tested twice for PLRV and BWYV using v i r u s -s p e c i f i c MAbs and TAS-ELISA, exactly as described i n section 2.2.4/3.2.4. 73 4.2.11 Aphid transmission tests As i n section 3.2.5 4.3 Results Approximately 200 transformants, containing cDNA sequences homologous to BWYV genomic RNA, were selected by colony hybridization. The majority of the inserts were less than 1000 bp as determined by agarose gel electrophoresis. The o r i g i n of one in s e r t of approximately 0.7 kb i n plasmid pBW7 9 was confirmed by northern blot analysis. A 3 2 P - l a b e l l e d probe synthesized from pBW7 9 was hybridized s p e c i f i c a l l y to BWYV RNA but did not hybridize with PLRV RNA or with t o t a l RNA p u r i f i e d from healthy potato or ground cherry. The s p e c i f i c i t y of pBW7 9 was demonstrated by i t s hybridization to RNA of f i v e BWYV is o l a t e s from Canada, USA, Germany, and the Netherlands; and by i t s f a i l u r e to hybridize to RNA of four PLRV i s o l a t e s and RNA from v i r u s - f r e e potato plants (Fig. 9A). The l i m i t of detection of BWYV RNA by a 3 2 P - o l i g o l a b e l l e d probe from pBW7 9 and random primed cDNA prepared from BWYV RNA was 100 pg i n a tenfold d i l u t i o n series (Fig. 9B and 9C). 74 Fig. 9. (A) Detection of beet western yellows virus (BWYV) isolates by nucleic acid spot hybridization using 3 2P-oligolabelled pBW79. Each spot represents nucleic acid extracted from 10 mg of leaf tissue. The samples are: la,b BWYV-BC; 2a,b PLRV-BC; 3a,b PLRV-ORE; 4a,b BWYV Sal(9); 5a,b BWYV RY-7; 6a,b PLRV ST4; 7a,b PLRV-ID; 8a,b BWYV RY-l-R; 9a,b BWYV—NETH; virus-free potato. The autoradiograph was exposed for 72 hr. (B) Detection of BWYV-BC RNA in a tenfold dilution series from 100 ng (lc) to 1 pg (6c) using 32P-labelled, random primed cDNA prepared from BWYV RNA. Equivalent amounts of RNA from virus-free potato and potato l e a f r o l l virus (PLRV) were spotted at l a to 6a and lb to 6b, respectively. Samples of v i r a l RNA were mixed with RNA from 10 mg of virus-free potato leaves before spotting. The autoradiograph was exposed for 24 hr. (C) The same as (B) except the probe was 32P-oligolabelled pBW79. 75 76 immunosorbent assay (DAS-ELISA) for beet western yellows virus (BWYV) in potato l e a f r o l l disease samples. Virus-free control samples were tested in wells 12a-e, BWYV-infected Physalis pubescens, the positive control, was in well 12f. No BWYV was detected and there was no cross-reaction with the potato l e a f r o l l virus positive control in well 12g. The rows are identified as a-h from top to bottom and the columns are numbered 1-12 from l e f t to right. 77 Fig. 11. Triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) for beet western yellows virus (BWYV) in potato l e a f r o l l disease samples. Virus-free control samples were tested in wells 12a-e. The positive control, BWYV-infected Physalis pubescens, was in well 12f. No BWYV was detected and there was no cross-reaction with the potato l e a f r o l l virus positive control in well 12g. The rows are identified as a-h from top to bottom and the columns are numbered from 1-12 from l e f t to right. 78 The probes did not cross-hybridize with PLRV RNA or RNA p u r i f i e d from v i r u s - f r e e potato leaves. None of the potato l e a f r o l l disease samples tested p o s i t i v e for BWYV by either DAS-or TAS-ELISA (Figs. 10 and 11) , or by NASH using either cloned (Fig. 12) or random primed cDNA probes (Fig. 13). BWYV was not transmitted, using aphids, from any of the potato l e a f r o l l samples to ground cherry or shepherd's purse indicator plants as determined by TAS-ELISA. In contrast, a l l of the potato l e a f r o l l disease samples tested p o s i t i v e for PLRV by TAS-ELISA and NASH using a cloned cDNA probe from pLP79. One f a l s e -negative r e s u l t was obtained by both DAS-ELISA and NASH using random primed cDNA prepared from PLRV RNA. PLRV was recovered from the potato l e a f r o l l disease samples that tested negative, using aphid transmission to ground cherry. A l l of the PLRV is o l a t e s infected ground cherry and most (119/165) infected shepherd's purse (Fig. 14). i Equivalent results (Fig. 15) were obtained by NASH using either Nytran or n i t r o c e l l u l o s e membranes except that the signals from Nytran were more di f f u s e than those from n i t r o c e l l u l o s e . 79 F i g . 12. Nucleic a c i d spot hybridization (NASH) of 20 potato l e a f r o l l disease samples. Each spot i s duplicated on both membranes and represents nucl e i c a c i d extracts from 10 mg of l e a f t i s s u e . V i r u s - f r e e control samples are located at positions 6- and 12a-f, and symptomless-potato control samples c o l l e c t e d at the winter t e s t plots are spotted at positions 3- and 9e-h. Potato l e a f r o l l v i r u s (PLRV) p o s i t i v e controls are at positions 6- and 12g, and beet western yellows v i r u s (BWYV) p o s i t i v e controls are at positions 6 and 12f. Positions 4-, 5-, 10- and 11a-h are blank. (A) Detection of PLRV with 3 2 P - o l i g o l a b e l l e d pLP79. (B) Detection of BWYV with 3 2 P - o l i g o l a b e l l e d pBW79. 80 81 F i g . 13. Nucleic a c i d spot hybridization (NASH) of potato l e a f r o l l disease samples. Each sample i s spotted on both membranes and represents nucleic a c i d extracts from 10 mg of l e a f t i s s u e . V i r u s - f r e e control samples are located at positions 12a-f, and symptomless-potato control samples c o l l e c t e d at the winter t e s t plots are spotted at the following p o s i t i o n s : l a ; 2f,g; 3c,f,g; 5g; 7g; 8d; and lOd. The potato l e a f r o l l v i r u s (PLRV) p o s i t i v e control i s at po s i t i o n 12g, and the beet western yellows v i r u s (BWYV) control i s at po s i t i o n 12f. Positions 10g,h; and l l a - h are blank. (A) Detection of PLRV with 3 2 P - l a b e l l e d random primed cDNA prepared from PLRV RNA. (B) Detection of BWYV with 3 2 P - l a b e l l e d random primed cDNA prepared from BWYV RNA. 82 1 2 3 4 5 6 7 8 9 10 11 12 C d e f g h 0 1 2 3 4 5 6 7 8 9 10 11 12 f g h 83 84 F i g . 15. Nucleic a c i d spot hybridization (NASH) of potato l e a f r o l l disease samples with 3 2 P - o l i g o l a b e l l e d pLP79. Each sample represents 10 mg of l e a f t i s s u e and i s duplicated on both the (A) n i t r o c e l l u l o s e membrane and (B) Nytran membrane. Vir u s - f r e e control samples are located at positions 6- and 12a-f, and symptomless-potato control samples c o l l e c t e d at the winter t e s t p l o t s are located at the following p o s i t i o n s : 2- and 8a; 2- and 8f; 4- and 10b; and 5-and l l f . Potato l e a f r o l l v i r u s p o s i t i v e controls are at positions 6- and 12g, and beet western yellows v i r u s p o s i t i v e controls are at positions 6- and 12f. 85 Q 1 2 3 4 5 6 7 8 9 10 11 12 a • ••• # • • • b • • • • • m 0 c ^ 0 ^ A 0 ^ 41 A A d • • • t) • e 0 ^ A A ^ A A A A Q J 1 2 3 4 5 6 7 8 9 10 11 12 b o* • # # o • • • c • • • • • • • • • • « O • • • • • • * ••••• €•#•• 86 4.4 Discussion The r e s u l t s show that BWYV was not detected i n potato l e a f r o l l disease samples from Canada and the United States by ELISA or NASH, whereas a l l of the samples tested p o s i t i v e for PLRV. Most of the PLRV i s o l a t e s infected shepherd's purse and i t i s now clear that t h i s plant i s not a r e l i a b l e diagnostic host for BWYV as was previously reported (Duffus, 1981a,b). Several research reports confirm that shepherd's purse i s a host for some stra i n s of PLRV (Fox et al., 1990; Sy l l e r , 1985; Thomas, 1984) and i t i s being investigated as a potent i a l overwintering host for PLRV i n some potato-producing areas i n Canada and the United States (Fox et al., 1990). In t h i s study, equivalent results were obtained by ELISA and NASH, although based on the detection of di f f e r e n t macromolecules. The results presented here show that both ELISA and NASH using either cloned or random primed cDNA, are useful and sen s i t i v e methods for screening plants for the presence of PLRV and BWYV. Under optimal conditions, approximately 1 ng/ml (100 pg/well) of antigen can be detected by ELISA (van Regenmortel, 1982) . NASH i s at least as se n s i t i v e . With random primed cDNA, 100 pg of p u r i f i e d BWYV RNA/spot was e a s i l y detected and 10 pg/spot was barely detectible a f t e r a 48 hr exposure. Cloned cDNA from pBW79 (approximately 0.7 kb) was less s e n s i t i v e but 100 pg of BWYV RNA could be e a s i l y detected. 87 The s e n s i t i v i t y of NASH could be increased by using a mixture of cloned probes, complementary to the entire RNA genome, or by using RNA probes. Salazar, Balbo, and Owens (1988) have demonstrated that as l i t t l e as 0.33 pg of PSTV RNA could be detected using RNA probes prepared by t r a n s c r i p t i o n of plasmid DNA templates containing a promotor for bacteriophage SP6 polymerase. When extreme s e n s i t i v i t y i s required, the polymerase chain reaction (PCR) provides an unusually simple method for making v i r t u a l l y an unlimited number of copies of cDNA from v i r a l RNA, before detection with nucleic acid probes (Kawasaki, 1990) . Byrne et al., (1988) used t h i s technique for the detection of r e t r o v i r a l RNA af t e r f i r s t producing cDNA, using M-MLV reverse transcriptase, and then allowing the standard Taqr-polymerase^ based system to amplify the cDNA. Although both ELISA and NASH allow sensitive and s p e c i f i c detection of PLRV and BWYV, at the present time ELISA i s the method of choice for routine t e s t i n g of plant samples, e s p e c i a l l y i f the laboratory i s not set up for handling radioactive isotopes. Storing and handling the reagents for ELISA are not te c h n i c a l l y demanding; MAbs can be stored as ascites f l u i d d i l u t e d 1:1 with saturated ammonium sulf a t e at 4 C for several years without loss of t i t e r . NASH requires considerable handling; using a sap press for t o t a l nucleic acid 88 preparation, several hundred samples can be processed i n a day compared to about one thousand samples for ELISA. Furthermore, autoradiography requires a dark room and i s more time-consuming than reading ELISA plates. The s p e c i f i c i t y and s e n s i t i v i t y of molecular hybridization makes the method a valuable adjunct to ELISA. I f sens i t i v e discrimination between v i r a l s trains i s required, the f l e x i b i l i t y of the hybridization step i n NASH allows control of the stringency of hybridization. Another major advantage of NASH i s that the method i s not r e s t r i c t e d to one region of the genome; for plant-pathogenic viruses, only about 2-5% of the nucleic acid of the v i r a l genome i s represented i n the antigenic determinants of the coat protein (Miller and Martin, 1988) . A major advantage of cloned probes i s t h e i r ease of preparation i n contrast to p u r i f y i n g virus and extracting RNA to make random primed cDNA probes. Furthermore, v i r a l RNA may not be 100% pure. Jayasena, Randies, and Barnett (1984) and Smith et al., (1988) have shown that host DNA may co-purify with luteovirus RNA either as a res u l t of attachment to, or encapsidation i n , luteovirus p a r t i c l e s . Contaminating DNA may also become l a b e l l e d during the preparation of random primed cDNA (Taylor et al., 1976) and may lead to f a l s e - p o s i t i v e r e s u l t s . 89 Nonradioactive probes are comparable to 3 2 P - l a b e l l e d probes i n s e n s i t i v i t y and have a d i s t i n c t advantage i n terms of safety, cost, and s t a b i l i t y . H a b i l i et al. (1987) have shown that photobiotin-labelled probes are stable for at least 8 months when stored at -20 C This technique shows considerable promise for the detection and diagnosis of the luteoviruses. Recent developments i n nonradioactive nucleic acid detection methods, r e s u l t i n g i n s e n s i t i v i t y i n the subpicogram range (Carlson et al., 1990), may lead the way for nucleic acid hybidization techniques to replace ELISA as the standard method for routine virus detection and diagnosis. The re s u l t s of ELISA, NASH, and aphid transmission tests reported here refute e a r l i e r reports that BWYV i s a common and important component of a complex causing potato l e a f r o l l disease. To the contrary, the results support the hypothesis that potato l e a f r o l l disease i s caused by one virus or a group of c l o s e l y related viruses properly c a l l e d PLRV. Keese et al. (1990) have recently reported that there i s l i t t l e genetic d i v e r s i t y among the viruses named PLRV. 90 Chapter 5 S u s c e p t i b i l i t y of potato to BWYV i s o l a t e s from Canada and the United States 5.1 Introduction There i s an obvious discrepancy between research that suggests that BWYV occurs widely i n North America and i s an important component of a potato l e a f r o l l disease complex (Duffus, 1981a,b; Sibara and Slack, 1985a,b; and Gallenberg et al., 1987); and experimental results that f i n d BWYV to be absent or undetectable i n potato l e a f r o l l - a f f e c t e d plants (Clarke, Powelson, and Beraha, 1983; E l l i s and Wieczorek, 1988; E l l i s , 1989) . Tamada et al. (1984) f a i l e d to f i n d virus i s o l a t e s resembling BWYV i n potato i n B r i t a i n ; Barker (198 6) f a i l e d to i n f e c t potato with B r i t i s h i s o l a t e s of BWYV; Marco (1984) could not in f e c t potato with BWYV is o l a t e s from I s r a e l , and Casper (1983) f a i l e d to transmit virus i s o l a t e s from l e a f r o l l infected potatoes to a host range t y p i c a l of BWYV. Despite these re s u l t s , the p o s s i b i l i t y exists that some is o l a t e s of BWYV i n North America d i f f e r enough from the European i s o l a t e s that they may infe c t potato. The objective of t h i s experiment was to determine the s u s c e p t i b i l i t y of potato to seven i s o l a t e s of BWYV from Canada and the United States. 91 5.2 Materials and methods 5.2.1 Virus i s o l a t e s and transmission tests Three i s o l a t e s of BWYV from Canada and four from the United States were used i n t h i s study. Two of these i s o l a t e s , IPTT752 and BW/PL-5.2, were i n plants with mixed infec t i o n s of BWYV and PLRV. Two i s o l a t e s of PLRV, one from B r i t i s h Columbia and one from Washington State were included as controls. The is o l a t e s of BWYV and PLRV used, the o r i g i n a l host from which they were isol a t e d , when known, and the donor of the i s o l a t e are l i s t e d i n Table VII. A l l of the is o l a t e s were maintained i n ground cherry (Physalis pubescens) . Table VII. Virus i s o l a t e s used in aphid transmission tests Virus i s o l a t e Origin Host Donor* BWYV-BC B.C., Canada sugar beet 1 BWYV-12H B.C., Canada common groundsel 2 BWYV-21OE B.C., Canada common groundsel 2 BWYV-CA Ca l i f o r n i a , USA common mallow 2 BWYV-D3 Washington, USA ground cherry 3 IPTT752 Washington, USA turnip 3 BW/PL-5.2 Washington, USA ground cherry 3 PLRV-LR7 Washington, USA potato 3 PLRV-BC B.C. Canada potato 4 1. H.R. MacCarthy, Agriculture Canada, Vancouver Research Station, Vancouver, B.C. 2. P.J. E l l i s , Agriculture Canada, as above. 3. P.E. Thomas, USDA, Prosser, Washington. 4. N.S. Wright, Agriculture Canada, Vancouver Research Station, Vancouver, B.C. 92 Virus-free GPA were reared on Chinese cabbage. Apterous aphids were allowed an ac q u i s i t i o n access feed of 72 hr or longer on ground cherry infected with each virus i s o l a t e . About 20 of these now v i r u l i f e r o u s aphids were transferred with camel hair brushes to each test plant and allowed an inoculation access feed of 72 hr, afte r which they were k i l l e d by spraying the plants with pirimicarb at 0.25 g ai/1. Two test plants each of shepherd's purse and ground cherry at the four true-leaf stage, and 10 Russet Burbank potato plants about 4-5 cm high were inoculated with each virus i s o l a t e . About six weeks afte r inoculation, v i r u s - f r e e GPA were allowed a 72 hr access feed on leaves of the potato test plants and then they were transferred to shepherd's purse and ground cherry indicator plants for inoculation as above. 5.2.2 ELISA Potato and the other indicator plants were assayed for PLRV and BWYV using TAS-ELISA as described i n section 3.2.4. Each plant was tested at least twice at six weeks following inoculation. 5.3 Results The re s u l t s of the transmission tests are presented i n Table VIII. Table VIII. Mean v i r u s - s p e c i f i c TAS-ELISA (A 4 0 5) re s u l t s of attempts to transmit beet western yellows virus (BWYV) and potato l e a f r o l l virus (PLRV) is o l a t e s to potato. Virus Shepherd's purse Ground cherry Potato i s o l a t e BWYV1 PLRV2 BWYV PLRV BWYV PLRV BWYV- 0 .375 0.027 1.203 0.017 0.005 0.008 BC (2/2) 3 (0/2) (2/2) (0/2) (0/10) (0/10) BWYV- 1.978 0.015 0.751 0.014 0.000 0.002 12H (0/2) (0/2) (2/2) (0/2) (0/10) (0/10) BWYV- 1.303 0.008 1.325 0.013 0.000 0.000 210E (2/2) (0/2) (2/2) (0/2) (0/10) (0/10) BWYV- 0.187 0.016 0.239 0.017 0.004 0.006 CA (2/2) (0/2) (2/2) (0/2) (0/10) (0/10) BWYV- 0.782 0.016 1.120 0.007 0.007 0.006 D3 (2/2) (0/2) (2/2) (0/2) (0/10) (0/10) IPTT- 1.250 0.141 0.273 2.640 0.000 0.4314 752 (2/2) . (2/2) (2/2) (2/2) (0/10) (6/10) BW/PL- 0.702 0.026 0.949 2.870 0.000 0.3645 5.2 (2/2) (0/2) (2/2) (2/2) (0/10) (5/10) PLRV- 0.000 2.887 0.000 3.047 0.001 1.934 BC (0/2) (2/2) (0/2) (2/2) (0/10) (10/10) PLRV- 0.010 1.212 0.009 3.084 0.005 2.473 LR7 (0/2) (2/2) (0/2) (10/10) (0/10) (10/10) Cont. 6 0 .000 0.022 0.000 0.031 0 .000 0.007 VF-GPA (0/2) (0/2) (0/2) (0/2) (0/10) (0/10) Cont. 7 0.007 0.021 0.001 0.007 0 .008 0.008 No GPA (0/4) (0/4) (0/4) (0/4) (0/10) (0/10) xMean BWYV TAS-ELISA (A405) reading 2Mean PLRV TAS-ELISA (A405) reading 3Numbers i n brackets indicate # po s i t i v e by TAS-ELISA/# tested "Mean PLRV TAS-ELISA (A405) reading for 6 of 10 potato plants rated p o s i t i v e by TAS-ELISA 5Mean PLRV TAS-ELISA (A405) reading for 5 of 10 potato plants rated p o s i t i v e by TAS-ELISA 6Virus-free control plants i n which 20 nonviruliferous GPA (VF-GPA) were allowed to feed for 72 hr 7Virus-free control plants maintained free of aphids 94 5.4 Discussion None of the potato plants tested p o s i t i v e by ELISA for BWYV, either when inoculated with a single BWYV i s o l a t e or when inoculated with a mixed i n f e c t i o n of PLRV and BWYV from ground cherry. The presence of virus i n the inoculating aphids was confirmed by inoculation and i n f e c t i o n of the susceptible ind i c a t o r plants, shepherd's purse and ground cherry. The TAS-ELISA res u l t s were confirmed by the back t e s t i n g of inoculated potatoes with aphid transmission to the indicator plants. BWYV could not be recovered by aphids from any (0/90) of the inoculated potatoes. In contrast, PLRV was re a d i l y recovered from many (31/40) of the plants inoculated with t h i s v i r u s . The aphid transmission results support the resu l t s of ser o l o g i c a l and nucleic acid hybridization tests that could not confirm the presence of BWYV i n l e a f r o l l - a f f e c t e d potatoes i n Canada and the United States ( E l l i s , 1989; E l l i s , 1990; E l l i s and Wieczorek, 1988). Therefore, l i k e Casper (1983), Marco (1985), and Barker (1986) I have not found any evidence that potato i s a host for BWYV. Although there i s a p o s s i b i l i t y that some BWYV is o l a t e s can i n f e c t potato, there may be an alternative explanation for the e a r l i e r reports that BWYV i s a widespread and important component of the potato l e a f r o l l disease i n North America. The evidence i s now overwhelming that PLRV does indeed i n f e c t shepherd's purse (Fox et al., 1990; Sy l l e r , 1985; and Thomas, 1984), and the use of t h i s plant as a d i f f e r e n t i a l host for BWYV 95 and PLRV i s an error. Because BWYV and PLRV are s e r o l o g i c a l l y r e l a t e d luteoviruses and because they have at least one epitope i n common (section 2.3.2), polyclonal antiserum may have led to questionable r e s u l t s and conclusions. The application of sens i t i v e and s p e c i f i c s e r o l o g i c a l tests (section 3.3) and nucleic acid hybridization tests (section 4.3) combined with the b i o l o g i c a l data presented here strongly suggest that BWYV i s not a threat to table stock or seed-potato production at the present time. 96 Chapter 6 A survey of weeds as reservoirs f o r BWYV and PLRV 6.1 Introduction BWYV occurs i n a wide range of crop and weed hosts i n the United States (Duffus, 1977; Timmerman, D'Arcy, and Spl i t t s t o e s s e r , 1985) and has recently been implicated as an important component of potato l e a f r o l l disease (Duffus, 1981a,b). In Canada, the importance of weeds as reservoirs of potato viruses has not been clo s e l y studied (Singh, 1987). Because of the known wide host range of BWYV and i t s p o t e n t i a l threat to both seed and table potato crops (Duffus, 1981a,b), a survey was conducted to evaluate common weeds, wild species and some volunteer crop plants as reservoirs of viruses inducing potato l e a f r o l l symptoms. An additional objective was to determine the occurrence of BWYV and PLRV i n weeds i n the potato-producing areas of B r i t i s h Columbia. In t h i s paper, native and naturalized f l o r a , and volunteer or abandoned c u l t i v a t e d crop plants are considered to be weeds. A preliminary report has been published ( E l l i s , 1988). 97 6.2 Materials and methods 6.2.1 C o l l e c t i o n of samples Foliage samples of common weeds found i n B r i t i s h Columbia, and some volunteer crop plants, were c o l l e c t e d i n the main potato-producing areas of the province. The samples were c o l l e c t e d from fence rows, i r r i g a t i o n ditches, and within both seed and table potato f i e l d s . The plants were c o l l e c t e d at many di f f e r e n t growth stages - from four expanded true leaves to the senescent growth stage (Lutman and Tucker, 1987) . Symptoms were not used as a c r i t e r i o n for selection of samples because luteovirus symptoms may be e a s i l y confused with those of drought, senescence, waterlogging, n u t r i t i o n a l imbalance, or herbicide injury (Duffus, 1977). Each sample consisted of a stem and leaves, i n good condition, from the middle of the plant canopy. Samples were kept i n p l a s t i c bags at 4 C u n t i l used. C o l l e c t i o n s i t e s were selected at random from a l i s t of potato producers provided by s t a f f of Agriculture Canada and the B r i t i s h Columbia Ministry of Agriculture and F i s h e r i e s . 6.2.2 Antisera and monoclonal antibodies As i n section 3.2.3. 98 6.2.3 ELISA Each sample was tested twice using the TAS-ELISA procedure described i n section 3.2.4. Positive and negative controls consisted of BWYV-infected ground cherry (Physalis pubescens), PLRV infected potato, and healthy greenhouse plants of the species being tested grown from seed. Infection with either BWYV or PLRV was indicated when absorbance readings (A405) of sample wells were greater than twice the mean absorbance reading of f i v e healthy controls (Timmerman et al., 1985) or 0.05, whichever was the greater. Plant samples rated p o s i t i v e by TAS-ELISA were further tested, by aphid transmission to indicator plants, to check the v a l i d i t y of ELISA r e s u l t s . 6.2.4 Aphid transmissions For aphid transmission tests, about 20 vi r u s - f r e e GPA were allowed a 48 hr access to leaf samples i n sealed p e t r i dishes. After a c q u i s i t i o n , the aphids were placed on healthy indicator plants (P. pubescens) for an inoculation access period of 72 hr. The aphids were then k i l l e d with pirimicarb at 0.25 g ai/1. The plants were kept i n an aphid-free greenhouse for six weeks and checked for symptoms. Leaf samples were taken from the indic a t o r plants at that time and assayed for BWYV and PLRV by TAS-ELISA. 99 6.3 Results A t o t a l of 10,182 weed samples, representing 98 species i n 22 families, were tested by ELISA for virus i n f e c t i o n (Table IX). BWYV was found i n 101 (1.0%) of the samples and PLRV i n six samples (0.06%). The species t e s t i n g p o s i t i v e are l i s t e d i n Table X. Aphid transmission tests were attempted on a l l of the samples that tested p o s i t i v e by ELISA except for a few samples that had become desiccated. Aphid transmission experiments confirmed the TAS-ELISA results i n a l l cases, except for the transmission of BWYV from 2 of 3 samples of p r i c k l y lettuce and 5 of 7 samples of scentless chamomile. 100 Table IX. Plants surveyed for beet western yellows virus and potato l e a f r o l l v i r u s i n the potato-producing areas of B r i t i s h Columbia Family Botanical name* Common name Amaranthaceae Amaranthus retroflexus L. redroot pigweed 2 9 0 Boraginaceae Lappula echinata G i l i b . bluebur Caryophyllaceae Spergula arvensis L. Stellaria media (L.) V i l l . corn spurry chickweed 3 6 4 6 2 6 Chenopodiaceae Seta vulgaris L. Chenopodium album L. Spinacia oleracea L. table beet lamb's-quarters garden spinach 7 2 6 3 Compositae Achillea millefolium L. Arctium minus (Hill) Bernh. Aster sp. Bellis perennis L. Chrysanthemum leucanthemum L, Cirsium arvense (L.) Scop. C. vulgare (Savi) Tenore Erigeron canadensis L. yarrow common burdock wild aster English daisy ox-eye daisy Canada t h i s t l e b u l l t h i s t l e Canada fleabane 1 2 6 5 6 9 2 6 0 3 5 Table IX. Continued Compositae Gnaphalium uliginosum L. low cudweed 1 Hypochoeris radicata L. spotted cat's-ear 230 Lactuca muralis (L.) Gaertn. wall lettuce 6 L. sativa L. garden lettuce 3 L. scariola L. p r i c k l y lettuce 297 Matricaria maritima L. scentless chamomile 209 M. matricarioides (Less.) Porter pineappleweed 65 Senecio jacobaea L. tansy ragwort 9 5. vulgaris L. common groundsel 459 Solidago canadensis L. Canada goldenrod 8 Sonchus arvensis L. smooth perennial sow-t h i s t l e 3 5. asper (L.) H i l l spiny annual sow-thistle 13 S. oleraceus L. annual sow-thistle 22 Tanacetum vulgare L. tansy 7 Taraxacum officinale Weber dandelion 456 Convolvulaceae Convolvulus arvensis L. f i e l d bindweed 10 Cruciferae Alyssum alyssoides L. small alyssum 1 Armoracia rusticana P. Goertn., B. Mey & Scherb. horseradish 1 Table IX. Continued Cruciferae B. napus var. napobrassica L. B. oleracea var. botrytis L. B. oleracea var. capitata L. B. pekinesis Rupr. Camelina microcarpa Andrz. Capsella bursa-pastoris (L.) Medic. Cardamine oligosperma Nutt. Cardaria draba (L.) Desv. Descurainia pinnata (Walt.) B r i t t . Lipidium perfoliatum L. Nasturtium officinale R. Br. Nestlia paniculata (L.) Desv. Raphanus raphanistrum L. Rorippa sylvestris (L.) Bess. Sinapis arvensis L. Sisymbrium altissimum L. S. officinale (L.) Scop. Thlaspi arvense L. rutabaga 49 b a l l cabbage 1 cauliflower 8 Chinese cabbage 1 small-seeded f a l s e f l a x 64 shepherd's purse 1309 l i t t l e western 100 b i t t e r c r e s s heart-podded hoary cress 31 green tansy mustard 125 clasping-leaved pepper- 1 grass water cress 177 b a l l mustard 2 wild radish 35 creeping yellow cress 190 wild mustard 255 tumble mustard 9 hedge mustard 233 stinkweed 653 Table IX. Continued Geraniaceae Erodium cicutarium (L.) L'Her. s t o r k ' s - b i l l 174 Geranium molle L. dovesfoot geranium 2 Labiatae Galeopsis tetrahit L. hemp-nettle 194 Lamium amplexicaule L. henbit 7 Melissa officinalis L. lemon-balm 6 Mentha arvensis L. f i e l d mint 9 Prunella vulgaris L. h e a l - a l l 3 Leguminosae Medicago lupulina L. black medic 10 M. sativa L. a l f a l f a 23 2 Pisum sativum L. garden pea 5 Trifolium pratense L. red clover 213 T. repens L. white clover 125 Vicia sp. vetch 2 Malvaceae Malva neglecta Wallr. common mallow 102 M. parviflora L. small-flowered mallow 3 Table IX. Continued Onagraceae Papaveraceae Plantaginaceae Polygonaceae Epilobium angustifolium L. E. minutum L i n d l . Oenothera erythrosepala Borb. Papaver nudicaule L. P. somniferum L. Plantago lanceolata L. P. major L. Polygonum aviculare L. P. convolvulus L. P. lapathifolium L. P. persicaria L. P. scabrum Moench Rumex acetosella L. jR. crispus L. fireweed 25 small-flowered willow- 15 herb red-sepaled evening- 1 primrose Iceland poppy 1 opium poppy 8 narrow-leaved plantain 158 broad-leaved plantain 63 prostrate knotweed 12 black bindweed 10 6 pale smartweed 264 lady's-thumb 221 green smartweed 8 sheep s o r r e l 190 curled dock 335 Table IX. Continued Portulacaceae Ranunculaceae Rosaceae Rubiaceae Scrophulariaceae Solanaceae Montia perfoliata (Donn) Howell Portulaca oleracea L. Ranunculus repens L. Fragaria vesca L. Geum macrophyllum Willd. Rosa sp. Rubus hispidus L. Gallium aparine L. G. boreale L. Digitalis purpurea L. VerJbascu/n thapsus L. Lycopersicon esculentum M i l l . Solanum nigrum L. S. tuberosum L. miner's lettuce 8 purslane 101 creeping buttercup 199 wild strawberry 73 large-leaved avens 4 0 wild rose 2 t r a i l i n g blackberry 2 cleavers 56 northern bedstraw 16 foxglove 4 common mullein 2 garden tomato 4 black nightshade 393 table potato 212 Table IX. Continued Umbelliferae Coriandrum sativum L. coriander 2 Daucus carota L. wild carrot 1 *Alex, Cayoutte, and Mulligan (1980) and Hitchcock and Cronquist (1973) Table X. Reservoir hosts of beet western yellows virus (BWYV) and potato l e a f r o l l v i r u s (PLRV) in southern B r i t i s h Columbia Test Host plant Infected /sample ELISA (A<05) positi v e samples ELISA (A 4 0 5) negative controls "Location BWYV Seneclo vulgaris 75/459 0.051-1.570 0.004 A,D,W Matricaria marltlma 7/209 0.111-0.538 0.010 V Capsella bursa-pastorls 5/1309 0.096-0.272 0.008 A, B Lactuca scarlola 3/297 0.114-0.528 0.034 B Cardamlne oligospermia 2/100 0.082-0.812 0.022 B Erodlum clcutarlum 2/174 0.285-0.436 0.010 A Polygonum lapathlfolium 2/264 0.610-1.187 0.030 P Stellarla media 1/626 0.213 0.005 W Sisymbrium officinale 1/233 0.218 0.008 B Cardarla draba 1/31 0.353 0.014 W Brasslca napus 1/49 0.149 0.015 P -Raphanus raphanlstrum 1/35 0.264 0.022 P PLRV Solarium tuberosum 3/212 1.996-2.478 0.001 L Capsella bursa-pastorls 2/1309 0.244-0.513 0.015 P Solarium nigrum 1/393 0.205 0.013 P 'Location A - Abbotsford B - Burnaby D - Delta L - Lumby P - Pemberton V - Vancouver Island W - Westham Island 6.4 Discussion TAS-ELISA was used to i d e n t i f y BWYV and PLRV reservoir hosts i n the potato-production areas of B r i t i s h Columbia. Confirmation of the ELISA results by aphid transmission tests validated TAS-ELISA as a rapid way of detecting luteoviruses i n plants. F a i l u r e to transmit virus from a l l ELISA-positive test samples may have resulted from poor acceptance of some hosts by laboratory aphids reared on Chinese cabbage (Brassica pekinensis) or to the poor condition of some samples that were stored for up to two weeks before the transmission t e s t s . Several reports of non-specific reactions i n ELISA (Gugerli, 1979; Nolan and Campbell, 1984; Timmerman et al., 1985; Gunn and Pares, 1988) emphasize the need to use high qu a l i t y antiserum and suitable controls (healthy plant sap for each species tested) and to v e r i f y p o s i t i v e r e s u l t s by another method. The two blocking steps, before and af t e r the addition of plant sample, and the use of monoclonal antibodies i n the TAS-ELISA protocol e f f e c t i v e l y reduced non-specific reactions (Table X). Weeds are important reservoirs of both vectors and viruses for several important crops (Duffus, 1971), e s p e c i a l l y BWYV, i n C a l i f o r n i a and the P a c i f i c Northwest (Wallis, 1967a,b) . The res u l t s show that both BWYV and PLRV are present i n the potato-109 producing areas of B r i t i s h Columbia but they do not indicate that weed reservoirs are important sources of potato l e a f r o l l -inducing viruses. Although BWYV was found i n about 1% of the samples tested, i t does not appear to present any threat to the potato crop i n B r i t i s h Columbia. I have not been successful i n many attempts to transmit BWYV to potato (section 5.3) and these r e s u l t s are i n agreement with those of MacCarthy (1969), Marco (1984), and Barker (1986). Weed reservoirs of BWYV are l i k e l y to be important sources of inoculum for the many crops that are susceptible to t h i s virus and many of the virus reservoirs are also good hosts for the most e f f i c i e n t vector, GPA. BWYV was f i r s t recorded i n B r i t i s h Columbia by MacCarthy (1969) when i t was recovered from sugar beet. Although sugar beets are no longer grown commercially in B r i t i s h Columbia, BWYV has survived in the weed population on Westham Island, near the location where i t was o r i g i n a l l y found. BWYV was also found i n weeds i n a g r i c u l t u r a l f i e l d s i n the Fraser Valley and near Pemberton. PLRV was found i n only six samples and three of these were volunteer potatoes. Infected volunteer potatoes have long been considered the most important source of inoculum (Banttari et al., 1990) . Finding a few black nightshade and shepherd's purse plants n a t u r a l l y infected with PLRV (Table X) suggests that weeds may occasionally play some part i n the epidemiology of potato l e a f r o l l disease. Shepherd's purse has been reported as an experimental host of PLRV but not to my knowledge as a 110 natural host. In the lower mainland of B r i t i s h Columbia and other areas of the P a c i f i c Northwest, shepherd's purse i s a winter annual that could quite possibly act as an overwintering host of PLRV. Thomas and Kaniewski (1986) reported that BWYV and PLRV could overwinter i n cruciferous weeds. Fox et al. (1990) have recently reported that PLRV was transmitted to and recovered from two c r u c i f e r s , tumble mustard and shepherd's purse by GPA under laboratory and greenhouse conditions. Although the authors suggested that these common weeds may be important sources of PLRV i n f e c t i o n of commercial potatoes, they also gave evidence that the weeds are r e l a t i v e l y poor sources of inoculum. Black nightshade i s an annual and, although not an overwintering host for either virus or vector, i t i s a preferred host of GPA (Tamaki, 1975) and may be a reservoir of virus and vectors for the spread of current season l e a f r o l l . Klein (1985) also reported black nightshade as a natural host for PLRV i n a weed survey of the San Luis Valley of Colorado. Reports of PLRV transmission to Cruciferae by Salaman and Wortly (1939) were not confirmed by the experiments of Helson and Norris (1943). Even as recently as 1981, shepherd's purse was thought to be a host for BWYV but not for PLRV (Duffus, 1981a,b). In section 4.3, I have shown that PLRV i s o l a t e s from many locations i n Canada and the United States are transmissible to shepherd's purse. Thomas (1981) and Hassan, Thomas, and Mink (1984) have also demonstrated that PLRV and the tomato s t r a i n of PLRV (TYTV) cause symptomless i n f e c t i o n of shepherd's purse. Nevertheless, i n much of Canada and the United States, most authorities would agree that PLRV-infected volunteer potato plants are the most important sources of primary inoculum of PLRV (Banttari et al., 1990; Thomas,1983). Another source i s low grade potatoes for planting material. A recent study by Vernon (1988) indicates that i n the densely s e t t l e d Fraser Valley of B r i t i s h Columbia, backyard gardens are almost c e r t a i n l y the most important reservoir of PLRV-infected potato. Many of these home gardeners plant ordinary commercial table potatoes, often from C a l i f o r n i a , do not apply i n s e c t i c i d e s , and are not aware that t h e i r potatoes are infected with a v i r u s . The r e s u l t s presented here represent a conservative estimate of virus i n f e c t i o n because ELISA i s not sensitive enough to detect virus at concentrations below 1 ng/ml (section 3.3) . If the virus concentration i n some of the samples was below t h i s l i m i t , due to recent i n f e c t i o n or to r e s t r i c t e d m u l t i p l i c a t i o n of the viruses i n some hosts, the sample would have been scored f a l s e l y negative. Fox et al. (1990) showed that the concentration of PLRV may be higher i n the root tissue than i n the fo l i a g e of tumble mustard and shepherd's purse, but another study (Eweida et al., 1988), showed that the d i s t r i b u t i o n of luteoviruses i n root and shoot tiss u e may be dependent on the growth stage at which the plants became infected. The i d e n t i f i c a t i o n of weed hosts of plant viruses can help i n the understanding of the ecological relationships that contribute to disease outbreaks. In Canada, more than a thousand d i f f e r e n t plants can be regarded as weeds but only about 230 species are considered economically important (Frankton and Mulligan, 1970) . U n t i l t h i s study, no report appears to have been published l i n k i n g potato virus disease spread with weed hosts i n Canada. Most of the weeds infected with BWYV are i n the Cruciferae or Compositae and are common i n B r i t i s h Columbia. Those that are winter annuals (fall-sprouted annuals) or perennials may serve as overwintering sources of BWYV or PLRV. When these plants grow i n protected areas, near the P a c i f i c Coast, they also serve as overwintering plants for the summer form of vector aphids, p a r t i c u l a r l y GPA. BWYV i s of considerable economic importance (Ashby, Bos, and Huijberts, 1979; Duffus, 1977; Walkey and Pink, 1990) . Its presence i n weeds i n a g r i c u l t u r a l areas of B r i t i s h Columbia indicates that a source of inoculum i s usually present. In years when GPA are abundant, BWYV probably i s a threat to many susceptible vegetable crops. MacCarthy (1969) has shown that 113 garden beet, spinach, lettuce, pea, b r o c c o l i , cauliflower, turnip, and Chinese cabbage are a l l susceptible to a l o c a l i s o l a t e . The presence of BWYV i n weeds i s not surprising considering the wide host range of t h i s virus and i t s occurrence i n the province for at least 20 years. 114 Chapter 7 Summary and conclusions 7.1 Summary of resu l t s The objectives outlined i n section 1.4 were achieved: 1. Several MAbs were produced that discriminate between PLRV and BWYV. Two MAbs, 510H and 112E, detected a l l of the strains of BWYV tested including the RPV i s o l a t e of BYDV now recognized as a s t r a i n of BWYV. MAb 510H was selected for large scale t e s t i n g because of i t s broad s p e c i f i c i t y to BWYV, detecting 16 is o l a t e s from six countries, and because i t was produced i n high t i t e r i n ascites f l u i d . MAbs 26BE and 41BC detected a l l of the PLRV is o l a t e s tested including the solanum yellows virus s t r a i n . One MAb detected an epitope common to both PLRV and BWYV. None of the MAbs reacted with other luteoviruses nor with healthy plant sap. 2. In t o t a l , 801 samples of potato l e a f r o l l disease were c o l l e c t e d and tested for PLRV and BWYV in 1986, 1987, and 1988 using TAS-ELISA and v i r u s - s p e c i f i c MAbs. The samples represented 32 c u l t i v a r s , o r i g i n a t i n g i n 8 Canadian provinces and 12 American states. None of the samples tested p o s i t i v e for 115 BWYV whereas 772 (96.4%) tested p o s i t i v e for PLRV. Neither PLRV nor BWYV could be recovered, by aphid transfers to indicator hosts, from 28 of the samples that tested negative for both viruses. One sample that was scored negative for both viruses by ELISA, tested p o s i t i v e with an aphid transmission test to P. pubescens; the indicator plant tested p o s i t i v e for PLRV by TAS-ELISA. 3. NASH was compared with ELISA and aphid transmission tests for detection of PLRV and BWYV i n 165 potato plants showing symptoms of potato l e a f r o l l disease. None of the plants tested p o s i t i v e for BWYV by DAS-ELISA, TAS-ELISA, or NASH using either a cloned BWYV probe or random primed cDNA prepared from BWYV RNA. The indicator plants a l l tested negative for BWYV by TAS-ELISA. A l l of the 165 potato l e a f r o l l disease samples tested p o s i t i v e for PLRV using TAS-ELISA and NASH with a cloned PLRV cDNA probe. TAS-ELISA, using MAb 371A, generally produced a stronger signal for PLRV than DAS-ELISA using PLRV polyclonal antiserum. DAS-ELISA produced one false-negative; the same sample tested p o s i t i v e by TAS-ELISA, NASH, and aphid transmission to P. jPuJbescens. NASH, using random primed cDNA prepared from PLRV RNA, also produced one false-negative. None of the f i v e v i r u s -free negative control samples or 18 symptomless f i e l d samples tested p o s i t i v e for either v i r u s . Seventy-two percent (119/165) of the PLRV is o l a t e s infected shepherd's purse. 116 4. The s u s c e p t i b i l i t y of potato to BWYV was tested by aphid inoculation of Russet Burbank plants with three i s o l a t e s of BWYV from Canada and four from the United States. Two of the is o l a t e s were i n a mixed i n f e c t i o n with PLRV. The potato plants were assayed for PLRV and BWYV by TAS-ELISA, and by aphid transmission to indicator plants six weeks af t e r inoculation. None of the plants tested p o s i t i v e for BWYV by ELISA. The ELISA res u l t s were confirmed by the aphid transmission t e s t s . BWYV could not be recovered, by aphid transfers to indicator plants, from any of the inoculated potato plants. PLRV was detected i n and recovered from plants inoculated with both PLRV and BWYV. 5. Common weeds were surveyed i n the potato-producing areas of B r i t i s h Columbia for PLRV and BWYV. In t o t a l , 10,098 weed samples, representing 98 species i n 22 plant families, were c o l l e c t e d and tested by TAS-ELISA from 1986 to 1989. BWYV was detected i n 1% of the plants tested; the hosts were: chickweed, common groundsel, heart-podded hoary cress, hedge mustard, l i t t l e western b i t t e r c r e s s , p r i c k l y lettuce, shepherd's purse, pale smartweed, rutabaga, scentless chamomile, s t o r k ' s - b i l l , and wild radish. PLRV was detected i n three volunteer potato plants, two samples of shepherd's purse, and one black nightshade plant. The low incidence of PLRV in hosts other than potato suggests that weeds are of minor importance i n the epidemiology of potato l e a f r o l l disease i n B r i t i s h Columbia. 117 7.2 Conclusions The r e s u l t s presented i n t h i s thesis provide an unambiguous picture of the ins i g n i f i c a n c e of BWYV i n potato l e a f r o l l disease. BWYV does not present a threat to either seed or table potato production. Potato l e a f r o l l disease i s caused by a single virus, PLRV. The virus exists as a number of b i o l o g i c a l variants that are ant i g e n i c a l l y i d e n t i c a l or cl o s e l y related (Stace-Smith, 1987). The natural host range of PLRV i s lim i t e d p r imarily to the Solanaceae but some species i n other plant families may become infected. At the present time there i s no convincing evidence that hosts other than potato are important reservoirs for PLRV i n B r i t i s h Columbia. However, the occurrence of PLRV i n a very few naturally infected winter annuals suggests that i n areas where there i s a s i g n i f i c a n t early f l i g h t of GPA, weeds may play a role i n the epidemiology of potato l e a f r o l l disease. With the development of ELISA as a routine test for detecting plant viruses and the a v a i l a b i l i t y of s p e c i f i c monoclonal antibodies to PLRV, we can now r e l i a b l y detect PLRV i n potato and other hosts. TAS-ELISA i s sensitive enough to detect PLRV i n single aphid vectors (Martin and E l l i s , 1987) and can be used to monitor aphid populations f or the virus, to allow assessment of ri s k s and more precise timing of potato t o p - k i l l dates. 118 Recently Kawchuck, Martin, and McPherson (1990) have succeeded i n producing transgenic potato plants, c u l t i v a r s Desiree and Russet Burbank, that express the coat protein gene of PLRV. This same strategy has been used to provide cross-protection from several plant viruses such as tobacco mosaic virus (Abel et al., 1986), a l f a l f a mosaic virus (Loesch-Fries et al., 1987), potato virus X (Hemenway et al., 1988), and cucumber mosaic virus (Cuozzo et al., 1988). However, by reducing v i r a l r e p l i c a t i o n , t h i s method may result i n plants that become infected with virus but at l e v e l s below the detection l i m i t of ELISA. Another new technology that w i l l have a s i g n i f i c a n t impact on detection and diagnosis of PLRV i s the polymerase chain reaction (PCR). The PCR technique allows v i r t u a l l y unlimited amplification of cDNA, from v i r a l RNA, before detection with nucleic acid probes (Kawasaki, 1990; Rotbart, 1990). As newer c u l t i v a r s of potato are released with better resistance to PLRV (Barker and Harrison, 1985) i t w i l l become increasingly d i f f i c u l t to detect the v i r u s . PCR and nucleic acid probes w i l l allow us to separate c u l t i v a r s which are c a r r i e r s of the virus from those which do not allow the virus to r e p l i c a t e . 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