Two strains of grapevine leafroll-associated virus 2 (GLRaV-2) were obtained by mechanical transmission from grapevines to Nicotiana benthantiana. The strains, designated 94/970 and 93/955, consistently differed with regard to the development of symptoms. The first induced chlorotic and occasional white-necrotic local lesions while the second induced chlorotic followed by metallic-opalescent, solid necrotic local lesions. The strains were indistinguishable with regard to the molecular weight of their capsid proteins or serologically. A difference in the pattern of minor dsRNA bands was consistently observed

Goszczynski, D. E.

Kasdorf, G. G. F.

Pietersen, G.

van Tonder, H.

English

JKI Open Journal Systems (Julius Kühn-Institut)

Vitis 35 (3), 133-135 (1996) Detection of two strains of grapevine leafroll-associated virus 2 by D. E. GoszczYNSKI, G. G. F. KAsooRF, G. PIETERSEN and H. VAN ToNDER Plant Protection Research Institute, Agricultural Research Council, Pretoria, Republic of South Africa Sum mar y : Two strains of grapevine leafroll-associated virus 2 (GLRaV-2) were obtained by mechanical transmission from grapevines to Nicotiana benthamiana. The strains, designated 94/970 and 93/955, consistently differed with regard to the develop-ment of symptoms. The first induced chlorotic and occasional white-necrotic local lesions while the second induced chlorotic followed by metallic-opalescent, solid necrotic local lesions. The strains were indistinguishable with regard to the molecular weight of their capsid proteins or serologically. A difference in the pattern of minor dsRNA bands was consistently observed. Key words : GLRaV-2, strains, symptoms, cytopathology, serology, dsRNA. Introduction Grapevine leafroll (GLR) is a viral disease known worldwide for many years but still of uncertain etiology (BovEY and MARTELLI 1992). Grapevine leafroll-associated virus 2 (GLRa V-2) is one of the 6 serologically distinct closteroviruses (GLRaV-1- 6) frequently detected in grape-vines affected by GLR (GuoERLI et al. 1984; RoscrouoNE and GUGERLI 1986; Hu et al. 1990; ZIMMERMANN et al. 1990; BovEY and MARTELLI 1992; GuoERLI and RAMEL 1993; BoscrA et al. 1995). It is the only grapevine closterovirus for which mechanical transmissibility to herbaceous spe-cies was confirmed by different laboratories (MONETTE and GooKIN 1993; BoscrA et al. 1995; GoszczYNSKI, unpub-lished). In this paper we report the existence of two biological strains of GLRaV-2. Materials and methods Virus isolates and their mechanical t ran s mission: The two isolates ofGLRaV-2 used in this study were recovered from GLR-affected Vitis vinifera L. cv. Muscat of Alexandria (Plant Quarantine Station, Stellenbosch) and hybrid LN33 (Nietvorbij Experimental Farm, Stellenbosch) by mechanical inoculation of N. benthamiana. Virus infection of LN 33 was achieved by chip-budding with buds from grapevine cv. Tinta Barocca (vine no. 10-27; ENGELBRECHT and KASDORF 1990). The viruses for mechanical transmission were prepared as follows: 4 g of freshly collected petioles from potted grape-vine plants were pulverised in liquid N2 using a pestle and mortar and mixed with 40 ml of 0.1 M Tris-HCl buffer pH 7.6 containing 0.01 M MgS04, 0.2 % 2-mercapto-ethanol, 2% Triton X-100, 0.5% bentonite and 4% poly-vinyl-polypyrrolidone (PVPP). After a low speed centri-fugation at 6000 g for 5 ruin the supematant was centrifuged through a 20 % sucrose cushion (in 0.1 M Tris-HCl buffer pH 7.6 with 0.01 M MgS04) at 26,000 rpm (TY30 rotor, Beckman) for 2 h 15 min at 8 °C. The pellet was re-suspended in 0.7 ml of buffer containing 0.01 M K2HP04, 0.01 M cysteine-HCl and 3 %nicotine (inoculation buffer) (BoscrA et al. 1993), mixed with celite, and used for in-oculation of N. benthamiana. Transmissions of viruses between N. benthamiana plants were done using sap of systemically infected plants extracted in inoculation buffer. Inoculations of N. benthamiana by isolates of GLRa V-2 were repeated more than 16 times over an 8-month period (January to September). d s R N A a n a 1 y s i s : GLRaV-2-infected N. benthamiana plants collected in February were used for extraction and analysis of dsRNA by the procedure of VALVERDE (1990). dsRNA of African horse sickness virus serotype 3 (AHSV-3) with Mr 3314, 3038, 2663, 2033, 1804, 1639, 1137, 1137, 1137 and 693 bp were used as Mr markers. Three dsRNA extractions and analyses were done. V i r u s p u r i f i c a t i o n : The virus was purified as follows: 80 g of systemically infected N. benthamiana plants were macerated using a pestle and mortar in 400 ml of0.1 MTris-HCl buffer pH 7.6 containing 0.01 M MgS04, 0.2 % 2-mercaptoethanol and 5 % Triton X-100 (extrac-tion buffer). This was expressed through cheesecloth and the extract thoroughly shaken with l/5 volume ofchloro-form, before centrifugation at 10,000 g for 15 min. The aqueous phase was collected, PEG 6000 and NaCl were added to a concentration of 8 % and 0.2 M respectively, and the solution was stirred on ice for 2 h. After centrifu-gation at 12,000 g for 25 min, the pellet was diluted in resuspension buffer (extraction buffer without 2-mercapto-ethanol) and resuspended slowly by stirring overnight at Correspondence to: Dr. D. E. GoszCZYNSKI, Plant Protection Research Institute, Agricultural Research Council, Private Bag Xl34, Pretoria 0001 , Republic of South Africa. Fax: (012) 329 3278. 134 D. E. GoszczYNSKI, G. G. F. KASDORF, G. PIETERSEN and H. VAN ToNDER 4 °C. The suspension was centrifuged at 6000 g for 5 min, then ultracentrifuged at 26,000 rpm (TY30 rotor, Beckman) for 2 h and 25 min at 8 oc through 20 % sucrose cushion prepared with dialysis buffer (resuspension buffer without Triton X-100). The resulting pellet was diluted in 40 ml resuspension buffer and shaken with a marble at 4 oc over-night. After centrifugation at 6000 g for 5 min (repeated twice) the supernatant was loaded on a Cs2S04 gradient (prepared from 1 ml of each 15 % and 25 % and 2 ml of 35 % Cs2S04 in dialysis buffer) and centrifuged at 28,000 rpm for 3 h and 45 min at 8 oc (SW 41 rotor, Beckman). Virus bands were collected and dialysed with dialysis buffer overnight at 4 °C. The preparations were then centrifuged at 12,000 g for 10 min. Supernatants were stored at -75 °C. A n t i s e r a p r o d u c t i o n : Antisera were produced by 4 intramuscular injections of rabbits, 2 weeks apart, with 0.5 ml preparations of purified virus emulsi-fied with complete (first injection) and incomplete (subse-quent injections) Freund's adjuvant. Blood was collected 2 weeks after the last injection. SDS-PAGE, Western blot and IEM were done as described by GoszczYNSKI et al. (1995) with some minor modifications. Viruses were denaturated with an equal volume of SDS-PAGE disruption buffer contain-ing 0.1 M Tris-HCl, pH 7.6, 0.01 M MgS04, 5 % SDS, 10% glycerol, 10 % 2-mercaptoethanol. Prestained SDS-PAGE standards (Bio Rad, low range) were phosphorylase B (112,000), bovine serum albumin (84,000), ovalbumin (53,200) , carbonic anhydrase (34,900), soybean trypsin inhibitor (28,700), and lysozyme (20,500). For IEM, the viruses were resuspended in 0.1 M sodium phosphate buffer pH 7.0. A Dual-mini vertical unit (American Bionetics, Inc.) and Mini trans-blot cell (Bio Rad) were used for electrophoresis (4.5% stacking and 12.5% resolving poly-acrylamide gels, at 180 V for 50 min) and electro-transfer (at lOO V for 60 min). Monoclonal antibody to GLRa V-2 (GuGERLI and RAMEL 1993) used in IEM was kindly donated by P. GuoERLI (Fed-eral Agricultural Research Station of Changins, Nyon, Switzerland). C y t o p a t h o l o g y : For ultrastructural studies, pieces of tissue were collected in February from systemi-cally virus-infected leaves of N. benthamiana showing vein clearing. They were double-fixed in 2.5 % glutaraldehyde and 1 % osmium tetroxide, en block stained with uranyl acetate, dehydrated in graded acetone series and embed-ded in Epon-Araldite resin (HAYAT 1972). Ultrathin sec-tions were stained with uranyl acetate and lead citrate and examined using a Joel JEM-100C electron microscope. Results and discussion Plants of N. benthamiana infected with viruses me-chanically transmitted from cv. Muscat of Alexandria and hybrid LN33 , contained only GLRaV-2 particles. This con-clusion was based on detailed IEM examination of leaf dips and purified virus preparations u'sing monoclonal an-tibody to this virus. The viruses were designated as iso-lates 94/970 and 93/955, respectively. The development of symptoms in inoculated N. benthamiana plants consistently differed between iso-lates . Those inoculated with isolate 94/970 developed chlorotic and occasional white-necrotic local lesions fol-lowed by systemic vein clearing. In some plants vein clear-ing turned to vein necrosis. N. benthamiana inoculated with isolate 93/955 developed chlorotic local lesions which turned to metallic-opalescent, solid necrotic local lesions (Fig. 1). These necrotic local lesions occurred only in N. benthamiana inoculated by isolate 93/955. Vein clear-ing induced by this isolate was followed by strong vein necrosis. Fig. I: Leaf of Nicotiana benthamiana with necrotic local le-sions induced by GLRaV-2 isolate 93/955 . Cytological alterations observed in N. benthamiana infected with isolates of GLRaV-2 generally agreed with those described by CASTELLANO et al. (1995). While we did not find cytological alterations characteristic for only one of the GLRaV-2 isolates, the relative intensity showed marked differences between isolates. Isolate 93/955 in-duced a greater number of cytoplasmic vesicles, as well as stronger appositions of a callose-like substance than iso-late 94/970. The extensive necrosis of vascular elements described by CASTELLANO et al. ( 1995) was observed only for isolate 93/955. EM observations indicated that the more severe cytological alterations induced by isolate 93/955 were not caused by higher numbers of viral particles present. In fact isolate 93/955 appeared to have a lower number of viral particles per cell than isolate 94/970. This was confirmed by analyses of leaf dips and purified virus preparations using IEM and SDS-PAGE. Fig. 2 A shows the typical difference in amount of purified capsid pro-teins between isolates of GLRaV-2 in SDS-PAGE. Electrophoretic mobilities of capsid proteins were the same for both isolates (Fig. 2 A). In addition, no notice-able serological differences between isolates were detected in IEM and Western blots using homologous antisera. Strains of GLRaV-2 135 A M 2 B M 2 Fig. 2: SDS-PAGE (A) and dsRNA (B) analyses of GLRaV-2 isolates 94/970 (lane 1) and 93/955 (lane 2). M = Mr markers. Polyacrylamide gel electrophoresis showed that both isolates contained the same pattern of 6 major dsRNA bands (Fig. 2 B). This was similar to the dsRNA pattern described for beet yellows virus (BYV) by Dooos and BAR-JosEPH (1983). Consistant differences between isolates were, how-ever, observed in minor dsRNA bands: dsRNA marked on Fig. 2 B as band "a" was clearly detected only in extracts of N. benthamiana infected with isolate 93/955, while dsRNA marked as band "b" was detected only for isolate 94/970. The consistent differences in external symptoms, in-tensity of cytological alterations and minor dsRNA band patterns observed for GLRaV-2 isolates 94/970 and 93/955 indicate that they represent two strains of this virus. Iso-late 93/955 is clearly more virulent for N. benthamiana than isolate 94/970. Whether a connection exists between dsRNAs differentiating these isolates and virulence has still to be determined. This study represents the first identification of two bio-logical strains of a grapevine closterovirus. References BovEY, R.; MARTELLI, G. P.; 1992: Directory of Major Virus and Virus-like Diseases of Grapevines. Mediterranean Fruit Improvement Council and International Council for the Study of Viruses and Virus Dis-eases of the Grapevine, Bari. BosciA, D.; GREIF, C.; GuoERLI, P.; MARTELu, G. P.; W ALTER, B.; GoNSALVES, D .; 1995: Nomenclature of grapevine leafroll-associated putative closteroviruses. Vitis 34, 171-17 5. - -; SAVINO, V.; MINAFRA, A.; NAMBA, S.; ELICIO, V; CASTELLANO, M. A.; GoNSALVES, D.; MARTELLI, G. P.; 1993: Properties of a filamentous virus isolated from grapevines affected by corky bark. Arch. Virol. 130, 109-120. CASTELLANO, M. A.; Asou-GHANEM, N.; MARTELLI, G . P.; BosCIA, D .; SAVINO, V; 1995: Cytopathology of two filamentous grapevine vi-ruses and their intracellular identification by gold immunolabelling. J . Plant Dis. Prot. 102,23-33. Dooos, J. A.; BAR-JosEPH, M.; 1983: Double-stranded RNA from plants infected with closteroviruses. Phytopathology 73, 419-423. ENGELBRECHT, D. J.; KASDORF, G. G. F. ; 1990: Field spread of corky bark, fleck, leafroll and Shiraz decline diseases and associated viruses in South African grapevines. Phytophylactica 22, 347-354. GoszCZYNSKI, D. E.; KASDORF, G. G. F.; PIETERSEN, G.; 1995: Production and use of antisera specific to grapevine leafroll-associated viruses following electrophoretic separation of their proteins and transfer to nitrocellulose. Afr. Plant Prot. 1, 1-8. GuoERLI, P.; RAMEL, M. E .; 1993: Grapevine leafroll associated virus 11 ana1yzed by monoclonal antibodies. Extended abstracts IIth Meet-ing ICVG, Montreux, Switzerland, 6-9 September 1993, 23-24. - -; BRUGGER, J . J.; BovEv, R.; 1984: L'enroulement de la vigne: mise en evidence de particules virales et developpement d'une methode immunoenzymatique pour le diagnostic rapide. Rev. Suisse Viticult. Arboricult. Horticult. 16, 299-304. HAYAT, M. A.; 1972: Basic Electron Microscopy Techniques. Van Nostrand Reinhold Company, New York. Hu, J. S.; GoNSALVES, D.; TELIZ, D.; 1990: Characterization of closterovirus-like particles associated with grapevine leafroll disease. J. Phytopathol. 128, 1-14. MONETTE, P. L .; GooKIN, S. E .; 1993: Mechanical transmission of closterovirus-like particles from a corky bark -affected grapevine to herbaceous species. Plant Pathol. (Trends Agricult. Sci.) 1, 7-12. ROSCIGLIONE, B.; GUGERLI, P.; 1986: Maladies de l'enroulement et du bois strie de la vigne: analyse microscopique el serologique. Rev. Suisse Viticu1t. Arboricult. Horticult. 18,207-211. VALVERDE, R. A. ; 1990: Analysis of double-stranded RNA for plant virus diagnosis. Plant Dis. 74, 255-258. ZIMMERMANN, D.; BAss, P.; LEGIN, R.; WALTER, B.; 1990: Characterization and serological detection of four closterovirus-like particles associ-ated with leafroll disease on grapevine. J. Phytopathol.130, 205-218. Received May 2, 1996 

Detection of two strains of grapevine leafroll-associated virus 2

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Detection of two strains of grapevine leafroll-associated virus 2

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