icvg 2009 part I pp 1-131.pdf - Cornell University

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— 46 — DETECTION OF VIRUSES IN SEEDS OF INFECTED GRAPEVINES WITH AN EMPHASIS ON GRAPEVINE RUPESTRIS STEM PITTING-ASSOCIATED VIRUS Nuredin HABILI 1 , Rodney DAVIES 1,2 , John RANDLES 1 1 Waite Diagnostics, School of Agriculture, Food and Wine, University of Adelaide, GLEN OSMOND, SA 5064, Australia 2 Clean Plant Technology, 167 Main Road, OAKBANK, South Australia 5243 *Corresp. author: Nuredin Habili, Telephone + 61883037426, Fax +61883037109 , Email: nuredin.habili@adelaide.edu.au Summary Three grapevine varieties, Cabernet Sauvignon clone SA125 infected with GRSPaV, GVA and GLRaV-9, and Grenache and Dolcetto, each infected with RSPaV, GVA and GLRaV-1 were selected to study the presence of each associated virus in their seeds following washing with water or water followed by 3.5% sodium hypochlorite bleach. RT-PCR assay of mature seeds showed that RSPaV and GVA were consistently retained after treatment with bleach while GLRaV-1 and -9 were not always detectable. Both embryonic and non-embryonic sections of the seeds tested positive for GRSPaV, indicating that the virus is present in all parts of the seed. INTRODUCTION Seeds from virus-infected grapevines need to be tested as a potential source of infection in vineyards. They are readily disseminated via machinery, cultural practices, animals and birds, and germinate to provide a population of feral grapevine seedlings both within and outside vineyards. This has prompted us to assay for four viruses in the seed of three infected varieties maintained at our research vinyard. We were particularly interested in Grapevine Rupestris stem pitting-associated virus (GRSPaV) a Foveavirus (Family: Flexiviridae) detected in over 90% of grapevine samples sent to Waite Diagnostics for testing (Symons et al., 2000). Although the virus is symptomless in most own-rooted vines, we have observed pitting symptoms on virus positive young Shiraz vines grafted onto Paulsen rootstock in a vineyard at McLaren Vale, South Australia. This virus has already been detected in seeds in one laboratory (Stewart & Nassuth, 2001), while the seed testing results were inconclusive in another laboratory, in which it was detected in the pollen (Rowhani et al, 2000). There are no reports of the distribution of the virus in sections of seeds. Invasion of the embryo by plant viruses is considered to be necessary for true seed transmission (Wang & Maule, 1994). We report here the results of tests to determine whether GRSPaV is inside seed and whether it occurs in different parts of dissected seeds. Other viruses studied included Grapevine virus A (GVA), a Vitivirus associated with Shiraz Disease in Australia (Habili & Randles, 2004), and two Ampeloviruses: Grapevine leafroll associated virus 1 (GLRaV-1) and GLRaV-9. The latter is associated with a mild leafroll symptom in infected grapevines. MATERIAL AND METHODS Vine varieties and seed preparation. Three grapevine varieties with known virus profiles planted in 1992 at the Waite Campus, South Australia, were selected for seed/virus studies. Mature seeds were collected from ripe berries over two seasons, cleaned by removal of pulp and either washed in water (water treatment) or washed in water followed by soaking in 3.5% sodium hypochlorite for 5 minutes (bleach treatment). The bleach treated seeds were then washed in 100 mM Na-acetate, pH 5.0, 1 mM EDTA for 30 minutes and rinsed in water before use. Total nucleic acid extraction. Samples of six complete seeds (cs) or 12 half seeds (cut transversely) were collected from each seed lot originating from a single bunch. Up to 10 bunches, each from a different vine of the same variety (clone) were used per treatment. Each half seed was classified as either containing the embryo section (es) or lacking the embryo (ne). The seeds or seed sections were crushed in 2 ml guanidine hydrochloride lysis buffer as described by Mackenzie et al. (1997), 1% Polyclar AT and 1% sodium metabisulphite. Nucleic acids were prepared after binding to silica, washing and eluting in 50 l TE. Further purification was carried out by passing the extract through a size-exclusive Sepharose mini-column (unpublished). RT-PCR assay. The method described by Mackenzie et al. (1997) was employed in a single-step RT-PCR with specific primers for each virus as described elsewhere (Habili & Randles, 2002). For the RT-PCR of GRSPaV the 48/49 primers of Zhang et al. (1998) were used. RESULTS AND DISCUSSION Virus status of the grapevine varieties: Viruses that we routinely detect in the three varieties planted in our research vineyard and studied here are listed in Table 1. These viruses are detectable in the cane shavings throughout the year, with no change in the profile for the last ten years. Viruses and seeds. GRSpaV and GVA were detected in the seeds of Cabernet and Grenache following washing with water alone, or with a subsequent bleach treatment (Table 2). In contrast, the two Ampeloviruses, GLRaV-1 and GLRaV-9 were more frequently removed by washing with water and there was an even lower frequency of detection following treatment with bleach (Table 2). This
— 47 — result suggests that the ampeloviruses adhere loosely to the seed surface, while GRSpaV and GVA may be either firmly attached or internal. To test whether GRSPaV was internal, seeds from each variety listed in Table 1 were treated with bleach and cut into two sections. Embryonic (es) and non-embryonic (ne) halves of the seed were tested separately. Both sections as well as the intact seed (cs) contained the GRSPaV RNA as revealed by RT-PCR (Fig. 1). Table 1. Virus status of the varieties which were the source of the seeds used in this study Var./ Virus GRSPaV GVA GLRaV-1 GLRaV-9 Cab Sauv. SA 125 yes yes no yes Grenache yes yes yes no Dolcetto yes yes yes no Figure 1. RT-PCR detection of GRSPaV in seeds of Cabernet Sauvignon (lanes 1-3), Grenache (lanes 4-6) and Dolceltto (lanes 7-9) following a bleach treatment. M, DNA ladder, pUC 19 Hpa II cut. Lane 10, positive control. cs, complete seed; es, embryo section; ne, non-embryo section. M 1 2 3 4 5 6 7 8 9 10 These results indicate that GRSPaV and GVA are internal but fail to distinguish between the possibilities that they are retained in the endosperm or vascular system of the seed only, or are localized in the embryo. The resolution of this question will depend on germinating and testing emerged seedlings for these viruses, as presence of virus in the seedling will confirm that it has entered the embryo from the vascular system of the seed (Wang & Maule, 1994). If viruses are shown to be seedborne, then progeny from infected parents could be infected. Hence breeders material is not necessarily free of embryo or pollen borne viruses. RT-PCR is shown here to be sensitive enough to detect virus in seed and to ensure that breeders release virus-tested lines to the industry. The high incidence of GRSPaV in Australian grapevines is being addressed by our 12 year old program of shoot tip culture and thermotherapy (R. Davies, unpublished). The most recalcitrant elongated virus for elimination is GRSPaV followed by GVA, while Ampeloviruses (leafroll viruses) are easily eliminated. GRSPaV requires an elevated temperature to be eliminated from grapevine tissue. We have noticed that once this virus is removed, the plantlet would appear to be free of all other viruses. Under such a harsh regime, it is only necessary to test for this virus as all other viruses have been eliminated. 350 bp cs es ne cs es ne cs es ne + Table 2. Viruses detected in seeds of Cabernet and Grenache after soaking in water or bleach Virus: RSPaV 1 GVA 1 GLRaV-1 2 GLRaV-9 1 Expt. No. water bleach water bleach water bleach water bleach 1 yes yes yes yes yes yes no no 2 yes yes yes yes yes no yes no 3 yes yes yes yes yes yes yes yes 4 yes yes yes no no no yes no 1 detected in Cabernet, 2 detected in Grenache LITERATURE HABILI, N. & RANDLES, J. 2002. Developing a standardised sampling protocol for consistent detection of grapevine viruses by the PCR assay. The Australian & New Zealand Grapegrower and Winemaker 464, 88-92. HABILI, N. & RANDLES, J. 2004. Descriptors for Grapevine Virus A- associated syndrome in Shiraz, Merlot and Ruby Cabernet in Australia, and its similarity to Shiraz Disease in South Africa. The Australian and New Zealand Grapegrower and Winemaker 488, 71-74. MACKENZIE, D., McLEAN, M., MUKERJI, S. & GREEN, M. 1997. Improved RNA extraction from woody plants for the detection of viral pathogens by reverse-transcription-polymerase chanin reaction. Plant Disease 81, 222-226. ROWHANI, A., ZHANG, Y., CHIN, J., MINAFRA, A., GOLINO, D. & UYEMOTO, J. 2000. Grapevine Rupestris stem pitting associated virus: population diversity, titer in the host, and possible transmission vector. Extended Abstracts 13th Meeting of ICVG, Adelaide, Australia, 37. SYMONS, R., HABILI, N. & BONFIGLIOLI, R. 2000. Waite Diagnostics – Development of a diagnostic service for the Australian viticultural industry. Extended Abstracts 13th Meeting of ICVG, Adelaide, Australia, 149. STEWART, S. & NASSUTH, A. 2001. RT-PCR based detection of Rupestris stem pitting associated virus within field-grown grapevines throughout the year. Plant Disease 85, 617-620. WANG, D.J. & MAULE, A. 1994. A model for seed transmission of a plant virus: genetic and structural analyses of pea embryo invasion by pea seed-borne mosaic virus. The Plant Cell 6, 777-787. ZHANG, Y., UYEMOTO, J., GOLINO, D. & ROWHANI, A. 1998. Nucleotide sequence and RT-PCR detection of a virus associated with grapevine rupestris stem pitting disease. Phytophathology 88, 1231-1237. ACKNOWLEDGEMENTS We would like to thank Mr. Tony Herve, the Director of Clean Plant Technology, South Australia, for support and advice. Progrès Agricole et Viticole, 2009, Hors Série – Extended abstracts 16 th Meeting of ICVG, Dijon, France, 31 Aug – 4 Sept 2009
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Page 5 and 6: AVANT-PROPOS Nous sommes très heur
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— 129 — THE VIRUS STATUS OF GRA
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— 131 — PRESENCE OF GLRAV 1, 2,
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