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PCR, Based on Degenerate Primers for the Simultaneous Detection of Viruses of the Closterovirus, Ampelovirus, Foveavirus, and Vitivirus Genera

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PCR, Based on Degenerate Primers for the Simultaneous Detection of Viruses of the Closterovirus, Ampelovirus, Foveavirus, and Vitivirus Genera Industry allocated project number SATI CFPA SAAPPA/SASPA DFTS Winetech [email protected] [email protected] [email protected] [email protected] [email protected] Tel: 021 872-1438 Tel: 021 872-1501 Tel: 021 882-8470 Tel: 021 870 2900 Tel: 021 807 3387 Indicate (X) client(s) to whom this final report is submitted. Replace any of these with other relevant clients if required . ______________________________________ FINAL REPORT 2014 Programme & Project Leader Information Research Or ganisation Project leader Programme leader Title, initials, surname Dr. I. Rong Dr. DE Goszczynski Present position Research Team Manager Senior Researcher Address PPRI, ARC PPRI, ARC P. Bag X134 P. Bag X134 Queenswood 0121 Queenswood 0121 Tel. / Cell no. 012 808 8138 012 808 8187 Fax 012 808 8299 012 808 8299 E-mail [email protected] [email protected] Project Information Research Organisation PPRI Gos 4 Project number Project title The investigation into the reliability and sensitivity of RT nested - PCR, based on degenerate primers for the simultaneous detection of viruses of the Closterovirus, Ampelovirus, Foveavirus, and Vitivirus genera. Fruit kind(s) Grapevines Start date (mm/yyyy) January 2012 End date January 2014 Project keywords RT nested-PCR; Closteroviridae, Flexiviridae; detection Approved by Research Organisation Programme leader (tick box) x This document is confidential and any unauthorised disclosure is prohibited. Final report 2 THIS REPORT MUST INCLUDE INFORMATION FROM THE ENTIREENTIRE PROJECT Executive Summary Give an executive summary of the total project. The reliability of RT nested-PCR technique designed for the simultaneous detection of viruses associated with leafroll (LR) and rugose wood (RW) diseases (Dovas and Katis, J. Vir. Meth. 109, 217-226, 2003) was investigated. Results of an analysis of the virus status of multivirus-infected grapevines of various cultivars proved that the technique has the capability to efficiently and specifically amplify all known and yet unknown viruses associated with these two economically important diseases. The technique was applied to the comparative analysis of viruses of grapevines cv. Shiraz with different Shiraz disease (SD) statuses. The study has led to the discovery of a new, highly divergent variant of Grapevine leafroll-associated virus 3 (GLRaV-3), and a new genetic variant of GVA. The results were published in the international journals. A broad spectrum, two-step RT-PCR technique based on a single primer with random hexanucleotide sequence was investigated. This technique is potentially able to amplify all grapevine infecting viruses. The replicative form of dsRNA of viruses was targeted for RT-PCR amplification. Results revealed that, in contrast to the virus-infected herbaceous host, N. benthamiana , the isolation of RF from grapevines is difficult and impractical. Also, the amplification of total dsRNA using this technique could not find an application for the routine diagnosis of virus infections owing to the fact that plant RNA sequences contaminated dsRNA samples, and were efficiently amplified. The technique, however, can be applied to the analysis of viruses of grapevines that exhibit virus-like symptoms, but which are negative for all known viruses. Problem identification and objectives State the problem being addressed and the ultimate aim of the project. According to recent data, grapevine is host to 60 viruses. The major concerns of grapevine industries world-wide are viruses of the Closteroviridae and Betaflexiviridae families, which include viruses associated with leafroll (LR) and rugose wood (RW) diseases. These diseases decrease productivity and longevity of vineyards, interfere with graft take, and negatively influence the quality of fruits and, ultimately, the quality of produced wines. Virus-infected grapevine can be cured by the application of virus elimination procedures, using heat treatment and in vitro tip cultures. However, the method is not 100% effective, and not all viruses are equally easy to eliminate. The propagation of virus-infected grapevines is then a possibility. Currently, the South African grapevine industry is using ELISA for simultaneous detection of GLRaV-1, -2 and -3. Although the technique is easy to use, it has limited sensitivity when compared with RT-PCR. In addition, it detects only three out of more than 10 viruses of the Closteroviridae family associated with leafroll disease (LR), and it does not detect viruses of the Betaflexiviridae family associated with rugose wood (RW) diseases. To ensure strict control of the the Closteroviridae and Betaflexivirida e-free status of grapevine propagative material, a sensitive virus detection technique that detects a broad range of viruses of these two families, is required. In 2003, Dovas and Katis (J. Vir. Meth. 109, 217-226, 2003) reported RT nested-PCR technique that can This document is confidential and any unauthorised disclosure is prohibited. Final report 3 simultaneously amplify all known members of Closteroviridae and Betaflexiviridae families. The technique is based on degenerate primers that overcome molecular differences between virus species, as well as molecular variability within a single species of virus. Thus, it can also amplify yet unknown members of these two virus families. The reliability of this technique was investigated in this study. Also, a broad spectrum two-step RT-PCR technique based on a single primer with random hexanucleotide sequence was investigated. This technique is potentially able to amplify all grapevine infecting viruses. Workplan (materials and methods) List trial sites, treatments, experimental layout and statistical detail, sampling detail, cold storage and examination stages and parameters. Viruses of the families Closteroviridae and Betaflexiviridae, which can be RT nested- PCR amplified using the technique of Dovas and Katis (2003), and which have been investigated in this study are listed in Table 1. Table 1. Virus Taxonomic classification (Family, Genus) Grapevine leafroll-associated virus – 1 (GLRaV- Closteroviridae, Ampelovirus (subgroup 1) 1) Grapevine leafroll-associated virus – 2 (GLRaV- Closteroviridae, Closterovirus 2) Grapevine leafroll-associated virus – 3 (GLRaV- Closteroviridae, Ampelovirus (subgroup 1) 3) Grapevine leafroll -associated virus - 4a Closteroviridae, Ampelovirus (subgroup 2) (GLRaV-4) Grapevine leafroll -associated virus – 5 Closteroviridae, Ampelovirus (subgroup 2) (GLRaV-5) Grapevine leafroll -associated virus – 6 Closteroviridae, Ampelovirus (subgroup 2) (GLRaV-6) Grapevine leafroll -associated virus – 9 Closteroviridae, Ampelovirus (subgroup 2) (GLRaV-9) Grapevine leafroll -associated virus – 10 Closteroviridae, Ampelovirus (subgroup 2) (GLRaV-10) Grapevine leafroll -associated virus – 11 Closteroviridae, Ampelovirus (subgroup 2) (GLRaV-11) Grapevine virus A (GVA) Betaflexiviridae, Vitivirus Grapevine virus B (GVB) Betaflexiviridae, Vitivirus Grapevine virus D (GVD) Betaflexiviridae, Vitivirus Grapevine virus E (GVE) Betaflexiviridae, Vitivirus Grapevine rupestris stem pitting -associated virus Betaflexiviridae, Foveavirus (GRSPaV) aHighlighted are Ampeloviruses of subgroup 2 which, according to recent molecular analysis, should be regarded as divergent genetic variants of the first discovered Ampelovirus, GLRaV-4 (Martelli et al. J.Pl.Path. 94, 7-19,2012). All members of this group are RT nested-PCR amplified using degenerate primers designed by Maliogka et al.(J. Virol. Meth. 154, 41-47, 2008). The technique was also used in this study (see Fig. 1). This document is confidential and any unauthorised disclosure is prohibited. Final report 4 Eight multiple virus-infected grapevine sources from PPRI collection which are shown in Table 2 were used to investigate the reliability of the technique. Table 2. Cultivar (code) Virus status a Viruses detected in this study d Waltham Cross (WX) GLRaV-1,-2,-3,-5, GVA, GLRaV-1,-2,-3,-10, GVA GRSPaV b GRSPaV, Pinot Noir (PN) GLRaV-1,-2,-3, GFkV NA Chasselas (CH) GLRaV-2,-6 GLRaV-2,-3,-5, GVB, GRSPaV Barlinka (BAR) GLRaV-4, GFkV GLRaV-3, -4, GVA, GRSPaV Ohanez (OH) GLRaV-5, GFkV GLRaV-3, -6 (-11), GVA, GVB Black Spanish (BS) GLRaV-1,-2,-3,-4,-5, GVA, GLRaV-1,-2,-3,-4, GVA, GFkV unknown Flexivirus Shiraz (SH) GLRaV-2 GLRaV-3,-4 LN33 (LN) c GLRaV-2, GVB GLRaV-2,-3 a Determined using IEM (GGF Kasdorf, PPRI), except grapevine cv. Waltham Cross and LN33. bSee Proser, et al. (Vir. Res. 124, 151-159, 2007). cThe grapevine is strongly CB-affected. A severe variant of GLRaV-2 was mechanically transmitted to N. benthamiana (Goszczynski et al. J.Phytopath 144, 581-583, 1996). The plant was also GVB positive in RT-PCR (Goszczynski, not published). dGrapevines LN33 and Shiraz were not analysed for Flexiviruses In the study, cane cuttings of four grapevines from Vititec’s nucleus collection (kindly supplied by Tobie Oosthuizen) were also used. The grapevines are shown in Fig. 1B as numbers 1, 2, 3 and 4, respectively. RT nested-PCR technique for the simultaneous amplification of viruses of the genera Foveavirus, Vitivirus and Closterovirus of the Betaflexiviridae and Closterovirdae families reported by Dovas and Katis (2003) was modified by carrying out reverse transcription (RT), and two rounds of PCR separately. The amplification of DNA from members of these two families was also carried out separately. In all these amplifications the degenerate primers designed by Dovas and Katis (2003) were used. This document is confidential and any unauthorised disclosure is
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