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

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— 48 — REAL TIME PCR FOR SENSITIVE RELIABLE GRAPEVINE VIRUS DETECTION Stefanie MALAN 1 , Michael-John FREEBOROUGH 1 , Johan BURGER 1 * 1 Department of Genetics, Stellenbosch University, Private Bag X1 MATIELAND, South Africa *Corresp. author : Johan Burger, +27218085858, jtb@sun.ac.za Summary Grapevine leafroll associated virus-3 (GLRaV-3) is an economicaly important virus in South Africa and throughout the grapevine growing world. Detection of the virus in rootstocks has proven to be problematic due to low virus titres. Sensitive, reproducible results were obtained with the use of SYBR Green reverse transcription real time PCR (qRT-PCR). When compared to ELISA reults, qRT-PCR proved to be more sensitive, resulting in reproducible detection at a higher frequency. increased from 20.6% to 29.4%. Reproducible results were obtained during the screening process. INTRODUCTION GLRaV-3 is one of the most important viruses threatening the sustainability of the South African grapevine industry. No known cure exists for viral diseases and one of the most effective ways to deal with viruses and associated diseases is to plant certified virus free material and to rogue infected material to reduce viral load and the spread of the virus. Scion material is routinely and successfully tested for various grapevine viruses. However, virus detection in rootstocks is problematic due to low and varying virus titres. Certified virus free scion material could thus be grafted on rootstock material of unknown viral status and lead to further spread of the disease. Recent developments in molecular diagnostic methods significantly improved detection of plant pathogens. qRT-PCR provides a highly sensitive, reliable and reproducible technique for virus detection in grapevine. MATERIAL AND METHODS Petioles from rootstocks were collected every two weeks throughout the growing season. Crude virus extractions were performed as described by Osman et al. 2007. After denaturation the samples were amplified with the Quantace OneStep qRT-PCR kit with primers specifically designed for all known variants of the GLRaV- 3 coat protein region. Amplification was performed in the RotorGene 6000 according to optimised conditions. Subsequent melting curve analysis was performed on amplified product to identify infected plants. The same crude virus extract was used for DAS-ELISA testing to compare efficiencies of the two different techniques. RESULTS AND DISCUSSION qRT-PCR detection of GLRaV-3. Amplified PCR product (Figure 1) were subjected to melting curve analysis. Samples infected with GLRaV-3 showed melting temperatures of 83°C – 83.8°C (Figure 2). Throughout the growing season (Jan - April) the percentage rootstock samples that tested positive for GLRaV-3 with qRT-PCR, Figure 1. Amplification plot of samples amplified via qRT-PCR Figure 2. Melting curves of amplified samples to identify infected samples Table 1. GLRaV-3 infected rootstock samples as detected with qRT-PCR and ELISA Sample Detection Method qRT-PCR ELISA 5.2 + + 5.3 + + 8.2 + 8.3 + + 9.2 + 11.2 + 15.2 + 16.2 + + 16.3 + 17.2 + + 17.4 + + GLRaV-3 detection via ELISA. The same rootstock samples were also subjected to ELISA. At optimal sampling time ELISA was able to detect 23.5% GLRaV-3 infected samples. However these results also include a sample that produced a false positive result. The actual accurate percentage detected was thus 20.5%. Discussion. The results of the rootstock screening shows that qRT-PCR delivered sensitive, reliable and reproducible detection of GLRaV-3. It also proved to be 8.9% more sensitive than the currently used method,
— 49 — ELISA. Rootstock samples are used as propagation material and sensitive reliable virus detection is of utmost importance for ensuring virus free phytosanitary status of grapevines. LITERATURE BEUVE, M., SEMPÉ, L. & LEMAIRE, O. 2007., A sensitive one-step real-time RT-PCR method for detecting Grapevine leafrollassociated virus 2 variants in grapevine. Journal of Virological Methods 141, 117–124. CABALEIRO, C. & SEGURA., A. 1997. Some characteristics of the transmission of grapevine leafroll associated virus 3 by Planococcus citri Risso. European Journal of Plant Pathology 103, 373–378. DOUGLAS N. & KRÜGER, K. 2008. Transmission efficiency of Grapevine leafroll-associated virus 3 (GLRaV-3) by the mealybugs Planococcus ficus and Pseudococcus longispinus (Hemiptera: Pseudococcidae). European Journal of Plant Pathology 122, 207- 212. MUROLO, S., ROMANAZZI, G., ROWHANI, A. & MINAFRA, A. 2008. Genetic variability and population structure. European Journal of Plant Pathology 120, 137–145. OSMAN, F., LEUTENEGGER, C., GOLINO, D. & ROWHANI, A. 2007. Real-time RT-PCR (TaqMan®) assays for the detection of Grapevine Leafroll associated viruses 1–5 and 9. Journal of Virological Methods 149, 292-299. MUMFORD, R., BOONHAM, N., TOMLINSON, J. & BARKER, I. 2006. Advances in molecular phytodiagnostics – new solutions for old problems. European Journal of Plant Pathology 116, 1–19. CHARLES, J.G., COHEN, D., WALKER, J.T.S., FORGIE, S.A., BELL, V.A. & BREEN, K.C., 2006. A review of Grapevine Leafroll. Hort Research Client Report 18447. Progrès Agricole et Viticole, 2009, Hors Série – Extended abstracts 16 th Meeting of ICVG, Dijon, France, 31 Aug – 4 Sept 2009
Page 3 and 4: 16th Meeting of the International C
Page 5 and 6: AVANT-PROPOS Nous sommes très heur
Page 7 and 8: Content - Sommaire Page Partners an
Page 9 and 10: Session IV - Epidemiology - Survey
Page 11 and 12: Poster 51 Poster 52 Poster 53 Poste
Page 13 and 14: Identification and distribution of
Page 15 and 16: — 15 — GRAPEVINE VIROLOGY HIGHL
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Page 19 and 20: — 19 — Likewise, an antibody bi
Page 21 and 22: — 21 — LITERATURE ABOU GHANEM-S
Page 23 and 24: — 23 — ORECCHIA, M., NOELKE, G.
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— 115 — Figure 2. Frequency of
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— 125 — Table 2. Sanitary statu
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— 127 — SANITARY STATUS OF AUTO
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— 129 — THE VIRUS STATUS OF GRA
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— 131 — PRESENCE OF GLRAV 1, 2,
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