WINETECH Technical Yearbook 2019

previously unclassified variant group of GRSPaV, IId, has been detected and remains to be further investigated. A noticeable difference in the distribution of virus variants was observed between previously certified and uncertified plants. The presence of mildly symptomatic or asymptomatic variants of GRSPaV in plants that tested negative by hardwood indexing indicates the need for development of improved detection and disease control methods, as non-symptom- causing variants may still play a role in virus pathogenesis (Alabi, et al ., 2010). Sequence data generated in this study can assist in the advancement of such methods. Although the results imply that the replicase area of the genome might be superior to the coat protein at discerning between virus variants, the ability of recombination to influence variant classification stresses the need to take these factors into account during assay design. SUMMARY Grapevine rupestris stem pitting-associated virus (GRSPaV) is linked to several grapevine d i seases . The ex i stence of mu l t i p l e strains of GRSPaV coupled with variable symptom expression and the occurrence of recombination has led to difficulties in detecting and classifying GRSPaV isolates.

Two surveys were conducted to investigate the genetic diversity of GRSPaV in South Africa, to compare the effectiveness of two genomic domains to classify virus variants and to investigate the impact of recombination on variant classification. GRSPaV variants identified in the surveys clustered into five of the six currently recognised lineages, and a seventh, previously unclassified lineage was detected. A correlation was observed between the detection of recombinant GRSPaV sequences and inconsistencies in classification when using different genome regions for analysis. Results from this study indicate that visual diagnosis on an indicator host is not always sensitive enough to detect mildly symptomatic or asymptomatic variants of GRSPaV, stressing the need for development of more sensitive detection assays to fulfil sanitary requirements. ACKNOWLEDGEMENTS The authors would like to acknowledge Winetech for project funding (Grant number: GenUS 15/2). We also want to thank the owners of the wine estates for permission to sample vineyards and for providing information on the ages and cultivars of sampled vines, the National Research Foundation of South Africa for personal funding, and Dr. Rachelle Bester and Dirk

Aldrich for their assistance in performing some of the lab work for this project. REFERENCES Meng, B. & Gonsalves, D., 2007. Grapevine rupestris stem pitting-associated virus: A decade of research and future perspectives. Plant Viruses 1: 52-62. Glasa, M., Predajňa, L. & Šoltys, K. et al. , 2017. Analysis of Grapevine rupestris stem pitting-associated virus in Slovakia reveals differences in intra-host population diversity and naturally occurring recombination events. Plant Pathology Journal 33(1): 34-42. doi:10.5423/PPJ.OA.07.2016.0158. Habili, N., Farrokhi, N., Lima, M.F., Nicholas, P. & Randles, J.W., 2006. Distribution of Rupestris stem pitting-associated virus variants in two Australian vineyards showing different symptoms. Annals of Applied Biology 148(1): 91-96. doi:10.1111/j.1744- 7348.2006.00041.x. Lima, M.F., Alkowni, R., Uyemoto, J.K., Golino, D., Osman, F. & Rowhani, A., 2006. Molecular analysis of a California strain of Rupestris stem pitting-associated virus isolated from declining Syrah grapevines. Archives of Virology 151(9): 1889-1894. doi:10.1007/s00705-006-0742-y. Meng, B., Li, C., Wang, W., Goszczynski, D.

& Gonsalves, D., 2005. Complete genome sequences of two new variants of Grapevine rupestris stem pitting-associated virus and comparative analyses. Journal of General Virology 86(Pt 5): 1555-1560. doi:10.1099/ vir.0.80815-0. Nakaune, R., Inoue, K. & Nasu, H. et al. , 2008. Detection of viruses associated with rugose wood in Japanese grapevines and analysis of genomic variability of Rupestris stem pitting- associated virus. Journal of General Plant Pathology. http://agris.fao.org/agris-search/ search.do?recordID=US201300878498 . Accessed July 5, 2017. Simon-Loriere, E. & Holmes, E.C., 2011. Why do RNA viruses recombine? Nature Reviews Microbiology 9(8): 617-626. doi:10.1038/ nrmicro2614. Martin, D.P., Murrell, B., Golden, M., Khoosal, A. & Muhire, B., 2015. RDP4: Detection and analysis of recombination patterns in virus genomes. Virus Evolution 1(1). doi:10.1093/ ve/vev003. Alabi, O.J., Martin, R.R. & Naidu, R.A., 2010. Sequence diversity, population genetics and potential recombination events in Grapevine rupestris stem pitting-associated virus in Pacific North-West vineyards. Journal of General Virology 91(Pt 1): 265-276. doi:10.1099/vir.0.014423-0.

– For more information, contact Johan Burger at jtb@sun.ac.za or Hano Maree at hjmaree@sun.ac.za.