WINETECH Technical Yearbook 2019

and other vitiviruses, have a special “affinity” for grapevine meristematic cells? All these questions can be answered only if we have established an easy model to study vitivirus- grapevine host interactions. The ideal is the model comprising a LN33 hybrid grapevine (Courderc 1613 x Thompson Seedless) and GVB-associated with corky bark disease (CBD). Although the RWD-associated viruses do not induce easily visible external symptoms in most grapevine cultivars, CBD is an exception. LN33 reacts to CBD infection with severe swelling and longitudinal cracking of canes between internodes (Photo 1). The symptoms are the result of the rapid and excessive proliferation of the secondary phloem cells (Beukman & Gifford, 1969). After CBD infection of LN33 grapevine the symptoms of the disease appear consistently every year in new growing canes. This secures a constant supply of genetically identical CBD-affected grapevine tissues for study. Also, it was found that young, in vitro- propagated LN33 plants are very susceptible to CBD. Transmission of CBD by micro grafting induced clearly visible swelling of stems of LN33 plants in 8-12 weeks (Tanne et al ., 1993). The result was confirmed by other laboratories (Pathirana & McKenzie, 2007). The second component of the GVB/LN33 model are cDNA clones of GVB variants. The construction of biologically active and stable cDNA clones of GVB is crucial. Although

GVB was isolated from grapevines by mechanical transmission of this vitivirus to its alternative herbaceous hosts, Nicotiana sp. (Boscia et al ., 1997), transmission of this virus back to grapevines is impossible with current knowledge. Our laboratory has made clear progress towards creating the model to investigate pathogenicity of GVB to LN33 grapevines. The virus is extensively genetically heterogenic (Shi et al ., 2004; Goszczynski, 2010A & 2018; Voncina et al ., 2011; Hu et al ., 2014A & B). Currently seven complete genome sequences of genetic variants of GVB, sharing 74.9- 85.2% nucleotide identity, are available in the GenBank/EMBL database. Four of these variants were identified in our laboratory (Goszczynski, 2010A & 2018; Goszczynski et al ., 1996). Also, we reported that the genetic variants of GVB differ in pathogenicity to LN33 grapevine. LN33 plants in our grapevine collection are infected with variant GVB-H1 and, over the years, consistently do not express CBD symptoms (Goszczynski, 2010A). The genome sequence of this variant, that is not pathogenic to LN33, is clearly divergent from other GVB variants. Finally, we constructed biologically viable cDNA clones of GVB (Goszczynski, 2015). The clones are crucial for the pathogenicity study of GVB variants, because constructing of clones is the only way to obtain a pure culture of genetic variants of this grapevine virus. In South Africa, CBD-affected LN33 grapevines are usually infected with a

mixture of various divergent genetic variants of GVB and other viruses, like GRSPaV and GLRaV-3 (Goszczynski, 2010A, B & 2018). Separating GVB from mixed virus infections is practically impossible. The GVB/LN33 model has enormous scientific potential. The severe and relatively quick reaction of LN33 to GVB infection, the easy manipulations in the genome of GVB using cDNA clones of this virus, and the comparative analysis of expression of the virus and molecular changes in grapevine cells infected with GVB genetic variants differing in pathogenicity to grapevines, may reveal which part of the GVB genome confers CBD-inducing pathogenicity determinants of the virus, and how the virus de-regulates cambium cell differentiation. This could lead to identification of grapevine genes targeted by GVB in CBD disease development and modification of these genes to make grapevines not susceptible to GVB, and possibly also to other vitiviruses. CONCLUSIONS The discovery of new viruses by researchers using the latest scientific diagnostic methods are inevitable. Although the grapevine industry cannot ignore these new findings, the importance of these emerging viruses must be confirmed by Koch’s postulates, or their etiological role must be confirmed by association with certain diseases. Both results will help to improve the phytosanitary status of our material.

ACKNOWLEDGEMENT The author thanks Winetech, South Africa, for the financial support of this study. REFERENCES Anonym, 2018A. State of the vitiviniculture world market. Available from: www.oiv. int/public/medias/5958/oiv-state-of-the- vitiviniculture-world-market-april-2018.pdf . Anonym, 2019. Phylloxera. Wikipedia. Available from: https://en.wikipedia.org/ wiki/Phylloxera . Melnyk, C.W., 2017. Plant grafting: insights into tissue regeneration. Regeneration 4, 3-14. Hunter, K., Volschenk, N., 2015. Factors impacting on graft union abnormality. WineLand , June 2015. Bonfiglioli, R.G., Habili, N., Green, M., Schleifert, L.F., Symons, R.H., 1998. The hidden problem – Rugose wood associated viruses in Australian viticulture. The Australian Grapegrower and Winemaker. December 1998, pp. 9-13. Goussard, P., 2013. A guide to grapevine abnormalities in South Africa: virus and virus-like diseases – stem grooving and corky bark (rugose wood complex) (Part 4.3). WineLand , February 2013. Anonym, 2014. Rugose wood complex. Journal of Plant Pathology 96 (1S), 73-88. Anonym, 2018B. ICTV Virus Taxonomy. Available from: https://talk.ictvonline.org/ taxonomy/ .