Early detection of grapevine leafroll virus in <Emphasis Type="Italic">Vitis vinifera</Emphasis> using <Emphasis Type="Italic">in vitro</Emphasis> micrografting

Micrografting of grapevine was investigated for its use as a tool in virus indexing of grapevine stock. Cabernet franc and Cabernet sauvignon scions infected with grapevine leafroll-associated closterovirus III (GLRaVIII) were grafted on to virus-free indicator rootstocks of LN 33 and Cabernet sauvignon growing in tissue culture. The two rootstocks and two scions were grafted in all four possible combinations along with two control grafts (virus-free scion on virus-free rootstock). A modified MS Murashige and Skoog (1962) tissue culture medium supplemented with 0.5 mg l^–1 6-benzylaminopurine was sufficient to induce multiple shoots. Shoots and micrografts readily produced roots in the basal medium. Micrografting gave an overall success rate of 77.8%, with no significant difference between LN 33 rootstock and Cabernet sauvignon. When leafroll infected scion material was micrografted on to virus-free rootstock, the rootstock leaf turned red (23.5% in LN 33 and 63.9% in Cabernet sauvignon) or it showed leafrolling (28.5%, no significant difference between LN 33 and Cabernet sauvignon) within 2–3 weeks. After 12 weeks in culture, the extent of viral symptoms in the micrografted material was high (81.3%), with no significant difference between LN 33 and Cabernet sauvignon; however, the expression of symptoms was more severe on Cabernet sauvignon than on LN 33 rootstock. Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) was used to validate the visual symptoms and the presence of virus was confirmed in 80% of the rootstock with visual symptoms of infection. Results indicate that micrografting is an effective method for viral indexing of grapevines. The method can be used in conjunction with wood indexing for post-entry quarantine to identify infected material and reject it much earlier than is currently possible.