Serological Detection of Grapevine Associated Closteroviruses in Infected Grapevine Cultivars

Grapevine leafroll (LR) and corky bark (CB) diseases occur wherever grapevines are grown and are associated with undesirable viticultural effects which include delayed ripening of fruit, reduced yield, altered fruit pigmentation, and reduced accumulation of sugar (6,24). Rootstockscion incompatibility (RSI) is a disease syndrome characterized by incompatibility at the graft union resulting in slow death of the scion (5). The etiology of LR disease is not clear. Different kinds of virus particles have been associated with symptomatic variants of LR disease (24), but only closterovirus-like particles have been consistently associated with this disease (14). Viruses found in LR infections are designated grapevine leafroll associated viruses (GLRaVs). Other closterovirus-like particles designated as grapevine corky bark associated viruses (GCBaVs) were found in CB infections (20). Many cultivars often carry latent infections of grapevine viruses and are asymptomatic until they are grafted onto a susceptible rootstock (5). Several laboratories have purified closterovirus-like particles from diseased vines and prepared monoclonal antibodies (MAb) and polyclonal antisera (PA) (8,10,11,20,26). Using sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the molecular weights of GLRaV-1, -3, -4, and GCBaV coat proteins have been reported to be approximately 38, 43, 36, and 26 kDa, respectively (8,10,11,20). Two different molecular weights (circa 26 and 36 kDa, respectively) have been reported for the coat proteins of the United States and French GLRaV-2 isolates (2,26), suggesting that these are different viruses. In addition, Gugerli and Ramel (8) reported that a MAb designated MCA 29-1 (prepared with GLRaV-2b) reacted to a polypeptide of approximately 26 kDa in a Swiss virus isolate. With the exception of GLRaV-2, GLRaV-2b, and GCBaV, the reported mass of the capsid proteins of grapevine associated closterovirus-like particles are greater than expected for closterovirus capsid proteins (circa 22 to 26 kDa). This information suggests that these viruses could represent a new virus group. Presently, LR is diagnosed by biological indexing and enzyme-linked immunosorbent assay (ELISA), while CB is diagnosed by biological indexing only. Symptom evaluation of an indicator host is subjective, non-specific, and time consuming. Discrepancies have been found between ELISA and woody indicator tests (18,22). GLRaV-1 and GLRaV-3 were detected by ELISA and Western blotting in previously registered foundation material that had been determined to be virus free by biological indexing (17,18). ELISA has also been unreliable, since the poor quality of the PA available would yield a positive reaction in response to non-specific reactivity or infection with one or more viruses. Furthermore, the distribution of GLRaVs is variable in vegetatively grown and dormant grapevines (18), making the choice of sampling strategies critical to successful detection of grapevine associated closteroviruses. In this study we show that a Western blot (WB) assay using various antibodies permits reliable and accurate detection of viral polypeptides, and hence diagnosis of viruses associated with RSI, leafroll, and corky bark diseases. Furthermore, the WB assay has allowed the characterization of PA reactive to several virus isolates.