Development of Methodologies for Rapid Detection of Grapevine Viruses

We have produced enough purified preparations of grapevine leafroll associated virus (GLRaV) type 4, grapevine fleck virus (GFkV) and grapevine virus B (GVB), and produced specific polyclonal antibodies. We are in the process of evaluating these antibodies and determining their usefulness for ELISA assays. In addition, we have produced enough purified preparations of GLRaV-1, grapevine virus A (GVA) and grapevine corky bark associated virus (GCBaV) to initiate immunizing rabbits for the production of specific polyclonal antibodies. We have developed a way to quickly obtain nucleotide sequence information for a specific conserved gene from diverse isolates and types of GLRaVs and demonstrated the usefulness of this sequence information in developing PCR-based diagnostic for these viruses. We have developed reliable immunocapture-polymerase chain reaction (IC-PCR) detection for GLRaV-4 and a sensitive and reliable reverse transcriptase-PCR (RT-PCR) for both GLRaV-4 and -5. We also have demonstrated some potential for developing diagnostics capable of simultaneous detection of GLRaV types. We have characterized a virus associated with rupestris stem pitting disease (RSP) and sequenced its entire genome. PCR primers were designed and used to develop a RT-PCR based detection assay. Using these PCR primers we tested 62 grapevines known to be infected with RSP based on biological assay on St. George indicator. Among these 62 vines, 60 tested positive for the virus by PCR. All 43 RSP negative vines were also tested negative by PCR. The above results show a good correlation between the isolated virus and rupestris stem pitting disease.