Molecular detection of Grapevine fleck virus-like viruses

Molecular reagents have been developed for virus-specific and simultaneous (virus-non-specific) detection of Grapevine fleck virus (GFkV) and allied viruses, ie. Grapevine asteroid mosaic-associated virus (GAMaV) and Grapevine red globe virus (GRGV). Degenerate primers designed on nucleotide sequences of the RNA-dependent RNA polymerase (RD) and methyltransferase (MTR) domains of the GFkV genome, were able to give amplification products of the expected size from total nucleic acid extracts of:vines infected with GFkV, GAMaV, and GRGV;a Californian grapevine accession infected by a marafi-like virus;Greek grapevine accessions infected by an unidentified agent that induced symptoms reminiscent of those elicited by GAMaV in Vitis rupestris.Degenerate primers designed on the nucleotide sequence of the helicase (HEL) domain of the GFLV genome recognized all the above viruses except for GAMaV and the unidentified Greek viral agent. RD primer set worked well also with crude grapevine cortical scrapings, thus constituting a useful universal reagent for the non-specific molecular identification of GFkV-like viruses in Vitis . The marafi-like virus from California was amplified by all sets of primers, but was recognized only by the GRGV-specific probe, suggesting that it is a likely isolate of GRGV: Likewise, the unidentified virus from Greek vines shared sequence homology with GFkV and allied viruses (GAMaV and GRGV) but exhibited differences relevant enough that call for further investigations to establish its taxonomic position. While GRGV was identified, though with a very low incidence, in some 11 southern Italian grapevine cultivars, no evidence was obtained for infection by GAMaV in any of 50 cultivars analyzed.

Production of monoclonal antibodies to Grapevine virus D and contribution to the study of its aetiological role in grapevine diseases

Six stable hybridoma cell lines secreting monoclonal antibodies (MAbs) to Grapevine virus D (GVD) were obtained by fusing spleen cells of immunized BALB/c mice with mouse myeloma cell line Sp 2/0-Ag 14, In ELISA all MAbs detected the virus in Nicotiana leaf extracts or cortical shavings from mature grapevine canes, The use of a polyclonal antiserum for coating plates and of monoclonal antibodies and antimouse-conjugated antibodies for antigen detection, gave highly efficient and reproducible results for identification of GVD in field-grown grapevines. The reliability of the ELISA kit was confirmed by GVD-transmission tests to herbaceous hosts, using in vitro explants as inoculum, 223 vines affected by one or more of the 4 syndroms of the rugose wood complex (Kober stem grooving, Corky bark, LN stem grooving and Rupestris stem pitting) were tested in ELISA for the detection of Grapevine virus A (GVA), Grapevine virus B (GVB) and GVD and by Western blot for the detection of Grapevine rupestris stem pitting associated virus (GRSPaV). The possible cause-effect relationship between GVA and KSG, GVB and Co, and GRSPaV and RSP was confirmed, but no consistent association was found between GVD and any of the 4 above syndromes, Intriguingly, a reduction in the expression of stem pitting symptoms in V. rupestris (from 90 % to 75 %) and of stem grooving symptoms in Kober 5BB (from 95 % to 70 %) was observed when vitiviruses and GRSPaV were contemporarily present in the same indicator. Preliminary data of a survey involving 676 grapevine samples showed a high incidence (31 %) of GVD, regardless of the geographical origin of samples.