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 G rapevine virus D (GVD) were obtained by fusing spleen cells of immunized BA LB/c mice with mouse myeloma cell line Sp 2/0-Ag 14, In ELISA all MAbs dete cted the virus in Nicotiana leaf extracts or cortical shavings from mature grapevine canes, The use of a polyclonal antiserum for coating plates and o f monoclonal antibodies and antimouse-conjugated antibodies for antigen det ection, gave highly efficient and reproducible results for identification o f GVD in field-grown grapevines. The reliability of the ELISA kit was confi rmed 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 ru gose wood complex (Kober stem grooving, Corky bark, LN stem grooving and Ru pestris stem pitting) were tested in ELISA for the detection of Grapevine v irus A (GVA), Grapevine virus B (GVB) and GVD and by Western blot for the d etection of Grapevine rupestris stem pitting associated virus (GRSPaV). The possible cause-effect relationship between GVA and KSG, GVB and Co, and GR SPaV and RSP was confirmed, but no consistent association was found between GVD and any of the 4 above syndromes, Intriguingly, a reduction in the exp ression 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. P reliminary data of a survey involving 676 grapevine samples showed a high i ncidence (31 %) of GVD, regardless of the geographical origin of samples.