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 v iruses, ie. Grapevine asteroid mosaic-associated virus (GAMaV) and Grapevin e red globe virus (GRGV). Degenerate primers designed on nucleotide sequenc es of the RNA-dependent RNA polymerase (RD) and methyltransferase (MTR) dom ains of the GFkV genome, were able to give amplification products of the ex pected size from total nucleic acid extracts of: (i) vines infected with GF kV, GAMaV, and GRGV; (ii) a Californian grapevine accession infected by a m arafi-like virus; (iii) Greek grapevine accessions infected by an unidentif ied agent that induced symptoms reminiscent of those elicited by GAMaV in V itis rupestris, Degenerate primers designed on the nucleotide sequence of t he helicase (HEL) domain of the GFLV genome recognized all the above viruse s except for GAMaV and the unidentified Greek viral agent. RD primer set wo rked 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 ampli fied by all sets of primers, but was recognized only by the GRGV-specific p robe, suggesting that it is a likely isolate of GRGV: Likewise, the unident ified virus from Greek vines shared sequence homology with GFkV and allied viruses (GAMaV and GRGV) but exhibited differences relevant enough that cal l for further investigations to establish its taxonomic position. While GRG V was identified, though with a very low incidence, in some 11 southern Ita lian grapevine cultivars, no evidence was obtained for infection by GAMaV i n any of 50 cultivars analyzed.