Grapevine fleck virus-like viruses in Vitis

Two sets of degenerate primers for the specific amplification of 572-575 nt and 386 nt segments of the methyltransferase and RNA- dependent RNA polyme rase cistrons of members of the genera Tymovirus and Marafivirus and of the unassigned virus Grapevine fleck virus (GFkV) were designed on the basis o f available sequences. These primers were used for amplifying and subsequen t cloning and sequencing part of the open reading frame 1 of the genome of GFkV, Grapevine asteroid mosaic-associated virus (GAMaV) and of another pre viously unreported virus, for which the name Grapevine red globe virus (GRG V) is proposed. Computer-assisted analysis of the amplified genome portions showed that the three grapevine viruses are phylogenetically related with one another and with sequenced tymoviruses and marafiviruses. The relations hips with tymoviruses was confirmed by the type of ultrastructural modifica tions induced in the host cells. RdRp-specific degenerate primers were succ essfully used for the aspecific detection of the three viruses in crude gra pevine sap extracts. Specific virus identification was obtained with RT-PCR using antisense virus-specific primers.