First report of Grapevine leafroll-associated virus 4 (GLRaV-4) in Spain

During phytosanitary certification of three grapevine clones (one of Gorgollosa and two of Mances de Tibus) received at IMIDA from the Balearic Islands (Spain), we observed discrepancies between symptoms on Cabernet Franc indicator plants and the results of ELISA testing for viruses associated with grapevine leafroll.According to DAS-ELISA analysis (Bioreba, Nyon, Switzerland), the three clones were free from GLRaV-1 and GLRaV-3 but positive for GLRaV-2. However, in four consecutive seasons on indicator rootstocks, symptom severity led to a suspicion that other leafroll viruses might be present; in our experience, symptoms produced by GLRaV-2 infection alone are rarely as severe as those that were observed. Testing of the clones with real-time RT-PCR using TaqMan® probes (Osman et al., 2007) detected Grapevine leafroll-associated virus 4.Using similar real-time PCR methods we confirmed the presence of GLRaV-2 but we did not detect either GLRaV-1 or GLRaV-3. Moreover we could also detect Grapevine fanleaf virus, GFLV (DAS-ELISA, Bioreba) in the Mances de Tibus vines; this was confirmed by symptom inspection in the indicator host Rupestris de Lot.

In order to confirm the presence of GLRaV-4, RNA extracts were also tested by conventional RT-PCR with consensus primers derived from the GLRaV-4 hsp70 gene: LR4H89 (5'-CATACCAACCGTTGTGGGTA-3') and LR4H327 (5'-TGCAATTACACTCCTTGTTTT-3'). Amplicons were of the expected size (238 bp) and were cloned using pGEMT-Easy (Promega).Comparisons showed that sequences were identical in all three isolates (GenBank Accession No. GU735409) and showed 99% nucleotide identity with the homologous hsp70 genes of GLRaV-4 isolates LS-24 from China (GQ849394) (Pei et al., 2010) and Cl-3642 from Chile (EU746619) (Escobar et al., 2008).

To our knowledge this is the first identification of GRLaV-4 in Spain. Current European regulations state that certified plant material cannot contain GLRaV-1 and GLRaV-3 but do not consider GLRaV-2 and other leafroll viruses (Anonymous, 2008).Our report suggests that other leafroll viruses should be included in the European regulations for grapevine certification. Further analysis of the GLRaV-4 isolates is underway and epidemiological investigations are currently in progress to establish the field incidence of these and other viruses in the provenance area of the grapevines within the Balearic Islands.