Detection of Grapevine virus B associated with rugose wood (corky bark) symptoms in grapevine cv. Napoleon in Murcia (Spain)

During a survey in early spring 2004, of table grapes cv. Napoleon in Murcia (Spain), symptoms of rugose wood and corky bark were observed in several plants. The causal agent of the rugose wood disease complex, which includes corky bark, has been attributed to members of the Vitivirus genus, such as Grapevine virus A (GVA) and Grapevine virus B (GVB) (Martelli et al., 2000). This last virus has been recognised as the primary cause of corky bark in grapevine (Nickel et al., 2002). A total of 18 plants from 9 different fields showing corky bark symptoms were analysed by RT-PCR with specific primers for GVA (H7038; C7273) and GVB (H6980; C7439) (MacKenzie et al., 1997). All samples tested positive for GVB, producing a single band of the expected size of 460 bp, but none were positive for GVA. Petiole extracts of two symptomatic positive plants, in phosphate buffer (pH 7.5) containing 2.5% nicotinic acid, were used as source for the mechanical inoculation of Nicotiana benthamiana and N. clevelandii plants (isolates M5 and M9). Only inoculated N. clevelandii plants showed symptoms: systemic stem necrotic spots and leaf malformation. These plants tested positive for GVB when tested by RT-PCR. The products were cloned in pGEMT-Easy vectors (Promega) and sequenced (EMBL Acc. No. AJ748850 and AJ748851 respectively). The nucleotide identity of both isolates (M5 and M9) was 81% in the 416 bp region corresponding to the partial ORF encoding the RNA binding protein, with respect to a published Grapevine virus B sequence (Acc. No. X75448; Saldarelli et al., 1996). The deduced amino acid sequences of isolates M5 and M9 were compared to X75448, the analysis revealed that they shared identities of 85 and 84% respectively and a similarity of 93% in both cases. These and other isolates are now being further characterised, by sequencing other genome regions and determining the host range with additional test plant species. Within Europe, GVB has previously been reported from Italy, France, Greece and Portugal, but to our knowledge this is the first report of GVB in Spain.