No abstract

The sequence of the genome of a Rupestris stem pitting-associated virus (RSPaV) isolated from a declining Syrah grapevine in California, designated the Syrah strain (RSPaV-SY) was determined. The genome of this strain had an overall nucleotide identity of 77% in comparison with RSPaV sequences in GenBank; the coat protein was the most conserved gene among RSPaV sequences and the replicase was the least conserved gene. Phylogenetic analysis of partial coat protein and replicase gene sequences showed RSPaV-SY clustered independently from the majority of RSPaV isolates.

Olive (Olea europaea) is host of 13 different viruses but, it is possible that other viruses, which are either non mechanically transmissible or occur in low concentration in plant tissues, are present in nature. This likelihood is supported by the widespread occurrence of double-stranded RNAs (dsRNAs) in plants negative to biological tests. As very little information is available on the geographical distribution of olive-infecting, investigations were conducted for gathering information on the prevalence and distribution of olive viruses in the Mediterranean basin based on the presence of double stranded RNA (dsRNA). A total of 527 samples were collected throughout the surveys covering 10 countries and 83 locations. Out of 286 tested samples collected from 6 Italian regions, 210 (73.4%) were dsRNA positive, whereas the average of viral infections in the Mediterranean basin was 64.5%. Molecular hybridisation tests, on 25 % of dsRNA-positive samples collected in Apulia, revealed the presence of the three nepoviruses (ArMV, CLRV and SLRSV), OLYaV and OLV-1.

In California, a novel closterovirus was detected in "Redglobe" grapevine, associated with graft incompatibility and given a trivial name "Grapevine rootstock stem lesion associated virus (GRSLaV)." The biological properties of the putative virus were ascertained when asymptomatic yet infected Redglobe scion buds were graft-inoculated onto test plants of Cabernet Sauvignon propagated on 18 different rootstocks. It proved lethal on test plants growing on rootstocks 1616C, 5BB, 5C, 3309C, and 1103 P, whereas latent infections occurred on the remaining scion-rootstock combinations. In contrast, GLRaV-2 type (type strain) produced only typical leafroll symptoms. In a different experiment, GLRaV-2 type was successfully sap-transmitted to N. benthamiana, whereas sap transmission of GRSLaV was unsuccessful. Double-stranded RNA was extracted from infected Redglobe grapevines, cloned, sequenced, and determined a genome length of 16,527 nucleotides. Computer-assisted analysis of open-reading frames (ORFs) revealed a genome organization typical of monopartite viruses in the genus Closterovirus with nine ORFs (range 71-79% identity) with GLRaV-2 type, the closest similar virus species within the family Closteroviridae. Also the 3'-UTR of GRSLaV consisted of 223 nucleotides with an extended oligo(A) tract similar to that of GLRaV-2 type, Beet yellow stunt virus, and Beet yellows virus. Recombinant GRSLaV coat protein was expressed in E. coli, purified, and immunized a rabbit to produce polyclonal antiserum. Serological data matched the molecular data, whereby exposed plant tissue extracts of grapevines infected by both viruses (GRSLaV and GLRaV-2) reacted positively with homologous and heterologous viral antisera but not with healthy grapevine extracts in ELISA and Western blot tests. Based on the comparative sequence data and shared antigens, GRSLaV is now considered a strain of GLRaV-2 and redesignated as Grapevine leafroll associated virus-2 Redglobe (GLRaV-2RG). Primers specific for GLRaV-2RG were developed, which did not amplify GLRaV-2 type strain. When both sets of specific primers were used in assays of different grapevine collections, the incidence of the respective viruses varied considerably, e.g., 1.7 and 13.5%, respectively, for GLRaV-2RG and GLRaV-2 type.

Olive hosts 13 viruses belonging in seven different genera. Additional non‐mechanically transmissible viruses probably infect olive in nature, as suggested by the widespread occurrence of double‐stranded RNAs (dsRNAs) in trees from which no viruses can be recovered by manual inoculation. Because sanitary selection appears to be the only measure for restraining virus dissemination through propagating material, detection methods are needed which are more sensitive and reliable than those currently available (biological and serological). The following molecular techniques have therefore been used and their efficiency compared: (1) dsRNA analysis; (2) dot‐blot hybridization with digoxigenin‐labelled riboprobes in separate reactions or in mixture; and (3) reverse transcription‐polymerase chain reaction (RT‐PCR). It was found that: (1) dsRNAs were detected in 210 out of 286 olive accessions (73.4%) coming from six different Italian regions; (2) one‐step RT‐PCR yielded much better results using TNA extracts than crude sap; and (3) dot‐blot hybridization of denatured dsRNAs with digoxigenin‐labelled virus‐specific riboprobes was the most reliable detection method available.