The effects of viruses on grape production and must quality are not fully understood. In this study, the evaluation of the impact of different virus infections on performance of the main autochthonous grapevine varieties of Mallorca (Callet, Manto Negro and Moll) was pursued. Therefore, a large number of vines were observed in field conditions over 4 years and tested by enzyme‐linked immunosorbent assay for viruses listed by the international certification programmes. In each variety, some specific virus infections resulted to be more effective than the others in inducing losses in production. In Callet, yield (Y) reduction was over 20% in plants infected by Grapevine fanleaf virus (GFLV). In Moll, plants subject to more than one infection showed over 40% Y decrease. In Manto Negro, the most surprising results were obtained, because plants showed almost 40% Y reduction because of Grapevine leafroll‐associated virus‐1 (GLRaV‐1) infection. In addition, virus infections were linked to some must quality parameter increase in Manto Negro and Moll, but in the majority of cases it was an indirect effect, because the decrease in production parameters played a predominant role by producing an important concentration effect. However, in Manto Negro, anthocyanin content decrease was directly related to GFLV infection.
Arabis mosaic virus (ArMV; genus Nepovirus, family Comoviridae) is one of several nepoviruses responsible for infectious degeneration disease of grapevines in Europe (3). The first occurrence in Spain, in the summer of 2007, was found in Val de Salnés, Rias Baixas appellation, Galice on 25-year-old vines of the Albariño variety grafted onto an unidentified rootstock and showing leaf yellowing. The second finding was in the spring of 2008 in Barriobusto, Rioja appellation, Basque Country on 30-year-old vines of Tempranillo variety grafted onto 41B rootstock. In this case, no obvious foliar symptoms were observed but fruit set was very poor. Positive ELISA results were obtained at two different laboratories using antibodies to ArMV obtained from two companies (BIOREBA, Reinach, Switzerland and Sediag, Longvic, France). At a third lab, the presence of ArMV was further confirmed by reverse transcription (RT)-nested PCR using the primers described by Bertolini et al. (1). External primers ArMV 1 and ArMV 2 amplified a fragment of 340 bp from the coat protein region of the virus and internal primers ArMV i1 and ArMV i2 amplified a fragment of 203 bp. The specificity of the amplicons was subsequently confirmed by sequencing and comparison with other ArMV isolates available in the GenBank, EMBL, and DDBJ databases. Alignment performed using Blastn showed 85% nucleotide sequence identity with ArMV isolate NW (Accession No. AY017339). ELISA revealed co-infection with GLRaV-1 in Galice, GLRaV-3 in Rioja, and GFkV at both sites; these other viruses being common in their respective appellations. ArMV could be mechanically transmitted from rooted cuttings onto Chenopodium amaranticolor with an average of a 46% success rate (1:10 tissue/buffer ratio; [2]), but the range was very wide (0 to 100%) and dependent on the individual source vine. No statistical differences were found between nicotine or phosphate buffer for extraction or when using shoot tips or root tips as a source of virus (Fisher's exact test). Infection in C. amaranticolor was symptomless, but detectable by ELISA, and systemic. The Galician grapevine was an isolated plant, replanted on the spot of a dead one. Xiphinema diversicaudatum, the nematode vector of ArMV, was found in the vineyard soil. Only two ArMV-positive vines were found among 1,993 plants analyzed in Galice from 2005 to 2007 (no field data available for the second finding). In Rioja, one positive vine was found in a random sample of 74 vines from two different vineyards. Further testing of the neighboring vines indicated that one of the adjacent plants was also infected. This minimal spread since the vineyard was planted is suggestive of a lack of vectored transmission. In Spain as a whole, the virus seems to be rare and associated with the Atlantic biogeographic region. Both vineyards were planted before certified material became widely available. Currently, statutory testing of grapevine propagation material should prevent further spread. References: (1) E. Bertolini et al. Phytopathology 93:286, 2003. (2) G. P. Martelli, ed. Graft-Transmissible Diseases of Grapevines. Handbook for Detection and Diagnosis, FAO, Rome, 1993. (3) G. P. Martelli and E. Boudon-Padieu. Directory of Infectious Diseases of Grapevines and Viruses and Virus-like Diseases of the Grapevine. Bibliographic Report 1998-2004, CIHEAM, Paris, 2006.
Grapevine leafroll-associated viruses (GLRaVs) cause significant reductions in yield and quality in the wine industry worldwide. At least nine different GLRaVs have been found in different regions of the world. In the process of virus indexing of candidate grapevine clones for certification, which includes grafting of scions onto rootstocks, we observed strong leafroll symptoms 1 year after grafting with one vine of cv. Estaladina in Castilla y León, Spain and one vine of cv. Tempranillo in La Rioja, Spain, collected in 2008 and 2007, respectively. Both vines tested positive by real-time reverse transcription (RT)-PCR with TaqMan probes specific for Grapevine leafroll-associated virus 5 and double-antibody sandwich (DAS)-ELISA with a mix of monoclonal antibodies that recognizes GLRaV-4, 5, 6, 7, and 9 (Bioreba, Reinach, Switzerland). RNA extracts of both GLRaV-5 positive vines were analyzed by conventional RT-PCR with a pair of consensus degenerated primers derived from GLRaV-5 hsp70 sequences available in GenBank: LR5HYF (5′-TGGGATGAAYAARTTCAATGC-3′) and LR5HYR (5′-TGAAATTCCTCATRTARGAGC-3′) that amplified a 250-bp fragment. Amplicons were cloned and the comparison of the amino acid sequences (Estaladina isolate, Est110: Accession No. HM208622; Tempranillo isolate, Tem020: Accession No. HM208618) showed in the case of the Est110 isolate, 100 and 82.6% identity, respectively, with the homologous genes of one GLRaV-5 isolate from the United States (AF233934 [3]) and Argentina (EU815935 [2]). For isolate Tem020, the hsp70 gene showed 97.1 and 81.2% amino acid identity with the homologous hsp70 genes of the United States and Argentina isolates. The coat protein (cp) genes of both isolates were also amplified and cloned using the specific GLRaV-5 primers, LR53413 (5′-CGTGATACAAGGTAGGACAACCGT-3′) and LR53843 (5′-CTTGCACTATCGCTGCCGTGAAT-3′), designed according to the sequence of AF233934. Fragments were of the expected size (430 bp) and the nucleotide sequences were obtained (Est110: Accession No. HM363522; Tem020: Accession No. HM363523) and used for pairwise nucleotide comparisons. The Est110 isolate showed 96.7 and 97.5% amino acid identity with the isolates from the United States and Argentina, respectively, while the Tem020 isolate showed 94.8 and 95.6% identity, respectively. Amino acid identity of Est110 and Tem020 cp genes was 100% when compared with the homologous genes of isolates AF233934 and EU815935. To our knowledge this is the first report of GRLaV-5 in Spain. Since 2008, we have detected eight additional vines positive for this virus in 200 clones analyzed for certification, suggesting that the incidence of GLRaV-5 in Spain could be widespread. This research indicates that virus indexing for GLRaV should be included in certification schemes for grapevine candidate clones (1) in Spain. References: (1) Anonymous. OEPP/EPPO Bull. 38:422, 2008. (2) S. Gomez Talquenca et al. Virus Genes 38:184, 2009. (3) F. Osman et al. J. Virol. Methods 141:22, 2007.
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 recognized as the primary cause of corky bark in grapevine (Nickel et al ., 2002). A total of 18 plants from nine 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 was positive for GVA. Petiole extracts of two positive plants with symptoms, in phosphate buffer (pH 7·5) containing 2·5% nicotinic acid, were used as sources for the mechanical inoculation of Nicotiana benthamiana and N. clevelandii plants (isolates M5 and M9). Only inoculated N. clevelandii plants showed symptoms: systemic necrotic stem 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 accession 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 (accession no. X75448; Saldarelli et al. , 1996). The deduced amino acid sequences of isolates M5 and M9 were compared with X75448, and 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 characterized, 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.
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