First Report on the Occurrence of Grapevine leafroll-associated virus 5 in Chilean Grapevines.

Grapevine leafroll is one of the most widespread and economically damaging viral diseases of grapevines. At least eight distinct Grapevine leafroll-associated viruses (GLRaVs), all members of the Closteroviridae family, have been associated with this disease (4). GLRaV-5 was recently reported in vineyards from Argentina (2). To determine if GLRaV-5 was present in Chilean grapevines, in addition to the previously reported GLRaV-1, -2, -3, -4, -7, and -9 (1), 45 dormant cane samples from 12 different cultivars were collected from different geographic regions of Chile and screened by reverse transcription-PCR. Two of the forty-five samples (cvs. Sauvignon Blanc and Superior) collected from the III (700 km north of Santiago) and VI (150 km south of Santiago) regions of Chile, respectively, were found to be infected with GLRaV-5 using two different pairs of virus-specific primers. The first pair of primers, LR5-1F: 5'-CCCGTGATACAAGGTAGGACA-3' and LR5-1R: 5'-CAGACTTCACCTCCTGTTAC-3' (3), was used to amplify a 690-bp fragment corresponding to a partial region of the coat protein gene. The sequences obtained from the two positive samples (GenBank Accession Nos. HM214148 and HM214149) shared 97 and 94% of nucleotide identities, respectively, with the corresponding fragment of a reference GLRaV-5 isolate (GenBank Accession No. EU815935). Both samples shared 99% of amino acid identity with the same reference isolate. A second pair of primers, LR5upF: 5'-CTCTGCTTTTCTGCTGGCA-3' and LR5doR: 5'-TATCTTTTATCTCCCGATAAACGAG-3' (4) that amplified a 160-bp fragment of the HSP70h gene was also used. The positive Chilean samples (GenBank Accession Nos. HM214150 and HM214151) shared in both cases 98% nucleotide and 98% amino acid identities with the corresponding fragment of a reference GLRaV-5 isolate (Accession No. AF039552). The two GLRaV-5-positive plants were additionally infected with other viruses previously reported in Chile (1). The cv. Sauvignon Blanc sample was also infected with GLRaV-2, Grapevine fleck virus, and Grapevine rupestris stem pitting-associated virus. The cv. Superior sample was also infected with GLRaV-3, GLRaV-4, and Grapevine virus A. References: (1) E. A. Engel et al. J. Virol. Methods 163:445, 2010. (2) S. Gomez et al. Virus Genes 38:184, 2009. (3) X. Good and J. Monis. Phytopathology 91:274, 2001. (4) V. I. Maliogka et al. J. Virol. Methods 154:41, 2008.

First Report of Grapevine Syrah virus-1 in Chilean Grapevines.

At least 58 viruses have been reported to infect grapevines, causing economic damage globally. Our lab has reported previously the presence of more than 10 viral species in Chilean grapevines (2,3). Grapevine Syrah virus-1 (GSyV-1) is a novel marafivirus recently described in California vineyards (1). Grapevine virus Q (GVQ) was described shortly after GSyV-1 and both genomes share more than 99% nucleotide identity (4). Since GSyV-1 and GVQ correspond to the same viral species, the name GSyV-1 will be used in the current note to avoid confusion. Forty dormant cane samples from 12 different cultivars were collected from different regions of Chile and screened by reverse transcription-PCR. One of the 40 samples (cv. Syrah) collected from the VI region of Chile was found to be infected with GSyV-1 using two different pairs of GSyV-1-specific primers. The first pair of primers GSyV-1Det-F: 5'-CAAGCCATCCGTGCATCTGG -3' and GSyV-1Det-R: 5'-GCCGATTTGGAACCCGATGG -3' (1), was used to amplify a 297-bp fragment corresponding to a partial region of the putative methyltransferase gene. The sequence (GenBank Accession No. GU566025) shared 87% nucleotide and 100% amino acid identities with the corresponding fragment of a Californian GSyV-1 isolate (GenBank Accession No. FJ436028). Since there are no commercial antibodies available for GSyV-1 detection, a second pair of primers, GVQCP-F: 5'-TCCCAGCTTCAGGGTGAATT -3' and GVQCP-R: 5'-GCATTGCTGCGCATTGGAGG -3' (4), that amplified a 720-bp fragment of the putative coat protein gene was also used. The sequence of 720 bp from the Chilean sample (GenBank Accession No. GU566024) shared 92% nucleotide and 98% amino acid identities with the corresponding fragment of a Californian GSyV-1 isolate (GenBank Accession No. FJ436028). The GSyV-1-positive sample was also infected with Grapevine fleck virus and Grapevine rupestris stem pitting-associated virus that have been reported previously in Chile. To our knowledge, this is the first report of GSyV-1 in Chile. Further studies will help to establish the incidence and effects of this virus in Chilean grapevines. References: (1) M. Al Rwahnih et al. Virology 387:395, 2009. (2) E. Engel et al. J. Virol. Methods. 163:445, 2010. (3) P. F. Escobar et al. Plant Dis. 92:1474, 2008. (4) S. Sabanadzovic et al. Virology 394:1, 2009.

First Report on the Occurrence of Grapevine leafroll-associated virus 7 and 9 in Chilean Grapevines.

Grapevine is one of the oldest horticultural crops and represents a highly valuable agricultural commodity. So far, nine distinct Grapevine leafroll-associated viruses (GLRaVs) within the Closteroviridae family have been found to be associated with grapevine leafroll disease (3). Previous studies have demonstrated a high incidence of GLRaV-1, -2, and -3 in Chile (2). To determine if other GLRaVs were present, 21 dormant cane samples were screened with a comprehensive 70-mer oligonucleotide microarray designed to simultaneously detect all grapevine viruses with total or partial genomic sequence available. The array contained 570 unique probes designed against specific regions of more than 40 viral genomes (E. Engel et al., 15th ICVG [Abstr.], 2006). One sample (cv. Black Seedless) showing a microarray hybridization pattern compatible with a mixed infection of GLRaV-7 and GLRaV-1 was analyzed by ELISA using GLRaV-7 specific antibodies (Agritest, Valenzano, Italy) and reverse transcription (RT)-PCR using virus-specific primers LR7-F: 5'- TAT ATC CCA ACG GAG ATG GC -3' and LR7-R: 5'- ATG TTC CTC CAC CAA AAT CG -3' (based on GenBank Accession No. Y15987). The serological analysis confirmed the presence of GLRaV-7 with further confirmation by the RT-PCR product of 502 bp corresponding to a fragment of the HSP70h gene that was cloned and sequenced. The Chilean GLRaV-7 sequence (GenBank Accession No. EU334662) showed 94% nucleotide and 95% amino acid identity when compared with a corresponding region of another GLRaV-7 isolate from Albania (GenBank Accession No. Y15987). GLRaV-1 infection was confirmed by ELISA (Bioreba AG, Reinach, Switzerland) and RT-PCR. A second sample (cv. Tintorera) showing microarray hybridization pattern compatible with a mixed infection of GLRaV-9 and Grapevine virus A (GVA) was analyzed by RT-PCR using virus-specific primers LR9-F: 5'- CGG CAT AAG AAA AGA TGG CAC -3' and LR9-R: 5'- TCA TTC ACC ACT GCT TGA AC -3' (1). The RT-PCR product of 393 bp corresponding to a fragment of the HSP70h gene was cloned and sequenced (GenBank Accession No. EU334663), showing 94% nucleotide and 95% amino acid identity when compared with a corresponding region of another GLRaV-9 isolate from the United States (GenBank Accession No. AY297819). Since there are no commercial antibodies available for GLRaV-9 detection, a second pair of primers, LR9-F1: 5'- AAA GGT TTC TGC TGG TTA CC -3' and LR9-R1: 5'- CTT TCA GAA CAG TCC TCC TC -3' that amplified a fragment of ORF1a was also used. The 301-bp product was cloned and sequenced (GenBank Accession No. EU588989) showing 93.7% nucleotide and 98% amino acid identity when compared with a corresponding region of another GLRaV-9 isolate (GenBank Accession No. AY297819). GVA infection was confirmed by ELISA (Bioreba AG) and RT-PCR. To our knowledge, this is the first report of GLRaV-7 and GLRaV-9 in Chile. Further studies will help determine the effect and incidence of these viruses in Chilean grapevines. References: (1) R. Alkowni et al. J. Plant Pathol. 86:123, 2004. (2) N. Fiore et al. J. Plant Pathol. 90:125, 2008. (3) G. P. Martelli and E. Boudon-Padieu. Options Méditerr. B55, 2006.

First Detection of Grapevine leafroll-associated virus 4 in Chilean Grapevines.

Grapevine leafroll is one of the most widespread and economically relevant viral diseases of grapevines. At least nine distinct Grapevine leafroll-associated viruses (GLRaVs), all members of the Closteroviridae family, have been associated with this disease in grapevine. Grapevine leafroll-associated virus 4 (GLRaV-4), currently classified as a Closteroviridae member under the Ampelovirus genus, was initially described in California. To determine if GLRaV-4 was present in Chilean grapevines, in addition to the previously reported GLRaV-1, -2, -3, -7, and -9 (1,2), 35 dormant cane samples from 12 different cultivars were collected from different regions of Chile and screened by reverse transcription-PCR. Two of the 35 samples (both cv. Thompson Seedless) collected from the III and VI regions of Chile were found to be infected with GLRaV-4 using two different pairs of GLRaV-4 specific primers. The first pair of primers, HSPV-F: 5'- ACA TTC TCC ACC TTG TGC TTT T -3' and HSPC-R: 5'- CAT ACA AGC GAG TGC AAT TAC -3' (3), was used to amplify a 321-bp fragment corresponding to a partial region of the HSP70h gene. The sequence (GenBank Accession Nos. EU746618 and EU746619) from both positive samples shared 98.4% nucleotide identity and approximately 99% identity with the corresponding fragment of a Californian GLRaV-4 isolate (GenBank Accession No. AF039553). Since there are no commercial antibodies available for GLRaV-4 detection, a second pair of primers, LR4CPINT-F: 5'- GAG AGT GAC AAG CAC CAG GTG C -3' and LR4CPFIN-R: 5'- TCA CCT CCT GTT GCC CA -3' (4), that amplified a 492-bp fragment of the coat protein gene was also used. The sequences of the 492-bp fragment from both Chilean samples (GenBank Accession Nos. EU746620 and EU746621) shared 99.6% nucleotide identity with one another and had 96.5% identity with an Israeli GLRaV-4 isolate (GenBank Accession No. AM176759). To our knowledge, this is the first report of GLRaV-4 in Chile. Further studies will help to establish the effects and incidence of this virus in Chilean grapevines. References: (1) E. Engel et al. Plant Dis. 92:1252, 2008 (2) N. Fiore et al. J. Plant Pathol. 90:125, 2008. (3) F. Osman et al. J. Virol. Methods 141:22, 2007. (4) P. Saldarelli et al. J. Plant Pathol. 88:203, 2006.