Engel E A, Escobar P, Montt C, Gómez-Talquenca S, Valenzuela P D T
Fundación Ciencia para la Vida and MIFAB, Zañartu 1482 and Universidad Andrés Bello, República 217, Santiago, Chile.
Fundación Ciencia para la Vida and MIFAB, Zañartu 1482, Santiago, Chile.
Plant Dis. 2008 Aug;92(8):1252. doi: 10.1094/PDIS-92-8-1252C.
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.
葡萄是最古老的园艺作物之一，也是一种极具价值的农产品。到目前为止，已发现隶属于长线形病毒科的9种不同的葡萄卷叶相关病毒（GLRaV）与葡萄卷叶病有关（3）。先前的研究表明，GLRaV-1、-2和-3在智利的发生率很高（2）。为了确定是否存在其他GLRaV，我们用一种全面的70聚体寡核苷酸微阵列对21个休眠茎样本进行了筛选，该微阵列旨在同时检测所有具有全部或部分基因组序列的葡萄病毒。该阵列包含570个独特的探针，针对40多种病毒基因组的特定区域设计（E. Engel等人，第15届国际葡萄病毒大会[摘要]，2006年）。对一个表现出与GLRaV-7和GLRaV-1混合感染相匹配的微阵列杂交模式的样本（品种：无核黑），使用GLRaV-7特异性抗体（意大利瓦伦扎诺的Agritest公司）进行ELISA分析，并使用病毒特异性引物LR7-F：5'-TAT ATC CCA ACG GAG ATG GC-3'和LR7-R：5'-ATG TTC CTC CAC CAA AAT CG-3'（基于GenBank登录号Y15987）进行逆转录（RT）-PCR分析。血清学分析证实了GLRaV-7的存在，通过对对应于HSP70h基因片段的502 bp RT-PCR产物进行克隆和测序进一步确认。与来自阿尔巴尼亚的另一个GLRaV-7分离株（GenBank登录号Y15987）的相应区域相比，智利GLRaV-7序列（GenBank登录号EU334662）显示出94%的核苷酸同一性和95%的氨基酸同一性。通过ELISA（瑞士雷纳赫的Bioreba公司）和RT-PCR确认了GLRaV-1感染。对另一个表现出与GLRaV-9和葡萄病毒A（GVA）混合感染相匹配的微阵列杂交模式的样本（品种：廷托雷拉），使用病毒特异性引物LR9-F：5'-CGG CAT AAG AAA AGA TGG CAC-3'和LR9-R：5'-TCA TTC ACC ACT GCT TGA AC-3'（1）进行RT-PCR分析。对对应于HSP70h基因片段的393 bp RT-PCR产物进行克隆和测序（GenBank登录号EU334663），与来自美国的另一个GLRaV-9分离株（GenBank登录号AY297819）的相应区域相比，显示出94%的核苷酸同一性和95%的氨基酸同一性。由于没有用于检测GLRaV-9的商业抗体，还使用了另一对引物LR9-F1：5'-AAA GGT TTC TGC TGG TTA CC-3'和LR9-R1：5'-CTT TCA GAA CAG TCC TCC TC-3'，它们扩增了ORF1a的一个片段。对301 bp的产物进行克隆和测序（GenBank登录号EU588989），与另一个GLRaV-9分离株（GenBank登录号AY297819）的相应区域相比，显示出93.7%的核苷酸同一性和98%的氨基酸同一性。通过ELISA（Bioreba公司）和RT-PCR确认了GVA感染。据我们所知，这是智利首次报道GLRaV-7和GLRaV-9。进一步的研究将有助于确定这些病毒对智利葡萄树的影响和发生率。参考文献：（1）R. Alkowni等人，《植物病理学杂志》86:123，2004年。（2）N. Fiore等人，《植物病理学杂志》90:125，2008年。（3）G. P. Martelli和E. Boudon-Padieu，《地中海选项》B55，2006年。
