Mahfoudhi N, Habili N, Masri S A, Dhouibi M H
Laboratoire de Protection des Végétaux, Institut National de la Recherche Agronomique de Tunisie, Rue Hedi Karray, 2049 Ariana, Tunisie.
Waite Diagnostics, School of Agriculture, Food and Wine, University of Adelaide, Glen Osmond, S.A. 5064, Australia.
Plant Dis. 2007 Oct;91(10):1359. doi: 10.1094/PDIS-91-10-1359A.
Grapevine leafroll disease is one of the most important diseases that occurs in cultivated grapevines in the world. So far, nine serologically distinct viruses of the family Closteroviridae have been isolated from diseased vines (3). A previous study (4) has shown that Grapevine leafroll-associated viruses (GLRaV) -1, -2, and -3 are present in Tunisian grapevines and GLRaV-3 is the predominant virus associated with leafroll disease. A survey was conducted in table grapes to identify other viruses associated with this disease. Samples of dormant canes were collected and screened by indirect Biotin Steptavidin ELISA with specific antibodies to GLRaV-5 (Bio-Rad, Sanofi, France) according to the manufacturer's instructions. Serological analysis revealed that nearly 47% of the samples were infected with GLRaV-5. To confirm GLRaV-5 identification and identify other leafroll viruses, vines with severe leafroll symptoms were collected and total RNA extracts were obtained from six samples and tested at Waite Diagnostics (University of Adelaide, Australia) by reverse transcription (RT)-PCR using primers for GLRaV-5 (2), LR5-1F 5'-CCCGTGATACAAGGTAGGACA-3' and LR5-1R 5'-CAGACTTCACCTCCTGTTAC-3' with a resulting amplicon size of 690 bp and primers for GLRaV-9, LR9F 5'-ACAGTGGTCGGCATAAGAAAAG-3' and LR9R 5'-ACACAAACATGCAGGCCAAAG-3' with a resulting amplicon size of 250 bp. Results showed that 1 of 6 and 5 of 6 of the samples were infected with GLRaV-5 and GLRaV-9, respectively, by RT-PCR and comparable results were obtained by ELISA. Amplicons were cloned and sequenced to confirm the identification of GLRaV-5 and GLRaV-9. The obtained sequences showed 99.1% nt identity and 94.8% amino acid similarity with an isolate of GLRaV-5 (GenBank Accession No. AF233934) and 97.6% nt identity and 94.8% amino acid similarity with an isolate of GLRaV-9 (GenBank Accession No. AY297819). The occurrence of GLRaV-9 has previously been reported in California and Australia (1). To our knowledge, this is the first report on the occurrence of GLRaV-5 and -9 in Tunisian grapevines. The widespread occurrence of GLRaV-5 and -9 is probably due either to the presence of their putative vectors, Planococcus ficus (Signoret) and Planococcus citri (Risso), or by propagation using infected local source material. Further studies are in progress to verify the implication of indigenous mealybugs in the spread of these viruses. References: (1) R. Alkowni et al. J. Plant Pathol. 86:123, 2004. (2) X. Good and J. Monis. Phytopathology 91:247, 2001. (3) P. Gugerli. ICVG, Extended Abstracts 14:23, 2003. (4) N. Mahfoudhi et al. EPPO Bulletin 28:197, 1998.
葡萄卷叶病是世界上栽培葡萄中发生的最重要病害之一。到目前为止，已从患病葡萄藤中分离出9种血清学上不同的长线形病毒科病毒(3)。先前的一项研究(4)表明，突尼斯葡萄藤中存在葡萄卷叶相关病毒(GLRaV)-1、-2和-3，且GLRaV-3是与卷叶病相关的主要病毒。对鲜食葡萄进行了一项调查，以确定与这种病害相关的其他病毒。采集休眠藤条样本，并根据制造商的说明，用针对GLRaV-5的特异性抗体（法国赛诺菲生物-拉德公司）通过间接生物素链霉亲和素酶联免疫吸附测定法进行筛选。血清学分析显示，近47%的样本感染了GLRaV-5。为了确认GLRaV-5的鉴定并鉴定其他卷叶病毒，采集了具有严重卷叶症状的葡萄藤，从6个样本中提取了总RNA提取物，并在澳大利亚阿德莱德大学韦特诊断中心使用GLRaV-5的引物(2)进行逆转录(RT)-PCR检测，引物为LR5-1F 5'-CCCGTGATACAAGGTAGGACA-3'和LR5-1R 5'-CAGACTTCACCTCCTGTTAC-3'，扩增产物大小为690 bp，以及用于GLRaV-9的引物，LR9F 5'-ACAGTGGTCGGCATAAGAAAAG-3'和LR9R 5'-ACACAAACATGCAGGCCAAAG-3'，扩增产物大小为250 bp。结果显示，通过RT-PCR，6个样本中有1个和6个样本中有5个分别感染了GLRaV-5和GLRaV-9，酶联免疫吸附测定法也得到了类似结果。对扩增产物进行克隆和测序，以确认GLRaV-5和GLRaV-9的鉴定。获得的序列与GLRaV-5的一个分离株（GenBank登录号AF233934）显示出99.1%的核苷酸同一性和94.8%的氨基酸相似性，与GLRaV-9的一个分离株（GenBank登录号AY297819）显示出97.6% 的核苷酸同一性和94.8%的氨基酸相似性。此前在加利福尼亚州和澳大利亚已报道过GLRaV-9的发生(1)。据我们所知，这是关于突尼斯葡萄藤中GLRaV-5和-9发生情况的首次报道。GLRaV-5和-9的广泛发生可能是由于其假定传播介体无花果粉蚧（西诺雷特）和柑橘粉蚧（里索）的存在，或者是使用受感染的本地源材料进行繁殖所致。正在进行进一步研究，以验证本地粉蚧在这些病毒传播中的作用。参考文献：(1)R. Alkowni等人，《植物病理学杂志》86:123，2004年。(2)X. Good和J. Monis，《植物病理学》91:247，2001年。(3)P. Gugerli，国际葡萄病毒大会，扩展摘要14:23，2003年。(4)N. Mahfoudhi等人，《欧洲和地中海植物保护组织通报》28:197，1998年。
