Marn M Viršček, Pleško I Mavrič, Zindović J
Agricultural Institute of Slovenia, Hacquetova Ulica 17, 1000 Ljubljana, Slovenia.
University of Montenegro, Biotechnical Faculty - Podgorica, Mihajla Lalića 1, 81 000 Podgorica, Montenegro.
Plant Dis. 2012 Apr;96(4):593. doi: 10.1094/PDIS-11-11-0994.
Numerous ornamental plants have been found to be symptomless hosts of various pospiviroids including Citrus exocortis viroid (CEVd), and hence, may serve as potential inoculum reservoirs for susceptible vegetable plants. Production of tomato, potato, and pepper, on which viroids from the genus Pospiviroid can cause severe damage, represents two-thirds of the vegetable production in Montenegro. We tested vegetable, ornamental, and weed host plants for the presence of pospiviroids in September 2011. Altogether 80 samples were taken. Samples of ornamental plants (15 of Petunia spp., 7 of Impatiens spp., 4 of Verbena spp., 3 of Dahlia spp., 3 of Pittosporum tobira, 3 of Vinca spp., 2 of Brugmansia spp., 2 of avocado, 2 of Portulaca spp., and 1 of Datura sp.) were taken from three places of production. One sample per species was collected from symptomless eggplants, tomatoes, sweet peppers, and avocados in the vicinity of one glasshouse with ornamental plants. Twenty-two samples from sweet pepper and seven samples from tomato, all grown under cover and all showing potential virus-like symptoms, were collected from three places of vegetable production. Two samples of Solanum nigrum and three samples of unidentified weed species belonging to genus Solanum were taken from two glasshouses. With the exception of weed plants, samples consisted of fully developed leaves collected from five plants. All sampled ornamental and weed plants were symptomless. RNA was extracted from approximately 15 mg of leaf tissue with the MagMAX-96 Total RNA Isolation Kit (Life Technologies, Foster City, CA) in accordance with the manufacturer's instructions for the MagMAX Express Magnetic Particle Processor. Samples were tested by reverse transcription (RT)-PCR using semi-universal pospiviroid primers (Pospi1-RE/FW and Vid-RE/FW [3]). None of the samples reacted with the Vid-RE/FW primer pair. An amplicon of an expected size (approximately 196 nt) was produced with the Pospi1-RE/FW primer pair from one Verbena sp. sample. Direct sequencing was performed by Macrogen (Amsterdam, The Netherlands). Sequence analysis indicated the presence of CEVd. This finding was confirmed by sequence analysis of the DNA product obtained by RT-PCR using Pospi1-FW/RE from a new extraction. Further analyses using primer pairs CEVd-AS/S (1) and CEVd-FW2/RE2 (4) were performed to obtain the full viroid sequence. The sequence of 372 nt was deposited in GenBank (Accession No. JN872140) and had 99% identity with two CEVd sequences from Verbena spp. (Accession Nos. EF192396 and DQ094297). To our knowledge, this is the first report of CEVd in a Verbena sp. in Montenegro and the second report in Europe (4). CEVd has been detected in Verbena spp. also in India and Canada and can be transmitted by seed (2). The infected Verbena sp. plants were not destroyed, since CEVd is not listed as a quarantine organism in Montenegro. The spread of CEVd infection to tomato could devastate the production of this crop in Montenegro. References: (1) A. Elleuch et al. Plant Protect. Sci. 39:139, 2003. (2) R. P. Singh et al. Eur. J. Plant Pathol. 124:691, 2009. (3) J. Th. J. Verhoeven et al. Eur. J. Plant Pathol. 110:823, 2004. (4) J. Th. J. Verhoeven et al. Plant Dis. 92:973, 2008.
人们发现许多观赏植物是包括柑橘裂皮类病毒（CEVd）在内的多种马铃薯纺锤块茎类病毒的无症状寄主，因此，它们可能成为易感蔬菜作物潜在的接种体库。番茄、马铃薯和辣椒是马铃薯纺锤块茎类病毒属病毒可造成严重损害的作物，其产量占黑山蔬菜总产量的三分之二。2011年9月，我们对蔬菜、观赏植物和杂草寄主植物进行了马铃薯纺锤块茎类病毒检测。共采集了80个样本。从三个生产地采集了观赏植物样本（矮牵牛属15个、凤仙花属7个、马鞭草属4个、大丽花属3个、海桐3个、长春花属3个、曼陀罗属2个、鳄梨2个、马齿苋属2个、曼陀罗1个）。从一个种植观赏植物的温室附近无症状的茄子、番茄、甜椒和鳄梨中，每个物种采集一个样本。从三个蔬菜生产地采集了22个来自甜椒和7个来自番茄的样本，这些样本均在保护设施下种植且均表现出潜在的类病毒症状。从两个温室中采集了2个龙葵样本和3个属于茄属的未鉴定杂草样本。除杂草植物外，样本由从五株植物上采集的完全展开的叶片组成。所有采样的观赏植物和杂草植物均无症状。按照适用于MagMAX Express磁珠处理器的制造商说明，使用MagMAX - 96总RNA提取试剂盒（Life Technologies公司，美国加利福尼亚州福斯特城）从约15毫克叶片组织中提取RNA。使用半通用马铃薯纺锤块茎类病毒引物（Pospi1 - RE/FW和Vid - RE/FW [3]）通过逆转录（RT）-PCR对样本进行检测。没有样本与Vid - RE/FW引物对发生反应。用Pospi1 - RE/FW引物对从一个马鞭草属样本中扩增出了预期大小（约196 nt）的扩增子。由Macrogen公司（荷兰阿姆斯特丹）进行直接测序。序列分析表明存在CEVd。通过对使用Pospi1 - FW/RE从新提取样本进行RT - PCR获得的DNA产物进行序列分析，证实了这一发现。使用引物对CEVd - AS/S（1）和CEVd - FW2/RE2（4）进行进一步分析以获得完整的类病毒序列。372 nt的序列已存入GenBank（登录号JN872140），与来自马鞭草属的两个CEVd序列（登录号EF192396和DQ094297）具有99%的同一性。据我们所知，这是CEVd在黑山的马鞭草属植物中的首次报道，也是在欧洲的第二次报道（4）。在印度和加拿大的马鞭草属植物中也检测到了CEVd，并且它可以通过种子传播（2）。由于CEVd在黑山未被列为检疫性生物，因此未销毁感染CEVd的马鞭草属植物。CEVd感染传播到番茄可能会摧毁黑山这种作物的产量。参考文献：（1）A. Elleuch等人，《植物保护科学》39:139，2003年。（2）R. P. Singh等人，《欧洲植物病理学报》124:691，2009年。（3）J. Th. J. Verhoeven等人，《欧洲植物病理学报》110:823，2004年。（4）J. Th. J. Verhoeven等人，《植物病害》92:973，2008年。
