David N, Mallik I, Gudmestad N C
Department of Plant Sciences, North Dakota State University, Fargo 58108.
Department of Plant Pathology, North Dakota State University, Fargo 58108.
Plant Dis. 2010 Jan;94(1):130. doi: 10.1094/PDIS-94-1-0130B.
Tobacco rattle virus (TRV) belongs to the genus Tobravirus and causes a stem mottle of potato (Solanum tuberosum) foliage and necrotic arcs and rings in tubers referred to as corky ringspot. This virus is generally transmitted by a number of species of stubby-root nematode. The virus is widespread and has been reported in California, Colorado, Florida, Idaho, Michigan, Oregon, Washington, Minnesota, and Wisconsin (2). In the spring of 2009, we received potato tubers of cv. Russet Burbank with internal necrotic arcs very similar to those caused by TRV from potato storages located in Grand Forks and Dickey counties of North Dakota. Total RNA was extracted from the necrotic lesions of two tubers from each location using the Total RNA Isolation kit (Promega Corp., Madison WI). These extracts were tested for TRV by reverse transcription (RT)-PCR using primers complementary to nucleotides 6555 to 6575 (Primer A) and identical to nucleotides 6113 to 6132 (Primer B) within the 3' terminus of TRV-SYM RNA-1 (GenBank Accession No. X06172) (3). The expected 463-bp amplicons from two separate tuber samples from each county were cloned (TOPO Cloning; Invitrogen, Carlsbad, CA) and sequenced. The sequences obtained from the four clones at both locations were found to be identical to each other and were 99% identical to the corresponding regions of TRV isolates from Michigan and Florida (GenBank Accession Nos. EU315226.1 and AF055912.1, respectively). Since sequences from all four clones were identical, only one of the sequences was submitted to Genbank (Accession No. GQ223114) and thus represents a consensus sequence. The extracts also tested positive in RT-PCR with a second set of primers corresponding to sequences in TRV RNA-2 yielding a 3.8-kbp amplicon (1). No evidence was found by RT-PCR for several other viruses that cause tuber necrosis in potato (Potato mop top virus, Tomato spotted wilt virus, Alfalfa mosaic virus, and tuber necrosis strains of Potato virus Y). The virus was mechanically transmitted by inoculating sap from symptomatic tubers from both counties to tobacco cv. Samsun NN, which showed typical bright yellow patches and spots on leaves 2 weeks postinoculation. TRV was confirmed in tobacco by RT-PCR from total RNA extracted from tobacco leaves with both sets of the aforementioned primers. To our knowledge, this is the first report of TRV in North Dakota and the first report of corky ringspot disease of potato in this state. References: (1) J. M. Crosslin et al. Virus Res. 96:99, 2003. (2) N. C. Gudmestad et al. Plant Dis. 92:1254, 2008. (3) D. J. Robinson. J. Virol. Methods 40:57, 1992.
烟草脆裂病毒（TRV）属于烟草脆裂病毒属，可导致马铃薯（Solanum tuberosum）叶片出现茎斑驳，块茎出现坏死弧和环，即所谓的木栓化环斑。该病毒通常由多种短根线虫传播。这种病毒分布广泛，已在加利福尼亚州、科罗拉多州、佛罗里达州、爱达荷州、密歇根州、俄勒冈州、华盛顿州、明尼苏达州和威斯康星州被报道（2）。2009年春季，我们从北达科他州大福克斯县和迪基县的马铃薯贮藏库收到了品种为褐皮伯班克的马铃薯块茎，其内部坏死弧与TRV引起的坏死弧非常相似。使用总RNA分离试剂盒（Promega公司，威斯康星州麦迪逊）从每个地点的两个块茎的坏死病变中提取总RNA。使用与TRV-SYM RNA-1（GenBank登录号X06172）3'末端核苷酸6555至6575互补的引物（引物A）和与核苷酸6113至6132相同的引物（引物B）通过逆转录（RT）-PCR对这些提取物进行TRV检测（3）。来自每个县的两个单独块茎样品的预期463bp扩增子被克隆（TOPO克隆；Invitrogen，加利福尼亚州卡尔斯巴德）并测序。在两个地点从四个克隆获得的序列彼此相同，并且与来自密歇根州和佛罗里达州的TRV分离株的相应区域有99%的同一性（分别为GenBank登录号EU315226.1和AF055912.1）。由于所有四个克隆的序列相同，因此仅将其中一个序列提交给Genbank（登录号GQ223114），因此代表一个共有序列。提取物在使用对应于TRV RNA-2序列的第二组引物进行的RT-PCR中也呈阳性，产生一个3.8kbp的扩增子（1）。通过RT-PCR未发现其他几种导致马铃薯块茎坏死的病毒的证据（马铃薯帚顶病毒、番茄斑萎病毒、苜蓿花叶病毒和马铃薯Y病毒的块茎坏死株系）。通过将来自两个县有症状块茎的汁液接种到烟草品种Samsun NN上进行机械传播，接种后2周，烟草叶片上出现典型的亮黄色斑块和斑点。通过使用上述两组引物从烟草叶片提取的总RNA进行RT-PCR，在烟草中确认了TRV。据我们所知，这是TRV在北达科他州的首次报道，也是该州马铃薯木栓化环斑病的首次报道。参考文献：（1）J.M.Crosslin等人，《病毒研究》96:99，2003。（2）N.C.Gudmestad等人，《植物疾病》92:1254，2008。（3）D.J.Robinson，《病毒学方法杂志》40:57，1992。
