Alfaro-Fernández A, Bese G, Córdoba-Sellés C, Cebrián M C, Herrera-Vásquez J A, Forray A, Jordá C
Instituto Agroforestal Mediterráneo, Universidad Politécnica de Valencia, Cno. de Vera s/n, 46022 Valencia, Spain.
Csongrád Megyei MgSzH Deak Ferenc U. 17, Szeged 6720, Hungary.
Plant Dis. 2009 May;93(5):554. doi: 10.1094/PDIS-93-5-0554C.
During the growing seasons of 2007 and 2008, in commercial greenhouses of tomato crops (Solanum lycopersicum L.) located in Szeged, Öcsöd, and Csongrád (southeastern regions of Hungary), unusual disease symptoms were observed, including necrotic spots in defined areas at the base of the leaflet, necrosis in the stems, and necrotic lines on the fruits surface. Affected plants appeared inside the greenhouses with a random distribution and the incidence recorded was at least 40%. These symptoms resembled those described for Tomato torrado virus (ToTV) infection in Spain (1) and Poland (3). To verify the identity of the disease, three symptomatic plants from commercial greenhouses of each geographic location were selected and analyzed by double-antibody sandwich-ELISA using polyclonal antibodies specific to Cucumber mosaic virus (CMV), Potato virus Y (PVY), Tomato mosaic virus (ToMV), Tomato spotted wilt virus (TSWV) (Loewe Biochemica, Sauerlach, Germany), and Pepino mosaic virus (PepMV) (DSMZ, Braunschweig, Germany). Total RNA was extracted and tested by reverse transcription (RT)-PCR with three pair of specific primers: one pair used to amplify the coat protein (CP) gene of PepMV (2) and the other two pairs specific to ToTV that amplify 580 bp of the polyprotein (4) and a fragment of 574 bp in the CP Vp23 (3). Nonisotopic dot-blot hybridization using a digoxygenin-labeled RNA probe complementary to the aforementioned fragment of the polyprotein was also performed. Tomato samples were negative for all the viruses tested by serological analysis and for PepMV by RT-PCR. However, all three samples were positive for ToTV by molecular hybridization and RT-PCR. RT-PCR products were purified and directly sequenced. The amplified fragments of the three Hungarian isolates, ToTV-H1, ToTV-H2, and ToTV-H3, for the polyprotein (GenBank Accession Nos. EU835496, FJ616995, and FJ616994, respectively) and the CP Vp23 (GenBank Accession Nos. FJ616996, FJ616997, and FJ616998, respectively) showed 99 to 98% nt identity with the polyprotein and the coat protein regions of ToTV from Spain and Poland (GenBank Accession Nos. DQ3888880 and EU563947, respectively). Whiteflies, commonly found in Hungarian greenhouses, have been reported to transmit ToTV (3), although the efficiency of transmission is unknown. To our knowledge, this is the first report of ToTV in Hungary. References: (1) A. Alfaro-Fernández et al. Plant Dis. 91:1060, 2007. (2) I. Pagán et al. Phytopathology 96:274, 2006. (3) H. Pospieszny et al. Plant Dis. 91:1364, 2007. (4) J. Van der Heuvel et al. Plant Virus Designated Tomato Torrado Virus. Online publication. World Intellectual Property Organization. WO/2006/085749, 2006.
在2007年和2008年的生长季节，位于匈牙利东南部塞格德、厄乔德和琼格拉德的番茄作物（Solanum lycopersicum L.）商业温室中，观察到异常的病害症状，包括小叶基部特定区域的坏死斑、茎部坏死以及果实表面的坏死线。受影响的植株在温室内随机出现，记录到的发病率至少为40%。这些症状与西班牙（1）和波兰（3）所描述的番茄托拉多病毒（ToTV）感染症状相似。为了验证病害的身份，从每个地理位置的商业温室中选取了三株有症状的植株，使用针对黄瓜花叶病毒（CMV）、马铃薯Y病毒（PVY）、番茄花叶病毒（ToMV）、番茄斑萎病毒（TSWV）（德国洛伊维生物化学公司，绍尔拉赫）和番木瓜花叶病毒（PepMV）（德国微生物菌种保藏中心，不伦瑞克）的多克隆抗体，通过双抗体夹心ELISA进行分析。提取总RNA，并使用三对特异性引物通过逆转录（RT）-PCR进行检测：一对用于扩增PepMV的外壳蛋白（CP）基因（2），另外两对特异性针对ToTV，分别扩增580 bp的多聚蛋白片段（4）和CP Vp23中574 bp的片段（3）。还使用与上述多聚蛋白片段互补的地高辛标记RNA探针进行了非同位素斑点杂交。通过血清学分析，番茄样品对所有测试病毒均呈阴性，通过RT-PCR对PepMV也呈阴性。然而，通过分子杂交和RT-PCR，所有三个样品对ToTV均呈阳性。RT-PCR产物经过纯化并直接测序。匈牙利的三个分离株ToTV-H1、ToTV-H2和ToTV-H3的多聚蛋白扩增片段（GenBank登录号分别为EU835496、FJ616995和FJ616994）以及CP Vp23扩增片段（GenBank登录号分别为FJ616996、FJ616997和FJ616998）与来自西班牙和波兰的ToTV的多聚蛋白和外壳蛋白区域（GenBank登录号分别为DQ3888880和EU563947）的核苷酸同一性为99%至98%。匈牙利温室中常见的粉虱已被报道可传播ToTV（3），尽管传播效率未知。据我们所知这是ToTV在匈牙利的首次报道。参考文献：（1）A. Alfaro-Fernández等人，《植物病害》91:1060，2007年。（2）I. Pagán等人，《植物病理学》96:274，2006年。（3）H. Pospieszny等人，《植物病害》91:1364，2007年。（4）J. Van der Heuvel等人，《植物病毒命名为番茄托拉多病毒》。在线出版物。世界知识产权组织。WO/2006/085749，2006年。
