Melzer M J, Sugano J S, Cabanas D, Dey K K, Kandouh B, Mauro D, Rushanaedy I, Srivastava S, Watanabe S, Borth W B, Tripathi S, Matsumoto T, Keith L, Gonsalves D, Hu J S
Plant and Environmental Protection Sciences, University of Hawaii, Honolulu96822.
USDA-ARS Pacific Basin Agricultural Research Center, Hilo, HI 96720.
Plant Dis. 2012 Jun;96(6):917. doi: 10.1094/PDIS-02-12-0147-PDN.
In August 2011, tomato (Solanum lycopersicum L.) fruit from a University of Hawaii field trial displayed mottling symptoms similar to that caused by Tomato spotted wilt virus (TSWV) or other tospoviruses. The foliage from affected plants, however, appeared symptomless. Fruit and leaf tissue from affected plants were negative for TSWV analyzed by double antibody sandwich (DAS)-ELISA and/or TSWV ImmunoStrips (Agdia, Elkhart, IN) when performed following the manufacturer's instructions. Total RNA from a symptomatic and an asymptomatic plant was isolated using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) and reverse transcribed using Invitrogen SuperScript III reverse transcriptase (Life Technologies, Grand Island, NY) and primer 900 (5'- CACTCCCTATTATCCAGG(T)-3') following the enzyme manufacturer's instructions. The cDNA was then used as template in a universal potyvirus PCR assay using primers 900 and Sprimer, which amplify sequences encoding the partial inclusion body protein (NIb), coat protein, and 3' untranslated region of potyviruses (1). A ~1,700-bp product was amplified from the cDNA of the symptomatic plant but not the asymptomatic plant. This product was cloned using pGEM-T Easy (Promega, Madison, WI) and three clones were sequenced at the University of Hawaii's Advanced Studies in Genomics, Proteomics, and Bioinformatics laboratory. The 1,747-bp consensus sequence of the three clones was deposited in GenBank (Accession No. JQ429788) and, following primer sequence trimming, found to be 97% identical to positions 7,934 through 9,640 of Pepper mottle virus (PepMoV; family Potyviridae, genus Potyvirus) accessions from Korea (isolate '217' from tomato; EU586126) and California (isolate 'C' from pepper; M96425). To determine the incidence of PepMoV in the field trial, all 292 plants representing 14 tomato cultivars were assayed for the virus 17 weeks after planting using a PepMoV-specific DAS-ELISA (Agdia) following the manufacturer's directions. Plants were considered positive if their mean absorbance at 405 nm was greater than the mean absorbance + 3 standard deviations + 10% of the negative control samples. The virus incidence ranged from 4.8 to 47.6% for the different varieties, with an overall incidence of 19.9%. Although plant growth was not noticeably impaired by PepMoV infection, the majority of fruit from infected plants was unsaleable, making PepMoV a considerable threat to tomato production in Hawaii. PepMoV has been reported to naturally infect tomato in Guatemala (3) and South Korea (2). To our knowledge, this is the first report of this virus in Hawaii and the first report of this virus naturally infecting tomato in the United States. References: (1) J. Chen et al. Arch. Virol. 146:757, 2001. (2) M.-K. Kim et al. Plant Pathol. J. 24:152, 2008. (3) J. Th. J. Verhoeven et al. Plant Dis. 86:186, 2002.
2011年8月,夏威夷大学田间试验中的番茄(Solanum lycopersicum L.)果实出现斑驳症状,类似于番茄斑萎病毒(TSWV)或其他番茄斑萎病毒属病毒引起的症状。然而,受影响植株的叶片没有症状。按照制造商的说明,采用双抗体夹心(DAS)-ELISA和/或TSWV免疫试纸条(Agdia公司,印第安纳州埃尔克哈特)对受影响植株的果实和叶片组织进行分析,结果显示未检测到TSWV。使用RNeasy植物小提试剂盒(Qiagen公司,加利福尼亚州瓦伦西亚)从一株有症状植株和一株无症状植株中提取总RNA,并按照酶制造商的说明,使用Invitrogen SuperScript III逆转录酶(Life Technologies公司,纽约州大岛)和引物900(5'-CACTCCCTATTATCCAGG(T)-3')进行逆转录。然后,将该cDNA用作模板,使用引物900和Sprimer进行通用马铃薯Y病毒属PCR检测,这两个引物可扩增编码马铃薯Y病毒属部分包涵体蛋白(NIb)、外壳蛋白和3'非翻译区的序列(1)。从有症状植株的cDNA中扩增出一条约1700 bp的产物,但无症状植株中未扩增出该产物。使用pGEM-T Easy载体(Promega公司,威斯康星州麦迪逊)对该产物进行克隆,并在夏威夷大学基因组学、蛋白质组学和生物信息学高级研究实验室对三个克隆进行测序。这三个克隆的1747 bp一致序列已存入GenBank(登录号JQ429788),去除引物序列后,发现其与来自韩国(番茄分离株“217”;EU586126)和加利福尼亚州(辣椒分离株“C”;M96425)的辣椒斑驳病毒(PepMoV;马铃薯Y病毒科马铃薯Y病毒属)登录号7934至9640位的序列有97%的同一性。为了确定田间试验中PepMoV的发病率,在种植17周后,按照制造商的说明,使用PepMoV特异性DAS-ELISA(Agdia公司)对代表14个番茄品种的所有292株植株进行病毒检测。如果植株在405 nm处的平均吸光度大于阴性对照样品的平均吸光度+3个标准差+10%,则认为该植株呈阳性。不同品种的病毒发病率在4.8%至47.6%之间,总体发病率为19.9%。虽然PepMoV感染并未明显损害植株生长,但受感染植株的大部分果实无法销售,这使得PepMoV对夏威夷的番茄生产构成了相当大的威胁。据报道,PepMoV在危地马拉(3)和韩国(2)可自然感染番茄。据我们所知,这是该病毒在夏威夷的首次报道,也是该病毒在美国自然感染番茄的首次报道。参考文献:(1)J. Chen等人,《病毒学档案》146:757,2001年。(2)M.-K. Kim等人,《植物病理学杂志》24:152,2008年。(3)J. Th. J. Verhoeven等人,《植物病害》86:186,2002年。