Hernandez C, Brown J K
Department of Plant Sciences, University of Arizona, Tucson 85721.
Plant Dis. 2010 Jul;94(7):917. doi: 10.1094/PDIS-94-7-0917B.
During April 2009, a commercial spinach field (1 km [250 acres]) in south-central Arizona developed geminivirus-like disease symptoms (4). Approximately 40 to 50% of the spinach plants exhibited extreme leaf distortion, foliar interveinal chlorosis, shortened internodes, and ~80% yield reduction. The beet leafhopper, Circulifer tenellus, the only known insect vector of curtoviruses in the United States, was observed on spinach plants. Total DNA was isolated (1) from three plant samples exhibiting the same symptom phenotype and used to PCR-amplify a 446-bp fragment of a suspected curtovirus, using primers F 5'-CTACCATCAGTAATGATGGG-3'and R 5' CATATTTGCCACCTCCAGTGTC-3' designed around the coat protein gene (Cp) for several known curtoviruses. DNA sequencing and BLAST analysis of the cloned fragments (n = 3 with 100% identity) revealed BLAST matches at 81 to 83% with the Cp for three isolates of Beet curly top Iran virus (BCTIV) (EU273816-18). To amplify the full-length curtovirus genome, total DNA from one of the three positive samples was used as the template in rolling circle amplification (RCA) employing the non-sequence specific TempliPhi 100 Amplification System (GE Healthcare) that amplifies circular DNA templates. The RCA products were linearized with PstI, yielding a ~3-Kbp fragment that was cloned into pGEM3zf+ (Promega, Madison, WI). To obtain the complete sequence, one plasmid (09-10-8) containing a full-length insert was selected and prepared for sequencing with the Template Generation System II Kit (Finnzymes, Espoo, Finland). The resultant 28 sequences were assembled into a contig using SeqMan software (DNASTAR, Madison, WI). Also, RCA clones (09.10-2, -3, and -4) from the same sample were subjected to DNA sequencing with universal M13F and M13R primers followed by primer walking (>300 bp overlap). The four 3,066-bp genomes shared 99 to 100% nt identity. An alignment (ClustalV; MegAlign, DNASTAR) with sequences of all curtovirus species available in GenBank indicated that the Arizona spinach isolates shared the highest nt sequence identity (59%) with Horseradish curly top virus (HrCTV). The next closest relatives were Beet mild curly top virus, Beet severe curly top virus, and Spinach curly top virus, at 50%. The genome consists of six open reading frames and lacks the AC3 gene, an arrangement most similar to HrCTV (3). The ICTV approved working cut-off for Curtovirus species demarcation at <89% nt identity (2) supports recognition of this isolate from spinach (GU734126) as a new, previously undescribed curtovirus species, for which we propose the name Spinach severe curly top virus (SSCTV-[Arizona:2009]). The curtovirus-like symptoms, presence of the curtovirus leafhopper vector, and isolation of a curtovirus-like genome from symptomatic spinach plants are highly suggestive of curtovirus etiology. To our knowledge, this is the first report of SSCTV worldwide and its association with diseased spinach in Arizona. Although a different curtovirus species was reported from the same infected spinach field (4), this study provides evidence that at least two curtoviruses were present in this spinach field in Arizona. References: (1) J. J. Doyle and J. L. Doyle. Focus 12:13, 1990. (2) C. M. Fauquet et al. Arch. Virol. 153:783, 2008. (3) K. A. Klute et al. J. Gen. Virol. 77:1369, 1996. (4) C. Nischwitz and M. W. Olsen. Online publication. doi:10.1094/PHP-2010-0216-01-BR. Plant Health Progress. 2010.
2009年4月期间，亚利桑那州中南部一片商用菠菜田（面积1平方千米[250英亩]）出现了类双生病毒病症状（4）。约40%至50%的菠菜植株表现出叶片极度扭曲、叶间脉间失绿、节间缩短，产量降低约80%。在菠菜植株上观察到甜菜叶蝉（Circulifer tenellus），它是美国已知的唯一一种曲顶病毒昆虫传播介体。从三个表现出相同症状表型的植株样本中提取总DNA（1），并使用围绕几种已知曲顶病毒外壳蛋白基因（Cp）设计的引物F 5'-CTACCATCAGTAATGATGGG-3'和R 5' CATATTTGCCACCTCCAGTGTC-3'，通过PCR扩增疑似曲顶病毒的446碱基对片段。对克隆片段（n = 3，一致性100%）进行DNA测序和BLAST分析，结果显示与伊朗甜菜曲顶病毒（BCTIV）三个分离株（EU273816 - 18）的Cp有81%至83%的BLAST匹配。为扩增曲顶病毒全基因组，以三个阳性样本之一的总DNA为模板，使用非序列特异性的TempliPhi 100扩增系统（GE Healthcare）进行滚环扩增（RCA），该系统可扩增环状DNA模板。RCA产物用PstI酶切线性化，得到一个约3千碱基对的片段，将其克隆到pGEM3zf +（Promega，麦迪逊，威斯康星州）中。为获得完整序列，选择一个含有全长插入片段的质粒（09 - 10 - 8），并用模板生成系统II试剂盒（Finnzymes，埃斯波，芬兰）进行测序准备。使用SeqMan软件（DNASTAR，麦迪逊，威斯康星州）将得到的28个序列组装成一个重叠群。此外，对来自同一样本的RCA克隆（09.10 - 2、-3和-4）用通用M13F和M13R引物进行DNA测序，随后进行引物步移（重叠>300碱基对）。这四个3066碱基对的基因组核苷酸一致性为99%至100%。与GenBank中所有曲顶病毒物种序列进行比对（ClustalV；MegAlign，DNASTAR）表明，亚利桑那州菠菜分离株与辣根曲顶病毒（HrCTV）的核苷酸序列一致性最高（59%）。其次亲缘关系较近的是甜菜轻性曲顶病毒、甜菜严重性曲顶病毒和菠菜曲顶病毒，一致性为50%。该基因组由六个开放阅读框组成，缺少AC3基因，这种排列与HrCTV最为相似（3）。国际病毒分类委员会（ICTV）批准的曲顶病毒物种划分工作阈值为核苷酸一致性<89%（2），这支持将从菠菜中分离得到的该分离株（GU734126）认定为一种新的、此前未描述过的曲顶病毒物种，我们将其命名为菠菜严重性曲顶病毒（SSCTV-[亚利桑那州：2009]）。类曲顶病毒症状、曲顶病毒叶蝉传播介体的存在以及从有症状的菠菜植株中分离出类曲顶病毒基因组，强烈提示了曲顶病毒病因。据我们所知，这是全球关于SSCTV的首次报道及其与亚利桑那州患病菠菜的关联。尽管此前报道了来自同一感染菠菜田的另一种曲顶病毒物种（4），但本研究提供了证据表明亚利桑那州这片菠菜田中至少存在两种曲顶病毒。参考文献：（1）J. J. Doyle和J. L. Doyle。焦点12：13，1990。（2）C. M. Fauquet等人。病毒学档案153：783，2008。（3）K. A. Klute等人。普通病毒学杂志77：1369，1996。（4）C. Nischwitz和M. W. Olsen。在线发表。doi：10.1094/PHP - 2010 - 0216 - 01 - BR。植物健康进展。2010年。
