Akad F, Webb S, Nyoike T W, Liburd O E, Turechek W, Adkins S, Polston J E
Department of Plant Pathology, University of Florida, Gainesville.
Department of Entomology and Nematology, University of Florida, Gainesville.
Plant Dis. 2008 Apr;92(4):648. doi: 10.1094/PDIS-92-4-0648C.
In October of 2006, yellow straightneck and zucchini squash plants (Cucurbita pepo L.) with crumpled, curled, thickened leaves were found in St. Johns and Marion counties in central Florida, respectively. Both locations had high populations of the whitefly, Bemisia tabaci. Incidences of symptomatic plants were greater than 95% in three squash fields (33 ha total) in St. Johns County and 35% in an experimental plot in Marion County. Twenty-three samples were collected from symptomatic plants (two from St. Johns County and 21 from Marion County). DNA was extracted for PCR and tested for the presence of begomoviruses using the following pairs of degenerate primers: AC1048/AV494, which amplifies a conserved region of the coat protein gene (2), PAR1c496/PAL1v1978, which amplifies a region of the begomovirus A component, and PBL1v2040/PCRc154, which amplifies a hypervariable region of the begomovirus B component (1). All squash samples yielded amplicons of sizes expected for a bipartite begomovirus: 1,159 nt with PAR1c496/PAL1v1978, 550 nt with AC1048/AV494, and 493 nt with PBL1v2040/PCRc154. The 1,159- and 493-nt amplicons obtained from two squash plants were cloned and sequenced. The 1,159 nt sequences from both plants shared 98% sequence identity with each other and 97% identity with equivalent regions of the A component of Cucurbit leaf crumple virus (CuLCrV) from Arizona and California (GenBank Accession Nos. AF256200 and AF224760, respectively). The 493-nt sequences amplified with PBL1v2040/PCRc154 were identical and shared a 96% identity with CuLCrV sequence (GenBank Accession No. AF327559) from Arizona and 97% identity with CuLCrV B component sequence (GenBank Accession No. AF224761) from California. Leaves were collected from eight symptomatic squash plants from Citra, FL and used for whitefly transmission assays. Approximately 100 adults of Bemisia tabaci biotype B were released onto each caged leaf and given a 24-h acquisition access period, after which a healthy squash seedling was introduced. Symptoms developed within 10 days on all test plants, and the presence of CuLCrV was confirmed by PCR assays, (primer pairs PAR1c496/PAL1v1978 and PBL1v2040/PCRc154) followed by sequencing. In 2007, similar symptoms were seen in several locations around the state. The same assays confirmed the presence of CuLCrV in watermelon (Citrullus lanatus L.) and squash in the following counties: Collier and Hendry in southwest Florida and Hillsborough, Manatee, and Sarasota in west-central Florida. To our knowledge, this is the first report of CuLCrV, and the first report of any begomovirus in cucurbits in Florida. References: (1) M. R. Rojas et al. Plant Dis. 77:340, 1993. (2) S. D. Wyatt and J. K. Brown. Phytopathology 86:1288, 1996.
2006年10月，分别在佛罗里达州中部的圣约翰斯县和马里恩县发现了叶片皱缩、卷曲、增厚的黄色直颈南瓜和西葫芦植株（西葫芦种）。这两个地方烟粉虱（烟粉虱）的数量都很多。在圣约翰斯县的三个南瓜田（总面积33公顷）中，有症状植株的发生率超过95%，在马里恩县的一个试验田中为35%。从有症状的植株上采集了23个样本（两个来自圣约翰斯县，21个来自马里恩县）。提取DNA进行PCR，并使用以下几对简并引物检测双生病毒的存在：AC1048/AV494，扩增外壳蛋白基因的保守区域（2）；PAR1c496/PAL1v1978，扩增双生病毒A组分的一个区域；PBL1v2040/PCRc154，扩增双生病毒B组分的一个高变区域（1）。所有南瓜样本均产生了双分体双生病毒预期大小的扩增子：PAR1c496/PAL1v1978扩增出1159 nt的片段，AC1048/AV494扩增出550 nt的片段，PBL1v2040/PCRc154扩增出493 nt的片段。从两棵南瓜植株上获得的1159 nt和493 nt的扩增子被克隆并测序。来自两棵植株的1159 nt序列彼此间的序列同一性为98%，与来自亚利桑那州和加利福尼亚州的葫芦叶皱缩病毒（CuLCrV）A组分的等效区域的同一性为97%（GenBank登录号分别为AF256200和AF224760）。用PBL1v2040/PCRc154扩增的493 nt序列相同，与来自亚利桑那州的CuLCrV序列（GenBank登录号AF327559）的同一性为96%，与来自加利福尼亚州的CuLCrV B组分序列（GenBank登录号AF224761）的同一性为97%。从佛罗里达州西特拉的8株有症状的南瓜植株上采集叶片，用于烟粉虱传播试验。将大约100只B型烟粉虱成虫释放到每个装有笼子的叶片上，并给予24小时的获毒时间，之后引入一株健康的南瓜幼苗。所有试验植株在10天内出现症状，并通过PCR检测（引物对PAR1c496/PAL1v1978和PBL1v2040/PCRc154）并测序确认存在CuLCrV。2007年，该州多个地方出现了类似症状。同样的检测方法确认了佛罗里达州西南部的科利尔县和亨德里县以及佛罗里达州中西部的希尔斯伯勒县、马纳蒂县和萨拉索塔县的西瓜（西瓜种）和南瓜中存在CuLCrV。据我们所知，这是CuLCrV的首次报道，也是佛罗里达州葫芦科作物中任何双生病毒的首次报道。参考文献：（1）M. R. Rojas等人，《植物病害》77:340，1993年。（2）S. D. Wyatt和J. K. Brown，《植物病理学》86:1288，1996年。
