双钩巢粉虱的种特异性SS-COⅠ检测技术  

Species-specific COⅠ primers to assist the molecular identification of the invasive whitefly,Paraleyrodes pseudonaranjae

作　　者：张桂芬[1,2] 郭建洋[1,2] 王瑞[1,2] 杨婷[1] 李小凤[1] 郭建英 曹凤勤[3] 张金良 万方浩[1,2] 

机构地区：[1]中国农业科学院植物保护研究所,植物病虫害生物学国家重点实验室,北京100193 [2]农业部外来入侵生物预防与控制研究中心,北京100081 [3]海南大学环境与植物保护学院,海南海口570228 [4]北京市植物保护站,北京100029

出　　处：《生物安全学报》2013年第3期157-162,共6页Journal of biosafety

基　　金：国家"973"计划项目(2009CB119200);公益性行业(农业)科研专项(200903034;201103026;201303019)

摘　　要：【背景】双钩巢粉虱是近年在我国南方新发现的一种入侵害虫，其寄主范围广，危害严重，进一步扩散蔓延趋势明显。【方法】针对粉虱类害虫体型微小、形态相似、难以准确快速识别的问题，以双钩巢粉虱为靶标，以我国常见的其他8种／隐种粉虱为参照，采用基于mtDNACOI基因的种特异性SS—COI方法，研究其快速分子检测技术。利用COI通用型引物LCO．1490／HCO．2198获得双钩巢粉虱及其他常见粉虱的COI序列，根据测序结果设计特异性SS—COI引物1对（PPZYFl／PPZYRl），其扩增片段大小为233bp。【结果】种特异性检验结果显示，该引物只对双钩巢粉虱的COI基因具有扩增能力，对其他种／隐种粉虱，包括烟粉虱的不同隐种（MEAMl隐种、MED隐种、AsiaⅡ3隐种和AsiaII1隐种）以及温室粉虱、柑橘粉虱、黑刺粉虱、螺旋粉虱等不具有扩增效果。该引物不仅对不同性别的成虫具有良好的扩增能力，对1—4龄若虫和单粒卵亦具有同样的扩增效能，其最低检测阈值为1／40960头雌性成虫。【结论与意义】本检测技术对口岸检疫以及观赏植物、果树及其种苗调运中的害虫检测、监测意义重大。[ Background ] Paraleyrodes pseudonaranjae Martin, a new invasive species in southern China, is a worldwide pest, causing serious damage to a wide range of host plants. Further spread of this whitefly species in China is expected. Most of whitefly species are small in size and similar in morphology, which makes them hard to be identified quickly. [ Method ] In the present study, we developed DNA markers to rapidly identify P. pseudonaranjae. The fragments of mitochondrial DNA cytochrome oxidase I （mtDNA COI） gene of P. pseudonaranjae and six other whitefly species were amplified （using COI gene 5＇ end universal primers LCO-ldg0/HCO-2198） and sequenced. A pair of P. pseudonaranjae species-specific COI（SS-COI） primers （ PPZYF1/PPZYR1 ） were developed. The target frag- ment amplified by the SS-COI primers PPZYF1/PPZYR1 was 233 bp long. [ Result ] The species specificity of the primer pair was val idated using eight other whitefly species, including different cryptic species of Bemisia tabaci （Gennadius） （MEAM1, MED, Asia 113 and AsiaⅡ 1 ） as well as Trialeurodes vaporariorum （Westwood） , Dialeurodes cirri （Ashmead） , Aleurocanthus spiniferus （Quaintance） and Aleurodicus dispersus （Russell）, all of them common in China. All P. pseudonaranjae specimens were detected, and no cross reac- tions with other whitefly species were obtained. The method was tested on single male and female adult, single red-eyed nymph, single 2nd- and 3rd-instar nymph, and even single egg and newly emerged nymph, and proved to be applicable for all of these life stages. When the dilution was 1/40960 of a whole female adult of P. pseudo naranjae, the 233 bp DNA fragment could be identified for all re-peats. [ Conclusion and significance ] The technique would be useful in quarantine at ports of entry and in pest detection and monitoring during transportation of ornamental plants, fruits and seedlings.

关 键 词：双钩巢粉虱 种特异性SS—COI引物 快速鉴定 分子检测 

分 类 号：S433[农业科学—农业昆虫与害虫防治]