利用种特异性COI引物(SS-COI)鉴别扶桑绵粉蚧

Identification of Phenacoccus solenopsis Tinsley ( Hemiptera:Pseudococcidae) with species-specific COI (SS-COI) primers

扶桑绵粉蚧Phenacoccus solenopsis Tinsley是我国近年新发现的一种严重威胁农林业生产的重要外来入侵害虫。针对扶桑绵粉蚧与其他粉蚧类昆虫难以准确快速识别且适生区广泛的问题,以扶桑绵粉蚧为靶标,以我国常见的其他7种粉蚧为参照,采用基于线粒体DNA细胞色素C氧化酶亚基Ⅰ(mtDNA COI)基因序列的种特异性(species-specific COI,SS-COI)PCR方法,研究其快速分子检测技术。通过已知粉蚧的COI基因序列设计通用型引物1对,获得扶桑绵粉蚧及其他7种粉蚧包括康氏粉蚧Pseudococcus comstocki Kuwana、南洋臀纹粉蚧Planococcus lilacius Cockerell、木槿曼粉蚧Maconellicoccus hirsutus(Green)、甘蔗红粉蚧Saccharicoccus sacchari(Cockerell)、新菠萝灰粉蚧Dysmicoccus neobrevipes Beardsley、番石榴粉蚧Planococcus minor Maskel和石蒜绵粉蚧Phenacoccus solani Ferris的COI基因序列,根据测序结果及数据库中已知粉蚧的COI基因序列设计SS-COI引物1对(PSZTF1/PSZTR1),其扩增片段大小为546bp。种特异性检验结果表明,该引物只对扶桑绵粉蚧的COI基因具有扩增能力,对其他7种粉蚧不具有扩增效果;该引物不仅对成虫具有良好的扩增能力,对不同虫态的扶桑绵粉蚧以及来自我国不同省市的14个地理种群和口岸截获的来自巴基斯坦的扶桑绵粉蚧亦具有同样的扩增效能。这些结果表明,该技术体系完全可用于扶桑绵粉蚧的准确识别及其检测监测,对有效阻截其进一步扩张蔓延意义重大。

Phenacoccus solenopsis Tinsley, a new invasive species in China, is a worldwide pest causing serious threat to the production of agriculture and forestry. Morphological identification of this mealybug species is limited by high degree of similarity and polymorphism. In the present study, a method was described for the development of DNA markers for rapidly identifying P. solenopsis. A pair of universal primers was designed based on published mitochondrial DNA cytochrome c oxidase subunit Ⅰ （mtDNA COI） gene sequences of mealybugs in GenBank. The mtDNA COI genes from P. solenopsis and seven other mealybug species common in China including Pseudococcus comstocki Kuwana, Planococcus lilacius Cockerell, Maconellicoccus hirsutus （Green）, Saccharicoccus sacchari （Cockerell）, Dysmicoccus neobrevipes Beardsley, Planococcus minor Maskel and Phenacoccus solani Ferris were amplified and sequenced. And then one pair of species-specific COI （SS-COI） primers was designed. The SS-COI primers amplified a single band of 546 bp from P. solenopsis. The specificity of the primer pair was validated using the seven other mealybug species mentioned above. The results showed that all and only P. solenopsis specimens were detected, and no cross reactions with other mealybugs were observed. The method was tested with individuals of the egg, 1st, 2nd and 3rd instar nymph and female adult, and demonstrated to be applicable for all life stages. Furthermore, the primer set was tested with one P. solenopsis population from Pakistan and fourteen P. solenopsis populations from invaded areas in China and proved to be applicable for all geographic populations. The diagnostic PCR assay developed here provides a quick, simple and reliable molecular technique for the identification and monitoring of P. solenopsis, which will be useful in intercepting and blocking the further spreading of P. solenopsis.