利用种特异性COI引物（SS-COI）鉴别扶桑绵粉蚧

›› 2013, Vol. 56 ›› Issue (6): 689-696.

利用种特异性COI引物（SS-COI）鉴别扶桑绵粉蚧

田虎¹, 李小凤¹, 万方浩^1,2, 张桂芬^1,2,*, 张金良³

(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. 北京市植保站, 北京 100029)

出版日期:2013-06-20 发布日期:2013-06-20

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

TIAN Hu¹, LI Xiao-Feng², WAN Fang-Hao^1,2, ZHANG Gui-Fen^1,2,*, ZHANG Jin-Liang³

(1. State Key Laboratory for the Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China; 2. Center for Management of Invasive Alien Species, Ministry of Agriculture, Beijing 100081, China; 3. Beijing Plant Protection Station, Beijing 100029, China)

Online:2013-06-20 Published:2013-06-20

摘要/Abstract

摘要： 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546 bp。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。

关键词: 扶桑绵粉蚧, 粉蚧, 种特异性COI引物, 分子检测, 快速鉴定

Abstract: 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.

Key words: Phenacoccus solenopsis, mealybugs, species-specific COI (SS-COI) primers, molecular detection, rapid identification

田虎, 李小凤, 万方浩, 张桂芬, 张金良. 利用种特异性COI引物（SS-COI）鉴别扶桑绵粉蚧[J]. , 2013, 56(6): 689-696.

TIAN Hu, LI Xiao-Feng, WAN Fang-Hao, ZHANG Gui-Fen, ZHANG Jin-Liang. Identification of Phenacoccus solenopsis Tinsley (Hemiptera: Pseudococcidae) with species-specific COI (SS-COI) primers[J]. , 2013, 56(6): 689-696.

[1]	黄俊, 智伏英, 吕要斌. 班氏跳小蜂对新寄主资源的寄生适合度[J]. 昆虫学报, 2019, 62(12): 1427-1434.
[2]	智伏英, 黄芳, 黄俊, 郦卫弟, 吕要斌. 石蒜绵粉蚧生物学特性[J]. , 2018, 61(7): 871-876.
[3]	李思怡, 王吉锐, 赖秋利, 张胜娟, 邵炜冬, 徐志宏. 温度对石蒜绵粉蚧生长发育和繁殖的影响[J]. , 2018, 61(10): 1170-1176.
[4]	姚志文, 杨帆, 吴月坤, 魏洪义, 陆宴辉. 放置时间和条件对空僵蚜中蚜虫和寄生蜂DNA检出率的影响[J]. , 2017, 60(6): 691-698.
[5]	胡俊杰, 孟翔, 周佳滨杨陆兴, 刘善海, 李润钊. 扶桑绵粉蚧转录组分析[J]. 昆虫学报, 2017, 60(1): 9-17.
[6]	王玉生, 周培, 田虎, 万方浩, 张桂芬. DNA条形码技术对重大潜在入侵害虫大洋臀纹粉蚧的鉴定有效性研究[J]. 昆虫学报, 2016, 59(7): 747-758.
[7]	陈苗苗, 郭荣, 张金良, 万方浩, 杨锦, 张桂芬. 基于种特异性COI标记的新入侵种甘蓝粉虱快速鉴定技术[J]. , 2015, 58(5): 579-586.
[8]	黄振, 陈韶萍, 谢婧, 郭琼霞. 应用种特异性PCR技术快速鉴定辣椒实蝇[J]. , 2015, 58(4): 460-466.
[9]	黄芳, 赵春玲, 吕要斌. 共生菌Tremblaya princeps在扶桑绵粉蚧个体发育中的动态变化[J]. , 2015, 58(10): 1140-1145.
[10]	黄芳，王飞飞，张治军，郦卫弟，吕要斌. 转换寄主前后扶桑绵粉蚧取食行为的EPG分析[J]. , 2014, 57(4): 503-508.
[11]	王飞飞，朱艺勇，黄芳，杜亮，吕要斌. 温度对扶桑绵粉蚧生长发育的影响[J]. , 2014, 57(4): 436-442.
[12]	王超，陈芳，陆永跃. 不同光周期条件下棉花粉蚧的生长发育和种群增长能力[J]. , 2014, 57(4): 428-434.
[13]	罗梅，张鹤，宾淑英，林进添. 基于转录组数据高通量发掘扶桑绵粉蚧微卫星引物[J]. , 2014, 57(4): 395-400.
[14]	陈芳, 陆永跃. 温度胁迫下棉花粉蚧热激蛋白基因的表达分析[J]. , 2014, 57(11): 1253-1264.
[15]	陈芳, 陆永跃. 热胁迫下棉花粉蚧内参基因的筛选[J]. , 2014, 57(10): 1146-1154.