福建新记录入侵害虫木瓜秀粉蚧的分子检测鉴定

A molecular detection and identification method for papaya mealybug (Paracoccus marginatus), the first recorded invasive pest in Fujian province of China

【目的】基于核糖体28S rDNA基因序列的种特异性PCR方法,快速鉴定福建新纪录入侵性害虫——木瓜秀粉蚧(Paracoccus marginatus Williams and Granara de Willink)。【方法】通过1对扩增粉蚧28S rDNA的通用引物(S3660/A335)扩增以木瓜秀粉蚧为靶标,以野外采集的另外10种粉蚧为对照的11种粉蚧的基因片段序列。根据所得基因序列结果并参照GeneBank数据库中已知粉蚧的28S rDNA基因序列,设计出木瓜秀粉蚧28S rDNA的种特异性引物(28S-ParF/28S-MarR),对该种特异性引物的效果进行检验。【结果】该种特异性引物对木瓜秀粉蚧的28S rDNA基因具有扩增能力,得到产物大小为446 bp的扩增片段,对其他10种粉蚧均无扩增效果。该引物不仅对木瓜秀粉蚧雌成虫具有稳定的扩增效果,对不同虫态、不同寄主来源以及不同地区来源的木瓜秀粉蚧均有稳定的扩增效果。【结论】对福建发现的木瓜秀粉蚧进行分子鉴定并建立了木瓜秀粉蚧快速分子检测技术,可用于识别和防控木瓜秀粉蚧,有助于遏制木瓜秀粉蚧的入侵危害。

【Objective】Paracoccus marginatus Williams and Granara de Willink is an invasive pest with strong diffusion and fecundity.It has caused serious damage to the papaya industry in Central America,Florida(USA),Guam(USA)and India.Pa.marginatus was first discovered in Fujian Province(Fuzhou and Zhangzhou)in 2017,showing great potential risks to papaya and other fruit crops,as well as flower industry in Fujian province.Because of the small body size and similar morphological characteristics,the morphological identification of mealybugs was inefficient.Rapid molecular identification of different species could be achieved through the use of DNA barcoding technology.Therefore,a technology for rapid molecular identification of Pa.marginatus was established based on the species-specific PCR method.【Methods】A species-specific PCR method based on ribosomal DNA-28S gene fragment(28S rDNA)was exploited to establish one technology for rapid detection and identification of Pa.marginatus.The additional 10 species of mealybugs(Phenacoccus solenopsis,Dysmicoccus boninsis,Nipaecoccus viridis,Phenacoccus solani,Pseudococcus comstocki,Pseudococcus cryptus,Planococcus lilacinus,Pseudococcus odermatti,Planococcus minor and Phenacoccus madeirensi)were collected in the fields as the contrast.In order to ensure the uniqueness of the source of DNA,the DNA templates were all extracted from one single female adult of these 11 species of mealybugs,respectively.28S rDNA of the 11 species was amplified by a pair of universal primers(S3660/A335).The obtained partial fragments of 28S rDNA were sequenced.And the phylogenetic tree was established by using a Neighbor-joining(NJ)method.According to the obtained 28S rDNA gene partial sequence of the 11 species and 28S rDNA gene sequences of Paracoccus galzerae in GeneBank database,the sequence alignment and analysis were performed on DNAMAN.28S rDNA species-specific primers(28S-ParF/28S-MarR)for Pa.marginatus were designed by selecting the sites with large differences in the sequence.And then,the specific effects,versatility and sensitivity of the specific primers were examined.【Results】The comparative results showed that the similarity between Pa.marginatus and Paracoccus marginatus isolate S3-668,KP692333 in the GenBank database was 100%.It was also indicated that the mealybugs were identified as Pa.marginatus by molecular identification.Phylogenetic analysis indicated that Pa.marginatus from Fuzhou and Zhangzhou was clustered in a clade.And that combined with Paracoccus galzerae(inter-species genetic distance is 0.058)to form a clade of the genus Paracoccus.The results of specificity tests showed that all Pa.marginatus specimens could be detected positively and a 446 bp fragment of the 28S rDNA of Pa.marginatus was obtained by the species-specific primers,while there was no cross reactions with other 10 species of mealybugs.The species-specific primers not only had a stable amplification effect on female adults,but also were proved to be applicable for the 2nd instar nymphs and the 3rd instar nymphs.Pa.marginatus from three different regions(Fuzhou and Zhangzhou in Fujian province,Jinghong in Yunnan province)and six different host plants(Carica papaya,Solanum melongena,Plumeria rubra,Solanum tuberosum,Tithonia diversifolia and Duranta erecta)was also successfully detected by the species-specific primers.【Conclusion】Molecular identification of Pa.marginatus first reported in Fujian province was carried out based on 28S rDNA molecular markers.It was proved by experiments that the 28S rDNA species-specific primers had ideal and stable specificity for Pa.marginatus and could be used to identify Pa.marginatus accurately.A rapid molecular detection technique for Pa.marginatus was established based on the species-specific PCR method.The technology has the characteristics of accuracy,rapidity,sensitivity and simplicity.Our present results indicated that the rapid detection technique should be useful in quarantine at ports,in pest detection and in monitoring during transportation of papaya and other fruit tree seedlings,as well as flowers.However,in view of the fact that no other mealybugs of the genus Paracoccus has been reported in China,this study can provide a reference for the molecular identification for the closely related species of Pa.marginatus.