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

Abstract: 【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-28 S gene fragment(28 S 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.28 S rDNA of the 11 species was amplified by a pair of universal primers(S3660/A335). The obtained partial fragments of 28 S rDNA were sequenced. And the phylogenetic tree was established by using a Neighbor-joining(NJ) method. According to the obtained 28 S rDNA gene partial sequence of the 11 species and 28 S rDNA gene sequences of Paracoccus galzerae in GeneBank database, the sequence alignment and analysis were performed on DNAMAN. 28 S rDNA species-specific primers(28 S-ParF/28 S-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 28 S 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 the2 ndinstar nymphs and the 3 rdinstar 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 28 S rDNA molecular markers. It was proved by experiments that the 28 S 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.