| Mealybugs(Hemiptera: Pseudococcidae) constitute a very diverse group, with about 2000 species belonging to 266 genera described worldwide. Unfortunately, they are one of the most important quarantine pest category that injury a wide range of agricultural and ornamental plants worldwide and may cause serious problems. Usually, mealybugs intercepted in the quarantine inspection are eggs, nymphs or debris, coupled with the effects of phenotypic variations, cryptic species and complex species, which make the identification difficult or even sometimes impossible until recently. It is imperative to establish rapid and reliable methods to identify invasive mealybugs in plant quarantine. In this study, DNA barcoding based on mitochondrial DNA cytochrome c oxidase subunit(mtDNA COI) gene and 28 S r RNA was developed to apply in identification of mealybugs. The efficacy of DNA barcoding in identification of Planococcus minor(Maskell), a significant, potential alien species of China, is also clarified. A species-specific COI(SS-COI) primers for the rapid identify of Phenacoccus manihoti Matile-Ferrero was designed based on the analysis of COI-5’ of mealybugs, subsequently. The main results are as follows.(1) Evaluation of total DNA extraction methods for DNA barcoding of mealybugs. The SDS method always shows the most satisfactory performance for extracting DNA from high-instar nymphs and female adults, while the De Barro method is best for eggs and low instar nymphs for handling rapidly and simply.(2) Identification of common mealybug species based on DNA barcoding. We coupled sequencing at COI gene and 28 S rRNA to generate multi-criterion identification of 461 mealybug individuals of 22 mealybug species belong to 9 genus, that collected from 11 provinces of China and 6 other countries. The sequences of 5’ end of COI gene recovered successful as well as 28 S D2-D3 for 366 specimens. 48 and 33 haplotypes are observed in these 22 mealybug species at the sequences of COI-5’ end and 28 S D2-D3, respectively. Based on the COI-5’ end, inter-specific genetic distance averaged 0.0921(range from 0.0220 to 0.1780) which is 27.1 times higher than the intra-specific variation averaged 0.0034(range from 0.0000 to 0.0160). Obviously, there was no overlap between the inter-species and intra-species distance. Phylogenetic analysis indicated that haplotypes differences between species clearly separated several taxa. While, overlap was observed between Pl. minor and Pl. citri(Risso) in genetic distance at 28 S D2-D3 sequences as well as 28 S D2. And few differences between closely related species belong to one genus appear at 28 S gene that lead to disability to distinguish them. The effects of cryptic species and complex species may be exist, polyphyletic at the genus Pseudococcus can be observed in any case. Overall, DNA barcoding based on the COI-5’ can provide a rapid and accurate identification of mealybug species in plant quarantine, more accurate when coupled with 28 S gene.(3) Efficacy of DNA barcoding in distinguishing Pl. minor, a significant, potential alien species of China, from the native related species Pl. citri. The 5’- and 3’-end of COI and 28 S rRNA of 36 individuals of Pl. minor intercepted during passenger inspection were sequenced. When the 5’- and 3’-end of COI of Pl. minor were sequenced and Blast, the identity between the fragments from GenBank and obtained in this study was 100% and 99-100%, respectively. The identity between Pl. minor and Pl. citri was respective 97-98% and 96-98%. There were 5 and 12 stable species-specific identification sets in the fragments of the 5’- and 3’-end of COI between Pl. minor and Pl. citri. Phylogenetic analysis indicated that Pl. minor of this study and those from the GenBank were clustered in a clade. When the 28 S D2-D3 was analyzed, the conservativeness between species belong to the genus Planococcus is strong. Pl. minor and Pl. citri could not be distinguished; genetic distance between two species was 0.004. Furthermore, the results based on the software of Species Identifier shown that the identification based on 5’- and 3’-end of COI was completely correct, however, that based on D2-D3 fragment resulted in 45.2-61.9% of fuzzy identification. Our results suggested that DNA barcoding based on 5’- and 3’-end of COI can be used to identify and detect Pl. minor rapidly and accurately. It will be significant in intercepting and blocking the further spreading of Pl. minor.(4) Identification of Ph. manihoti with SS-COI primers. Ph. manihoti, a common invasive species in Asia and Africa, also a high risk to invade more countries in this region, is the main pest on Manihot esculenta Grantz, causing devastating loss to cassava production. Unfortunately, morphological identification of it is limited by high degree of similarity and polymorphism of related species. In this study, the SS-COI primers pair(PMSSZW-1F/PMSSZW-1R) for the rapid identify of Ph. manihoti was designed based on COI-5’ sequences of mealybugs. The SS-COI primers amplified a single band of 353 bp of Ph. manihoti. The species specificity of the primers was validated using eight other common mealybug species. The results showed that all and only Ph. manihoti specimens were correctly identified, and no cross reactions with other mealybugs were observed. The method was tested with single individuals of the egg, 1st, 2nd and 3rd instar nymphs and female adult, adult debris(head, thorax, abdomen, leg) and host plant(M. esculenta and Cucurbita moschata Duch) of Ph. manihoti. Evidently, this method is applicable for different development stages/adult debris and host plant of Ph. manihoti. In addition, the 353 bp section could be clearly identified for all repeats even at the dilution as low as 135.22±10.41 pg total DNA equal to 1/20480 of a whole female adult of Ph. manihoti. This method is promising, effective and reliable for the identification of Ph. manihoti, which should be applied in the customs quarantine, monitoring and management of this invasive pest, also is of great significance to the firmly cassava production.This work provides a solidly rapid and accurate technology of mealybugs identifying and will be used as basis for the identification at entry-exit inspection and quarantine as well as remote monitoring of mealybugs, combined with SS-PCR or other molecular tools derived from DNA barcoding to establishment comprehensive identifying system which will facilitate the import and export, ensuring the safety of agriculture industry, and provide the technical support to the “One Belt and One Road”. |
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