煙草粉蝨生物小種之分子鑑定及其二級內共生菌之初探

Abstract

煙草粉蝨為一種群 (species complex)，自命名以來，迄今於世界各地已有 24 個以上的生物小種 (biotype)，而生物小種間並無明顯的形態特徵可供區分，隨著分子生物技術的發展，應用分子標示來區分煙草粉蝨生物小種的研究日益增加。本研究主要根據 DNA 逢機增幅多態型聚合酶連鎖反應 (RAPD-PCR) 及粒線體 COI 序列為基礎，開發能有效鑑別煙草粉蝨 A、B、Q、Nauru、AN 及 S 生物小種之專一性引子組。結果顯示，逢機引子 OPA15 及 OPO18 可分別針對煙草粉蝨 A 及 Q 生物小種，增幅出特異性片段，依據此片段所設計之引子組 BaAF/BaAR 及 BaQF/BaQR，則可分別針對煙草粉蝨 A 及 Q 生物小種增幅出具鑑別力之 DNA 片段；此外，4 個正向引子 BaBF、BaNaF、BaANF 及 BaSF 則根據比對不同煙草粉蝨生物小種之粒線體 COI 序列設計，分別與粒線體 COI 之反向引子 L2-N-3014 配合，可針對煙草粉蝨 B、Na、AN 及 S 生物小種增幅出專一性片段。另一方面，臺灣地區所採集之 202 個煙草粉蝨族群，利用二級內共生菌之 16S rDNA 及Wolbachia 之 wsp 基因進行內共生菌的偵測，結果顯示，55.4% 的族群受二級內共生菌的感染，38.1% 的族群受 Wolbachia 的感染，再經由類緣關係分析，感染臺灣地區煙草粉蝨之二級內共生菌皆為 Proteobacteria γ-3 亞綱的種類，而 Wolbachia 則屬於 B 群的 Con/Rug 亞群的種類。由各生物小種感染之比例顯示，此二類內共生菌之分布與生物小種有密切的相關，然而影響其分布的因素仍需後續的研究以進一步釐清。

Bemisia tabaci is a worldwide serious pest possessing more than 24 biotypes; these biotypes are difficult to distinguish by morphological characters only. In this study, we designed the specific primer sets for each of 6 B. tabaci biotypes (A, B, Q, Na, AN, S). Two primer sets, BaAF/BaAR and BaQF/BaQR, for A and Q biotypes, respectively, were based on the sequences of RAPD specific fragments. Four forward primers, BaBF, BaNaF, BaANF, and BaSF, with a common reverse primer, L2-N-3014, for other 4 biotypes, B, Nauru, AN, and S, respectively, were based on their own COI sequence. The 16S ribosomal DNA (rDNA) of the secondary endosymbiont and Wolbachia-specific gene, wsp (cell surface protein of Wolbachia), were amplified and sequenced. Based on the occurrences of 16S rDNA and wsp by PCR, more than 55% and 38% of examined B. tabaci (202 populations) harbored secondary endosymbiont and Wolbachia, respectively. Molecular phylogenetic analysis of 16S rDNA sequences showed that the secondary endosymbiont was located in the clade of γ-3 subdivision of the Proteobacteria. wsp gene sequences, revealed that the Wolbachia found in B. tabaci populations of Taiwan belongs to the group B and subgroup Con/Rug. The results also showed that the carrying/infection rate of secondary endosymbiont/Wolbachia might be related to the biotype of B. tabaci.