绿僵菌属4个生防潜力菌株的多基因鉴定

Multigene Identification of Four Potential Biocontrol Strains in Metarhizium Genus

传统的绿僵菌分类鉴定以形态特征为主要依据,其局限性使种内仍包含着复杂的类群。DNA分子鉴定使绿僵菌属、种分类更为细化和准确,对其育种、杀虫机理、生态学等的研究非常重要。本文对绿僵菌4个有生防潜力的菌株IPPM010202、IPPM2029、IMI330189和M200614的5个持家基因即核糖体转录间隔区序列(ITS1-4)、β微管蛋白(beta-tubulin)、RNA聚合酶Ⅱ大亚基(RPB1)、RNA聚合酶Ⅱ第二大亚基(RPB2)和翻译延伸因子(EF-1α)的同源序列进行了克隆和测序,通过构建系统发育树进行了多基因进化分析。结果表明,4个生防菌株分别位于进化树的3个分支上,鉴定菌株IPPM010202和IPPM2029同属于平沙绿僵菌Metarhizium pingshaense,IMI330189为蝗绿僵菌M.acridum,M200614属于低温绿僵菌M.frigidum,它们之间亲缘关系很近,5个基因的遗传分化距离只有0.021~0.036。ITS1-4序列有较高变异频率和变异幅度,分类效率优于其他基因;EF-1α序列在鉴别菌株和分析亲缘性上有优势。多基因的系统进化分析能够弥补单基因分析的局限,取得更为准确的鉴定和更为细化的分类结果。

The classical identification of Metarhizium spp. is based on the morphological characteristics, which usually results in complex groups within a species. DNA molecular identification can offer a more refined and accurate classification of the species in Metarhizium genus, which is very important for studies on breeding, insecticide mechanism and ecology of the fungus. Here, four potential biocontrol strains of MetarhiziumIPPM010202, IPPM2029, IMI330189 and M200614 were identified by phylogenetic analysis of five identified genes, i.e., ribosomal transcribed spacer sequences（ITS1-4）, beta-tubulin, RNA polymerase Ⅱ first, second subunit（RPB1 and RPB2）, and translation extension factor（EF-1α）. Based on the phylogenetic tree constructed from the four candidate strains and twenty-two reference strains, the four strains were located on three branches of the phylogenetic tree. The strain IPPM010202 and IPPM2029 belong to M. pingshaense, IMI330189 is identified as M. acridum and M200614, as M. frigidum, a species adaptive to low temperature. They have close genetic relationship between each other, with genetic distance of 0.021―0.036. Also, the results showed that the ITS1-4 sequence had higher mutation frequency and mutation range, which makes it a superior classification gene. The EF-1α sequence had the advantage in identifying the strain and analyzing genetic relationship. The multigene phylogeny can compensate for the limitations of single gene analysis, so that it can obtain a more accurate identification and more detailed classification.