白色拟青霉分离鉴定及优势毒力菌株筛选

Isolation, Identification and Screen ofDominant Virulent Strains of Simplicillium lanosoniveum

用选择性培养方法从土壤中分离纯化6株真菌,经形态学初步鉴定为拟青霉属Simplicillium.采用PCR扩增、克隆了6株真菌的核糖体ITS序列,并进行系统进化分析,以明确菌株的分类地位.结果显示,6株真菌的序列长度为616 bp.系统进化分析发现,6株真菌与白色拟青霉Simplicillium lanosoniveum(GeneBank NO. KT878334、 MT001191)在同一进化分支上,相似性为99.9%~100%.形态学和分子鉴定表明,6株真菌为S.lanosoniveum.采用孢子浸渍法,用含量为1×108个孢子/mL的孢子悬液侵染家蚕3龄幼虫进行优势毒株筛选(球孢白僵菌Beauveria bassiana和金龟子绿僵菌Metarhizium anisopliae为阳性对照组).结果显示,各处理组累积校正死亡率最高为92.30%,最低为30.78%.其中,S.lanosoniveum-Sl05,Sl06菌株接种7 d后累积校正死亡率分别为84.62%和89.29%,致死中时LT50分别为3.96 d和3.73 d.S.lanosoniveum-Sl05,Sl06菌株为家蚕3龄幼虫致病的优势毒力菌株.本研究为S.lanosoniveum菌株的生物防治奠定了基础.

Six fungal strains were isolated and purified from the soil by selective culture method,and morphologically preliminarily identified as Simplicillium genus.Then the ribosomal ITS sequences of six fungal strains were amplified and cloned by PCR,and phylogenetic tree was constructed to clarify the taxonomic position of fungal strains.The results showed that the ITS sequences of the six strains were 616 bp in length.Phylogenetic analysis showed that the six fungal strains were on the same evolutionary branch and 99.9%-100%homology with the S.lanosoniveum(GeneBank NO.KT878334 and MT001191).Combined with morphological and molecular identification,the six isolates of fungi were identified as S.lanosoniveum.The dominant virulent strain was selected by spore impregnation method by infesting the 3rd instar of Bombyx mori with a concentration of 1×108 spores/mL(Beauveria bassiana and Metarhizium anisopliae were used as positive controls).The results showed that the highest cumulative corrected mortality rate was 92.30%and the lowest was 30.78%for each treatment.Among them,the cumulative corrected mortality of S.lanosoniveum-Sl05 and Sl06 strains after 7 days of inoculation were 84.62%and 89.29%,respectively,and the median mortality(LT 50)was 3.96 days and 3.73 days,respectively.Therefore,the S.lanosoniveum-Sl05 and Sl06 strains were the dominant virulent strains for the third instar larvae of the B.mori.The study lays the foundation for the biological control of S.lanosoniveum strains.