食用菌异迟眼蕈蚊苏云金芽孢杆菌筛选及杀虫蛋白基因鉴定  

Screening of Bacillus thuringiensis and identification of insecticidal crystal protein gene against Bradysia difformis in mushroom cultivation

作　　者：王帆帆 曲绍轩[2] 林金盛[2] 李辉平[2] 侯立娟[2] 蒋宁[2] 骆昕 马林[2,3] 韩巨才 WANG Fanfan;QU Shaoxuan;LIN Jinsheng;LI Huiping;HOU Lijuan;JIANG Ning;LUO Xin;MA Lin;HAN Jucai(College of Agriculture, Shanxi Agricultural University, Taigu 030801, Shanxi, China;Vegetable Research Institute, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China;Jiangsu Key Laboratory for Horticultural Crop Genetic Improvement, Nanjing 210014, China)

机构地区：[1]山西农业大学农学院,山西太谷030801 [2]江苏省农业科学院蔬菜研究所,南京210014 [3]江苏省高效园艺作物遗传改良重点实验室,南京210014

出　　处：《浙江大学学报（农业与生命科学版）》2019年第2期189-195,共7页Journal of Zhejiang University:Agriculture and Life Sciences

基　　金：国家现代农业产业技术体系建设专项资金(CARS20)

摘　　要：苏云金芽孢杆菌(Bacillus thuringiensis, Bt)杀虫谱广,活性高,可以很好地应用于双翅目眼蕈蚊科的防治。为了筛选出对食用菌异迟眼蕈蚊(Bradysia difformis)有高毒力的苏云金芽孢杆菌菌株,并进行杀虫蛋白基因鉴定,采用醋酸钠-抗生素法,从江苏省南京市紫金山土壤中分离产晶体芽孢杆菌,通过室内毒力测定和对食用菌菌丝影响试验逐步进行筛选,同时进行Bt杀虫蛋白基因型聚合酶链式反应-限制性片段长度多态性(polymerase chain reaction-restricted fragment length polymorphism, PCR-RFLP)扩增鉴定。结果从分离到的17株产晶体芽孢杆菌中筛选出7个对异迟眼蕈蚊杀虫活性的校正死亡率在50%以上且不影响食用菌菌丝生长的菌株。经16S rDNA基因序列同源性分析,这7株菌与公共数据库中苏云金芽孢杆菌菌株的同源性可达99%以上,初步认定为苏云金芽孢杆菌(B. thuringiensis)。Bt杀虫蛋白基因型鉴定发现,这7个菌株含有cry4/10、cry11、cyt1、cyt2等4个不同的毒蛋白基因型。筛选出的这7株Bt菌株对于防治食用菌异迟眼蕈蚊具有较好的开发利用潜力。Bacillus thuringiensis (Bt) is exhibiting the high ability and wide insecticidal spectrum in the Sciaridae pest control. This experiment was conducted to screen out effective Bt strains and identify their insecticidal crystal protein (ICP) genotypes against Bradysia difformis in the mushroom cultivation. The bacillus strains that produced crystals were separated from the soil of Zijin Mountain of Nanjing by sodium acetateantibiotic method. The strains that showed insecticidal activity against larvae of B. difformis and inhibitory effect to the hyphae of mushroom were screened by using indoor toxicity measurement method and effect test on hyphae, separately. Then ICPs were identified based on polymerase chain reaction-restricted fragment length polymorphism (PCR-RFLP) analysis. Seven strains that showed high insecticidal activity (with the corrected mortality over 50%) against B. difformis and no influence on Pleurotus ostreatus were screened from 17 strains obtained from Nanjing. The 16S rDNA analysis results showed that the seven strains were homologous to B. thuringiensis with 99% identity. The PCR-RFLP system results revealed that the seven strains contain cry4/10, cry11, cyt1, and cyt2 genes. The Bt strains have potential for application to the biocontrol of B. difformis in mushroom cultivation.

关 键 词：异迟眼蕈蚊 苏云金芽孢杆菌 杀虫活性 杀虫晶体蛋白基因 

分 类 号：S436.462[农业科学—农业昆虫与害虫防治]