对二点委夜蛾高毒力苏云金芽胞杆菌的筛选及其基因型分析

Selection and genotype analysis of Bt isolates with high virulence against Athetis lepigone (Mschler)

【目的】获得对二点委夜蛾Athetis lepigone(M(o|¨)schler)高毒力的苏云金芽胞杆菌(Bt)菌株,寻找对该虫具有特异杀虫活性的蛋白毒素,探索Bt菌株或其杀虫基因应用于二点委夜蛾防治的可行性。【方法】通过生物测定方法比较了36株苏云金芽胞杆菌和一株恶臭假单胞工程菌PHB-cry1Ab对二点委夜蛾幼虫的杀虫活性,同时利用PCR-RFLP方法对这些菌株的基因型进行了分析。【结果】不同Bt菌株对二点委夜蛾幼虫的杀虫活性差别很大,杀虫活性高的菌株都含有cry1Ac基因。饲毒72 h后含单基因的BtHD-73菌株(cry1Ac)对二点委夜蛾2龄幼虫的毒力(LC_(50)值为188.51μg/g)明显高于含多基因的Bt SC-40菌株(cry1Ac,cry2Ac,cry1I,vip3A)的毒力(LC_(50)值为418.13μg/g)。含有vip3A基因的Bt SC-40和BtHD-13营养期上清液对二点委夜蛾2龄幼虫表现出一定的杀虫活性(72 h死亡率分别达到42.5%和57.4%),而无vip3A基因的Bt HD-73营养期上清液未表现出明显的杀虫活性。【结论】由cry1Ac基因编码的Cry1Ac蛋白对二点委夜蛾幼虫具有特异杀虫活性,Vip3A蛋白对二点委夜蛾幼虫可能也有一定的杀虫活性。

[Objectives] To obtain Bt strains and insecticidal proteins with high insecticidal activity against Athetis lepigone （Moschler）. [Methodsl The insecticidal activities of 36 Bt strains and an engineered Pseudomonas putida with a crylAb gene were evaluated on second instar larvae of A. lepigone by bioassay. PCR-RFLP analysis was performed to verify the genotypes of all Bt isolates. [Results] The bioassay results indicated obvious differences in the insecticidal activities of the different Bt isolates tested. The results also revealed that the Bt strains with high insecticidal activity all contained the crylAc gene. Furthermore, the results of a comparison of the toxicities of Bt SC-40 （crylAc, cry2Ac, cryll, vip3A） and Bt HD-73 （crylAc） showed that the LC50 of BT HD-73 （188.51μg/g） was lower than that of Bt SC-40 （418.13μg/g）. The supematants from the vegetative stage of Bt SC-40 and Bt HD-1 with the vip3A gene showed certain insecticidal activities （42.5 % and 57.4 % mortality, respectively） to second instar larvae ofA. epigone. However, the supematants of Bt HD-73 with no vip3A gene did not show obvious insecticidal activity. [Conclusion] These results imply that the CrylAc toxin encoded by the crylAc gene play an important role in the resistance of plants to A. lepigone larvae. Vip3A may also have some insecticidal properties with regard to this pest.