苏云金芽孢杆菌的分离鉴定及伴孢晶体蛋白生物毒力效果分析

Study on Isolation and Identification of Bacillus thuringiensis and the Effect of Parasporal Crystal Protein Against Lasioderma serricorne

为发掘苏云金芽孢杆菌伴孢晶体蛋白对烟草甲幼虫生物毒力活性。以复烤片烟云烟87为供试材料,通过醋酸钠+抗生素的方法从烟叶表面分离菌株,采用生理生化指标对比和16S rDNA测序鉴定出苏云金芽孢杆菌。确定了其伴孢晶体的形态和基因型,考察了Bt菌株生长周期,分析了伴孢晶体蛋白并对烟草甲幼虫的毒力活性。结果表明:①分离得到的Bt菌株能产生菱形的cry1型和球形的cry8型伴孢晶体蛋白,其伴孢晶体蛋白对3~4周龄的烟草甲幼虫有很高的毒力活性。②分别使用不同浓度晶体蛋白处理后的烟叶感染烟草甲幼虫,1 d后幼虫出现死亡现象,形态变为黑褐色,生物毒力分别随感染时间和蛋白浓度梯度的增加而逐步提高。③感染4 d后150,10 mg/L的死亡率分别达(96.67±4.22)%,(51.11±5.02)%。结果不仅表明分离得到的苏云金芽孢杆菌伴孢晶体蛋白对烟草甲幼虫具有较强的生物毒性,且为进一步利用生物性技术防治烟叶仓储害虫提供理论参考。

To discovery of the biological virulence activity of Bacillus thuringiensis spore crystal protein on the Lasioderma serricorne.Using Yunyan No.87 as the test material,the bacterial strain was isolated from the tobacco leaf surface by sodium acetate+antibiotics,and the Bacillus thuringiensis was identified by comparison of physiological and biochemical indexes and 16S rDNA sequencing.The morphology and genotype of its companion parasporal crystal protein were determined,the growth period of Bt strain was investigated,the parasporal crystal protein was isolated,and the toxicity activity of the Lasioderma serricorne was analyzed.The results showed that:the isolated Bt strain could produce the rhomboid cry1 type and the spherical cry8 type parasporal crystal protein,and the parasporal crystal protein had a high toxic activity to the 3-4 week old Lasioderma serricorne.After the treatment with different concentrations of parasporal crystal protein,the Lasioderma serricorne wsa infected after 1 days,the Lasioderma serricorne died and the morphology turned to black and brown.The biological virulence respectively increased with the increase of the infection time and protein concentration gradient.The mortality rates of 150,10 mg/L after 4 days of infection were(96.67±4.22)%and(51.11±5.02)%.These results not only indicated that Bacillus thuringiensis spore crystal protein had a strong biological toxicity to the Lasioderma serricorne,but also provided a technical reference for the further use of biological technology to control tobacco storage pests.