黑龙江省苏云金杆菌的分离及对夜蛾科昆虫高毒力菌株的筛选

Screening and identification highly virulent strain of Bacillus thuringiensis from the soil of Heilongjiang Province to control Noctuidae insects

【目的】为了发掘新的苏云金杆菌(Bacillus thuringiensis)的资源,在黑龙江省不同地区采集不同类型的土壤样品分离出对夜蛾科具有高毒力的菌株。【方法】采用醋酸钠选择性筛选法筛选Bt菌株,利用10对通用引物对分离株进行基因型分析,SDS-PAGE进行杀虫晶体蛋白分析,同时测定苏云金杆菌分离株对棉铃虫Helicoverpa armigera(Hübner)、甘蓝夜蛾Mamestra brassicae(Linnaeus)、斜纹夜蛾Spodoptera litura(Fabricius)的杀虫活性。【结果】从黑龙江省不同地区采集的352份不同类型的土壤样品中,共分离出46株苏云金芽孢杆菌野生菌株,出菌率为13.06%。油镜下可观察到伴孢晶体的形态有菱形、球形、镶嵌形及不规则形。结果表明产菱形晶体的菌株多含有cry1类基因,而同时产生菱形、球形及不规则形晶体的菌株则含有多种基因型。SDS-PAGE蛋白分析发现这些菌株主要表达130、90、60 ku蛋白。对其中的部分菌株进行毒力测定,结果表明有4株菌株对3种夜蛾科昆虫具有高毒力。【结论】黑龙江省苏云金芽孢杆菌分布广泛,类型多样,已获得对夜蛾科昆虫有高毒力的菌株,这对夜蛾科害虫的绿色防控及延缓其抗性具有重要意义。

[Objectives] To find new, highly insecticidal, Bacillus thuringiensis stains with which to control Noctuid insects. [Methods] B. thuringiensis strains were isolated from soil in Heilongjiang province using Sodium acetate selective screening. The genotypes of B. thuringiensis isolates were identified with 10 general primers. SDS-PAGE was used to analyze insecticidal crystal proteins. The insecticidal activity of these isolates against Helicoverpa armigera（Hübner）, Mamestra brassicae（Linnaeus） and Spodoptera litura（Fabricius） was assayed. [Results] A total of 46 strains of B. thuringiensis were isolated from different soils from various regions in Heilongjiang province. The percentage of B. thuringiensis strains isolated was 13.06%. Observed under an oil lens, the shapes of parasporal crystals were bipyramidal, spherical, mosaic and irregular. Strains with bipyramidal crystals contained cry1 genes whereas those with spherical and irregular shapes contained other genes. The result of SDS-PAGE showed that these strains expressed 130, 90 and 60 ku proteins. In toxicity tests, four B. thuringiensis strains were high toxic to Noctuid insects. [Conclusion] Different strains of B. thuringiensis are widely distributed in Heilongjiang province including those that are highly toxic to Noctuid insects. It is important to develop environmentallyfriendly methods of control for Noctuid insects and to delay the development of resistance in these pests.