利用双亲灭活原生质体融合技术选育高效苏云金芽胞杆菌和枯草芽胞杆菌融合菌株

摘要

苏云金芽胞杆菌B7发酵液在药后96 h对小菜蛾2龄幼虫致死率高达85.06％;枯草芽胞杆菌TL2对黄瓜枯萎病和辣椒疫霉病有显著拮抗活性(对峙培养5d对病原菌的平均抑制率在60％以上).本文对菌株B7和TL2的原生质体形成、完全灭活及融合的最佳条件分别进行了筛选,研究结果表明菌株B7在溶菌酶1.0 mg/mL、酶解70 min的条件下,原生质体形成率和再生率最佳,分别为85.0％和29.41％;菌株TL2在溶菌酶1.25 mg/mL、酶解50 min的条件下,原生质体形成率和再生率最佳,分别为80.00％和25.00％.菌株TL2原生质体完全灭活的最佳条件为60℃水浴35 min,菌株B7原生质体完全灭活的最佳条件为紫外照射25 min.灭活后的两亲本融合最佳条件为30℃水浴融合20 min后涂布再生培养基,用影印法连续传代10次以上,最后筛选出17株稳定的融合子,经过杀虫防病检测最后筛选到一株兼具两亲本特性的高效融合子TLB15. TLB15发酵液在药后96 h对小菜蛾2龄幼虫校正死亡率为93.44％,TLB15发酵液对黄瓜枯萎病和辣椒疫霉病(对峙培养5d)的平均抑制率分别为60.00％和62.79％.%Fermentation broth of Bacillus thuringiensis B7 caused 85.06％ mortality ofPlutella xylostella 2nd instar larvae at 96 h after treatment.Bacillus subtilis TL2 showed antagonistic effects on pathogenic fungi Fusarium oxysporum and Phytophthora capsici,which reduced more than 60％ of mycelial growth of these fungi at 5 d after confronting incubation.The best condition for protoplast obtaining,complete inactivation,and protoplast fusion of these two bacterial strains were determined.The best protoplast formation rate and regeneration rate were 85.00％ and 29.41％,respectively,for strain B7 under 1.0 mg/L of lysozyme for 70 min.The best protoplast formation rate and regeneration rate were 80.00％ and 25.00％,respectively,for strain TL2 under 1.25 mg/L of lysozyme for 50 min.The best protoplasts inactivation condition for strain TL2 was water bath at 60 ℃ for 35 min,and for strain B7,ultraviolet radiation for 25min.The best protoplast fusion condition was water bath at 30 ℃ for 20 min.Then the mixture was spread on regeneration medium plates.Seventeen fusants were obtained after 10 times of subcultures,and the fusant TLB 15 showed both high insecticidal and fungicidal activity.Fermentation broth of TLB 15 resulted in 93.44％ mortality of P.xylostella 2nd instar larvae at 96 h after treatment and inhibited 60.00％ and 62.79％ of mycelial growth of F.oxysporum and P capsici,respectively (5 d of confrontation cultivation).