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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5 d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/m L、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/m L、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5 d)的平均抑制率分别为60.00%和62.79%。
The lethal rate of B. thuringiensis B7 fermentation broth to the 2nd instar larvae of Plutella xylostella 96 h was up to 85.06%; B. subtilis TL2 had significant antagonistic activity against Fusarium oxysporum and Phytophthora capsici (average inhibition of pathogenic bacteria for 5 d Rate above 60%). In this paper, the optimal conditions of protoplast formation, complete inactivation and fusion of strains B7 and TL2 were respectively screened. The results showed that protoplasts formed under the conditions of lysozyme 1.0 mg / mL and enzymolysis 70 min The rate and regeneration rate were the best (85.0% and 29.41%, respectively). The highest rate of protoplast formation and regeneration was found in strain TL2 with lysozyme 1.25 mg / m L and enzymatic hydrolysis for 50 min, which were 80.00% and 25.00 %. The optimal condition for inactivating the protoplast of strain TL2 was 35 min in water bath at 60 ℃. The optimal conditions for complete inactivation of strain B7 protoplasts were UV irradiation for 25 min. The optimal conditions of inactivation of the amphipathic fusion were as follows: 30 ℃ fusion in water bath for 20 min, then coated with regeneration medium, and then passaged more than 10 times by photoprinting. Finally, 17 stable fusants were screened, A highly efficient fusion TLB15 with both amphipathic features was screened. The corrected mortality of TLB15 fermentation broth to 2nd instar larvae of Plutella xylostella 96 h after treatment was 93.44%. The average inhibitory rates of TLB15 fermentation broth against cucumber blight and Phytophthora capsici (confrontation culture 5 d) were 60.00% and 62.79% .