抗烟草疫霉活性木霉与芽孢杆菌共培养体系的构建与优化

Construction and Optimization of a Co-culture System of Anti-Phytophthora nicotianae Trichoderma asperellum HG1 and Bacillus subtilis Tpb55

为减轻化学药剂防治烟草疫霉带来的弊端,提高生防菌剂效果,通过琼脂糖扩散法筛选抗性木霉并构建木霉和枯草芽孢杆菌Tpb55共培养体系,采用菌丝生长速率法评价了种间互作防治烟草疫霉的效果,利用单因素法优化共培养体系的培养基成分和发酵条件,并通过盆栽试验验证其对烟草黑胫病的防治效果。结果表明,棘孢木霉HG1具有良好的抗烟草疫霉活性,与枯草芽孢杆菌Tpb55的共培养体系如下:HG1(10^(6)CFU/mL)接种量为2%,与Tpb55(10^(6)CFU/mL)接种比例为10∶1,采用序列共培养方式,即先接种HG1,24 h后接种Tpb55。优化后的最适培养基成分为木糖10 g/L,蛋白胨和酵母粉(质量比为2∶1)5 g/L;最佳发酵为温度25℃,初始pH 7.5,转速140 r/min。共培养发酵液盆栽防病效果显著优于单培养,防治效果达到74.72%。该体系发酵后能够明显提升两株生防菌抑制烟草疫霉的活性,并对烟草黑胫病具有显著防病效果,为后续多菌株共发酵生防菌剂的开发提供基础。

In order to reduce the disadvantages of chemical control of Phytophthora nicotianae and improve the effect of biocontrol agents,Trichoderma asperellum HG1 with obvious anti-Phytophthora nicotianae activity was obtained by the agarose diffusion method and the a co-culture system of Trichoderma asperellum HG1 and Bacillus subtilis Tpb55 was constructed to evaluate the inhibition effect of interspecific interaction against Phytophthora nicotianae by the mycelial growth rate method.The composition of medium media and fermentation conditions were optimized by single factor experiment.The control effects of co-culture and single-culture fermentation liquors of Trichoderma asperellum HG1 and Bacillus subtilis Tpb55 on tobacco black shank were compared by pot experiments.The suitable co-culture system was determined as follows:the inoculation ratio of HG1(10^(6) CFU/mL) and Tpb55(10^(6) CFU/mL) was 10:1.Sequential inoculation was adopted as HG1 was inoculated first,and Tpb55 was inoculated after 24 h.The fermentation broth composition and culture conditions of the co-culture system were optimized by single factor method,guiding by the inhibition rate of Phytophthora nicotianae.The optimum medium composition was determined as follows:xylose 10 g/L,peptone and yeast powder(the mass ratio was 2:1) 5 g/L;The optimal fermentation conditions were as follows:temperature 25 ℃,initial pH 7.5,rotating speed 140 r/min.The disease control effect of co-culture fermentation liquors in pot experiments was 74.72%,which was significantly better than that of single-culture.The system could significantly improve the inhibition activity of the two biocontrol strains against Phytophthora nicotianae,and the fermentation liquors of the system had significant disease control effect on tobacco black shank,which provides a basis for the development of subsequent multi strain co-culture biocontrol agents.