响应面法优化梨黑斑病生防链霉菌SZF-179发酵培养基

Optimization of Fermentation Media through Response Surface Methodology for Streptomyces odonnellii SZF-179 against Alternaria alternata

由链格孢引起的梨黑斑病是梨树生长及采后贮藏过程中的重要病害,其防治主要依赖于化学杀菌剂,亟需开发安全高效的生物杀菌剂。我们前期筛选获得的奥唐纳链霉菌Streptomyces odonnellii SZF-179菌株对梨黑斑病菌具有较强拮抗活性。为提高其发酵水平,探究其在梨黑斑病生物防控中的应用潜力,采用响应面法对该菌株的发酵培养基进行了优化。通过单因素试验确定最佳摇瓶发酵条件、最适碳氮源与无机盐组合,Plackett-Burman试验筛选出影响菌株发酵上清液抑菌活性的3个显著因子:麦芽糖、酵母膏和碳酸钙;再结合最陡爬坡试验、Box-Behnken设计及响应面法拟合显著因子与菌株发酵上清液抑菌率的非线性方程求解,得到优化的发酵培养基配方,其组成为麦芽糖15.86 g/L、土豆淀粉10.00 g/L、酵母膏8.15 g/L、芝麻粕5.00 g/L、硫酸铵0.50 g/L和碳酸钙0.20 g/L。此模型预测发酵液上清液50倍稀释液抑菌率为70.39%,验证值为70.51%,预测值与验证值之间吻合较好,较优化前发酵液上清液50倍稀释液抑菌率(30.70%)提高了约1.3倍。优化后发酵培养物对梨黑斑病的田间防治效果达到61.21%,并使梨树的落叶率降至14.56%,显著低于对照的44.25%。本研究为新型绿色高效梨黑斑病生物杀菌剂产品的研制提供重要的技术支撑。

Pear black spot caused by Alternaria alternata is an important disease of pear during the cultivation and post-harvest storage,and its control relies on chemical fungicides,therefore it is urgent to develop safer and more efficient biofungicides.An actinomycetes isolate,Streptomyces odonnellii SZF-179 obtained from our previous study showed high antagonistic activity against A.alternata.To enhance the fermentation level of this strain and determine its application potential to control pear black spot,the fermentation media were optimized through response surface methodology.The optimal shake-flask fermentation conditions and the proper combination of carbon sources,nitrogen sources and mineral salts were achieved through single-factor experiment.Three factors,such as maltose,yeast extract and calcium carbonate significantly affecting the growth inhibition of A.alternata by supernatants from SZF-179 fermentation broth,were determined by Plackett-Burman design.By combining the steepest ascending experiment and Box-Behnken design and response-surface methodology to fit the nonlinear equation between the significant factors and the inhibiting rate of the strain's fermentation broth supernatant,an optimized fermentation medium formula was obtained with the composition of 15.86 g/L maltose,10.00 g/L potato starch,8.15 g/L yeast extract,5.00 g/L sesame meal,0.50 g/L ammonium sulfate,and 0.20 g/L calcium carbonate.The constructed model predicted that the inhibiting rate of the 50 fold dilution of the fermentation broth supernatant from the optimized medium was 70.39%,with a validation value of 70.51%.The predicted value was in good agreement with the validation value,and the inhibiting rate was about 1.3 times higher than that of the 50 fold dilution from the fermentation broth supernatant(30.70%)before the optimization of the culture medium.The optimized culture of S.odonnellii SZF-179 achieved a control efficacy of 61.21%on pear black spot in the field trial,and reduced the defoliation rate to 14.56%which was significantly lower than the defoliation rate of blank control,Which was 44.25%.This study will support technically the development of new microbial fungicides to control pear black spot.