梨火疫病菌拮抗细菌FX1培养基及摇瓶发酵条件优化

Optimization of Culture Medium and Flask Fermentation Conditions for Bacillus velezensis FX1 against Erwinia amylovora

贝莱斯芽胞杆菌Bacillus velezensis FX1是从新疆库尔勒香梨生产区果蔬天然酵素发酵液中分离筛选出的对梨火疫病菌Erwinia amylovora具有较强拮抗作用的菌株。为提高其抑菌活性物质的产量和防病效果,本研究以平板抑菌活性和发酵液活菌浓度作为检测指标,采用单因素试验、Plackett-Burman试验和响应曲面法对培养基成分(碳源、氮源、无机盐)及摇瓶发酵条件(温度、转速以及初始pH)进行筛选和优化,确定FX1菌株的最适培养基和发酵条件。结果表明,FX1的最适培养基为:玉米淀粉10.0 g/L,酵母膏25.1 g/L,KH2PO40.5 g/L,MnSO40.001 g/L;最适发酵条件为初始pH 7.0,转速150 r/min,温度30.9℃,装液量100 mL/500 mL,培养时间48 h。优化后FX1菌株活菌数可达1.53×109 CFU/mL,抑菌圈直径可达26.6 mm,相比优化前分别增加了3.10倍和1.32倍。苹果离体花序的防效试验结果显示,喷施FX1菌体发酵液、无菌发酵滤液和脂肽类物质粗提取液能显著降低花腐率(P<0.05),对梨火疫病的保护性防效分别可达68.53%、71.47%和76.30%,均高于优化前。研究结果为贝莱斯芽孢杆菌FX1菌剂的开发及其在梨火疫病生物防治中的应用提供了依据。

Bacillus velezensis FX1 was isolated and screened from the natural enzyme fermentation broth of fruits and vegetables in the pear production area of Kulle,Xinjiang,as a strain with strong antagonistic effect on the Erwinia amylovora.In order to improve the bacterial inhibitory activity and disease control effect,the medium components(carbon source,nitrogen source,inorganic salt)and shake flask fermentation conditions(temperature,rotation speed and initial pH)were screened and optimized by single-factor test,Plackett-Burman test and response surface method.The results showed that the optimal medium for FX1 was corn starch 10.0 g/L,yeast paste 25.1 g/L,KH2PO40.5 g/L,MnSO40.001 g/L.The optimal fermentation conditions were initial pH 7.0,rotation speed 150 r/min,temperature 30.9℃,loading volume 100 mL/500 mL,and cultivation time 48 h.After optimization,the viable bacterial count of FX1 strain could reach 1.53×109 CFU/mL and the diameter of inhibition circle could reach 26.6 mm,which increased 3.10 times and 1.32 times,respectively.Spraying of FX1 fermentation solution,sterile fermentation filtrate and crude extract of lipopeptides could significantly reduce the flower rot rate(P<0.05),and the protective efficacy against fire blight could reach 68.53%,71.47%and 76.30%,respectively,which were higher than that before optimization.The above results provide a basis for the development of Bacillus Bellais FX1 bacterial agent and its application in biological control of fire blight.