番茄灰霉病生防细菌TD-7的鉴定、发酵条件优化及其防治效果

通过平板稀释分离法，从多年生草本植物薄荷根际土壤中分离到一株对灰葡萄孢Botrytis cinerea抑菌活性良好的细菌TD-7。通过形态特征和16S rDNA、ITS序列分析，鉴定该菌为解淀粉芽胞杆菌Bacillus amyloliquefaciens，并对其发酵滤液抑菌机理、最优发酵条件、最优发酵培养液组分及防治效果进行了研究。研究发现，菌株TD-7发酵滤液对番茄灰霉菌孢子的萌发有强烈的抑制作用，对菌丝生长抑制率在95%以上。皿内酶分泌试验发现，该菌可分泌几丁质酶、纤维素酶和蛋白酶，未检测到β-1，3-葡聚糖酶活性。发酵滤液抑菌活性最强的条件为在250 mL三角瓶中，将种子液按3%的体积比接种到50~75 mL培养液中，初始pH 8.0，温度32~36℃，转速200 r/min，发酵5 d。通过正交优化，得到最佳发酵培养液配方为玉米粉3%、蛋白胨1.5%、KH₂PO₄ 0.05%。经摇床发酵条件及发酵培养液组分优化，菌株TD-7发酵滤液抑菌活性提高了26.39%。该菌的发酵液对温室盆栽及田间大棚中的番茄灰霉病均具有良好的防治效果，分别为80.61%和87.70%。滤液稳定性试验结果表明，菌株TD-7发酵滤液中活性物质具有较广的pH适应范围（pH 4.0~9.0），在高温和紫外光照射下较稳定，有制成生物制剂应用于番茄灰霉病生物防治的潜力。

Identification,Fermentation Condition Optimization and Control Efficiency of Biocontrol Bacterium TD-7 against the Tomato Grey Mould

The bacterial strain TD-7, which was isolated from the rhizoplane soil of the perennial plant peppermint by plate dilution method, had a good inhibitory effect on the tomato grey mould Botrytis cinerea. The bacterium was identified as Bacillus amyloliquefaciens based on morphological features and sequence analysis of 16S rDNA and ITS. We investigated the inhibition mechanism, optimal fermentation condition, optimal fermentation medium composition and control effect of the TD-7 fermentation filtrate. The results show that the fermentation filtrate had a strong inhibition effect on the spore germination of B. cinerea with the inhibition rate of the mycelium growth over 95%. In vitro, strain TD-7 can secrete chitinase, cellulase and protease, while the activity of beta-1,3-glucanase was undetectable. The optimal fermentation conditions for producing antimicrobial substance were 250 mL flask, volume ratio of seed liquid 3%, fermentation medium 50-75 mL, initial pH 8.0, temperature 32-36℃, rotation speed 200 r/min and fermentation time 5 d. Orthogonal test demonstrated that the optimal medium was composed of 3.0% corn meal, 1.5% peptone and 0.05% KH₂PO₄. By optimizing fermentation conditions of shake cultivation and components of fermentation medium, the inhibitory activity of TD-7 fermentation filtrate was increased by 26.39%. The TD-7 fermentation broth had remarkable control efficacies on the tomato grey mould in the pot and greenhouse experiment as 80.61% and 87.70%, respectively. The stability experiment of the TD-7 fermentation filtrate shows that the active substance had a broad adaption range to pH value (pH 4.0-9.0), and was stable under high temperatures and UV-treatment. This suggests that the strain TD-7 of B. amyloliquefaciens is promising to be developed into a biocontrol formulation for controlling the tomato grey mould.