嗜酸性PGPR菌株CLB-17的筛选、鉴定及其对烟草青枯病菌的生防活性

Screening, identification and evaluation of acidophilic Bacillus subtilis CLB-17 for biocontrol of Ralstonia solanacearum causing tobacco wilt

为提高生防菌在酸性土壤中对烟草青枯病的生防效果,采用室内酸性平板快速筛选法从烟草根际土壤中筛选嗜酸性拮抗细菌,应用Biolog GENⅢ自动微生物鉴定系统结合16S rDNA序列分析进行种类鉴定,并采用室内琼脂扩散法在pH 5.5和pH 7.0培养条件下比较其对烟草青枯病菌Ralstonia solanacearum的抑制活性,通过盆栽试验测定其对烟草青枯病的防效及对烟草的促生作用,通过测定胞外酶活性和铁载体含量分析其生防特性。结果表明:从烟草根际土壤中筛选获得1株具有高拮抗活性的嗜酸性细菌,命名为CLB-17,并将其鉴定为枯草芽胞杆菌Bacillus subtilis。在pH 5.5条件下,菌株CLB-17的拮抗活性最强,其发酵液对烟草青枯病菌的抑菌带宽度为7.11 mm,而在pH 7.0条件下抑菌带宽度为6.37 mm;在pH 5.5条件下,CLB-17菌株的铁载体相对含量(2.12)及蛋白酶活性(40.12 U/mL)、葡聚糖酶活性(142.52 U/mL)和酸性磷酸酯酶活性(6.42 U/mL)均高于pH 7.0条件下各指标(1.51、36.14 U/mL、110.24 U/mL和6.11 U/mL);该菌株在pH 5.5条件下对烟草青枯病的防效为76.99%,高于在pH 7.0条件下的防效66.18%;且烟草株高、整株干重、根干重和叶绿素含量均显著高于pH 7.0处理和空白对照。表明枯草芽胞杆菌CLB-17在酸性条件下的生防活性高,在酸性土壤中对烟草青枯病的生防效果更好,且对烟草生长具有促进作用。

To search for biocontrol agent for tobacco bacterial wilt in acid soils, an antagonistic bacterium against Ralstonia solanacearum causing tobacco bacterial wilt was screened out from tobacco rhizosphere based on a rapid screening method using a pH 5.5 nutrient agar(NA) medium and identified by Biolog GEN Ⅲ combined with 16S r DNA sequence analysis. Its efficacy against tobacco bacterial wilt and growth promoting effect on tobacco were evaluated in p H 5.5 and pH 7.0 soils in greenhouse, and the biocontrol related siderophore contents and enzyme activities were characterized by biochemical test. The results showed that an acidophilic antagonistic bacterium was obtained and named as CLB-17,identified as Bacillus subtilis. Bacillus subtilis CLB-17 had a higher inhibitory activity against R. solanacearum in acid conditions than in neutral conditions. At pH 5.5, the fermentation broth of CLB-17had a 7.11 mm inhibitory bandwidth on NA plates, which was wider than the 6.37 mm inhibitory bandwidth at p H 7.0. The B. subtilis CLB-17 could secret siderophore, protease, glucanase and acid phosphatase. The relative content of siderophore(2.12), protease activity(40.12 U/mL), glucanase activity(142.52 U/mL) and acid phosphatase activity(6.42 U/mL) of CLB-17 at pH 5.5 were higher than those at pH 7.0(1.51, 36.14 U/mL, 110.24 U/mL and 6.11 U/mL). In pot experiments, the control efficacy of B. subtilis CLB-17 on tobacco bacterial wilt was 76.99% at p H 5.5, which was higher than the control efficacy of 66.18% at pH 7.0. Similarly, CLB-17 significantly increased tobacco plant height, whole plant dry weight, root dry weight and chlorophyll content more in p H 5.5 soil than in pH 7.0 soil. These results suggested that an efficient plant growth promoting rhizobacteria(PGPR) strain was obtained for further improving the biocontrol effect on tobacco bacterial wilt and the growth promotion of tobacco in acid soils.