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    梁丽琼, 黄少莉, 邵杭, 等. 水稻基腐病菌拮抗菌解淀粉芽孢杆菌E3菌株的鉴定及抑菌活性[J]. 华南农业大学学报, 2021, 42(4): 51-62. doi: 10.7671/j.issn.1001-411X.202009039
    引用本文: 梁丽琼, 黄少莉, 邵杭, 等. 水稻基腐病菌拮抗菌解淀粉芽孢杆菌E3菌株的鉴定及抑菌活性[J]. 华南农业大学学报, 2021, 42(4): 51-62. doi: 10.7671/j.issn.1001-411X.202009039
    shu
    LIANG Liqiong, HUANG Shaoli, SHAO Hang, et al. Identification of an antagonistic strain Bacillus amyloliquefaciens E3 against Dickeya zeae and its antimicrobial activity[J]. Journal of South China Agricultural University, 2021, 42(4): 51-62. doi: 10.7671/j.issn.1001-411X.202009039
    Citation: LIANG Liqiong, HUANG Shaoli, SHAO Hang, et al. Identification of an antagonistic strain Bacillus amyloliquefaciens E3 against Dickeya zeae and its antimicrobial activity[J]. Journal of South China Agricultural University, 2021, 42(4): 51-62. doi: 10.7671/j.issn.1001-411X.202009039
    shu

    水稻基腐病菌拮抗菌解淀粉芽孢杆菌E3菌株的鉴定及抑菌活性

    Identification of an antagonistic strain Bacillus amyloliquefaciens E3 against Dickeya zeae and its antimicrobial activity

      摘要
    • 摘要:
      目的  纯化和鉴定从柑橘根际土壤分离得到的可抑制水稻基腐病菌Dickeya zeae EC1生长的解淀粉芽孢杆菌Bacillus amyloliquefaciens，并解析其抑菌机制。
      方法  采用平板扩散法从根际土壤中筛选出1株拮抗D. zeae EC1的B. amyloliquefaciens E3菌株；根据细菌菌落表型、生理生化特征结合16S rDNA序列分析等方法对E3菌株进行分类鉴定；检测无菌E3培养液对D. zeae EC1侵染水稻种子能力的影响；盐酸沉淀结合丙酮抽提法提取E3菌株脂肽粗提物；采用平板对峙法测定E3菌株脂肽粗提物对病原真菌的抑菌活性，琼脂扩散法测定E3菌株脂肽粗提物对病原细菌的抑菌活性及最小抑菌浓度(Minimum inhibitory concentration, MIC)；用Durashell C18柱对脂肽粗提物进行HPLC分离纯化；通过液相色谱−质谱联用(LC-ESI-MS)分析，鉴定抑菌物质的相对分子质量并推测其化学组成。
      结果  B. amyloliquefaciens E3菌株对茄病镰刀菌Fusarium solani、香蕉基腐病菌、茄科劳尔氏菌Ralstonia solanacearum等多种植物病原菌生长具有抑制作用，表现为广谱抗性；B. amyloliquefaciens E3菌株的无菌培养液能够抑制D. zeae EC1侵染萌发的水稻种子，显著提高水稻种子的萌芽率；B. amyloliquefaciens E3菌株的脂肽粗提物可以抑制D. zeae EC1的生长，其MIC为348.97 µg/mL；HPLC分离纯化结合LC-ESI-MS分析发现，B. amyloliquefaciens E3菌株分泌的主要抑菌活性物质包括Surfactin、Fengycin和Iturin 3类脂肽类抗生素。
      结论  B. amyloliquefaciens E3菌株具有作为生防菌的潜力，它可能通过分泌Surfactin、Fengycin和Iturin 3种脂肽类抑菌活性物质，拮抗水稻基腐病菌、茄科劳尔氏菌、茄病镰刀菌等多种植物病原菌。研究结果可为该菌的生物防治应用提供理论依据。

       

      Abstract:
      Objective  To isolate and identify Bacillus amyloliquefaciens strain which can inhibit the growth of Dickeya zeae EC1 from the rhizosphere soil of citrus plants, and analyze its antibacterial mechanism.
      Method  The B. amyloliquefaciens E3 strain antagonizing the growth of D. zeae EC1 was screened from the rhizosphere soil by plate diffusion method. The E3 strain was classified and identified based on colony phenotype, physiological and biochemical characteristics of bacteria and 16S rDNA sequence analysis. Effects of E3 sterile culture medium on the seeds germination ability of rice infected by D. zeae EC1 was detected. The crude lipopeptides of E3 strain was extracted by hydrochloric acid precipitation combined with acetone extraction. The antimicrobial activity of crude lipopeptides against pathogenic fungi were determined by plate confrontation method. The antimicrobial activity of crude lipopetides against pathogenic bacteria were determined by agar diffusion method, and the minimum inhibitory concentration (MIC) was obtained. The crude lipopeptides were separated and purified by HPLC with Durashell C18 column. Liquid chromatography-mass spectrometry (LC-ESI-MS) analysis was used to identify the molecular weights of the antimicrobial substances and speculate its chemical composition.
      Result  B. amyloliquefaciens E3 strain had an inhibitory effect against Fusarium solani, D. zeae MS1 and Ralstonia solanacearum, etc, indicating that it had broad-spectrum resistance. The sterile culture medium of B. amyloliquefaciens E3 strain inhibited the infection of germinating rice seeds by D. zeae EC1 and significantly increased the seed germination rate. The crude lipopeptides of E3 strain inhibited the growth of D. zeae EC1 with the MIC of 348.97 µg/mL. HPLC combined with LC-ESI-MS analysis showed that the main antibacterial substances secreted by E3 strain included surfactin, fengycin and iturin.
      Conclusion  The B. amyloliquefaciens E3 strain is potential to be a biocontrol agent. It can antigonize a variety of plant pathogens such as D. zeae EC1, R. solanacearum and F. solani by secreting three kinds of lipopeptides including surfactin, fengycin and iturin. The results provide a theoretical basis for the application of B. amyloliquefaciens E3 in biological control.

       

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