生物技术通报 ›› 2022, Vol. 38 ›› Issue (2): 57-66.doi: 10.13560/j.cnki.biotech.bull.1985.2021-0337

• 研究报告 • 上一篇    下一篇

牡丹根腐病原菌拮抗细菌抑菌活性物质分析

杨瑞先(), 刘萍, 王祖华, 阮宝硕, 汪智达   

  1. 洛阳理工学院环境工程与化学学院,河南 洛阳 471023
  • 收稿日期:2021-03-18 出版日期:2022-02-26 发布日期:2022-03-09
  • 作者简介:杨瑞先,女,博士,副教授,研究方向:植物内生细菌及病害生物防治;E-mail: fairy19790805@163.com
  • 基金资助:
    河南省高等学校重点科研项目(20A210028);河南省高校青年骨干教师培养计划(2019GGJS242)

Analysis of Antimicrobial Active Metabolites from Antagonistic Strains Against Fusarium solani

YANG Rui-xian(), LIU Ping, WANG Zu-hua, RUAN Bao-shuo, WANG Zhi-da   

  1. School of Environmental Engineering and Chemistry,Luoyang Institute of Science and Technology,Luoyang 471023
  • Received:2021-03-18 Published:2022-02-26 Online:2022-03-09

摘要:

前期研究发现解淀粉芽胞杆菌(Bacillus amyloliquefaciens)md8和md9对牡丹根腐病原菌具有较好的抑制作用,但其抑菌物质的组成尚不清楚。本文首先明确了2个菌株3种脂肽类物质合成的基因片段,利用酸沉淀和葡聚糖凝胶层析法进行抑菌物质的分离纯化,牛津杯对峙法检测脂肽类粗提物和凝胶层析分离组分的抑菌活性;进一步利用实时荧光定量PCR(RT-qPCR)检测菌株在拮抗根腐病原菌过程中脂肽类物质合成基因相对表达量的变化,基质辅助激光解吸飞行时间质谱(MALDI-TOF-MS)分析抑菌物质的种类。结果表明,2个菌株的脂肽类粗提物和凝胶层析分离组分对根腐病原菌均具有良好的平板抑菌效果,RT-qPCR结果表明2个菌株合成伊枯草菌素(iturin)的基因ituD在对峙培养过程中相对表达量显著增加,芬荠素(fengycin)合成基因fenA也呈现上调表达。MALDI-TOF-MS分析表明2个菌株中具有抑菌作用的分离组分主要为伊枯草菌素类物质Iturin B和Bacillomycins D,结合实时荧光定量PCR结果,推测菌株md8和md9在拮抗根腐病原菌过程中发挥主要生防作用的物质为伊枯草菌素。

关键词: 牡丹, 根腐病原菌, 解淀粉芽胞杆菌, 脂肽类物质合成基因, 抑菌活性物质

Abstract:

Previous studies revealed that two stains Bacillus amyloliquefaciens md8 and md9 have strong antagonism to Fusarium solani causing tree peony root rot;however,the components of antimicrobial substances are not clear yet. First,the biosynthesis gene fragments of 3 lipopeptides were identified. Acid precipitation and the Sephadex gel chromatography column were used to separate and purify the antimicrobial substances. Then Oxford Cup method was adapted to detect the inhibitory activity of the crude lipopeptides and separation components. RT-qPCR was employed to detect the expression variations of lipopetide biosynthesis genes in the strains md8 and md9 during inhibiting F. solani process in the dual culture. And finally MALDI-TOF-MS technique was used to analyze the types of antimicrobial substances. The results showed that the crude lipopeptide extracts and separation components via Sephadex gel chromatography had favorable effect against the pathogen of root rot. The RT-qPCR results indicated that gene ituD of md8 and md9 for iturin biosynthesis was constantly expressed in high amounts throughout the antagonism. The gene fenA for fengycin biosynthesis were up-regulated when md8 and md9 were confronted with F. solani. The MALDI-TOF-MS results further verified that iturin B and bacillomycin D were the main antimicrobial components of md8 and md9 against F. solani. Combined with RT-qPCR results,it was speculated that iturin produced by the strains md8 and md9 played an important role in the inhibitory effect on F. solani.

Key words: Paeonia suffruticosa, Fusarium solani, Bacillus amyloliquefaciens, lipopeptide biosynthesis genes, antimicrobial active metabolites