杨树溃疡病拮抗菌Brevibacillus brevis XDH抗菌活性成分及其作用机制研究

Poplar canker disease is a major biological disaster of poplar plantation in China, and so the exploring of the biological drugs and biocontrol methods is of great significance. Brevibacillus brevis XDH is a novel antagonistic bacteria isolated from poplar rhizosphere (GenBank Accession No. DQ279738). Preliminary research showed that this strain has high antagonistic activities against the Botryosphaeria dothidea that caused poplar canker. The antimicrobial substances produced by XDH are of excellent fermentation characteristics, as well as stable and safe, which demonstrated that the strain and its active substances have great potential for development and application. The purification procedures for the four active constituents of the antimicrobial substances have been established already, among which the component C has been identified as a novel non- ribosomal peptide. In this research, the molecular structures and the physicochemical properties of the active compounds will be elucidated via ultraviolet spectroscopy, infrared spectroscopy, MS, and NMR. The antagonistic mechanism will be illustrated by the application of fluorescence labeling, molecular probe labeling, electron microscopy and laser scanning confocal microscopy observation. It hopes to provide a theoretical basis for the further development of strain XDH and its antimicrobial active substances.

杨树溃疡病是我国杨树人工林的重大生物灾害，探寻其生物防治药物及生物控制方法意义重大。短短芽孢杆菌(Brevibacillus brevis)XDH是我们从杨树根际分离获得的一株新的拮抗菌（GenBank注册号为DQ279738），前期研究结果表明：该菌株对杨树溃疡病菌（Botryosphaeria dothidea）拮抗作用强，发酵性状优异，抗菌物质性质稳定，使用安全，极具研发潜力。该菌株产生的抗菌物质4个活性组分的分离纯化技术路线已经确立，其中，C 组分的一级结构骨架已经获得，为一新的芽孢杆菌非核糖体肽。本研究拟在前期研究的基础上，运用紫外光谱、红外光谱、质谱、核磁共振谱分析等，究明各活性组分的结构与理化性质；运用荧光染色、电镜、激光共聚焦显微镜、电感耦合等离子体质谱分析等技术，探明抗菌物质对杨树溃疡病菌的作用机制。以期为进一步开发该菌株及其抗菌物质提供理论依据。

通过硫酸铵分级盐析，CM Sepharose FF离子交换层析、Sephadex G-25分子筛层析以及制备HPLC等方法，从杨树溃疡病拮抗菌Brevibacillus brevis XDH的发酵液中分离纯化了两种抗菌活性组分A、B。其中组分B的发酵产量高，且抑菌活性强。通过元素组成分析，傅立叶变换高分辨质谱仪（FTIR-MS）一级质谱分析分子量，Orbitrap Elite高分辨质谱仪二级质谱分析氨基酸序列，JNM ECA-600超导脉冲傅立叶变换核磁共振谱分析组分B的H谱和C谱，对组分B进行结构解析，发现组分B的结构为：N、C端分别为由氨基酸Ile、Val修饰而成的Hmp、Vol，中间部分为18个氨基酸组成的线型非核糖体肽，其精确分子量为1569.06396，其一级结构式为：Hmp-Aba-Val-Orn-Ile-Val-Val-Lys-Val-Leu- Lys-Tyr-Leu-Vol。.对组分B的抗菌谱测定和部分敏感菌株的最低抑菌浓度MIC的测定发现，组分B对杨树溃疡病菌（Botryosphaeria dothidea）和杨树细菌性溃疡病菌（Ervvinia herbicola）均具有很强的抑菌活性，而且，对某些动植物病原真菌和细菌也具有较强的抑菌作用。.组分B对杨树溃疡病菌的抑菌作用，主要作用于细胞壁和细胞膜。拮抗菌Brevibacillus brevis XDH及其产生的活性成分在杨树溃疡病的防治上，具有重要研究和应用价值。