拮抗三七根腐病病原真菌的放线菌代谢产物及其作用机制

Root rot disease is a kind of fatal diseases for Panax notoginseng, causes crop failure, even to no harvest, seriously affects the qualities and quantities of products. It becomes a serious obstacle in development of plantation and related industries of P. notoginseng, which plays very important roles in traditional Chinese medicines and clinical usages. There are still no effective practices to treat the serious root diseases, and biocontrol methods are waited to investigate and develop urgently. In pioneer studies, we developed screening on bioactive microbial strains against phytopathogenic microbes of Panax notoginseng from established actinomycetes library in Yunnan Institute of Microbiology. Herein in this project, we will investigate antagonistic metabolites from selected actinomycetes strains with higher inhibition or fungicide activities. Combined with antagonisitic activities, single factor test, orthogonal experimental design and response surface analysis (SRA) will be employed to optimize media and culture conditions for selected actinomycetes strains. Extracts from optimized culture will be analysed by HPLC-DAD and isolated by all kinds of chromatographic methods, including preparative HPLC. Portions and purified molecules will be screened for antagonistic activities during the procedure of isolation. Structures will be determined by spectrum analysis, such as MS, NMR, UV and IR. Antagonistic mechanisms will be analyzed by morphological observation combined with localized vital staining, isotope or fluorescene labeling methods, et al. The aims of this proposal are to find: (1) novel molecules from actinomycetes for control of Panax notoginseng root rot disease in future, (2) antagonistic functions of the active compounds.

三七根腐病是三七种植产业中的重要病害，可致欠收或绝收，严重影响着三七的产量和质量，阻碍着三七产业的健康发展；但对三七根腐病的防治缺少有效的生物防治措施。本项目在前期大量筛选三七病害生防菌株的基础上（从云南省微生物研究所放线菌资源库中筛选），以具有拮抗三七根腐病病原真菌的放线菌为研究材料，采用用单因素控制、正交试验设计和响应面分析法探索适合的发酵培养基和培养条件，以拮抗三七根腐病原真菌活性筛选模型，从发酵提取物中筛选活性成分，运用现代色谱技术和光谱技术对活性成分进行分离和结构鉴定，并利用形态观察法、同位素或荧光标记法等分析其拮抗病原菌的作用机制。研究目的：探索从放线菌资源中发现拮抗三七根腐病原真菌的新的活性代谢产物及作用机理，为开发新型生物农药控制三七病害提供物质基础和理论基础。

微生物及其次生代谢产物在农业生产中具有广泛的应用。在本项目经费的支持下，我们以三七根腐病的病原真菌为靶标，开展从云南省微生物研究所建设的特色放线菌资源库筛选活性菌株、优化培养基和培养条件、分离与鉴定活性新化合物等方面的研究工作。.在课题组师生的共同努力，经过抗菌活性的初筛和复筛，共筛选到高活性的微生物菌株12株。采用多项分类技术与方法对活性菌株进行了准确的分类与定位，获得3株微生物新物种（已发表2个）。以抗菌活性为评价指标，采用单因素控制法、正交试验法等对活性菌株的培养基及培养条件进行了优化；对其中6株高活性菌株进行了40-60L的扩大发酵，以活性跟踪方法对其产物进行分离、纯化。目前为止，已经从其中4株活性菌株的发酵代谢产物中分离鉴定了化合物103个，其中新化合物27个、新天然产物2个。以抗菌、抗炎等模型对部分化合物进行了活性复测，其作用机制方面的研究正在进行中。其他活性菌株的代谢产物的分离、结构鉴定与活性测定工作仍在继续进行中。.到2019年底，团队依托该项目共发表科研论文9篇（其中2篇为新物种，未标注基金号），均为SCI收录期刊；获得中国发明专利2项。团队共培养毕业硕士7名，本科毕业生3名。在读研究生5名。