三株海南红树林来源微生物抗香蕉枯萎病菌的化学成分研究

Mangroves are the trees and shrubs which grows in saline coastal habitats in the tropics and subtropics. Owing to their unique ecological environment, they are potential sources for the isolation of microorganisms which can supply a large quantity of novel bioactive compounds. Three microorganism strains including fungi and actinomycetes isolated from Sonneratia hainanensis and its rhizospheric soil samples were obtained in our pilot work. Their secondary metabolites both exhibited inhibitory activities against of Fusarium oxysporum f. sp. cubense which induces the destructive disease for banana and has severely damaged banana industry of Hainan. Yet there are few reports about the study on the secondary metabolites of microorganisms from Sonneratia hainanensis an endemic mangrove of Hainan. In this study, talented strains will be selected from the three strains and the fermentation conditions of them will be optimized. The optimal conditions will be selected using the chemical and biological activity screening phases. The chemical screening will focus on the diversity and types of the secondary metabolites with HPLC and TLC methods. Anti-fusarium oxysporum f. sp. cubense activity will be selected for biological activity screening phases. After fermentations of the talented strains by the fermentation conditions before and after optimization, the secondary metabolites will be purified by modern separation technology and identified with various structural authentication methods under the guide of anti-fusarium oxysporum f. sp. cubense bioassay. And the structure-activity relationship will be investigated after bioactivity test against plant pathogens. Furthermore, according to the structure-activity relationship, the derivatives of novel bioactive compounds will be prepared. The study results will offer new active compounds for controlling banana fusarium wilt and provide valuable theoretical foundation for the development of pesticide from secondary metabolites of mangrove microorganisms.

红树林是热带亚热带海岸潮间带的特殊植物群落，其特殊的生态环境使其共附生微生物成为了新活性天然产物的重要来源。海南海桑（Sonneratia hainanensis）是海南特有的红树植物，目前关于该植物共附生微生物活性天然产物的研究鲜有报道。香蕉枯萎病是香蕉的毁灭性病害，严重危害海南香蕉产业，而目前尚无有效防治该病的药剂。本课题将对申请者所在课题组前期从海南海桑及其根际土壤来源的微生物中筛选得到的3株具有抗香蕉枯萎病菌的菌株进行进一步生物活性及化学多样性筛选，获得目标菌株。通过改变目标菌株发酵方式，培养基成份、pH及盐度等优化菌株的发酵条件，利用HPLC和TLC进行化学筛选，同时兼顾生物活性；以优化前后的发酵条件进行规模发酵，利用现代分离技术及结构鉴定方法对菌株代谢产物进行分离鉴定，并进行化合物生物活性、结构修饰及构效关系等方面的研究，为防治香蕉枯萎病的生物源农药研发提供活性新化合物，同时为红树林来源微生物天然产物在农药开发方面提供理论依据。

香蕉枯萎病是香蕉枯萎病菌引发的毁灭性真菌病害，严重危害我国香蕉产业，目前尚无防治该病的有效药剂。天然产物具有低毒、高选择性等特点，是药物先导结构的主要来源。红树林特殊的生态环境使其共附生微生物成为新活性天然产物的重要来源。本项目从红树林来源微生物中筛选获得化学成分多样性丰富且具有抗香蕉病菌活性的2株红树林来源放线菌和2株红树林来源真菌作为目标菌株，对其代谢产物及生物活性进行研究，为防治香蕉枯萎病的生物源农药研发提供先导化合物，取得了以下研究结果：1) 通过改变菌株发酵方式，培养基成分等优化目标菌发酵条件，以最优发酵条件进行规模发酵，利用现代分离技术及结构鉴定方法对菌株代谢产物进行分离鉴定，共分离化合物74个，鉴定了其中60个化合物结构，新化合物16个；抗菌活性测试结果表明其中具有抗香蕉枯萎病菌活性的化合物4个，具有抗芒果炭疽菌活性化合物3个，具有抗烟草青枯病菌活性化合物1个，并对化合物的构效关系进行了初步讨论，同时获得抗香蕉枯萎病菌活性组分1个。2) 所分离鉴定的化合物中，已知化合物星孢菌素对香蕉枯萎病菌具有强效抗菌活性（MIC为6.25 μg/ml），以星孢菌素作用于香蕉枯萎病菌，研究其对该致病菌菌丝生长影响，并在显微镜下观察该致病菌孢子型态变化，初步研究其作用机制，结果表明星孢菌素可抑制香蕉枯萎病菌菌丝的生长，同时其抑制该致病菌孢子萌发，最终使其变形死亡。3) 对以星孢菌素为主要成分的活性组分进行抗香蕉枯萎病菌活性的植株抗病仿效实验，结果表明其在实际植株生长过程中对香蕉枯萎病菌具有良好的抑制作用。本项目研究结果为防治香蕉枯萎病的生物源农药研发提供先导化合物，为红树林来源微生物天然产物在农药开发方面提供理论依据。