稻曲病菌次生代谢产物及其生物活性

False smut,caused by Ustilaginoidea virens (teleomorph: Villosiclava virens), is one of the most serious fungal diseases of rice. Ustiloxins, which are cyclopeptide mycotoxins produced by rice false smut pathogen, have been proved to be toxic to plants and animals especially for their cytotoxic activity by inhibiting microtuble assembly and cell skeleton formation of the eucaryotic organisms. Our previous results showed that there were a vast diversity of the false smut pathogenic isolates collected from the different areas of China with their secondary metabolite composition and pathogenicity. Based on the previous research of the ustiloxins and ustilaginoidins from the rice false smut balls (mainly as chlamydospores) by Japanese scientists, the purposes of this proposal attempt to comprehensively clarify the secondary metabolites from the pathogen materials including rice false smut balls and mycelial fermentation cultures collected from the different areas of China, establishment of the standard sample system for the ustiloxins, isolation and identification of the unknown ustiloxins and ustilaginoidins, evaluation of the biological activities of the secondary metabolites. Other aspects such as high-performance liquid chromatography-mass spectrometry (HPLC-MS) analysis of the main secondary metabolites, biosynthetic regulation of the main metabolites in mycelial fermentation culture, structure-activity relationships, mechanisms of action, physiological effects of the metabolites on the host rice (including its seedlings and cultured cells), as well as the potential application of the metabolites will also been studied. It aims to provide the basis for rice false smut disease conctrol, food security, as well as the creation of new medicines and agrochemicals.

稻曲病是危害水稻的一种重要真菌病害，由稻曲病菌（或称稻绿核菌）产生的稻曲菌素能够抑制真核生物微管蛋白的组装与细胞骨架形成，抑制细胞有丝分裂，导致植物生长异常和动物器官病变。之前，仅日本科学家对稻曲球（主要为厚垣孢子）中的两类次生代谢产物（稻曲菌素和稻黑粉菌素）进行了初步研究，稻曲病菌中其他次生代谢产物尚未被揭示。本课题组前期研究结果表明，来源于我国的稻曲病菌在次生代谢产物和致病性方面存在多样性。本申请项目试图了解我国的代表性稻曲病菌（包括稻曲球和稻曲菌发酵培养物）中次生代谢产物的种类，建立稻曲菌素的标样体系，分离和鉴定未知的稻曲菌素和稻黑粉菌素，评价稻曲菌中次生代谢产物的生物活性，进行主要次生代谢产物的液相色谱-质谱分析和发酵培养中代谢产物的合成调控研究，探讨构效关系、作用机制、对宿主的生理效应和应用潜力，为有效防治稻曲病、保障粮食安全和新药创制提供依据。

本项目详细研究了稻曲球和稻曲病菌发酵培养物中次生代谢产物的种类和结构；评价了次生代谢产物的生物活性和对水稻培养细胞的生理效应；稻曲球等样品中主要次生代谢产物的液相色谱和液相色谱-质谱分析；采用免疫学方法检测稻曲球等样品中的稻曲病菌毒素；采用大孔吸附树脂富集主要的次生代谢产物；采用高速逆流色谱的方法制备代谢产物；还研究了稻曲球中真菌的多样性；测定了不同发育时期的稻曲球中稻曲菌素和稻绿核菌素的含量和种类的动态变化；开展了稻曲病菌中稻曲菌素、稻绿核菌素、山梨素等次生代谢产物的生物合成调控研究。主要成果如下：（1）从稻曲球和稻曲病菌发酵培养物中共分离并鉴定出代谢产物72个，其中新化合物28个（包括1个稻曲菌素、15个稻绿核菌素、10个山梨素、2个生物碱类）；（2）稻曲菌素、稻绿核菌素和山梨素均表现出一定的植物毒、细胞毒、抗菌和抗氧化活性，以及对水稻培养细胞的生理效应；（3）建立了稻绿核菌素和稻曲菌素的HPLC和LC-MS分析方法；（4）研制出用于检测稻曲菌素A和B的酶联免疫试剂盒和试纸条；（5）建立了稻曲菌素和稻绿核菌素的大孔树脂富集和逆流色谱制备工艺；（6）建立了稻曲菌素和稻绿核菌素的标样体系和HPLC指纹图谱；（7）揭示了稻曲球中可培养真菌的多样性；（8）发现早期稻曲球中稻曲菌素A和B的含量和总量明显高于中期和晚期稻曲球样品中的含量和总量，2,3,2’,3’-位羟基化的稻绿核菌素在晚期稻曲球中的含量要明显高于早期和中期的稻曲球样品。研究结果为揭示稻曲病菌的代谢网络和生物合成调控规律、代谢产物的生物活性和生理生态功能、稻曲病菌与水稻之间的相互关系，同时为有效防治稻曲病、保障粮食和饲料安全，以及新农药和医药创制提供了依据。本项目已发表SCI刊源的论文12篇、国内核心期刊论文3篇、获授权专利2项（其中1项已进行了成果转化），培养研究生16名，其中2人获得硕士学位，3人获得博士学位。