海藻内生真菌农用生物活性物质及其合成的研究

Marine fungi metabolites are the new resources and more important for discovery of novel structure and environment-friendly agricultural antimicrobial active substances. CCH-1, with strange morphology, isolated from marine red alga Chondria Crassicaulis on Qingdao coastline showed rich metabolic and significant farm antimicrobial activity screening in nearly 100 marine fungi. For dominant alga, Chondria Crassicaulis contained a large number of secondary metabolites with strong biological activities, its endophytic fungi and active metabolites may have potential biological control abilities to plant diseases. So it is worthy of further study. Based on the previous results, the present project focused on the work of bioactivity-guided isolation, structural elucidation of secondary metabolites of two static fermentation extracts on 2 kinds of cultural mediums, discovery unique structure compounds as well as strong activities, investigation the structure-activity relationship, establishment characteristic spectral library of agricultural antifungal activity substances, initial speculation biogenesis pathway, and then providing template compounds for the agricultural chemicals research. In addition, too small amount of natural products are also attracted considerable attention on chemical synthesis of strong agricultural antimicrobial compounds, according to their biogenesis and computer-aided design the chemical synthesis routes on the basis of theoretical and computational chemistry, using modern spectroscopic techniques to structural identification, then will provide more experimental material in vivo plant experiments.

新资源海洋真菌代谢产物的研究对具有新颖结构和环境友好型农用抑菌活性物质的发现具有重要意义。在近100株海洋真菌农用抑菌活性筛选中发现粗枝软骨藻的内生真菌(CCH-1)生长形态奇异、代谢丰富、活性显著，且粗枝软骨藻为青岛海域优势藻种，自身代谢过程可产生多种高生物活性物质，因此其内生菌及其活性代谢物质具有潜在的植物病害生防能力，值得深入研究。本课题拟通过现代色谱技术对两种不同优化培养的CCH-1次级代谢产物进行活性跟踪分离纯化，使用现代波谱技术鉴定单体化合物结构，发现结构新颖、活性显著的代谢产物，探讨构效关系，建立特征农用抑菌活性物质谱图库，初步推测生源合成途径，为新型农药研发提供模板化合物；鉴于天然产物获得量过少，本项目拟对活性显著物质结合其生源合成和理论化学计算辅助设计合成路线进行化学合成，使用现代波谱技术进行结构、性能表征，为植物活体抑菌实验提供充足的实验材料。

目标：从青岛近海海域海洋生物中筛选农抗抑菌杀虫活性菌株，自粗枝软骨藻中分离纯化出优势海洋内生真菌菌株，经规模发酵培养后，分离并鉴定农抗活性菌株的活性成分，并进行化学合成应用初探。.方法：以细菌、农业病原真菌、卤虫及农业生产中常见且危害严重的线虫等为供试菌株和供试害虫，从海洋真菌资源库中筛选得到抑菌杀虫活性菌株；应用 OSMAC培养策略，结合抑菌杀虫活性追踪，综合运用硅胶柱层析、凝胶柱层析、反相柱层析与现代色谱法分离并鉴定抑菌杀虫活性菌株不同培养条件下产生的抑菌杀虫活性成分。.结果如下：.1、CCH-1菌株经MH1培养基种发酵后分离纯化获得单体化合物进行活性筛选，结果发现化合物FT-32和FT-33对BS的抑菌圈分别高达40mm和44mm，抑制活性均高于阳性对照（32mm），选择性较高。.2、CCH-2菌株经RICE培养基发酵后，对分离纯化获得的部分化合物进行活性筛选，结果发现化合物WT-7对耐药菌S.A.、WT-10对B. T.、WT-11对B.S.抑菌圈直径均约为15mm，阳性对照为24mm。对于卤虫致死活性，化合物WT-11和WT-10抑制活性较高，其半致死浓度LD50分别为0.939，1.320ug/ml。.3、对CCH-3菌株的RICE培养基获得的部分化合物活性筛选结果，发现化合物AS-7的抑菌圈高达45mm，抑菌活性明显高于阳性对照（32mm），进行农业病原菌生长率抑制活性筛选发现，对棉花枯萎病菌（MHKW）和柑橘炭疽病菌(GJTJ)表现出一定的抑制活性，半抑制浓度分别为：135 ug/ml 和273 ug/ ml。.4、对其共生海洋红藻次级代谢产物的活性进行筛选，结果发现其抑菌活性均较弱，但是Z4，Z12，Z19，Z22，Z30对红线虫的抑制活性较强，其LD50依次在39.01358.683ug/mL之间；且化合物Z4和Z12对卤虫表现出强烈的抑制作用，LD50分别为3.337ug/mL，2.852ug/mL。.除此之外，利用天然产物中获得量较大的简单易追踪物质，与化学合成中的催化、光电材料等合作，发表论文5篇，其中SCI论文3篇。