三株红树植物内生真菌农用杀虫活性次级代谢产物的发现

Mangroves grow in tropical and subtropical intertidal estuarine zones, with numerous stress factors in their habitat, such as high salinity, high temperatures, anoxic sludge and pests. To adapt the environment, mangrove plants and their endophytic fungi can produce varieties of specific secondary metabolites to build their own denfense system. Many mangrove plants present strong ability to resist pests. This makes their endophytic fungi a potential source to exploit novel natural drugs with anti-insect activity. In our previous work, we screened 499 endophytic fungal strains isolated from 12 mangrove plants, and found that 3 fungi displayed significant mosquitocidal activity and produced abundant secondary metabolites. In this project, we will further characterize these three strains. Based on the feature of salt tolerance of mangrove endophytic fungi, salinity stress and other tests will be performed with mosquitocidal activity as a guide. Fermentation conditions will be optimized for these three fungi, to get crude extract with better mosquitocidal activity. Compounds with mosquitocidal activity will be isolated and elucidated by spectroscopic and chemical methods. Then these compounds will be prepared directly on a larger scale. Their indoor insecticidal activity will be tested by feeding assay with Plutella xylostella, Leucania separata, Helicoverpa armigera, and Pierisrapae Linne. In addition, safety evaluation will be conducted by acute toxicity experiment of Bombyx mori. Through this project, we try to get some active agro-insecticide compounds with high efficiency and low toxicity, and establish a solid foundation to develop bio-pesticides with independent intellectual property rights.

红树植物为了适应和抵抗高盐、高温、缺氧、虫害等特殊生态环境，植物及其内生真菌可产生特殊的次级代谢产物来构建自身防御体系。许多红树植物具有较强的抗虫害能力，从其内生真菌着手，有望获得杀虫活性次级代谢产物。我们在前期筛选获得三株分离自木榄和海芒果的目标菌株，其发酵培养物粗提物具有显著杀蚊活性，并且次级代谢产物非常丰富。本项目拟进一步对三株目标菌株分类鉴定；在杀蚊活性导向下，利用红树植物内生真菌耐盐性的特点，设计高盐胁迫等试验，优化三株目标菌株的发酵条件，获得活性良好的发酵培养物粗提物；运用现代色谱学的分离手段和波谱学的结构鉴定技术，快速分离、鉴定和定向制备抗蚊活性次级代谢产物。采用小菜蛾、粘虫、菜缢管蚜致死活性筛选模型验证相关化合物的室内杀虫活性，采用家蚕急性毒性试验进行环境安全性评价，以便寻找到高效低毒的农用杀虫活性化合物，为开发具有自主知识产权的绿色环保的生物农药奠定基础。

红树植物为了适应和抵抗高盐、高温、缺氧、虫害等特殊生态环境，植物及其内生真菌可 产生特殊的次级代谢产物来构建自身防御体系。许多红树植物具有较强的抗虫害能力，从其内生真菌着手，有望获得杀虫活性次级代谢产物。我们在前期筛选获得三株分离自木榄 和海芒果的目标菌株，其发酵培养物粗提物具有显著杀蚊活性，并且次级代谢产物非常丰富。本项目在杀蚊活性导向下，优化三株目标菌株的发酵条件，扩大规模发酵并对发酵培养物的提取物进行分离纯化以获得具有抗虫活性的化合物。从该三株目标菌株分离纯化获得38个化合物，从另三株红树植物内生真菌分离获得63个化合物，其中10个新化合物，其中有一个为具有中等抗虫活性的新骨架化合物。完成了390株红树植物内生真菌（其中包括已完成次级代谢产物系统研究的所有红树植物内生真菌）的ITS 区域序列的PCR扩增和测序，并完成了部分相关菌株的形态鉴定。另外，从分离自深海沉积物、海藻、海绵等的真菌菌株的发酵产物中，分离纯化获得了24个新结构化合物，其中包括2种新骨架化合物。这些新结构化合物具有抗肿瘤、COX-2抑制、抗病毒、抗菌等生物学活性。本项目研究成果，为开发具有自主知识产权的抗虫生物农药以及抗肿瘤、抗炎、抗病毒和抗菌药物奠定了基础。本项目发表SCI 论文9篇，中文核心期刊论文1篇，申请国家发明专利1项。