广谱植物内生真菌促进连作花生结瘤与氮素积累的机理

Peanut is an important oil crop. Continuous cropping of peanut will cause cropping obstacle. Our previous studies showed that the application of a strain of broad spectrum endophytic fungus Phomopsis liquidambari B3 could significantly promote peanut nodulation, increase peanut yield and the content of grain protein. Promoting peanut nodulation is the key factor to overcome the cropping obstacle by adding endophytic fungus, but the detail of the mechanism is not clear. We presume that optimizing the soil environment, changing the peanut physiological condition, promoting the peanut growth and photosynthesis by adding endophytic fungus maybe the main reasons. In this project, denaturing gel gradient electrophresis, physical and chemical analysis, DNA sequencing and expression profiling analysis and so on will be used to investigate. Firstly, the relations of allelochemicals degradation and rhizobia diversity will be investigated to explain the reason of soil environment tendency to be benefit for peanut nodulation. Secondly, physiological responses and signal processes of peanut nodulation stimulated by endophytic fungal invasion will be analyzed. Additionally, the gene expression and plant physiological processes of the peanut nodulation will be investigated by expression profiling analysis. Thirdly, the relations of energy supplement for peanut nodulation, nitrogen fixation, nitrogen accumulation and promoting plant photosynthesis or growth of peanut by inoculating endophytic fungus will be explored. This study will provide a new perspective in overcoming peanut continuous cropping obstacle. It will be benefit to further explain the ecological relationships of endophytes and hosts or other symbiotic microorganism in theory.

花生是我国重要油料作物，多年连作会导致连作障碍。前期研究表明，向土壤中施加广谱内生真菌拟茎点霉B3菌株可以显著增加花生产量并提高结瘤能力和籽粒蛋白含量。提高花生结瘤能力是克服连作障碍的关键原因之一，而其机理不清楚。推测其可能原因是B3菌改善了土壤环境，改变了花生生理状态并促进生长与光合作用而有利于结瘤和固氮。本项目拟采用变性胶梯度电泳、理化分析、DNA测序与表达谱分析等手段进行研究。首先探讨B3菌添加后化感物质分解与根瘤菌多样性关系，分析土壤环境向有利于花生结瘤状态改变的原因。其次，分析B3菌刺激花生产生有利于结瘤的生理反应和信号过程，探讨促进结瘤的基因表达基础，阐述内生真菌有利于结瘤的生理过程。第三，探讨B3菌促进花生生长和光合作用为根瘤形成、固氮提供能量并导致籽粒氮素积累和增产的机制。本研究将为克服花生连作障碍提供新的视角，理论上有利于进一步阐述内生菌与宿主及其它共生微生物的生态关系。

花生是我国重要油料作物，多年连作会导致连作障碍。前期研究表明，向土壤中施加广谱内生真菌枫香拟茎点霉 B3 菌株可以显著增加花生产量并提高结瘤能力和籽粒蛋白含量。提高花生结瘤能力是克服连作障碍的关键原因之一，而其机理不清楚，本项目对此开展研究。研究结果表明，B3菌施加到土壤中，可以分解土壤中的化感物质，如阿魏酸、肉桂酸、芥子酸、木犀草素、白藜芦醇等，从而有利于细菌的增殖。B3菌施加到连作花生土壤中，可以提高根际土壤中固氮菌丰度，改变其群落结构。B3菌施加也有利于根瘤内可培养细菌及具有促生潜力的细菌多样性增加。阐明了B3菌降解上述化感物质的途径，克隆其降解关键酶之一漆酶的基因，在粟酒裂殖酵母中异源表达，证明了其功能。B3菌接种到花生，可以在其体内有效定殖，并引起其防御反应相关酶活力的升高，根际分泌物的增加。这有利于根瘤菌与花生建立共生关系。B3菌共生组花生根系分泌物能够显著增加根瘤菌的生长、被膜形成、趋化性、nodC 结瘤基因表达和结瘤数量。对根系分泌物组分分析表明，这种结瘤能力的增强与根系分泌物中酚酸、黄酮、有机酸和氨基酸组分含量的提高密切相关。花生体内钙离子、钙调蛋白、H2O2、 NO、生长素，介导了B3和宿主之间的信号过程。B3 菌与慢生根瘤菌共接种促进了连作花生根瘤原基和成熟根瘤的形成，同时增强了根瘤的生长发育和固氮效率。B3菌处理短暂地诱导了花生的防御响应，激活了根部黄酮合成相关酶的活性，促进了叶片光合活性和总糖含量的提高。同时，共接种组花生根瘤内碳代谢物含量以及碳代谢相关酶的活性比对照组高。本研究对阐明花生与微生物复合共生关系以及缓解其连作障碍有重要参考价值。总体上按期完成了研究工作，实现预期目的。已发表17篇研究论文，其中SCI论文11篇，中文期刊论文6篇，另有部分论文在整理中。申请发明专利2件。博士生2人，硕士生6人参加本项目研究并撰写学位论文，获得相应学位。