胜红蓟增强枯草芽孢杆菌在石榴根际定殖的机理探究

Application of Bacillus subtilis to control pomegranate wilt is a safe and environmental biological method. Wherever, difficulties of colonization in soil and instability of control efficiency continually restricted its application in production. Our previous studies found that planting the predominant weed Ageratum conyzoides around young pomegranate trees greatly improved colonization level of Bacillus subtilis B9601 in soil Application of Bacillus subtilis to control pomegranate wilt, caused by devastating fungus pathogen Ceratocystis fimbriata, is a safe and environment friendly method. However, the practical application of B. subtilis is hampered by its unsuccessful colonization and inconsistent efficacy in rhizosphere of pomegranate.. Our previous studies found that the weed Ageratum conyzoides planted around young pomegranate trees greatly improved colonization of B. subtilis B9601 in soil and efficacy of biocontrol against pomegranate wilt by pot experiment. We also found that nitrogen was an important factor influencing B9601’s colonization. Based on our preliminary results, we hypothesize that nitrogen stress caused by excessive fertilization was one of important reasons leading to unsuccessful colonization of strain B9601 in soil. We speculate it was A. conyzoides that help B9601 colonize in soil and promote biocontrol efficacy by eliminating the nitrogen stress and secreting root exudates. The study will be conducted to monitor colonization of green fluorescence protein (GFP) labeled strain B9601 in soil with or without A. conyzoides, , to purify and identify the root exudates, which can promote the growth of strain B9601, secreted by A. conyzoides using methods of HPLC and GC-MS. This project will provide more refined understanding how weed root exudates support colonization of strain B9601 in rhizosphere niche. This project will also uncover the relationship between soil beneficially ecological function provided by weed and soil-borne disease control by antagonistic bacteria, and provide new insight into extending comprehensive utilization of biodiversity.

利用拮抗菌枯草芽孢杆菌防治石榴枯萎病是一种安全、环保的生物防治方法，但枯草芽孢杆菌在土壤中定殖困难、防效不稳定限制了其在生产上的应用。前期试验发现，石榴园优势杂草胜红蓟促进了枯草芽孢杆菌B9601在土壤中的定殖并提高对石榴枯萎病的防效。施肥过量是石榴园根际土壤胁迫因素之一。据此我们假定：施肥过量造成的氮胁迫是造成菌株B9601在根际定殖难的重要原因。而胜红蓟通过根系的吸收作用解除氮胁迫，并释放根系分泌物促进B9601在土壤中的定殖，进而增强控制枯萎病的效果。我们拟采用绿色荧光蛋白基因标记和定量检测B9601在根际的定殖率，结合HPLC、GC-MS等色谱技术纯化鉴定胜红蓟根系分泌物的活性等技术手段，探明胜红蓟根系如何优化菌株B9601的定殖环境、增强对石榴枯萎病防效的原因。本项目的实施对揭示杂草调节土壤微生态环境、增强生防菌控制土传病害的作用具有重要意义，为深化生物多样的利用提供理论依据。

石榴枯萎菌是造成云南蒙自石榴园毁园的土传病害，曾给蒙自的石榴产业造成重创。采用化学农药处理，并不能控制石榴枯萎病的扩展和蔓延。利用生防菌控制土传病害的发生蔓延是一种安全环保的土传病害控制方式。然而，生防菌株进入土壤以后，不能有效定殖，导致至今未有商业化的应用于石榴枯萎病控制的生防制剂进入市场。. 项目前期研究发现，石榴园优势杂草胜红蓟具有提高生防菌株定殖水平的作用。为深入提示胜红蓟如何提高生防菌定殖水平的机理，本项目假定由于田间氮肥施用过多，恶化了生防菌的定殖环境，分布于果园中的杂草胜红蓟通过吸收作用降低氮胁迫，同时释放根系分泌物质从而改善定殖环境。. 为更清楚明确生防菌在土壤中的定殖状况，本研究采用基因工程技术，将荧光质粒载体将荧光基因导入到生防菌bp4201之中，形成用了本研究的工程菌bp4201-gfp。经检验该工程菌可稳定遗传、可满足本研究的需要。. 通过构建具有荧光标记基因的生防菌株bp4201-gfp，然后在有、无土壤吸附剂的条件下向土壤中添加生防菌株bp4201-gfp，测定有无胜红蓟条件下生防菌株bp4201的定殖水平。结果表明，在没有胜红蓟的土壤中，生防菌株bp4201始终维持在较低的水平，而且氮素表现出明显的抵制效应；土壤中有胜红蓟的土壤中，添加氮素并不会影响生防菌bp4201的定殖，反而提高了生防菌bp4201在土壤中的定殖水平；在土壤中添加吸附剂的情况下，生防菌bp4201的定殖水平有0~30%的下降。. 在室内，单独用胜红蓟根系分泌物处理生防菌bp4201，根系分泌物表现出显著的促进 bp4201生长的作用，可提高生防菌bp4201定殖水平达30%~100%。为明确根系分泌物中的活性成分，本项目研究了在氮胁迫作用下，根系分沁物中主要成分的变化，结果发现上调差异代谢物有131种，下调差异代谢物有75种。在离体条件下，各选定5种变化最大的差异代谢物，结果发现这些差异代谢物均对生防菌bp4201的增殖均有促进作用。. 本研究完成了项目预设的所有内容，明确了胜红蓟强生防菌定殖能力控制石榴枯萎菌的机理，该研究结果为农业生物多样性的应用和土传病害的防治提供了新思路。