生防芽孢杆菌B579田间定殖动态及其微生态效应研究

With the development of eco-agriculture, interest in biological control of soil-borne plant pathogens has increased considerably in the last few decades, because it could provide control of diseases that cannot or only partly be managed by other control strategies. Therefore, the stratagem of biological control has become an important approach to create a more long-lasting effect and facilitating sustainable agriculture. And colonization is an essential step for biocontrol agents to suppress pathogens in rhizosphere. Biocontrol agents are often effective in the laboratory, while the biocontrol effect achieved in the field is sometimes unpredictable. Some of these failures can be attributed to inadequate colonization of microbial inocula. And a better understanding of the unwanted and unspecific actions of the introduced beneficial microorganisms against non-target organisms is required. In our previous study, Bacillus subtilis B579, which was isolated from rhizosphere of cucumber, could effectively suppress Fusarium wilt of cucumber and induce systemic resistance under greenhouse conditions. In the present study, the root colonization of cucumber by B. subtilis B579 would be tested using the fluorescence in situ hybridization method with oligonucleotide probes and specific gfp-tagging of the inoculant strain in combination with confocal laser scanning microscopy. The colonization dynamic state of B579 in rhizosphere, root, stem and leaf blade would be clarified. Besides, the best inoculation method and inoculation cycle would be definited. . In the present study, the non-target effects of B579 on the microbial communities in rhizosphere of cucumber would be analyzed using PCR, DGGE and T-RFLP methods. The variation of rhizospheric microorganism communities was often leads to a variation of soil enzyme activity. So, the rhizosphere soil enzyme activity would be tested, which inclued urase, esterase, phosphotase and chitinase. The effect of B579 on soil microecosystem would be clarified. This study would play an important role in agricultural application and commercialization of biocontrol bacteria B579.

近年来，随着现代生态农业的发展，环境友好的病虫害防治方法日益受到人们的关注，因此利用有益微生物及其代谢产物防治植物病害成为农业可持续发展的重要途径。生防菌的有效定殖是发挥生防效果的关键因素，但影响生防菌有效定殖的关键因素尚不清晰以及生防菌的大量施入对土壤微生态系统的影响尚缺乏可预见性，严重制约着生防菌的大田应用。本研究以前期筛选到的优良生防芽孢杆菌B579为研究对象，采用寡核苷酸探针原位标记、绿色荧光蛋白标记技术对生防菌B579的大田定殖行为进行原位及定性定量分析，明确其在根际及植物不同部位的定殖动态及定殖周期，从而阐明影响其有效定殖的关键因素；同时利用PCR、DGGE和T-RFLP方法评价其对土壤微生物多样性的影响，结合土壤关键酶活力（脲酶、酯酶、磷酸酶和几丁质酶）的变化，确定生防菌对土壤微生态系统的影响，为生防菌B579的大田有效应用提供理论依据。

近年来，随着现代生态农业的发展，环境友好的病虫害防治方法日益受到人们的关注，因此利用有益微生物及其代谢产物防治植物病害成为农业可持续发展的重要途径。但影响生防菌的有效定殖的关键因素不清晰以及有益微生物的大量施入对土壤微生态系统的影响尚缺乏可预见性，制约着生防菌在大田中的应用。本研究以前期筛选到的优良生防枯草芽孢杆菌B579为研究对象，采用绿色荧光蛋白标记技术对生防菌B579的大田定殖行为进行了定性定量分析，明确了其在植物根际的定殖动态，并确定了最佳的使用方式为浸种和灌根处理，最佳使用周期为两个月。利用根际土壤总DNA高通量测序分析的方法，对细菌和真菌群落分别进行了基于OUT（Operational Taxonomic Units）的微生物物种丰度分析、Alpha 多样性分析和Beta多样性分析，明确了生防菌的有效定殖能显著提高土壤微生物群落的丰度和多样性；同时结合土壤关键酶活力（脲酶、过氧化氢酶、碱性磷酸酶和几丁质酶）的变化，阐明了生防菌可显著提高土壤关键酶活力，进而提高土壤微生物总活力及土壤生产力，进一步明确了生防菌对土壤微生态系统的有益影响，为今后生防菌B579的大田应用提供了理论支持。