土壤对根结线虫生防细菌的抑菌机制及解除研究

Soil fungistasis and soil bacteriostasis are the vital factors which reducing control efficacies of microbial agents against soil-borne diseases. By focusing on the mechanism and annulment of soil bacteriostasis against bacterial antagonist of root-knot nematode using Bacillus megaterium YMF3.25 as target, our project will be carried out to characterize the following issues. Firstly, B. megaterium YMF3.25 will be tagged by gfp gene and dual-resistance genes, and used to establish a method for measuring soil bacteriostasis. Secondly, we will characterize the relatiohship between soil bacteriostasis and the physicochemical properties of soils and soil bacterial community. Thirdly, we will determine the bacterial species and their volatiles involving in soil bacteriostasis. Finally, we attempt to find the method to annul the soil bacteriostasis against B. megaterium YMF3.25 basing on our patent technology. Results of our project will provide novel knowledge to understand the mechanism of soil bacteriostasis, and promote the progress of bioncontrol of soil-borne diseases using bacterial agents.

土壤抑菌作用（包括抑真菌作用和抑细菌作用）是影响土传病害生防制剂防效的关键因素之一。项目围绕土壤对细菌的抑菌作用机制及其解除主题，以生防细菌Bacillus megaterium YMF3.25为靶标菌；首先，通过双抗和绿色荧光蛋白基因标记目标菌，建立土壤抑细菌作用的检测方法。其次，以B. megaterium YMF3.25应用中防效波动地块为参考，代表性选取防效高、中、低不同的地块，明确土壤抑细菌程度与土壤理化因子间的相互关系；进一步明确土壤抑菌程度与土壤细菌区系组成特征的关系。第三，明确强抑制性土壤中的关键细菌类群，鉴定其参与土壤抑菌作用的主要物质。最后，利用项目组的专利技术，结合双抗平板检测和根际定殖荧光检测方法，探究土壤抑制B. megaterium YMF3.25的解除方法。本项研究有助于认识土壤抑细菌作用的本质，对促进细菌型微生物菌剂的田间生态学研究及其应用具有重要意义。

基于GFP和抗性标记，建立了土壤抑制生防细菌的检测方法，评估了采自全国7个省份、25种作物的135种农业土壤对B. megaterium YMF3.25的抑菌作用。结果表明土壤抑细菌作用广泛存在，供测土壤的抑菌率在7.81%-53.67%之间，平均抑制率为17.58%。供测土样的pH值、有机质含量、微生物多样性指数与抑菌率呈正相关关系，相关系数r分别为0.73、0.91和0.84。以线虫生防菌B. megaterium YMF3.25为靶标，明确了中国农业土壤通过产生挥发性物质参与抑菌作用的主要细菌类群，以及通过非挥发性物质参与抑菌作用的细菌类群；鉴定了参与土壤抑菌作用的细菌产生的5种挥发性抑菌物质。揭示了葡萄糖解除土壤抑菌作用机制是通过激活自噬、DNA复制和修复、RNA选择性剪接和降解途径等细胞过程进而缓解土壤对生防菌的抑菌作用。本项研究有助于认识土壤抑细菌作用的本质，对促进细菌型微生物菌剂的田间生态学研究及其应用具有重要意义。