产尿素细菌的生物多样性及其防控根结线虫病的机制研究

Basing on the knowledges of ammonia as the nematicidal virulence factor and a signal molecule to induce trap-formation in nematode-trapping fungi, this project focuses on the biodiversity of urea-producing bacteria (UPB) and their control potential against roo-knot nematodes, and will carry out the studies as the follow. Firstly, we will identify UPB from virous substances, characterize their species diversity, and evaluate theri nematicidal activities. Secondly, we will identify the nematicidal virulence factors from the representatives of UPB based on the methods of genomics, comparative genomic and transcriptomics. Thirdly, we will evaluate the control effecacy of the combination of UPB and urease-positive bacteria in controlling root-knot nematodes, and the safety towards crops and soil microbe when producing the NH3. Finally, on the basis of UPB can mobilize nematode-trapping fungi to kill nematodes, we will study on the potential and vital fators of UPB and nematode-trapping fungi in the process of control roo-knot nematodes. As a producer of nitrogen (urea) and potential novel resource against nematode, the scientific significance and application value of UPB are extremely significant. Results of this study will provide a new strategy for the reduction of pesticide and chemical fertilizer.

基于NH3的杀线虫活性及其作为诱导捕食线虫真菌捕器形成的信号分子，本项目围绕产尿素细菌生物多样性及其对根结线虫病的生物防控机制，拟开展以下研究：⑴在项目组前期工作基础上进一步发掘产尿素细菌资源，明确其物种多样性，评估其杀线虫活性；⑵基于基因组学，比较基因组学及转录组学研究，分析产尿素细菌主要类群侵染线虫的毒力因子；⑶评估产尿素细菌与脲酶活性细菌(脲酶)配伍时释放NH3防控根结线虫病的生防潜力及对作物、土著微生物的安全性；⑷基于细菌驱动捕食线虫真菌捕食线虫的生防学机制，研究产尿素细菌及捕食线虫真菌协同防控根结线虫病的潜力及必要的生防条件。产尿素细菌作为氮肥（尿素）的生产者和潜在的线虫生防新资源，研究的科学意义和应用价值极为显著，研究结果将为国家“减农药、减化肥”目标的实现提供新策略。

本项目对产尿素细菌的生物多样性及其防控根结线虫病的机制进行了深入的研究，获得的主要研究结果包括：（1）首次报道122种分离自烟草内生菌、蚯蚓堆肥、植烟土壤、动物粪便的细菌可以通过其生物合成产生尿素，分属Actinobacteria、Firmicutes、Proteobacteria、Bacteroidetes和Deinococcus-Thermus五个类群；Bacillus为优势属；报道细菌新物种8个。（2）发现14株产尿素细菌的杀线虫活性大于70%， Myroides xuanwuensis F4301的杀线虫效果最好，致死率为83.78%。在10株产尿素细菌发酵液中添加脲酶时，其对J2的致死率提高了1.13-22.72%，对番茄根结线虫病均有良好的防控效果，根瘤须根占比在3.19-21%，显著低于对照（75.23%）；产尿素细菌Brevibacillus brevis YMF3.469与富含脲酶的黑豆粉配合对根结线虫的防效达76.22%。（3）分析了产尿素细菌Bacillus zhangzhouensis 1.4517 的基因组及其产尿素机制，发现该菌株存在精氨酸酶基因（rocF），精氨酸由谷氨酸在多个合成酶、激酶的作用下生成，遵循线性途径，存在精氨酸酶代谢途径；该细菌的精氨酸通过精氨酸酶途径代谢生成尿素。（4）优化了产尿素细菌B. haikouensis MNJ12的发酵条件，在此优化条件下MNJ12菌株的产尿素能力从最初的490.03μg/mL提高到744.51μg/mL。（5）发现来自杏鲍菇的脲酶与产尿素细菌互作产生氨气，在土壤中添加1%的蚓粪能显著提高土壤中捕食线虫真菌的物种多样性，对烟草根结线虫病的防效达61%。 (6) 发表SCI论文12篇；培养硕士研究生2名、指导1名本科生完成毕业论文。