根腐线虫发病蕉园根际微生物群落和功能特征解析

Root-lesion nematodes (Pratylenchus spp) rank second only to root knot and cyst nematodes as having the greatest economic impact on crops worldwide. The main life history and infection cycle of Pratylenchus spp were completed in the rhizosphere environment. The occurrence of Pratylenchus spp is closely related to the composition and diversity of the rhizosphere microorganisms. P. goodeyi was firstly identified on Banana in China by our group. At present, there is a lack of research on the relationship between P. goodeyi and their rhizosphere microorganisms. Is there any bacterial groups that closely related to the occurrence of P. goodeyi? In this study, two treatments will be set up on the diease group and the healthy group of banana. With Illumina Miseq sequencing technology, 16S/ITS rDNA sequencing will be carried out to statistical analyze the structure changes of rhizosphere microbial community. Some bacterial groups that closely related to the occurrence of P. goodeyi will be cultured, and then mined to be the microbial resources for controlling P. goodeyi. By metaproteomics technology, some functional proteins and pathways which show significant toxicity against P. goodeyi will also be identified to analyze the function changes of rhizosphere microbial community. And then Functional genes will be mined for controlling P. goodeyi. Finally, Metagenomic library from the rhizosphere soils was constructed, the highly nematicidal clones were isolated from this library, and then nematicidal gene was obtained through clone sequencing. Our study has important implications in designing a transgenic resistance approach to control banana parasitic nematodes.

短体线虫（根腐线虫）是危害仅次于根结线虫和胞囊线虫的世界第三大植物寄生线虫，短体线虫在根际环境中完成其主要生活史和侵染循环。短体线虫的发生与根际微生物的组成和多样性存在关联。本课题组在国内首次鉴定了危害香蕉的短体线虫新种：古氏短体线虫。目前，该线虫与寄主植物根际微生物相互关系的研究还是一片空白。是否普遍性存在某些菌群与该线虫密切相关？本研究拟对海南香蕉田设发病组和健康组，利用Illumina Miseq测序技术进行16S/ITS rDNA测序，分析根腐线虫发病蕉园微生物群落结构的改变，为有针对性地发掘该线虫生防资源提供策略。利用宏蛋白质组学技术，分析对该线虫具有杀虫作用的功能蛋白和通路，揭示根腐线虫发病蕉园根际微生物群落功能变化规律，为有针对性地发掘杀虫基因提供策略。构建蕉田土壤大片段宏基因组文库，筛选对该线虫具有杀虫活性的功能基因。本研究将为新型生物农药创制和转基因植物培育提供理论依据。

根腐线虫是世界分布最为广泛和最具破坏性的迁徙性植物内寄生线虫之一。根腐线虫的发生与根际微生物的组成和多样性存在关联。本项目研究根腐线虫发病蕉园微生物群落和功能基因，从而有针对性地发掘线虫生防资源和杀虫基因，同时鉴定对线虫具有杀虫活性的功能基因。通过本项目的实施，目前分离得到25株对线虫具有较高活性的拮抗菌，对这些拮抗菌的进一步分析鉴定结果表明，在这25株菌株中，20株为芽胞杆菌菌株，2株为类芽孢杆菌属，一株为Burkholderia. sp。对其中的部分菌株进行了基因组测序，预测其潜在的杀虫基因。通过在植物体内表达高效杀线虫基因，验证其抗线虫功能。本研究获得了一系列转芽胞杆菌杀虫基因的烟草植物，选取表达量比较高的株系进行抗线虫活性测定。盆栽试验证明转基因烟草能够明显的抑制线虫在植物体内的发育，显著降低线虫的侵染和繁殖。线虫侵染转GFP烟草实验进一步从细胞生物学水平证明线虫能够取食到根部表达的蛋白。在分离微生物的同时，我们意外得到了另外的植物寄生线虫。分别是螺旋线虫，肾形线虫等。同时对部分线虫进行了基因组测序和分析。这些结果表明多种病原线虫的复合感染比较普遍。在项目执行过程中，已发表SCI一区论文1篇，拟投稿SCI期刊论文2篇。同时培养了包括项目负责人在内的诸多年轻人。本项目获得了抗线虫的转基因植物，在防治线虫病害中具有较大应用价值，为培育抗植物寄生线虫植物新品种奠定理论基础，为抗线虫转基因作物的商业化提供新的视角，研究结果具有重要的理论和实践意义。