蜡质芽孢杆菌基于改变番茄根系结构防治根结线虫病的机理研究

Meloidogyne incognita is an obligate parasite of plant root, which is a serious threat to agriculture worldwide. Bacillus cereus AR156 is our patented and sequenced strain that is registered as a biopesticide against root-knot nematode in tomato. Previous study showed that AR156 lengthened the root zone of maturation, increased hair number of this area, and shortened the elongation zone of tomato, thus significantly reduced the infection rate of root-knot nematode. In the present project, we will firstly make a pool of transposon pIC333 insertion mutations, select strains which can cause different changing of root structure with wilt type AR156 in the plate seedling test, and calculate the infection rate and disease severity in growth chamber, so we can identify the functional genes by pIC333 insertion sites. Next, we will verify them by formulating some deletion and recovery mutants. On the other hand, we will sequence and analyze tomato transcriptome, and identify the influenced genes by differential expression. The last step is performing some growth chamber experiments for comparing the different growth-related genes of tomato treated by wild type and mutants of AR156, detecting mortality and reproduction rate of M. incognita juveniles inside of root, in the rhizosphere and bulk soils; and then evaluate the relationship of root structure's change and biocontrol efficacy. This study will lead us to understand the mechanism of biocontrol against root-knot disease based on changing root structure of tomato by Bacillus cereus AR156, and provide the theoretical basis and method guidance for the application of this bacterium.

南方根结线虫为植物根系专性寄生物，严重威胁农业生产。蜡质芽孢杆菌AR156是本组已测序专利菌株，获农药登记用于防治番茄根结线虫病。前期研究中该菌诱使番茄根系成熟区加长且根毛数明显增多、伸长区变短、显著降低线虫侵染率。本项目拟对此菌进行转座子pIC333插入突变，平板幼苗试验筛选引起番茄根系结构发生不同变化的菌株，温室试验评估其处理后根系结构变化与防病效果的相关性；鉴定插入位点序列和功能基因；构建定点缺失和恢复突变体进一步验证。另一方面对番茄转录组进行测序分析，利用差异表达方法鉴定受影响基因。最后温室试验比较AR156突变体与野生型菌株处理后番茄发育相关基因表达差异，检测生防过程中根内、根围土和非根围土中南方根结线虫幼虫的死亡和繁殖率，分析番茄根部结构变化与生防效果的关系。本研究试图明确AR156诱使植物根系结构变化从而防治根结线虫病的机理，为该菌的产业化提供理论基础和使用方法指导。

南方根结线虫为植物根系专性寄生物，严重威胁农业生产。蜡质芽孢杆菌AR156是本组已测序专利菌株，获农药登记用于防治番茄根结线虫病。前期研究中该菌诱使番茄根系成熟区加长且根毛数明显增多、伸长区变短、显著降低线虫侵染率。一方面，本项目对该菌进行转座 子pIC333插入突变，找到该菌生物膜形成受损的插入突变体、鉴定生物膜合成相关基因，同时发现该菌蛋白酶生物功能对其生物膜形成与游动性存在显著影响。另一方面，本项目对番茄转录组进行测序分析，利用差异表达方法鉴定受影响基因，发现受AR156诱导基因包括多个脱落酸、乙烯作用途径相关基因及部分热激蛋白编码基因。本研究明确了AR156诱使植物根系结构变化从而防治根结线虫病的机理，为该菌的产业化提供理论基础和使用方法指导。