榨菜（茎瘤芥）根肿菌休眠孢子萌发温度响应分子生态机制研究

The main diseases of tumorous stem mustard in cultivation is clubroot, Which is a serious threat to mustard production. The dormant spore germination of Plasmodiophora brassicae is the basic condition for infection of tumorous stem mustard and disease occurrence. Temperature and root exudates of tumorous stem mustard are the main factors inducing p. brassicae dormant spore germination, in the process of cultivation. In this project, P. brassicae dormant spore would treat with whether to add tumorous stem mustard root exudates at different temperatures, and analysis the transcriptome data based on genome annotation information. Temperature regulating genes during the germination of dormant spores would be Screened, and temperature signal pathway would be established. Target gene siRNA in temperature signal pathway would be synthetised, which use to validate target gene function on p. brassicae dormant spore germination. Genes that promote the germination of dormant spores would be screened. Double strands RNA of genes promoting the germination of dormant spore would be expressed in biocontrol fungi Trichoderma longibrachiatum. SiRNA cut by DICER enzyme and Secretion of T. longibrachiatum would be collected to analysis of polymerization prevention effect to clubroot. This project can clarify temperature regulation mechanism during dormancy spore germination from the perspective of molecular ecology, preliminary develop molecular vaccine, Lay the theoretical foundation and technical support for the prevention and treatment of disease tumorous stem mustard clubroot.

根肿病是榨菜（茎瘤芥）栽培中的主要病害严重威胁榨菜产业。根肿菌休眠孢子萌发是其侵染榨菜造成病害的前提，在栽培过程中温度和榨菜根系分泌物是诱导根肿菌休眠孢子萌发的主要因素。本项目拟在不同温度下对根肿菌休眠进行是否添加榨菜根系分泌物处理，结合根肿菌基因组注释信息分析转录组数据，筛选在休眠孢子萌发过程中温度调控基因，建立休眠孢子萌发过程中温度信号通路；利用温度信号通路中关键基因siRNA验证靶标基因功能，筛选对休眠孢子萌发有促进作用的基因；利用生防真菌长枝木霉表达多种促进根肿菌休眠孢子萌发基因的dsRNA，收集长枝木霉菌中被DICER酶剪切成的siRNA和长枝木霉菌分泌物，分析长枝木霉菌分泌物聚合靶标基因siRNA对榨菜根肿病的防治效果。本项目能够在分子生态角度阐明榨菜根肿菌休眠孢子萌发过程中温度调控机理，初步开发分子疫苗，为榨菜根肿病防治奠定理论基础和技术支撑。

根肿病是茎瘤芥栽培中的主要病害，严重威胁榨菜产业。根肿菌休眠孢子萌发是其侵染茎瘤芥造成病害的前提，在栽培过程中温度和榨菜根系分泌物是诱导根肿菌休眠孢子萌发的 主要因素。本项目开展了根肿菌中存在基于外源siRNA吸收的RNAi现象验证，根肿菌休眠孢子在不同温度下萌发过程中差异表达基因筛选，根肿菌PBRA_6677、PBRA_2550、PBRA_3405基因在根肿菌侵染致病过程中功能分析，根肿菌调控茎瘤芥根内微生物规律分析，以及利用长枝木霉表达根肿菌mapk1、mapk2、mapk3基因dsRNA对茎瘤芥根肿病防治作用研究。结果表明，根肿菌中存在基于吸收外源siRNA的RNAi现象；不同温度下根肿菌休眠孢子萌发差异显著，筛选到mapk通路以及24个效应子基因参与根肿菌萌发和侵染；利用基于siRNA的RNAi技术分析返现PBRA_2550、PBRA_3405基因与根肿菌侵染致病相关；PBRA_6677基因的粗蛋白能够明显降低根肿病的致病能力和病情指数，同时该基因粗蛋白对茎瘤芥根际微生物具有调控作用；根肿菌对茎瘤芥根内微生物的调控作用，受根肿菌抑制真菌种类比较少细菌种类以嗜甲基菌属等主体；长枝木霉军表达的根肿菌3个mapk基因的融合片段，并能够在长枝木霉中表达，对根肿病具有抑制作用。本项目能够在分子生态角度阐明榨菜根肿菌休眠孢子萌发过程中温度调控通路，阐述了3个重要效应子与根肿病致病性关系，PBRA_6677蛋白具有开发为分子疫苗潜力，相关研究结果为榨菜根肿病防治奠定理论基础和技术支撑。