生防改良菌株F1-35对西瓜尖镰孢枯萎病的防治机理

Fusarium oxysporum exists in the soil and the plant roots extensively. The pathogenic strains can infect in the vascular system, resulting the wilt of plants, causing great economic losses. Non pathogenic strain Fo47 has been used to control the Fusarium wilt diseases of cotton, potato, watermelon, cucumber which are caused by pathogenic Fusarium oxysporum. At the same time, Fo47 can accelerate the growth rate of the plants and improve the resistant to environmental stress. Through protoplast fusion with Fo47 and biocontrol actinomycetes, we obtained the strain F1-35. The biocontrol effect to Fusarium oxysporum f. sp. niveum, colonization ability and accelerate growth rate to watermelon of the recombinant F1-35 and were all better than that of Fo47. F1-35 played a major role in controlling Fon disease. In order to illustrate the molecular mechanism of the F1-35 for biological control on Fon, we investigated the induced systemic resistance elicited by F1-35 in watermelon plant defense response to Fon and influence on Fon infection process using the methods of GFP/RFP mark, fluorescent microscope,Quantitative Competitive Polymerase Chain Reaction(QC-PCR), Western blot and qRT-PCR and scanning electronmicroscope. The signal transduction pathway and expression of related defense genes in F1-35 Induced Systemic Resistance to Fon in watermelon plants was studied, and the mechanism of F1-35 acted on the colonization, infection and reproduction of Fon were assayed too. Above results will reveal the biocontrol mechanism of F1-35 to Fon. The whole result will be beneficial to research and develop the resource of protoplast fusion between different kingdom, and provide theoretical principle for a novel biological pesticide against plant Fusarium wilt diseases.

尖镰孢(Fusarium oxysporum)广泛存在于土壤和植物根际，其中的致病菌株能侵入维管束系统导致植物萎蔫枯死，造成严重的经济损失。非致病性尖镰孢 Fo47已成功应用于防治棉花、番茄等尖镰孢枯萎病，同时具有促生和抗逆作用。本实验室通过原生质体跨界融合技术，用生防放线菌153改良Fo47，获得了生防效果、定殖能力和促生作用均优于Fo47的菌株F1-35。本项目拟采用GFP/RFP标记、QC-PCR、Western blot、qRT-PCR、扫描电镜和荧光显微镜观察等技术手段，从F1-35对西瓜的诱导抗性及其对西瓜枯萎病菌侵染过程的影响两个方面，系统研究F1-35诱导西瓜植株抗病性的信号传导途径及相关防卫基因表达差异，及F1-35对病菌定殖、侵染等环节的作用方式，揭示F1-35对西瓜枯萎病的生防机理。研究将为进一步挖掘具有生防潜力的融合菌株及新型抗植物枯萎病生物农药的创制提供理论基础。

本项目以生防放线菌153和Fo47跨界融合获得的改良生防菌株F1-35为基础，采用GFP/RFP标记、荧光显微镜观察、蛋白组学、qRT-PCR等技术手段，从F1-35对西瓜的诱导抗性及其对西瓜枯萎病菌侵染过程的影响两个方面，系统研究F1-35诱导西瓜植株抗病性的信号传导途径及其相关防卫基因表达差异，及F1-35对病菌定殖、侵染等环节的作用方式，揭示F1-35对西瓜枯萎病的生防机理。主要取得了一下研究结果：.通过GFP/RFP分别标记生防菌株F1-35和致病菌株，研究F1-35对西瓜枯萎菌定殖和侵染的影响，同时研究F1-35对西瓜幼苗在枯萎病菌胁迫下相关防御酶的活性变化，结果表明F1-35相对于Fon可在西瓜幼苗根部提前48h大量定殖，具有占位作用，同时可提高西瓜幼苗体内相关防御酶的活性。.通过蛋白质组学开展生防菌处理西瓜幼苗后根系蛋白与对照根系蛋白差异比较，发现差异蛋白主要集中在糖代谢通路，碳代谢，氨基酸合成，丙酮酸途径，苯丙素合成途径，RNA转运，嘌呤代谢，植物病原互作，mRNA检测途径，核糖体，丙氨酸，天冬氨酸和谷氨酸代谢，PI3K-Akt信号通路，精氨酸和脯氨酸代谢。其中显著上调表达蛋白参与的抗病途径主要集中在JA和SA相关蛋白，PR5和3-磷酸甘油醛脱氢酶。而下调蛋白为赤霉素结合蛋白（GIP）、生长素抑制蛋白（ARP）和JA负调控蛋白WRKY 17。表明生防菌F1-35处理可激发西瓜幼苗中SA和JA途径提高对西瓜枯萎病的抗性。.以qRT-PCR技术分析SA、JA途径相关的关键基因PR1、PR3、PAL、LOX1、CTR1及NPR1的表达量，结果表明在根、茎、叶组织与SA、JA正相关的关键酶基因PR1、PR3、PAL、LOX1、NPR1均上调表达，与ET负相关的关键酶基因CTR1基因表达量下调，说明生防菌F1-35处理后，西瓜幼苗的SA、JA/ET抗病途径均被刺激上调表达。.综上所述，生防菌F1-35在西瓜幼苗根部具有占位作用，生防菌F1-35处理后刺激西瓜植株SA、JA途径增强对病原菌Fon的抗性。研究结果可为进一步挖掘具有生防潜力的融合菌株以及新型抗植物枯萎病害生物农药的创制提供理论基础，具有较好的科学意义和应用前景。