丛枝菌根真菌提高桉树抗青枯菌的机制研究

The Eucalyptus bacterial wilt disease causing by Ralstonia solanacearum is a devastating soil borne bacterial disaster disease. This disease seriously threatens the healthy development of Eucalyptus plantation and causes huge economic losses. Previous studies have shown that arbuscular mycorrhizal fungi can effectively control the R. solanacearum of Eucalyptus. However, the interaction mechanism between arbuscular mycorrhizal fungi，R. solanacearum and Eucalyptus is unclear. This is greatly restricted the application of arbuscular mycorrhizal fungi to control Eucalyptus bacterial wilt disease. Therefore, this project is going to build symbiotic systems between the arbuscular mycorrhizal fungi and Eucalyptus, and inoculation with R. solanacearum. Growth and development, nutrient absorption and biochemical characteristics of these interactions will be observed and measure, which are involving in the host defense response. Then, specific expression genes related to the resistance in arbuscular mycorrhizal fungi, pathogenicity genes of R. solanacearum, and defense genes of Eucalyptus will be screened using the RNA-seq sequencing. The expression patterns of Eucalyptus defense genes inducing by arbuscular mycorhizal fungi will be analyzed, which are resistant against R. solanacearum. Meanwhile, the key inducing defense gene of these will be isolated and subcellular localized. In addition, the Eucalyptus transformation system will be constructed. The biological function of the target gene will be validated by using over expression, Crispr/Cas9 or RNAi reverse genetic techniques. This project will reveal mechanism involved in enhancing the resistance of Eucalyptus against R. solanacearum by using the arbuscular mycorrhizal fungi. The result will provide direct theoretical basis and technical support to control the bacterial wilt of forestry by using the mycorrhizal fungi.

由青枯菌引起的桉树青枯病是一种毁灭性的土传细菌性灾害，严重威胁桉树人工林的健康发展，造成巨大的经济损失。前期研究表明，丛枝菌根真菌能有效防治桉树青枯菌。然而，由于丛枝菌根真菌-桉树-青枯菌互作机制不清楚，严重制约了丛枝菌根真菌防控桉树青枯病的应用。因此，本项目拟构建丛枝菌根真菌和桉树的共生体系，接种青枯菌，研究丛枝菌根真菌-桉树-青枯菌互作后，桉树的生长发育、养分吸收和防御相关生理生化指标；利用RNA-seq测序技术筛选特异性表达的丛枝菌根真菌抗病原菌胁迫相关基因、青枯菌致病相关基因、桉树防御相关基因，分析丛枝菌根真菌诱导的桉树抗青枯菌相关基因的表达模式，分离其中关键基因并进行亚细胞定位；构建桉树遗传转化体系，利用过表达、Crispr/Cas9或RNAi反向遗传技术研究目的基因的生物学功能。本项目将揭示丛枝菌根真菌提高桉树抗青枯病的机制，为利用菌根真菌防治林木青枯病提供理论依据及技术支撑。

近年来，由青枯菌引起的土传病害桉树青枯病在全球大面积发生，青枯菌变异速度快，防控难度大，严重制约桉树产业的健康发展。本研究分离鉴定获得桉树最新青枯菌菌株YD，明确其基本特征，筛选了较抗病桉树品系，基因组插入获得红色荧光报告菌株，能便捷地观测青枯菌在桉树中的致病进程。分别构建了丛枝菌根真菌异形根孢囊霉和摩西管柄囊霉、青枯菌与巨桉之间不同的互作体系，接种AMF后第8-12周在巨桉幼苗根系形成较稳定的共生结构，菌根化显著提高巨桉幼苗叶片光合能力、增加养分积累、促进植株健壮生长。随着青枯菌侵染，AMF介导巨桉根系与叶片组织中防御相关物质迅速积累和防御相关酶活性显著提高以提高桉树对青枯菌的防御，对桉树青枯病防控效果达到74.00%以上。其次，研究AMF、青枯菌与桉树不同互作体系的转录水平特征。青枯菌侵染后，AMF介导桉树激素信号转导、淀粉和蔗糖代谢、MAPK信号等途径防御青枯菌。基于青枯菌侵染菌根化桉树根系水杨酸和茉莉酸含量显著高于非菌根化处理，外源喷施不同浓度的不同激素显示1 mMol/L SA处理对桉树青枯病的抗性效果最好，筛选出AMF介导的桉树SA途径EgMLP、EgNPR1、ICS1、EDS1、PR-1、PR-5、BGL2、WRKYs关键基因，明确其表达特征。AMF介导EgMLP34和EgMLP328_5基因显著上调，积累SA增强寄主对青枯菌的防御，初步构建了巨桉毛根转化与AMF共培养体系。同时，AMF响应青枯菌显著差异表达基因主要富集在碳氮代谢、磷酸戊糖等通路，二者可能存在能量的竞争。鉴定出575个巨桉根系miRNA，AMF介导桉树miR319、miRNA482和miR399等可能负调控激素信号传导、养分代谢等以调控桉树的生长、共生和防卫反应。此外，结合桉树人工林短期施肥、灌水等生产措施对土壤真菌群落结构及其环境因子的影响，发现施肥灌水下桉树生产力最高，显著改变土壤真菌群落结构和功能尤其是共生型菌根真菌，分离鉴定获得其中优势菌马勃菌并构建了二者的共生体系。研究结果为利用菌根真菌提高桉树生产力和防控桉树青枯病提供了一定的资源，也为阐明AMF提高桉树抗青枯菌能力的机制奠定一定理论基础。