柑橘内生细菌菌群与宿主品种抗/感溃疡病能力的相关性研究

Many endophyte strains reported is able to enhance the resistance of plant to host currently. However, it is few researched that there is relativity between the endophytic community and the resistance or susceptibility of host variety. Citrus canker is an important disease of orange, and it is essential to control the disease that the mechanism of citrus resistance is known thoroughly. On base of the former work, the research will be done involving two contents by using Citrus unchiu Marc. (variety with resistance to citrus canker) and Citrus sinensis Osbeck (variety with susceptibility to citrus canker). ① Detecting all endophytic bacteria from orange by Pyrosequencing, and combining with the correlative informations of cultured endophytic bacteria obtained, the relativity will be analysed. ② After inoculating the main endophytic bacteria from host and (or) Xanthomonas axonopodis pv. citri to two orange sterile seedlings, the disease will be graded, and the cellar ultrastructure and the distributing of pathogen will be observed by using electronmicroscope, and the gene express of Plant Defensin 1.2 (PDF1.2) in citrus is inspected by applying the Quantitative Real Time Polymerase Chain Reaction (qRT-PCR), all which will cleared the influence and the action of the main endophytic bacteria from orange on the resistance of host variety, and the relativity of endophytic bacteria community with the ability of orange variety against the citrus canker will be validated. The results will be able to provide a theoretical evidence to reveal the nature of the varietal resistance or susceptibility of orange to citrus canker, and establish a theoretical foundation to understand further the interaction between endophytic bacteria community and their hosts.

目前已报道的很多内生菌株对植物抗病性具增强作用。但内生菌群与宿主品种抗/感病能力间的相关性研究较少。溃疡病是威胁柑橘生产的重要病害之一，充分了解寄主的抗病机制是防治该病的前提。本项目在前期工作的基础上，拟选用温州蜜柑（抗溃疡品种）和赣南纽荷尔脐橙（感溃疡品种）为材料，①应用454焦磷酸测序技术分别研究两种柑橘在不同生长期体内细菌菌群结构特征与功能差异，结合可培养菌株的相关信息系统分析内生细菌菌群与宿主品种抗/感溃疡病能力的相关性；②接种优势内生细菌和（或）溃疡病菌于两种柑橘无菌苗，通过测定植株发病程度、电镜观察病菌形态和分布、qRT-PCR检测植株中PDF 1.2基因表达情况，明确优势内生细菌对宿主抗/感病能力的影响和作用，验证内生细菌菌群与柑橘品种抗/感溃疡病能力的相关性。研究结果可为充分认识柑橘品种抗/感溃疡病的本质提供理论依据，亦可为深入了解内生菌群体与宿主的互作关系奠定理论基础。

溃疡病是威胁柑橘生产的重要病害之一，充分了解寄主的抗病机制是防治该病的前提。本项目选用温州蜜柑（抗溃疡病品种）和纽荷尔脐橙（感溃疡病品种）为材料，通过富集内生细菌、检测内生细菌菌群和分离可培养内生细菌，分析了柑橘内生细菌与寄主品种抗/感溃疡病的相关性，并在构建无菌苗的基础上进行了一些验证，取得了以下研究结果。.1、采用高通量测序对不同柑橘样品的内生细菌富集方法进行了研究，筛选出了适当的酶组合处理不同柑橘组织以便富集其体内细菌，从而有效减少叶绿体DNA对内生细菌研究的干扰。这为后续实验及柑橘内生细菌菌群的分子研究提供了技术支持。.2、应用高通量测序技术分别测定了两种柑橘在不同生长期体内细菌菌群情况，通过软件分析菌群结构特征与功能差异得出，柑橘抗病和感病品种中的内生细菌菌群存在一定差异，尤其优势菌属差异较大，温州蜜柑存在较多具有拮抗特性的内生细菌，而纽荷尔脐橙中同时存在拮抗细菌和感病协同细菌。.3、通过分离柑橘可培养内生细菌发现，相比于感病品种，抗病品种中内生细菌的种类少但数量多，尤其是拮抗菌株抑菌能力强。进一步的活体接种试验筛选出了几株对柑橘溃疡病防治效果较好的内生菌株，可为柑橘溃疡病的生物防治提供素材。.4、通过一系列的组织培养试验进行了柑橘无菌苗的构建，成功获得长势良好的柑橘组培苗，为后续试验奠定了基础。.5、接种几株优势内生细菌和（或）溃疡病菌于柑橘无菌苗，通过测定发现，优势细菌能稳定地在柑橘体内定殖；脐橙优势内生细菌部分能减轻溃疡病的发生，而蜜柑优势内生细菌则均能抑制溃疡病的发生；PDF1.2基因未曾扩增出来，可能是因为内生细菌激发柑橘系统抗性由其他基因调控或抗性并未被激发，需要进一步研究。.由以上结果可以得出，柑橘组织中的细菌能在一定程度上影响寄主抗溃疡病的能力。研究结果可为充分认识柑橘品种抗/感溃疡病的本质提供理论依据。