杏褪绿卷叶植原体宿主植物内生细菌的群落变化及与宿主抗/感病性的相关性研究

Apricot chlorotic Leaf roll (ACLR) was first discovered in southern Xinjiang autonomous region, which is one of the strong pathogenicity disease associated with phytoplasma, a quarantine pathogen, and consided to cause significant disasters for fruit industry if the pathogen infects and spreads wildly. But there is a distinct lack of knowledge about it in our country. In this project, we propose to study that (ⅰ) investigating the disease distribution in the main producing areas of Apricot, and molecular classification of ACLR associated phytoplasma, cloning and bioinfomatics analysis of its multilocus genes including 16S rDNA, tuf, rp, secY and nusA to determin its phylogenetic relationships and molecular variability. (ⅱ) describing the temporal and spatial variations of community structure of endophtic bacteria living in different tissues of healthy and the phytoplasma infected trees by culture-dependent method and culture-independent method including PCR-DGGE and RT- PCR-DGGE, and analyze specific community composition and proportion, reveal dominant bacterial species which may interact with ACLR phytoplasma. (ⅲ) revealing the correlation between endophtic bacteria diversity and ability of apricot cultivars with different resistance (susceptibility) to ACLR. The objective of this research is to in-depth understand the interaction of phytoplasma pathogen, host plant and companion bacteria, provide theoretic basis for studying function of pathogenic mechanism, also would contribute to selecting and developping resistant varieties, and obtain information for future studies focused on developing sustainable control strategies.

杏褪绿卷叶病是新疆杏产区首次发现的高致死、检疫性植原体病害，对近年快速发展的新疆林果产业带来巨大隐患。国内目前对其研究极少。本项目提出：(ⅰ)对新疆主要杏产区的杏褪绿卷叶病进行广泛调查、采样鉴定，获取植原体病原多位点基因的序列和分子特征信息，分析其系统发育关系、遗传变异和株系分化。(ⅱ)采用纯培养和免培养手段研究比较杏褪绿卷叶植原体宿主感病和健康植株内生细菌多样性和群落结构的时空动态变化特征，发现影响植原体侵染、消长的内生细菌关键种群。(ⅲ) 解析内生细菌与植原体的互作关系，探讨内生细菌群落多样性与杏树抗/感病能力的相关性。研究结果将为深入了解植原体-内生细菌-宿主的相互作用，阐明植原体的致病机理提供基础。同时为抗病种质资源的筛选和抗病品种选育，探求植原体病害防控的新策略提供新的启发与依据，不仅具有重要的科学意义，而且对充分发掘内生细菌的应用潜能起到积极的促进作用。

【主要研究内容】本项目旨在确定杏褪绿卷叶植原体的分类地位，获取其分子生物学信息。在此基础上研究感病植株体内植原体病原消长对内生细菌群落结构的影响，解析内生细菌群落多样性与宿主抗/感病性的相关性。【重要结果】（1）通过对植原体16S rDNA、secY、rp多位点基因扩增、克隆、测序和分子特征分析，确定杏褪绿卷叶植原体新疆分离物与16SrⅤ-B亚组成员枣疯病植原体、樱桃绿化植原体亲缘关系最近，但对不同保守基因的虚拟酶切分析结果发现，杏褪绿卷叶植原体新疆分离物属于16SrⅤ组的1个新亚组。（2）针对杏褪绿卷叶植原体新疆分离物16S rDNA基因和杏树18S rDNA基因设计并筛选出1套特异性引物和探针，建立了双重荧光定量PCR检测方法，同时针对杏褪绿卷叶植原体新疆分离物tuf基因保守区域设计了一套特异性LAMP引物，建立了LAMP检测方法。（3）通过对田间不同杏树品种的病情、症状调查同时结合嫁接鉴定，发现被鉴定杏树品种多数为感病品种，但也有部分品种表现出较强的抗病性。（4）通过对抗/感病杏树、同株显症及无症组织中可培养内生细菌的分离、多样进行分析和菌种鉴定，发现不同抗/感病杏树、同株显症/无症组织内生细菌的种类和丰度差异明显，植原体的侵染改变了寄主内生细菌菌群结构。通过定量分析结果发现，植原体与绝对优势菌群假单胞菌的种群数量呈负相关关系，而与细菌总数并无相关性。（5）利用16S rDNA高通量测序技术对不同抗/感病杏树、同株显症及无症组织中内生细菌多样性进行分析，发现植原体的消长对内生细菌群落结构产生了显著影响。研究结果表明植原体的消长与总细菌的含量及绝对优势菌群并无直接的相关性，但与部分次优势菌群则有着密切的关系。【科学意义】研究结果将为深入了解植原体与内生细菌的相互作用，阐明植原体的致病机理提供基础，对充分发掘内生细菌应用潜能起到积极的促进作用。