库尔勒香梨气调贮藏期间黑头病的发病机制研究

Korla fragrant pear is one of the special fruit resources in Xinjiang. The black-head disease, which significantly occurred in the process of Controlled Atmosphere Storage (CA) in post-harvest period, has become a main problems restricting the industrial development of the fragrant pear. Because of the differences of environment and infective carrier and the complexity of pathogen mutation, research on the control technology of black-head disease of Korla fragrant pear has the specificity of regional urgency, and the pathogenesis of black-head disease is yet to be clarified. Based on the survey of black-head disease of fragrant pear in Xinjiang orchards and gas storehouse, the control samples with different gas storage will be set up, and the correlation between the physical-chemical property of the pears and the dynamic variation of black-head disease will be investigated; Combined with the experimental techniques including traditional cultivation, Polymerase Chain Reaction (PCR) and Biolog identification system, the pathogen of black-head disease will be separated and identified; At the same time, the mass spectrum fingerprint of the peptides from different fruit protein samples will be analyzed, using the techniques such as Two-Dimensional Electrophoresis (2-DE) and Matrix-Assisted Laser Desorption Ionization Time of Flight Mass-Spectrometry (MALDI-TOF/MS), to study the changing of protein composition and abundance of the diseased pear at different periods, and then, to reveal the pathogenesis of black-head disease of Korla fragrant pear, combining with principal component (PCA) analysis and randomized control group validation. Expected results of the study will provide strong theoretical basis for the development of prevention and control technology toward the black-head disease of Korla fragrant pear.

库尔勒香梨为新疆特色水果资源，其采后气调贮藏过程中显著的黑头病发病现象已成为制约香梨鲜果产业发展的主要问题。基于环境与侵染载体的差异性及病原菌变异的复杂性，库尔勒香梨黑头病专一控制技术研究具有区域迫切性，目前库尔勒香梨黑头病发病机制尚待明晰。本项目在对新疆果园及气调贮藏库香梨黑头病调研统计的基础上，设置不同气调贮藏样品对照组，分析果品理化指标与黑头病动态变化的相关性；结合传统培养方法、PCR技术及Biolog鉴定系统，对黑头病病原菌进行分离与鉴定；同时采用二维电泳(2-DE)、基质辅助激光解析电离飞行时间质谱(MALDI-TOF/MS)等分析技术对不同果实蛋白样品进行肽段质谱指纹分析，研究不同时期发病果实蛋白质组成和丰度变化，结合主成(PCA)分析法及随机对照组验证，深入揭示库尔勒香梨黑头病的发病机制。预期研究成果为库尔勒香梨黑头病控制技术研究提供有力的理论依据。

库尔勒香梨为新疆特色水果资源，其采后气调贮藏过程中显著的黑头病发病现象已成为制约香梨鲜果产业发展的主要问题。本项目通过对气调贮藏条件下库尔勒香梨黑头病病原菌进行分离、鉴定，进而构建分离菌株的系统发育关系；通过研究库尔勒香梨采后贮藏期间黑头病发病与理化指标之间的相关性；其次基于蛋白质组学（iTRAQ）分析技术，研究病原菌接种后不同时期发病果实蛋白质组成和丰度变化，验证并揭示库尔勒香梨黑头病的发病机制。主要研究结果如下：.1. 结合形态学和分子生物学等手段对致病菌进行鉴定，从病梨萼端表面及香梨生长环境中共分离出11株菌株，综合病原菌菌落形态、孢子显微结构及回接试验，筛选出2株候选菌株(SY－6、XL－6)。通过CTAB法提取病原菌DNA，经rDNA 内转录间隔区(ITS－rDNA) 基因片段测序及美国国立生物技术信息中心NCBI网站的同源性比对，鉴定得出SY－6、XL－6均属于链格孢属。.2.通过接种链格孢菌，测定香梨在气调贮藏和冷库贮藏的条件下理化指标的变化规律，研究表明黑头病在发病中主要影响了香梨的细胞壁代谢以及活性氧代谢的程度，使得库尔勒香梨在感染黑头病后果肉内生理生化反应以及代谢发生失调，从而严重影响香梨的品质。另一方面由于香梨黑头病发病期间致病菌侵染导致香梨抗性下降，抗性的下降又增大了香梨黑头病的发病速度，同时黑头病也影响香梨的矿物质元素，使其生理性病害加剧。香梨病斑部位的褐变与黑头病菌的侵染、Ca含量的下降以及酶促褐变的加剧都有着关联。而气调贮藏能更好的保证香梨品质，从而提高香梨的抗病性，使其不易遭受病原菌的侵染。.3. 研究病原菌接种后不同时期发病果实蛋白质组成和丰度变化，并对差异表达蛋白进行分析鉴定.结合GO功能注释和KEGG代谢通路分析，挖掘并验证与香梨黑头病发病相关的蛋白，明晰了差异表达蛋白与黑头病致病之间的相关性，初步解析了库尔勒香梨黑头病发病的分子机制，为库尔勒香梨的贮藏保鲜和黑头病的防治提供坚实理论基础。