梨果皮绿原酸介导的Alternaria alternata活性氧代谢对其侵染的调控

Latent infection is a major cause for postharvest disease of fruit and vegetables, peel structure and chemical compounds of immature fruit involed in regulation of infection and quiescence of postharvest pathogens. Previous reports showed that phenolic compounds in fruit peel was related to fungal infection, but detail regulatory mechanism is still unknown. Effect of chlorogenic acid based on concentration level in fruit peel of Pingguoli pear on reactive oxygen species production (production of O2- , H2O2 and related enzymes activities), antioxidant system (GHS content and related enzymes activities), extracellular hydrolytic enzymes (cutinase and PG enzymes) gene express of Alternaria alternata, the cause agent of pear black rot, will be analyzed by scanning electron,fluorescence microscopic technique and biochemical and molecular methods. In addition, effect of exogenous chlorogenic acid and H2O2 on infection process and disease incidence will also be evaluated in vivo test. Expected results of the project will elucidate the regulatory role of chlorogenic acid on reactive oxygen metabolism of A. alternata, and mechanism of chlorogenic acid mediated reactive oxygen metabolism in regulation of fungal infection, and also provide theoretical basis for further understanding of latent infection mechanism and effectively controlling postharvest diseases of fruit and vegetables.

潜伏侵染是导致果蔬采后病害发生的主要原因，未成熟果皮结构和化学组分对病原物侵入和潜伏具有重要调控作用。已有研究表明果皮酚类物质与病原物的侵染有关，但具体调控机理尚不清楚。本研究以Alternaria alternata为研究对象，采用扫描及荧光显微技术、生化及分子生物学等方法，通过体外培养介质转换试验，系统研究基于苹果梨果皮含量水平的绿原酸处理对A. alternata活性氧产生（O2-和H2O2的产生及相关酶活性）、抗氧化系统（GHS含量及相关酶活性）及降解酶类（角质酶和多聚半乳糖醛酸酶）基因表达的影响；同时观察分析外源绿原酸及活性氧H2O2处理对A. alternata侵染过程及发病率的影响。以揭示梨果皮绿原酸对A. alternata活性氧代谢的调节作用，并系统阐明梨果皮绿原酸介导的活性氧代谢对病原物侵染的调控机理, 以期为进一步系统揭示潜伏侵染机制及采后病害的有效控制提供理论依据。

酚类物质作为植物重要的次生代谢产物，对果实品质、抗氧化及抗病性均具有调控作用，但其在病原物与寄主互作中的具体调控机理尚不清楚。项目采用HPLC分析、显微技术、生化及分子生物学等方法，分析比较了发育及贮藏期间早酥梨、皇冠梨和苹果梨果皮酚类物质含量及其抗氧化性变化；并通过体外试验研究了基于梨果皮含量的绿原酸等酚类物质对黑斑病菌Alternaria alternata活性氧代谢、致病因子产生及侵染过程的影响。结果表明：.1. 发育期三种梨果皮总酚和总黄酮含量呈先上升后降低的趋势，而在藏期间其含量均下降趋势。梨果皮中8种主要酚类物质含量差异较大，其中熊果苷的含量最高、绿原酸次之；发育及贮藏期梨果皮提取物均表现出较强的抗氧化性，且其抗氧化能力与总酚、阿魏酸、表儿茶素含量呈显著正相关。.2. A. alternata在梨果上的潜伏侵染率随发育期呈上升趋势，且其潜伏侵染率与梨果皮绿原酸、咖啡酸和对香豆酸含量呈显著负相关。绿原酸、咖啡酸、阿魏酸和对香豆酸对A. alternata的生长具有显著的抑制作用。.3. 基于梨果皮含量的绿原酸和咖啡酸显著提高了A. alternata菌体内H2O2和O2-.产生的产生及NOX活性。且其处理后A. alternata SOD酶及AsA-GSH循环中的抗氧化酶GR、GPX、APX和MDHAR活性、基因表达量及GSH、AsA、DHA含量和GSH/GSSG比率均显著增加，而硫氧还蛋白系统中Trx含量和TrxR活性显著降低。.4. 梨果皮含量水平的绿原酸、咖啡酸和儿茶素在体外和体内条件下均显著抑制了A.alternata角质酶、PG、PMG、PGTE、PMTE、Cx以及β-葡萄糖苷酶等胞外降解酶的活性，同时显著的降低了AOH、AME、ALT、Ten四种A. alternata毒素的产生，但刺激了A. alternata黑素色的形成。.5. 基于果皮含量水平的绿原酸、咖啡酸和儿茶素处理均能显著的延缓或抑制A. alternata在梨果皮和洋葱表皮上的侵染过程，其抑制效果随酚类物质浓度的增大而显著提高。同时外源酚类物质处理均能延缓梨果黑斑病的扩展。. 综上所述，绿原酸等梨果皮酚类物质及其氧化性随果实的发育及贮藏期而变化，且其对A. alternata生长、侵入过程、侵染结构形成及致病性均具有重要的调控作用。