苹果多酚对采后荔枝霜疫霉菌的抑制作用机理研究

Control of postharvest disease by plant-derived natural products, as a safe and environment-friendly approach, has been received much attention in recent years. Apple polyphenols (APP) extracted from apple fruit have exhibited strong inhibitory effect on some pathogenic microbes. However, the inhibitory mechanism of APP against pathogenic microbes has not been understood yet, to the best of our knowledge, also, there is no available information regarding the effect of APP on postharvest disease and pathogens. On the basis of preliminary results, in this project, Peronophythora litchii, a pathogen causing postharvest decay of litchi fruit, will be chosen as the biological material, and multidisciplinary approaches involving the postharvest pathology, cell biology, physiological and biochemistry, molecular biology and proteomics in combination with multiple techniques including laser confocal microscope, scanning/transmission electron microscope, DNA gel electrophoresis and imaging, circular dichroism spectroscopy, and quantitative proteomics will be applied. We will analyze the effects of APP on P. litchii involving cell ultrastructure, cell wall and cell membranes, mitochondria, nucleic acid, and pathogenicity-related physiological and biochemical metabolisms, and nucleic acid; meanwhile we will identify and validate the differentially expressed proteins of P. litchii in response to APP, and further discern the biochemical pathways, and physiological and pathogenic processes in association with these changed proteins. These studies would serve to reveal the inhibitory mechanism of APP against P. litchii. The results would provide a theoretical evidence for application of APP in postharvest storage and preservation.

利用植物源天然产物控制果蔬采后病害，作为一种较为安全且环境友好的新型保鲜技术，受到了国内外学者的广泛关注。提取自苹果果实的苹果多酚对多种病原菌表现出显著的抑制作用，然而目前关于苹果多酚抑菌机理方面的研究十分匮乏，且尚未见有关苹果多酚对采后病害控制的报道。本项目在前期试验的基础上，拟以导致荔枝采后腐烂变质的真菌霜疫霉菌为生物试材，综合采后病理学、生理生化、细胞生物学、分子生物学和蛋白质组学等学科方法，利用扫描和透射电子显微镜、激光共聚焦显微镜、凝胶电泳及成像、圆二色谱、定量蛋白质组学等技术，分析苹果多酚对荔枝霜疫霉菌的细胞超微结构、细胞壁与细胞膜、线粒体、核酸及与致病力相关生理生化代谢的影响；鉴定并验证苹果多酚处理的病原菌差异表达的蛋白质，解析差异蛋白涉及的代谢途径及生理病理过程，从不同层次揭示苹果多酚抑制荔枝霜疫菌的作用机制。该项目研究可为苹果活性活性成分用于果蔬采后防腐保鲜奠定理论基础。

由荔枝霜疫霉菌（Peronophythora litchii）引发的荔枝霜疫霉病是采后荔枝最常见的病害之一，导致果实严重腐烂，造成巨大的经济损失。利用植物源天然产物控制果蔬采后病害，作为一种较为安全且环境友好的新型保鲜技术，备受国内外学者关注。本研究通过天然产物苹果多酚对P. litchii进行处理，分析其抑菌作用及其机理。结果显示，苹果多酚可有效抑制P. litchii体外菌丝生长和孢子囊萌发，且抑制效率呈浓度依赖关系，以5 g L-1和10 g L-1为最优，进而选择5 g L-1进行后续研究。P. litchii损伤接种果实的实验结果表明，苹果多酚处理可推迟荔枝果实在贮藏期间霜疫霉病病斑的扩展。超微结构观察结果显示，苹果多酚能够导致P. litchii菌丝扭曲及断裂、孢子囊表面皱缩与塌陷、菌丝细胞结构严重受损（如液泡和线粒体等细胞器被破坏或消失，细胞质空泡化严重并出现质壁分离）。碘化丙啶染色实验结果显示，P. litchii质膜完整性受苹果多酚处理而破坏，导致菌体还原糖和可溶性蛋白外渗。苹果多酚处理后，P. litchii线粒体呼吸链复合酶（NADH-Q还原酶、琥珀酸-Q还原酶、细胞色素C氧化酶、FoF₁-ATP酶等）活性显著降低，造成呼吸速率和ATP生成量降低、活性氧积累，进而对细胞生物大分子造成氧化损伤。苹果多酚处理可体外抑制P. litchii菌丝体胞外水解酶（角质酶、多聚半乳糖醛酸酶、果胶甲基转移消除酶、纤维素酶等）活性，从而能够降低其病原菌致病力。定量蛋白质组学分析结果表明，苹果多酚可抑制P. litchii菌丝体内氧化磷酸化、抗氧化、膜脂合成、应激响应等相关蛋白表达，进而影响P. litchii的能量代谢与物质合成，造成细胞结构和功能改变。本项目研究结果为苹果活性活性成分用于果蔬采后防腐保鲜奠定了理论基础。