桂枝抑制柑橘青霉病和绿霉病菌活性成分分离鉴定及抑菌机理研究

Blue and green mold caused by the pathogen Penicillium italicum and P.digitatum, respectively, are the most common postharvest disease of citrus fruits. This project will be aimed to isolate and identify the active constituents, Structure-Activity Relationships and antibacterial mechanism of Cinnamomi Ramulus against P.italicum and P.digitatum in order to control of the two major Citrus Postharvest Disease (CPD). Various kinds of chromatography columns and spectroscopy methods will be used to isolation and identification of the antibacterial constituents from the Cinnamomi Ramulus extracts. Citrus infected the two pathogens will be used to verified the antibacterial activity of Cinnamomi Ramulus extracts in vivo. Furthermore, the bacteriostatic quality control standards will be made and the efficient method of enrichment the bacteriostatic compositions will be optimized. As for antimicrobial mechanism, the influences of Cinnamomi Ramulus and active constituents on the hyphae and spores superfine morphology, cell membrane permeability, plasma membrane integrity and spore germination inhibition rate of the two pathogens will observed by optical microscope method. The influences of Cinnamomi Ramulus and its active constituents on the macromolecular content (including protein, total sugar, DNA and RNA), metabolic enzyme activity will determinated by the methods of biochemistry and molecular biology. All of those will help us to explore the antibacterial mechanism of Cinnamomi Ramulus and isolation its active constituents. The successful of this project will provide feasible methods and theoretical basis for screening and exploiting of other natural bacteriostatic agent in the control of CPD. And it will also provide scientific basis for deep development and utilization of Cinnamomi Ramulus. So this project has important academic value and application prospect.

由意大利青霉和指状青霉导致的青霉病和绿霉病是柑橘采后最常见的两种病害。本研究以这两种病原菌为靶标菌株，研究中草药桂枝的抑菌活性成分的分离、结构鉴定、构效关系及抑菌机理。采用多种柱色谱分离技术，从桂枝中分离抑菌活性成分，通过各种谱学技术鉴定其结构；优化抑菌活性组分的富集和分离方法，制定桂枝防治柑橘采后病害的质量控制标准；对接种病原菌的柑橘进行"活体"保鲜验证。抑菌机理方面，用光学显微镜观察桂枝活性物质对两种菌株的菌丝形态、孢子超微形态、细胞膜通透性、质膜完整性，孢子萌发抑制率的影响；用生物化学和分子生物学技术测定活性物质作用菌丝后对菌体生物大分子(蛋白质，总糖，DNA和RNA)，代谢关键酶活性的影响，阐明抑菌机理。研究成果为阐明天然植物防治果蔬采后病害活性物质分离、鉴定及作用机理研究提供可靠的实验方法和理论依据，为桂枝的深开发和利用提供科学依据，具有重要的学术意义和潜在的社会经济价值。

由意大利青霉和指状青霉导致的青霉病和绿霉病是柑橘采后最常见的两种病害。本研究以这两种病原菌为靶标菌株，研究了中草药桂枝的抑菌活性成分的提取、分离、结构鉴定、抑菌机理，主要抑菌成分肉桂醛的结构修饰，桂枝及抑菌活性成分对柑橘的贮藏保鲜效果。从桂枝中分离鉴定了两个抑菌活性成分肉桂醛和肉桂酸，他们对包括意大利青霉和指状青霉等15种植物病原菌的抑菌活性。肉桂醛对意大利青霉的最小抑菌浓度(MIC)、最小杀菌浓度(MFC)分别为0.025 mg/mL和0.1 mg/mL，肉桂酸分别为0.1 mg/mL和0.4 mg/mL；肉桂醛和肉桂酸处理的菌体，其孢子萌发率降低，菌丝扭曲膨大或断裂，细胞膜渗透率增强，菌体内可溶性糖及蛋白质含量减少，琥珀酸脱氢酶（Succinate dehydrogenase , SDH）和苹果酸脱氢酶（Malate dehydrogenase, MDH）活性降低。肉桂醛和肉桂酸能有效抑制意大利青霉菌的孢子萌发，阻碍菌体生长，破坏细胞膜致使通透性增加，内含物外渗，且能有效破坏三羧酸循环（Tricarboxylic acid cycle, TCA）过程，菌体能量代谢途径受阻，从而抑制其正常的生长发育。1H-NMR代谢组学揭示肉桂醛对意大利青霉菌的抑制作用可能通过影响能量代谢，氨基酸代谢和核酸代谢等途径。新余蜜橘损伤接种实验发现肉桂醛和肉桂酸处理均能降低新余蜜橘的MDA和H2O2含量，显著提高并维持SOD、PPO等防御酶的活性，以25 mg/mL肉桂醛和30 mg/mL肉桂酸处理效果最佳。肉桂醛和肉桂酸能有效增强果实活体防护效果以及诱导果实的抗病性，且抗性的产生可能与果实内相关防御酶活性提高有关。桂枝及肉桂醛对柑橘的贮藏保鲜效果有较好的效果，可以显著减少柑橘腐烂率，降低果实失重率和丙二醛(MDA)含量，延缓可溶性固形物(TSS)、可滴定酸(TA)、VC及总糖含量的降解速率，维持贮藏后期较高的超氧化物岐化酶(SOD)、苯丙氨酸解氨酶(PAL)、过氧化物酶(POD)以及多酚氧化酶(PPO)活性，从而能有效保持果实采后的贮藏特性，延长保鲜期。