茶树精油对果实采后主要致病菌抑制差异化的物质基础及作用机制研究

The fungal disease is the main reason for postharvest losses of fruit and vegetable. Essential oils can significantly against fungi and control these diseases, however, the antifungal component and mechanism is unclear. Based on our preliminary studies, tea tree oil (TTO) can play different inhibition effect on the Botrytis Cinerea, Rhizopus stolonifer and Penicillium expansum. First of all, in order to find the the material base of different inhibition effect on these fungi, the effects of TTO and its single component againt fungi will be tested in vitro. Then, the antifungal activtity of random combined component will be measured to investigating the relationship between. Then, fungi will be treated by TTO and main antifungal component, transmission electron microscopy (TEM) will be used to observe the changes of fungal ultrastructure. Construction of liposomes as a membrane model, study the permeability and composition changes of liposomes and fungal cell membrane. Observing the DNA breakage, determine components with DNA binding or conformational change by gel electrophoresis, atomic force microscopy and circular dichroism. Extraction of mitochondria from fungi, determination the respiratory control rate and oxidative phosphorylation efficiency, measurment the activity of mitochondrial complex enzyme and ATP enzyme. By these study, it will be revealed that the action target and mechnism of TTO and main antifungal component on these fungi and the reason of the relationship between. Finally, the antifungal molecular mechanism will be revealed by using the the gene chip technology to analyzing the changes of fungal gene expression profiles. These conclusion will enrich the theory basis and promote the commercial application of essential oils on the fruit and vegetable preservation.

真菌病害是果蔬采后损失的主要原因，植物精油抗菌效果好，但抗菌组分和机理有待阐明。前期研究表明茶树精油（TTO）对灰葡萄孢霉、匍枝根霉和扩展青霉的抑制存在差异。本项目首先采用TTO、单组分标准品对3种真菌开展体外抗菌试验，明确主要抗菌组分；并将不同组分进行复合抗菌，分析组分之间的关系，阐明抑菌差异的物质基础。以TTO、主要抗菌组分处理真菌，通过投射电镜观察超微结构变化；构建脂质体细胞膜模型，研究脂质体、真菌细胞膜透性和结构改变；借助凝胶电泳、原子力显微镜和圆二色谱观察DNA断裂、判断组份与DNA结合或其构象变化；提取线粒体，测定呼吸控制速率和氧化磷酸化效率、线粒体复合酶和ATP酶活性，阐明主要组分、TTO对不同真菌的作用靶点和机制，揭示组分之间存在关系的原因。最后通过基因芯片技术分析基因表达谱变化，揭示TTO抗真菌的分子机制。研究结论势必能丰富精油抗真菌的基础理论，促进其商业化应用。

本项目主要研究茶树精油(TTO)及其各单组份对采后3种主要腐败真菌（B.cinerea，P. expansum，R. stolonifer）的抑制作用，明确主要抑菌组分、差异组分及组分间的相关作用。通过GC-MS分析表明，TTO主成分为松油烯-4-醇、γ-松油烯、-松油烯、1,8-桉叶素、松油烯、ρ-伞花烃、-松油醇，具有较好的抗氧化和抗癌能力。TTO对B.cinerea、P. expansum、R. stolonifer的EC50分别为0.698、1.18和0.83mL/L，MIC分别为2.5、6.5和2.5 mL/L，说明其对B.cinerea的抑制效果最强，对P. expansum的抑制效果最弱。组分中的松油烯-4-醇和a-松油烯对3种真菌都表现出较强的抑菌能力，抑菌能力差异的主要原因是由于精油中占50%的松油烯-4-醇对B.cinerea和R. stolonifer抑制能力最强，且其他组分也能发挥作用；但是P. expansum仅对占精油中8%的a-松油烯最敏感，对其他多个组分不敏感。各单组分都造成B.cinerea膜渗透性增加，松油烯-4-醇的影响远高于精油本身，但是1,8-桉叶素的影响并不明显。将松油烯-4-醇与其他任何单组份进行复合，都能显著提高抑菌能力，尤其是与1,8-桉叶素的明显增效作用值得关注，我们推测2者是精油中的主要差异组分，前者主要作用与细胞膜、后者可穿膜后作用于胞内靶点，这也通过受复合处理的真菌光镜观察得到了证实。电镜观察发现，TTO处理的B.cinerea细胞壁增厚，细胞膜轮廓不清，细胞质不均匀，细胞器漏失；菌体细胞壁细胞膜完整性被破坏，细胞通透性增加从而造成菌悬液的A260、AKP酶含量及电导率上升；膜脂肪酸组成分析发现，处理组不饱和与饱和脂肪酸比值下降，细胞膜流动性受到抑制。可见，TTO主要以一种膜破坏剂的形式发挥抑真菌作用。草莓果实经过合适的精油熏蒸处理能有利于保持品质，在处理后的12-24h内也可显著诱导提高抗病性关键酶活性。目前已经发表SCI 2篇、EI 1篇、中文核心2篇，参加国内会议报告1次；培养硕士研究生3名。