柠檬醛抑制柑橘采后绿霉的作用机制

Green mold caused by Penicillium digitatum is the most damaging postharvest disease of citrus fruit. The plant volatile component such as citral present in citrus peel oil has been elucidated to have significant inhibitory role in controlling the growth of P. digitatum, while the underlying mechanism remains undetermined. In present research, the effect of citral at different concentrations on the membrane permeability of P. digitatum will be determined. The morphological and ultrastructural changes of P. digitatum hyphae treated by citral will be determined by scanning electron microscopy and transmission electron microscopy, respectively. The cell compositions such as lipid content, fatty acid content, ergosterol content, ions leakage, extracellular pH and extracellular ATP contents will be also determined. The effects of citral on green mold on citrus fruit and on the fruit quality of the fruit inoculated with P. digitatum will be determined. In addition,the suppression subtractive hybridization (SSH) combining with gene chip technique will be adapted to reveal the molecular clues of response of P. digitatum to citral, and to obtain the key genes closely related to the resistance to citral. All these are to explore the potential inhibitory mechanism of citral on P. digitatum. Our present research can not only provide a good opportunity for better understanding of the inhibitory mechanism of citral on postharvest pathogens in citrus fruit, but also has important theoretical and practical guidance for controlling the postharvest diseases in citrus fruit by plant volatile components, prolonging the shelf life and reducing postharvest losses of citrus fruit in future.

绿霉（指状青霉，P. digitatum）侵染引起的绿霉病是最严重的柑橘采后病害。柑橘果皮精油所含芳香物质柠檬醛已被证实可有效抑制指状青霉生长，但对应的抑菌机制尚不明了。本研究拟以指状青霉为对象，分析不同浓度柠檬醛对指状青霉细胞膜通透性的影响；通过扫描电镜和透射电镜观察柠檬醛对指状青霉菌丝形态和细胞超微结构的影响；测定柠檬醛对指状青霉脂质含量、脂肪酸含量、麦角固醇含量、离子泄露、胞外pH和ATP 含量等细胞组成的影响；分析浸泡、熏蒸和涂膜柠檬醛对柑橘绿霉病的作用效果及对果实品质的影响；利用抑制性扣除杂交（SSH）结合基因芯片杂交技术获得指状青霉应答柠檬醛毒害的分子证据，发掘关键基因；旨在探讨柠檬醛抑制柑橘采后绿霉的作用机制。项目的开展不仅能丰富人们对柠檬醛抑制柑橘采后致病菌的认识，也为今后应用植物芳香物质控制柑橘采后病害、延长柑橘货架期、减少柑橘采后损失等具有重要的理论和现实意义。

本研究探讨了柠檬醛抑制指状青霉的作用机理。结果表明，柠檬醛对指状青霉菌丝体的最小抑菌浓度（MIC）和最小杀菌浓度（MFC）分别为2.00μL/mL和4.00μL/mL。SEM和TEM结果表明，MIC和MFC柠檬醛处理可导致指状青霉菌丝体形态和超微结构发生显著改变。MIC和MFC柠檬醛处理后，指状青霉细胞膜通透性增加，胞内核酸、K+和Mg2+发生泄露，胞内出现酸化现象，脂质和麦角固醇含量降低，胞内MDA和H2O2积累，POD和LOX酶活性上升，暗示着发生了膜脂过氧化伤害。MIC和MFC柠檬醛处理可改变指状青霉线粒体的形态，导致ATP泄露，损伤糖酵解和三羧酸循环。果蜡中添加10×MFC柠檬醛可显著降低接种指状青霉柑橘果实绿霉病的发生，对果实外观和内在品质无明显影响。RNA-seq结果表明，经1/2MIC柠檬醛处理后，6335个基因差异表达，包括2322个上调和4033 个下调基因，这些基因可注释到ABC转运、糖酵解/糖异生、三羧酸循环、氧化磷酸化、脂肪酸代谢和类固醇生物合成等155个KEGG通路。荧光定量PCR结果显示，柠檬醛处理影响5个麦角固醇生物合成途径相关基因（ERG7、ERG11、ERG6、ERG3和ERG5）的表达，与固醇的种类变化、麦角固醇的含量降低以及羊毛甾醇的大量累积相符。iTRAQ结果显示，1/2MIC柠檬醛处理可诱导125个蛋白差异表达，这些蛋白可主要注释到包括氧化磷酸化、脂肪酸代谢等在内的42个KEGG通路。酶活实验证明，参与氧化磷酸化途径的复合体酶I、复合体酶II、复合体酶III、复合体酶IV和 复合体酶V均受到柠檬醛的显著影响，与iTRAQ的结果相吻合。此外，1/2MIC柠檬醛处理可降低线粒体膜电位。综上，本研究结果表明，柠檬醛对指状青霉的抑菌机理主要与膜损伤有关。