特基拉芽孢杆菌XK29挥发物2-甲基丁酸对甘薯长喙壳菌的抑制作用研究

Inhibitory effects of volatile 2-methylbutyric acid produced by Bacillus tequilensis XK29 on Ceratocystis fimbriata

摘要:

【目的】研究特基拉芽孢杆菌挥发物2-甲基丁酸对甘薯长喙壳菌的抑制作用，评价2-甲基丁酸对甘薯黑斑病的防治效果。【方法】采用I-分隔平皿和气相抑菌体系，研究不同剂量的2-甲基丁酸对甘薯长喙壳菌菌丝生长和孢子萌发的抑制作用；使用乳酸酚棉蓝染色观察2-甲基丁酸对甘薯长喙壳菌显微形态的影响；利用荧光探针钙荧光白和溴化丙锭检测2-甲基丁酸对甘薯长喙壳菌细胞壁结构与细胞膜通透性的影响；使用荧光探针2,7-二氯荧光素二乙酸酯检测甘薯长喙壳菌胞内活性氧含量变化；测定谷胱甘肽含量分析病原菌应对氧化损伤能力的改变；通过线粒体脱氢酶活力和丙酮酸含量的检测，分析2-甲基丁酸对甘薯长喙壳菌线粒体功能和能量代谢的影响；评价2-甲基丁酸在甘薯黑斑病防治中的使用效果。【结果】2-甲基丁酸显著抑制甘薯长喙壳菌菌丝生长和孢子萌发，降低其产孢能力，导致菌丝折叠弯曲并形成不连续的空腔。2-甲基丁酸使甘薯长喙壳菌的细胞壁结构改变，细胞膜通透性增加，胞内活性氧含量升高，谷胱甘肽含量显著降低，使病原菌应对氧化损伤的能力下降，线粒体脱氢酶活力和丙酮酸含量显著降低，诱发线粒体功能障碍，干扰细胞能量代谢，最终导致细胞死亡。此外，2-甲基丁酸对甘薯黑斑病也具有良好的防治作用。【结论】2-甲基丁酸对甘薯长喙壳菌具有显著的抑制作用，可作为安全高效的气相抑菌材料用于新型熏蒸制剂的研发。

关键词:挥发性有机物;2-甲基丁酸;甘薯长喙壳菌;线粒体;活性氧

Abstract:

[Objective] To investigate the inhibitory effects of 2-methylbutyric acid produced by Bacillus tequilensis XK29 on Ceratocystis fimbriata, and to evaluate its control effects on sweet potato black rot. [Methods] I-plates (2-section) and gas-phase antimicrobial system were used to study the inhibitory effects of 2-methylbutyric acid on mycelial growth and spore germination of C.fimbriata, and its effect on the micro-morphology of C.fimbriata was observed by lactophenol cotton blue staining. Fluorescent probes calcofluor white and propidium iodide were adopted to detect the influences of 2-methylbutyric acid on cell wall structure and cell membrane permeability of C.fimbriata, and the change of intracellular reactive oxygen species of C.fimbriata was explored by fluorescence probe 2ʹ,7ʹ-dichlorodihydrofluorescein diacetate. Additionally, the content of glutathione was determined to analyze the ability of the pathogenic fungus to deal with oxidative damage, and the activity of mitochondrial dehydrogenase and the content of pyruvic acid were determined to clarify the effects of 2-methylbutyric acid on mitochondrial function and energy metabolism of C.fimbriata. In the end, we evaluated the application of 2-methylbutyric acid to the control of sweet potato black rot. [Results] 2-methylbutyric acid significantly inhibited the mycelial growth and spore germination of C.fimbriata, thus reducing the sporulation ability of C.fimbriata and resulting in folded mycelia and discontinuous cavities. The cell wall structure was changed, and the membrane permeability and the content of intracellular reactive oxygen species were increased. 2-methylbutyric acid markedly reduced the content of glutathione, thereby decreasing the ability of pathogens to deal with oxidative damages. Furthermore, the activity of mitochondrial dehydrogenase and the content of pyruvic acid dropped, which induced mitochondrial dysfunction and interfered with cell energy metabolism, thus leading to cell death. In addition, 2-methylbutyric acid controlled the sweet potato black rot.[Conclusion] 2-methylbutyric acid had remarkable inhibitory effects on C.fimbriata. It could be used as a safe and efficient gas-phase antimicrobial material for the development of new fumigants.

Key words:volatile organic compounds;2-methylbutyric acid;Ceratocystis fimbriata;mitochondria;reactive oxygen species