香芹酚和丁香酚对腐皮镰刀菌的抑菌活性及抑菌机理

Antagonistic activity and defense mechanism of carvacrol and eugenol against Fusarium solani

摘要:

【背景】腐皮镰刀菌(Fusarium solani)是世界上最具破坏性的土传病原菌之一，严重影响作物的产量及品质。因此寻找并开发广谱的可持续生物防治药剂迫在眉睫，而植物次级代谢物为自然界筛选生物源天然杀菌剂提供了丰富的原料。【目的】研究香芹酚和丁香酚对腐皮镰刀菌的抑菌活性，探究其可能涉及的抑菌机理。【方法】采用菌丝生长速率法、十字交叉法和孢子萌发法分析香芹酚和丁香酚对菌丝和孢子的抑制活性，通过扫描电镜(scanning electron microscope，SEM)观察菌丝微观形态的变化，利用荧光染料碘化丙啶(propidium iodide，PI)观察细胞膜的损伤情况，并测定其对腐皮镰刀菌细胞膜胞外电导率、蛋白质含量和麦角固醇合成的影响。【结果】香芹酚和丁香酚对腐皮镰刀菌菌丝生长和孢子萌发均有显著的抑制效果，并呈现剂量依赖效应，EC₅₀值分别为92.39 μL/L和263.00μL/L。SEM结果表明，2种精油处理腐皮镰刀菌后，其细胞壁和细胞膜均遭到破坏从而不能维持菌丝正常的线性形态，表现出不同程度的弯曲、褶皱和凹陷。PI染色结果发现，2种精油处理严重破坏了腐皮镰刀菌细胞膜的完整性和通透性，导致胞浆流失、胞外电导率和蛋白质含量急剧增加。高浓度的香芹酚(400 μL/L)和丁香酚(800μL/L)处理菌体48 h后，腐皮镰刀菌中麦角固醇含量分别降低了78.61%和67.73%。【结论】香芹酚和丁香酚能抑制腐皮镰刀菌菌丝生长和孢子萌发，破坏腐皮镰刀菌细胞膜的完整性和通透性，干扰细胞膜上麦角固醇的合成来发挥其抗菌功效。本研究为腐皮镰刀菌生物防治药剂的筛选提供了理论基础。

Abstract:

[Background] Fusarium solani, one of the most destructive soil-borne pathogens worldwide, seriously affects the yield and quality of crops. Therefore, it is urgent to develop broad-spectrum sustainable biocontrol agents and the abundant secondary metabolites of plants are natural sources. [Objective] To explore the activity of carvacrol and eugenol against F. solani and the possible underlying mechanism. [Methods] With the mycelium growth rate method, crossing method, and spore germination method, the inhibitory effect of carvacrol and eugenol on mycelium growth and spore germination of F. solani was analyzed. The scanning electron microscope (SEM) was used for observing the mycelial morphology of F. solani, and propidium iodide (PI) staining was used for observing the damage to F. solani cell membrane. Moreover, the extracellular conductivity, protein content, and ergosterol biosynthesis were analyzed. [Results] Carvacrol and eugenol significantly suppressed the mycelial growth and spore germination of F. solani in a dose-dependent manner with the median effective concentration (EC₅₀) values of 92.39 μL/L and 263.00 μL/L, respectively. According to the observation under SEM, the cell wall and cell membrane of F. solani were damaged after exposed to carvacrol and eugenol. As a result, the mycelia had bends, folds, and depressions rather than the normal linear morphology. PI staining revealed that two essential oils broke F. solani cell membrane and enhanced the membrane permeability, resulting in the outflow of cytoplasmic contents and the surge in extracellular conductivity and protein content. After treatment with high concentration of carvacrol (400 μL/L) and eugenol (800 μL/L) for 48 h, the ergosterol content in F. solani decreased by 78.61% and 67.73%, respectively. [Conclusion] Carvacrol and eugenol inhibit the mycelial growth and spore germination of F. solani, damage the cell membrane, enhance membrane permeability, and disrupt the ergosterol biosynthesis on cell membrane, thereby exerting the antifungal activity. In conclusion, this study lays a theoretical basis for the screening of biocontrol agents of F. solani.