地肤子提取物对苹果树腐烂病菌的抑制作用及对其菌体结构和功能的影响

为开发新型安全高效的植物源苹果树腐烂病防治剂,采用菌丝生长速率法测定中草药地肤子乙醇提取物及各层析流分L1～L9对苹果树腐烂病菌Valsa mali的抑制作用,同时通过室内常规分析方法观测流分L7对该菌菌丝形态结构、细胞膜通透性及物质吸收和代谢的影响。结果表明,地肤子乙醇粗提物浓度为2 mg/mL时对苹果树腐烂病菌具有较强的离体抑制活性,处理后96 h的抑菌率达到92.16%。分别用石油醚、氯仿、正丁醇萃取醇提物,石油醚萃取物对苹果树腐烂病菌的抑制效果最为明显,其EC_(50)为0.07 mg/mL;在石油醚萃取物通过硅胶柱层析分离得到的9种流分中,流分L7对苹果树腐烂病菌的抑制作用显著,离体抑菌率高达96.73%;且在流分L7处理下,病菌菌丝体出现肿胀、膨大或畸形等现象,细胞膜通透性增大,可溶性蛋白、还原糖以及丙酮酸的含量随流分L7浓度的升高而持续降低,当浓度为100μg/mL时,菌丝体可溶性蛋白、还原糖及丙酮酸的含量分别降低70.78%、71.74%和78.68%。表明在离体培养条件下地肤子乙醇提取物能明显抑制苹果树腐烂病菌的生长,并对菌丝体细胞膜结构和功能稳定性产生显著影响,具有进一步开发为新型植物源苹果树腐烂病防治剂的潜力。

Antifungal activity of the extracts from fructus kochiae against apple canker pathogen Valsa mali and their impacts on the cell structure and function

To develop novel botanical fungicides against apple canker pathogen, Valsa mali, the inhibitory activity of both the crude ethanol extract and sub-fractions (L1-L9) of fructus kochiae was tested against V. mali with measuring mycelium growth rate. The mechanism of L7, the most effective subfraction, was preliminarily studied by using conventional analysis methods, and the effect of L7 on mycelial morphology, cell membrane permeability, and rate of essential substance absorption and metabolism was determined. The results showed that the ethanol extracts of fructus kochiae strongly inhibited the growth of V. mali at the concentration of 2 mg/mL, with an in vitro inhibitory rate of 92.16% after 96 h. Crude extraction was performed with petroleum ether, chloroform and n-butanol, followed by inhibition assay with each extract fraction. Petroleum ether extract displayed significantly higher inhibition activity against V. mali with the EC₅₀ at 0.07 mg/mL. Silica gel column chromatography was used to further purify petroleum ether extract and a total of nine sub-fractions (L1-L9) were obtained. The most pronounced antifungal activity was detected in L7 fraction with an inhibitory rate of 96.73%. Morphological alterations of V. mali such as swelling, deformity induced by L7 were observed. Cell membrane permeability was positively reacted to the increase of L7 fraction concentration, while the content of soluble protein, reducing sugar, and pyruvate in mycelia were decreased with the increase of the concentration of L7, which were reduced by 70.78%, 71.74%, and 78.68%, respectively, at the concentration of 100 mg/mL. The study indicated that the ethanol extracts of fructus kochiae significantly inhibited the growth of V. mali in vitro, and negatively impacted cell membrane structure and functional stability of the mycelium. These extracts from plants could be developed as potential fungicides against apple canker.