Maung Chaw Ei Htwe, Lee Hyung Gwon, Cho Jeong-Yong, Kim Kil Yong
Department of Agricultural and Biological Chemistry, Environmentally-Friendly Agricultural Research Center, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Republic of Korea.
Department of Food Science and Technology, College of Agriculture and Life Sciences, Chonnam National University, Gwangju, Republic of Korea.
World J Microbiol Biotechnol. 2021 Aug 22;37(9):159. doi: 10.1007/s11274-021-03046-x.
Botrytis cinerea, the causal agent of gray mold is one of the major devastating fungal pathogens that occurs in strawberry cultivation and leads to massive losses. Due to the rapid emergence of resistant strains in recent years, an ecofriendly disease management strategy needs to be developed to control this aggressive pathogen. Bacillus velezensis CE 100 exhibited strong antagonistic activity with 53.05% against B. cinerea by dual culture method. In the present study, 50% of culture filtrate supplemented into PDA medium absolutely inhibited mycelial growth of B. cinerea whereas the highest concentration (960 mg/L) of different crude extracts including ethyl acetate, chloroform, and n-butanol crude extracts of B. velezensis CE 100, strongly inhibited mycelial growth of B. cinerea with the highest inhibition of 79.26%, 70.21% and 69.59% respectively, resulting in severe damage to hyphal structures with bulging and swellings. Hence, the antifungal compound responsible was progressively separated from ethyl acetate crude extract using medium pressure liquid chromatography. The purified compound was identified as methyl hippurate by nuclear magnetic resonance and mass spectrometry. The inhibitory effect of methyl hippurate on both spore germination and mycelial growth of B. cinerea was revealed by its dose-dependent pattern. The spore germination rate was completely restricted at a concentration of 3 mg/mL of methyl hippurate whereas no mycelial growth was observed in agar medium supplemented with 4 mg/mL and 6 mg/mL of methyl hippurate by poisoned food method. Microscopic imaging revealed that the morphologies of spores were severely altered by long-time exposure to methyl hippurate at concentrations of 1 mg/mL, 2 mg/mL and 3 mg/mL and hyphae of B. cinerea were severely deformed by exposure to methyl hippurate at concentrations of 2 mg/mL, 4 mg/mL and 6 mg/mL. No significant inhibition on tomato seed germination was observed in treatments with methyl hippurate (2 mg/mL) for both 6 h and 12 h soaking period as compared to the controls. Based on these results, B. velezensis CE 100 could be considered a potential agent for development of environmentally friendly disease control strategies as a consequence of the synergetic interactions of diverse crude metabolites and methyl hippurate.
灰葡萄孢菌是灰霉病的病原菌，是草莓种植中主要的毁灭性真菌病原体之一，会导致巨大损失。由于近年来抗性菌株迅速出现，需要制定一种生态友好型病害管理策略来控制这种具有侵袭性的病原体。贝莱斯芽孢杆菌CE 100通过对峙培养法对灰葡萄孢菌表现出较强的拮抗活性，抑菌率为53.05%。在本研究中，将50%的培养滤液添加到PDA培养基中可完全抑制灰葡萄孢菌的菌丝生长，而贝莱斯芽孢杆菌CE 100的不同粗提物（包括乙酸乙酯、氯仿和正丁醇粗提物）的最高浓度（960 mg/L）对灰葡萄孢菌的菌丝生长有强烈抑制作用，最高抑制率分别为79.26%、70.21%和69.59%，导致菌丝结构严重受损，出现凸起和肿胀。因此，使用中压液相色谱法从乙酸乙酯粗提物中逐步分离出负责的抗真菌化合物。通过核磁共振和质谱鉴定纯化后的化合物为马尿酸甲酯。马尿酸甲酯对灰葡萄孢菌的孢子萌发和菌丝生长的抑制作用呈剂量依赖性。在马尿酸甲酯浓度为3 mg/mL时，孢子萌发率完全受到抑制，而通过毒饵法在添加了4 mg/mL和6 mg/mL马尿酸甲酯的琼脂培养基中未观察到菌丝生长。显微镜成像显示，在1 mg/mL、2 mg/mL和3 mg/mL浓度下长时间暴露于马尿酸甲酯会严重改变孢子形态，在2 mg/mL、4 mg/mL和6 mg/mL浓度下暴露于马尿酸甲酯会使灰葡萄孢菌的菌丝严重变形。与对照相比，在浸泡6小时和12小时的处理中，马尿酸甲酯（2 mg/mL）对番茄种子萌发没有显著抑制作用。基于这些结果，由于多种粗代谢产物和马尿酸甲酯的协同相互作用，贝莱斯芽孢杆菌CE 100可被视为开发环境友好型病害控制策略的潜在菌株。
