防御假单胞菌FD6抗菌代谢物鉴定及其对灰霉病菌的抑制作用

Identification of an antifungal compound produced by Pseudomonas protegens FD6 and determination of its inhibitory effect against Botrytis cinerea

摘要:

目的 研究防御假单胞菌(Pseudomonas protegens) FD6发酵粗提物的抑菌活性及其作用机制，解析粗提物中主要的活性物质。方法 通过平板对峙试验筛选发酵最佳的培养基、萃取剂、溶剂，以及发酵粗提物抑菌活性的稳定性。利用MALDI-TOF/TOF质谱鉴定粗提物中主要抗菌物质，并探究粗提物的抑菌谱。通过菌丝形态观察、孢子萌发试验、PI染色及电导率测定，探究粗提物对番茄灰霉病菌(Botrytis cinerea)的抑菌作用机制。结果 平板对峙试验表明，菌株FD6以不添加碳源马铃薯汁培养基进行发酵的粗提物抑菌活性最强。以乙酸乙酯作为萃取剂，通过甲醇溶解所得的粗提物抑菌活性最强。质谱分析表明，粗提物中含有环脂肽orfamide A。进一步检测粗提物抑菌谱，发现其对桃枝枯病菌(Phomopsis amygdali)、小麦赤霉病菌(Fusarium graminearum)、梨炭疽病菌(Colletotrichum gloeosporioides)和桃褐腐病菌(Monilinia fructicola)均具有抑制作用。稳定性试验发现，粗提物在121 ℃处理20 min以及pH 4.0-10.0条件下抑菌活性稳定，且蛋白酶K和胰蛋白酶处理不影响其抑菌活性。粗提物处理B. cinerea菌丝可导致其形态异常，B. cinerea分生孢子经2.0 mg/mL粗提物处理后萌发被完全抑制，50%的细胞膜受到破坏，电导率增加，细胞内核酸与蛋白外渗。结论 防御假单胞菌FD6产生的代谢物抑菌谱广且稳定性强，为发掘新型绿色农药提供先导化合物。

Abstract:

Objective To explore the antifungal activity and inhibitory mechanism and identify the main antagonistic substances of the crude extract of Pseudomonas protegens FD6 in fermentation.Methods The plate confrontation test was employed to screen the optimal medium, extractant, and solvent and determine the antifungal activity stability of the crude extract. The main antifungal substances in the crude extract were identified by MALDI-TOF/TOF mass spectrometry, and the antifungal spectrum of the crude extract was explored. The inhibition mechanism of the crude extract against Botrytis cinerea was investigated by mycelial morphology observation, spore germination test, PI staining, and conductivity determination.Results The crude extract of FD6 in the potato juice medium without any carbon source displayed the strongest antifungal activity. The methanol-solved crude extract obtained with ethyl acetate as the extractant had the strongest inhibitory activity. The main antifungal substance of the crude extract was identified as a cyclic lipopeptide, orfamide A, based on mass spectrometry data. In addition, the crude extract displayed broad-spectrum inhibitory activities against Phomopsis amygdali, Fusarium graminearum, Colletotrichum gloeosporioides, and Monilinia fructicola. The antifungal activity of the crude extract was stable after treatment at 121 ℃ for 20 min, within the range of pH 4.0-10.0, and with protease K and trypsin. The mycelial morphology of B. cinerea became abnormal after treatment with the crude extract. The treatment with 2.0 mg/mL crude extract completely inhibited the conidial germination of B. cinerea, caused a 50% damage of the cell membrane, increased the conductivity, and led to leakage of intracellular nucleic acids and proteins.Conclusion The metabolites produced by P. protegens FD6 have broad-spectrum and stable antifungal activities, serving as lead compounds for exploration of new green fungicides.