地衣芽胞杆菌FJAT-4脂肽结构鉴定及其对尖孢镰刀菌的抑制作用

Characterization of lipopeptides produced by Bacillus licheniformis FJAT-4 and their effect on Fusarium oxysporum

摘要:

[目的] 地衣芽胞杆菌FJAT-4产生的脂肽能够有效抑制尖孢镰刀菌的生长，本研究的目的在于探究地衣芽胞杆菌FJAT-4脂肽结构，分析培养基组分和培养温度对FJAT-4产抑菌脂肽的影响，阐述脂肽对尖孢镰刀菌的抑制作用，为菌株抑菌机理的阐释及其在枯萎病防治中的推广应用奠定基础。[方法] 通过酸沉醇提法提取地衣芽胞杆菌FJAT-4产生的脂肽；利用液相色谱串联四极杆飞行时间质谱进行地衣芽胞杆菌FJAT-4脂肽组成分析及结构鉴定；以抑菌圈大小为指标评估地衣芽胞杆菌FJAT-4脂肽对尖孢镰刀菌的抑制效果；通过扫描电镜观察地衣芽胞杆菌FJAT-4粗脂肽对尖孢镰刀菌的抑制作用。[结果] 地衣芽胞杆菌FJAT-4产生的抑菌脂肽由C₁₇ fengycin A、C₁₇ fengycin B、C₁₇ fengycin B₂、C₁₆ fengycin A衍生物、C₁₆ fengycin B衍生物、C₁₃-C₁₅ surfactin及C₁₃-C₁₅ surfactin衍生物组成，其中C₁₃-C₁₅ surfactin衍生物（m/z [M+Na]⁺=1048.6/1062.6/1076.6）为新化合物。培养基成分不同对菌株FJAT-4脂肽组成影响较小，但温度对菌株FJAT-4产生抑菌脂肽的影响很大，该菌株在较低温度（20-25 ℃）下培养不产生脂肽，30-40 ℃下培养能产生抑菌脂肽，且高温有利于提高脂肽中surfactin的比例。该脂肽类物质对辣椒、番茄、香蕉和甜瓜尖孢镰刀菌等多种植物病原真菌均具有很好的抑制效果，且呈剂量依赖性。扫描电镜结果表明地衣芽胞杆菌FJAT-4所产的脂肽会严重影响辣椒、番茄、香蕉和甜瓜尖孢镰刀菌菌丝的正常生长，导致菌丝断裂变形、孢子变形或显著抑制了孢子的生长。[结论] 地衣芽胞杆菌FJAT-4产生的抑菌脂肽为fengycin和surfactin类物质，该抑菌脂肽会致使尖孢镰刀菌菌丝体发育畸形，影响尖孢镰刀菌的正常生长。

Abstract:

[Objective] Lipopeptides produced by Bacillus licheniformis FJAT-4 can strongly inhibit the mycelia growth of Fusarium oxysporum. The aim of this study was to identify the structure of antifungal lipopeptide, analyze the effect of culture medium and temperature on the antifungal lipopeptide production, and explain antifungal effect of lipopeptide against F. oxysporum. This study is helpful to understand the mechanisms of Bacillus licheniformis FJAT-4 in controlling F. oxysporum.[Methods] Crude lipopeptides were extracted by acid precipitation and resolved in methanol. Their structures were identified using liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry. Their antifungal activities were determined through the assay of inhibition zone experiment. Their effects against F. oxysporum were observed using scanning electron microscopy.[Results] Lipopeptides produced by B. licheniformis FJAT-4 were identified as C₁₇ fengycin A, C₁₇ fengycin B, C₁₇ fengycin B₂, C₁₆ fengycin A variant, C₁₆ fengycin B variant, C₁₃-C₁₅ surfactin and C₁₃-C₁₅ surfactin variants. Among them, C₁₃-C₁₅ surfactin variants with m/z 1048.6/1062.6/1076.6 were new compounds. Results showed that the culture mediums have relatively little influence on the composition of lipopeptides produced by FJAT-4. However, the culture temperature presented obviously impact on the production of antifungal lipopeptides, which can only be produced by strain FJAT-4 growing at higher temperature (30-40℃) in comparison to temperature between 20-25℃. The higher culture temperature was in favour of increasing the proportion of surfactins in lipopeptide mixture. The lipopeptides exhibited strong antifungal activity against several plant pathogenic F. oxysporum strains in a dosage-dependent mode. Observation under scanning electron microscopy showed that the antagonistic lipopeptides secreted by B. licheniformis FJAT-4 significantly led to abnormal mycelia and spore growth of F. oxysporum.[Conclusion] The lipopeptides produced by FJAT-4 was composed of fengycins and surfactins. These lipopeptides display strong antifungal activity against several strains of plant pathogenic F. oxysporum, and destroyed the mycelial structure of the pathogenic F. oxysporum.