伯克霍尔德菌HQB-1抑制香蕉枯萎病菌的活性化合物分离鉴定

Isolation and identification of bioactive compounds of Burkholderia HQB-1 strain inhibiting banana wilt

摘要:

【背景】伯克霍尔德菌HQB-1对香蕉枯萎病菌(Fusarium oxysporum f. sp. cubense Tropical Race 4，Foc TR4)具有良好的防治效果。【目的】从菌株HQB-1发酵液中分离活性化合物并通过超高效液相联用质谱进行检测，获得该菌株具有良好生防作用的单体化合物。【方法】以HQB-1为目标菌株，大批量发酵并进行发酵液分离、提纯，通过超高效液相联用质谱法与核磁共振波谱法鉴定活性化合物。【结果】 HQB-1菌株发酵液中的蛋白对Foc TR4无抑制作用；HQB-1菌株产生儿茶酚型铁载体，而不产生异羟肟酸型铁载体；对HQB-1菌株发酵液离心、浓缩、干燥，获得乙酸乙酯浸膏(粗提物)，经过大孔树脂柱的充分吸附，在30%、60%及无水甲醇的洗脱下获得组分1?3，对Foc TR4的抑菌率分别为10.06%、27.82%和51.40%；选择抑菌率最大的组分3过硅胶柱层析，获得黄绿色晶体。该化合物在365 nm波长下具有最大吸收峰，将核磁共振图谱与SciFinder和SDBS信息数据库进行图谱比对，将该抑菌活性化合物鉴定为吩嗪-1-羧酸(Phenazine-1-Carboxylic Acid，PCA)。PCA对Foc TR4的最小抑菌浓度最低，仅为1.563 μg/mL，说明PCA对Foc TR4的抑制效果较强。【结论】从HQB-1菌株中分离得到活性化合物PCA，PCA的发现为香蕉枯萎病的生物防治奠定了良好的理论基础。

Abstract:

[Background] Burkholderia HQB-1 has good control effect on banana fusarium wilt (Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4)). [Objective] The active compound was separated from the fermentation broth of HQB-1 strain, and the active compound was detected by HPLC-MS to obtain the monomer compound with good biocontrol effect. [Methods] HQB-1 strain was fermented in large quantities, and then the fermentation broth was separated and purified. The active compounds were identified by HPLC-MS and NMR. [Results] The protein from fermentation broth of HQB-1 strain had no inhibitory effect on Foc TR4. The catechol type iron carrier rather than hydroxamic acid type iron carrier was produced from HQB-1 strain. HQB-1 strain fermentation broth was centrifuged, concentrated and dried to obtain ethyl acetate extract (crude extract), which was then fully adsorbed by macroporous resin column. The components 1?3 were obtained after eluting by 30%, 60% and anhydrous methanol, and the corresponding inhibitory rate to Foc TR4 was 10.06%, 27.82% and 51.40% respectively. The component with the highest inhibitory rate was selected, and the yellow green crystal was obtained through silica gel column chromatography. The compound had a maximum absorption peak at 365 nm wavelength. The antibacterial activity compound was identified as phenazine-1-carboxylic acid (PCA), after the comparative analysis of NMR spectrum with SciFinder and SDBS information database. The minimum inhibitory concentration (MIC) of PCA to Foc TR4 was the lowest, only 1.563 μg/mL, indicating that PCA had a strong inhibitory effect on Foc TR4. [Conclusion] The active compound PCA isolated from HQB-1 strain had the biocontrol effect on banana fusarium wilt.