两株香蕉枯萎病拮抗细菌的筛选及抑菌机理

Inhibition of banana Fusarium wilt by two biocontrol agents

摘要:

【目的】从发病蕉园中的健康香蕉根际筛选能有效抑制香蕉枯萎病病原菌生长的拮抗菌，进一步研究其抑菌机理。【方法】应用双层平板初筛和平板对峙实验复筛具有抑菌效果的拮抗菌；经生理生化试验、16S rRNA基因测序和特异引物扩增对拮抗菌进行鉴定；酸沉淀方法提取拮抗菌发酵液抑菌物质粗提液，基于比色法和HPLC测定粗提液对菌丝蛋白质含量、脂质过氧化、麦角甾醇和果胶酶活性的影响。【结果】筛选获得两株拮抗细菌H-2和H-7，初步鉴定为枯草芽孢杆菌和解淀粉芽孢杆菌，GenBank登录号分别为KX791428和KX791430；温室盆栽试验显示，两株拮抗菌对香蕉枯萎病的生防效率分别为59.1%和53.0%；H-2和H-7粗提液处理病原菌菌丝后，因脂质过氧化产生的丙二醛含量显著增加，分别达0.55 μmol/L和0.48 μmol/L；而蛋白含量、麦角甾醇含量和果胶酶活性均显著下降，其中H-2处理的抑制幅度更大，三项指标分别为0.15 mg/g、1.31 mg/g和0.008 7 U/mL，显著低于对照的0.25 mg/g、1.96 mg/g和0.035 U/mL。【结论】从健康香蕉根际筛选到两株拮抗细菌，两者可能通过增强病原菌菌丝的脂质过氧化和降低细胞代谢产物合成的方式抑制病原菌生长，可为两株拮抗菌的生防应用提供理论依据。

关键词:香蕉枯萎病，拮抗菌，筛选，抑菌机理

Abstract:

[Objective] To screen effective biocontrol agents from healthy banana rhizosphere in a diseased field and further study the inhibition mechanism. [Methods] Double-deck plates and fermentation antagonism study were used as primary- and secondary-screening for antagonists. Observing strains were identified based on physiological and biochemical tests, 16S rRNA gene sequencing and specific gene amplification. Crude extracts of fermentation were observed by using acid precipitate method and then added to the fermentation of pathogen for 5 days. Concentration of protein, malondialdehyde (MDA), ergosterol and pectinase activities were analyzed based on colorimetric method and HPLC to find out the effect of antagonists crude extracts on the growth of pathogen. [Results] Two antagonists, named as H-2 and H-7, were observed and identified as Bacillus subtilis (GenBank: KX791428) and Bacillus amyloliquefaciens (GenBank: KX791430), respectively. Fusarium wilt of banana was suppressed by these two strains and the biocontrol efficacies were 59.1% and 53.0%, respectively, based on the greenhouse condition. Concentration of MDA was significantly increased to 0.55 μmol/L and 0.48 μmol/L by treating the pathogen hyphae with suspension of H-2 and H-7, respectively. Meanwhile, concentration of protein, ergosterol and pectinase activities were significantly decreased in both H-2 and H-7 treatments and the lower index values were observed by H-2 by showing as 0.15 mg/g, 1.31 mg/g and 0.008 7 U/mL, respectively, which were significantly lower than those of CK (0.25 mg/g, 1.96 mg/g and 0.035 U/mL). [Conclusion] In conclusion, two antagonists screened from healthy banana rhizosphere soil were able to inhibit the growth of pathogen by increasing the lipid peroxidation of pathogen hyphae and decreasing the synthesis of metabolism products, which provide theoretical basis on the biocontrol application of two antagonists.

Key words:Fusarium wilt of banana, Antagonistic strain, Screening, Inhibition mechanism