丁香内生死亡谷芽孢杆菌DX78的分离鉴定及其抑菌成分

Clove endophyte Bacillus vallismortis DX78: isolation, identification, and preliminary study of antimicrobial components

摘要:

【背景】植物内生菌往往产生与植物相同、相似或新颖的次生代谢产物，丁香具有广谱优异的抗菌活性，可从中分离到强抑菌作用的内生细菌。【目的】筛选抑制姜瘟病菌的丁香内生细菌并分离其活性成分。【方法】牛津杯法筛选拮抗内生细菌；根据16S rRNA基因序列鉴定菌株；有机溶剂萃取、硅胶柱层析和薄层制备色谱分离活性成分；测定¹H NMR、¹³C NMR和DEPT (135°)并对分离的活性成分进行结构鉴定；滤纸片法和菌丝生长速率法测定活性成分抑菌活性。【结果】共分离到112株丁香内生细菌，从叶中分离最多，占37.4%。其中17株对姜瘟病菌有抑制，DX78菌株抑制作用最好，经鉴定为死亡谷芽孢杆菌，并从其发酵液中追踪分离到邻苯二甲酸二丁酯(dibutyl phthalate, DBP)。DBP对姜瘟病菌、猕猴桃溃疡病菌的MIC分别为0.3 mg/disc、0.25 mg/disc；其还可抑制多种植物病原真菌，特别对苹果炭疽叶枯病菌、番茄灰霉病菌和苹果腐烂病菌的抑制EC₅₀仅分别为3.751、18.568和22.019 µg/mL。以苹果炭疽叶枯病菌为靶标菌，戊唑醇抑制毒力约为DBP抑制毒力的4.5倍，DBP抑制毒力约为多菌灵抑制毒力的12.5倍。【结论】丁香抗病内生细菌丰富，从死亡谷芽孢杆菌DX78中分离的DBP对苹果炭疽叶枯病菌抑制作用强，值得进一步研究。

Abstract:

[Background] Endophytic microorganisms often produce the same, similar or novel secondary metabolites as host plants. Clove has excellent antimicrobial activities, from which the endophytic bacteria with strong antimicrobial effects could be isolated. [Objective] To screen out clove endophytic bacteria against Ralstonia solanacearum and isolate active components. [Methods] The antimicrobial endophytic bacteria were screened out by oxford-cup tests and identified by 16S rRNA sequence analysis. The active components were isolated by organic solvent extraction, silica-gel column chromatography, and preparative thin layer chromatography, and identified by NMR. The inhibitory effects of the active component on pathogens were determined by filtering paper method and mycelial growth rate method. [Results] A total of 112 strains of endophytic bacteria were isolated, and most strains were obtained from leaves, accounting for 37.4%. Seventeen strains showed inhibitory effect on R. solanacearum. Among these strains, DX78 demonstrated the strongest activity and was identified as Bacillus vallismortis. Dibutyl phthalate (DBP) was isolated from the fermentation broth of DX78. The minimum inhibitory concentrations of DBP against R. solanacearum and Pseudomonas syringae were 0.3 mg/disc and 0.25 mg/disc, respectively. Furthermore, DBP inhibited plant pathogenic fungi, with the EC₅₀ values of 3.751, 18.568, and 22.019 µg/mL against Glomerella cingulata, Botrytis cinerea, and Valsa mali, respectively. With G. cingulata as the target fungus, the inhibitory toxicity of tebuconazole was about 4.5 times that of DBP, and the inhibitory toxicity of DBP was about 12.5 times that of carbendazim. [Conclusion] Clove harbors rich antimicrobial endophytic bacteria, and DBP isolated from B. vallismortis DX78 has strong inhibitory effect on G. cingulata, which is worthy of further study.