高寒特境中甸黄芪植物根际微生物物种多样性及其抗生物膜活性成分

Species diversity and anti-biofilm components of rhizosphere microorganisms derived from Astragalus forrestii in special high-cold environments

摘要:

滇西北高寒地区分布着丰富的黄芪属植物资源，该属植物“根际效应”明显，其根际微生物极具抗菌药用资源研究价值。【目的】认知滇西北高寒特境中甸黄芪根际微生物的物种多样性，探究其可培养菌株次生代谢产物的化学多样性及抗菌、抗生物膜活性。【方法】采用宏基因组和微生物纯培养方法对中甸黄芪植物根际微生物进行物种多样性分析，同时采用高效液相色谱（high performance liquid chromatography，HPLC）、超高效液相色谱-质谱联用法（ultra-performance liquid chromatography-mass spectrometry，UPLC-MS）结合“微量肉汤稀释法” “孔板法”等多级联合筛选策略综合评估可培养菌株的抗菌活性药源研究价值。【结果】对中甸黄芪根际土壤样本的微生物分类操作单元（operational taxonomic units，OTU）进行分类注释，得到22门54纲105目187科316属856种微生物，其中优势菌群为慢生根瘤菌属。纯培养共获得27属54种95株可培养菌株，包括20属33种54株细菌和7属21种41株真菌，优势属分别为芽孢杆菌属和青霉属。其中，1株细菌Pseudomonas tolaasii ZTB4和3株真菌Aspergillus tabacinus ZNF17、Lecanicillium aphanocladii ZNF15、Umbelopsis nana ZTF31的次生代谢产物具有广谱抗菌活性。同时，菌株ZTB4和ZNF17的次生代谢产物也显示出优秀的抗耐甲氧西林金黄色葡萄球菌（methicillin-resistant Staphylococcus aureus，MRSA）生物膜活性，并已验证这2株菌株的主要活性成分分别为环脂肽类与黄酮类。【结论】中甸黄芪植物根际微生物物种多样性较为丰富，其可培养菌株次生代谢产物有较好的化学多样性和抗菌、抗生物膜活性。研究结果为我国特境特色微生物药用资源的开发利用提供理论依据。

Abstract:

There are abundant plant resources of Astragalus in the high-cold region of northwestern Yunnan. These plants have obvious rhizosphere effect and their rhizosphere microorganisms are considered medicinal resources with great antimicrobial potential. [Objective] To understand the species diversity of rhizosphere microorganisms of Astragalusforrestii growing in the special high-cold environment of northwestern Yunnan, and the chemical diversity and the antimicrobial and anti-biofilm activities of the secondary metabolites produced by cultivable strains. [Methods] Metagenomic sequencing and culture-dependent method were employed to explore the microbial diversity in the rhizosphere soil of A. forrestii. High performance liquid chromatography (HPLC) and ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) were combined with broth microdilution and microplate assays to evaluate the chemical diversity and antimicrobial and anti-biofilm activities of the ethyl acetate (EA) extracts of strain fermentation broths. [Results] The operational taxonomic units of the rhizosphere soil samples of A. forrestii were classified and annotated to 856 species belonging to 316 genera, 187 families, 105 orders, 54 classes, and 22 phyla. Bradyrhizobium was the dominant genus. A total of 95 cultivable strains of 54 species belonging to 27 genera were obtained, including 54 bacterial strains of 33 species belonging to 20 genera and 41 fungal strains of 21 species belonging to 7 genera, among which Bacillus and Penicillium were predominant. The secondary metabolites of the bacterial strain Pseudomonas tolaasii ZTB4 and the fungal strains Aspergillus tabacinus ZNF17, Lecanicillium aphanocladii ZNF15, and Umbelopsis nana ZTF31 displayed broad-spectrum antimicrobial activities. The secondary metabolites of strains ZTB4 and ZNF17 showed strong inhibitory activity against the biofilm formation of methicillin-resistantStaphylococcus aureus (MRSA). The main active components of these two strains were cyclic lipopeptides and flavonoids, respectively. [Conclusion] The rhizosphere soil of A. forrestii harbors rich microorganism, and the secondary metabolites of some cultivable strains have high chemical diversity and strong antimicrobial and anti-biofilm activities. The results provide a theoretical basis for the development and utilization of characteristic microbial medicinal resources in special environments in China.