Abstract:[Background] Bacterial biofilm is a major factor causing bacterial resistance and recurrent nosocomial infections. But up to now there have been no effective anti-biofilm drugs. Rhizosphere microorganisms of some plants in high-cold, special environment can produce large quantities of active components which can improve the host immunity, and are thought to be a medicinal resource with great anti-biofilm potential. [Objective] To understand the rhizosphere microbial diversity of Astragalus yunnanensis and A. tatsienensis var. incanus growing in Baima Snow Mountain, and to screen cultivable strains with antibacterial and anti-biofilm activities. [Methods] In this study, metagenomics and traditional culture-dependent methods were employed to explore the rhizosphere microbial diversity of A. yunnanensis and A. tatsienensis var. incanus from Baima Snow Mountain in Deqin County, Diqing Tibetan Autonomous Prefecture, Yunnan Province. The microplate assay was conducted for determining the antibacterial and anti-biofilm activity of these cultivable microbes. [Results] Metagenomic sequencing indicated that the rhizosphere microorganisms of A. yunnanensis samples belonged to 6 phyla, 7 classes, 8 orders, 8 families, 9 genera and 10 species, and Thermus was the dominant microflora. The rhizosphere microbes of A. tatsienensis var. incanus samples belonged to 6 phyla, 8 classes, 10 orders, 11 families, 14 genera and 15 species, and Bradyrhizobium was the dominant microflora. A total of 145 cultivable strains were obtained by pure cultivation, including 112 bacteria and 33 fungi. Among them, 59 bacterial strains of 16 genera and 35 species and 19 fungal strains of 4 genera and 5 species were isolated from the rhizosphere soil of A. yunnanensis, with Streptomyces, Pseudomonas and Aspergillus being predominant. The other 53 bacterial isolates of 16 genera and 29 species and 14 fungal isolates of 3 genera and 4 species were derived from the rhizosphere soil of A. tatsienensis var. incanus, among which Bacillus, Stenotrophomonas and Aspergillus were the most abundant genera. For the screened microbes with antibacterial and anti-biofilm activity, 51 bacterial strains and 7 fungal strains were chosen at different species levels as representatives to explore their potential as medicinal resources of natural antibiotics. The crude ethyl acetate extract of the fermentation broth of 5 bacteria and 1 fungus were demonstrated to have moderate to strong activity against Gram-positive pathogens, and 4 strains of them showed anti-biofilm activity against methicillin-resistant Staphylococcus aureus (MRSA). Finally, two microbial strains Streptomyces fulvissimus KTA1 and Aspergillus fumigatus YNF5 were considered as the promising bioactive strains. [Conclusion] It is reported for the first time that there is abundant species community composition in the rhizosphere microorganisms of Astragalus plants distributed in northwestern Yunnan province. These cultivable microorganisms could be regarded as a great medicinal source for the exploitation of natural antibiotics. This study was significant for utilization and protection of characteristic plants-associated microorganisms in high-cold, special environment of northwestern Yunnan.
