Phylogenetic analysis of dominant microorganisms of the genera Bacillus and Phyllobacterium, isolated from the rhizosphere of spring barley

Abstract

Goal. To determine the taxonomic position of dominant microorganisms in the rhizosphere of spring barley — the representatives of the genera Bacillus and Phyllobacterium — based on phylogenetic analysis of the nucleotide sequence of the 16S rRNA gene. Methods. To identify the bacteria they used the analysis of the nucleotide sequence of the 16S rRNA gene. Bacterial DNA was extracted from the suspension of bacterial cells using GeneJet Genomic DNA Purification Kit (Thermo Scientific) according to the protocols of the manufacturer. Amplification of the 16S rRNA gene was performed with primers 27f (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492r (5′-CGGTTACCTTGTTACGACTT-3′). Results. The taxonomic position is determined by dominant microorganisms in the rhizosphere of spring barley (strains of Phyllobacterium ifriqiyense 1 and Bacillus methylotrophicus 10 from working collection of the Department of ecobiotechnology and biodiversity, NULES of Ukraine) based on phylogenetic analysis of the nucleotide sequence of the 16S rRNA gene. The nucleotide sequence of a fragment of the gene 16S pPHK of the mentioned above strains was registered in the international database GenBank (NCBI) with numbers: MK947049, MK947055, and MK947050, MK947056 respectively. Obtained amplicon in size of ~1500 BP was cut out from the gel and purified using GeneJet PCR Purification Kit (Thermo Scientific). The DNA concentration was determined on spectrophotometer DS11FX+ (DeNovix, USA). The purified PCK-product was sequenced in two directions on the device 3130 «Genetic Analyzer» (Applied Biosystems, USA) using a set of reagents «BigDye Terminator v 3.1 Cycle Sequencing Kit». Conclusions. Analysis of the isolated strains of Phyllobacterium ifriqiyense 1 and Bacillus methylotrophicus 10 for the similarity of the nucleotide sequences of the 16S rRNA gene revealed 99% similarity with sequences of typical representatives of the species concerned. Promising strain Phyllobacterium ifriqiyense 1 and Bacillus methylotrophicus 10 can be successfully introduced in the metagenome of aboriginal groups of the soil as biological agents of microbial preparations. They can provide metabolic functions of biological systems of the rhizosphere of barley, and be practically valuable agents of bioprotector action, induction of systemic resistance of plants against phytopathogens.