Use of rpoB gene phylogenetic marker-based distinction of abiotic stress tolerant and plant-growth promoting Bacillus paralicheniformis isolates from their closely related Bacillus licheniformis

Abstract

Bacillus paralicheniformis is a new identified species, which was distinguished from Bacillus licheniformis in 2015 through extensive phylogenomic and phylogenetic analyses. In this context, this study aimed to achieve a clear identification of the active plant-growth promoting rhizobacteria (PGPR) B. paralicheniformis isolates among the closely related B. licheniformis through molecular typing, helping for the development of clearly-identified PGPR isolates to be used as biofertilizers. A total of 15 rhizobacteria were isolated from the olive rhizosphere soils. These bacterial isolates exhibited various proprieties in terms of abiotic stress tolerance, biofilm formation under stress conditions, and enzyme activities (i.e., lipases, cellulases, and proteases). In addition, several PGP traits such as phosphate solubilization and the production of siderophores and indol-3 acetic acid were also observed. Molecular identification through 16S rRNA sequencing initially identified all the isolates as Bacillus spp. The multi-locus sequence typing (MLST) scheme, using six housekeeping genes (adk, ccpA, recF, rpoB, sucC, and spo0A) had unveiled distinct allelic profiles resulting in 13 unique sequence types (ST). Notably, a comprehensive analysis indicated no exact allele matches existed between the examined isolates and those documented in the PubMLST database. Among the six housekeeping genes, we noticed that rpoB gene (RNA polymerase, subunit beta) had multiple polymorphic sites that were bordered by conserved sequences. Furthermore, the phylogenetic analysis based on rpoB had identified clusters indicating distinct phylogenetic relationships between B. paralicheniformis and B. licheniformis, and successfully differentiated the two species from a pool of 90 strains. The rpoB partial sequence proved to be effective for accurate species discrimination.