Molecular identification of Azospirillum spp.: Limitations of 16S rRNA and qualities of rpoD as genetic markers

Abstract

Since their discovery, plant-growth promoting rhizobacteria from the genus Azospirillum have been subjected to intensive research due to their biotechnological potential as crop inoculants. Phylogenetic analysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsistencies and low confidence often arise when working with close species. In this work, it was observed that these difficulties might be explained by a high number of rRNA operons with considerable inter-genic variability within Azospirillum genomes. To search for alternative genetic markers from a list of housekeeping genes, the correlation between pairwise gene and whole-genome similarities was examined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for the PCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 type strains of different species. The sequences obtained were used for inferring a phylogenetic tree of the genus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla isolated from many different locations of Argentine. In addition, several strains that might represent novel species were detected. The results indicate that the sequencing of rpoD is a suitable alternative method for a confident molecular identification in Azospirillum spp.