Osmotic variations in the soil can affect bacterial growth diminishing the number of inoculated bacteria. In a scenario of water deficit having tolerant bacteria would be beneficial to achieve a better response of the plant to stress. Thus, selection of more resistant bacteria could be useful to design new inoculants to be used in arid zones. In this sense, a group of Azospirillum isolates deposited in INTA collection was characterized in order to select strains tolerant to osmotic stress. The results obtained demonstrated that Az19 strain has similar in vitro PGPR characteristics to Az39, the most used strain in Argentina for inoculants industries, with the advantage of a better tolerance to osmotic and salt stress. Inoculation of maize plants with this strain resulted in a better response against water deficit compared to Az39 strain, encouraging us to further study the behavior of this strain in greenhouse and field trials in view of developing new inoculants suitable for areas with water deficit.

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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.

Strain Az39T of Azospirillum is a diazotrophic plant growth-promoting bacterium isolated in 1982 from the roots of wheat plants growing in Marcos Juárez, Córdoba, Argentina. It produces indole-3-acetic acid in the presence of l-tryptophan as a precursor, grows at 20–38 °C (optimal 38 °C), and the cells are curved or spiral-shaped, with diameters ranging from 0.5–0.9 to 1.8–2.2 µm. They contain C16 : 0, C18 : 0 and C18 : 1  ω7c/ω6c as the main fatty acids. Phylogenetic analysis of its 16S rRNA gene sequence confirmed that this strain belongs to the genus Azospirillum , showing a close relationship with Azospirillum baldaniorum Sp245T, Azospirillum brasilense Sp7T and Azospirillum formosense CC-Nfb-7T. Housekeeping gene analysis revealed that Az39T, together with five strains of the genus (Az19, REC3, BR 11975, MTCC4035 and MTCC4036), form a cluster apart from A. baldaniorum Sp245T, A. brasilense Sp7T and A. formosense CC-Nfb-7T. Average nucleotide identity (ANI) and digital DNA–DNA hybridization (dDDH) between Az39T and the aforementioned type strains revealed values below 96 %, the circumscription limit for the species delineation (ANI: 95.3, 94.1 and 94.0 %; dDDH: 62.9, 56.3 and 55.6 %). Furthermore, a phylogeny evaluation of the core proteome, including 809 common shared proteins, showed an independent grouping of Az39T, Az19, REC3, BR 11975, MTCC4035 and MTCC4036. The G+C content in the genomic DNA of these six strains varied from 68.3 to 68.5 %. Based on the combined phylogenetic, genomic and phenotypic characterization presented here, we consider that strain Az39T, along with strains Az19, REC3, BR 11975, MTCC4035 and MTCC4036, are members of a new Azospirillum species, for which the name Azospirillum argentinense sp. nov. is proposed. The type strain is Az39T (=LBPCV39T=BR 148428T=CCCT 22.01T).

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