Molecular identification of Azospirillum spp.: Limitations of 16S rRNA and qualities of rpo D 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.

In vitro PGPR properties and osmotic tolerance of different Azospirillum native strains and their effects on growth of maize under drought stress

“…Notably, previous results on whole genome comparisons indicated that Az39, which has been historically considered as an A. brasilense strain, might be part of a different species (Maroniche et al, 2017). Thus, taken together, all the evidences suggest that Az39, as well as strains Az3, Az19 and Az63, do not belong to A. brasilense species.…”

“…Further studies will be needed to determine the behavior of this strain in field trials in view of developing new inoculants suitable for areas with water deficit. Table 2 or elsewhere (Maroniche et al 2017). …”

“…The resulting sequences were deposited in the Genbank database (Table 2). Phylogenetic analyses using the obtained 16S rRNA sequences and available rpoD sequences (Maroniche et al, 2017) were carried out with MEGA 6 software (Tamura et al, 2013) using the maximum likelihood method, Tamura-Nei substitution model and a bootstrap phylogeny test of 1000 iterations. Sequences of other Azospirillum strains were retrieved from Genbank.…”

“…The 16S rRNA gene data was obtained by PCRamplification with universal primers 27F and 1496R and sequencing, while rpoD sequences already available from a previous characterization of the IMyZA-INTA collection (Maroniche et al, 2017) were retrieved from Genbank. The obtained sequences indicated that strains Az3, Az19 and Az63 are closely related to A. brasilense Az39 according to both 16S rRNA (99.8-99.9 % identity) and rpoD (99.6-99.9 % identity) genes ( Fig.…”

Ureide metabolism in plant-associated bacteria: purine plant-bacteria interactive scenarios under nitrogen deficiency

“…Due to the complexity of the microbiome associated with plants, the analyses of the purine metabolism will concentrate mainly on bacteria, with emphasis on the bacterial species or isolates fully identified with 98-100% similarity with reference strains. The names of the bacterial species are mentioned as in the cited references, although we are aware of the recent re-classification of a number of Pseudomonas and Azospirillum strains into different genospecies (Tran et al 2017;Maroniche et al 2017) as well as of the split of the genera Burkholderia and Paraburkholderia (Martínez-Hidalgo and Hirsch 2017).…”