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

Since their discovery, plant-growth promoting rhizobacteria from the genus Azospirillum have been sub-jected to intensive research due to their biotechnological potential as crop inoculants. Phylogeneticanalysis of Azospirillum spp. is carried out by 16S rRNA sequencing almost exclusively, but inconsis-tencies and low confidence often arise when working with close species. In this work, it was observedthat 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 ofhousekeeping genes, the correlation between pairwise gene and whole-genome similarities was exam-ined. Due to its good performance, rpoD was selected for further analyses. Genus-specific primers for thePCR-amplification and sequencing of rpoD from Azospirillum spp. were designed and tested on 16 typestrains of different species. The sequences obtained were used for inferring a phylogenetic tree of thegenus, which was in turn used as a reference to successfully identify a collection of 31 azospirilla iso-lated from many different locations of Argentine. In addition, several strains that might represent novelspecies were detected. The results indicate that the sequencing of rpoD is a suitable alternative methodfor a confident molecular identification in Azospirillum spp.

Inst. de Microbiología y Zoología Agrícola IMyZA

Fil: Maroniche, Guillermo Andrés. Unidad Integrada Balcarce-Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias-Instituto Nacional de Tecnología Agropecuaria (INTA). Estación Experimental Agropecuaria Balcarce; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Garcia, Julia Elena. Instituto Nacional de Tecnología Agropecuaria (INTA). Instituto de Microbiología y Zoología Agrícola; Argentina

Fil: Salcedo, Florencia. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

Fil: Creus, Cecilia Mónica. Universidad Nacional de Mar del Plata. Facultad de Ciencias Agrarias; Argentina