One Hundred Years of Progress Dedicated to Understanding the Evolution of Tools and Knowledge in Azospirillum Taxonomy

Abstract

Azospirillum is one of the most extensively studied genera of plant growth-promoting rhizobacteria (PGPR), widely recognized for its biological nitro-gen fixation (BNF) ability and phytohormone production. The genus currently includes 36 proposed species, 25 of which have been validly published. Despite its agronomic relevance, the taxonomy of Azospirillum remains incomplete and, in some cases, inconsistent. This chapter traces the historical development of Azospirillum taxonomy and highlights how genome-based tools are reshaping our understanding of species boundaries within the genus. Using a curated dataset of 69 unique genomes, including two newly sequenced strains (A. largimobile Sp A3a, A. zeae N7), we applied whole-genome comparisons, average nucleotide identity (ANI), and core genome phylogeny to evaluate interspecies relationships. Results indicate high genomic plasticity, an open pangenome (γ = 0.85), and support multiple taxonomic reassignments. In addition, we examined key functional traits linked to plant growth promotion, such as nitrogen fixation and indole-3-acetic acid (IAA) biosynthesis, and analyzed their distribution across phylogenetic groups. These findings underscore the limitations of classical taxonomical approaches and emphasize the importance of using high-resolution genomic tools for taxonomic studies. As genome sequencing becomes faster, more accurate, and economically accessible, its routine application should become the standard for defining new Azospirillum species. To facilitate similar studies integrating bacterial genomics and taxonomy, we report a reproducible protocol covering the full workflow from Azospirillum cell culture to the generation of high quality, annotated, and complete genomes.