Three aerobic, asymbiotic, N2-fixing bacterial strains, designated P205T, P204 and P207, were isolated from a paddy soil in Yanting County, China. Based on 16S rRNA gene sequences, the three strains were closely related to Azotobacter chroococcum IAM 12666T (=ATCC 9043T) (99.00-99.79 % similarities). Strain P205T formed an individual branch distinct from the other two newly isolated strains and other related type strains in phylogenetic analyses based on 16S rRNA gene and 92 core genes. The average nucleotide identity (ANI), average amino acid identity (AAI) and digital DNA-DNA hybridization (dDDH) values based on genome sequences of strain P205T and A. chroococcum ATCC 9043T, P204, P207 were near or slightly higher than the thresholds for species circumscription (95-96, 95-96 and 70 %, respectively), and the dDDH values were significantly lower than the threshold for delineating subspecies (79-80 %), which strongly supported that strain P205T belonged to A. chroococcum but was a novel subspecies distinct from the type strain of A. chroococcum. This finding was further corroborated by distinct phenotypic characteristics such as growth in Luria-Bertani (LB) medium, carbon source utilization and chemical sensitivity to vancomycin. Therefore, strain P205T represents a novel subspecies of Azotobacter chroococcum, for which the name Azotobacter chroococcum subsp. isscasi subsp. nov. is proposed with the type strain P205T (=KCTC 72233T=CGMCC 1.16846T=CCTCC AB 2019080T). The subspecies Azotobacter chroococcum subsp. chroococcum subsp. nov. is created automatically with the type strain ATCC 9043T (=DSM 2286T=JCM 20725T=JCM 21503T=LMG 8756T=NBRC 102613T=NCAIM B.01391T=NRRL B-14346T=VKM B-1616T).
Keywords: Azotobacter chroococcum; average nucleotide identity (ANI); biological nitrogen fixation; digital DNA–DNA hybridization (dDDH); taxonomy.