Identification and molecular characterization of Azotobacter chroococcum and Azotobacter salinestris using ARDRA,...

Azotobacter chroococcum and A. salinestris do not possess significant and distinct morphological and physiological differences and are often mistaken with each other in microbiological research. In this study, 12 isolates of Azotobacter isolated by standard protocol from soils were identified morphologically and physiologically as A. chroococcum. The isolates were more closely investigated for the molecular differentiation and diversity of A. chroococcum and A. salinestris. For this purpose, the ARDRA technique including HpaII, RsaI, and AluI restriction enzymes, and REP, ERIC, and BOX markers were used. The nifD and nifH genes were also utilized to evaluate the molecular identification of these two species. The 16S rDNA evaluation showed that only four out of the 12 isolates were identified as A. chroococcum and the rest were A. salinestris. The results revealed that HpaII was able to differentiate A. chroococcum from A. salinestris whereas RsaI and AluI were not able to separate them. Moreover, BOX and REP markers were able to differentiate between A. chroococcum and A. salinestris. However, ERIC marker and nifD and nifH genes were unable to separate these species. According to the results, HpaII restriction enzyme is suggested to save time and cost. BOX and REP markers are recommended for differentiation and clear discrimination not only between A. chroococcum and A. salinestris but also among their strains.