This study evaluated DNA restriction fingerprinting as a method to differentiate strains of Azospirillum, soil bacteria capable of nitrogen fixation and used as potential biological fertilizers. Researchers analyzed 18 Azospirillum strains representing four established species (A. amazonense, A. brasilense, A. halopraeferens, and A. lipoferum) plus unidentified strains. Total DNA was digested with restriction endonucleases HindIII or BglII, and resulting fragments were separated using SDS-PAGE and visualized with silver nitrate staining. Each strain produced a unique, reproducible restriction pattern with both enzymes. Fragment patterns were analyzed using densitometry and computer-assisted numerical analysis, with similarity coefficients calculated using the Dice method. UPGMA clustering identified four main clusters and two single-member clusters. The results demonstrated that DNA restriction fingerprinting successfully distinguished individual strains and provided information about species relationships, particularly confirming that A. amazonense is heterogeneous and distinct from other azospirilla. The method proved valuable for strain identification, though clear species separation between A. brasilense and A. lipoferum was not achieved, likely due to atypical strains and inherent genomic heterogeneity.

Key findings

• Each of the 18 Azospirillum strains possessed a unique and completely reproducible DNA restriction fingerprint pattern with both HindIII and BglII enzymes
• UPGMA clustering using Dice similarity coefficients at 0.65 cut-off value separated strains into four main clusters plus two single-member clusters, broadly correlating with known species
• A. amazonense strains showed low similarity values (SD < 0.50) with other species, supporting previous evidence that this species is heterogeneous and evolutionarily distant
• DNA restriction fingerprinting enables differentiation of closely related Azospirillum strains and could supplement routine identification tests for strain recognition and tracing of soil inoculants