Nitrogen Fixation Through Acetylene Reduction Assay (ARA)

Nitrogenase activity of all strains was studied by the ARA method (Hardy et al., 1968). A pure rhizobacterial colony was inoculated on 10 mL semi-solid NFb medium (Supplementary Table S1) in a test tube and incubated at 32 ± 2°C for 36–48 h. Then, under sterile conditions air was removed from the tubes and replaced with acetylene gas (5 mL), and the test tube was kept for another 24 h at 32 ± 2°C. At the end of the incubation, 0.5 mL of headspace gas was carefully extracted from each tube and analyzed in a GC-17A gas chromatograph (Shimadzu, Japan) with DB-1,701 column (Agilent, Santa Clara, United States) set using the flame ionization detector (FID) at 80°C and the injector at 110°C, with 35 mL min^–1 flow rate of carrier gas. The quantity of ethylene (C₂ H₄) produced by each strain was calculated and presented as nmol C₂H₄ produced mg protein^–1 h^–1.