Isolation and Characterization of Diazotrophs

Cultures from the DNA-SIP assay at day 35 were used for the isolation of the diazotrophic bacteria. Briefly, the cultures were serially diluted in 0.9% NaCl solution (up to 10⁻⁵) and then screened on agar plates containing the N-free Jensen's medium (M710). The plates were incubated at 30 °C for 3–5 days, and colonies were further purified and characterized. Then, PCR amplification of the nifH gene was performed to screen for diazotrophs [43 (link)]. An acetylene reduction assay was conducted to measure the nitrogenase activity to confirm the capability for nitrogen fixation [43 (link)]. The near full-length 16S rRNA gene was amplified with the primer pair 27F/1492R and sequenced, and sequences were annotated against the NCBI database. The neighbor-joining phylogenetic tree was constructed with MEGA. The morphology of the isolated strains was examined by scanning electron microscopy (Phenom proX, Phenom-World BV, Netherlands).