Isolation of N-Fixing Bacteria from Warm-Season Pasture Grasses and the Evaluation of Nitrogen Effects on the Bacterial Communities Present in Cenchrus clandestinus

Nitrogen is essential for plant growth. Reliance on synthetic nitrogen fertilisers, however, is costly and contributes to soil degradation. Utilising nitrogen-fixing bacteria as biofertilisers may offer a sustainable alternative, reducing fertiliser costs and environmental impact. In this study, we attempted to isolate nitrogen-fixing bacteria from 14 seed batches of warm-season pasture grasses and successfully isolated bacteria from three of these batches. Whole genome sequencing confirmed the presence of the nif operon within all three isolates. Two seed batches of Cenchrus clandestinus (Hochst. ex Chiov.) Morrone from which nif-containing bacteria were isolated, along with two ‘nif’-negative C. clandestinus seed batches, were used in nitrogen-limiting growth assays. This was done to evaluate the effect of the presence of seed-associated nitrogen-fixing bacteria within a seed batch on nitrogen-limited plant growth and the associated plant microbiome composition, using 16S amplicon sequencing of root and shoot samples. Initial plant growth assays revealed significantly reduced root length between plants grown from seed batches harbouring nitrogen-fixing bacteria and those without, under limiting nitrogen availability, but no resulting shoot biomass reduction was observed. The plant microbiomes of these nif-positive seed batches were also statistically similar to each other, compared to the nif-negative seed batch plants. Plant microbiomes of all four C. clandestinus seed batches were significantly different from their original seed microbiomes, showing shifts in community composition. This study demonstrates the presence of potential nitrogen-fixing bacteria associated with warm-season pasture grass seeds at low abundance and reveals differences in plant-associated bacterial community composition between seed batches harbouring and lacking these bacteria.