Evidence for nitrogen fixation in switchgrass

Background/Question/Methods

Nitrogen (N) fixation occurs in some non-leguminous plant species, via associations with endophytic or free-living diazotrophs, but it has remained largely unexplored in temperate grassland systems. Switchgrass (Panicum virgatum), a North American prairie grass, often shows no yield response to N fertilizer addition, even where N outputs exceed N inputs (e.g., N removal via harvest exceeds fertilizer inputs). We hypothesized that this lack of response is a result of switchgrass’ obtaining N via associative N fixation. We injected ¹⁵N₂ gas into the rhizosphere of switchgrass grown without fertilizer addition, and then analyzed the ¹⁵N composition of the actively-growing leaves, and compared their composition to a non-enriched control plant. In addition, we extracted and sequenced DNA from switchgrass leaves, stems, and roots, to test for the presence of microbial diazotrophs and determine their identity.

 Results/Conclusions

We found that switchgrass leaves and stems had an elevated ¹⁵N concentration after ¹⁵N₂ injection (2.2‰ compared to 0.51‰ in the control). This increase was detectable immediately after the enrichment phase, indicating that switchgrass incorporated fixed N into its tissues. We amplified fragments of nitrogen reductase (nifH) from the leaves, stems, and roots of switchgrass, revealing that N-fixing bacteria were present.  The amplicon sequences were most similar (>95% amino acid identity) to those of Bradyrhizobium, Azospirillum, Burkholderia, and Xanthobacter. Bradyrhizobium and Burkholderia are known to associate with nodulating plants, while Azospirillum and Xanthobacter are free-living N fixers that can be found in close association with plant roots. Overall, our results suggest that switchgrass can form associations with microbial N fixers to augment its N supply. Future work will focus on quantifying the N fixation rate and clarifying the relationship between switchgrass and associated N-fixing bacteria.