Multisubstrate DNA-SIP of the switchgrass rhizosphere with 13CO2 and 15N2

Associative nitrogen fixation (ANF) is a spatially and temporally variable phenomenon in which plant C is used to fuel the activity of root-associated diazotrophs. Though this association can provide valuable N to the plant, the magnitude of these N inputs and the nature of this potential symbiosis is poorly understood. Potential associative nitrogen fixing bacteria (ANFB) have long been observed in rhizosphere communities of switchgrass, yet there have been few studies with direct evidence of associative diazotrophs in situ. To investigate the coupling of plant derived carbon inputs to nitrogen fixation in the rhizosphere we performed a multisubstrate DNA-SIP experiment using whole plant switchgrass mesocosms labeled with either 13CO2 or 15N2. Labeled plant photosynthate enters the rhizosphere community by way of exudation or rhizodeposition, while labeled nitrogen enters the community as a result of diazotrophic nitrogen fixation. We identified isotopically labeled DNA from 772 taxa, including 365 that were 13C-labeled, 192 that were 15N-labeled, and 215 that were dual 13C- and 15N-labeled. The dual incorporators included Actinomycetota (20.76%), Pseudomonadota (20.47%), Acidobacteriota (18.9%), Planctomycetota (11.29%), Verrucomicrobiota (9.3%), Bacillota (6.85%), Myxococcota (3.38%), and Nitrososphaerota (~2%), among others. We find Vicinamibacteraceae and Chthoniobacteraceae to be abundant and frequently labeled, and surprisingly, we find at least 15 other families that are typically found associated with cyanobacterial biological soil crusts. It is plausible that biocrust communities might establish on the soil surface below switchgrass plants and that diffuse cyanobacterial crusts might provide a source of fixed N to the plant.