A novel technique utilizing enriched 15N2 to trace nitrogen transfer in grass and legume mixtures

Authors: QUEIROZ, LUANA - University Of Florida; DUBEUX, JOSE - University Of Florida; SOLLENBERGER, LYNN - University Of Florida; VENDRAMINI, JOAO - University Of Florida; LIAO, HUI-LING - University Of Florida; Jaramillo, David; SANTOS, ERICK - University Of Alberta; ABREU, DACIELE - University Of Florida; LIRA, MARIO - Federal Rural University Of Pernambuco; CASAGRANDE, DANIEL - University Of Florida; MACKOWIAK, CHERYL - University Of Florida; RUIZ-MORENO, MARTIN - University Of Florida

Submitted to: Plant and Soil
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/24/2025
Publication Date: 7/8/2025
Citation: Queiroz, L., Dubeux, J., Sollenberger, L., Vendramini, J., Liao, H., Jaramillo, D.M., Santos, E., Abreu, D., Lira, M., Casagrande, D., Mackowiak, C., Ruiz-Moreno, M. 2025. A novel methodology to track nitrogen transfer in a grass-legume mixture using enriched 15N2. Plant and Soil. https://doi.org/10.1038/s41598-025-08988-y.
DOI: https://doi.org/10.1038/s41598-025-08988-y

Interpretive Summary: Legumes are a potentially important N source in pasture systems, but quantifying the transfer of biologically fixed N from the legume to the grass component is difficult. To address this limitation, a greenhouse system was developed to directly estimate biological nitrogen fixation (BNF) using isotopically labeled nitrogen gas (15N2). This system was tested with annual ryegrass and crimson clover. This study showed that the H-pot construction allows for the evaluation of belowground transmission of nitrogen, an important mechanism of transfer between grasses and legumes. Moreover, this technique showed that supplying 15N2 directly to the root system reduces the isotopically-labelled gas resources required, with minimal disruption of the natural system. Moving forward, this technique will allow for measurements of plant BNF and nitrogen transfer through mechanisms such as free-living, mycorrhizal symbionts, or other endophytic diazotrophs.

Technical Abstract: Legumes are a potentially important N source in pasture systems, but quantifying the transfer of biologically fixed N from the legume to the grass component is difficult. To address this limitation, a greenhouse H-pot system was developed to directly estimate biological N2 fixation (BNF) using 15N2. The system was tested with annual ryegrass (Lolium multiflorum L.) and crimson clover (Trifolium incarnatum L.). Legume and grass root systems growing in either H pots or individual pots were exposed to 15N2. Control H pots were separated by mesh to prevent contact between roots and mycorrhizae from each side of the pot. To reduce volume demand and avoid cross-contamination in the greenhouse, the gas was supplied through underground tubes. Annual ryegrass and crimson clover were significantly enriched in 15N when the respective root system was supplied with 15N2. Annual ryegrass was also significantly enriched when crimson clover roots were supplied with the gas and there was free root and mycorrhizal contact between both sides of the H pot, but not when this contact was precluded. Plants grown in single pots did not become enriched when the gas was not supplied to their root systems, indicating the overall air was not enriched. The H-pot construction allows the evaluation of belowground transmission, an important mechanism of transfer. Supplying 15N2 directly to the root system strongly reduces gas consumption, with minimal disruption of the natural system. The lack of enrichment when plants were not directly supplied indicates negligible atmospheric enrichment. Annual ryegrass enrichment, when supplied with the gas, suggests BNF either by free-living, mycorrhizal symbionts, or endophytic diazotrophs.