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

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. A greenhouse H-pot system was developed to directly estimate belowground N transfer from biological N₂ fixation (BNF) using ¹⁵N₂. The system was tested with 'Prine' annual ryegrass (Lolium multiflorum L.) and 'Dixie' crimson clover (Trifolium incarnatum L.). Legume and grass root systems growing in either individual or H pots were exposed to ¹⁵N₂. Control H pots were separated by mesh to prevent contact between roots from each side of the pot. To reduce enriched gas volume demand and avoid cross-contamination in the greenhouse, the gas was supplied through underground tubes in the root zone. Ryegrass and clover exhibited an enrichment of ¹⁵N₂ when their respective root systems were supplied with ¹⁵N₂. Additionally, ryegrass also showed enrichment when clover roots received the gas, provided there was direct contact between the root systems on both sides of the H pot; however, this enrichment did not occur when such contact was prevented. Plants cultivated in monoculture without the application of ¹⁵N₂ did not present enrichment. The H-pot facilitates the evaluation of belowground transmission, an essential mechanism for N transfer. The technique of gradually supplying ¹⁵N₂ directly to the root system may serve as a valuable labeling method for tracking nitrogen transfer. The absence of enrichment when plants were not directly supplied indicates negligible atmospheric enrichment. However, the enrichment observed in ryegrass when supplied with the gas suggests BNF through alternative pathways.