Residue mulching shifts nitrogen-source partitioning from atmospheric to non-fixed N during red clover regrowth

Introduction Biological nitrogen fixation (BNF) introduces new nitrogen (N) into legume-based systems, whereas plant-residue mulching primarily recycles N already present within the production system. We investigated whether N returned as mulch supplements atmospheric N acquisition or substitutes for it during red-clover ( Trifolium pratense L .) regrowth. Methods Following the first cut, red clover received no N input (TP-0), mineral N as a readily available-N reference (TP-N), red-clover mulch (TP-T), or Italian ryegrass ( Lolium multiflorum Lam .) mulch (TP-L), with three replicate pots per treatment. After 55 d of regrowth, the proportion of aboveground N derived from the atmosphere (pNdfa) was estimated using the ¹⁵ N natural-abundance method with treatment-specific non-legume references. Results Aboveground dry matter and total N showed no detectable treatment differences (p = 0.835 and p = 0.840, respectively). In contrast, pNdfa declined from 63.0% in TP-0 to 41.5% in TP-N, 18.7% in TP-T, and 14.8% in TP-L (p < 0.001). Fixed-N accumulation was 0.771, 0.529, 0.223, and 0.190 g N pot ⁻¹ , respectively, whereas non-fixed N increased from 0.459 to 0.745, 0.948, and 1.028 g N pot ⁻¹ . Thus, N addition altered the source composition of the aboveground N pool much more strongly than its total size. Discussion Under mulch, lower atmospheric-N contribution coincided with greater accumulation of non-fixed N without a detectable increase in total aboveground N. These findings provide quantitative evidence that residue return can shift short-term N-source partitioning in regrowing red clover, with greater reliance on non-fixed N coinciding with reduced reliance on newly fixed atmospheric N.