Use of different plant parts to study N 2 fixation with 15 N techniques in field‐grown red clover ( Trifolium pratense )

Red clover, Trifolium pratense L., is the dominant forage legume in Sweden and is usually harvested twice per year, once in June and once in August. Two 15 N‐based methods – 15 N isotopic dilution (ID) and 15 N natural abundance (NA) – were used to study N 2 fixation from spring until first harvest in late June, from first to second harvest in late August, and from second harvest until first frost in autumn in Umeå, Sweden. The material studied comprized three neighbouring fields carrying a first year ley, a second year ley and a third year ley. For the 15 N ID method, small amounts of highly enriched 15 N‐nitrate were added to experimental plots. The non‐legumes in the plots, essentially Phleum pratense L. together with Festuca pratensis L., served as reference plants for both the ID and 15 N NA measurements. Dry matter, N and 15 N were separately analysed in leaves (laminae), stems (including petioles), stubble and roots. The proportion of N derived from air (pNdfa) was then calculated for each plant part and for whole plants. Estimates of the proportion of N derived from N 2 fixation (pNdfa) were always very high, usually ≥0.8. Generally, estimates of pNdfa obtained by the ID and NA methods were similar, but the ID method gave higher estimates of pNdfa than the NA method when the highest N 2 fixation levels were recorded, at the August harvest. Regression analyses suggest that estimates of pNdfa in leaves could provide useful indications of pNdfa in shoots and whole T. pratense plants, thus avoiding the need for time‐consuming root analyses.