Biological nitrogen fixation estimated by 15N dilution, natural 15N abundance, and N difference techniques in a subterranean clover-grass sward under Mediterranean conditions

Pasture legumes are used as a source of biologically fixed N-2. Several methods have been used to evaluate the amount of N-2 fixed in mixed pastures, but none is considered clearly superior. In an Eutric Leptosol of southern Portugal, N-2 fixation by subterranean clover mixed with grasses was evaluated over 2 years and subjected to several cuts by N-15 dilution (ID), natural N-15 abundance (NA) and N difference (ND) techniques. The amount of fixed N-2 in subterranean clover determined by natural N-15 abundance with correction for isotopic fractionation ranged from 32 kg N ha(-1) in 1992/1993 to 37 kg N ha(-1) in 1993/1994. Assuming that no isotopic fractionation occurred during fixation (B = 0%), fixation capacities at around 80% of nitrogen derived from the atmosphere (Ndfa) were found by the natural N-15 abundance technique. However, with correction for isotopic fractionation during N-2 fixation (B = -1.13%), fixation capacities closer to 50% Ndfa were obtained, similar to the value obtained in the second-year experiment with the isotopic dilution method. In year 1, the fixation capacity, as estimated by the isotopic dilution method, was about 37% Ndfa. The nitrogen difference (clover N-grass N) calculations underestimated the amount of N-2 fixed in 1992/1993. This method assumes that both legumes and non-legumes absorb the same amount of N from the soil, which may not be true. Also, only the above-ground legume herbage was analyzed in this experiment. From the results, either ID or NA methods, particularly with a correction for the B-value, can be used to estimate N-2 fixation in mixed pastures, rather than the ND method. The natural N-15 abundance (NA) technique can be more versatile than the ID technique, allowing frequent sampling in undisturbed grassland ecosystems with reduced costs. Nitrogen fixation decreased in June, in both years, probably due to a lower soil water content and higher soil temperature. (C) 1999 Elsevier Science B.V. All rights reserved.