Biological nitrogen fixation by two tropical forage legumes assessed from the relative ureide abundance of stem solutes: N-15 calibration of the technique in sand culture

The use of the relative ureide abundance (RUA) in the sap of mainly tropica l ureide-producing legumes as a means to estimate the contribution of biolo gical nitrogen fixation (BNF) is potentially an useful technique as it does not require the use of reference plants or additions of N-15-labelled fert ilizer, and the analyses necessitate only relatively simple equipment. Howe ver, one problem in the application of the technique arises from the diffic ulty of obtaining sap samples from such legumes, especially small-stemmed f orage legumes under field conditions. This study was conducted to investiga te the possibility of using RUA in hot-water extracts of the stems of two f orage legumes, Desmodium ovalifolium and a Centrosema hybrid, to estimate t he contribution of BNF. In this case only ureide and nitrate are analysed t o calculate RUA (100 x ureide-N/(ureide-N + nitrate-N)). The technique was calibrated with the N-15 isotope dilution technique in sand culture where t he plants were fed with 5 different levels of nitrate (0, 12.5, 25, 50 and 100 mg N pot(-1)). Despite the fact that in many stem extracts more than 90 % of the N was neither nitrate or ureide, the colorimetric techniques utili sed proved reliable and relatively immune to interference from other solute s in the extracts. One problem with the use of the N-15 dilution technique to calibrate the RUA technique is that the former gives an integrated estim ate of the BNF contribution since planting (or between harvests) and the la tter is a point estimate at the time of sampling. This was overcome by usin g a 'plant to plant simulation technique' where estimates of BNF are calcul ated from the daily accumulation of total N and the labelled N derived from the growth medium by the legumes using a curve-fitting strategy. These est imates of BNF for the days when stem extracts were analysed for nitrate and ureide showed linear correlations (r(2) = 0.82 and 0.90 for the D. ovalifo lium and Centrosema hybrid, respectively). This indicated that RUA of stem extracts of these two legumes was a reliable indicator of the BNF contribut ion, at least under controlled conditions.