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

Abstract

The use of the relative ureide abundance (RUA) in the sap of mainly tropical ureide-producing legumes as a means to estimate the contribution of biological nitrogen fixation (BNF) is potentially an useful technique as it does not require the use of reference plants or additions of 15N-labelled fertilizer, and the analyses necessitate only relatively simple equipment. However, one problem in the application of the technique arises from the difficulty of obtaining sap samples from such legumes, especially small-stemmed forage legumes under field conditions. This study was conducted to investigate the possibility of using RUA in hot-water extracts of the stems of two forage legumes, Desmodium ovalifolium and a Centrosema hybrid, to estimate the contribution of BNF. In this case only ureide and nitrate are analysed to calculate RUA (100 × ureide-N/(ureide-N + nitrate-N)). The technique was calibrated with the 15N isotope dilution technique in sand culture where the 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 utilised proved reliable and relatively immune to interference from other solutes in the extracts. One problem with the use of the 15N dilution technique to calibrate the RUA technique is that the former gives an integrated estimate of the BNF contribution since planting (or between harvests) and the latter is a point estimate at the time of sampling. This was overcome by using a `plant to plant simulation technique' where estimates of BNF are calculated 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 estimates 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. ovalifoliumand Centrosema hybrid, respectively). This indicated that RUA of stem extracts of these two legumes was a reliable indicator of the BNF contribution, at least under controlled conditions.