Effects of Nitrate and Ammonium on Nitrogenase (C₂H₂ Reduction) Activity of Swards of Subterranean Clover, Trifolium subterraneum L

Abstract

Small swards of subterranean clover plants were grown under controlled conditions without mineral nitrogen and allowed to establish an effective nitrogen fixation system. Nutrient solutions containing nitrate from 0 to 16 mM or of ammonium from 0 to 5 mM were then applied and changes in nitrogenase activity (NA) estimated by acetylene reduction assay (AR) and the rate of hydrogen evolution (HE) for periods of up to 35 days. In two experiments a split-root system was used to enable mineral nitrogen to be applied to only one-half of a nodulated root system whilst the NA of both halves was monitored.

NA by subterranean clover was very sensitive to exogenous mineral nitrogen, concentrations as low as 0.5 mM NO₃^- suppressing activity significantly, and 3-5 mM stopping it almost completely within 7 days. The degree of inhibition induced by concentrations between 0.5 and 3 mM NO₃^- was less at a photon irradiance of 1000 compared with 300 µmol quanta s^-1 m^-2 . Under some conditions NA continued at a reduced but steady rate in the presence of nitrate. NH₄⁺ also markedly depressed NA but a concentration greater than 5 mM was needed to effect the same response. After NO₃^- was applied to an active symbiosis, nitrate reductase activity increased as NA decreased. Our results do not support the hypothesis of a direct effect of NO₃^- on nitrogenase due to the accumulation of toxic NO₂^-. Although our results allow that assimilate might be diverted from the nodules after the application of NO₃^- thus reducing N₂-fixation, an alternative hypothesis is proposed: that nitrogenase and nitrate reductase work in a complementary manner in supplying reduced nitrogen to whole plants, and NO₃^- depresses N₂-fixation through a regulatory system involving the level of soluble nitrogen in the plant.

We conclude that nitrogen fixation by subterranean clover in the field may be depressed below its potential due to the presence of soil mineral nitrogen.