Effect of reference plant on estimation of nitrogen fixation by subterranean clover using 15N methods

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Abstract
Subterranean clover (Trifolium subterraneum L.) was grown in separate associations with annual ryegrass (Lolium rigidum Gaud.) and with phalaris (Phalaris aguatica L.) in reconstituted soil profiles (0-400 mm depth), and N2 fixation was measured by 15N isotope dilution and 15N natural abundance methods. In all experiments, two values were determined, viz. P, the percentage of clover nitrogen (N) iixed from atmospheric N2, and R, the ratio of N assimilated from added 15N-labelled N to N assimilated from indigenous soil N. Estimates of P that were negative were obtained for the clover/phalaris association, using 15N isotope dilution, during the first 16 days after 15N addition. This was due to the R value being higher for clover than for phalaris, which in turn was due to differences in their temporal pattern of growth interacting with a declining ISN enrichment of the plant-available soil N. The R values for clover and ryegrass, when grown together, were similar throughout the experiment, and estimates of P by 15N isotope dilution and natural 15N abundance were similar. The activity of plant roots in different soil layers was examined by injecting a solution of 15N-labelled nitrate at 50, 150 and 300 mm depths. Uptake of 15N was similar at all depths for phalaris and clover, whereas ryegrass assimilated a greater amount at 150 and 300 mrn. However, since the amounts of roots and indigenous plant-available soil N were small in the 100-400 mm zone relative to the 0-100 mm zone, the greater activity of ryegrass roots at depth had no significant effect on the estimates of P. In both plant associations, the estimates of P, by both of the 15N methods, increased with time, and were higher in the clover/ryegrass association than with clover/phalaris. Since this was associated with lower levels of inorganic soil N in the clover/ryegrass association, it must be recognized that the reference plant can induce real changes in P by influencing the soil N status, in the association, as well as causing erroneous estimates of P by the 15N isotope technique.