Estimating Symbolic Nitrogen Fixation by Trifolium subterraneum L. during Regrowth1

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Abstract
Two complementary experiments were conducted to determine whether symbiotic N2 fixation could be estimated by applying 15N technologies to Trifolium subterraneum L. organs that would remain after various grazing treatments. Both experiments analyzed monocultures and 50:50 mixtures of T. subterraneum L. and Bromus mollis L. planted at 2000 seeds/m2 in 15N‐supplemented Laughlin loam, a member of the fine‐loamy, mixed, mesic family of Ultic Haploxerolls. In one study, lysimeters were monitored through two growing seasons, while plant shoots were clipped once or three times during each growing season. In the other study, plants were grown in unleached pods under glasshouse conditions with various clipping treatments. Symbiotic N2 fixation was estimated by the 15N‐dilution method and compared with the value determined from a total N‐difference method that included leached N changes in the case of lysimeters. Clipping treatments had similar effects on both estimates of N2 fixation in clover monocultures when shoot 15N concentrations at the final harvest were used for the 15N‐dilution calculations. Shoot 15N values also produced estimates similar to the N‐difTerence method in clover‐grass mixtures harvested only at the end of the season, but such 15N‐dilution calculations consistently underestimated N2 fixation relative to the N‐difference method for clover‐grass mixtures clipped more than once. Using root or crown 15N concentrations to estimate N2‐fixation produced lower values than the N‐difference method in all clipping regimes and species compositions. A major difficulty in using either the 15N‐dilution technique or the N‐difference method to estimate N2 fixation in pastoral systems is that a measure of total reduced N harvested in the legume is required. This requirement can be met in clipping trials but not in continuous grazing studies. Efforts to calculate N2‐derived N in forage from 15N concentration in shoots, crowns, or roots remaining after previous clippings and reduced N in herbage of unclipped plants, a realistic technique for grazing studies, were inaccurate relative to the N‐difference method in this study. Thus one must conclude that although 15N technologies can estimate N2 fixation with the same relative accuracy as the N‐difference method in unclipped plants, neither technique can be applied with certainty when undetermined amounts of forage are removed by animals.