Selection of Reference Plants and Methods for Estimating N2 Fixation in Mycorrhizal Leguminous Plants

Abstract

AbstractThe effects of plant species and mycorrhizae on reference plant suitability were examined with regard to estimating N2 fixation in two leguminous plants via the 15N isotope dilution technique (ID). Atom percent excess (A % E) 15N values of ryegrass (Lolium perenne L. ‘PennSinn’), nonnodulated big trefoil (Lotus pedunculatus Cav. ‘Marshfield’), and nonnodulated subterranean clover (Trifolium subterraneum L. ‘Mt. Barker’), each with or without mycorrhizae, were compared at fertilizer N rates of 10 and 100 kg N ha−1. Precision of the ID method was compared to the total nitrogen difference (ND) method. Atom percent excess 15N values of the reference plants had lower coefficients of variation than total N yields (mg plant−1), and were unaffected by mycorrhizal treatment. Overall shoot values of A % E 15N were similar in trefoil and ryegrass, but were lower in clover in the 100 kg N ha−1 fertilizer treatment. In roots, the A % E 15N values for clover were significantly larger than those for ryegrass. There were no differences between the values of N derived from the atmosphere (Ndfa) (percent and amount for shoot, root, and total plant) in clover, as estimated by the ID method, using nonnodulated clover and ryegrass as reference plants. Values for Ndfa (percent and amount), as estimated by the ND method, differed significantly from the ID values. The ND values were either smaller or larger than ID values, depending on N rate and inoculation treatment. In the 100 kg N ha−1 rate, the ND method resulted in negative values for root Ndfa. Given the similarity of A % E 15N shoot values in all three reference plants, and the insensitivity of Ndfa to the reference plant used for its calculation, ryegrass was found to be an appropriate reference plant for determining N2 fixation via the ID method in both big trefoil and subterranean clover.