Evaluation of the15N Natural Abundance Method and Xylem Sap Analysis for Assessing N2Fixation of Understorey Legumes in Jarrah (Eucalyptus marginataDonn ex Sm.) Forest in S.W. Australia

Hansen, A. P. and Pate, J. S. 1987. Evaluation of the 15N natural abundance method and xylem sap analysis for assessing N2 fixation of understorey legumes in jarrah (Eucalyptus marginata Donn ex Sm.) forest in S.W. Australia.—J. exp. Bot 38: 1446–1458. Nodulated seedlings of Acacia pulchella, A. alata and A. extensa were grown in glasshouse sand culture under a range of levels (0–16 mol m⊟3) of nitrate, supplied as 15NO⊟3, or as unenriched laboratory grade nitrate (δ15N value 5·5%o). Nitrate at 8·0 mol m ⊟3 or above was highly inhibitory to growth of all species. Using 15N dilution analysis of the 15N enriched cultures to measure symbiotic dependency, it was shown that δ15N values of the parallel unenriched cultures increased in near linear fashion from close to zero in fully symbiotic plants to values close to that of the supplied NO⊟3 in plants experiencing nitrate levels (4·0 mol m⊟3 or above) inhibiting N2 fixation by over 90%. Xylem sap analyses (0·4 mol m⊟3 NO⊟3 treatments) showed asparagine as the major nitrogenous solute, relatively little spill-over of free nitrate, and no evidence of major shifts in balance of amino compounds with increasing dependence on nitrate. This essentially invalidated use of the technique as a field assay for N2 fixation by the species. δ15N values for total N of soil sampled at 64 widely distributed sites in jarrah forest ranged from – 2·15 to +5·4 (mean +2·1). Comparable values for soil mineral N (NH+4 and NO⊟3) were +0·3 to + 14·2 (mean +5·1). δ15N values of the total plant N of the legumes and of non-N2-fixing reference species were also highly variable between sites, with little evidence of reference plant N accurately reflecting the 15N abundance of soil nitrogen, or of visibly well nodulated legume components showing consistently lower δ15N values than their companion reference plants. At one site it was possible to compare δ15N values of first season seedling legumes with previously published estimates of their progressive N2 fixation using C2H2 reduction assays. It was concluded that heterogeneity in 15N discrimination of soil within the ecosystem precluded effective use of the 15N natural abundance technique for assessing legume N2 fixation.