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

Hansen, A. P. and Pate, J. S. 1987. Evaluation of the ¹⁵N naturalabundance method and xylem sap analysis for assessing N₂ fixationof understorey legumes in jarrah (Eucalyptus marginata Donnex Sm.) forest in S.W. Australia.—J. exp. Bot 38: 1446–1458. Nodulated seedlings of Acacia pulchella, A. alata and A. extensawere grown in glasshouse sand culture under a range of levels(0–16 mol m³) of nitrate, supplied as ¹⁵NO₃, or as unenrichedlaboratory grade nitrate (¹⁵N value 5·5%_o). Nitrate at8·0 mol m ³ or above was highly inhibitory to growthof all species. Using ¹⁵N dilution analysis of the ¹⁵N enrichedcultures to measure symbiotic dependency, it was shown that¹⁵N values of the parallel unenriched cultures increased innear linear fashion from close to zero in fully symbiotic plantsto values close to that of the supplied NO₃ in plants experiencingnitrate levels (4·0 mol m³ or above) inhibiting N₂ fixationby over 90%. Xylem sap analyses (0·4 mol m³ NO₃ treatments)showed asparagine as the major nitrogenous solute, relativelylittle spill-over of free nitrate, and no evidence of majorshifts in balance of amino compounds with increasing dependenceon nitrate. This essentially invalidated use of the techniqueas a field assay for N₂ fixation by the species. ¹⁵N values for total N of soil sampled at 64 widely distributedsites in jarrah forest ranged from – 2·15 to +5·4(mean +2·1). Comparable values for soil mineral N (NH⁺₄and NO₃) were +0·3 to + 14·2 (mean +5·1).¹⁵N values of the total plant N of the legumes and of non-N₂-fixingreference species were also highly variable between sites, withlittle evidence of reference plant N accurately reflecting the¹⁵N abundance of soil nitrogen, or of visibly well nodulatedlegume components showing consistently lower ¹⁵N values thantheir companion reference plants. At one site it was possibleto compare ¹⁵N values of first season seedling legumes withpreviously published estimates of their progressive N₂ fixationusing C₂H₂ reduction assays. It was concluded that heterogeneity in ¹⁵N discrimination ofsoil within the ecosystem precluded effective use of the ¹⁵Nnatural abundance technique for assessing legume N₂ fixation. Key words: Acacia spp., ¹⁵N natural abundance,, xylem sap analysis,, nitrogen fixation.