Application of N-15 and xylem ureide methods for assessing N-2 fixation of three shrub legumes periodically pruned for forage

The apparently diminished capacity for N-2 fixation by the shrub legume Calliandra calothyrsus (Calliandra) relative to other woody perennial legumes was investigated in a field experiment in northern Queensland, Australia. In this trial, (i) the proportion of plant nitrogen (N) derived from symbiotic N-2 fixation (%Pfix) and the amounts of N-2 fixed were compared in Calliandra, Gliricidia sepium (Gliricidia) and Codariocalyx gyroides (Codariocalyx), (ii) variations in N-2 fixation due to season or tree age were determined, (iii) estimates of Pfix derived with the N-15 natural abundance technique were compared with values obtained from N-15 enrichment or xylem sap ureide procedures to determine whether the previous conclusions about Calliandra's ability to fix N had resulted from specific problems with the natural abundance methodology used in the earlier studies. Inoculated seedlings of each of the three shrub legume species were planted in dense stands (1.5 m rows, 0.5 m between trees) in two randomised blocks. The northern block was used solely for natural abundance measurements, while N-15-enriched KNO3 (10 atom % N-15 excess) was applied four times over a 52 week period to plots in the southern block. The non-nodulating tree legume Senna spectabilis (formally Cassia spectabilis) was used as a non-N-2-fixing reference for the N-15-based procedures, with Guinea grass (Panicum maximum) included as an additional non-fixing check. Growth by the trees above 75 cm was first cut and removed after 22 weeks and regrowth was subsequently pruned periodically for another 95 weeks. Sampling for dry matter production, N yield and estimates of Pfix were restricted to the central four of the 32 plants which constituted each replicate plot. Information generated during the 117 week study indicated that estimates of Pfix by N-15 natural abundance were closely similar to values derived with N-15-enrichment or sap ureides. The data indicated that Calliandra had a reduced reliance upon N-2 fixation relative to Gliricidia and Codariocalyx for the first 65 weeks after establishment. This appeared to be due to more prolifc root growth by Calliandra than either of the other Nz-fixing species and an ability to extract a greater proportion of its N requirements from soil mineral N. However, after week 65 and for the remainder of the experiment, estimates of Pfix for Calliandra were similar to the other shrub legumes. Over 117 weeks, prunings from Calliandra and Gliricidia had removed 52-58 t dry matter ha(-1), and between 1471 and 1678 kg N ha(-1), of which 1026-1063 kg N ha(-1) was estimated to have been derived from N-2 fixation. At the time of final harvest, 65-73% of the fixed N was present in shoot regrowth of the N-2 fixing shrubs, 9-18% in the roots, 15% in the trunk, and 2-6% in fallen leaves.