Use of in situ 15 N-labelling to estimate the total below-ground nitrogen of pasture legumes in intact soil–plant systems

A leaf-feeding technique for in situ 15N-labelling of intact soil–pasture plant systems was assessed, using subterranean clover ( Trifolium subterraneum L.) and serradella ( Ornithopus compressus L.) grown under glasshouse conditions. Total recoveries of fed 15N were 87–100% following leaf-feeding of plants at flowering but were lower (74–84%) following the feed at the vegetative stage. Below-ground recovery of fed 15N ranged from 7 to 26%, with serradella partitioning a greater proportion of labelled N below ground than subterranean clover. Additionally, plants of both species fed at the vegetative stage accumulated a greater proportion of the 15N label below ground than did those fed at flowering. Dry sampling procedures, which utilised freeze-drying, enabled fractionation of the below-ground portion of the system into ‘clean’ nodulated macro-roots with no adhering soil, residual uncleaned root, rhizosphere, and bulk soil. Calculated specific enrichment for the ‘clean’ roots at different depths demonstrated a relatively uniform distribution of 15N label in the subterranean clover roots, whereas the presence of large indeterminate nodules in the crown region of serradella roots contributed to apparent uneven distribution of label. Approximately half of the N in the residual fraction of both species consisted of labelled material, postulated to be mostly fine root. Additionally, 5–20% of the rhizosphere N and 0·5–3% of the N in bulk soil was legume root-derived, with some 15N detected in the extractable total soluble N and microbial N pools. Rhizodeposition of N represented approximately 10% of total plant N and 17–24% of total below-ground N for subterranean clover, whereas values for serradella were 20 and 34–37%, respectively. Estimated total below-ground N of subterranean clover reached a maximum value of 177 mg N/plant at 98 days after sowing, which corresponded with a peak shoot N of 243 mg N. Maximum below-ground N for serradella attained 196 mg N/plant 84 days after sowing with a corresponding shoot biomass of 225 mg N. There was a decline in the total below-ground N of serradella at maturity. Overall, recovered clean root N represented 30–62% of estimated total below-ground N, so it was concluded that standard root recovery procedures might be likely to underestimate severely the total below-ground N accretion and N turnover by legumes. The implications of these results for field estimation of total legume N yield, biological N fixation, and the N benefit from legumes in rotations are discussed.