Changes in dinitrogen fixation in maturing stands of Casuarina equisetifolia and Leucaena leucocephala

Biological dinitrogen fixation in Casuarina equisetifolia J.R. & G. Forst. and Leucaena leucocephala (Lam.) de Wit was evaluated using the 15N-enrichment technique under field conditions in single-species and mixed-species plantings (with a nonfixing reference species, Eucalyptus x robusta J.E. Smith) between 1.0 and 3.5 years of age in Puerto Rico. Following periodic labelling of trenched and untrenched plantation quadrats with 15N-enriched ammonium sulfate, analyses of foliar and whole-tree (weighted average) N-isotopic ratios and total biomass N were used to estimate the proportion of nitrogen derived from biological dinitrogen fixation (PNDFA) and total nitrogen derived from fixation (TNDFA) in C. equisetifolia and L. leucocephala. The 15N-enrichment technique yielded accurate estimates of dinitrogen fixation in maturing stands of these two tree species provided the reference species (Eucalyptus) was grown in close proximity to the N-fixing species in trenched, mixed-species plots. Changes in the 15N/14N ratio of soil-available nitrogen in single-species plots of the N-fixing and reference were found to yield inaccurate estimates of dinitrogen fixation in the single-species plots of C. equisetifolia and L. leucocephala after 2 years of age. The results confirm earlier findings that foliar sampling is a useful nondestructive alternative to whole-tree biomass sampling for the 15N-enrichment protocol. Between 1.0 and 3.5 years after plantation establishment, PNDFA in C. equisetifolia remained relatively constant between 50 and 60%, while PNDFA in L. leucocephala declined from nearly 100% at 1 year to less than 40% at 3.5 years. The rate of dinitrogen fixation (kg.ha-1.year-1) did not decline as the stands matured. Cumulative dinitrogen fixation (TNDFA) estimates at 3.5 years were very similar between species: 73 in C. equisetifolia and 74 kg N.ha-1 year-1 in L. leucocephala.