Estimating biological N2 fixation by a tropical legume tree using the non-nodulating phenophase as the reference in the 15N natural abundance method

The non-nodulating phenophase of a legume tree was tested as a non-N₂-fixing reference in application of the ¹⁵N natural abundance method for estimating the N₂ fixation. We applied this method to study the effects of three pruning intensities, complete pruning every 6 months (T-6), ca. 50% pruning every 3 months (P-3) and intact control (C), on N₂ fixation in Erythrina lanceolata (Papilionaceae) planted as shade and support trees for vanilla (Vanilla planifolia) in a subhumid tropical site in Quepos, Costa Rica. We measured nodulation and N₂ fixation for 12 months. The trees under the C regime nodulated abundantly during the rainy season vegetative growth but did not nodulate during the dry season and flowering. A linear regression (r²=0.76) was observed between the nodule biomass and δ¹⁵N values under the C regime, suggesting quite a stable specific N₂ fixation rate. The stable δ¹⁵N values throughout the year in the non-N₂-fixing Morus nigra (Moraceae) growing on the same soil indicated that the temporal variation in the plant available soil ¹⁵N was low. We used the intercept term of the regression (δ¹⁵N=3.5 when nodule biomass was 0) as the non-N₂-fixing δ¹⁵N value when estimating the percentage of N fixed from atmosphere out of tree total N. The percentage varied from 0 during the driest period to 53% during rainy season. Pruning reduced rainy season nodulation under the T-6 and P-3 regimes almost to nil, and the δ¹⁵N values were high. Our results suggest that the conditions for using the non-nodulating phenophase as the non-N₂-fixing reference required in the ¹⁵N natural abundance method were fulfilled. The C trees showed a clear phenological cycle in N₂ fixation, while pruning severely disturbed the N₂ fixation under the T-6 and P-3 regimes, indicating that E. lanceolata is better suited for agroforestry systems in which the trees are not managed by periodic prunings.