Use of the 15N natural abundance technique to quantify biological nitrogen fixation by woody perennials

Biological nitrogen fixation (BNF) associated with trees and shrubs plays a major role in the functioning of many ecosystems, from natural woodlands to plantations and agroforestry systems, but it is surprisingly difficult to quantify the amounts of N-2 fixed. Some of the problems involved in measuring N-2 fixation by woody perennials include: (a) diversity in occurrence, and large plant-to-plant variation in growth and nodulation status of N-2-fixing species, especially in natural ecosystems; (b) long-term, perennial nature of growth and the seasonal or year-to-year changes in patterns of N assimilation; and (c) logistical limitations of working with mature trees which are generally impossible to harvest in their entirety. The methodology which holds most promise to quantify the contributions of N-2 fixation to trees is the so-called 'N-15 natural abundance' technique which exploits naturally occurring differences in N-15 composition between plant-available N sources in the soil and that of atmospheric N-2. In this review we discuss probable explanations for the origin of the small differences in N-15 abundance found in different N pools in both natural and man-made ecosystems and utilise previously published information and unpublished data to examine the potential advantages and limitations inherent in the application of the technique to study N-2 fixation by woody perennials. Calculation of the proportion of the plant N derived from atmospheric N-2 (%Ndfa) using the natural abundance procedure requires that both the N-15 natural abundance of the N derived from BNF and that derived from the soil by the target N-2-fixing species be determined. It is then assumed that the N-15 abundance of the N-2-fixing species reflects the relative contributions of the N derived from these two sources. The N-15 abundance of the N derived from BNF (B) can vary with micro-symbiont, plant species/provenance and growth stage, all of which create considerable difficulties for its precise evaluation. If the %Ndfa is large and the N-15 abundance of the N acquired from other sources is not several delta(15)N units higher or lower than B, then this can be a major source of error. Further difficulties can arise in determining the N-15 abundance of the N derived from soil (and plant litter, etc.) by the target plant as it is usually impossible to predict which, if any, non-N-2-fixing reference species will obtain N from the same N sources in the same proportions with the same temporal and spatial patterns as the N-2-fixing perennial. The compromise solution is to evaluate the N-15 abundance of a diverse range of neighbouring non-N-2-fixing plants and to compare these values with that of the N-2-fixing species and the estimate of B. Only then can it be determined whether the contribution of BNF to the target species can be quantified with any degree of confidence. This review of the literature suggests that while the natural abundance technique appears to provide quantitative measures of BNF in tree plantation and agroforestry systems, particular difficulties may arise which can often limit its application in natural ecosystems.