Bedard-Haughn et al. (2003) reviewed the use of 15N as a tracer for N cycling in landscapes. They concluded that natural 15N abundance can provide valuable estimates of the contribution of different sources or identify variation in landscape processes. Unfortunately, the large 15N spatial and temporal variabilities can limit source discrimination by masking sources' differences, whereas 15N-enriched materials are widely accepted as tracers. The use of N-enriched materials is limited by their excessive cost, and, in natural ecosystems, by possible disturbances due to an uncommon introduction of N in the environment. Overall, it has been recommended the d15N approach in semi-quantitative studies of N patterns to generate new hypotheses on N cycling, while the 15N-enriched method can be used to quantitatively test hypotheses (Bedard-Haughn et al. 2003).
The 15N tracer technique has been widely applied to estimate the leguminous N2 fixation in natural and agricultural ecosystems (Bedard-Haughn et al. 2003; Shearer and Kohl 1993). Both the d15N- and the 15N-enriched methods can be applied.
The NAM can be easily applied when the soil 15N abundance available for plant uptake is sufficiently different from atmospheric N2. The isotope effect associated with N2 fixation usually alters the 15N abundance of atmospheric N2 by no more than 2% (Bedard-Haughn et al. 2003; Shearer and Kohl 1993). Moreover, due to isotope discrimination, the 15N abundance of plant-available soil N is different from soil total N. For a correct quantification, a non-N2-fixing reference plant can be grown on the same soil and its 15N abundance analysed. Similarly, isotopic discrimination during N2 fixation can be assessed by growing a legume hydroponically (Shearer and Kohl 1993).
The 15N-enriched method can be applied following two different procedures depending on the objective of the research experiment (1) growing the legume in an 15N2-enriched atmosphere or (2) on an 15N-enriched soil after fertilization. If an 15N2-enriched atmosphere is selected, plants fix N2 with a greater 15N content than that soil. The extent to which soil N is diluted by this 15N enrichment in a fixing plant reflects the magnitude of fixation. The 15N2-enriched atmosphere method has limitations in terms of being (a) technically difficult (to prevent leaks and maintain normal environmental conditions); (b) a short-term kinetic measurement that is not useful for long-term integrated quantification of N2 fixation (Warembourg 1993).
The 15N-enriched fertilizer method, also known as 15N isotope dilution method, consists in adding 15N to soil. Unlike the 15N2-enriched atmosphere method, plants absorb N from soil containing more 15N content than atmosphere. The 15N dilution of atmospheric N reflects the magnitude of fixation. The main limitation is the need to estimate the 15N abundance of the N uptake by plants from soil through the analysis of 15N abundance in a reference plant. This kind of quantification is necessary since the addition of a labelled fertilizer makes difficult to assume a uniform mixing with soil N.
Various calculations are available when natural abundance or 15N isotope dilution methods are applied. When a reference plant is used, the following model is applied (Shearer and Kohl 1993; Hauggaard-Nielsen et al. 2003, 2009):
„ d15N reference plant — d15N fixing plant
d15N reference plant — d15N fixing plant grown hydroponically x 100.
A simplified calculation for the isotope dilution method accounts for the isotope discrimination (Warembourg 1993):
%Nf N atom% 15N fixing plant ^^ atom%15N reference plant
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