Measuring N₂ Fixation in Legumes Using ¹⁵N Natural Abundance: Some Methodological Problems Associated with Choice of Reference Plants

Nodulated legumes contribute significantly to the N economy of soils and, in so doing, increase agricultural yields of food crops (Dakora and Keya, 1997). The amount of N fixed in symbiotic legumes by root-nodule bacteria (species of Rhizobium, Bradyrhizobium, Allorhizobium, Agrorhizobium, Mesorhizobium and Sinorhizobium) has generally been assessed under field conditions using the ¹⁵N natural-abundance technique and/or the N difference method. Although the ¹⁵N natural-abundance technique has been used successfully for field measurements of N₂ fixation in cultivated food grain and pasture legumes, the method is beset by problems associated with the choice of reference plants, especially in natural settings. When using this method, the N derived from atmospheric nitrogen fixation (%Ndfa or P_fix) is usually estimated (Shearer and Kohl, 1986) as:

$$P_{fix}=\frac{{\delta }^{15}{N}_{\mathrm{ref}}-\delta N_{\mathrm{leg}}15}{\delta N_{\mathrm{ref}}-\mathrm{Bvalue}15}\times 100$$

where δ¹⁵N_ref is the mean δ¹⁵N value of the reference plant, δ¹⁵N_leg is the mean δ¹⁵N value of the test legume, and the B value is the δ¹⁵N of the legume wholly dependent on N₂ fixation for its N nutrition. The B value represents the value of atmospheric N₂ and usually incorporates the isotopic fractionation associated with N₂ reduction in root nodules (Shearer and Kohl, 1986).