Abstract

This paper has been written in the context of the Italian national research project (PRIN) "Physiology of production and nitrogen fixation of forage legumes in eco-compatible farming systems", with the aim of analysing the state of the art on the main methods to estimate symbiotic nitrogen fixation of legumes in the field. Assumptions, procedures, advantages and limits of the following methods are illustrated: nitrogen balance (NB), acetylene reduction assay (ARA), ¹⁵N₂ isotope reduction (IR), ¹⁵N isotopic dilution (ID), ¹⁵N natural abundance (NA) and xylem sap analysis. Some applications on the estimate of below-ground nitrogen of legumes and the implications on rotations and mixtures are also described.
The developrnent of methods based on the nitrogen isotopic discrimination, particularly ID and NA methods, have substantially contributed to overcome the limitations of old methods such as ARA and NB. However, new methods can be misused if fundamental assumptions are not satisfied, such as the time-space uniformity of soil ¹⁵N enrichment and the similar N uptake dynamics of the fixing and reference species. Modelling approaches can help to mitigate the assumption violations.
It is not available a standard methodology to assess the below-ground fixed mtrogen of any legume species, as specific procedures should be developed for each species and experimental site. As this is a relevant point from an agronomic perspective, further research is needed to assess below-ground nitrogen of legumes and the amount of symbiotic fixed nitrogen which enters the nitrogen cycle. Particular attention should also be deserved to the development of adequate methods to exploit the results of the field experiments involving main stakeholders of the legume-based production systems.

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