An overview of the role and significance of 15N methodologies in quantifying biological N2 fixation (BNF) and BNF dynamics in agro-ecosystems

Quantitative estimates of BNF are needed to improve our understanding of the ecology of N in the environment and aid efforts to improve agricultural N management. Static models based on the principle of 15N isotope dilution have been proposed to estimate the proportion of N in a N2-fixing species that is derived from the atmosphere via biological N2 fixation. Furthermore, equations have been developed to quantify the movement of biologically fixed N between neighboring species or from legumes to cereals in crop rotations. The present paper is structured to provide a comprehensive overview of these methods in a logical and systematic manner. While the relevant literature is vast, some aspects have fortunately been covered by recent in-depth reviews which will be identified and briefly summarized. The overview will emphasize the more practical indirect methodologies based either on artificial 15N enrichment or 15N depletion, or alternatively on 15N natural abundance. In considering methods used to estimate symbiotic dependence, the major structural division is whether or not a non-N2-fixing reference plant is employed, and approaches taken to remove this source of error are described. Four examples are provided to illustrate the contemporary success of 15N-based methods, one in basic research involving endophytic BNF, and three in applied research involving legume breeding for enhanced BNF, the response of legumes to climate change and biotic and abiotic factors affecting legume symbiotic performance