Influence of growth stage and seed nitrogen on B values and potential contributions to error in estimating biological N2 fixation using the 15N natural abundance method

Abstract

The 15N natural abundance technique is increasingly being used to biological nitrogen (N2) fixation by legume crops, but requires the determination of a ‘B value’ which describes the isotopic fractionation for a given plant species and strain of rhizobium. Most B values in the literature are derived for shoot tissue harvested at mid pod filling, when plant N content is generally at its maximum. However, if legumes are grown as green manure inter-crops and growth is terminated at earlier stages, to obtain accurate values for total N2 fixed growth-stage specific B values and correction for seed N contributions may be necessary. This study aimed to evaluate the magnitude of the impact of different values of the N content and 15N abundance of seeds and of B values on the estimates of the proportion of legume N derived from N2 fixation. We investigated whether B values at pod filling differ from those at earlier growth stages by growing chickpea (Cicer arietinum), white lupin (Lupinus albus), field pea (Pisum sativum) and two faba bean (Vicia faba) cultivars, in sand-culture in a glasshouse without N, and quantifying the δ15N values of nodules, roots and shoots at five growth stages including mid pod filling as a control. We then used simulations to investigate the degree of error associated with the use of growth stage-inappropriate or B values uncorrected for seed N on estimates of % N derived from atmosphere (%Ndfa). While the differences in B values between flowering and pod filling were only statistically significant at P < 0.1 for white lupin, there was a trend of declining B value with plant age for chickpea and faba beans. Simulations using data from large (Aguadulce) and small (Fiesta)-seeded faba bean cultivars indicated that the magnitude of potential errors in estimating %Ndfa using B values derived at inappropriate growth stages or uncorrected for seed N content were around 10% at early flowering in both faba bean cultivars and were negligible by late flowering (69 days after sowing). Where legumes are grown until flowering or beyond, the magnitude of potential error in estimation of %Ndfa from the use of B values derived at inappropriate growth stages or uncorrected for seed N content is relatively low compared to other sources of error encountered when employing the 15N natural abundance technique.