B value and isotopic fractionation in N₂ fixation by chickpea (Cicer arietinum L.) and faba bean (Vicia faba L.)

A prerequisite for the calculation of the proportion of a legume's nitrogen derived from the atmosphere (%Ndfa) using the ¹⁵N natural abundance technique (NA) is the determination of the B value (δ¹⁵N value for a legume when completely dependent on N₂ fixation for growth). With this objective, Kabuli-type chickpea seeds were inoculated with Mesorhizobium ciceri, strain ISC-6, and an indigenous strain mixture (ISM) from the Guadalquivir Valley (southern Spain); and mayor-equina-type faba bean seeds were inoculated with Rhizobium leguminosarum strain ISL-19. Both legumes were grown in perlite with an N-free nutrient solution and in a growth chamber; uninoculated plants were also grown under the same conditions. Samples of the inoculated plants were collected at full flowering and maturity, analyzing total N and ¹⁵N content of the nodules, root, shoot and seeds. The seeds sown and uninoculated plants at wilting were also analyzed. The uninoculated chickpea and faba bean plants showed strong ¹⁵N discrimination in the remobilization of reserve proteins from the cotyledons to the shoot. Only 52% of the ¹⁵N from the chickpea seed and 46% from the faba bean seed were translocated to the shoot. The Bwhole plant value for both inoculated species (−1.44‰ in chickpea and −1.33‰ in faba bean) demonstrated the existence of a significant isotopic discrimination of N in the fixation of atmospheric N₂. The Bshoot value did not show significant differences between full flowering and seed maturity for either species, which allowed field sampling to be performed for the calculation of %Ndfa between both growth stages. Each species presented different ¹⁵N discrimination for the different parts analyzed. In chickpea, strong discrimination was observed between the nodules (+3.11‰) and the rest of the plant (−1.67‰); this discrimination was less pronounced in faba bean and always with negative values between the underground parts (nodules and root, with a mean value of −0.75‰) and the shoot (−1.71‰). The Bshoot value obtained was −1.80‰ in chickpea and −1.71‰ in faba bean. However, given the importance of the amount of seed N in chickpea and faba bean, seed ¹⁵N contribution should be considered in the calculation of the Bshoot value using a mass balance model which considers 50% of this amount. In this case, the adjusted Bshoot values would increase to −2.05‰ and −1.89‰ in chickpea and faba bean, respectively.