15N tracer chemical form underlies divergent estimates of symbiotic N2 fixation and nitrogen transfer

Abstract The 15N tracer technique is pivotal for quantifying nitrogen (N) dynamics in intercropping, but the assumption that results are independent of the tracer’s chemical form remains untested. We demonstrated that the choice of tracer form (NO3– vs. NH4+) systematically affected quantitative estimates of both symbiotic N2 fixation (SNF) and interspecific N transfer. In a pot experiment with maize and three leguminous green manures, the 15N-dilution technique revealed that the use of 15NH4+ as a tracer resulted in higher estimates of the proportion of N derived from the atmosphere (%Ndfa) by an average of 18.0% compared to 15NO3–. Concurrently, a 15N foliar labeling experiment showed that the tracer form assimilated by the donor plant strongly altered the observed interspecific transfer pattern: legumes transferred 2.2 times more N derived from 15NO3– than from 15NH4+ to maize, while maize transferred 1.6 times more N derived from 15NH4+ than from 15NO3– to legumes. This bidirectional transfer pattern can be best explained by the distinct biogeochemical behaviors of the two N forms and their divergent metabolic assimilation pathways within plants. Our findings exposed a critical, yet previously unquantified, methodological effect. We contend that the chemical identity of the tracer should be reported as a mandatory methodological parameter, as estimates are not absolute but represent methodology-dependent perspectives. This necessitates a critical reevaluation of data across studies, cautioning against direct comparisons of results obtained with different tracer forms.