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.

Key words: ¹⁵N-dilution, ¹⁵N-foliar feeding, green manure, isotopic fractionation, methodological effect, nitrogen cycling, tracer chemistry