Technical note: Methodological choices influence root carbon exudation measurements

Root exudation is a significant pathway for belowground carbon (C) allocation in forest ecosystems, with profound implications for soil processes, nutrient cycling, and overall ecosystem functioning. Despite its importance, quantifying root exudation from mature trees in situ remains technically challenging, and methodological inconsistencies among studies hinder the synthesis and upscaling of findings. Here, we evaluated how variations in commonly used exudate collection protocols influence measured C fluxes. Specifically, we tested the effects of root resting, trap moisture, and trap solution composition on exudation rates in two contrasting ecosystems: a temperate forest in Germany and a Mediterranean forest in Israel. By incorporating both inter- and intraspecific root combinations, we also accounted for potential species-interaction effects.Our results highlight several methodological sensitivities. Omitting root resting can streamline sampling. Moisture conditions within cuvettes strongly affect flux estimates, with saturated traps yielding higher values than moist traps. Exudation responses were further influenced by soil phosphorus availability in the trap solutions, with elevated root C exudation under P-deficiency.Together, these findings emphasize that methodological variation can substantially alter root exudation C flux rates. We conclude that while some streamlining of protocols is feasible, careful attention to incubation procedures and the use of second flush samples yield more reliable results. Standardized approaches- or, at a minimum, transparent and detailed reporting - are essential to improve comparability across studies. Addressing these methodological challenges will allow more accurate quantification of root exudation, strengthen its integration into terrestrial C models, and ultimately refine our understanding of belowground C allocation under global change.