Technical note: methodological choices influence root carbon exudation measurements

Root exudation is an important pathway of belowground carbon (C) allocation in forest ecosystems, with profound implications for soil processes, nutrient cycling, and ecosystem functioning. Despite its importance, quantifying root exudation from mature trees in situ remains technically challenging, and methodological inconsistencies among studies hinder synthesis and upscaling. In this technical note, we systematically 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 biomes: 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 reveal several methodological sensitivities. Omitting a root-resting phase can streamline sampling without compromising measurements. Moisture conditions within cuvettes strongly influenced flux estimates, with saturated traps yielding up to six-times higher values than moist traps. Root exudation was further affected by trap solution composition, with initially increased exudation under P-deficient solutions in both biomes. Roots showed an adaptive response to the syringe environment, particularly to differences in trap-solution composition and moisture conditions; with effects partially diminishing after 48 hrs when a second flush was collected. However, the magnitude and direction of these responses differed between biomes. Within our experimental framework, first-flush sampling using a standard trap solution provided the most consistent basis for cross-biome comparison of root exudation fluxes. Species mixing had no detectable effect on exudation rates. Together, these findings emphasize that methodological variation can substantially alter root exudation C flux rates. Standardized approaches – or, at a minimum, transparent and detailed reporting – are essential to improve comparability across studies. Addressing 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.