Estimation of the amount of root exudates and simultaneous identification of their composition in non‐sterile soil is a challenging objective in rhizosphere research. We coupled 3 methods: (1) labeling of corn in 14 CO 2 atmosphere to separate root‐derived and soil‐derived organic substances in the rhizosphere, (2) a previously developed leaching method to collect rhizodeposits, and (3) pyrolysis field ionization mass spectrometry (Py‐FIMS) to investigate the molecular‐chemical composition of rhizodeposits. Eluted rhizodeposits accounted for 2.8 % (Loam) and 0.97 % (nutrient solution in quartz sand) of recovered 14 C and showed clear differences in composition between the growth substrates. The 14 CO 2 evolved mostly by root respiration accounted for 3.5–4.0 % without significant differences according to growth substrate or diurnal dynamics. Principal component analysis of the Py‐FI mass spectra of leachates showed a clear diurnal dynamics of the amount and the composition of corn rhizodeposits collected during day‐time and night‐time. Differences originated mostly from signals assigned to carbohydrates, sterols, and peptides. This approach is recommended for forthcoming studies of rhizodeposition in different soil substrates, crops grown, and time‐series of exudate sampling.
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