**Background:** Plants release a wide variety of metabolites through their roots to shape rhizosphere processes, including nutrient mobilization, microbial communication, and defense. Hydroponic systems are commonly used for root exudate sampling due to ease of access, but it remains unclear how well these reflect exudates from soil-grown plants. Cover crops are valuable for sustainable agriculture, and understanding their root exudate profiles under realistic conditions is important for leveraging their beneficial traits.
**Methods:** Four cover crop species (white mustard, lacy phacelia, bristle oat, Egyptian clover) were grown both in the field (Asendorf, Germany; Stagnic-Cambisol, silty loam, pH 6.0-6.4) and in hydroponic culture (phytochamber, 16h light/8h dark, 25°C/20°C). Field plants were grown in 20 µm polyamide mesh bags to allow intact root recovery. Root exudates were collected after 29 days (hydroponics) or ~5 weeks (field) at comparable developmental stages. Field exudates were sampled for 2 hours in 1 L deionized water with Micropur® forte to suppress microbial activity; hydroponic exudates were sampled for 4 hours in 4.5 L deionized water. Total carbon was measured by elemental analysis. Primary metabolites (20 amino acids by UPLC-fluorescence, 3 sugars by enzymatic assays, 5 organic acids by IC-MS/MS) were quantified. Secondary metabolites were profiled by untargeted LC-ESI-Q-ToF-MS with feature detection and annotation against multiple databases. Statistical analyses included linear models, constrained correspondence analysis, and PCA.
**Key Results:** Total carbon recovery was significantly higher in hydroponics for all species: mustard showed ~27-fold more C, phacelia ~5-fold, oat ~2.5-fold, and clover ~4-fold compared to field conditions. However, chemical richness of secondary metabolites was higher in field-grown phacelia (2.4-fold) and clover (3.8-fold), while mustard showed 849 features in hydroponics vs. 207 in the field. Oat had similar feature counts under both conditions. The variance explained by cultivation condition (Var[C]) was higher for secondary metabolites (59-67%) than for primary metabolites (36-57%). PCA of secondary metabolites showed clear separation by species and cultivation condition, while primary metabolite patterns were less discriminative. Chemical classification of the top 100 features per species/condition revealed that lipids and lipid-like molecules were highly abundant in field samples across all species. Clover exudates were dominated by phenylpropanoids and polyketides (especially isoflavonoids like biochanin A, formononetin, genistein, kaempferol) regardless of cultivation condition. Mustard exudates were characterized by organoheterocyclic compounds, particularly indole derivatives. Oat exudates showed characteristic benzenoids (methylanthranilic acid-related compounds). Phacelia showed more diverse chemical classes, with field samples enriched in lipids and hydroponic samples enriched in organic acids and derivatives. At the compound level, the majority of detected metabolites were specific to either field or hydroponic conditions for each species.
**Clinical Implications:** This study demonstrates that hydroponic root exudate sampling provides a valid but incomplete picture of the metabolic complexity of root exudates from field-grown plants. For identifying broad metabolite superclasses characteristic of a species, hydroponic systems may be sufficient. However, for understanding ecologically relevant exudation patterns—particularly the release of lipids, terpenoids, and defense-related compounds that respond to soil conditions, microbial communities, and abiotic stress—field sampling is essential. The species-specific metabolite fingerprints identified (e.g., isoflavonoids in clover, indoles in mustard, benzenoids in oat) could serve as biomarkers for root activity in soil ecology studies. These findings have implications for designing cover crop mixtures and breeding programs aimed at improving nutrient cycling, pathogen suppression, and soil health through root exudate-mediated processes.