Disentangling hyphal- and root-derived contributions to dissolved organic carbon in mixed tree systems

Soils represent the largest terrestrial reservoir of organic carbon, with dissolved organic matter (DOM) acting as its most mobile and reactive fraction and the immediate precursor to mineral-associated organic matter, the dominant long-term carbon pool. While DOM dynamics have been extensively studied in bulk soil and the rhizosphere, the hyphosphere—soil influenced by fungal hyphae—remains comparatively understudied, despite the extraordinary spatial reach, rapid turnover, and mineral surface interactions of mycorrhizal fungi. Disentangling root- versus hyphal-derived dissolved organic carbon (DOC) inputs is therefore critical for understanding how recent plant carbon is redistributed and stabilized in soils.

Here, we applied a nested ingrowth core system to experimentally separate rhizosphere and hyphosphere DOC pools under semi-controlled greenhouse conditions. The system consisted of an outer mesh core permitting root and hyphal access and an inner fine-mesh core allowing hyphal ingrowth only, both filled with inert sand. Ingrowth cores were installed in pots containing native tree species planted in monocultures and mixtures. At harvest, distinct sand fractions representing bulk sand, rhizosphere, and hyphosphere subsets were recovered and extracted for total organic carbon (TOC) analysis; samples are being further characterized using untargeted metabolomics.

Preliminary results indicate clear differences in TOC concentrations among compartments, with highest values in rhizosphere samples, intermediate values in the hyphosphere, and lowest concentrations in bulk sand. Species composition exerted a strong influence on total TOC concentrations, and root ingrowth into the outer cores varied markedly among species. Metabolomic analyses are currently in progress and will be used to further assess compositional differences between rhizosphere- and hyphosphere-derived DOC.

Together, this work highlights the hyphosphere as a distinct and experimentally accessible domain of DOC production and underscores the need to explicitly consider fungal pathways when linking fresh carbon inputs to persistent soil organic matter formation.