A culturomics-inspired multi-medium workflow for soil fungal isolation and ITS metabarcoding comparison

This method article describes a reproducible workflow for recovering, identifying, and comparing soil-associated fungi using complementary culture-dependent and sequence-based approaches. The workflow was designed for environmental mycology studies in which fungal DNA detection needs to be complemented by the recovery of living isolates. It combines standardized soil suspension processing, parallel plating on five culture media, daily morphotype selection and purification, MALDI-TOF MS screening, targeted multilocus sequencing when required, and comparison with ITS1/ITS2 metabarcoding profiles. In its application to soil samples, the workflow recovered a diverse viable isolate collection, supporting its usefulness for expanding fungal recovery, improving lower-level taxonomic anchoring, enriching identification resources, and distinguishing culture-recovered from sequence-detected fungal fractions.•Five culture media are used in parallel to broaden the spectrum of viable soil fungi recovered under standardized incubation conditions.•Recovered isolates are identified using MALDI-TOF MS and targeted multilocus sequencing when necessary.•Culture-recovered taxa are compared with ITS1/ITS2 metabarcoding profiles to distinguish shared and method-specific fungal fractions.