Culturable Fungi Recovered from Deep-Sea Sediments: Diversity, Phylogenetic Placement, and In Vitro Antagonistic...

Deep-sea sediment-derived fungal isolates remain underrepresented in organized living collections for phenotype-based screening. We applied 12 complementary isolation protocols-dilution plating, ethylenediaminetetraacetic acid (EDTA) pretreatment, and plate stamping-to four deep-sea sediment materials (2595-6000 m) representing three sampling units from the Mariana Trench slope region and the South China Sea. After morphotype-based purification, isolates were characterized by internal transcribed spacer (ITS) closest-match analysis and phylogenetic placement; selected representatives were further screened against five phytopathogenic strains using dual-culture, culture filtrate, and volatile organic compound (VOC) assays. In total, 159 isolates were placed within Ascomycota, Basidiomycota, and Mucoromycota, representing 35 closest-match genus-level groups and 22 low-similarity ITS phylotypes (<95% similarity) preserved as living cultures. Cladosporium, Penicillium, and Aspergillus were most frequent. Recovered diversity profiles varied among sediment materials and protocols, with plate stamping capturing a large, compositionally distinct subset of the collection. In dual culture, all 23 tested isolates inhibited at least one pathogen, with maximum mycelial growth inhibition reaching 98.74%. Culture filtrate and VOC assays produced partially discordant activity profiles, highlighting assay-format-dependent effects. This study establishes an ITS-organized living fungal collection and provides a phenotype-based framework for targeted taxonomic, chemical, and biocontrol-oriented follow-up.