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Development of a novel approach involving culturomics coupled to MALDI - TOF mass spectrometry for the characterization of soil fungal communities and comparison to metabarcoding analysis.

Charbel Elias, Laure Malleret, Stéphane Ranque
LCE, VITROME, FRANCE

Fungi are key actors in soil ecology and emerge as potential bioremediation agents. Yet, knowledge regarding soil fungal diversity and community structure remains relatively scarce. This gap is partly due to both extended fungal diversity and limitations of current fungal identification methods. While classical microbiology methods are inadequate in a high-throughput laboratory workflow, metagenomics, which is the current approach used in environmental sciences, is limited by within-fungal species genetic heterogeneity, the relatively high proportion of unidentified nucleotide sequences obtained from fungi that are absent from the reference nucleotide databases, and the detection of nucleotide sequences obtained from "non-viable" species. Culturomics is a high-throughput microorganisms' culture method, coupled to Matrix Assisted Laser Desorption Ionization - Time of Flight mass spectrometry (MALDI – TOF MS), that aims to comprehensively identify strains or species in biological samples. Initially applied to the study of the human gut bacterial community structure, this approach can be theoretically extended to the analysis of any ecological niche. We aim at applying this pioneering approach in environmental sciences to characterize polluted soil samples fungal community structure.
We analyzed the fungal communities of characterized soil samples contaminated with Polycyclic Aromatic Hydrocarbons (PAHs). Samples were suspended in PBS, diluted, and plated onto five culture media. Fungal colonies were identified via MALDI-TOF MS and/or DNA barcode sequencing. We compared the fungal diversity indices and community structure, assessed via culturomics or ITS metabarcoding.
In total, 49 fungal species were isolated and identified from PAH-contaminated soil samples displaying different pretreatment profiles. The isolated strains belonged to 18 distinct genera of the Ascomycota and Mucoromycota divisions. The species Aspergillus felis, Aspergillus niger, Aspergillus udagawae, and Curvularia inaequalis, were strongly associated with PAH contamination, making them good candidates for remediation processes. In addition, a possible novel species of the genus Mortierella was cultured and identified through this new approach.
These encouraging results establish the proof of concept and validate the feasibility of culturomics-based soil fungal community characterization. Culture–based approaches are central to isolating and characterizing native strains from contaminated sites, with the aim to further assess their bioremediation potential following a nature-inspired engineering strategy. Novel species identified through this approach will extend our MALDI-TOF MS reference spectra database, thus contributing to filling the knowledge gap of the metagenomics approach.