Isotope Labeling Reveals Complex Microbial Interactions during Agaricus bisporus Compost Colonization

Zenodo (CERN European Organization for Nuclear Research)

Abstract

Study abstract Microbial interactions strongly influence carbon and nitrogen flows in mushroom compost, yet their functional roles during Agaricus bisporus colonization remain unresolved. We combined PLFA-SIP and nanoSIMS imaging with ITS amplicon sequencing to follow resource flows and microbial activity across spatial scales. Stable-isotope tracers (¹³C-glucose and ¹⁵N-ammonium) revealed that A. bisporus simultaneously facilitates and suppresses bacterial populations: fungal activity increased glucose assimilation by bacteria yet reduced overall bacterial biomass. NanoSIMS visualized nutrient-rich microenvironments along hyphae where bacterial ¹³C and ¹⁵N assimilation was elevated. Sequencing showed the fungal community to comprise essentially two organisms, A. bisporus and Mycothermus thermophilus, which differ approximately elevenfold in their content of the fungal biomarker C18:2ω6,9c. Total fungal PLFA therefore tracks which of the two dominates as much as it tracks fungal biomass. Together these findings reveal coupled fungal–bacterial nutrient processing and show that biomarker-based estimates of fungal biomass require community composition to be known. Multi-scale isotope probing provides a framework for resolving microbial interactions in complex detrital systems. Contents. This repository contains the R scripts underlying the statistics and figures of the above study, organized by method in three folders. DNA_SIP/ holds the CsCl density‑gradient ITS2 pipeline (run 00→03) and the community‑over‑time figure. Bulk_PLFA_SIP/ holds the PLFA/bulk/CO₂ figure scripts and one consolidated per‑timepoint one‑way ANOVA script that reproduces Supplementary Tables S2–S5. nanoSIMS/ holds the single‑cell ROI and correlation plots. Each script has a header describing the figure or table it produces; README.txt explains the folder structure and the DNA‑SIP methodological choices, and README_figure_map.txt gives a one‑line script→figure/table map. Scripts use relative input paths (edit the path near the top of each). Raw ITS2 reads are archived at NCBI (BioProject PRJNA1503199); the processed data these scripts operate on are in the companion data deposit.

Authors 9

1. Madalina M. Vita

   Utrecht University · Westerdijk Fungal Biodiversity Institute

   Affiliation as printed

   Westerdijk Fungal Biodiversity Institute

   Utrecht University

2. Femke van Dam

   Linnaeus University

   Affiliation as printed

   Linnaeus University

3. Michiel V. M. Kienhuis

   Utrecht University

   Affiliation as printed

   Utrecht University

4. Desmond D. Eefting

   Utrecht University

   Affiliation as printed

   Utrecht University

5. Klaas G.J. Nierop

   Utrecht University

   Affiliation as printed

   Utrecht University

6. S. Emilia Hannula Aachen

   Leiden University

   Affiliation as printed

   Leiden University

7. Lùbos Polerecký

   Utrecht University

   Affiliation as printed

   Utrecht University

8. Francien Peterse

   Utrecht University

   Affiliation as printed

   Utrecht University

9. Jack J. Middelburg

   Utrecht University

   Affiliation as printed

   Utrecht University