bordering with the Pannonia region are inhabited by diverse riparian forests that support production of different truffle species, predominantly the most prized white truffle of Piedmont (Tuber magnatum Pico), but also other commercial species (T. macrosporum Vitt., T. aestivum Vitt.). Surprisingly, little is known about the native root-associated mycobiome (RAM) of these lowland truffle-producing forests. Therefore, in this study we aim at exploring and comparing the RAMs of three different truffle-producing forests from Kolubara river plane in Serbia. Molecular methods based on next generation sequencing (NGS) were used to evaluate the diversity of root-associated fungal communities and to elucidate the influence of environmental factors on their differentiation. To our knowledge, this is the first study from such habitats with a particular focus on comparative analysis of the RAM in different truffle-producing habitats using a high-throughput sequencing approach. Our results indicated that the alpha diversity of investigated fungal communities was not significantly different between different truffle-producing forests and within a specific forest type, while the seasonal differences in the alpha diversity were only observed in the white truffle-producing forests. Taxonomic profiling at phylum level indicated the dominance of fungal OTUs belonging to phylum Ascomycota and Basidiomycota, with very minor presence of other phyla. Distinct community structures of root-associated mycobiomes were observed for white, mixed, and black truffle-producing forests. The core mycobiome analysis indicated a fair share of fungal genera present exclusively in white and black truffle-producing forest, while the core genera of mixed truffle-producing forests were shared with both white and black truffle-producing forests. The majority of detected fungal OTUs in all three forest types were symbiotrophs, with ectomycorrhizal fungi being a dominant functional guild. Apart from assumed vegetation factor, differentiation of fungal communities was driven by factors connected to the distance from the river and exposure to fluvial activities, soil age, structure, and pH. Overall, Pannonian riparian forests appear to host diverse root-associated fungal communities that are strongly shaped by variation in soil conditions.
