Metabarcoding Unveils Seasonal Soil Microbiota Shifts and Their Influence on Boletus edulis and Boletus reticulatus Mycelium in Quercus robur Stands

Forest ecosystems undergo seasonal shifts in bacterial and fungal communities, but little is known about the specific microbiota associated with <i>Quercus robur</i>-<i>Boletus edulis</i> systems. This study represents the first examination of seasonal changes in soil microbiota in pedunculate oak habitats in Galicia (NW Spain) and their relationship with <i>Boletus edulis</i> and <i>Boletus reticulatus</i> mycelium prevalence and concentration. Soil microbiota richness, diversity, and composition, as well as seasonal variation in <i>Boletus</i> mycelium, were assessed using DNA metabarcoding and qPCR, respectively. Sampling was conducted in autumn at two 30-40-year-old <i>Q. robur</i> stands. Bacterial communities were dominated by <i>Acidobacteria</i> (34%) and <i>Proteobacteria</i> (33%), with <i>Acidobacterium</i> (12%), <i>Paludibaculum</i> (9%), and <i>Edaphobacter</i> (7%) identified as most abundant. Fungal communities were primarily Basidiomycota (93%), led by <i>Russula</i> (46%). For both bacteria and fungi, the highest OTU richness was observed in September, followed by a significant decrease in October and a partial recovery in November. <i>Boletus</i> species were found to exhibit positive correlations with specific bacteria (e.g., <i>Massilia</i>, <i>Rhizobium</i>) and fungi (e.g., <i>Amanita</i>, <i>Clavaria</i>, <i>Inocybe</i>, <i>Scleroderma</i>, <i>Suillus</i> and <i>Mortierella</i>), suggesting a potential influence of these microbes on mycelium development. This study provides novel insights into the seasonal dynamics of soil microbiota and their potential role in <i>Boletus</i> ecology, thereby advancing understanding of host-microbe interactions in temperate forests.