Responses of ectomycorrhizal extraradical mycelium and associated bacteria to drought and warming

In temperate forest ecosystems, ectomycorrhizal fungi (EMF) are key regulators of global carbon cycling. Up to 70% of the carbon accumulation in the organic soil layer originates from roots and mycorrhizal fungi rather than from aboveground litter deposition. A significant fraction of the photosynthetically fixed carbon that EMF receive from trees is directed toward production of extraradical mycelium, the main fungal component responsible for nutrient uptake. Despite the importance of EMF mycelium, the processes regulating its growth and functioning remain poorly understood in the face of climate change. Even less is known about the role of bacteria in regulating ectomycorrhizal physiology under altered environmental conditions. To address this, carbon-free in-growth sand bags used to "bait" ECM fungi were buried in the soil at two beech forests in Switzerland in an experimental climate warming x drought field study. In-growth mesh bags provide a powerful method for investigating this critical component of the belowground carbon stock, allowing targeted assessment of extraradical mycelium biomass production and turnover, and to study the bacteria associated with EMF mycelium. We quantified fungal and associated bacterial biomass within the sand bags using phospholipid fatty acids (PLFA) analysis, and we compared the composition and potential functions of the bacterial biome using DNA metabarcoding and metatranscriptomics. Across treatments and sampling windows, bacterial biomass was positively correlated with EMF biomass, indicating a tight coupling between extraradical mycelium and associated bacterial communities under climate stress. After the entire growing season, drought reduced the fungal-to-bacterial biomass ratio, while warming and the combined warming × drought treatment had weaker effects. This suggests that bacteria associated with the extraradical mycelium of ectomycorrhizal fungi are relatively more tolerant to drought stress than the fungi themselves. At finer taxonomic resolution, response ratios revealed group-specific and site-dependent responses of bacterial and fungal functional groups to climate treatments. While single stressors usually reduced the biomass of Gram-positive, Gram-negative, actinobacteria and fungi in both experimental sites, the combined warming × drought treatment frequently resulted in contrasting non-additive responses, especially in one of the two sites, indicating complex interactions between climate drivers. Overall, our results highlight that extraradical mycelium-associated bacterial communities remain tightly linked to ectomycorrhizal fungi under climate stress but with distinct tolerances that may shift bacterial contributions which support EMF functioning.