Anthropogenically enhanced atmospheric sulphur (S) and nitrogen (N) deposition has acidified and eutrophied forest ecosystems worldwide. However, both S and N mechanisms have an impact on microbial communities and the consequences for microbially driven soil functioning differ. We conducted a two-forest stand (Norway spruce and European beech) field experiment involving acidification (sulphuric acid addition) and N (ammonium nitrate) loading and their combination. For 4 years, we monitored separate responses of soil microbial communities to the treatments and investigated the relationship to changes in the activity of extracellular enzymes. We observed that acidification selected for acidotolerant and oligotrophic taxa of Acidobacteria and Actinobacteria decreased bacterial community richness and diversity in both stands in parallel, disregarding their original dissimilarities in soil chemistry and composition of microbial communities. The shifts in bacterial community influenced the stoichiometry and magnitude of enzymatic activity. The bacterial response to experimental N addition was much weaker, likely due to historically enhanced N availability. Fungi were not influenced by any treatment during 4-year manipulation. We suggest that in the onset of acidification when fungi remain irresponsive, bacterial reaction might govern the changes in soil enzymatic activity.

The Archaeorhizomycetes are recently discovered fungi with poorly resolved ecology. Even their abundance in soil fungal communities is currently disputed. Here we applied a PCR-independent, RNA-based metatranscriptomic approach to determine their abundance among fungi in eleven different soils across Europe. Using small subunit (SSU) ribosomal RNA transcripts as marker, we detected Archaeorhizomycetes in 17 out of 28 soil metatranscriptomes. They had average relative SSU rRNA abundance of 2.0% with a maximum of 9.4% among fungal SSU rRNAs. Network analysis revealed that they co-occur with arbuscular mycorrhizal Glomerales, which is in line with their previously suggested association with plant roots. Moreover, Archaeorhizomycetes ranked among the potential keystone taxa. This metatranscriptomic survey exemplifies the usage of non-targeted molecular approaches for the study of soil fungi. It provides PCR- and DNA-independent evidence for the low abundance of Archaeorhizomycetes in soil fungal communities, although they might be non-negligible players despite their low abundance.