Environmental surveys on soil viruses are still rare and mostly anecdotal, i. e., they mostly report on viruses at one location or for only a few sampling dates. Detailed time-series analysis with multiple samples can reveal the spatio-temporal dynamics of viral communities and provide important input as to how viruses interact with their potential hosts and the environment. Such surveys, however, require fast, easy-to-apply and reliable methods. In the present study we surveyed monthly across 13 months the abundance of virus-like particles (VLP) and the structure of the viral communities in soils along a land use transect (i.e., forest, pasture, and cropland). We evaluated 32 procedures to extract VLP from soil using different buffers and mechanical methods. The most efficient extraction was achieved with 1 × saline magnesium buffer in combination with 20 min vortexing. For community structure analysis we developed an optimized fingerprinting approach (fluorescent RAPD-PCR; fRAPD) by combining RAPD-PCR with fluorescently labeled primers in order to size the obtained fragments on a capillary sequencing machine. With the concomitantly collected data of soil specific factors and weather data, we were able to find correlations of viral abundance and community structure with environmental variables and sampling site. More specifically, we found that soil specific factors such as pH and total nitrogen content played a significant role in shaping both soil viral abundance and community structure. The fRAPD analysis revealed high temporal changes and clustered the viral communities according to sampling sites. In particular we observed that temperature and rainfall shaped soil viral communities in non-forest sites. In summary our findings suggest that sampling site was a key factor for shaping the abundance and community structure of soil viruses, and when site vegetation was reduced, temperature and rainfall were also important factors.

Summary: There is limited knowledge on how the association of trees with different mycorrhizal types shapes soil microbial communities in the context of changing tree diversity levels. We used arbuscular (AM) and ectomycorrhizal (EcM) tree species as con‐ and heterospecific tree species pairs (TSPs), which were established in plots of three tree diversity levels including monocultures, two‐species mixtures and multi‐tree species mixtures in a tree diversity experiment in subtropical China. We found that the tree mycorrhizal type had a significant effect on fungal but not bacterial alpha diversity. Furthermore, only EcM but not AM TSPs fungal alpha diversity increased with tree diversity, and the differences between AM and EcM TSPs disappeared in multi‐species mixtures. Tree mycorrhizal type, tree diversity and their interaction had significant effects on fungal community composition. Neither fungi nor bacteria showed any significant compositional variation in TSPs located in multi‐species mixtures. Accordingly, the most influential taxa driving the tree mycorrhizal differences at low tree diversity were not significant in multi‐tree species mixtures. Collectively, our results indicate that tree mycorrhizal type is an important factor determining the diversity and community composition of soil microbes, and higher tree diversity levels promote convergence of the soil microbial communities. Significance statement: More than 90% of terrestrial plants have symbiotic associations with mycorrhizal fungi which could influence the coexisting microbiota. Systematic understanding of the individual and interactive effects of tree mycorrhizal type and tree species diversity on the soil microbiota is crucial for the mechanistic comprehension of the role of microbes in forest soil ecological processes. Our tree species pair (TSP) concept coupled with random sampling within and across the plots, allowed us the unbiased assessment of tree mycorrhizal type and tree diversity effects on the tree‐tree interaction zone soil microbiota. Unlike in monocultures and two‐species mixtures, we identified species‐rich and converging fungal and bacterial communities in multi‐tree species mixtures. Consequently, we recommend planting species‐rich mixtures of EcM and AM trees, for afforestation and reforestation regimes. Specifically, our findings highlight the significance of tree mycorrhizal type in studying ‘tree diversity – microbial diversity – ecosystem function’ relationships.

Although fungi play important roles in biogeochemical cycling in aquatic ecosystems and have received a great deal of attention, much remains unknown about the living fractions of fungal communities in aquifers of the terrestrial subsurface in terms of diversity, community dynamics, functional roles, the impact of environmental factors and presence of fungal pathogens. Here we address this gap in knowledge by using RNA-based high throughput pair-end illumina sequencing analysis of fungal internal transcribed spacer (ITS) gene markers, to target the living fractions of groundwater fungal communities from fractured alternating carbonate-/siliciclastic-rock aquifers of the Hainich Critical Zone Exploratory. The probed levels of the hillslope multi-storey aquifer system differ primarily in their oxygen and nitrogen content due to their different connections to the surface. We discovered highly diverse living fungal communities (384 Operational Taxonomic Units, OTUs) with different taxonomic affiliations and ecological functions. The observed fungal communities primarily belonged to three phyla: Ascomycota, Basidiomycota and Chytridiomycota. Perceived dynamics in the composition of living fungal communities were significantly shaped by the concentration of ammonium in the moderately agriculturally impacted aquifer system. Apart from fungal saprotrophs, we also detected living plant and animal pathogens for the first time in this aquifer system. This work also demonstrates that the RNA-based high throughput pair-end illumina sequencing method can be used in future for water quality monitoring in terms of living fungal load and subsequent risk assessments. In general, this study contributes towards the growing knowledge of aquatic fungi in terrestrial subsurface biogeosphere.

Groundwater environments provide habitats for diverse microbial communities, and although Archaea usually represent a minor fraction of communities, they are involved in key biogeochemical cycles. We analysed the archaeal diversity within a mixed carbonate-rock/siliciclastic-rock aquifer system, vertically from surface soils to subsurface groundwater including aquifer and aquitard rocks. Archaeal diversity was also characterized along a monitoring well transect that spanned surface land uses from forest/woodland to grassland and cropland. Sequencing of 16S rRNA genes showed that only a few surface soil-inhabiting Archaea were present in the groundwater suggesting a restricted input from the surface. Dominant groups in the groundwater belonged to the marine group I (MG-I) Thaumarchaeota and the Woesearchaeota. Most of the groups detected in the aquitard and aquifer rock samples belonged to either cultured or predicted lithoautotrophs (e.g., Thaumarchaeota or Hadesarchaea). Furthermore, to target autotrophs, a series of¹³CO₂stable isotope-probing experiments were conducted using filter pieces obtained after filtration of 10,000 L of groundwater to concentrate cells. These incubations identified the SAGMCG Thaumarchaeota and Bathyarchaeota as groundwater autotrophs. Overall, the results suggest that the majority of Archaea on rocks are fixing CO₂, while archaeal autotrophy seems to be limited in the groundwater.

Summary: Understanding the complex interactions between trees and fungi is crucial for forest ecosystem management, yet the influence of tree mycorrhizal types, species identity, and diversity on tree‐tree interactions and their root‐associated fungal communities remains poorly understood. Our study addresses this gap by investigating root‐associated fungal communities of different arbuscular mycorrhizal (AM) and ectomycorrhizal (EcM) tree species pairs (TSPs) in a subtropical tree diversity experiment, spanning monospecific, two‐species, and multi‐species mixtures, utilizing Illumina sequencing of the ITS2 region. The study reveals that tree mycorrhizal type significantly impacts the alpha diversity of root‐associated fungi in monospecific stands. Meanwhile, tree species identity's influence is modulated by overall tree diversity. Tree‐related variables and spatial distance emerged as major drivers of variations in fungal community composition. Notably, in multi‐species mixtures, compositional differences between root fungal communities of AM and EcM trees diminish, indicating a convergence of fungal communities irrespective of mycorrhizal type. Interestingly, dual mycorrhizal fungal communities were observed in these multi‐species mixtures. This research underscores the pivotal role of mycorrhizal partnerships and the interplay of biotic and abiotic factors in shaping root fungal communities, particularly in varied tree diversity settings, and its implications for effective forest management and biodiversity conservation.