biostudies-arrayexpressMetabolomicsUnknownTranscriptomicsGenomicsProteomicsStefan van de RuitenbeekMinIONDNA-seqmixed samplemixed samplehttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15101Anthropogenic nutrient inputs alter soil biodiversity; however, it remains largely unknown whether changes in soil microeukaryotes (fungi and protists) are primarily driven by direct effects, such as modifications in soil properties, or by indirect effects, such as plant diversity loss. To disentangle these mechanisms, we investigated the long-term effects (11 years) of fertilization and manipulated plant diversity (1, 2, or 4 plant species) on soil microeukaryote communities in a temperate grassland experiment using long-amplicon rRNA sequencing. Our results indicate that fertilization generally had a stronger influence on microeukaryote communities than plant species richness. Fertilization altered the community composition of fungi and protists, increased OTU richness by 20.8% and 52.7%, respectively, and shifted community dominance from fungi to protists. Regarding plant diversity, we observed an effect exclusively on the protist community. Changes were primarily explained by increased plant biomass (driven by both fertilization and plant diversity) and by higher soil phosphorus and lower soil pH levels (driven exclusively by fertilization). Regarding life strategies, we observed synergistic treatment effects: fertilization primarily enhanced fungal saprophytes (only richness), fungal animal pathogens, and protist consumers, whereas plant diversity affected phototrophic protists (reduction) and protist animal pathogens (enhancement). Notably, fertilization and plant diversity decline together led to a cumulative increase in fungal plant pathogens. In conclusion, we highlight that fertilisation alone has a significant effect on soil microeukaryotes, while the additional decline in plant diversity affects different soil groups that are not directly affected by fertilisation. This synergistic pattern indicates that fertilization can influence the entire microeukaryote community through direct and indirect mechanisms, with a cumulative enhancement on certain groups, such as plant pathogens.biostudies-arrayexpressNucleic Acid Extraction - DNA was extracted from 1 g of soil according to Harkes et al. (2025).Library Construction - Sequencing libraries were generated by pooling PCR products of the samples in equimolar ratio’s to ensure even sequencing depth. The libraries were then cleaned using NucleoMag NGS Clean-up and Size Select beads (0.6X bead:sample ratio) to discard unwanted contaminants and short DNA fragments (<600 bp). Of each library, 150 fmol was prepared for nanopore sequencing using kit SQK-LSK112 (Oxford Nanopore Technologies Plc., UK) following instructions of the manufacturer.Sequencing - Following library preparation, 17 fmol of each of the prepared libraries was loaded on a MinION R9.4.1 flow cell. Sequencing was performed on a MinION Mk1C (MinKNOW v22.11.2).Sample Collection - On 10 June 2022, during peak biomass season, we took six soil cores from each of the 48 plots using a 1-cm-diameter soil auger (each 10 cm deep). We did this in all 4 corners (each 30 cm from the edges of the plot) and twice in the center (50 cm from south to north edge and either 40 cm from west or east edge). Soil samples per plot were combined in a plastic bag and stored in a cool box until transported to the laboratory. Samples were sieved using a 2-cm mesh sieve to remove stones, plant material and larger animals. A representative subsample of the sieved soil from each plot was frozen at -20°C for later DNA analyses, the rest was stored at 4°C for soil property analyses.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesStefan van de RuitenbeekArne SchwelmRobbert van HimbeeckPeter DietrichfalseDisentangling the impact of long-term fertilization and plant diversity reduction on soil micro-eukaryotic communities using long-read sequencing.Anthropogenic nutrient inputs alter soil biodiversity; however, it remains largely unknown whether changes in soil microeukaryotes (fungi and protists) are primarily driven by direct effects, such as modifications in soil properties, or by indirect effects, such as plant diversity loss. To disentangle these mechanisms, we investigated the long-term effects (11 years) of fertilization and manipulated plant diversity (1, 2, or 4 plant species) on soil microeukaryote communities in a temperate grassland experiment using long-amplicon rRNA sequencing. Our results indicate that fertilization generally had a stronger influence on microeukaryote communities than plant species richness. Fertilization altered the community composition of fungi and protists, increased OTU richness by 20.8% and 52.7%, respectively, and shifted community dominance from fungi to protists. Regarding plant diversity, we observed an effect exclusively on the protist community. Changes were primarily explained by increased plant biomass (driven by both fertilization and plant diversity) and by higher soil phosphorus and lower soil pH levels (driven exclusively by fertilization). Regarding life strategies, we observed synergistic treatment effects: fertilization primarily enhanced fungal saprophytes (only richness), fungal animal pathogens, and protist consumers, whereas plant diversity affected phototrophic protists (reduction) and protist animal pathogens (enhancement). Notably, fertilization and plant diversity decline together led to a cumulative increase in fungal plant pathogens. In conclusion, we highlight that fertilisation alone has a significant effect on soil microeukaryotes, while the additional decline in plant diversity affects different soil groups that are not directly affected by fertilisation. This synergistic pattern indicates that fertilization can influence the entire microeukaryote community through direct and indirect mechanisms, with a cumulative enhancement on certain groups, such as plant pathogens.2026-07-06T00:00:00Z2026-07-06T15:45:29.26Z2025-04-29T22:54:42.396ZE-MTAB-15101ERP172092EFO_0002944EFO_0004170EFO_0002693EFO_0005518EFO_0004184