Differential Effects of Long-Term Fertilization and Plant Species Richness on Soil Fungi and Protists

Anthropogenic nutrient enrichment and plant diversity loss reshape soil biodiversity, yet disentangling their individual and combined effects on key groups such as fungi and protists remains a major challenge. Here, we investigated soil microeukaryote communities using long-read amplicon rRNA gene sequencing in a temperate grassland experiment with 11 years of moderate NPK fertilization and manipulated plant diversity (1, 2, or 4 plant species). 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, with an increase in OTU richness by 20.8% and 52.7%, respectively, and shifted community dominance from fungi to protists. Plant diversity exclusively affected protists with a shift in community composition. Community changes were largely driven by increases in plant biomass (resulting from both fertilization and plant diversity), alongside higher soil phosphorus and lower soil pH, which were exclusively influenced by fertilization. Moreover, the experimental treatments exerted distinct effects on the different life strategies of fungi and protists. Fertilization enhanced fungal saprophytes (only richness), fungal animal pathogens, and protist consumers, whereas a decline in plant diversity increased phototrophic protists and decreased protist animal pathogens. Notably, fertilization and the decline in plant diversity together led to a cumulative increase in fungal plant pathogens. In conclusion, our results show that fertilization and reduced plant species richness exert distinct yet interacting effects on soil microeukaryotic communities. This highlights the need for integrated assessments of these two factors, rather than studying them in isolation, when evaluating global change effects.