Biochar amendment enhances the diversity of phagotrophic and phototrophic protists in citrus orchard soils via fungal-mediated pathways

Soil acidification severely constrains citrus production across the globe. Biochar, as a soil conditioner, demonstrates potential for enhancing citrus yields and mitigating soil acidity; however, its influence on protist communities in citrus orchard soils remains poorly understood, particularly under field conditions. A field experiment was conducted with different biochar amendments (derived from swine manure, rice straw, and wood) in this study. Soil properties and the biotic community (fungi, bacteria, and metazoa) were analyzed. High-throughput sequencing of the 18S rRNA gene was used to characterize the protist community. Biochar amendment significantly increased soil pH, electrical conductivity, organic matter, and a few traits of citrus (ANOVA, P < 0.05 for all variables), and manure and straw biochar exhibited relatively greater effectiveness in improving these parameters. It altered the diversity and composition of soil protists, enhancing the richness of phagotrophic and phototrophic protists but not parasitic protists. Path analysis indicated that fungal-mediated pathways were the primary drivers of such changes. Specifically, increased fungal diversity led to the subsequent enhancement of protist diversity through bottom-up control mechanisms. Biochar amendment fostered more complex and interconnected microbial communities, strengthening the associations between protists and fungi. Our findings suggest that the biochar-induced improvement of citrus traits in acidic orchards is statistically associated with fungal-mediated restructuring of the soil protist community, although causal confirmation requires further experimental validation. This study provides critical insights into the ecological mechanisms underlying biochar-induced soil health improvements, highlighting the importance of interactions between high-level phylogenetic branches of eukaryotic microbes in sustainable orchard management.