Metabarcoding Analysis of Rhizosphere and Bulk Soils in Bulgaria Reveals Fungal Community Shifts Under Oat–Vetch Intercropping Versus Sole Oat Cultivation

Fungal communities in the rhizosphere are crucial in maintaining soil health, driving nutrient cycling, and enhancing plant productivity. This study examined the role of intercropping of oats (<i>Avena sativa</i> L.) with vetch (<i>Vicia sativa</i> L.) and their subsequent use as green manure (incorporating fresh plant biomass into soil to enhance nutrient cycling and microbial activity) on fungal diversity and community structure. Three field treatments were organized as follows: (i) unplanted control, (ii) single-oat cultivation, and (iii) oat-vetch intercropping. In the ripening stage of oats development, the plants in the intercropping treatment were ploughed at a depth of 30 cm as green manure. Soil samples at ripening stage and 3 months after ploughing were analyzed. High-throughput sequencing of the ITS2 region, combined with multivariate diversity analyses (alpha and beta diversity, PCA, NMDS, and UniFrac), revealed distinct fungal community profiles across treatments. Ascomycota dominated under conventional and untreated conditions, while Basidiomycota, Mortierellomycota, and Glomeromycota were enriched in intercropped and organically amended plots, notably at intercropping. Intercropping and green manuring significantly increased species richness, evenness, and phylogenetic fungal diversity. These treatments also supported higher abundances of beneficial fungi such as <i>Mortierella</i>, <i>Glomus</i>, and <i>Trichoderma</i>, while reducing potentially pathogenic taxa like <i>Fusarium</i>. Rank-abundance curves and rarefaction analysis confirmed that diversified systems hosted more balanced and complex fungal assemblages. Beta diversity metrics and ordination analyses indicated strong dissimilarities between the conventionally managed and diversified systems. The results showed that intercropping and organic inputs alter fungal community composition and promote microbial resilience and ecological functionality in the rhizosphere. These practices promoted the development of stable and diverse fungal networks essential for sustainable soil management and crop production.