The family Polymycoviridae comprises small viruses with multi-segmented, double-stranded RNA (dsRNA) genomes. These viruses usually have four genomic segments, though some species may have up to eight. Detected in fungi and oomycetes, they often alter host development, impacting morphology, growth, sporulation, and virulence. As a relatively recent addition to the ICTV, its organization and taxonomic classification remains challenging. These difficulties are exacerbated by the fact that many newly discovered mycoviruses originate from metagenomic studies, complicating the association of genomic segments and the host. In this study, we screened a collection of fungal endophytes to explore mycoviral diversity. Sequencing of extracted dsRNA revealed three new polymycoviral genomes from different fungal species, each containing six to seven segments. One represents a completely new viral species, while the other two provide crucial host and genomic information for viruses previously identified only through metagenomic studies. Phylogenetic analyses grouped these genomes into three distinct clades within the genus Polymycovirus. To evaluate the biological impact of these viruses, isogenic host lines were generated and compared under controlled conditions. Phenotypic assays revealed no significant differences in radial growth, sporulation rate, or biomass production between infected and cured lines of Cladosporium ramotenellum K. Schub., Zalar, Crous & U. Braun and C. cladosporioides (Fresen.) G.A. de Vries, but the infected line of Fusarium redolens Wollenw. exhibited a larger mycelium compared to the cured lines. These findings expand the known diversity of polymycoviruses and highlight the specificity of virus-fungus interactions and variability in associated phenotypic effects. They emphasize the importance of studying individual virus-fungus pairs in axenic culture systems to better understand the complex nature of their interactions in the Polymycoviridae family.
