A collection of isolates from natural environments in Sardinia reveals a complex virome that includes negative-sense fungal viruses with unprecedented genome organizations.

genus includes soil-inhabiting fungi that provide important ecosystem services in their interaction with plants and other fungi, as well as biocontrol of fungal plant diseases. A collection of isolates from Sardinia has been previously characterized, but here we selected 113 isolates, representatives of the collection, and characterized their viral components. We carried out high-throughput sequencing of ribosome-depleted total RNA following a bioinformatics pipeline that detects virus-derived RNA-directed RNA polymerases (RdRps) and other conserved viral protein sequences. This pipeline detected seventeen viral RdRps with two of them corresponding to viruses already detected in other regions of the world and the remaining fifteen representing isolates of new putative virus species. Surprisingly, eight of them are from new negative-sense RNA viruses, a first in the genus . Among them is a cogu-like virus, closely related to plant-infecting viruses. Regarding the positive-sense viruses, we report the presence of an 'ormycovirus' belonging to a recently characterized group of bisegmented single-stranded RNA viruses with uncertain phylogenetic assignment. Finally, for the first time, we report a bisegmented member of which infects fungi. The proteins encoded by the second genomic RNA of this virus were used to re-evaluate several viruses in the and genera, here shown to be bisegmented and encoding a conserved polypeptide that has structural conservation with the nucleocapsid domain of rhabdoviruses.