Heterobasidion root rot fungi represent a major threat to conifer forest stands, and virocontrol (biocontrol) has been proposed as an alternative strategy of disease management in recent years. Here, we investigated the occurrence of RNA viruses and viroid-like genomes in Heterobasidion annosum sensu lato in near-natural forests of Bosnia and Herzegovina (Dinaric Alps), a region previously unexplored in this regard. Seventeen H. annosum s.l. isolates were screened for virus presence by RNA Sequencing and bioinformatic analyses. In total, 32 distinct mycoviruses were discovered in the datasets, 26 of which were previously unknown. The detected viruses represent two dsRNA (Partitiviridae and Curvulaviridae), six linear ssRNA (Mitoviridae, Narnaviridae, Botourmiaviridae, Virgaviridae, Benyviridae, and Deltaflexiviridae) and three circular ssRNA (Dumbiviridae, Quambiviridae, and Trimbiviridae) virus families. In addition to the known circular ambiviruses with their hammerhead (HHRz) and hairpin (HPRz) ribozymes, two other smaller non-coding circular RNAs of ca. 910 bp each were identified encoding HHRz and deltavirus (DVRz) ribozymes in both polarities of their genomes. This study documents the first report of a putative viroid-like RNA agent in Heterobasidion, along with beny-like and deltaflexivirus-like viruses in Heterobasidion abietinum, and expands the known virosphere of Heterobasidion species in Southeastern European forests.

Department of Forest Ecology Faculty of Forestry and Wood Technology Mendel University in Brno Zemědělská 1 613 00 Brno Czech Republic

Department of Forest Protection and Wildlife Management Faculty of Forestry and Wood Technology Mendel University in Brno Zemědělská 3 613 00 Brno Czech Republic

Department of Genetics and Biotechnologies Faculty of Agriculture and Technology University of South Bohemia in České Budějovice Na Sádkách 1780 370 05 České Budějovice Czech Republic

Department of Integrated Protection of Forest Ecosystems Faculty of Forestry University of Banja Luka Vojvode Stepe Stepanovića Blvd 75a 78 000 Banja Luka Bosnia and Herzegovina

Instituto de Biología Molecular y Celular de Plantas Universidad Politécnica de Valencia CSIC 46022 Valencia Spain

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