Diversity of RNA Viruses and Circular Viroid-like Elements in Heterobasidion spp. in Near-Natural Forests of Bosnia and Herzegovina

<i>Heterobasidion</i> 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 <i>Heterobasidion annosum</i> sensu lato in near-natural forests of Bosnia and Herzegovina (Dinaric Alps), a region previously unexplored in this regard. Seventeen <i>H. annosum</i> 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 (<i>Partitiviridae</i> and <i>Curvulaviridae</i>), six linear ssRNA (<i>Mitoviridae</i>, <i>Narnaviridae</i>, <i>Botourmiaviridae</i>, <i>Virgaviridae</i>, <i>Benyviridae</i>, and <i>Deltaflexiviridae</i>) and three circular ssRNA (<i>Dumbiviridae</i>, <i>Quambiviridae</i>, and <i>Trimbiviridae</i>) 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 <i>Heterobasidion</i>, along with beny-like and deltaflexivirus-like viruses in <i>Heterobasidion abietinum</i>, and expands the known virosphere of <i>Heterobasidion</i> species in Southeastern European forests.