Diversity and impact of single-stranded RNA viruses in Czech <i>Heterobasidion</i> populations

<i>Heterobasidion annosum sensu lato</i> comprises some of the most devastating pathogens of conifers. Exploring virocontrol as a potential strategy to mitigate economic losses caused by these fungi holds promise for the future. In this study, we conducted a comprehensive screening for viruses in 98 <i>H</i>. <i>annosum</i> s.l. specimens from different regions of Czechia aiming to identify viruses inducing hypovirulence. Initial examination for dsRNA presence was followed by RNA-seq analyses using pooled RNA libraries constructed from <i>H. annosum</i> and <i>Heterobasidion parviporum</i>, with diverse bioinformatic pipelines employed for virus discovery. Our study uncovered 25 distinct ssRNA viruses, including two ourmia-like viruses, one mitovirus, one fusarivirus, one tobamo-like virus, one cogu-like virus, one bisegmented narna-like virus and one segment of another narna-like virus, and 17 ambi-like viruses, for which hairpin and hammerhead ribozymes were detected. Coinfections of up to 10 viruses were observed in six <i>Heterobasidion</i> isolates, whereas another six harbored a single virus. Seventy-three percent of the isolates analyzed by RNA-seq were virus-free. These findings show that the virome of <i>Heterobasidion</i> populations in Czechia is highly diverse and differs from that in the boreal region. We further investigated the host effects of certain identified viruses through comparisons of the mycelial growth rate and proteomic analyses and found that certain tested viruses caused growth reductions of up to 22% and significant alterations in the host proteome profile. Their intraspecific transmission rates ranged from 0% to 33%. Further studies are needed to fully understand the biocontrol potential of these viruses <i>in planta</i>.IMPORTANCE<i>Heterobasidion annosum sensu lato</i> is a major pathogen causing significant damage to conifer forests, resulting in substantial economic losses. This study is significant as it explores the potential of using viruses (virocontrol) to combat these fungal pathogens. By identifying and characterizing a diverse array of viruses in <i>H. annosum</i> populations from Czechia, the research opens new avenues for biocontrol strategies. The discovery of 25 distinct ssRNA viruses, some of which reduce fungal growth and alter proteome profiles, suggests that these viruses could be harnessed to mitigate the impact of <i>Heterobasidion</i>. Understanding the interactions between these viruses and their fungal hosts is crucial for developing effective, environmentally friendly methods to protect conifer forests and maintain ecosystem health. This study lays the groundwork for future research on the application of mycoviruses in forest disease management.