The fungal species complex Heterobasidion annosum sensu lato (s.l.) is considered one of the most devastating conifer pathogens in the boreal forest region. They affect European coniferous forests with root and butt rot, causing annual economic losses of €800 million. Despite several efforts in practical forestry to control the disease, the economic loss remains considerable. Therefore, it is still necessary to introduce alternate control measures for Heterobasidion infection. Heterobasidion spp. are infected by a diverse community of mycoviruses, mostly partitiviruses. Here, these viruses were studied to find potential viruses for biocontrol purposes. We described six novel Heterobasidion partitivirus (HetPV) species phylogenetically related to Helicobasidium mompa partitivirus V70 that infect four pathogenic Heterobasidion species. Interestingly, our study revealed that HetPV13-an1 causes severe phenotypic debilitation in its native and exotic fungal host. The RNA sequencing of isogenic virus infected and cured fungal strains showed that HetPV13-an1 affected the transcription of 683 genes. The RT-qPCR analysis showed that the response toward HetPV13-an1 infection varied between H. annosum and H. parviporum. Moreover, the wood colonization efficacy of H. parviporum infected by HetPV13-an1 was restricted in living Norway spruce trees. The ratio of polymerase and coat protein genome segments/transcripts of eight partitiviruses analysed was highly variable in mycelia. All the virus species had unique ratios of the genome segments, which were stable over different temperatures and hosts. The co-infection with HetPV13-an1 and HetPV15-pa1 reduced host growth up to 95%. Regarding the transmission efficacy of mycoviruses, HetPV15-pa1 transmission to a pre-infected host was elevated from zero to 50% by the presence of HetPV13-an1, and a double infection of these viruses in the donor resulted in an overall transmission rate of 90%. Altogether, the study demonstrated that the interplay between co-infecting viruses and their host is highly complex and that partitiviruses show potential for biocontrol
