Complex transmission of partiti-, ambi- and ourmiaviruses in the forest pathogen Heterobasidion parviporum.

Utilizing Heterobasidion partitivirus 13 strain an1 (HetPV13-an1) and 15 strain pa1 (HetPV15-pa1) in co-infection is considered a potential biocontrol approach against Heterobasidion root and butt rot. Both partitiviruses mediate debilitating effects in most Heterobasidion host isolates and are generally transmitted efficiently between host strains. In this investigation, we conducted transmission experiments in the laboratory (in vitro) using several H. parviporum isolates to test whether using dual partitivirus infections is a more efficient way of transmitting viruses to new hosts compared to using single partitivirus infections, and whether co-occurring single-stranded RNA (ssRNA) viruses are co-transmitted during the process. The results showed that H. parviporum donors carrying both partitiviruses, HetPV13-an1 and HetPV15-pa1, transmitted HetPV15-pa1 more efficiently to recipients than the same donors infected with only HetPV15-pa1. In contrast, the transmission of HetPV13-an1 did not differ significantly between donors infected with both or only one partitivirus. Altogether, the transmission rates of HetPV13-an1 and HetPV15-pa1 were high on artificial media. Moreover, the transmission of the ssRNA viruses Heterobasidion ourmia-like virus 1(HetOlV1-pa7) and 4 (HetOlV4-an1) as well as Heterobasidion ambi-like virus 3 (HetAlV3-pa4) across different recipients were found to be variable. This study demonstrated for the first time the transmission of ambi- and ourmiaviruses between H. parviporum isolates in dual cultures and showed that H. parviporum mycelia can be cured of these ssRNA viruses using heat treatment.

Virus transmission frequencies in the pine root rot pathogen Heterobasidion annosum.

The combined use of Heterobasidion partitiviruses 13 and 15 (HetPV13-an1 and HetPV15-pa1) is considered a promising biocontrol approach against Heterobasidion root and butt rot. In a previous study, the transmission frequency of HetPV15-pa1 was found to be higher from a double partitivirus-infected donor than from a single partitivirus-infected donor. In this study, we included a wider array of recipient isolates to assess whether the phenomenon is widespread across different host strains and conducted transmission experiments on artificial media (in vitro) using a total of 45 different H. annosum donor-recipient pairs. In addition to investigating whether double partitivirus infection improves the transmission of HetPV13-an1 and HetPV15-pa1, we examined for the first time how efficiently co-infecting ssRNA viruses are concomitantly transmitted with the partitiviruses, and whether pre-existing ssRNA viruses in the recipients affect virus transmission. Generally, the transmission rates of HetPV13-an1 and HetPV15-pa1 were high from both single partitivirus-infected and double partitivirus-infected donors to most of the H. annosum recipient strains, with few exceptions. However, in contrast to previous experiments, the transmission frequency was not higher from the double partitivirus-infected donors. Also, ourmiavirus was transmitted between H. annosum strains, but the presence of another ourmiavirus in the recipient might affect the efficacy.

Efficacy of Biological and Chemical Control Agents against Heterobasidion Spore Infections of Norway Spruce and Scots Pine Stumps on Drained Peatland.

Treatment of conifer stumps with a control agent effectively prevents Heterobasidion spore infections in summer cuttings and protects the residual stand and the next tree generation from damage caused by Heterobasidion root rot. Thus far, stump treatment experiments have been carried out in mineral soils, and no information is available on the efficacy of stump treatment agents in boreal peatland conditions. In the present study, biological and chemical control agents (Phlebiopsis gigantea and urea, respectively) were tested in Scots pine and Norway spruce stands subjected to thinning, cap cutting, and clearcutting on drained peatland in Central Finland. The control efficacy of urea was high in both spruce and pine stumps (on average 99.5 and 85.3%, respectively), while the efficacy of P. gigantea was highly variable on both tree species and ranged from full protection down to negative control effect, i.e., there were more Heterobasidion infections on the treated than untreated half of the stumps. The moisture content of the stump wood or the thickness of the peat layer did not affect the control efficacy of either control agent. These results emphasize a need for further studies to determine the reasons for the unsteadiness of the biological control in peatland conditions.

Discovery and Community Dynamics of Novel ssRNA Mycoviruses in the Conifer Pathogen Heterobasidion parviporum.

Heterobasidion species are highly destructive basidiomycetous conifer pathogens of the Boreal forest region. Earlier studies have revealed dsRNA virus infections of families Curvulaviridae and Partitiviridae in Heterobasidion strains, and small RNA deep sequencing has also identified infections of Mitoviridae members in these fungi. In this study, the virome of Heterobasidion parviporum was examined for the first time by RNA-Seq using total RNA depleted of rRNA. This method successfully revealed new viruses representing two established (+)ssRNA virus families not found earlier in Heterobasidion: Narnaviridae and Botourmiaviridae. In addition, we identified the presence of a recently described virus group tentatively named "ambiviruses" in H. parviporum. The H. parviporum isolates included in the study originated from experimental forest sites located within 0.7 km range from each other, and a population analysis including 43 isolates was conducted at one of the experimental plots to establish the prevalence of the newly identified viruses in clonally spreading H. parviporum individuals. Our results indicate that viral infections are considerably more diverse and common among Heterobasidion isolates than known earlier and include ssRNA viruses with high prevalence and interspecies variation.

Heterobasidion Partitivirus 13 Mediates Severe Growth Debilitation and Major Alterations in the Gene Expression of a Fungal Forest Pathogen.

The fungal genus Heterobasidion includes some of the most devastating conifer pathogens in the boreal forest region. In this study, we showed that the alphapartitivirus Heterobasidion partitivirus 13 from Heterobasidion annosum (HetPV13-an1) is the main causal agent of severe phenotypic debilitation in the host fungus. Based on RNA sequencing using isogenic virus-infected and cured fungal strains, HetPV13-an1 affected the transcription of 683 genes, of which 60% were downregulated and 40% upregulated. Alterations observed in carbohydrate and amino acid metabolism suggest that the virus causes a state of starvation, which is compensated for by alternative synthesis routes. We used dual cultures to transmit HetPV13-an1 into new strains of H. annosum and Heterobasidion parviporum The three strains of H. parviporum that acquired the virus showed noticeable growth reduction on rich culturing medium, while only two of six H. annosum isolates tested showed significant debilitation. Based on reverse transcription-quantitative PCR (RT-qPCR) analysis, the response toward HetPV13-an1 infection was somewhat different in H. annosum and H. parviporum We assessed the effects of HetPV13-an1 on the wood colonization efficacy of H. parviporum in a field experiment where 46 Norway spruce trees were inoculated with isogenic strains with or without the virus. The virus-infected H. parviporum strain showed considerably less growth within living trees than the isolate without HetPV13-an1, indicating that the virus also causes growth debilitation in natural substrates.IMPORTANCE A biocontrol method restricting the spread of Heterobasidion species would be highly beneficial to forestry, as these fungi are difficult to eradicate from diseased forest stands and cause approximate annual losses of €800 million in Europe. We used virus curing and reintroduction experiments and RNA sequencing to show that the alphapartitivirus HetPV13-an1 affects many basic cellular functions of the white rot wood decay fungus Heterobasidion annosum, which results in aberrant hyphal morphology and a low growth rate. Dual fungal cultures were used to introduce HetPV13-an1 into a new host species, Heterobasidion parviporum, and field experiments confirmed the capability of the virus to reduce the growth of H. parviporum in living spruce wood. Taken together, our results suggest that HetPV13-an1 shows potential for the development of a future biocontrol agent against Heterobasidion fungi.

Distribution of Viruses Inhabiting Heterobasidion annosum in a Pine-Dominated Forest Plot in Southern Finland.

We investigated the diversity and spatial distribution of viruses infecting strains of the root rot fungus Heterobasidion annosum collected from pine stumps at a heavily infected forest site. Four different partitiviruses were detected in 14 H. annosum isolates at the study site, constituting approximately 29% of all Heterobasidion isolates investigated (N = 48). Two of the viruses detected were new partitiviruses designated here as Heterobasidion partitivirus 16 (HetPV16) and HetPV20, and two were previously known partitiviruses: HetPV7 and HetPV13. The two new partitiviruses found, HetPV16-an1 and HetPV20-an1, shared ~70% RdRp nucleotide sequence identity with the alphapartitivirus Rosellinia necatrix partitivirus 2, and less than 40% identity with known viruses of Heterobasidion spp. HetPV7-an1 was closely similar to HetPV7-pa1 isolated earlier from Heterobasidion parviporum, supporting the view of conspecific virus pools in different Heterobasidion species. Three fungal isolates were found to be co-infected with two different partitivirus strains (HetPV7-an1 and HetPV13-an2 or HetPV16-an1 and HetPV20-an1). Different isolates representing each host clone had variable virus compositions, and virus strains occurring in more than one host clone showed minor sequence variations between clones.

Viruses accumulate in aging infection centers of a fungal forest pathogen.

Fungal viruses (mycoviruses) with RNA genomes are believed to lack extracellular infective particles. These viruses are transmitted laterally among fungal strains through mycelial anastomoses or vertically via their infected spores, but little is known regarding their prevalence and patterns of dispersal under natural conditions. Here, we examined, in detail, the spatial and temporal changes in a mycovirus community and its host fungus Heterobasidion parviporum, the most devastating fungal pathogen of conifers in the Boreal forest region. During the 7-year sampling period, viruses accumulated in clonal host individuals as a result of indigenous viruses spreading within and between clones as well as novel strains arriving via airborne spores. Viral community changes produced pockets of heterogeneity within large H. parviporum clones. The appearance of novel viral infections in aging clones indicated that transient cell-to-cell contacts between Heterobasidion strains are likely to occur more frequently than what was inferred from genotypic analyses. Intraspecific variation was low among the three partitivirus species at the study site, whereas the unassigned viral species HetRV6 was highly polymorphic. The accumulation of point mutations during persistent infections resulted in viral diversification, that is, the presence of nearly identical viral sequence variants within single clones. Our results also suggest that co-infections by distantly related viral species are more stable than those between conspecific strains, and mutual exclusion may play a role in determining mycoviral communities.

Virus community dynamics in the conifer pathogenic fungus Heterobasidion parviporum following an artificial introduction of a partitivirus.

Viruses infecting the conifer pathogenic fungus Heterobasidion annosum sensu lato are intracellular and spread via anastomosis contacts. In the laboratory, these viruses transmit readily even between somatically incompatible isolates, but their dispersal capacity in natural conditions has not been previously studied. We introduced a mycovirus to a heavily diseased forest site by inoculating Norway spruce stumps with heartrot decay using a mycelial suspension of Heterobasidion parviporum strain RT3.49C hosting the partitivirus strain HetRV4-pa1. The Heterobasidion population at the sample plot was screened for mycoviruses prior to and after the inoculation. Based on sequence analysis, the resident H. parviporum strains harbored six different strains of the virus species Heterobasidion RNA virus 6 (HetRV6) and one strain of HetRV4 prior to the inoculation. After three growth seasons, the inoculated H. parviporum host strain was not detected, but the introduced virus had infected two resident H. parviporum genets. The presence of a preexisting HetRV6 infection did not hinder spread of the introduced partitivirus but resulted in coinfections instead. The resident HetRV6 virus population seemed to be highly stable during the incubation period, while the single indigenous HetRV4 infection was not detected after the inoculation. In laboratory infection experiments, the introduced virus could be transmitted successfully into all of the resident H. parviporum genets. This study shows for the first time transmission of a Heterobasidion virus between somatically incompatible hosts in natural conditions.

Population structure of a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato.

We describe a novel putative mycovirus infecting the conifer root-rot fungus Heterobasidion annosum sensu lato. This virus, designated as Heterobasidion RNA virus 6 (HetRV6), is taxonomically distant from all previously known viruses of Heterobasidion species, but somewhat related to the Curvularia thermal tolerance virus and the Fusarium graminearum virus 4. Based on a population analysis including 35 virus strains from Heterobasidion abietinum, Heterobasidion parviporum, Heterobasidion annosum sensu stricto and Heterobasidion occidentale, HetRV6 showed a considerable degree of geographical and host-related differentiation. The North American and Eurasian virus populations were clearly separated. In Eurasia, we observed cases of discrepancy between virus and host taxonomy, suggesting interspecies virus transfer. HetRV6 was also successfully transmitted between the three European species H. abietinum, H. annosum and H. parviporum. Based on growth rate tests on agar plates and spruce stem pieces, HetRV6 seemed to be cryptic or slightly mutualistic to its host.