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.

Bunyaviruses Affect Growth, Sporulation, and Elicitin Production in Phytophthora cactorum.

Phytophthora cactorum is an important oomycetous plant pathogen with numerous host plant species, including garden strawberry (Fragaria × ananassa) and silver birch (Betula pendula). P. cactorum also hosts mycoviruses, but their phenotypic effects on the host oomycete have not been studied earlier. In the present study, we tested polyethylene glycol (PEG)-induced water stress for virus curing and created an isogenic virus-free isolate for testing viral effects in pair with the original isolate. Phytophthora cactorum bunya-like viruses 1 and 2 (PcBV1 & 2) significantly reduced hyphal growth of the P. cactorum host isolate, as well as sporangia production and size. Transcriptomic and proteomic analyses revealed an increase in the production of elicitins due to bunyavirus infection. However, the presence of bunyaviruses did not seem to alter the pathogenicity of P. cactorum. Virus transmission through anastomosis was unsuccessful in vitro.

RNA-Dependent RNA Polymerase from Heterobasidion RNA Virus 6 Is an Active Replicase In Vitro.

Heterobasidion RNA virus 6 (HetRV6) is a double-stranded (ds)RNA mycovirus and a member of the recently established genus Orthocurvulavirus within the family Orthocurvulaviridae. The purpose of the study was to determine the biochemical requirements for RNA synthesis catalyzed by HetRV6 RNA-dependent RNA polymerase (RdRp). HetRV6 RdRp was expressed in Escherichia coli and isolated to near homogeneity using liquid chromatography. The enzyme activities were studied in vitro using radiolabeled UTP. The HetRV6 RdRp was able to initiate RNA synthesis in a primer-independent manner using both virus-related and heterologous single-stranded (ss)RNA templates, with a polymerization rate of about 46 nt/min under optimal NTP concentration and temperature. NTPs with 2'-fluoro modifications were also accepted as substrates in the HetRV6 RdRp-catalyzed RNA polymerization reaction. HetRV6 RdRp transcribed viral RNA genome via semi-conservative mechanism. Furthermore, the enzyme demonstrated terminal nucleotidyl transferase (TNTase) activity. Presence of Mn2+ was required for the HetRV6 RdRp catalyzed enzymatic activities. In summary, our study shows that HetRV6 RdRp is an active replicase in vitro that can be potentially used in biotechnological applications, molecular biology, and biomedicine.

Armillaria root rot fungi host single-stranded RNA viruses.

Species of Armillaria are distributed globally and include some of the most important pathogens of forest and ornamental trees. Some of them form large long-living clones that are considered as one of the largest organisms on earth and are capable of long-range spore-mediated transfer as well as vegetative spread by drought-resistant hyphal cords called rhizomorphs. However, the virus community infecting these species has remained unknown. In this study we used dsRNA screening and high-throughput sequencing to search for possible virus infections in a collection of Armillaria isolates representing three different species: Armillaria mellea from South Africa, A. borealis from Finland and Russia (Siberia) and A. cepistipes from Finland. Our analysis revealed the presence of both negative-sense RNA viruses and positive-sense RNA viruses, while no dsRNA viruses were detected. The viruses included putative new members of virus families Mymonaviridae, Botourmiaviridae and Virgaviridae and members of a recently discovered virus group tentatively named "ambiviruses" with ambisense bicistronic genomic organization. We demonstrated that Armillaria isolates can be cured of viruses by thermal treatment, which enables the examination of virus effects on host growth and phenotype using isogenic virus-infected and virus-free strains.

Viral diversity in Phytophthora cactorum population infecting strawberry.

Eighty-eight Phytophthora cactorum strains isolated from crown or leather rot of strawberry in 1971-2019 were screened for viruses using RNA-seq and RT-PCR. Remarkably, all but one isolate were virus-infected, most of them harbouring more than one virus of different genera or species. The most common virus occurring in 94% of the isolates was the Phytophthora cactorum RNA virus 1 (PcRV1) resembling members of Totiviridae. Novel viruses related to members of Endornaviridae, named Phytophthora cactorum alphaendornaviruses 1-3 (PcAEV1-3), were found in 57% of the isolates. Four isolates hosted viruses with affinities to Bunyaviridae, named Phytophthora cactorum bunyaviruses 1-3 (PcBV1-3), and a virus resembling members of the proposed genus 'Ustivirus', named Phytophthora cactorum usti-like virus (PcUV1), was found in a single isolate. Most of the virus species were represented by several distinct strains sharing ≥81.4% aa sequence identity. We found no evidence of spatial differentiation but some temporal changes in the P. cactorum virus community were observed. Some isolates harboured two or more closely related strains of the same virus (PcAEV1 or PcRV1) sharing 86.6%-96.4% nt identity in their polymerase sequence. This was surprising as viruses with such a high similarity are typically mutually exclusive.

Role and function of Chondrostereum purpureum in biocontrol of trees.

A decay fungus, Chondrostereum purpureum (Pers. Ex Fr.) Pouzar, has been investigated in Europe, Northern America and New Zealand for its ability to decay hardwood stumps and thus prevent sprouting. The aim of these investigations has been to find an alternative to mechanical (cutting only) and chemical sprout control (cutting and applying chemicals to stumps in order to prevent sprouting). Mechanical sprout control is not an efficient option due to hardwood tree species' ability to re-sprout efficiently after cutting, and therefore management costs are high. Chemicals would be efficient but due to their harmful effects on the environment, alternatives are needed. The fungal treatment, i.e., cutting accompanied with C. purpureum inoculum is an environmentally friendly and efficient option for sprout control. This mini-review comprises the role and function of C. purpureum in biocontrol of trees: the ecology of C. purpureum, its sprout control efficacy, factors affecting sprout control efficacy, devices in biological sprout control, potential risks, and the future perspectives of biological sprout control. KEY POINTS: • A fungus Chondrostereum purpureum is efficient in preventing sprouting of hardwoods • C. purpureum is not sensitive to environmental conditions • Devices should be developed for cost-efficient biological sprout control.

Phenotypic Recovery of a Heterobasidion Isolate Infected by a Debilitation-Associated Virus Is Related to Altered Host Gene Expression and Reduced Virus Titer.

The fungal genus Heterobasidion includes forest pathogenic species hosting a diverse group of partitiviruses. They include the host debilitating Heterobasidion partitivirus 13 strain an1 (HetPV13-an1), which was originally observed in a slowly growing H. annosum strain 94233. In this study, a relatively fast-growing sector strain 94233-RC3 was isolated from a highly debilitated mycelial culture of 94233, and its gene expression and virus transcript quantities as well as the genomic sequence of HetPV13-an1 were examined. The sequence of HetPV13-an1 genome in 94233-RC3 was identical to that in the original 94233, and thus not the reason for the partial phenotypic recovery. According to RNA-seq analysis, the HetPV13-an1 infected 94233-RC3 transcribed eight genes differently from the partitivirus-free 94233-32D. Three of these genes were downregulated and five upregulated. The number of differentially expressed genes was considerably lower and the changes in their expression were small compared to those of the highly debilitated original strain 94233 with the exception of the most highly upregulated ones, and therefore viral effects on the host transcriptome correlated with the degree of the virus-caused debilitation. The amounts of RdRp and CP transcripts of HetPV13-an1 were considerably lower in 94233-RC3 and also in 94233 strain infected by a closely related mildly debilitating virus HetPV13-an2, suggesting that the virus titer would have a role in determining the effect of HetPV13 viruses on their hosts.

Rainstorm effects on the biocontrol efficacy of the decay fungus Chondrostereum purpureum against birch sprouting in boreal forests.

In forest regeneration areas, alongside roads and railways, under electric power lines and above gas pipe lines, there is a need for regular sprout control. A biocontrol method against broadleaved sprouting with formulations including the decay fungus Chondrostereum purpureum (Pers. Ex Fr.) Pouzar has been shown to be effective. Yet, heavy rain during spreading of this fungal inoculum on freshly cut stumps may affect the efficacy of the treatment, i.e., stump mortality during the following years. Thus, we performed an experiment where freshly cut birch stump surfaces (Betula pendula Roth and Betula pubescens Ehrh.) were treated with fungal inoculum under heavy irrigation and without it. Furthermore, two different adjuvants which aimed to fix the fungal inoculum to freshly cut stumps during irrigation and to protect against solar radiation were tested. Our results revealed that the artificial rainstorm treatment caused a delay in the efficacy of C. purpureum, but after three growing seasons, there was no significant difference in the mortality of birch stumps treated under irrigation or without it (stump mortalities 74 and 86%, respectively). Adjuvants did not improve the efficacy in stumps treated under irrigation nor in those treated without irrigation. KEY POINTS: • Heavy rain delayed the sprout control efficacy of a fungus Chondrostereum purpureum. • Final efficacy of formulations was the same in wet and dry conditions. • No additional adjuvants are needed to improve formulations.

Multiple virus infections on Heterobasidion sp.

Heterobasidion viruses have previously been shown to affect each other's transmission and phenotypic effects on their hosts in a complex way. In this work, Heterobasidion parviporum strains hosting five coinfecting viruses simultaneously were constructed and used as donors in transmission experiments. They showed that viruses move more frequently between the mycelia of the same species than between the mycelia of H. parviporum and Heterobasidionannosum. One of the strains was used to show that coinfection of five viruses is relatively unstable in a natural environment and analyses of the growth rate and competitive ability of Heterobasidion strains hosting various virus combinations revealed that viral effects are not additive. The results also supported the view that the transmission of the promising virocontrol agent HetPV13-an1 may be enhanced by coinfecting viruses in the donor mycelium. However, its detrimental effects may be blocked by the presence of other viruses in the same mycelium. REPOSITORIES: GenBank accession number MN058080.

Alphapartitiviruses of Heterobasidion Wood Decay Fungi Affect Each Other's Transmission and Host Growth.

Heterobasidion spp. root rot fungi are highly destructive forest pathogens of the northern boreal forests, and are known to host a diverse community of partitiviruses. The transmission of these mycoviruses occurs horizontally among host strains via mycelial anastomoses. We revealed using dual cultures that virus transmission rates are affected by pre-existing virus infections among two strains of H. annosum. The transmission efficacy of mycovirus HetPV15-pa1 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% to a partitivirus-free recipient. On contrary, pre-existing virus infections of two closely related strains of HetPV11 hindered each other's transmission, but had unexpectedly dissimilar effects on the transmission of more distantly related viruses. The co-infection of HetPV13-an1 and HetPV15-pa1 significantly reduced host growth, whereas double infections including HetPV11 strains had variable effects. Moreover, the results showed that RdRp transcripts are generally more abundant than capsid protein (CP) transcripts and the four different virus strains express unique transcripts ratios of RdRp and CP. Taken together, the results show that the interplay between co-infecting viruses and their host is extremely complex and highly unpredictable.

Description, Distribution, and Relevance of Viruses of the Forest Pathogen Gremmeniella abietina.

The European race of the ascomycetous species Gremmeniella abietina (Lagerberg) Morelet includes causal agents of shoot blight and stem canker of several conifers in Europe and North America, which are known to host a diverse virome. GaRV6 is the latest and sixth mycovirus species reported within G. abietina. Before its description, one victorivirus and one gammapartitivirus species were described in biotype A, two mitoviruses in both biotypes A and B and a betaendornavirus in biotype B. Possible phenotypic changes produced by mycoviruses on G. abietina mycelial growth have been reported in Spanish mitovirus-free and GaRV6-hosting G. abietina isolates, which had higher growth rates at the optimal temperature of 15 °C, but no other major differences have been observed between partitivirus-like dsRNA and dsRNA-free isolates. In this review, we reappraise the diversity of viruses found in G. abietina so far, and their relevance in clarifying the taxonomy of G. abietina. We also provide evidence for the presence of two new viruses belonging to the families Fusariviridae and Endornaviridae in Spanish isolates.

Cadophora margaritata sp. nov. and other fungi associated with the longhorn beetles Anoplophora glabripennis and Saperda carcharias in Finland.

Symbiosis with microbes is crucial for survival and development of wood-inhabiting longhorn beetles (Coleoptera: Cerambycidae). Thus, knowledge of the endemic fungal associates of insects would facilitate risk assessment in cases where a new invasive pest occupies the same ecological niche. However, the diversity of fungi associated with insects remains poorly understood. The aim of this study was to investigate fungi associated with the native large poplar longhorn beetle (Saperda carcharias) and the recently introduced Asian longhorn beetle (Anoplophora glabripennis) infesting hardwood trees in Finland. We studied the cultivable fungal associates obtained from Populus tremula colonised by S. carcharias, and Betula pendula and Salix caprea infested by A. glabripennis, and compared these to the samples collected from intact wood material. This study detected a number of plant pathogenic and saprotrophic fungi, and species with known potential for enzymatic degradation of wood components. Phylogenetic analyses of the most commonly encountered fungi isolated from the longhorn beetles revealed an association with fungi residing in the Cadophora-Mollisia species complex. A commonly encountered fungus was Cadophora spadicis, a recently described fungus associated with wood-decay. In addition, a novel species of Cadophora, for which the name Cadophora margaritata sp. nov. is provided, was isolated from the colonised wood.

Interfertility and genetic variability among European and North American isolates of the basidiomycete fungus Chondrostereum purpureum.

The conspecificity of Finnish and western Canadian isolates of the decay fungus Chondrostereum purpureum was investigated by several approaches, including the assessment of genetic variability, mating and progeny analysis, and the analysis of selected phenotypic traits. Eight second-generation single spore strains per fungal isolate pairing were investigated with specific genetic markers developed for both Finnish and Canadian parental isolates. Tests of linkage disequilibrium were used to analyze whether these markers assorted independently among single spore strains. This procedure was similarly applied to the third-generation spore progeny. Finally, global non-metric multidimensional scaling was used to analyze independent random amplified microsatellite marker data to assess the genetic variability of the parental Finnish and Canadian isolates, and their second- and third-generation progeny. Our results revealed that the parental isolates from Finland and western Canada were genetically divergent, but no interfertility barriers were identified between these geographically distant fungi. Furthermore, parental genetic markers used in mating studies demonstrated that second- and third-generation spore progenies underwent normal meiosis and genetic recombination without linkage disequilibrium. Based on this work, the studied C. purpureum isolates from Finland and Canada can be considered as belonging to a single biological species, although genetic and limited phenotypic differentiation was observed.

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.

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.

Looking for relationships between the populations of Dothistroma septosporum in northern Europe and Asia.

Dothistroma septosporum, a notorious pine needle pathogen with an unknown historical geographic origin and poorly known distribution pathways, is nowadays found almost in all areas inhabited by pines (Pinus spp.). The main aim of this study was to determine the relationship between North European and East Asian populations. In total, 238 Eurasian D. septosporum isolates from 11 countries, including 211 isolates from northern Europe, 16 isolates from Russian Far East and 11 isolates from Bhutan were analysed using 11 species-specific microsatellite and mating type markers. The most diverse populations were found in northern Europe, including the Baltic countries, Finland and European Russia. Notably, D. septosporum has not caused heavy damage to P. sylvestris in northern Europe, which may suggest a long co-existence of the host and the pathogen. No indication was obtained that the Russian Far East or Bhutan could be the indigenous area of D. septosporum, as the genetic diversity of the fungus there was low and evidence suggests gene flow from northern Europe to Russian Far East. On the western coast of Norway, a unique genetic pattern was observed, which differed from haplotypes dominating other Fennoscandian populations. As an agent of dothistroma needle blight, only D. septosporum was documented in northern Europe and Asia, while D. pini was found in Ukraine and Serbia.

Occurrence of similar mycoviruses in pathogenic, saprotrophic and mycorrhizal fungi inhabiting the same forest stand.

Fungal viruses (mycoviruses) are considered to be highly host specific, but phylogenetic analysis supports the occasional occurrence of horizontal transmission between species. We used an extensive sampling strategy to investigate whether similar viruses occur in more than one fungal species of the same forest habitat. Mycelial samples were collected from in-growth mesh bags (N = 259), fruiting bodies (N = 173) and cultured isolates (N = 68) at a forest site where the spatial distribution of viral infections in clonal individuals of the wood decay fungus Heterobasidion parviporum was mapped in detail earlier. The investigation revealed previously known Heterobasidion viruses in ∼2% of the single or pooled mycorrhizal samples from mesh bags, ∼3% of the fruiting body samples and none of the fungal cultures analyzed. Novel virus strains distinct from known Heterobasidion viruses were detected in cultures of ectomycorrhizal fungi (Lactarius tabidus, L. rufus) and saprotrophic fungi (Megacollybia platyphylla, Mucoraceae spp.). Overall, our results support the view that mycoviruses do not readily cross species borders. Regarding potential virocontrol applications, the introduction of Heterobasidion viruses into natural habitats is not expected to cause a major infection pressure towards the indigenous fungal community. However, the ecological consequences of the putative interspecies virus transmission events detected require further investigation.

Tree invasions and biosecurity: eco-evolutionary dynamics of hitchhiking fungi.

When non-native plants reach novel environments, they typically arrive with hidden microbiomes. In general, most of these hitchhikers remain on their co-evolved hosts, some contribute to the invasiveness of their hosts, and a small number can undergo host shifts and move onto native hosts. Invasion success can vary depending upon the different categories of fungal associates. When an invader tree relies on a fungal mutualism to survive in the new environment, there is a fundamentally lower likelihood of either the tree, or the fungus, establishing novel associations. In contrast, parasitic hitchhikers could merely use their host plants to move through the landscape and to become established on new hosts (host shifts). Evidence suggests the frequency of these host shifts is low and depends upon the fungal functional group. However, epidemics caused by invasive pathogens in native ecosystems have occurred globally. Thus, elucidating the potential for hidden non-native fungi to form novel host associations in a new environment is important for biodiversity conservation.

The efficacy of six elite isolates of the fungus Chondrostereum purpureum against the sprouting of European aspen.

The sprouting of broad-leaved trees after cutting is problematic in forest regeneration areas, along roads and railways, under electric power and above gas pipe lines. In Finland, one of the most difficult species to control in these areas is the European aspen (Populus tremula), which produces both stump sprouts and root suckers after saplings have been cut. In this study, we investigated whether a decay fungus of broad-leaved trees, Chondrostereum purpureum, could be used as a biological control agent against aspen sprouting. The efficacy of six elite strains of C. purpureum (improved earlier in a breeding process) was investigated on aspen for three years. The most efficient C. purpureum strain, R53, tested earlier on birch (Betula pendula and B. pubescens), was efficient in causing mortality of aspen stumps and preventing the development of root suckers. With this strain, stump mortality was 78%, while significantly lower in control stumps which were cut only (47%). Aspen trees in the vicinity of the treatments (within a 10 m radius around each sapling) decreased the efficacy of C. purpureum. This study shows that the decay fungus C. purpureum can successfully be used in the sprout control of aspen saplings.