Heterobasidion Species Research Articles

The relationship between trampling damages on roots and wood decay in Norway spruce forests used for silvopasture

Forest grazing by free-roaming livestock is a common practice in many countries. The forestry sector sees the practice as unfortunate owing to several reasons, such as damages inflicted by grazing in young plantations. Concerning Norway spruce forests, a tree species known to develop wood decay with high frequency followed from stem bark damage, there is a strong perception among foresters that the trampling damage caused by livestock on the superficial root system of this tree leads to decay. Because of the very limited scientific documentation available on this topic, we pursued a clarification by investigating three 38- to 56-year-old Norway spruce forests used for silvopasture. Two types of injuries were observed on exposed roots: bark cracks characterized by resin exudation, and injuries involving localized bark peeling and exposure of the underlying wood. These injuries occurred up to 250 cm away from the root collar, with the sector 50–150 cm away from the root collar showing the highest incidence of injuries. In two of the forest stands, wood within the injured root areas was primarily colonized by the wound parasite Corinectria fuckeliana or species of the order Helotiales, fungi that do not cause wood decay. Wood colonization of injured roots by Heterobasidion species, the most frequent wood decay fungi of Norway spruce, was common in the third stand, but only in a few cases it was possible to deduce that the colonization had probably initiated via trampling injuries on roots. In a few cases, an injury was located at stem base at the root collar height along paths used by animals, and in such cases, it was obvious that stem colonization by Heterobasidion species had initiated via the wound. The relatively small amount of data warrants caution when drawing conclusions. Considering the high establishment frequency of decay via stem bark wounds of Norway spruce observed in previous studies, our data would suggest that roots are generally better equipped to defend themselves upon infliction of superficial wounds than stem of this tree species. The likelihood of trampling injuries leading to decay may vary considerably between different stands, this presumably depending on the level of local propagule pressure by pathogenic wood decay fungi and the frequency of damages close to root collar.

Multiple rearrangements and low inter- and intra-species mitogenome sequence variation in the Heterobasidion annosum s.l. species complex.

Mitochondria are essential organelles in the eukaryotic cells and responsible for the energy production but are also involved in many other functions including virulence of some fungal species. Although the evolution of fungal mitogenomes have been studied at some taxonomic levels there are still many things to be learned from studies of closely related species. In this study, we have analyzed 60 mitogenomes in the five species of the Heterobasidion annosum sensu lato complex that all are necrotrophic pathogens on conifers. Compared to other fungal genera the genomic and genetic variation between and within species in the complex was low except for multiple rearrangements. Several translocations of large blocks with core genes have occurred between the five species and rearrangements were frequent in intergenic areas. Mitogenome lengths ranged between 108 878 to 116 176 bp, mostly as a result of intron variation. There was a high degree of homology of introns, homing endonuclease genes, and intergenic ORFs among the five Heterobasidion species. Three intergenic ORFs with unknown function (uORF6, uORF8 and uORF9) were found in all five species and was located in conserved synteny blocks. A 13 bp long GC-containing self-complementary palindrome was discovered in many places in the five species that were optional in presence/absence. The within species variation is very low, among 48 H. parviporum mitogenomes, there was only one single intron exchange, and SNP frequency was 0.28% and indel frequency 0.043%. The overall low variation in the Heterobasidion annosum sensu lato complex suggests a slow evolution of the mitogenome.

In vitro screening of Latvian isolates of Bjerkandera adusta and Sistotrema brinkmannii as potential biocontrol agents against Heterobasidion root and butt rots

Root and butt rots caused by Heterobasidion spp. are destructive diseases of conifer forest stands that may be controlled by spraying freshly cut stumps with chemical or biological treatments, including the effective and widely used biological control agent Phlebiopsis gigantea. In this study, we tested in vitro different Latvian isolates of the fungal species Bjerkandera adusta and Sistotrema brinkmannii for their antagonistic activity against both Heterobasidion species present in Northern Europe and Baltic countries, namely H. annosum and H. parviporum, using Latvian isolates of P. gigantea and the Rotstop® strain as controls. The growth rate of isolates on agar media and their ability to produce oidia or conidia were also assessed. In general, B. adusta performed better than S. brinkmannii. Some isolates of B. adusta ranked similarly to those of P. gigantea in terms of oidia production, growth rate and ability to overgrow the mycelium of both H. annosum and H. parviporum. These isolates will be further tested in field conditions. Key words: biological control, antagonistic fungi, fungal isolates

Pyoluteorin Produced by the Biocontrol Agent Pseudomonas protegens Is Involved in the Inhibition of Heterobasidion Species Present in Europe.

Pseudomonas protegens (strain DSMZ 13134) is a biocontrol agent with promising antagonistic activity hinging on antibiosis against the fungal forest pathogens Heterobasidion spp. Here, by using High-Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS), we assessed whether monocultures of P. protegens (strain DSMZ 13134) produce the three major determinants of biocontrol activity known for the genus Pseudomonas: 2,4-diacetylphloroglucinol (2,4-DAPG), pyoluteorin (PLT), and pyrrolnitrin (PRN). At the tested culture conditions, we observed the production of PLT at concentrations ranging from 0.01 to 10.21 mg/L and 2,4-DAPG at a concentration not exceeding 0.5 mg/L. Variations of culture conditions involving culture medium, incubation temperature, and incubation period had no consistent influence on PLT production by the bacterium. Assays using culture medium amended with PLT at the same concentration of that present in cell-free filtrate of the bacterium, i.e., 3.77 mg/L, previously documented as effective against Heterobasidion spp., showed a remarkable activity of PLT against genotypes of all the four Heterobasidion species present in Europe, including the non-native invasive H. irregulare. However, such antifungal activity decreased over time, and this may be a constraint for using this molecule as a pesticide against Heterobasidion spp. When the bacterium was co-cultured in liquid medium with genotypes of the different Heterobasidion species, an increased production of PLT was observed at 4 °C, suggesting the bacterium may perform better as a PLT producer in field applications under similar environmental conditions, i.e., at low temperatures. Our results demonstrated the role of PLT in the inhibition of Heterobasidion spp., although all lines of evidence suggest that antibiosis does not rely on a single constitutively produced metabolite, but rather on a plethora of secondary metabolites. Findings presented in this study will help to optimize treatments based on Pseudomonas protegens (strain DSMZ 13134) against Heterobasidion spp.

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.

Comparative Efficacy of State-of-the-Art and New Biological Stump Treatments in Forests Infested by the Native and the Alien Invasive Heterobasidion Species Present in Europe.

The Heterobasidion annosum species complex includes major fungal pathogens of conifers worldwide. State-of-the-art preventative stump treatments with urea or with commercial formulations of the fungal biological control agent Phlebiopsis gigantea (i.e., Rotstop®) may become no longer available or are not approved for use in many areas of Europe infested by the three native Heterobasidion species and by the North American invasive H. irregulare, making the development of new treatments timely. The efficacy of Proradix® (based on Pseudomonas protegens strain DSMZ 13134), the cell-free filtrate (CFF) of the same bacterium, a strain of P. gigantea (MUT 6212) collected in the invasion area of H. irregulare in Italy, Rotstop®, and urea was comparatively investigated on a total of 542 stumps of Abies alba, Picea abies, Pinus pinea, and P. sylvestris in forest stands infested by the host-associated Heterobasidion species. Additionally, 139 logs of P. pinea were also treated. Results support the good performances of Rotstop®, and especially of urea against the native Heterobasidion species on stumps of their preferential hosts and, for the first time, towards the invasive North American H. irregulare on stumps of P. pinea. In some experiments, the effectiveness of Proradix® and of the strain of P. gigantea was weak, whereas the CFF of P. protegens strain DSMZ 13134 performed as a valid alternative to urea and Rotstop®. The mechanism of action of this treatment hinges on antibiosis; therefore, further improvements could be possible by identifying the active molecules and/or by optimizing their production. Generally, the performance of the tested treatments is not correlated with the stump size.

Biocontrol potential of Pseudomonas protegens against Heterobasidion species attacking conifers in Europe

The biocontrol potential of a commercial product based on the bacterium Pseudomonas protegens (strain DSMZ 13134) and of its cell-free filtrate (CFF) was tested under controlled conditions in vitro and on wood discs against genotypes of the three native European Heterobasidion species and on the non-native H. irregulare, all reported as destructive forest pathogens in Europe. In vitro experiments through traditional dual culture assays and by incorporating CFF into the media showed that treatments were effective in reducing significantly and regardless of the culture medium and incubation temperature both mycelial growth and rate of conidial germination of Heterobasidion spp., and that antibiosis could be the main mechanism involved in the inhibition of Heterobasidion spp. Outcomes of dual culture assays performed on two-divided Petri plates further suggest that antibiosis operates through the production of diffusible rather than volatile compounds. Based on comparative experiments on wood discs of preferential hosts of Heterobasidion spp., CFF performed significantly better than the commercial product against most of Heterobasidion spp., further confirming that the antagonistic activity hinges on antibiosis rather than on other mechanisms. While on wood discs the tested treatments seem poorly effective against the non-native H. irregulare, CFF significantly and substantially reduced infections of the native European Heterobasidion species compared to controls mostly to the level of the state of the art chemical treatment urea and of the biological treatment based on Phlebiopsis gigantea.

An Updated Global Species Diversity and Phylogeny in the Forest Pathogenic Genus Heterobasidion (Basidiomycota, Russulales).

Heterobasidion species are amongst the most intensively studied polypores because several species are aggressive white rot pathogens of managed coniferous forests mainly in Europe and North America. In the present study, both morphological and multilocus phylogenetic analyses were carried out on Heterobasidion samples from Asia, Oceania, Europe and North America. Three new taxa were found, i.e., H. armandii, H. subinsulare, and H. subparviporum are from Asia and are described as new species. H. ecrustosum is treated as a synonym of H. insulare. So far, six taxa in the H. annosum species complex are recognized. Heterobasidion abietinum, H. annosum, and H. parviporum occur in Europe, H. irregulare, and H. occidentale in North America, and H. subparviporum in East Asia. The North American H. irregulare was introduced to Italy during the Second World War. Species in the H. annosum complex are pathogens of coniferous trees, except H. subparviporum that seems to be a saprotroph. Ten species are found in the H. insulare species complex, all of them are saprotrophs. The pathogenic species are distributed in Europe and North America; the Asian countries should consider the European and North American species as entry plant quarantine fungi. Parallelly, European countries should consider the American H. occidentale and H. irregulare as entry plant quarantine fungi although the latter species is already in Italy, while North America should treat H. abietinum, H. annosum s.s., and H. parviporum as entry plant quarantine fungi. Eight Heterobasidion species found in the Himalayas suggest that the ancestral Heterobasidion species may have occurred in Asia.

New Taxon-Specific Heterobasidion PCR Primers Detect and Differentiate North American Heterobasidion spp. in Various Substrates and Led to the Discovery of Heterobasidion irregulare in British Columbia, Canada.

Heterobasidion annosum sensu lato is a species complex of pathogenic white-rot wood decay fungi which cause root and butt rot in conifer and hardwood species across the Northern hemisphere. Annual losses to forest managers are valued in the billions of dollars, due to tree mortality, reduction in timber yield, and wood decay. In North America, H. irregulare and H. occidentale have a partially overlapping host and geographic range, cause similar disease symptoms and produce similar fruiting bodies, making discrimination between the two of them often difficult. We developed two sets of primers that bind specifically to conserved, but species-specific portions of glyceraldehyde 3-phosphate dehydrogenase and elongation factor 1α alleles. The method is sensitive enough to detect either species from infected wood. Analysis of North American isolates has further clarified the distribution of both species on this continent, including the detection of H. irregulare for the first time on ponderosa pine (Pinus ponderosa) and eastern white pine (Pinus strobus) in British Columbia. This method has the potential to be a valuable tool for the detection of the pathogen in exported/imported wood products, as well as for the further identification and assessment of the distribution of North American Heterobasidion species.

Multiplex real‐time PCR assays for the detection and identification of Heterobasidion species attacking conifers in Europe

Four species of the destructive forest pathogen Heterobasidion annosum sensu lato (s.l.) are present in Europe: H. annosum sensu stricto (s.s.), H. abietinum and H. parviporum are native species, while H. irregulare is a non‐native invasive species currently reported only in Italy, yet recommended for regulation throughout Europe. In this study, real‐time PCR detection tests were developed for each of the four species, which can be used simultaneously or individually thanks to probes labelled with species‐specific fluorescent dyes. The different performance criteria of each assay were evaluated, and it was determined that they were theoretically capable of detecting amounts of DNA corresponding to 311, 29 and 29 cell nuclei in H. annosum s.s., H. irregulare and H. parviporum, respectively. The specificity of each assay was assessed with a wide set of strains. Real‐time PCR tests successfully detected Heterobasidion species from 36 fruiting bodies taken from the forest, as well as from artificially inoculated or naturally infected wood samples. The multiplex real‐time PCR assays developed in this study could have practical applications both in forest management and in phytosanitary monitoring.

Heterobasidion species detected using High Throughput Sequencing (HTS) methods on British Columbia nursery plants

A total of 15 out of 26 local and imported plant samples from British Columbia nurseries were obtained, and following DNA extraction, PCR was conducted to amplify the fungal ribosomal ITS region which was sequenced using High Throughput Sequencing methods. Bioinformatics analysis of these sequences revealed the presence of Heterobasidion irregulare and H. occidentale. A total of 529 Heterobasidion Illumina DNA sequence reads were found in the 13/15 successfully PCR amplified plant samples, with H. irregulare comprising 86% of the Heterobasidion DNA reads and was more commonly found (13/15 samples) than H. occidentale (10/15 samples). Previously, H. irregulare has been mentioned only once before as being present in BC. The presence of Heterobasidion DNA in nearly all successfully PCR amplified samples from plants grown locally or recently imported, is interpreted as the result of local spore dispersal, likely originating from nearby infected trees. This raises the question whether asymptomatic nursery plants can participate in the spread of H. irregulare in BC and elsewhere in Canada.

Molecular analyses identify hybridization-mediated nuclear evolution in newly discovered fungal hybrids.

Hybridization may be a major driver in the evolution of plant pathogens. In a high elevation Alpine larch stand in Montana, a novel hybrid fungal pathogen of trees originating from the mating of Heterobasidion irregulare with H. occidentale has been recently discovered. In this study, sequence analyses of one mitochondrial and four nuclear loci from 11 Heterobasidion genotypes collected in the same Alpine larch stand indicated that hybridization has increased allelic diversity by generating novel polymorphisms unreported in either parental species. Sequence data and ploidy analysis through flow cytometry confirmed that heterokaryotic (n + n) genotypes were not first‐generation hybrids, but were the result of multiple backcrosses, indicating hybrids are fertile. Additionally, all admixed genotypes possessed the H. occidentale mitochondrion, indicating that the hybrid progeny may have been backcrossing mostly with H. occidentale. Based on reticulate phylogenetic network analysis by PhyloNet, Bayesian assignment, and ordination tests, alleles can be defined as H. irregulare‐like or H. occidentale‐like. H. irregulare‐like alleles are clearly distinct from all known H. irregulare alleles and are derived from the admixing of both Heterobasidion species. Instead, all but one H. occidentale alleles found in hybrids, although novel, were not clearly distinct from alleles found in the parental H. occidentale population. This discovery demonstrates that Alpine larch can be a universal host favouring the interspecific hybridization between H. irregulare and H. occidentale and the hybridization‐mediated evolution of a nucleus, derived from H. irregulare parental species but clearly distinct from it.

Heterobasidion-growth inhibiting Bacillus subtilis A18 exhibits medium- and age-dependent production of lipopeptides

Heterobasidion annosum s.s. and H. parviporum are severe pathogens of conifers causing butt rot and root rot thus reducing the economic value of timber. Here, the antifungal activity of Bacillus subtilis isolate A18 against these two Heterobasidion species was investigated. Five different culture media with different culture age were investigated to study the effect of substrate composition and culture age for metabolite production. Bacterial cultures and cell-free culture filtrates were tested for antifungal activity. Inhibition of fungal growth was analysed using the agar disc-diffusion method. MALDI-TOF and LC-HRMS analyses were used to identify the antifungal metabolites. Substrate composition and age of culture were found to be active variables with direct effect on the antifungal activity of bacterial culture extracts. High anti-fungal activity was observed when B. subtilis was cultured in PDB, SGB and LB media for four days. Mass-spectrometry analysis showed the presence of lipopeptides in culture filtrates identified as members of the surfactins, polymixins, kurstakins and fengycins. A culture filtrate containing fengycin-type lipopeptides showed the highest bioactivity against Heterobasidion species. Bacterial cultures had higher bioactivity compared to their respective cell free culture filtrates. The results of the present study suggest that B. subtilis A18 is a powerful biocontrol agent against Heterobasidion infections of tree wounds and stumps.

Screening for Exotic Forest Pathogens to Increase Survey Capacity Using Metagenomics.

Anthropogenic activities have a major impact on the global environment. Canada's natural resources are threatened by the spread of fungal pathogens, which is facilitated by agricultural practices and international trade. Fungi are introduced to new environments and sometimes become established, in which case they can cause disease outbreaks resulting in extensive forest decline. Here, we describe how a nationwide sample collection strategy coupled to next-generation sequencing (NGS) (i.e., metagenomics) can achieve fast and comprehensive screening for exotic invasive species. This methodology can help provide guidance to phytopathology stakeholders such as regulatory agencies. Several regulated invasive species were monitored by processing field samples collected over 3 years (2013 to 2015) near high-risk areas across Canada. Fifteen sequencing runs were required on the Ion Torrent platform to process 398 samples that yielded 45 million reads. High-throughput screening of fungal and oomycete operational taxonomic units using customized fungi-specific ribosomal internal transcribed spacer 1 barcoded primers was performed. Likewise, Phytophthora-specific barcoded primers were used to amplify the adenosine triphosphate synthase subunit 9-nicotinamide adenine dinucleotide dehydrogenase subunit 9 spacer. Several Phytophthora spp. were detected by NGS and confirmed by species-specific quantitative polymerase chain reaction (qPCR) assays. The target species Heterobasidion annosum sensu stricto could be detected only through metagenomics. We demonstrated that screening target species using a variety of sampling techniques and NGS-the results of which were validated by qPCR-has the potential to increase survey capacity and detection sensitivity, reduce hands-on time and costs, and assist regulatory agencies to identify ports of entry. Considering that early detection and prevention are the keys in mitigating invasive species damage, our method represents a substantial asset in plant pathology management.

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.

The complete genome sequence of HetPV20-an1, an alphapartitivirus infecting the conifer-pathogenic fungus Heterobasidion annosum.

Root rot fungi of the genus Heterobasidion are highly destructive conifer pathogens in the northern Boreal forest region. This report describes the complete genome sequence of Heterobasidion partitivirus 20 infecting a Finnish strain of Heterobasidion annosum. The bisegmented dsRNA genome of HetPV20-an1 encodes a predicted RNA-dependent RNA polymerase of 605 amino acids (aa) and a capsid protein of 536 aa. Based on sequence similarity and phylogenetic analysis, this virus is a new member of the genus Alphapartitivirus. HetPV20-an1 shares ~65% RdRP aa sequence identity with the most similar virus strain, Rosellinia necatrix partitivirus 2, whereas the CP of HetPV20-an1 is most similar to that of rose partitivirus with ~27% overall aa sequence identity. HetPV20-an1 is only distantly related to previously known partitiviruses of Heterobasidion species and shares ~29% RdRP aa sequence identity and ~16% CP aa sequence identity with Heterobasidion partitivirus 1 from H. abietinum.

Erratum to: 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.

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.

Comparative Genomics of Sibling Fungal Pathogenic Taxa Identifies Adaptive Evolution without Divergence in Pathogenicity Genes or Genomic Structure.

It has been estimated that the sister plant pathogenic fungal species Heterobasidion irregulare and Heterobasidion annosum may have been allopatrically isolated for 34–41 Myr. They are now sympatric due to the introduction of the first species from North America into Italy, where they freely hybridize. We used a comparative genomic approach to 1) confirm that the two species are distinct at the genomic level; 2) determine which gene groups have diverged the most and the least between species; 3) show that their overall genomic structures are similar, as predicted by the viability of hybrids, and identify genomic regions that instead are incongruent; and 4) test the previously formulated hypothesis that genes involved in pathogenicity may be less divergent between the two species than genes involved in saprobic decay and sporulation. Results based on the sequencing of three genomes per species identified a high level of interspecific similarity, but clearly confirmed the status of the two as distinct taxa. Genes involved in pathogenicity were more conserved between species than genes involved in saprobic growth and sporulation, corroborating at the genomic level that invasiveness may be determined by the two latter traits, as documented by field and inoculation studies. Additionally, the majority of genes under positive selection and the majority of genes bearing interspecific structural variations were involved either in transcriptional or in mitochondrial functions. This study provides genomic-level evidence that invasiveness of pathogenic microbes can be attained without the high levels of pathogenicity presumed to exist for pathogens challenging naïve hosts.

Identification of genes differentially expressed during the interaction between the plant symbiont Suillus luteus and two plant pathogenic allopatric Heterobasidion species

The effects of biological invasions by non-native species have been widely studied in terms of environmental, economic, and human health impacts. However, little is known about the consequences that non-native plant pathogens may determine on host plant symbionts, such as ectomycorrhizal (ECM) fungi. In this study, interactions between Suillus luteus, an ECM fungus of pine trees, and the allopatrically differentiated fungal pathogens of pines Heterobasidion irregulare and H. annosum were investigated in dual culture by morphological and gene expression analyses. Growth of S. luteus was inhibited by both Heterobasidion species, but based on statistical analysis, growth inhibition was due to the isolate rather than to the species. The expression analysis on genes related to cell wall hydrolytic enzymes and hydrophobins, putatively involved in the fungus–fungus interaction, allowed to identify significantly up- and down-regulated genes both in the symbiont and in the pathogens. Based on the transcript analysis, it was not possible to distinguish the impact of the two pathogenic species on the ECM fungus. The only exception was a S. luteus gene coding for a putative chitinase (SlGH18_8356) that was found to be differentially regulated during interaction with H. irregulare compared to H. annosum.