Heterobasidion Annosum Sensu Lato Research Articles

Diversity and impact of single-stranded RNA viruses in Czech Heterobasidion populations.

Heterobasidion annosum sensu lato 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 H. annosum 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 H. annosum and Heterobasidion parviporum, 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 Heterobasidion 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 Heterobasidion 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 in planta.IMPORTANCEHeterobasidion annosum sensu lato 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 H. annosum 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 Heterobasidion. 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.

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.

Changes to relative stand composition after almost 50 years of Heterobasidion root disease in California true fir and pine forests

AbstractThe Agaricomycete Heterobasidion annosum sensu lato (s.l.), a species complex of fungal pathogens, causes root and butt rot on conifers throughout the northern hemisphere, thereby shaping structure, composition, and evolution of vast and diverse forest ecosystems. We analyze forest change 48–49 years following Heterobasidion root disease emergence in 63 permanent plots first established in 1970–1972 and measured at least once every decade through 2020. We use this dataset to infer the long‐term consequences of Heterobasidion root disease in a set of common forest types that reflect some of the most important hosts for this set of diseases in western North America. We contrasted three host–pathogen systems located within the Sierra Nevada, southern Cascades Mountain Range and the Modoc Plateau: (1) H. irregulare in host communities dominated by several Pinus (pine) species and Calocedrus decurrens (incense cedar) in Yosemite Valley, on the Sierra Nevada Range western slope; (2) H. irregulare in a largely single species (Pinus jeffreyi) host disease system on the eastern slopes of the Sierra Nevada and southern Cascades as well as the Modoc Plateau; and (3) H. occidentale—also on the western slope of the Sierra Nevada—in stands with a mixture of susceptible Abies (true fir) and a diversity of non‐hosts for this Heterobasidion species. Approximately 50 years after disease emergence, relative basal area and stem density were significantly reduced within disease centres in all three pathosystems, but changes to forest composition and relative species dominance were determined by pre‐disease host and non‐host diversity. In the western‐slope H. occidentale system, the disease increased the dominance of non‐susceptible species, Pinus species and C. decurrens. In the multi‐genus Yosemite pathosystem, H. irregulare did not significantly shift species dominance, and as expected, species shifts did not occur in the largely single‐genus eastern‐slope forests. In these widespread California forest ecosystems, two factors appeared to determine forest conditions almost 50 years after Heterobasidion root disease monitoring: the size of the disease centre and the initial diversity of non‐hosts. Along with pathogen species, these factors appear to affect the local severity of disease as well as the degree of species shifts relative to initial plot compositions, reaffirming host susceptibility classifications associated with these forest types.

Phlebiopsis gigantea strains from Estonia show potential as native biocontrol agents against Heterobasidion root rot and contain diverse dsRNA and ssRNA viruses

Coniferous forests, especially Norway spruce (Picea abies L.) stands, are threatened by root rot disease caused by Heterobasidion annosum sensu lato (Fr.) Bref. The basidiomycetous fungus, Phlebiopsis gigantea (Fr.) Donk, is used as a biocontrol agent against this pathogen in several countries. In Estonia, stump treatments are currently conducted using the Rotstop preparation, which consists of a P. gigantea strain isolated in Finland. However, for ecological reasons it would be preferable to use an indigenous biocontrol strain. In this study, we tested new strains of P. gigantea isolated from Norway spruce stumps in Estonia for their growth rate and competitive ability against H. parviporum and H. annosum, and identified P. gigantea strains promising for biocontrol purposes. As viruses may cause phenotypic debilitation in fungi, we analyzed the virome of these strains by high-throughput sequencing (RNA-Seq) and assessed whether the presence of mycoviruses affects their performance as compared to virus-free strains of P. gigantea. Viruses related to members of Partitiviridae and Curvulaviridae, as well as unclassified ambi-like, aspi-like and “fusariviruses” were identified for the first time in P. gigantea.

The Profitability of Cross-Cutting Practices in Butt-Rotten Picea abies Final-Felling Stands

Research Highlights: This study offers new information on the cross cutting of decayed stems with the sounding of short (0.5 m) offcuts and the bucking of longer (3.0 m) butt-rotten poles. Background and Objectives: The root and butt-rot fungus Heterobasidion annosum sensu lato (Fr.) Bref. causes wood quality damage to trees in softwood forests. When timber is harvested in butt-rotten forests, it is essential that the decayed part of the tree is recognized and cut away from a stem, while the healthy and good quality log section of a stem is cross cut with precision sawlogs. The objective of the study was to investigate the impact of two off-cutting methods on stem processing time, cutting productivity, sawlog volume, and commercial value at the roadside landing when harvesting timber from the butt-rotten Norway spruce (Picea abies (L.) Karst.) final-felling forests. Materials and Methods: The length of the short offcuts used was 0.5 m. The results of the cross-cutting practices were compared to the decayed pulpwood poles of 3 m from the butt of the rotten stems. Time and motion studies were carried out in stands before the profitability calculations. The study data consisted of 1980 Norway spruce sawlog stems. Results: Sounding of the short offcuts added significantly to the stem processing time of butt-rotten stems, but the sawlog volume and the timber value recovery of the stems were higher than those of the decayed pulpwood poles of 3 m. Conclusions: The study concluded that sounding of butt-rotten Norway spruce stems with one to three offcuts is economically profitable if the diameter of the decayed column at the stem stump’s height is small (≤5 cm). In contrast, when the width of the decay is larger (>5 cm), it is more profitable to first cross cut the decayed pulpwood pole of 3 m and then to observe the height of the decayed part of the stem.

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.

Frequency of stump infections by Heterobasidion annosum s.l. and benefits from urea treatments vary with tree species and season in European Alpine forests

The destructive forest pathogen Heterobasidion annosum sensu lato (s.l.) may be controlled either by timing logging operations in periods of the year characterised by a low risk of airborne stump infection or by treating stump surfaces immediately after felling. 1616 stumps of four of the most widely-distributed European host tree species, either untreated or manually and operationally treated with urea, were sampled from 29 pure and mixed forest stands of the western Italian Alps and analysed for the presence of the pathogen. The aims of the study were to (i) compare the overall and seasonal frequency of stump infection of different tree species; (ii) assess the efficacy of treatments on different tree species; (iii) explore if the efficacy of treatment is affected by the season of application, and (iv) estimate the benefits resulting from treatments at different time periods. Overall, the frequency of infection of untreated stumps was significantly higher (P < 0.05) in Norway spruce (Picea abies) than in European larch (Larix decidua), silver fir (Abies alba) and Scots pine (Pinus sylvestris). In general, the frequency of stump infection of each tree species increased throughout the year, reaching a maximum in autumn, possibly in relation to the higher availability of airborne inoculum in that season. Manual operational stump treatment with urea reduced the frequency of infected stumps compared to controls both overall and for every tree species, although the reduction was significant only in the overall analysis and in the analysis performed separately for Norway spruce and larch. Binary logistic regression models point to the general efficacy of treatments regardless of the season of application, although the frequency of infection of treated stumps is predicted to increase slightly in autumn. Despite the efficacy of treatments being slightly reduced in autumn, the predicted reduction of stump infection probability due to treatments, hence the benefits provided by treatments, is expected to vary in magnitude depending on the species but to increase similarly according to a roughly sigmoid curve throughout the year, approaching a maximum in autumn. The information provided by this paper may help forest managers to design profitable and/or sustainable tactics to control the disease.

Characterization of a Heterobasidion irregulare endo‐rhamnogalacturonase that mediate growth on pectin

AbstractHeterobasidion irregulare is one of five Heterobasidion annosum sensu lato (s.l.) species, which are destructive pathogens in boreal and temperate forests of the northern hemisphere that causes root and butt rot in conifer. A gene encoding endo‐rhamnogalacturonase (HIRHG), which belongs to the glycoside hydrolase family 28 (GH28), was found in a quantitative trait loci (QTL) region for virulence in Heterobasidion. In this study, we showed that HIRHG is highly upregulated during necrotrophic infection of Norway spruce compared with growth in liquid culture and that the HIRHG encoded protein is produced during fungal growth on complex carbon sources. Phylogenetic analysis of endo‐rhamnogalacturonases revealed that rhamnogalacturonase genes have been lost in most of the biotrophic and hemibiotrophic plant pathogens investigated but were common in necrotrophic pathogens and saprophytic fungi. Heterologous expression of the HIRHG gene in the hemibiotrophic fungus Magnaporthe oryzae increased its capacity to grow on pectin; however, the transformed M. oryzae isolates showed significant less infection of rice leaves compared to the wild type.

Small secreted proteins from the necrotrophic conifer pathogen Heterobasidion annosum s.l. (HaSSPs) induce cell death in Nicotiana benthamiana

The basidiomycete Heterobasidion annosum sensu lato (s.l.) is considered to be one of the most destructive conifer pathogens in the temperate forests of the northern hemisphere. H. annosum is characterized by a dual fungal lifestyle. The fungus grows necrotrophically on living plant cells and saprotrophically on dead wood material. In this study, we screened the H. annosum genome for small secreted proteins (HaSSPs) that could potentially be involved in promoting necrotrophic growth during the fungal infection process. The final list included 58 HaSSPs that lacked predictable protein domains. The transient expression of HaSSP encoding genes revealed the ability of 8 HaSSPs to induce cell chlorosis and cell death in Nicotiana benthamiana. In particular, one protein (HaSSP30) could induce a rapid, strong, and consistent cell death within 2 days post-infiltration. HaSSP30 also increased the transcription of host-defence-related genes in N. benthamiana, which suggested a necrotrophic-specific immune response. This is the first line of evidence demonstrating that the H. annosum genome encodes HaSSPs with the capability to induce plant cell death in a non-host plant.

Heterotrimeric G-proteins in Picea abies and their regulation in response to Heterobasidion annosum s.l. infection.

BackgroundHeterotrimeric G-proteins are important signalling switches, present in all eukaryotic kingdoms. In plants they regulate several developmental functions and play an important role in plant-microbe interactions. The current knowledge on plant G-proteins is mostly based on model angiosperms and little is known about the G-protein repertoire and function in other lineages. In this study we investigate the heterotrimeric G-protein subunit repertoire in Pinaceae, including phylogenetic relationships, radiation and sequence diversity levels in relation to other plant linages. We also investigate functional diversification of the G-protein complex in Picea abies by analysing transcriptional regulation of the G-protein subunits in different tissues and in response to pathogen infection.ResultsA full repertoire of G-protein subunits in several conifer species were identified in silico. The full-length P. abies coding regions of one Gα-, one Gβ- and four Gγ-subunits were cloned and sequenced. The phylogenetic analysis of the Gγ-subunits showed that PaGG1 clustered with A-type-like subunits, PaGG3 and PaGG4 clustered with C-type-like subunits, while PaGG2 and its orthologs represented a novel conifer-specific putative Gγ-subunit type. Gene expression analyses by quantitative PCR of P. abies G-protein subunits showed specific up-regulation of the Gα-subunit gene PaGPA1 and the Gγ-subunit gene PaGG1 in response to Heterobasidion annosum sensu lato infection.ConclusionsConifers possess a full repertoire of G-protein subunits. The differential regulation of PaGPA1 and PaGG1 indicates that the heterotrimeric G-protein complex represents a critical linchpin in Heterobasidion annosum s.l. perception and downstream signaling in P. abies.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0676-1) contains supplementary material, which is available to authorized users.

Risk of False Positives during Sampling for Heterobasidion annosum s.l.

A standard method to detect infection by Heterobasidion annosum sensu lato (s.l.) in stumps or stems is to cut a disc and examine it under a microscope. Concerns have been raised that spores can be transferred from the bark to the cut surface, thus contaminating the sample. The aims of this study were to test whether viable basidiospores of H. annosum s.l. can be transferred from the bark onto disc surfaces by a chainsaw and to investigate the impacts of different sampling procedures on the extent of contaminations. Logs were cut with or without adding basidiospores to the bark prior to the cut. Infection measurements were significantly greater for discs with treated bark (100% infected, infection coverage 40 cm2 dm-2 of disc area) compared with control discs (47% infected, infection coverage 0.2 to 0.3 cm2 dm-2). In addition, trees were sampled under authentic field conditions using different procedures. The infection measurements differed significantly depending on the procedure; sampling involving debarking or disinfection of the bark with 70% ethanol prior to cutting had lower measurements (6 to 19% and 13% infected, respectively) compared with leaving the bark on untreated (63 to 75% infected). Consideration of the contamination risk is warranted when evaluating the results of earlier studies and when planning new experiments.

Transcriptional Responses Associated with Virulence and Defence in the Interaction between Heterobasidion annosum s.s. and Norway Spruce.

Heterobasidion annosum sensu lato is a serious pathogen causing root and stem rot to conifers in the northern hemisphere and rendering the timber defective for sawing and pulping. In this study we applied next-generation sequencing to i) identify transcriptional responses unique to Heterobasidion-inoculated Norway spruce and ii) investigate the H. annosum transcripts to identify putative virulence factors. To address these objectives we wounded or inoculated 30-year-old Norway spruce clones with H. annosum and 454-sequenced the transcriptome of the interaction at 0, 5 and 15 days post inoculation. The 491860 high-quality reads were de novo assembled and the relative expression was analysed. Overall, very few H. annosum transcripts were represented in our dataset. Three delta-12 fatty acid desaturase transcripts and one Clavaminate synthase-like transcript, both associated with virulence in other pathosystems, were found among the significantly induced transcripts. The analysis of the Norway spruce transcriptional responses produced a handful of differentially expressed transcripts. Most of these transcripts originated from genes known to respond to H. annosum. However, three genes that had not previously been reported to respond to H. annosum showed specific induction to inoculation: an oxophytodienoic acid–reductase (OPR), a beta–glucosidase and a germin-like protein (GLP2) gene. Even in a small data set like ours, five novel highly expressed Norway spruce transcripts without significant alignment to any previously annotated protein in Genbank but present in the P. abies (v1.0) gene catalogue were identified. Their expression pattern suggests a role in defence. Therefore a more complete survey of the transcriptional responses in the interactions between Norway spruce and its major pathogen H. annosum would probably provide a better understanding of gymnosperm defence than accumulated until now.

Accumulation of iron-binding compounds in root of Pinus sylvestris challenged by Heterobasidion annosum sensu lato

Accumulation of iron-binding compounds in root of Pinus sylvestris challenged by Heterobasidion annosum sensu lato

Underground spaces as neglected niche for occurrence ofHeterobasidion annosumcomplex

Summary The serious forest pathogen Heterobasidion annosum sensu lato was found during mycological exploration in caves and underground tunnels. Seventeen isolates of Heterobasidion annosum s.l. were obtained in these habitats in the Czech Republic, Slovakia, Romania and Spain during 2008–2012. Three species, H. abietinum, H. annosum sensu stricto, and H. parviporum, were identified by analyses of DNA sequences of three gene regions: glyceraldehyde 3-phosphate dehydrogenase, translation elongation factor 1-alfa and transcription factor. Isolates of H. parviporum were obtained from moonmilk, air and underground gallery wall. H. parviporum recorded from Spain, Castaňar de Ibor Cave, was obtained far from the natural distribution of its main host, Picea abies. Heterobasidion annosum was obtained from cave air and sediment, isopod faeces and underground gallery wall. Heterobasidion abietinum was isolated from bat guano and outdoor air. The origin and ways of fungal dispersal are discussed. The forest stands surrounding caves and tunnels are hypothesized as the potential source of inoculum to inhabit these unusual spaces, although the real paths of the pathogen to the habitats remain unclear.

A molecular diagnostic assay for the detection and identification of wood decay fungi of conifers

SummaryTen taxon‐specific primers were designed to amplify the Internal Transcribed Spacer of the rRNA operon of several important decay fungi of coniferous wood, including Armillaria spp., Echinodontium spp., Fomitopsis pinicola, Fuscoporia torulosa, Heterobasidion annosum sensu lato (s.l.), Onnia spp., Phaeolus schweinitzii, Phellinus weirii s.l., Pholiota spp. and Porodaedalea spp. Primers designed in this study and in a previous one for the identification of Laetiporus sulphureus and Stereum spp. were combined in two multiplex PCRs, which were tested for efficiency and specificity, and detected at least 1 pg of fungal target DNA. Target DNA at concentrations of 10−1 pg or lower can be detected with this assay using SYBR® Green Real‐Time PCR. Validation assays performed on 129 naturally infected wood samples or fruiting bodies confirmed the reliability of the multiplex PCR‐based diagnostic method. This method represents a simple and rapid diagnostic tool for the detection of a number of destructive wood decay fungi of conifer wood.

Effects of the use of biocontrol agent (Phlebiopsis gigantea) on fungal communities on the surface of Picea abies stumps

One of the main objectives of sustainable forestry policies is to phase out, if possible, the use of chemical agents in forestry. The saprotrophic fungus Phlebiopsis gigantea has for several years been used as a biocontrol agent against the pathogen Heterobasidion annosum sensu lato. This pathogen is the major cause of root rot disease in conifers that results in economic losses estimated at 50 million euros to Finnish forestry industry annually. A major problem is that, although the effectiveness of P. gigantea as a bio-control agent has empirically been shown, the long term biological effect of this fungus on conifer trees as well as on other wood microflora has not been empirically proven. We investigated the impact of P. gigantea treatment on stump mycobiota using 454-pyrosequencing approach as this has not done before. Samples from forest sites pre-treated with P. gigantea for 1, 6 and 13years ago were collected, DNA was isolated and the ITS regions were pyrosequenced. Similarly samples were also collected from untreated stumps within the same forest site over the same period of time. A total of 53,117 fungal sequences were generated by 454-pyrosequencing from 18 wood samples. After data cleaning we had 26,127 sequences representing 49% of the original sequences. Possible impact of the treatment on fungal communities in different study sites was analyzed using Principal Component Analysis (PCA). Also diversity indexes and similarity indexes between controls and treated stumps were calculated. Biocontrol P. gigantea represented 0.43% of all fungal sequences and was found only from stumps after one year post-treatment. The pathogen H. annosum s.l. was not observed in this study. Numbers of different OTUs were always higher in the control stumps. However there was no statistical difference noted between treated and non-treated stumps. Based on our results we can conclude that stump treatment should continue as there is no obvious adverse effect on the other stump mycobiota.

Biology, Epidemiology, and Control ofHeterobasidionSpecies Worldwide

Heterobasidion annosum sensu lato is a species complex comprising five species that are widely distributed in coniferous forests of the Northern Hemisphere and are each characterized by a distinct host preference. More than 1,700 papers have been published on these fungi in the past four decades, making them perhaps the most widely studied forest fungi. Heterobasidion species are at different levels on the saprotroph-necrotroph gradient, and the same individual can switch from one mode to the other. This offers a unique opportunity to study how genomic structure, gene expression, and genetic trade-offs may all interact with environmental factors to determine the life mode of the organism. The abilities of Heterobasidion spp. to infect stumps as saprotrophs and to spread to neighboring trees as pathogens have resulted in significant damages to timber production in managed forests. This review focuses on the current knowledge of the biology, ecology, evolution, and management of these species and is based on classical and modern studies.

Heartwood stump colonisation by Heterobasidion parviporum and H. annosum s.s. in Norway spruce (Picea abies) stands

Stump-to-tree spread is a key step in the root and butt-rot disease caused by Heterobasidion annosum sensu lato. In Norway spruce stands, Heterobasidion parviporum is more likely to spread to neighbouring trees from stumps with a large diameter than from small stumps. However, H. annosum sensu stricto does not appear to share this pattern of infection. Given that the ratio of heartwood to sapwood varies depending on the size of the stump, we investigated the role of heartwood and sapwood in stump infection by H. parviporum and H. annosum s.s. To determine whether heartwood–sapwood susceptibility was different in different host species, both Norway spruce and Scots pine stumps were included in the study. Sapwood, heartwood and the border zone between sapwood and heartwood of spruce stumps and the heartwood and sapwood of pine stumps were artificially inoculated with different genotypes of H. annosum s.s. and H. parviporum. Only one zone was inoculated per stump. Infection was assessed 2 and 10months after inoculation at 5 and 30cm below the point of inoculation. On spruce stumps, H. parviporum and H. annosum s.s. were mostly confined to the heartwood. The area infected by H. annosum s.s. was smaller than that infected by H. parviporum. Both sapwood and border infections tended to grow towards the heartwood, whereas heartwood inoculations tended to remain in the heartwood, indicating that heartwood was more favourable for H. parviporum and H. annosum s.s. colonisation in spruce stumps. On pine, heartwood colonisation failed and only H. annosum s.s. was found in the sapwood. Our results suggest a possible association between the size of the stump and the spread of H. annosum s.s. to neighbouring trees mediated by the heartwood content, suggesting that screening for heartwood resistance could be a potentially interesting direction for future research. Treating stumps at final felling would be advisable owing to the large area of heartwood exposed by this operation.

Biosynthesis of fomannoxin in the root rotting pathogen Heterobasidion occidentale

Fomannoxin is a biologically active benzohydrofuran, which has been suggested to be involved in the pathogenicity of the root rotting fungus Heterobasidion annosum sensu lato. The biosynthesis of fomannoxin was investigated through an isotopic enrichment study utilizing [1-¹³C]glucose as metabolic tracer. ¹³C NMR spectroscopic analysis revealed the labeling pattern and showed that the isoprene building block originates from the mevalonic acid pathway, whereas the aromatic motif is formed via the shikimic acid route by elimination of pyruvate from chorismic acid. A natural product, 4-hydroxy-3-(3-methylbut-2-enyl)benzaldehyde, was isolated and characterized, and was suggested to be a key intermediate in the biosynthesis of fomannoxin and related secondary metabolites previously identified from the H. annosum fungal species complex.