Search

Root and butt rot caused by pathogenic fungi in the genera Heterobasidion and Armillaria is a pressing issue in managed Norway spruce forests. The disease results in financial losses for the forest owners and reduces the volume of wood that can be used in long‐lived products. Pathogenic wood decay fungi spread either with the aid of airborne spores or via mycelial growth among neighbouring trees, the latter leading to clustering (tendency of decayed trees to be in close proximity relative to their neighbouring trees) of decay‐affected trees in forests. Understanding the spatial patterns of the decay‐affected trees at the forest stand level is vital for designing management strategies to address this problem.We examined decay clustering in 273 clear‐cut Norway spruce stands in Norway using harvester‐recorded data on spatial occurrence of decayed and healthy Norway spruce trees. We tested clustering using three global‐cluster tests that account for population density and distribution, evaluating clustering without identifying specific cluster locations.The proportions of clustered and non‐clustered stands differed depending on the statistical test used for clustering assessment, resulting in overall agreement of 32.8% for clustered and 36.9% for non‐clustered. Clustered stands exhibited a median cluster distance (maximum distance between the decay‐affected trees within a cluster) of 12 m (Inter‐Quantile Range, IQR, 6–20 m) and a median of 6 (IQR 3–16) nearest neighbour trees (number of decayed trees forming a cluster), estimates comparable with prior studies focused on assessment of trees infected by mycelial spread of the same fungal individual. The decay incidence in the clustered stands was 16.24%, while the non‐clustered stands had a butt‐rot incidence of 20.97%. In clustered stands the average number of trees per hectare was higher (693) than in non‐clustered stands (553).Synthesis and applications: Our study demonstrates that Norway spruce stands display a diverse range of spatial patterns of butt rotted trees. We found that higher densities of Norway spruce trees probably facilitate the vegetative spread of pathogenic wood decay fungi, leading to clustering of decay‐affected trees. To disrupt the spread of decay fungi between tree generations, precision planting of trees other than Norway spruce around infested stumps of prior generation trees has been recommended by earlier studies. We discussed the potential of using harvester‐derived geoposition data for butt‐rotted trees upon planning and execution of forest regeneration. [ABSTRACT FROM AUTHOR]

Heterobasidion, a significant forest pathogen affecting coniferous forests in the Northern Hemisphere, can infect 27 species of coniferous trees, leading to widespread forest mortality. It has already caused considerable damage to both natural and plantation forests in Europe. As essential components of the global ecosystem, forests are increasingly affected by ongoing environmental changes. The ability to accurately predict and effectively respond to pathogen outbreaks across different habitats is becoming increasingly critical. This study employs an optimized MaxEnt model in conjunction with six Global Climate Models (GCMs) to simulate and predict the potentially suitable distributions and changes of three Heterobasidion species in Europe (Heterobasidion abietinum, Heterobasidion annosum sensu stricto, Heterobasidion parviporum) under current conditions and four future climate scenarios (SSP126, SSP245, SSP370, and SSP585) for the period 2081–2100. The objective of this analysis is to assess the potential effects of climate change on the distribution of Heterobasidion species. The results indicate that the distributions of the three Heterobasidion species are influenced by factors such as temperature and precipitation. By 2081–2100, under most climate scenarios, except for the SSP585 scenario, the distribution areas of the three Heterobasidion species show an expansion trend. Notably, Heterobasidion abietinum showed the most significant expansion of its suitable habitat, while the expansion of low-suitability areas for Heterobasidion parviporum and Heterobasidion annosum sensu stricto was more pronounced. Heterobasidion abietinum is projected to shift southward due to factors such as precipitation, while Heterobasidion parviporum and Heterobasidion annosum sensu stricto are expected to migrate northward, influenced by factors such as temperature and host tree species. [ABSTRACT FROM AUTHOR]

Heterobasidion parviporum is a destructive forest pathogen causing root and stem rot of Norway spruce (Picea abies) in northern hemisphere. Previous research has suggested that a gene form of PaLAR3 that encodes for the leucoanthocyanidin reductase enzyme, may increase resistance to H. parviporum in Norway spruce, whether heterozygous or homozygous. Our experiment was initially focused on testing this assertion with materials from the Finnish Norway spruce breeding program, comprising 52 full-sib families. Additionally, we investigated the influence of mycoviruses on the growth of H. parviporum. The frequencies of the three PaLAR3 genotypes AA, AB, and BB were 64%, 30%, and 6%, respectively. We found no significant differences in the necrotic response to H. parviporum between the homozygous (BB) and heterozygous (AB) genotypes vs. the homozygous (AA) genotype across the two fungal strains tested, suggesting the gene is rather ineffective as a marker for overall H. parviporum resistance. The two H. parviporum strains used in the study showed significantly different necrosis responses from the control. Mycovirus infection in H. parviporum strains led to a significant reduction in lesion width in phloem and sapwood. Furthermore, the growth of the fungus was notably restricted when hyphae hosted mycoviruses, indicating a high potential of mycoviruses to reduce fungal growth and necrosis development.

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.

The Heterobasidion annosum species complex is a major threat to the forest industry in the northern temperate regions of the world. The fungal and bacterial biota communities in Heterobasidion-infected trees have been extensively studied, but less is known about the interkingdom and intrakingdom interactions among the microbiome. The bacteria-fungi interkingdom analysis has revealed significant findings compared to the intrakingdom analysis in recent studies. In this study, we aimed to (i) unravel the contribution and development of the bacteria and other fungi in the Heterobasidioninfected wood decay process; (ii) uncover how environmental factors and the conifer pathogen drive the microbiome community structure and function changes; and (iii) identify the core microbiome during the Heterobasidion-mediated wood decay process. Internal transcribed spacer and 16S amplicon sequencing data from the Heterobasidion fruiting body and its associated woody tissue were applied in this analysis. Samples collected from managed forest and nature reserve forest were classified into four decay classes based on the extent of decay of the wood. Our result showed that bacteria and fungi have different abundances and activities in both study materials. The Heterobasidion ecological correlation network is conserved, especially in the fruiting body. The core microbiome defined in our analysis showed huge potential for the microbiome community’s functioning. A particular Heterobasidion Operational Taxonomic Units (OTU) had the most important impact on the community and disease development. Forest management has the biggest impact on the microbiome assembly, and the fungal community is more sensitive to environmental changes. IMPORTANCE We applied macro- (forest stand and forest management) and microscale (bacterial and fungal community) analyses for a better understanding of the Heterobasidion pathosystem and associated wood decay process. The core microbiome, as defined by hierarchy analysis and a consistent model, and environmental factors correlation with the community assembly were found to be novel. [ABSTRACT FROM AUTHOR]

• Norway spruce had 64%, 30%, and 6% of PaLAR3 genotypes AA, AB, and BB, respectively. • Two H. parviporum strains caused significantly variable necrosis responses. • The two mycovirus strains tested significantly reduced lesion width in Norway spruce. • The two mycovirus strains in H. parviporum reduced fungal growth in sapwood up to 41% Heterobasidion parviporum is a destructive forest pathogen causing root and stem rot of Norway spruce (Picea abies) in northern hemisphere. Previous research has suggested that a gene form of PaLAR3 that encodes for the leucoanthocyanidin reductase enzyme, may increase resistance to H. parviporum in Norway spruce, whether heterozygous or homozygous. Our experiment was initially focused on testing this assertion with materials from the Finnish Norway spruce breeding program, comprising 52 full-sib families. Additionally, we investigated the influence of mycoviruses on the growth of H. parviporum. The frequencies of the three PaLAR3 genotypes AA, AB, and BB were 64%, 30%, and 6%, respectively. We found no significant differences in the necrotic response to H. parviporum between the homozygous (BB) and heterozygous (AB) genotypes vs. the homozygous (AA) genotype across the two fungal strains tested, suggesting the gene is rather ineffective as a marker for overall H. parviporum resistance. The two H. parviporum strains used in the study showed significantly different necrosis responses from the control. Mycovirus infection in H. parviporum strains led to a significant reduction in lesion width in phloem and sapwood. Furthermore, the growth of the fungus was notably restricted when hyphae hosted mycoviruses, indicating a high potential of mycoviruses to reduce fungal growth and necrosis development. [ABSTRACT FROM AUTHOR]

Prophylactic stump treatments against the conifer root rot fungus Heterobasidion annosum s.l. should take into account the airborne inoculum density of the pathogen, in order to be economically feasible. Our objective was to test if an automatic volumetric spore trap (AVST) could be used as a sensitive alternative of passive traps for quantifying Heterobasidion airspora in forest stands. The routinely used wood disc exposure method (WDE) was implemented simultaneously with AVST in five coniferous monocultures and three near-natural stands without silvicultural management in Czechia. Air sampling took place for 24-h intervals in five months of the main sporulation period. The atmospheric concentration of H. annosum s.l. spores (cHa) was determined by qPCR with generic and species-specific primers. AVST detected more target species than WDE on 77% of sampling occasions. When comparing the relative abundance of the three European H. annosum s.l. species detected by AVST and WDE, H. parviporum and H. abietinum were found to be overrepresented on wood discs. cHa in November was significantly higher than in May and June, confirming the seasonal pattern typical for temperate Europe. For an accurate and rapid estimation of Heterobasidion primary infection risk, the usage of AVST is highly recommended. [ABSTRACT FROM AUTHOR]

Utilizing Heterobasidion partitivirus 13 strain an1 (HetPV13-an1) and 15 strain pa1 (HetPV15-pa1) in co-infection is considered a potential biocontrol approach against Heterobasidion root and butt rot. Both partitiviruses mediate debilitating effects in most Heterobasidion host isolates and are generally transmitted efficiently between host strains. In this investigation, we conducted transmission experiments in the laboratory (in vitro) using several H. parviporum isolates to test whether using dual partitivirus infections is a more efficient way of transmitting viruses to new hosts compared to using single partitivirus infections, and whether co-occurring single-stranded RNA (ssRNA) viruses are co-transmitted during the process. The results showed that H. parviporum donors carrying both partitiviruses, HetPV13-an1 and HetPV15-pa1, transmitted HetPV15-pa1 more efficiently to recipients than the same donors infected with only HetPV15-pa1. In contrast, the transmission of HetPV13-an1 did not differ significantly between donors infected with both or only one partitivirus. Altogether, the transmission rates of HetPV13-an1 and HetPV15-pa1 were high on artificial media. Moreover, the transmission of the ssRNA viruses Heterobasidion ourmia-like virus 1(HetOlV1-pa7) and 4 (HetOlV4-an1) as well as Heterobasidion ambi-like virus 3 (HetAlV3-pa4) across different recipients were found to be variable. This study demonstrated for the first time the transmission of ambi- and ourmiaviruses between H. parviporum isolates in dual cultures and showed that H. parviporum mycelia can be cured of these ssRNA viruses using heat treatment., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

The combined use of Heterobasidion partitiviruses 13 and 15 (HetPV13-an1 and HetPV15-pa1) is considered a promising biocontrol approach against Heterobasidion root and butt rot. In a previous study, the transmission frequency of HetPV15-pa1 was found to be higher from a double partitivirus-infected donor than from a single partitivirus-infected donor. In this study, we included a wider array of recipient isolates to assess whether the phenomenon is widespread across different host strains and conducted transmission experiments on artificial media (in vitro) using a total of 45 different H. annosum donor-recipient pairs. In addition to investigating whether double partitivirus infection improves the transmission of HetPV13-an1 and HetPV15-pa1, we examined for the first time how efficiently co-infecting ssRNA viruses are concomitantly transmitted with the partitiviruses, and whether pre-existing ssRNA viruses in the recipients affect virus transmission. Generally, the transmission rates of HetPV13-an1 and HetPV15-pa1 were high from both single partitivirus-infected and double partitivirus-infected donors to most of the H. annosum recipient strains, with few exceptions. However, in contrast to previous experiments, the transmission frequency was not higher from the double partitivirus-infected donors. Also, ourmiavirus was transmitted between H. annosum strains, but the presence of another ourmiavirus in the recipient might affect the efficacy., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Elina Roininen reports financial support was provided by The Finnish Society of Forest Science. Jarkko Hantula reports financial support was provided by Academy of Finland. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Numerous species of pathogenic wood decay fungi, including members of the genera Heterobasidion and Armillaria, exist in forests in the northern hemisphere. Detection of these fungi through field surveys is often difficult due to a lack of visual symptoms and is cost-prohibitive for most applications. Remotely sensed data can offer a lower-cost alternative for collecting information about vegetation health. This study used hyperspectral imagery collected from unmanned aerial vehicles (UAVs) to detect the presence of wood decay in Norway spruce (Picea abies L. Karst) at two sites in Norway. UAV-based sensors were tested as they offer flexibility and potential cost advantages for small landowners. Ground reference data regarding pathogenic wood decay were collected by harvest machine operators and field crews after harvest. Support vector machines were used to classify the presence of root, butt, and stem rot infection. Classification accuracies as high as 76% with a kappa value of 0.24 were obtained with 490-band hyperspectral imagery, while 29-band imagery provided a lower classification accuracy (~60%, kappa = 0.13). [ABSTRACT FROM AUTHOR]

Tree mortality plays a vital role in the accuracy of growth and yield calculations. Economic loss caused by Heterobasidion sp. and Armillaria sp. is a common issue in forestry. Estonian forests, which are mostly managed, are susceptible to fungal infection due to freshly exposed wood surfaces, such as stumps and mechanical wounds. External signs of infection are often scarce and may lead to incorrect stand vitality valuation. Modern devices, such as the PiCUS 3 Sonic Tomograph, can be used for non-destructive decay assessment. We assessed decay in two intensively managed Norway spruce (Picea abies L. Karst.) stands in order to identify the reliability of sonic tomography in tree vitality assessment. We hypothesize that the tomograph assessment is more accurate than the visual assessment for detecting the extent of decay damage in Norway spruce stems. The sample trees were first visually assessed without additional equipment. In the second phase, the same sample trees were measured with the PiCUS 3 Sonic Tomograph. In the last part of the study, the sample trees were assessed from the tree stumps following the clear-cut. We identified a relationship (p-value < 0.001) between the tomograph assessment and the stump assessment when major decay was present. We did not discover a relationship between the visual assessment and stump assessment, indicating that evaluating the decay from external signs is inaccurate according to our results. Our data also indicate that the tomograph is not able to detect the early stages of decay damage, since it has no substantial effect on the wood structure. [ABSTRACT FROM AUTHOR]

The microbiome of Heterobasidion -induced wood decay of living trees has been previously studied; however, less is known about the bacteria biota of its perennial fruiting body and the adhering wood tissue. In this study, we investigated the bacteria biota of the Heterobasidion fruiting body and its adhering deadwood. Out of 7,462 operational taxonomic units (OTUs), about 5,918 OTUs were obtained from the fruiting body and 5,469 OTUs were obtained from the associated dead wood. Interestingly, an average of 52.6% of bacteria biota in the fruiting body was shared with the associated dead wood. The overall and unique OTUs had trends of decreasing from decay classes 1 to 3 but increasing in decay class 4. The fruiting body had the highest overall and unique OTUs number in the fourth decay class, whereas wood had the highest OTU in decay class 1. Sphingomonas spp. was significantly higher in the fruiting body, and phylum Firmicutes was more dominant in wood tissue. The FAPROTAX functional structure analysis revealed nutrition, energy, degradation, and plant-pathogen-related functions of the communities. Our results also showed that bacteria communities in both substrates experienced a process of a new community reconstruction through the various decay stages. The process was not synchronic in the two substrates, but the community structures and functions were well-differentiated in the final decay class. The bacteria community was highly dynamic; the microbiota activeness, community stability, and functions changed with the decay process. The third decay class was an important turning point for community restructuring. Host properties, environmental factors, and microbial interactions jointly influenced the final community structure. Bacteria community in the fruiting body attached to the living standing tree was suppressed compared with those associated with dead wood. Bacteria appear to spread from wood tissue of the standing living tree to the fruiting body, but after the tree is killed, bacteria moved from fruiting body to wood. It is most likely that some of the resident endophytic bacteria within the fruiting body are either parasitic, depending on it for their nutrition, or are mutualistic symbionts. [ABSTRACT FROM AUTHOR]

The microbiome of Heterobasidion-induced wood decay of living trees has been previously studied; however, less is known about the bacteria biota of its perennial fruiting body and the adhering wood tissue. In this study, we investigated the bacteria biota of the Heterobasidion fruiting body and its adhering deadwood. Out of 7,462 operational taxonomic units (OTUs), about 5,918 OTUs were obtained from the fruiting body and 5,469 OTUs were obtained from the associated dead wood. Interestingly, an average of 52.6% of bacteria biota in the fruiting body was shared with the associated dead wood. The overall and unique OTUs had trends of decreasing from decay classes 1 to 3 but increasing in decay class 4. The fruiting body had the highest overall and unique OTUs number in the fourth decay class, whereas wood had the highest OTU in decay class 1. Sphingomonas spp. was significantly higher in the fruiting body, and phylum Firmicutes was more dominant in wood tissue. The FAPROTAX functional structure analysis revealed nutrition, energy, degradation, and plant-pathogen-related functions of the communities. Our results also showed that bacteria communities in both substrates experienced a process of a new community reconstruction through the various decay stages. The process was not synchronic in the two substrates, but the community structures and functions were well-differentiated in the final decay class. The bacteria community was highly dynamic; the microbiota activeness, community stability, and functions changed with the decay process. The third decay class was an important turning point for community restructuring. Host properties, environmental factors, and microbial interactions jointly influenced the final community structure. Bacteria community in the fruiting body attached to the living standing tree was suppressed compared with those associated with dead wood. Bacteria appear to spread from wood tissue of the standing living tree to the fruiting body, but after the tree is killed, bacteria moved from fruiting body to wood. It is most likely that some of the resident endophytic bacteria within the fruiting body are either parasitic, depending on it for their nutrition, or are mutualistic symbionts.

Abstract 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.

Abstract Background Root and butt rot of conifer trees caused by fungi belonging to the Heterobasidion annosum species complex is one of the most economically important fungal diseases in commercial conifer plantations throughout the Northern hemisphere. We investigated the interactions between Heterobasidion fungi and their host by conducting dual RNA-seq and chemical analysis on Norway spruce trees naturally infected by Heterobasidion spp. We analyzed host and pathogen transcriptome and phenolic and terpenoid contents of the spruce trees. Results Presented results emphasize the role of the phenylpropanoid and flavonoid pathways in the chemical defense of Norway spruce trees. Accumulation of lignans was observed in trees displaying symptoms of wood decay. A number of candidate genes with a predicted role in the higher level regulation of spruce defense responses were identified. Our data indicate a possible role of abscisic acid (ABA) signaling in the spruce defense against Heterobasidion infection. Fungal transcripts corresponding to genes encoding carbohydrate- and lignin-degrading enzymes, secondary metabolism genes and effector-like genes were expressed during the host colonization. Conclusions Our results provide additional insight into defense strategies employed by Norway spruce trees against Heterobasidion infection. The potential applications of the identified candidate genes as markers for higher resistance against root and butt rot deserve further evaluation.

Numerous species of pathogenic wood decay fungi, including members of the genera Heterobasidion and Armillaria, exist in forests in the northern hemisphere. Detection of these fungi through field surveys is often difficult due to a lack of visual symptoms and is cost-prohibitive for most applications. Remotely sensed data can offer a lower-cost alternative for collecting information about vegetation health. This study used hyperspectral imagery collected from unmanned aerial vehicles (UAVs) to detect the presence of wood decay in Norway spruce (Picea abies L. Karst) at two sites in Norway. UAV-based sensors were tested as they offer flexibility and potential cost advantages for small landowners. Ground reference data regarding pathogenic wood decay were collected by harvest machine operators and field crews after harvest. Support vector machines were used to classify the presence of root, butt, and stem rot infection. Classification accuracies as high as 76% with a kappa value of 0.24 were obtained with 490-band hyperspectral imagery, while 29-band imagery provided a lower classification accuracy (~60%, kappa = 0.13).

Volatile organic compounds (VOCs) play an important role in the communication among organisms, including plants, beneficial or pathogenic microbes, and pests. In vitro, we observed that the growth of seven out of eight Basidiomycete species tested was inhibited by the VOCs of the biocontrol agent Pseudomonas protegens strain CHA0. In the Ascomycota phylum, only some species were sensitive (e.g., Sclerotinia sclerotiorum, Botrytis cinerea, etc.) but others were resistant (e.g., Fusarium oxysporum f. sp. cubense, Verticillium dahliae, etc.). We further discovered that CHA0 as well as other ten beneficial or phytopathogenic bacterial strains were all able to inhibit Heterobasidion abietinum, which was used in this research as a model species. Moreover, such an inhibition occurred only when bacteria grew on media containing digested proteins like peptone or tryptone (e.g., Luria-Bertani agar or LBA). Also, the inhibition co-occurred with a pH increase of the agar medium where the fungus grew. Therefore, biogenic ammonia originating from protein degradation by bacteria was hypothesized to play a major role in fungus inhibition. Indeed, when tested as a synthetic compound, it was highly toxic to H. abietinum (effective concentration 50% or EC50 = 1.18 M; minimum inhibitory concentration or MIC = 2.14 M). Using gas chromatography coupled to mass spectrometry (GC/MS), eight VOCs were found specifically emitted by CHA0 grown on LBA compared to the bacterium grown on potato dextrose agar (PDA). Among them, two compounds were even more toxic than ammonia against H. abietinum: dimethyl trisulfide had EC50 = 0.02 M and MIC = 0.2 M, and 2-ethylhexanol had EC50 = 0.33 M and MIC = 0.77 M. The fungus growth inhibition was the result of severe cellular and sub-cellular alterations of hyphae occurring as early as 15 min of exposure to VOCs, as evidenced by transmission and scanning electron microscopy observations. Transcriptome reprogramming of H. abietinum induced by CHA0’s VOCs pointed out that detrimental effects occurred on ribosomes and protein synthesis while the cells tried to react by activating defense mechanisms, which required a lot of energy diverted from the growth and development (fitness cost).

Volatile organic compounds (VOCs) play an important role in the communication among organisms, including plants, beneficial or pathogenic microbes, and pests. In vitro , we observed that the growth of seven out of eight Basidiomycete species tested was inhibited by the VOCs of the biocontrol agent Pseudomonas protegens strain CHA0. In the Ascomycota phylum, only some species were sensitive (e.g., Sclerotinia sclerotiorum , Botrytis cinerea , etc.) but others were resistant (e.g., Fusarium oxysporum f. sp. cubense , Verticillium dahliae , etc.). We further discovered that CHA0 as well as other ten beneficial or phytopathogenic bacterial strains were all able to inhibit Heterobasidion abietinum , which was used in this research as a model species. Moreover, such an inhibition occurred only when bacteria grew on media containing digested proteins like peptone or tryptone (e.g., Luria-Bertani agar or LBA). Also, the inhibition co-occurred with a pH increase of the agar medium where the fungus grew. Therefore, biogenic ammonia originating from protein degradation by bacteria was hypothesized to play a major role in fungus inhibition. Indeed, when tested as a synthetic compound, it was highly toxic to H. abietinum (effective concentration 50% or EC50 = 1.18 M; minimum inhibitory concentration or MIC = 2.14 M). Using gas chromatography coupled to mass spectrometry (GC/MS), eight VOCs were found specifically emitted by CHA0 grown on LBA compared to the bacterium grown on potato dextrose agar (PDA). Among them, two compounds were even more toxic than ammonia against H. abietinum : dimethyl trisulfide had EC50 = 0.02 M and MIC = 0.2 M, and 2-ethylhexanol had EC50 = 0.33 M and MIC = 0.77 M. The fungus growth inhibition was the result of severe cellular and sub-cellular alterations of hyphae occurring as early as 15 min of exposure to VOCs, as evidenced by transmission and scanning electron microscopy observations. Transcriptome reprogramming of H. abietinum induced by CHA0's VOCs pointed out that detrimental effects occurred on ribosomes and protein synthesis while the cells tried to react by activating defense mechanisms, which required a lot of energy diverted from the growth and development (fitness cost). [ABSTRACT FROM AUTHOR]

Significant hybridization between the invasive North American fungal plant pathogen Heterobasidion irregulare and its Eurasian sister species H. annosum is ongoing in Italy. Whole genomes of nine natural hybrids were sequenced, assembled and compared with those of three genotypes each of the two parental species. Genetic relationships among hybrids and their level of admixture were determined. A multi-approach pipe- line was used to assign introgressed genomic blocks to each of the two species. Alleles that introgressed from H. irregulare to H. annosum were associated with pathways putatively related to saprobic processes, while alleles that introgressed from the native to the invasive species were mainly linked to gene regulation. There was no overlap of allele categories introgressed in the two directions. Phenotypic experiments documented a fitness increase in H. annosum genotypes characterized by introgression of alleles from the invasive species, supporting the hypothesis that hybridization results in putatively adaptive introgression. Conversely, intro- gression from the native into the exotic species appeared to be driven by selection on genes favoring genome stability. Since the introgression of specific alleles from the exotic H. irregulare into the native H. annosum increased the invasiveness of the latter species, we propose that two invasions may be co-occurring: the first one by genotypes of the exotic species, and the second one by alleles belonging to the exotic species. Given that H. irregulare represents a threat to European forests, monitoring programs need to track not only exotic genotypes in native forest stands, but also exotic alleles introgressed in native genotypes. [ABSTRACT FROM AUTHOR]

Heterobasidion irregulare and H. occidentale are two closely related conifer root rot pathogens in the H. annosum sensu lato (s.l.) species complex. The two species H. irregulare and H. occidentale have different host preference with pine and non-pine tree species favored, respectively. The comparison of transcriptomes of H. irregulare and H. occidentale growing in Norway spruce bark, a susceptible host non-native to North America, showed large differences in gene expression. Heterobasidion irregulare induced more genes involved in detoxification of host compounds and in production of secondary metabolites, while the transcriptome induced in H. occidentale was more oriented towards carbohydrate degradation. Along with their separated evolutionary history, the difference might be driven by their host preferences as indicated by the differentially expressed genes enriched in particular Gene Ontology terms. [ABSTRACT FROM AUTHOR]

Southern pine forests are important fiber and wood sources, and critical to local, regional, and national economies in the United States. Recently, certain areas of southern pine forests, especially those dominated by loblolly pine (Pinus taeda), have been reported to exhibit abnormally high levels of tree dieback and mortality. However, causal agents either have not been well defined or are controversial in their impacts on tree health. We assessed various abiotic (e.g., slope, aspect, soil characteristics) and biotic (e.g., tree species, stand characteristics, presence of root fungi) factors in 37 healthy (asymptomatic) and unhealthy (symptomatic) sites to elucidate specific factors affecting loblolly pine health in Alabama and Georgia. Soil nutrient content did not differ statistically between healthy and unhealthy sites, but manganese contents were slightly greater, and nitrogen and carbon contents were slightly lower in healthy sites. Unhealthy sites did have a higher silt content than did healthy sites. Pine stems and basal area were greater on unhealthy than on healthy sites, whereas opposite trends were observed for the incidence of stem cankers and mechanical damage. An increased incidence of the root fungal pathogen Heterobasidion irregulare , the causal agent of Heterobasidion root disease, was found on unhealthy sites, but incidence of Leptographium spp. did not differ between the two site types. Thus, soil attributes, stand structure, and management history seem to be the most critical factors affecting loblolly pine health, at least at the local level. Further, some of these factors may be improved through appropriate silvicultural techniques, emphasizing the importance of silviculture in maintaining pine health throughout the southern region. [ABSTRACT FROM AUTHOR]

Main conclusion: Two terpene compounds and four genes were identified as potential biomarkers for further evaluation for Scots pine susceptibility or tolerance against Heterobasidion annosum. Scots pine (Pinus sylvestris) is one of the main sources of timber in the boreal zone of Eurasia. Commercial pine plantations are vulnerable to root and butt rot disease caused by the fungus Heterobasidion annosum. The pathogen affects host growth rate, causes higher mortality and decreases in timber quality, resulting in considerable economic losses to forest owners. Genetic and biochemical factors contributing to Scots pine tolerance against H. annosum infection are not well understood. We assessed the predictive values of a set of potential genetic and chemical markers in a field experiment. We determined the expression levels of 25 genes and the concentrations of 36 terpenoid compounds in needles of 16 Scots pine trees randomly selected from a natural population prior to artificial infection. Stems of the same trees were artificially inoculated with H. annosum, and the length of necrotic lesions was documented 5 months post inoculation. Higher expression level of four genes included in our analysis and encoding predicted α-pinene synthase (two genes), geranyl diphosphate synthase (GPPS), and metacaspase 5 (MC5), could be associated with trees exhibiting increased levels of necrotic lesion formation in response to fungal inoculation. In contrast, concentrations of two terpenoid compounds, β-caryophyllene and α-humulene, showed significant negative correlations with the lesion size. Further studies with larger sample size will help to elucidate new biomarkers or clarify the potential of the evaluated markers for use in Scots pine disease resistance breeding programs. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Plants live in close association with microbial symbionts, which may affect the host fitness, productivity, and tolerance against biotic and abiotic stressors. The composition of plant microbial communities is influenced by many biotic and abiotic factors, but little is known about the effect of plant pathogens on the structure of these communities. In this study, we investigated the structure of bacterial communities associated with different tissues of asymptomatic and symptomatic (Heterobasidion-rotten) Norway spruce (Picea abies (L.) Karst.) trees. Our results demonstrated that each of the investigated anatomic tissues (root, bark, down stem, upper stem, and needles) harbored a unique bacterial assemblage. However, the health status of the host trees had little effect on the structure of bacterial communities, as the only significant differences among asymptomatic and symptomatic trees were found in the composition of the bacterial communities of needles. Proteobacteria was predominant in all anatomic regions with the highest abundance in needles (86.7%), whereas Actinobacteria showed an opposite trend, being more abundant in the woody tissues than in needles. Additionally, we performed profiling of terpenoid compounds present in spruce xylem and phloem. Total concentrations of monoterpenes and sesquiterpenes were considerably higher in asymptomatic trees. However, we found no significant correlations between terpenoid profiles of spruce trees and the composition of their bacterial communities. Our results provide an insight into the diversity of bacteria associated with Norway spruce tree tissues. At the same time, the health status and terpenoid content of host trees had a limited effect on the composition of bacterial communities in our survey. [ABSTRACT FROM AUTHOR]

Heterobasidion irregulare is a causal agent of root and butt-rot disease in conifers, and is native to North America. In 1944 it was introduced in central Italy in a Pinus pinea stand, where it shares the same niche with the native species Heterobasidion annosum. The introduction of a non-native pathogen may have significant negative effects on a naïve host tree and the ecosystem in which it resides, requiring a better understanding of the system. We compared the spatio-temporal phenotypic, transcriptional and metabolic host responses to inoculation with the two Heterobasidion species in a large experiment with P. pinea seedlings. Differences in length of lesions at the inoculation site (IS), expression of host genes involved in lignin pathway and in cell rescue and defence, and analysis of terpenes at both IS and 12 cm above the IS (distal site, DS), were assessed at 3, 14 and 35 days post inoculation (dpi). Results clearly showed that both species elicit similar physiological and biochemical responses in P. pinea seedlings. The analysis of host transcripts and total terpenes showed differences between inoculation sites and between pathogen and mock inoculated plants. Both pathogen and mock inoculations induced antimicrobial peptide and phenylalanine ammonia-lyase overexpression at IS beginning at 3 dpi; while at DS all the analysed genes, except for peroxidase, were overexpressed at 14 dpi. A significantly higher accumulation of terpenoids was observed at 14 dpi at IS, and at 35 dpi at DS. The terpene blend at IS showed significant variation among treatments and sampling times, while no significant differences were ever observed in DS tissues. Based on our results, H. irregulare does not seem to have competitive advantages over the native species H. annosum in terms of pathogenicity towards P. pinea trees; this may explain why the non-native species has not widely spread over the 73 years since its putative year of introduction into central Italy. [ABSTRACT FROM AUTHOR]

We investigated the wood fungal community composition in stems of living Norway spruce (Picea abies (L.) Karst.) trees in 20–40 year-old forest stands from Latvia that differed in recent management history (stands with or without thinning) and former land use (former agricultural or former forest lands). Fungal internal transcribed spacer (ITS2) amplicons of DNA extracted from wood were sequenced to assess wood fungal communities. Alternaria, Ascocoryne, Didymella, Heterobasidion, Ophiostoma, Orbilia, Pesotum, Phoma and Pseudocercosporella were the dominant wood pathogen and wood saprotroph genera in the analysed samples. PERMANOVA analysis identified some differences in fungal communities among site types analysed (p < 0.001); tree height and presence/absence of rot in the wood samples (mainly Heterobasidion rot) significantly influenced fungal community composition (p < 0.001). Significant negative co-occurrence (p < 0.05) was observed between Heterobasidion and Ascocoryne genera indicating the differing wood colonizing niches of these taxa. Both stand level factors, such as management history and former land use, and tree level parameters are significant for wood inhabiting fungal communities of living spruces. [ABSTRACT FROM AUTHOR]

Root and butt rot caused by members of the Heterobasidion annosum species complex is the most economically important disease of conifer trees in boreal forests. Wood decay in the infected trees dramatically decreases their value and causes considerable losses to forest owners. Trees vary in their susceptibility to Heterobasidion infection, but the genetic determinants underlying the variation in the susceptibility are not well-understood. We performed the identification of Norway spruce genes associated with the resistance to Heterobasidion parviporum infection using genome-wide exon-capture approach. Sixty-four clonal Norway spruce lines were phenotyped, and their responses to H. parviporum inoculation were determined by lesion length measurements. Afterwards, the spruce lines were genotyped by targeted resequencing and identification of genetic variants (SNPs). Genome-wide association analysis identified 10 SNPs located within 8 genes as significantly associated with the larger necrotic lesions in response to H. parviporum inoculation. The genetic variants identified in our analysis are potential marker candidates for future screening programs aiming at the differentiation of disease-susceptible and resistant trees.

Effects of different pre-planting soil preparations and post-harvest wood debris applications in a clear-cut Scots pine plantation, on the abundance, diversity, and activity of culturable microorganisms were investigated. The investigation was done 9 years after the re-plantings had been done. This formed part of an investigation of silvicultural practices for conservation and the biological control of Armillaria and Heterobasidion in northern temperate forests (Poland). The treatments being compared, were expected to have altered the soil’s physical and chemical properties, and consequently, its biological properties. Only soft-rot microfungi from the Ascomycota and Zygomycota were detected in the soil. Fungi, including those antagonistic to Armillaria and Heterobasidion, were more abundant after shallow ploughing than after deep ploughing or ridging, and where chipped rather than coarse wood debris was left on the soil surface or incorporated. Scots pine trees had the most biomass and the least mortality after ridging and leaving coarse wood debris on the surface (associated with only a relatively moderate abundance of fungi).

Scots pine sawdust, composted bark or coarse, post-harvest woody debris from conifers had been spread over the surface of barren forest soil before planting with Scots pine. The effects of the Scots pine sawdust, composted bark or coarse, post-harvest woody debris from conifers on the abundance and diversity of culturable fungi were investigated. The amendments were aimed at increasing the soil suppressiveness to Armillaria and Heterobasidion. The classical soil-dilution method was chosen for qualitative and quantitative analyses of fungal communities in soils because of its proven reliability and consistency. The soil was inhabited by saprotrophic fungi from Ascomycota and Zygomycota, including species known to be potential antagonists of Armillaria or H. annosum (i.e. Clonostachys + Trichoderma spp., Penicillium commune, P. daleae, P. janczewskii) or stimulants of Armillaria (i.e. Pseudogymnoascus roseus, Trichocladium opacum). Eleven years after treatment, the abundance and diversity of fungi, the abundance of P. commune, and locally the abundance of P. janczewskii increased, while Clonostachys + Trichoderma spp., and locally, P. daleae and T. opacum decreased. Amending the barren soil with organic matter does not guarantee effective, long-term suppressiveness of the sandy loam soil to Armillaria and Heterobasidion. Increased abundance of entomopathogenic and nematophagous species, 11 years after treatment, does suggest the long-term possibility of insect or nematode control in soil.

Summary: Plant microbiome plays an important role in maintaining the host fitness. Despite a significant progress in our understanding of the plant microbiome achieved in the recent years, very little is known about the effect of plant pathogens on composition of microbial communities associated with trees. In this study, we analysed the mycobiome of different anatomic parts of asymptomatic and symptomatic Norway spruce trees naturally infected by Heterobasidion spp. We also investigated the primary impact of the disease on the fungal communities, which are associated with Norway spruce trees. Our results demonstrate that the structure of fungal communities residing in the wood differed significantly among symptomatic and asymptomatic Heterobasidion‐infected trees. However, no significant differences were found in the other anatomic regions of the trees. The results also show that not only each of individual tree tissues (wood, bark, needles and roots) harbours a unique fungal community, but also that symptomatic trees were more susceptible to co‐infection by other wood‐degrading fungi compared to the asymptomatic ones. [ABSTRACT FROM AUTHOR]

Heterobasidion parviporum and H. annosum are the most damaging root and butt rot pathogens of conifers in southern Fennoscandia where their distribution and frequency are reasonably well described in literature. However, in the most northern boreal forests of Europe the occurrence and significance of these pathogens is poorly known. In the future global warming may increase root and butt rot damages in the North. In this research the distribution of Heterobasidion spp. in northern Finland was compiled from literature and various available databases and from a field study in two counties of northern Finland, Northern Ostrobothnia and Kainuu. Conifer stumps from 101 regeneration cutting sites were inspected and sample discs from stumps with signs of decay were investigated for the presence of Heterobasidion spp. In all, 11,846 stumps were investigated. Decay was found in 429 Norway spruce stumps and 50 Scots pine stumps. H. parviporum was isolated from five spruce discs, each from a different site. H. annosum was not detected. According to previous records H. annosum has been identified only twice in northern Finland and these locations are over 600 km south of the northernmost Scots pine stands in Fennoscandia. Instead, H. parviporum occurs sporadically in the whole distribution area of Norway spruce in northern Finland. Most probably H. parviporum can be found in overmature dense stands at fertile sites but even there only at low frequencies. In northern Finland butt rot of conifers is caused mainly by other decay fungi than Heterobasidion . Low pH of the organic soil layer is probably a significant factor hampering H. parviporum from spreading in the North. [ABSTRACT FROM AUTHOR]

Heterobasidion butt rot causes considerable losses to forestry in coniferous forests in many parts of Europe including southern Fennoscandia. In northern Fennoscandia, including central and northern Finland, both Heterobasidion annosum and H. parviporum are rare. This study aimed to determine the incidence of butt rot in Scots pine ( Pinus sylvestris L.) and Norway spruce ( Picea abies (L.) Karst.) north of the area commonly infested by Heterobasidion spp. in Finland. The incidence of butt rot in these conifers was investigated in Northern Ostrobothnia and Kainuu using two data sets: stump inspections in 101 regeneration cutting sites and the Finnish National Forest Inventory (NFI) data. According to stump inspections butt rot occurred in 1% of Scots pine trees and 10% of Norway spruce trees. In the most recent NFI data from 2009 to 2011 the incidence of trees with rot in mature managed stands of these two counties was 1% and 5% for Scots pine and Norway spruce, respectively. According to NFI, these values are nearly equal to the average incidence of butt rot in mature pines and spruces of the whole of Finland. Consequently, the low incidence of Heterobasidion butt rot in these parts of Finland does not mean lower occurrence of butt rot. Butt rot incidence varied greatly between sites: 0–24% of Norway spruce stumps in spruce dominated sites and 0–13% of Scots pine stumps in pine dominated sites were infected. High butt rot incidence in Norway spruce was associated with high site fertility, stand age, low organic matter content of soil and low temperature sum of the site. In Scots pine butt rot appeared after stand age exceeded 120 years while decay occurred already in 40–80 years old Norway spruces. High butt rot incidence in Scots pine was associated also with high flow accumulation of the site. Precipitation, plot slope, plot aspect, ditches, stand density and elevation were insignificant variables in models explaining decay incidence. During 1991–2013 butt rot incidence in Scots pine or Norway spruce has not changed in the study area. Similarly, to the more southern areas with high Heterobasidion risk, butt rot control in the North should focus especially on fertile mineral soil sites. [ABSTRACT FROM AUTHOR]

Our comparative studies reveal that the two lytic polysaccharide monooxygenases HiLPMO9B and HiLPMO9I from the white-rot conifer pathogen Heterobasidion irregulare display clear difference with respect to their activity against crystalline cellulose and glucomannan. HiLPMO9I produced very little soluble sugar on bacterial microcrystalline cellulose (BMCC). In contrast, HiLPMO9B was much more active against BMCC and even released more soluble sugar than the H. irregulare cellobiohydrolase I, HiCel7A. Furthermore, HiLPMO9B was shown to cooperate with and stimulate the activity of HiCel7A, both when the BMCC was first pretreated with HiLPMO9B, as well as when HiLPMO9B and HiCel7A were added together. No such stimulation was shown by HiLPMO9I. On the other hand, HiLPMO9I was shown to degrade glucomannan, using a C4-oxidizing mechanism, whereas no oxidative cleavage activity of glucomannan was detected for HiLPMO9B. Structural modeling and comparison with other glucomannan-active LPMOs suggest that conserved sugar-interacting residues on the L2, L3 and LC loops may be essential for glucomannan binding, where 4 out of 7 residues are shared by HiLPMO9I, but only one is found in HiLPMO9B. The difference shown between these two H. irregulare LPMOs may reflect distinct biological roles of these enzymes within deconstruction of different plant cell wall polysaccharides during fungal colonization of softwood. [ABSTRACT FROM AUTHOR]

To study simultaneous biotic and abiotic stress effects on monoterpene mediated defence response, four-year-old seedlings of two clones of Picea abies (c386: “relatively resistant” c386; c171: “relatively susceptible” clone 171) were inoculated with an isolate of rot fungus Heterobasidion parviporum and simultaneously subjected to a drought stress recovery cycle. Fungal growth into the wood was generally higher in well-watered (W) than in drought-stressed (D) plants. As predawn water potential decreased, gas exchange and maximal photochemical efficiency of PSII also declined. Water potential and chlorophyll fluorescence showed differences between clones in D plants, c386 being more tolerant than c171, while stomatal conductance and net photosynthesis differed between clones in W plants; c171 showing higher values than c386. A recovery of physiological functions was observed after re-watering. The clones showed different constitutive monoterpene profiles. The infection generally caused some extensive compositional changes in the relative contents of several monoterpenes and their enantiomers; also, sterile infection (wounded only samples) affected the relative proportions of monoterpenes, although to a lesser extent than reactions elicited by the fungus. The trend in monoterpene response to disease treatments was the same in D and W plants of both clones. In particular, higher proportion of δ-3-carene was found constitutively in c386 in comparison with c171; besides, infection with H. parviporum increased the relative contents of δ-3-carene in all the samples, even if proportions of this monoterpene in wounded plus inoculated tissues of c386 were significantly higher than cortical tissues from non-inoculated branches only in W seedlings 44 days after treatment. Although drought stress decreased the total absolute contents of monoterpenes, total monoterpene concentrations significantly increased in response to infection by H. parviporum . [ABSTRACT FROM AUTHOR]

Infections with Heterobasidion parviporum devalue the Norway spruce timber as the decayed wood does not meet the necessary quality requirements for sawing. To evaluate the incorporation of disease resistance in the Norway spruce breeding strategy, an inoculation experiment with H. parviporum on 2-year-old progenies of 466 open-pollinated families was conducted under greenhouse (nursery) conditions. Lesion length in the phloem (LL), fungal growth in sapwood (FG) and growth (D) were measured on an average of 10 seedlings for each family. The genetic variation and genetic correlations between both LL, FG and growth in the nursery trial and wood quality traits measured previously from 21-year old trees in two progeny trials, including solid-wood quality traits (wood density, and modulus of elasticity) and fiber properties traits (radial fiber width, tangential fiber width, fiber wall thickness, fiber coarseness, microfibril angle and fiber length). For both LL and FG, large coefficients of phenotypic variation (> 26%) and genetic variation (> 46%) were detected. Heritabilities of LL and FG were 0.33 and 0.42, respectively. We found no significant correlations between wood quality traits and growth in the field progeny trials with neither LL nor FG in the nursery trial. Our data suggest that the genetic gains may reach 41 and 52% from mass selection by LL and FG, respectively. Early selection for resistance to H. parviporum based on assessments of fungal spread in the sapwood in nursery material, FG, will not adversely affect growth and wood quality traits in late-age performance. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMOs) are a group of recently discovered enzymes that play important roles in the decomposition of recalcitrant polysaccharides. Here, we report the biochemical, structural, and computational characterization of an LPMO from the white‐rot fungus Heterobasidion irregulare (HiLPMO9B). This enzyme oxidizes cellulose at the C1 carbon of glycosidic linkages. The crystal structure of HiLPMO9B was determined at 2.1 Å resolution using X‐ray crystallography. Unlike the majority of the currently available C1‐specific LPMO structures, the HiLPMO9B structure contains an extended L2 loop, connecting β‐strands β2 and β3 of the β‐sandwich structure. Molecular dynamics (MD) simulations suggest roles for both aromatic and acidic residues in the substrate binding of HiLPMO9B, with the main contribution from the residues located on the extended region of the L2 loop (Tyr20) and the LC loop (Asp205, Tyr207, and Glu210). Asp205 and Glu210 were found to be involved in the hydrogen bonding with the hydroxyl group of the C6 carbon of glucose moieties directly or via a water molecule. Two different binding orientations were observed over the course of the MD simulations. In each orientation, the active‐site copper of this LPMO preferentially skewed toward the pyranose C1 of the glycosidic linkage over the targeted glycosidic bond. This study provides additional insight into cellulose binding by C1‐specific LPMOs, giving a molecular‐level picture of active site substrate interactions. Database: The atomic coordinates and structure factors for HiLPMO9B have been deposited in the Protein Data Bank with accession code 5NNS. [ABSTRACT FROM AUTHOR]

Summary: Characterization of genes involved in differentiation of pathogen species and isolates with variations of virulence traits provides valuable information to control tree diseases for meeting the challenges of sustainable forest health and phytosanitary trade issues. Lack of genetic knowledge and genomic resources hinders novel gene discovery, molecular mechanism studies and development of diagnostic tools in the management of forest pathogens. Here, we report on transcriptome profiling of Heterobasidion occidentale isolates with contrasting virulence levels. Comparative transcriptomic analysis identified orthologous groups exclusive to H. occidentale and its isolates, revealing biological processes involved in the differentiation of isolates. Further bioinformatics analyses identified an H. occidentale secretome, CYPome and other candidate effectors, from which genes with species‐ and isolate‐specific expression were characterized. A large proportion of differentially expressed genes were revealed to have putative activities as cell wall modification enzymes and transcription factors, suggesting their potential roles in virulence and fungal pathogenesis. Next, large numbers of simple sequence repeats (SSRs) and single nucleotide polymorphisms (SNPs) were detected, including more than 14 000 interisolate non‐synonymous SNPs. These polymorphic loci and species/isolate‐specific genes may contribute to virulence variations and provide ideal DNA markers for development of diagnostic tools and investigation of genetic diversity. [ABSTRACT FROM AUTHOR]

Summary: Heterobasidion irregulare is one of the most destructive fungal pathogens of pines in North America and was accidentally introduced into central Italy, where it has become invasive. The fungus is currently recommended for regulation by the European and Mediterranean Plant Protection Organisation (EPPO). In this work, an efficient diagnostic tool for the early detection of H. irregulare based on Loop‐mediated isothermal AMPlification (LAMP) coupled with two different DNA extraction methods was developed. The LAMP assay showed high specificity and good sensitivity, with a limit of detection of about 20 picograms of target DNA and time of detection of less than 40 min. The assay was successfully tested on a variety of different samples, including fungal fruiting bodies, infected plants and colonized wood. A survey on environmental samples collected in the field was also performed using the LAMP assay coupled with a rapid DNA extraction method. The possible applications of this molecular diagnostic tool encompass the monitoring of pine forests surrounding the current invasion area, laboratory or in‐field analyses of samples from suspected trees, and the surveillance in the ports of entry of wood imported from North America. [ABSTRACT FROM AUTHOR]

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 virusinfected 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. [ABSTRACT FROM AUTHOR]

Heterobasidion spp. root rot causes severe damage to forests throughout the northern temperate zone. In order to prevent Heterobasidion infection in summertime cuttings, stumps can be treated with urea or Phlebiopsis gigantea. In this study, the consumption of stump treatment materials and the quality of stump treatment work were investigated. A total of 46 harvesters were examined in May-November 2016 in Finland. The average stem size of softwood removal and softwood removal per hectare explained the consumption of stump treatment material. The quality of stump treatment work was good in the study. The best coverage was achieved with the stumps of 20-39 cm diameter at stump height (d0). It can be recommended that the harvester operator self-monitors and actively controls his/her treatment result in cutting work and sets the stump treatment equipment in a harvester if needed. The results also suggested that when cutting mostly small- and medium-diameter (d0 ≤ 39 cm) conifers, the stump treatment guide bars with relatively few (<18) open holes are used, and at the harvesting sites of large-diameter trees, the guide bars with a relatively great (>27) number of open holes are applied. [ABSTRACT FROM AUTHOR]

Summary: The fungal pathogen Heterobasidion irregulare has caused substantial damage to pine species in much of the south‐eastern United States, but the potential for damage from Heterobasidion root disease to forest tree species of the Great Lakes Region has not been thoroughly investigated. In each of three trials in two infested stands, fresh stem discs of six native conifer species were exposed to natural inoculum and examined for the asexual Spiniger stage of H. irregulare after incubation in the laboratory. In the first trial, the fungus was infrequently observed on discs, and differences among species were not found. But in the other two trials, detection of the pathogen was much more frequent on discs of tamarack (Larix laricina), red pine (Pinus resinosa) and white spruce (Picea glauca) than those of northern white cedar (Thuja occidentalis), eastern hemlock (Tsuga canadensis) and balsam fir (Abies balsamea). In a second study, the ability of an isolate of H. irregulare to decay root sapwood of these same species was compared using a soil‐block decay test. After 16 weeks of incubation, weight loss due to decay was greatest for red pine (23.2%) and larch (19.1%) and least for balsam fir (1.3%). Differences in the ability of H. irregulare to infect and saprophytically utilize substrate of these important tree species should prompt additional investigation of the relative risk posed to forests of the Great Lakes Region. [ABSTRACT FROM AUTHOR]

We tested if root colonisation by ectomycorrhizal fungi (EMF) could alter the susceptibility of Norway spruce ( Picea abies ) seedlings to root rot infection or necrotic foliar pathogens. Firstly, spruce seedlings were inoculated by various EMF and challenged with Heterobasidion isolates in triaxenix tubes. The ascomycete EMF Meliniomyces bicolor , that had showed strong antagonistic properties towards root rot causing Heterobasidion in vitro , protected spruce seedlings effectively against root rot. Secondly, spruce seedlings, inoculated with M. bicolor or the forest humus, were subjected to necrotrophic foliar pathogens in conventional forest nursery conditions on peat substrates. Botrytis cinerea infection after winter was mild and the level of needle damage was independent of substrate and EMF colonisation. Needle damage severity caused by Gremminiella abietina was high in seedlings grown in substrates with high nutrient availability as well as in seedlings with well-established EMF communities. These results show that albeit M. bicolor is able to protect spruce seedlings against Heterobasidion root rot in axenic cultures it fails to induce systemic protection against foliar pathogens. We also point out that unsterile inoculum sources, such as the forest humus, should not be considered for use in greenhouse conditions as they might predispose seedlings to unintended needle damages. [ABSTRACT FROM AUTHOR]

The goal of this study was to assess the long-term effects of partial harvesting and supplementary soil scarification on the frequency of root and butt rot in managed uneven-sized Norway spruce stands. Frequency of rot and the population structure of the rot fungi were assessed on 1353 stumps after clear-cutting 21 years after a selection harvesting experiment. The initial experiment was comprised of three harvest strength (low, intermediate and high) of single-tree selection, removing approximately 25, 45 and 65% of the stand basal area. Uncut control plots were established at the same time. Supplementary soil scarification was applied in subplots within the single-tree selection plots, using a medium-sized excavator. After clear-cutting the stumps were analyzed with respect to rot caused by Heterobasidion parviporum , Armillaria spp., Stereum sanguinolentum as well as other rot fungi. Rot caused by Armillaria spp. was most common (8.6% of the stumps), while infection by H. parviporum (2.9%) or S. sanguinolentum (3.0%) was less frequent. The group “other rot” (5.4%) comprised 21 identified taxa, each occurring in 1–15 stumps. Significantly lower rot frequencies were found for the uncut control (16.3%) and intermediate harvest strength (15.7%), compared with low harvest strength (23.6%). A rot frequency of 21.0% was found in the high harvest strength. In two of three harvest strengths, the rot frequency was higher than for the uncut control. As the observed rot frequencies did not increase consistently with increasing harvest strength, the results do not completely support the initial expectations of increased rot after single-tree selection compared with the uncut control. However, since the probability of rot in individual stumps on plots treated with single-tree selection was significantly affected by the distance to the nearest strip road ( H. parviporum ) as well as dependent on the size of and distance to the nearest stump of trees cut during the experimental harvest ( H. parviporum , S. sanguinolentum and total rot), it is evident that the single-tree selection harvesting was partially responsible for some of the observed rot. One of the selection criteria in the initial harvest was a sanitary removal of trees of poor vitality. Varying degrees of sanitation felling may therefore have offset the effects of new infections in wounds or spread of rot fungi through adjacent stumps. Supplementary soil scarification in small gaps of the residual stand had no significant effect on the frequency of rot, suggesting that such treatment may be used to facilitate regeneration in uneven-sized spruce stands on similar sites. [ABSTRACT FROM AUTHOR]

Heterobasidion 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. [ABSTRACT FROM AUTHOR]

Heterobasidion irregulare(Underw.) Garbel. & Otrosina is an important fungal parasitic basidiomycete of coniferous trees throughout temperate regions of the world. In Canada, the disease was first reported on red pine in Ontario in 1955, then in Quebec in 1989. As infection probability is proportional to the density of its aerial basidiospores infecting freshly cut pine stumps, the purpose of this study was to quantify theH. irregulareaerial basidiospore deposition along a southern Quebec transect. Spore counts from automated rotary arm collectors were determined by using a ribosomal ITS TaqMan real-time PCR detection assay. Total count was performed on rods at each location, with the highest score of 706.3 spores in Harrington, then 459.6 spores in Cowansville, rapidly decreasing to 52.3 spores in Compton and reaching lows in St-Aubert and Rimouski, with an eastward direction on the transect. Clearly, plantations in southwestern Quebec are at a higher risk of infection than anywhere else in the province. With few exceptions, weekly spore depositions at all sites other than Harrington were greatly below 0.2 per m−2 h−1. Despite this very low spore deposition rate observed in one season, the large number of trees thinned annually warrants that stump treatment with Rotstop® C is still the best strategy, especially in valuable plantations, to prevent against infection byHeterobasidion. Heterobasidion irregulare(Underw.) Garbel. & Otrosina est un important basidiomycète parasite des conifères des régions tempérées de la planète. Au Canada, la maladie a été détectée pour la première fois en Ontario, en 1955, sur le pin rouge, puis au Québec en 1989. Étant donné que la probabilité d’infection est proportionnelle à la densité des basidiospores dispersées par voie aérienne infectant les souches des arbres qui viennent d’être coupés, l’objectif de cette étude était de quantifier le dépôt des basidiospores deH. irregularedispersées par voie aérienne le long d’un transect traversant le sud du Québec. Les d&#xe9.comptes de spores résultant des collecteurs automatiques à bras rotatifs ont été déterminés par analyse de détection basée sur une PCR en temps réel de type TaqMan de l’ITS ribosomique. Le d&#xe9.compte total a été effectué sur des tiges à chaque location, le plus haut compte étant de 706.3 spores, provenant de Harrington, puis de 459.6 spores, de Cowansville, décroissant rapidement à 52.3 spores à Compton pour atteindre les plus bas comptes à Saint-Aubert et à Rimouski, en direction est en suivant le transect. De toute évidence, les plantations du Sud-Ouest québécois sont plus susceptibles d’être infectées que celles des autres régions de la province. À quelques exceptions près, les dépôts hebdomadaires de spores à tous les sites, sauf à Harrington, étaient bien sous les 0.2/m−2h−1. Malgré ce très faible taux de dépôt de spores observé en une saison, le grand nombre d’arbres prélevés chaque année garantit que le traitement des souches avec Rotstop C est encore la meilleure stratégie, spécialement dans les plantations de grande valeur, pour prévenir l’infection causée parHeterobasidion. [ABSTRACT FROM AUTHOR]

Lytic polysaccharide monooxygenases (LPMO) are important redox enzymes produced by microorganisms for the degradation of recalcitrant natural polysaccharides. Heterobasidion irregulare is a white-rot phytopathogenic fungus that causes wood decay in conifers. The genome of this fungus encodes 10 putative Auxiliary Activity family 9 (AA9) LPMOs. We describe the first biochemical characterization of H. irregulare LPMOs through heterologous expression of two CBM-containing LPMOs from this fungus (HiLPMO9H, HiLPMO9I) in Pichia pastoris. The oxidization preferences and substrate specificities of these two enzymes were determined. The two LPMOs were shown to cleave different carbohydrate components of plant cell walls. HiLPMO9H was active on cellulose and oxidized the substrate at the C1 carbon of the pyranose ring at β-1,4-glycosidic linkages, whereas HiLPMO9I cleaved cellulose with strict oxidization at the C4 carbon of glucose unit at internal bonds, and also showed activity against glucomannan. We propose that the two LPMOs play different roles in the plant-cell-wall degrading system of H. irregulare for degradation of softwood and that the lignocellulose degradation mediated by this white-rot fungus may require collective efforts from multi-types of LPMOs. [ABSTRACT FROM AUTHOR]