The common ash (Fraxinus excelsior) populations in Europe are suffering from ‘ash dieback’ caused by the fungal pathogen Hymenoscyphus fraxineus. The disease was introduced to north-eastern Europe more than three decades ago and has spread throughout the continent since then. Hope that common ash will not completely disappear is provided by findings that a small percentage of ash trees remain healthy despite being exposed to high pathogen pressure and that this reduced disease susceptibility has a heritable, genetic component. Here, we review recent studies related to the mechanisms underlying the resistance and tolerance of common ash to H. fraxineus. Our aim is to provide up-to-date information relevant for ash conservation and breeding programmes, and to identify knowledge gaps where research would provide further insights. To date, the identification of resistant and/or tolerant trees is mostly based on crown health assessments of common ash trees exposed to high infection pressure in forests or in experimental nurseries. Other assays are being developed, and lesion length in artificial stem inoculation experiments seems to correlate well with crown health in the field. The extent to which phenological, physiological, and morphological traits may contribute to resistance and tolerance is still unclear. The availability of genomic resources for common ash has led to the identification of molecular markers putatively associated with resistance and/or tolerance. Similarly, phytochemical markers in bark/stem and leaf tissue with a putative association with resistance and/or tolerance have been identified. However, a thorough validation of these markers to clarify their utility for breeding programmes is still lacking. Although recent research attributes high importance to the defence mechanisms acting in the bark or stem tissues of common ash, the ability of ash to prevent foliar infections or inhibit fungal growth from petioles to shoots requires further investigation. Overall, current research shows that the mechanisms underlying resistance and tolerance are highly complex and have not yet been fully explored.

BACKGROUND:Culturing of fungi is labor-intensive and reveals limited diversity, while high-throughput sequencing of barcodes (i.e., metabarcoding) enables a simultaneous detection of fungi from multiple environmental samples. Metabarcoding using short-read sequencers, such as Illumina platforms, provides high sequencing depths but results in many unidentified taxa. Long-read sequencing can improve species and genus assignments but might encompass lower sequencing depth and limit diversity coverage. In this study, fungi in seeds of eleven angiosperm and gymnosperm tree species were assessed using traditional culturing, Illumina short-read metabarcoding, and Oxford Nanopore Technologies long-read metabarcoding. We focused on seed-borne fungi as understanding their diversity and potential impacts on seedlings is crucial for securing plant health. We compared (1) the number and identity of fungal genera and species between metabarcoding approaches and traditional culturing and (2) fungal alpha- and beta-diversity between metabarcoding methods, considering different hosts and fungal lifestyles. RESULTS:In both short- and long-read metabarcoding datasets, similar numbers of fungal reads and operational taxonomic units were assigned to comparable numbers of fungal genera and species. About one-third of the identified genera were plant pathogens, followed by saprotrophs and endophytes. Culturing overall revealed fewer fungal genera, while most of the fungal reads in short-read metabarcoding datasets stemmed from cultured taxa. Long-read metabarcoding revealed lower per-sample diversity than short-read metabarcoding and distinct fungal communities compared to those from the short-read datasets. Host-dependent patterns in alpha- and beta-diversity were observed across methods, with angiosperms harboring more fungal taxa than gymnosperms, and distinct community structuring across host tree groups and species, although the differences were stronger in short-read than long-read metabarcoding datasets. CONCLUSIONS:Illumina and Oxford Nanopore Technologies metabarcoding captured similar host-dependent diversity patterns despite observed differences in numbers and composition of fungi. Short-read metabarcoding might be optimal for fungal biodiversity studies due to higher sequencing depths and resultant breadth of diversity. As error rates are continuing to decrease, reference databases expand, and throughput improves, long-read metabarcoding is becoming a strong candidate for future diagnostic studies of fungi. Traditional culturing captures most of the fungi from short-read metabarcoding and remains valuable for obtaining isolates for further research.

Climate significantly influences the distribution, composition, and diversity of fungal communities, impacting the growth, spread, and virulence of fungal forest pathogens. This study employs advanced landscape genomics methods to explore the genomic adaptations of three major fungal pathogens: Those responsible for Dutch elm disease, dothistroma needle blight, and Swiss needle cast. Our findings reveal that precipitation and humidity are primary drivers of adaptation in these species. We use these insights to forecast potential adaptations under future climate scenarios (genomic offset) and identify specific genes and pathways associated with climate responses in each pathogen. Notably, we detect a convergence in moisture adaptation across these distantly related species, particularly in genes related to the cytoskeleton and transporters. This study enhances our understanding of fungal pathogen evolution in response to climate change, offering crucial insights for forest disease management.

Since the early 1990s, the invasive pathogen Hymenoscyphus fraxineus has been spreading in Europe causing severe dieback of common ash (Fraxinus excelsior) and narrow-leaved ash (Fraxinus angustifolia). H. fraxineus also causes necrotic lesions at the stem base and on roots of ash trees, which frequently serve as an entry point of secondary wood decay fungi, like Armillaria spp. Rot of the stem base and roots leads to structural weaknesses of ash trees, which makes them prone to uprooting or stem fracturing during storm events. To prevent fatalities and damage to infrastructure it is crucial to timely identify and remove potentially hazardous ash trees. Here, we investigated the synergistic effects of H. fraxineus and Armillaria spp. on the health status (crown defoliation, presence, and extent of basal stem necroses) of mature trees of common ash in ten mixed forest stands in Switzerland over a four-year period (2018-2022). In addition, we conducted non-destructive static load tests on a set of 30 ash trees to assess their breaking and tipping stability so that stability weakness at the stem base could be related to tree health data. The health of the monitored ash trees declined rapidly during the monitoring period, indicating that also mature ash trees in mixed forests may be heavily impacted by ash dieback after prolonged exposure to H. fraxineus (here 12-13 years) and subsequent colonization by root rot pathogens. At the end of the monitoring, only 4.1 % of ash trees with a healthy crown (defoliation <= 25 %) remained and 75.4 % of ash trees showed basal stem necroses, which were, with a few exceptions, all colonized by Armillaria. Although the results from the static load tests indicated that predicting tree stability based on crown defoliation level and stem base damage level is not straightforward, i.e. also trees with advanced crown defoliation and stem necrosis can still be stable, our study shows that ash trees with necroses affecting at least 20 % of the basal stem circumference and trees with more than 75 % crown defoliation are likely to suffer from a weakness at the stem base. Building on the new findings and previous research, guidelines for the management of mature ash trees affected by ash dieback are suggested.

The globally distributed basidiomycete genus Armillaria includes wood decomposers that can act as opportunistic parasites, causing deadly root rot on woody plants. To test whether RNA viruses are involved in this opportunistic behaviour, a large isolate collection of five Armillaria species collected over 40 years in Switzerland from trees, dead wood and soil was analysed. De novo assembly of RNA-Seq data revealed 21 viruses, 14 of which belong to putative new species. Two dsRNA viruses and an unclassified Tymovirales are formally described for the first time for Armillaria. One mitovirus occurred with a high prevalence of 71.1%, while all other viruses were much less prevalent (0.6%-16.9%). About half of all viruses were found only in one fungal species, others occurred in 2-6 fungal species. Co-infections of 2-7 viruses per isolate were not uncommon (34.9%), and most viruses persisted circulating within fungal populations for decades. Some viruses were related to viruses associated with other Armillaria species, supporting the hypothesis that virus transmission can occur between different fungal species. Although no specific correlation between viruses and the fungal trophic strategy was found, this study opens new insights into viral diversity hidden in the soil microbiome.

Background Culturing of fungi is labor-intensive and reveals limited diversity, while high-throughput sequencing of barcodes (i.e., metabarcoding) enables simultaneous detection of fungi from multiple environmental samples. Short-read metabarcoding, using for example Illumina platforms, provides high sequencing depths but results in many unidentified taxa. Long-read sequencing may improve species and genus assignments but encompasses lower sequencing depth, limiting diversity coverage. In this study, we assessed fungi in seeds of eleven angiosperm and gymnosperm tree species using traditional culturing, Illumina short-read metabarcoding, and Oxford Nanopore Technologies long-read metabarcoding. We focused on seed-borne fungi as understanding their diversity and potential impacts on seedlings is crucial for securing plant health. We compared 1) the number and identity of fungal genera and species between metabarcoding approaches and traditional culturing and 2) fungal alpha- and beta-diversity between metabarcoding methods, considering different hosts and fungal lifestyles.Results In both short- and long-read metabarcoding datasets, similar numbers of fungal reads and operational taxonomic units were assigned to comparable numbers of fungal genera and species. About one-third of the identified genera were plant pathogens, followed by saprotrophs and endophytes. Culturing overall revealed fewer fungal genera, while most of the fungal reads in short-read metabarcoding datasets stemmed from cultured taxa. Long-read metabarcoding revealed lower per-sample diversity than short-read metabarcoding and distinct fungal communities. Host-dependent patterns in alpha- and beta-diversity were observed across all methods, with angiosperms harboring more fungal taxa than gymnosperms, and distinct community structuring across host tree groups and species, although the differences were stronger in short-read than long-read metabarcoding datasets.Conclusions Illumina and Oxford Nanopore Technologies metabarcoding captured similar host-dependent diversity patterns despite observed differences in numbers and composition of fungi. Short-read metabarcoding might be optimal for fungal biodiversity studies due to higher sequencing depths and resultant breadth of diversity. As error rates decrease, reference databases expand, and throughput improves, long-read metabarcoding may become a strong candidate for future diagnostic studies of fungi. Traditional culturing captures most of the fungi from short-read metabarcoding and remains valuable for obtaining isolates for further research.

Fungal bioluminescence is mystifying humans since ancient times. Nevertheless, the biosynthetic pathway behind this phenomenon was only very recently resolved. Fungal bioluminescence occurs in five distantly related linages (Omphalotaceae lineage, Armillaria lineage, mycenoid lineage, Lucentipes lineage and Eoscyphella lineage) of the basidiomycete order Agaricales. Recent research suggests fungal bioluminescence has emerged 160 million years ago in the most common ancestor of the mycenoid and marasmioid clade and is maintained since then. Surprisingly, in the mycenoid linage, primarily represented by the genus Mycena, most species are considered non-luminescent, implying that many mycenoid species have lost their bioluminescent ability. Here, we report evidence for bioluminescence in Mycena crocata and show that the genome of this species is fully equipped with the genes associated with fungal bioluminescence. Mycena crocata is a long-known species frequently reported from Europe and Japan, which was considered non-luminescent until now. The low light emission intensity and the restriction of the luminescence to the vegetative mycelium and the base of the basidiome may be reasons why bioluminescence was not perceived earlier. We assume there might be other known Mycena species whose luminescent properties are not yet discovered, and that therefore the number of bioluminescent Mycena species is currently underestimated.

Invasive exotic pathogens pose a threat to trees and forest ecosystems worldwide, hampering the provision of essential ecosystem services such as carbon sequestration and water purification. Hybridization is a major evolutionary force that can drive the emergence of pathogens. Phytophthora ramorum , an emergent pathogen that causes the sudden oak and larch death, spreads as reproductively isolated divergent clonal lineages. We use a genomic biosurveillance approach by sequencing genomes of P. ramorum from survey and inspection samples and report the discovery of variants of P. ramorum that are the result of hybridization via sexual recombination between North American and European lineages. We show that these hybrids are viable, can infect a host and produce spores for long-term survival and propagation. Genome sequencing revealed genotypic combinations at 54,515 single nucleotide polymorphism loci not present in parental lineages. More than 6,000 of those genotypes are predicted to have a functional impact in genes associated with host infection, including effectors, carbohydrate-active enzymes and proteases. We also observed post-meiotic mitotic recombination that could generate additional genotypic and phenotypic variation and contribute to homoploid hybrid speciation. Our study highlights the importance of plant pathogen biosurveillance to detect variants, including hybrids, and inform management and control.

Armillaria root disease, caused by diverse species from the Agaricomycete genus Armillaria s.l. (including the closely related genus Desarmillaria), is a primary cause of mortality and growth loss of widely ranging horticultural, urban, and forest trees and shrubs in diverse boreal, temperate, and tropical/subtropical regions where woody plants grow around the world. Whether behaving as a pathogen or saprotroph, Armillaria causes white rot, in which the three main polymeric constituents of woody tissues (lignin, cellulose, hemicellulose) are degraded during the decay process. This chapter provides a compendium of Armillaria root diseases on diverse trees in wide-spread global regions and includes topics such as species identification, disease spread, geographical distribution, ecology, infection biology, and disease management.

The ascomycete fungi Dothistroma septosporum (Dorogin) M. Morelet and D. pini Hulbary are both responsible for Dothistroma needle blight disease (DNB) on pines. Using a DNA-barcoding approach, we confirmed that the DNB infection of lodgepole pine (Pinus contorta Dougl. var. latifolia Engelm.), jack pine (P. banksiana Lamb.) and their hybrid in four research sites in northern Alberta (Canada) was caused by D. septosporum, confirming that the range of D. septosporum extends east of the Rocky Mountains into Alberta. Analysis of mating-type (MAT) genes showed the presence of both mating types in each of the pine plantations, indicating that the pathogen has the potential to complete its lifecycle in this region. Controlled inoculation confirmed that the two native pines found in this area (P. contorta var. latifolia and P. banksiana) and their hybrid are susceptible to D. septosporum, suggesting that continuous monitoring of the disease needs to be carried out to prevent any future outbreak in natural stands.

A Loop-Mediated Isothermal Amplification (LAMP) assay was developed for the detection of the pine pathogen Dothistroma septosporum (G. Dorog.) M. Morelet. The specificity of the LAMP assay was tested using a selection of pine needle fungi, including Dothistroma pini Hulbary, and Lecanosticta acicola (Thüm.) Syd.; only D. septosporum DNA was amplified by the test. In terms of sensitivity, the assay was able to detect as little as 1 pg of total D. septosporum DNA. This assay enables DNA extracted from diseased host needles to be rapidly tested for the presence of D. septosporum using relatively simple to operate equipment away from a fully equipped molecular biology laboratory.

Dothistroma septosporum, the primary causal agent of Dothistroma needle blight, is one of the most significant foliar pathogens of pine worldwide. Its wide host and environmental ranges have led to its global success as a pathogen and severe economic damage to pine forests in many regions. This comprehensive global population study elucidated the historical migration pathways of the pathogen to reveal the Eurasian origin of the fungus. When over 3800 isolates were examined, three major population clusters were revealed: North America, Western Europe, and Eastern Europe, with distinct subclusters in the highly diverse Eastern European cluster. Modeling of historical scenarios using approximate Bayesian computation revealed the North American cluster was derived from an ancestral population in Eurasia. The Northeastern European subcluster was shown to be ancestral to all other European clusters and subclusters. The Turkish subcluster diverged first, followed by the Central European subcluster, then the Western European cluster, which has subsequently spread to much of the Southern Hemisphere. All clusters and subclusters contained both mating-types of the fungus, indicating the potential for sexual reproduction, although asexual reproduction remained the primary mode of reproduction. The study strongly suggests the native range of D. septosporum to be in Eastern Europe (i.e., the Baltic and Western Russia) and Western Asia.

Many current tree improvement programs are incorporating assisted gene flow strategies to match reforestation efforts with future climates. This is the case for the lodgepole pine (Pinus contorta var. latifolia), the most extensively planted tree in western Canada. Knowledge of the structure and origin of pathogen populations associated with this tree would help improve the breeding effort. Recent outbreaks of the Dothistroma needle blight (DNB) pathogen Dothistroma septosporum on lodgepole pine in British Columbia and its discovery in Alberta plantations raised questions about the diversity and population structure of this pathogen in western Canada. Using genotyping-by-sequencing on 119 D. septosporum isolates from 16 natural pine populations and plantations from this area, we identified four genetic lineages, all distinct from the other DNB lineages from outside of North America. Modeling of the population history indicated that these lineages diverged between 31.4 and 7.2 thousand years ago, coinciding with the last glacial maximum and the postglacial recolonization of lodgepole pine in western North America. The lineage found in the Kispiox Valley from British Columbia, where an unprecedented DNB epidemic occurred in the 1990s, was close to demographic equilibrium and displayed a high level of haplotypic diversity. Two lineages found in Alberta and Prince George (British Columbia) showed departure from random mating and contemporary gene flow, likely resulting from pine breeding activities and material exchanges in these areas. The increased movement of planting material could have some major consequences by facilitating secondary contact between genetically isolated DNB lineages, possibly resulting in new epidemics.

Recombination shapes the evolutionary trajectory of populations and plays an important role in the faithful transmission of chromosomes during meiosis. Levels of sexual reproduction and recombination are important properties of host–pathogen interactions because the speed of antagonistic co-evolution depends on the ability of hosts and pathogens to generate genetic variation. However, our understanding of the importance of recombination is limited because large taxonomic groups remain poorly investigated. Here, we analyze recombination rate variation in the basidiomycete fungus Armillaria ostoyae , which is an aggressive pathogen on a broad range of conifers and other trees. We analyzed a previously constructed, dense genetic map based on 198 single basidiospore progeny from a cross. Progeny were genotyped at a genome-wide set of single-nucleotide polymorphism (SNP) markers using double digest restriction site associated DNA sequencing. Based on a linkage map of on 11,700 SNPs spanning 1007.5 cM, we assembled genomic scaffolds into 11 putative chromosomes of a total genome size of 56.6 Mb. We identified 1984 crossover events among all progeny and found that recombination rates were highly variable along chromosomes. Recombination hotspots tended to be in regions close to the telomeres and were more gene-poor than the genomic background. Genes in proximity to recombination hotspots were encoding on average shorter proteins and were enriched for pectin degrading enzymes. Our analyses enable more powerful population and genome-scale studies of a major tree pathogen.

The basidiomycete genus Armillaria s.l. (Armillaria s.s. and Desarmillaria) has a worldwide distribution and plays a central role in the dynamics of numerous woody ecosystems, including natural forests, tree plantations for timber production, orchards, vineyards and gardens. Early studies have shown that all Armillaria species are capable of degrading dead woody substrates, causing white rot. Moreover, most species exhibit a parasitic ability, and can be considered as facultative necrotrophs. Although over the years extensive research has been conducted on the phylogeny, biology and ecology of different Armillaria species, numerous theoretical and applied questions remain open. Recently published studies have provided new perspectives, the most significant of which we present in this review. First, new investigations have highlighted the importance of a multilocus approach for depicting the phylogeny of the genus Armillaria. Second, the importance of clonality and sexuality for the different species is now better described, enabling a more accurate prediction of population dynamics in various environments. Third, genome sequencing has provided new insights into genome evolution and the genetic basis of pathogenicity and wood degradation ability. Fourth, several new studies have pointed out the possible influence of climate change on Armillaria distribution, biology and ecology, raising questions regarding the future evolution of Armillaria species and their effect on ecosystems. In this review, we also give a state-of-the-art overview of the control possibilities of parasitic Armillaria species. Finally, we outline some still open questions in Armillaria research, the investigation of which will strongly benefit from recent methodological advances.

Very little is known about the biology and ecology of haploid Armillaria strains in nature. In this outdoor inoculation experiment, we assessed the virulence of six haploid Armillaria ostoyae strains along with their diploid parent towards 2-year-old seedlings and 4-year-old saplings of Norway spruce (Picea abies), and determined their ability to colonise freshly cut stumps. As inoculum source an Armillaria-colonised hazelnut (Corylus avellana) stem segment was inserted into the soil substrate. Re-isolations from mycelial fans at the root collar of infected trees or stumps were made. Surprisingly, not a single haploid re-isolate could be recovered. Microsatellite genotyping of 133 re-isolates suggests that the inoculated haploid strains were diploidised either by mating propagules (basidiospores or haploid mycelia) already present in the soil substrate or naturally disseminated in the course of the experiment from nearby forests. Consequently, no conclusion about the infectious ability of haploid Armillaria mycelia under natural conditions can be drawn. Nonetheless, the diploid half-sib families resulting from the diploidisation showed varying degrees of virulence, with a high correlation between the experiment with 2-year-old seedlings and 4-year-old saplings. Despite extensive genotyping of re-isolates, no evidence for somatic recombination between haploid mating propagules and diploidised mycelia was detected, suggesting that this is an uncommon phenomenon in A. ostoyae.

Although Armillaria borealis is one of the closest relatives of the aggressive root rot pathogen A. ostoyae, little is known about its ecology. In central and northern Europe, A. borealis often co-occurs with A. ostoyae or A. cepistipes, a weak pathogen, in conifer or mixed-forest stands. In this study, the virulence of 10 A. borealis, nine A. ostoyae, and five A. cepistipes isolates toward 2- and 4-year-old potted Norway spruce (Picea abies) seedlings was assessed. In addition, the ability of all isolates to colonize fresh stumps cut from 6-year-old Norway spruce seedlings was tested. All inoculations were done by insertion of Armillaria-colonized hazelnut stem segments into the soil substrate. On the 2-year-old seedlings, A. borealis and A. ostoyae showed, overall, a similar virulence 29 months after inoculation whereas, on the 4-year-old seedlings, A. ostoyae was more virulent. The third species in the experiment, A. cepistipes, caused almost no seedling mortality. Six months after cutting, the highest percentage of stumps was colonized by A. ostoyae (94.3%), followed by A. borealis (85.2%), and A. cepistipes (78.4%). Our inoculation experiments show that A. borealis has a clear pathogenic potential toward Norway spruce seedlings. However, compared with A. ostoyae, damage caused by A. borealis may decrease more rapidly with increasing tree age. Similar to other Armillaria species, A. borealis seems to be an efficient colonizer of fresh stumps.

Filamentous fungi exhibit a broad spectrum of heritable growth patterns and morphological variations reflecting the adaptation of the different species to distinct ecological niches. But also within species, isolates show considerable variation in growth rates and other morphological characteristics. The genetic basis of this intraspecific variation in mycelial growth and morphology is currently poorly understood. By chance, a growth mutant in the root rot pathogen Armillaria ostoyae was discovered. The mutant phenotype was characterized by extremely compact and slow growth, as well as shorter aerial hyphae and hyphal compartments in comparison to the wildtype phenotype. Genetic analysis revealed that the abnormal phenotype is caused by a recessive mutation, which segregates asa single locus in sexual crosses. In order to identify the genetic basis of the mutant phenotype, we performed a quantitative trait locus (QTL) analysis. A mapping population of 198 haploid progeny was genotyped at 11,700 genome-wide single nucleotide polymorphisms (SNPs) making use of double digest restriction site associated DNA sequencing (ddRADseq). In accordance with the genetic analysis, a single significant QTL was identified for the abnormal growth phenotype. The QTL confidence interval spans a narrow, gene dense region of 87kb in the A. ostoyae genome which contains 37 genes. Overall, our study reports the first high-density genetic map for an Armillaria species and shows its successful application in forward genetics by resolving the genetic basis of a mutant phenotype with a severe defect in hyphal growth.