Dr. Renate Heinzelmann

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.

Vom Triebsterben betroffene Eschen sind zusätzlich oft mit Hallimasch infiziert, was zu einem gefährlichen Stabilitätsverlust führt. In der Schweiz ausgearbeitete Richtlinien sollen dabei helfen, die Kipp- und Bruchgefahr einer Esche einzuschätzen.

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.

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.

Les frênes atteints de chalarose présentent souvent une infection des racines et du pied du tronc par l'armillaire. La dégradation des racines conduit à une perte de stabilité. Quelques règles devraient aider à évaluer les dangers et les minimiser.

Vom Eschentriebsterben betroffene Eschen zeigen oft eine Infektion des Wurzelsystems und des Stammfusses mit Hallimasch. Die Wurzelzersetzung führt zu einem Stabilitätsverlust. Richtlinien sollen helfen, Gefahren richtig einzuschätzen und zu minimieren.

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.

Der Hallimasch ist ein in der Schweiz weit verbreiteter Pilz. Er lebt versteckt das ganze Jahr über in errottendem Holz oder im Boden und wird erst im Herbst anhand seiner massenhaft an Baumstümpfen und abgestorbenen Bäumen heranwachsenden Fruchtkörper sichtbar. Die meiste Zeit führt er ein unscheinbares Leben als Zersetzer von Totholz. Einige Arten sind jedoch dazu in der Lage, lebende Bäume zu befallen und sind daher wirtschaftlich relevant. Auch in Gärten oder in Parks tritt der Hallimasch als ungeliebter Gast auf.

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.

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.

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 as a 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 87 kb 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.

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.

Development of molecular markers for phylogenetic, population genetics and phylogeographic studies remains arduous in non-model species with low or no genomic resources. Sequencing the whole or a large part of the genome of the target species using next-generation sequencing technologies is considered a promising method, although it still needs a large investment in bioinformatics. To quickly find polymorphic markers in fungal species, we tested an alternative method, named PHYLORPH. This method allows users to quickly target polymorphic regions of single copy genes in fungi using public databases. We applied this method to Armillaria species, which are important fungal pathogens and saprophytes playing a central role in the dynamics of forest ecosystems worldwide. We isolated 32 single copy genes with numerous single nucleotide polymorphism (SNP) sites. A genetic analysis of two French populations validated the polymorphism of 80 among 92 SNPs tested, and seven of these sequences exactly reconstructed the known phylogenetic tree of four tested Armillaria species. These results confirmed that the PHYLORPH method is efficient to identify various markers at both the intra- and interspecific levels for fungal species with no or few previous genetic markers.

Der Hallimasch ist in den Wäldern der Schweiz weit verbreitet. Er ist ein ungeliebter Gast in Gärten und Parks, weil er nahezu alle Baum- und Straucharten befallen und abtöten kann. Auch im Wald kann der Hallimasch als Schädling auftreten. Die meiste Zeit führt er jedoch ein unauffälliges Leben als Zersetzer von Totholz.

Mycelial fan formation was studied in five Armillaria cepistipes, ten A. borealis and ten diploid and six haploid A. ostoyae strains on excised stem segments of Picea abies. Stem segments were either non-autoclaved or autoclaved, representing dying and dead wood, respectively. To confirm the identity of mycelial fans on non-autoclaved stem segments, re-isolations were made and isolates characterized with microsatellite markers. Mycelial fan formation on autoclaved stem segments was fast and reliable for most of the tested Armillaria strains. On non-autoclaved stem segments, mycelial fan formation was slower, more erratic and less predictable. Mycelial fan formation was fastest in A. cepistipes closely followed by A. borealis and was slowest in A. ostoyae. For two A. cepistipes and four A. ostoyae strains (all diploid), growth rates of mycelial fans were estimated in a time course experiment. They ranged between 5.1 and 8.7 mm/day for autoclaved and between 1.4 and 4.7 mm/day for non-autoclaved stem segments. The haploid A. ostoyae strains also formed mycelial fans on autoclaved stem segments, but typically slower and less reliably than the diploid strains. Whether haploid strains are able to produce mycelial fans on non-autoclaved stem segments remains unknown because of accidental diploidization of the original haploid strains which was likely caused by basidiospores introduced into the study system on the non-autoclaved stems. Overall, the method developed in this study maybe useful for further investigations into the genetic, physiological and biochemical nature of mycelial fan formation in the genus Armillaria.

Armillaria cepistipes is a common wood-rotting basidiomycete fungus found in most forests in Central Europe. In Switzerland, the habitat of A. cepistipes is fragmented because of the presence of major geographical barriers, in particular the Alps, and past deforestation. We analysed the impact of habitat fragmentation on the current spatial genetic structure of the Swiss A. cepistipes population. A total of 167 isolates were sampled across an area of 41 000 km² and genotyped at seven microsatellite and four single nucleotide polymorphism (SNP) loci. All isolates belonged to different genotypes which, according to the Bayesian clustering algorithm implemented in TESS, originated from a single gene pool. Our analyses indicate that the overall A. cepistipes population shows little, but significant (F_ST=0.02), genetic differentiation. Such a situation suggests gene flow is strong, possibly due to long-distance dispersal of airborne basidiospores. This hypothesis is supported by the fact that we could not detect a pattern of isolation by distance. Gene flow is partially restricted by the high mountain ranges of the Alps, as indicated by a signal of spatial autocorrelation detected among genotypes separated by less than about 80-130 km. In contrast, past deforestation seems to have no significant effect on the current spatial population structure of A. cepistipes. This might indicate the existence of a time lag between the current spatial genetic structure and the processes that have induced this specific structure.