Ink disease, caused by the two oomycetes Phytophthora cinnamomi and Phytophthora x cambivora, is one of the most devastating diseases of sweet chestnut (Castanea sativa) in Europe. Its control is particularly difficult, with few fungicides showing a satisfactory effect, but being progressively banned in forest ecosystems, or in forest-like plantations because of ecological concerns. Therefore, alternative, nature-friendly control methods are urgently needed. The bacterial genus Pseudomonas includes many species with antifungal and antimicrobial properties. In this study, we isolated Pseudomonas bacteria from the rhizosphere of healthy and diseased chestnut trees in southern Switzerland. Out of 166 isolated Pseudomonas strains, five showed significant growth inhibition of P. cinnamomi and/or P. x cambivora in vitro. Three strains that significantly inhibited the growth of both pathogens were tested against a collection of 20 isolates of each species, which showed that the in vitro inhibition is independent of the pathogen isolate. The mechanisms underlying this inhibition remain unclear, since none of the three strains harbored biosynthesis genes coding for well-known antimicrobial secondary metabolites. Phylogenetic analysis on whole genome sequences showed that two of the strains belong to the P. fluorescens subgroup, while one strain probably belongs to a previously unrecognized species closely related to the P. putida subgroup. The results of this study indicate that Pseudomonas have the potential to be used as biocontrol agents against ink disease. Further experiments are needed to confirm their inhibitory effect against P. cinnamomi and P. x cambivora in vivo.

Background Tree seeds harbor diverse fungal communities, including both pathogens and mutualists, that can influence plant health. These communities comprise living, metabolically active organisms as well as dormant or dead cells. Because only active fungi interact with their hosts, distinguishing active from inactive taxa is crucial, especially for environmental and phytosanitary monitoring. Traditional culturing methods capture living fungi but account for only a small fraction of the total fungal diversity. Currently, these methods are increasingly replaced by high-throughput DNA metabarcoding, which detects a broader range of taxa. However, DNA persists after cell death and occurs in dormant cells, preventing distinction between active and inactive fungi. In contrast, RNA metabarcoding detects metabolically active organisms and may better reflect living fungal communities than the other two methods, though its use in assessing plant-associated fungi remains underexplored. We used culturing, DNA-, and RNA-based metabarcoding to compare fungal communities associated with seeds of three key European tree species ( Fagus sylvatica , Abies alba , Pinus sylvestris ). Results DNA and RNA metabarcoding detected largely distinct, non-overlapping fungal communities, with differences primarily driven by rare active taxa in the RNA dataset. Several cultured genera—likely representing abundant and metabolically active taxa—were shared between both metabarcoding approaches. Conclusions These results highlight the complementary nature of the three methods for characterising seed-associated fungi. Combining culturing, DNA- and RNA-based metabarcoding may provide the most comprehensive assessment of fungal diversity, while RNA metabarcoding alone offers a promising opportunity to identify the active members of fungal communities for improved environmental and phytosanitary monitoring.

Within Phytophthora alni, an invasive pathogen of alders (Alnus spp.), three species have been identified. The most frequent and pathogenic species is P. × alni. It has a variable intraspecific structure, with dominance of the Pxa-1 genotype and the presence of dozens of rare genotypes (in most cases derived from Pxa-1). Its local populations are highly variable, and their population structure and development remain unknown. We compared two sets of strains isolated from identical sites during the epidemic (2005 to 2010) and post-epidemic (2020 to 2024) phases of the disease in the Vltava River basin (Czech Republic) and studied them using microsatellite marker analysis and fitness tests (sporangia production, growth, and virulence). We acquired 151 P. × alni isolates of 23 multilocus genotypes. We found that during the post-epidemic phase, genetic diversity decreased, and the dominance and incidence of the Pxa-1 genotype increased. Only the dominant genotype (Pxa-1) was repeatedly isolated from the same sites, whereas the rare genotypes were replaced. During the post-epidemic phase, both the incidence of rare genotypes and the degree of their derivation from Pxa-1 decreased. The rare genotypes had lower fitness than Pxa-1 (the more changes there were, the worse the fitness was). These results allow us to hypothesize the evolution of local populations of P. × alni in Europe, as the most pathogenic genotype, Pxa-1, will also prevail during the late phases of the disease, and the risk of further damage to the surviving host populations will persist.

Global increases in connectivity have greatly accelerated the frequency of biological invasions across most of Earth’s ecosystems, including forests. Once invasive organisms become established in a naïve environment, they are difficult to eradicate or contain; thus, management strategies often focus on mitigating their impacts. As the use of chemical pesticides in forests is increasingly prohibited, biological control of pests and diseases has gained importance as an environmentally friendly alternative. Virus-mediated hypovirulence in the chestnut blight fungus Cryphonectria parasitica is one of the few successful examples of biological control of an invasive forest pathogen. However, experiments testing the stability of this system in situ are still missing. In this study, we conducted a field experiment in chestnut stands with naturally established hypovirulence in Switzerland, Croatia, and North Macedonia to evaluate the effectiveness of CHV1-mediated biocontrol of chestnut blight under different vegetative compatibility (vc) type population structures. Our results demonstrate that CHV1 is highly effective as a biological control agent against C. parasitica. Artificially initiated bark cankers of various vc types were rapidly infected by resident CHV1 strains, which significantly reduced canker growth and sporulation, thereby increasing the survival chances of the infected chestnut sprouts. Under field conditions, vegetative incompatibility barriers proved to be far less restrictive for virus transmission than predicted in vitro. Furthermore, our study demonstrates that the immigration of new fungal genotypes into existing cankers is an inherent component of the epidemiology of C. parasitica, which significantly contributes to the spread of CHV1. These results are particularly favourable for ensuring the success of hypovirulence-mediated biocontrol of chestnut blight in Europe. However, our conclusions cannot be automatically translated to genetically distant vc types from outside Europe, whose accidental introduction should be further avoided.

Tree seeds harbor diverse fungal communities, including both pathogens and mutualists, that can influence plant health. These communities comprise living, metabolically active organisms as well as dormant or dead cells. Because only active fungi interact with their hosts, distinguishing active from inactive taxa is crucial, especially for environmental and phytosanitary monitoring. Traditional culturing methods capture living fungi but account for only a small fraction of the total fungal diversity. Currently, these methods are increasingly replaced by high-throughput DNA metabarcoding, which detects a broader range of taxa. However, DNA persists after cell death and occurs in dormant cells, preventing distinction between active and inactive fungi. In contrast, RNA metabarcoding may better reflect living fungal communities than the other two methods, though its use in assessing plant-associated fungi remains underexplored. We used culturing, DNA-, and RNA-based metabarcoding to compare fungal communities associated with seeds of three key European tree species (Fagus sylvatica, Abies alba, Pinus sylvestris). Dominant fungal communities in seeds were strongly shaped by host species identity and were largely shared across DNA and RNA metabarcoding datasets, with roughly half of the most abundant genera detected by both methods. Differences between DNA- and RNA-derived communities were predominantly associated with rare taxa in the RNA dataset, although distinguishing true biological signals from noise introduced by different methodological workflows remains challenging. Several cultured genera, likely both abundant and metabolically active, were consistently detected by both approaches. These results highlight the complementary nature of the three methods for characterising seed-associated fungi. Combining culturing, DNA- and RNA-based metabarcoding may provide the most comprehensive assessment of fungal diversity, while RNA metabarcoding alone offers a promising opportunity to identify the active members of fungal communities for improved environmental and phytosanitary monitoring.

Monitoring regulated quarantine plant pathogens and, when relevant, their vectors is compulsory in the European Union. Local Monochamus species (Coleoptera, Cerambycidae) vector the pine wood nematode, Bursaphelenchus xylophilus, a non-native pest already established in most of Portugal. Only 29 M. galloprovincialis individuals were trapped in Belgium in ten years (2013–2022), despite a dense coverage of pheromone-baited traps, suggesting absence or a very rare local occurrence in the country. In the northern neighbouring countries, only one single established population is known in The Netherlands and one in Denmark. A species distribution model based on pheromone-trap catches (negative and positive) of M. galloprovincialis from 4,914 traps in 29 European countries between 2008 and 2019 was developed, using the overall climate conditions and the distribution of seven pine tree species as explanatory variables. The effect of spatial scale was tested with a multi-scale approach. With a 225*225 km spatial grain, the major explanatory variables were the mean diurnal temperature range and, to a lesser extent, the presence of Pinus spp. The model predicted a low probability of presence in Belgium, the Netherlands, Great Britain and north-western Germany compared to southern Europe. Genotyping allowed to conclude that at least some of the beetles caught in Belgium originated from foreign locations. All catches were located close to entry points, suggesting introduction with imported material. The small size of most of the Belgian pine stands may also explain the absence or apparently transient status, or rareness of Monochamus spp. This study thus suggests that surveys in Belgium should privilege entry points rather than local forest stands.

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.

In this study, we assessed the occurrence and diversity of four oomycete genera ( Phytophthora , Phytopythium , Pythium , and Globisporangium ) in 13 declining alder ( Alnus glutinosa and A. incana ) stands in Switzerland. For this, we sampled and analyzed soil from tree rhizosphere, water from streams and rivers along which the stands were located, and symptomatic alder bark. The overall isolation rate was 47.2%, with a total of 400 oomycete isolates recovered at all 13 sites. The highest incidence of oomycete isolates was in soil samples (baiting, 82.5% isolation rate), followed by water (baiting, 14.7%), and bark (direct isolation, 2.7%). Of all recovered oomycete isolates, 90.3% could be successfully assigned to a known species, for a total of 23 species identified, including both preferential saprotrophs and pathogens. Among all genera, Phytophthora was the most abundant with 273 isolates (75.6%), followed by Phytopythium , Pythium , and Globisporangium . Oomycete species diversity showed a significant variation among substrates. Only one species— Phytophthora lacustris —was abundant in all substrates, while 16 species were restricted to a specific substrate, mainly soil. The rhizosphere of symptomatic alder trees harbored the most diverse oomycete community, highlighting once again the importance of soil as a reservoir for these microorganisms. Only two Phytophthora species were isolated from alder bark lesions, namely, P. × alni , the known causal agent of alder decline, and P. lacustris . The low recovery rate of P. × alni might be due to attempts to isolate it from old, inactive lesions, but may also suggest that alder decline might be caused by other oomycetes infecting the root system of the trees.

Recombination suppression can evolve in sex or mating-type chromosomes, or in autosomal supergenes, with different haplotypes being maintained by balancing selection. In the invasive chestnut blight fungus Cryphonectria parasitica, a genomic region was suggested to lack recombination and to be partially physically linked to the mating-type (MAT) locus based on segregation analyses. Using hundreds of available C. parasitica genomes and generating new high-quality genome assemblies, we show that a ca. 1.2 Mb genomic region proximal to the mating-type locus lacks recombination, with the segregation of two highly differentiated haplotypes in balanced proportions in invasive populations. High-quality genome assemblies further revealed an inversion in one of the haplotypes in the invaded range. The two haplotypes were estimated to have diverged 1.5 million years ago, and each harboured specific genes, some of which likely belonging to Starships. These are large transposable elements, mobilized by tyrosine recombinases, able to move accessory genes, and involved in adaptation in multiple fungi. The MAT-proximal region carried genes upregulated under virus infection or vegetative incompatibility reaction. In the native range, the MAT-proximal region also appeared to have a different evolutionary history than the rest of the genome. In all continents, the MAT-Proximal region was enriched in nonsynonymous substitutions, in gene presence/absence polymorphism, in tyrosine recombinases and in transposable elements. This study thus sheds light on a case of a large nonrecombining region partially linked to a mating compatibility locus, likely maintained by balancing selection on differentiated haplotypes, possibly involved in adaptation in a devastating tree pathogen.

Hylurgus ligniperda (F.) is a highly successful invader among bark beetles (Scolytinae) and forest insects in general. Native to the western Palearctic region, it has become established in every continent where its host plants ( Pinus spp.) occur. Especially in southern hemisphere regions with large pine plantations, it is often highly abundant. As a repeat invader with a wealth of information on various aspects relevant for biological invasions, it is highly suitable as a model organism for studying the role of international trade, the planting of non-native trees, and the potential occurrence of bridgehead invasions (where abundant non-native populations precipitate further invasions). In the present study, our specific objectives were to reconstruct the worldwide invasions of H. ligniperda and the pathways involved by using a multi-pronged approach including population genetics, analysis of historic interception data generated from inspections of imports, and records of establishments in the literature. Our review of the native and non-native ranges of H. ligniperda and the chronology of establishments revealed at least 13 separate invasions of non-native regions, beginning with Madeira (Portugal) before 1850, and, most recently, eastern China in 2019. We compared the population genetics of 464 specimens from eight countries in the native range and eight countries in the non-native range. Sequencing of the mitochondrial COI gene revealed the presence of 29 haplotypes in six well-supported clades, based on a Bayesian analysis. Non-native populations had significantly lower haplotype diversity (mean h = 0.219) than populations in the native range (mean h = 0.691). Countries in the non-native range had an average of about two haplotypes compared with about four haplotypes in native countries. In the non-native range, only one or two haplotypes were dominant, and these differed among invaded regions except for haplotype HL-H3 which occurred in Australia, New Zealand and China as well as in four countries in southern Europe, and HL-H4 which was dominant in New Zealand, California, New York State, and eastern China as well as two countries in southern Europe. Analyses of interceptions of H. ligniperda with imports arriving in five countries revealed that between 74% and 99% of interceptions originated from other non-native regions while in the USA, most interceptions were linked to imports from the native range beginning in the 1970s. Based on the combined evidence of the chronology of invasions, interception data, and analysis of haplotype distribution, we conclude that the early invasions (before 1950) probably all originated from the native range, while several of the more recent invasions probably originated from parts of the non-native range (suggestive of a bridgehead effect). However, it cannot be determined with certainty what the original sources of each of the invading populations were. ### Competing Interest Statement The authors have declared no competing interest. National Science Foundation, DBI-1639145 Ministry of Agriculture of the Czech Republic, MZE-RO0123 Russian Science Foundation, https://ror.org/03y2gwe85, 22-16-00075

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.

Biological invasions, driven mainly by human activities, pose significant threats to global ecosystems and economies, with fungi and fungal-like oomycetes playing a pivotal role. Ink disease, caused by Phytophthora cinnamomi and P. × cambivora, is a growing concern for sweet chestnut stands (Castanea sativa) in Europe. Since both pathogens are thermophilic organisms, ongoing climate change will likely exacerbate their impact. In this study, we applied species distribution modeling techniques to identify potential substitutive species for sweet chestnut in the light of future climate scenarios SSP126 and SSP370 in southern Switzerland. Using the presence-only machine learning algorithm MaxEnt and leveraging occurrence data from the global dataset GBIF, we delineated the current and projected (2070–2100) distribution of 28 tree species. Several exotic species emerged as valuable alternatives to sweet chestnut, although careful consideration of all potential ecological consequences is required. We also identified several native tree species as promising substitutes, offering ecological benefits and potential adaptability to climatic conditions. Since species diversification fosters forest resilience, we also determined communities of alternative species that can be grown together. Our findings represent a valuable decision tool for forest managers confronted with the challenges posed by ink disease and climate change. Given that, even in absence of disease, sweet chestnut is not a future-proof tree species in the study region, the identified species could offer a pathway toward resilient and sustainable forests within the entire chestnut belt.

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.

Invasive fungal diseases represent a major threat to forest ecosystems worldwide. As the application of fungicides is often unfeasible and not a sustainable solution, only a few other control options are available, including biological control. In this context, the use of parasitic mycoviruses as biocontrol agents of fungal pathogens has recently gained particular attention. Since the 1990s, the Asian fungus Hymenoscyphus fraxineus has been causing lethal ash dieback across Europe. In the present study, we investigated the biocontrol potential of the mitovirus Hymenoscyphus fraxineus mitovirus 2 (HfMV2) previously identified in Japanese populations of the pathogen. HfMV2 could be successfully introduced via co-culturing into 16 of 105 HfMV2-free isolates. Infection with HfMV2 had contrasting effects on fungal growth in vitro, from cryptic to detrimental or beneficial. Virus-infected H. fraxineus isolates whose growth was reduced by HfMV2 showed overall a lower virulence on ash (Fraxinus excelsior) saplings as compared with their isogenic HfMV2-free lines. The results suggest that mycoviruses exist in the native populations of H. fraxineus in Asia that have the potential for biological control of ash dieback in Europe. [Formula: see text] Copyright © 2024 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Phytophthora species are a cause for concern due to their invasive potential and the damage they can cause in agriculture, forestry, and natural ecosystems worldwide. Since water plays a crucial role in their dispersal, stream and river baiting is commonly used to survey risk areas for the presence of quarantine Phytophthora species. However, our understanding of the distribution and diversity of Phytophthora species in European watercourses remains incomplete. This study investigated the presence and diversity of Phytophthora species in Swiss watercourses, with a focus on the highly urbanized Swiss Plateau. Over the period 2012–2016, we sampled 32 watercourses, including major rivers and smaller streams. We isolated Phytophthora on selective media and sequenced the internal transcribed spacer region to identify the species. We recovered 241 Phytophthora isolates, representing 11 species from five major clades. Phytophthora clade 6 prevailed, with P. lacustris being the most common, found in 94.7% of the watercourses. The number of Phytophthora species per watercourse ranged from one to five, with no correlation to watercourse complexity. Our study reveals the presence of six previously unreported species in Switzerland, while known invasive species were not found. Watercourses appear less suited to detect invasive pathogenic Phytophthora species with a still limited distribution in the environment.

Ambrosia beetles are highly successful as invaders because they are often transported internationally with wood packaging and other wood products and because their inbreeding mating systems facilitates establishment of invading populations. In 2022, two independent insect surveys in canton Ticino (southern Switzerland) revealed the widespread occurrence of the invasive ambrosia beetle Anisandrus maiche (Kurentzov, 1941) from southern to central-upper Ticino. This species is native to east Asia and has previously been found as a non-native invasive species in the United States, Canada, western Russia, Ukraine and, in 2021, in northern Italy. Here, we present the results of several trapping studies using different trap types (bottle traps, funnel traps and Polytrap intercept traps) and attractants and a map of the distribution of the species. In total, 715 specimens of A. maiche, all female, were trapped, and the identity of selected individuals was confirmed by morphological and molecular identification based on three mitochondrial and nuclear markers (COI, 28S and CAD). Trap samples from early April to early September 2022 in intervals of two to four weeks showed that flights of A. maiche occurred mainly from June to mid-August. Isolation of fungal associates of A. maiche from beetles trapped alive revealed the presence of four fungal species, including the ambrosia fungus Ambrosiella cleistominuta, the known mutualist of A. maiche. The identity of A. cleistominuta was confirmed by comparing DNA sequences of its nuclear, internal transcribed spacer (ITS) gene with reference sequences in NCBI and BOLDSYSTEMS. This represents the first record of A. cleistominuta in Europe. Of the other fungal associates isolated from A. maiche in Ticino, Fusarium lateritium is of note as there is a possibility that A. maiche could act as a vector of this plant pathogen. We highlight several research needs that should be addressed to gain insight into the potential impact of these non-native species and to overcome problems with heteroplasmy in COI sequences in studies of invasion and population genetics of ambrosia beetles.

Recombination suppression often evolves in sex chromosomes and around mating-type loci. In the invasive chestnut blight fungus Cryphonectria parasitica (Ascomycota), a genomic region was previously suggested to lack recombination and to be partially linked to the mating-type (MAT) locus based on the analysis of a few progenies. Using hundreds of available C. parasitica genomes and generating several new high-quality genome assemblies from the native and introduced range of the pathogen, we show that a ca. 1.2 Mb genomic region proximal to the mating-type locus lacks recombination worldwide. In invasive populations, this MAT-proximal region displayed two highly differentiated haplotypes, that were strongly associated to mating types, but not completely. High-quality assemblies revealed an inversion in one of the haplotypes and footprints of degeneration worldwide, the MAT-proximal region being enriched in gene disruptions, non-synonymous substitutions and transposable elements in both haplotypes. The divergence between the two haplotypes was estimated to have occurred at least 1.5 million years ago and two haplotypes segregate in all continents, including the native range. High differentiation between haplotypes, their occurrence on different continents, their balanced frequencies within populations, their genomic rearrangements and degeneration worldwide, altogether suggest an ancient recombination suppression maintained by selection. The MAT-Proximal region carries multiple genes upregulated under virus infection or vegetative incompatibility reaction. This study sheds light on a case of a large non-recombining region partially linked to a mating compatibility locus, and on balancing selection maintaining differentiated haplotypes, possibly involving deleterious mutations and/or host or virus adaptation in a devastating tree pathogen.### Competing Interest StatementThe authors have declared no competing interest.

AbstractIn 2022, two independent insect surveys in canton Ticino (southern Switzerland) revealed the widespread occurrence of the invasive ambrosia beetleAnisandrus maichefrom southern to central-upper Ticino. This species is native to east Asia and has previously been found as a non-native invasive species in the United States, Canada, western Russia, Ukraine and, in 2021, in northern Italy. Here, we present the results of several trapping studies using different trap types (bottle traps, funnel traps and Polytrap intercept traps) and attractants and a map of the distribution of the species. In total, 685 specimens ofA. maiche, all female, were trapped, and the identity of selected individuals was confirmed by morphological and molecular identification based on three mitochondrial and nuclear markers (COI, 28S and CAD). Traps checked from early April to early September 2022 in intervals of two to four weeks showed that flights ofA. maicheoccurred mainly from June to mid-August. Isolation of fungal associates ofA. maichefrom beetles trapped alive revealed the presence of four fungal species, including the ambrosia fungusAmbrosiella cleistominuta, the known mutualists ofA. maiche. The identity ofA. cleistominutawas confirmed by comparing DNA sequences of its nuclear, internal transcribed spacer (ITS) gene with reference sequences in NCBI and BOLDSYSTEMS. This represents the first record ofA. cleistominutain Europe.Ambrosiella cleistominutawas also found in association with another non-native invasive ambrosia beetle,Xylosandrus crassiusculus, at a botanic garden in central Ticino. As ambrosia beetles usually show a high degree of fidelity with only one mutualistic fungus (in the case ofX. crassiusculusnormallyAmbrosiella roeperi), this association is highly unusual and probably the result of lateral transfer among these non-native invasive species. Of the other fungal associates isolated fromA. maichein Ticino,Fusarium lateritiumis of note as there is a possibility thatA. maichecould act as a vector of this plant pathogen. We highlight several research needs that should be addressed to gain insight into the potential impact of these non-native species and to overcome problems with heteroplasmy in COI sequences in studies of invasion and population genetics of ambrosia beetles.