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Phytophthora plurivora can affect a range of ecologically and silviculturally important tree species, including European beech (Fagus sylvatica), a common late successional tree species native to Europe. Here, we report on the high-quality genome of P. plurivora strain TJ71 (CBS 124093). We sequenced it using Oxford Nanopore MinION and PacBio Sequel II long-read sequencing with 80x coverage, chromatin conformation capture (Hi-C) sequencing with 400x coverage and DNBSEQ 150bp paired-end short reads sequencing with 200x coverage. This complex sequencing approach allowed assembly of the genome at the chromosome level. Specifically, the Phytophthora plurivora genome resulted in 18 scaffolds of 47 Mbp total size with 95% completeness of the eukaryotic gene set as implemented in BUSCO. This is a considerable improvement relative to the previous NCBI reference genomes P. plurivora (NMPK00000000.1) with ~41 Mbp organized in 1,898 scaffolds with 93.8% eukaryotic BUSCO completeness. This high quality genome provides a valuable resource for further evolutionary, epidemiological and population genomic studies.

In 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.

Invasive fungal diseases represent a major threat to forest ecosystems worldwide. As fungicides are 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 fungusHymenoscyphus fraxineushas 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 out of 105 virus-free isolates. A virus infection had contrasting effects on fungal growthin vitro, from cryptic to detrimental or beneficial. Virus-infectedH. fraxineusisolates whose growth was reduced by HfMV2 showed a lower virulence on ash (Fraxinus excelsior) saplings compared to their isogenic virus-free isolates. The results suggest that mycoviruses exist in the native populations ofH. fraxineusin Asia that have the potential for biological control of ash dieback in Europe.

This chapter describes the use of polymorphic microsatellite (simple sequence repeats, SSR) markers for genotyping isolates of Cryphonectria parasitica, the causal agent of chestnut blight. The SSR presented are particularly useful to characterize the genetic population structure of this invasive fungal pathogen, including invasion history (e.g., possible source population, introduced genotypes) and reproduction mode (sexual vs. asexual). Microsatellite markers can also be used to track fungal strains in laboratory and field experiments.

Hyperparasites can affect the evolution of pathosystems by influencing the stability of both pathogen and host populations. However, how pathogens of perennial hosts evolve in the presence of a hyperparasite has rarely been studied. Here, we investigated temporal changes in genetic diversity of the invasive chestnut blight pathogen Cryphonectria parasitica in the presence of its parasitic mycovirus Cryphonectria hypovirus 1 (CHV1). The virus reduces fungal virulence and represents an effective natural biocontrol agent against chestnut blight in Europe. We analysed genome-wide diversity and CHV1 prevalence in C. parasitica populations in southern Switzerland that were sampled twice at an interval of about 30 years. Overall, we found that both pathogen population structure and CHV1 prevalence were retained over time. The results suggest that recent bottlenecks have influenced the structure of C. parasitica populations in southern Switzerland. Strong balancing selection signals were found at a single vegetative incompatibility (vic) locus, consistent with negative frequency-dependent selection imposed by the vegetative incompatibility system. High levels of mating among related individuals (i.e., inbreeding) and genetic drift are probably at the origin of imbalanced allele ratios at vic loci and subsequently low vc type diversity. Virus infection rates were stable at ~30% over the study period and we found no significant impact of the virus on fungal population diversity. Consequently, the efficacy of CHV1-mediated biocontrol was probably retained.

International audience; Biological control (BC) is defined as the use of living natural enemies, antagonists, or competitors (biological control agents) to control other living organisms. In the second half of the last century, the general interest in BC has increased considerably because greater environmental awareness in society and the implementation of integrated pest management (IPM) strategies have pushed towards the development of environmentally friendly control approaches. However, BC is still only rarely used for pathogens (fungi, bacteria, viruses, nematodes, and phytoplasmas) of forest trees. Here, we present and discuss the biological specificities of both the hosts and the fungal pathogens which may account for this situation. To increase the likelihood of BC success, we suggest a holistic approach involving the use of top-down regulators, competitors and amensalists, all exerting pressure on the pathogen, as well as bottom-up forces helping the host (e.g., endophytes, mycorrhiza). Moreover, BC to mitigate emerging forest diseases should be fully integrated into other sustainable management strategies. Finally, we propose guidelines for developing an efficient BC of emerging fungal pathogens of forest trees.

global trade in living plants and plant material has significantly increased the geographic distribution of many plant pathogens. As a consequence, several pathogens have been first found and described in their introduced range where they may cause severe damage on naïve host species. Knowing the center of origin and the pathways of spread of a pathogen is of importance for several reasons, including identifying natural enemies and reducing further spread. Several Phytophthora species are well-known invasive pathogens of natural ecosystems, including Phytophthora multivora. Following the description of P. multivora from dying native vegetation in Australia in 2009, the species was subsequently found to be common in South Africa where it does not cause any remarkable disease. There are now reports of P. multivora from many other countries worldwide, but not as a commonly encountered species in natural environments.a global collection of 335 isolates from North America, Europe, Africa, Australia, the Canary Islands, and New Zealand was used to unravel the worldwide invasion history of P. multivora, using 10 microsatellite markers for all isolates and sequence data from five loci from 94 representative isolates. Our population genetic analysis revealed an extremely low heterozygosity, significant non-random association of loci and substantial genotypic diversity suggesting the spread of P. multivora readily by both asexual and sexual propagules. The P. multivora populations in South Africa, Australia, and New Zealand show the most complex genetic structure, are well established and evolutionary older than those in Europe, North America and the Canary Islands.according to the conducted analyses, the world invasion of P. multivora most likely commenced from South Africa, which can be considered the center of origin of the species. The pathogen was then introduced to Australia, which acted as bridgehead population for Europe and North America. Our study highlights a complex global invasion pattern of P. multivora, including both direct introductions from the native population and secondary spread/introductions from bridgehead populations.

Intra-host dynamics are a core component of virus evolution but most intra-host data come from a narrow range of hosts or experimental infections. Gaining broader information on the intra-host diversity and dynamics of naturally occurring virus infections is essential to our understanding of evolution across the virosphere. Here we used PacBio long-read HiFi sequencing to characterize the intra-host populations of natural infections of the RNA mycovirus

In a pilot study, a biological control with hypovirus-infected Cryphonectria parasitica was applied in 3 study plots in Turkey, in June 2013. The aim of this study was to evaluate the success of the applied biological control by verifying the establishment of the released hypovirus (CHV-1) strains using molecular markers. C. parasitica isolates were sampled from cankers at 3 different time points: before the biological control treatments (April 2013), and 5 months (October 2013) and 11 months (May 2014) after the treatment. In total, 255 C. parasitica isolates were recovered and characterized. First, the culture morphology and vegetative compatibility type of these isolates were assessed. Next, the presence of hypoviruses in white isolates was checked by RNA extraction and subsequent RTPCR. Finally, a sequence analysis was performed to compare the hypoviruses to the released biological control hypoviruses by examining single nucleotide markers and reconstructing their phylogenetic relationship. The study sites in Izmir and Kutahya were found to be free of hypoviruses prior to the biological control, whereas in Sinop, the occurrence of hypoviruses of the Italian subtype was observed. Reisolations of the treated cankers and subsequent molecular analysis resulted in the detection of the released biocontrol hypovirus strains in all 3 study sites. The reisolated hypoviruses in Izmir and Kutahya originated from either one of the released biocontrol strains. In Sinop, both natural and artificially introduced hypoviruses were detected. Our study showed that the released biological control hypoviruses persisted in the treated cankers, which is promising for the biological control of chestnut blight in Turkey.

Intraspecific cryptic invasions may occur when new strains of an invasive species are introduced into an area where this species had already been introduced previously. In plant pathogens, such invasions are not well studied, even if, potentially, they can have severe consequences. Here, we investigated the effects of a potential intraspecific invasion in Europe of Cryphonectria parasitica, the causal agent of chestnut blight. Specifically, we tested the hypotheses that (i) non-European strains are more virulent on Castanea sativa than those already present in Europe because they have never encountered this new host, and (ii) the variation in virulence among strains is higher within native than within introduced populations. In a greenhouse, 2-year-old C. sativa seedlings were inoculated with Cryphonectria parasitica strains from South Korea, the United States, and Switzerland, and lesion development and seedling mortality were recorded weekly. Additionally, growth and sporulation of the strains were measured in vitro on agar medium at 15 and 24°C. Although lesion growth was similar for all strains, seedlings inoculated with strains from South Korea and Switzerland died faster than seedlings inoculated with strains from the United States. Moreover, in vitro strains from South Korea grew faster and produced more spores at both temperatures than the strains from the other two countries. In conclusion, our results did not support the two hypotheses. All strains, regardless of their origin, were found to be highly virulent on the inoculated chestnut seedlings. Nevertheless, current phytosanitary measures to avoid the introduction of new genotypes of C. parasitica into Europe should be further implemented.

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch) The highly destructive chestnut blight disease can be successfully controlled by infecting the virulent strain of C. parasitica with the hyperparasitic mycovirus CHV-1. The artificial application of the virus-induced hypovirulence, however, requires that the vegetative compatibility (vc) diversity in the target C. parasitica population be determined beforehand. Conventional vc type determination by pairing the unknown isolate with tester strains of known vc types is time consuming and not always reliable. A genotyping assay was developed that uses DNA from C. parasitica isolates and discriminates the six known di-allelic vic loci and the two mating idiomorphs in two fluorescence-labeled multiplex PCRs. Validation was conducted by successfully genotyping the 74 known European vc tester strains and a conventionally characterized natural population of C. parasitica. Cross-species amplification tests with three Cryphonectria species (C. japonica, C. naterciae, and C. radicalis) revealed a high amplification specificity for C. parasitica, while the analytical sensitivity of the assay was established at 20 pg per reaction of genomic DNA. In conclusion, this fluorescent multiplex genotyping assay offers a simple and high-throughput tool for the characterization of the vegetative compatibility and mating type of C. parasitica at population level, which is particularly important for the application of mycovirus-mediated biological control of chestnut blight.

The invasive fungus Cryphonectria parasitica, the causal agent of chestnut blight, is able to survive and sporulate on the bark of fresh dead Castanea sativa wood for at least 2 years. Here, we experimentally investigated the role of fresh dead wood in the epidemiology of chestnut blight, specifically in the spread of the hyperparasitic virus Cryphonectria hypovirus 1, which acts as biocontrol agent of C. parasitica. A total of 152 artificially initiated, virulent bark cankers in four chestnut stands were treated with virus-infected asexual spores originating either from sporulating dead wood or from a spore suspension. Molecular markers for both the virus and the fungal carrier were used to examine the spread of the applied biocontrol virus. Fourteen months after treatment, 42 to 76% of the conidial spray-treated cankers and 50 to 60% of the cankers exposed to a sporulating dead stem had been virus infected by the applied hypovirulent conidia in all four study sites. Virus infection reduced canker expansion and promoted canker healing (callusing). Thus, fresh chestnut dead wood may play an important role in supporting the successful spread of natural hypovirulence in chestnut forests. Further, combined with the application of virus-infected conidial suspensions, it may help promote the establishment of artificially released hypoviruses in chestnut stands to control chestnut blight.

Chestnut blight has spread throughout Europe since the introduction of its causal agent, Cryphonectria parasitica, >70 years ago. In our study, we analyzed the diversity of vegetative compatibility (vc) and microsatellite genotypes of C. parasitica, as well as sequence diversity of Cryphonectria hypovirus 1 (CHV1) in six populations from Switzerland, Croatia, and North Macedonia. Resampling of local populations that were already investigated more than a decade ago allowed us to analyze the spatial and temporal population structure across an invasive range of the pathogen in Europe. Regardless of which genetic marker was used, the >60-year-old Swiss and Croatian populations had high population diversity, whereas more recent North Macedonian populations were mostly clonal. These diversity differences between the investigated populations remained stable over time. A high diversity of CHV1 was observed in all three countries, with North Macedonian strains forming a separate cluster from strains obtained in other countries. No correlation between vc diversity and CHV1 prevalence was observed, suggesting a well-established and maintained natural hypovirulence in all countries, further corroborated by an observed increase in genetic diversity of Croatian C. parasitica populations over time, without collapse of CHV1 prevalence.

In pathogenic fungi and oomycetes, interspecific hybridization may lead to the formation of new species having a greater impact on natural ecosystems than the parental species. From the early 1990s, a severe alder (

International audience; 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.

Invasive microbial species constitute a major threat to biodiversity, agricultural production and human health. Invasions are often dominated by one or a small number of genotypes, yet the underlying factors driving invasions are poorly understood. A prime example for a successful global invasion is the recent outbreak of the chestnut blight fungus Cryphonectria parasitica . Native to East Asia, the pathogen colonized North America and Europe during the first half of the 20 th century. After decimation of the American chestnut, the pathogen threatens European chestnut trees. To unravel the mechanisms underpinning the most recent invasion of south-eastern Europe over the past decades, we sequenced genomes of 188 predominantly European strains. Genotypes outside of the invasion zone showed high levels of diversity with evidence for frequent and ongoing recombination. The invasive lineage emerged from the highly diverse European genotype pool rather than a secondary introduction from Asia. The expansion across south-eastern Europe was mostly clonal and shows distinct signs of mutation accumulation. The lineage is also dominated by a single mating type suggesting a fitness advantage to switch to asexual reproduction. However, we show experimentally that the lineage retained the ability to undergo mating consistent with the low degree of recombination detected among strains within the lineage and possibly closely related strains. Our findings show how an intermediary, highly diverse bridgehead population gave rise to an invasive, largely clonally expanding pathogen.

International audience; Gnomoniopsis castaneae is an emerging fungal pathogen currently scored as the major nut rot agent on chestnut, although it is also associated with cankers on both chestnut and hazelnut, as well as with necrosis on chestnut galls and leaves. Described for the first time in 2012, G. castaneae has been reported in several countries across Europe, Asia and Australasia, often in relation to severe outbreaks. The goal of this review is to provide a comprehensive summary of the state of the art about G. castaneae, highlighting the main results achieved by the research and stressing the most relevant knowledge gaps that still need to be filled. This overview includes topics encompassing the taxonomy of the fungal pathogen, its host range and geographic distribution, the symptomatology and the diagnostic methods available for its detection, its impact, biology, ecology and epidemiology. The main interactions between G. castaneae and other organisms are also discussed, as well as the possible control strategies. In these past few years, relevant progresses in the knowledge of G. castaneae have been achieved, yet the complexity of the challenges that this pathogen poses to chestnut growers and to the scientific community advocates for further 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 Armillariaostoyae 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.

International audience; During the last years, simple sequence repeats (SSRs, also known as microsatellites) and single-nucleotide polymorphisms (SNPs) have become the most popular molecular markers for describing neutral genetic variation in populations of a wide range of organisms. However, only a limited number of studies has focused on comparing the performance of these two types of markers for describing the underlying genetic structure of wild populations. Moreover, none of these studies targeted fungi, the group of organisms with one of the most complex reproductive strategies. We evaluated the utility of SSRs and SNPs for inferring the neutral genetic structure of Armillaria cepistipes (basidiomycetes) at different spatial scales. For that, 407 samples were collected across a small (150 km(2)) area in the Ukrainian Carpathians and a large (41 000 km(2)) area in the Swiss Alps. All isolates were analyzed at 17 SSR loci distributed throughout the whole genome and at 24 SNP loci located in different single-copy conserved genes. The two markers showed different patterns of structure within the two spatial scales studied. The multi-allelic SSR markers seemed to be best suited for detecting genetic structure in indigenous fungal populations at a rather small spatial scale (radius of ~50-100 km). The pattern observed at SNP markers rather reflected ancient divergence of distant (~1000 km) populations that in addition are separated by mountain ranges. Despite these differences, both marker types were suitable for detecting the weak genetic structure of the two A. cepistipes populations investigated.

In this paper we resolve the taxonomic status of the fungus Diplodina castaneae (Ascomycetes, Diaporthales, Gnomoniaceae) which occurs on the European chestnut (Castanea sativa) as endophyte and as the causal agent of Javart disease. Specimens from Switzerland, Spain, and Azerbaijan were sequenced at five nuclear loci (β-tubulin, EF-1α, ITS, LSU, and RPB2). Phylogenies were inferred to place D. castaneae in the Gnomoniaceae family. Moreover, growth rates and morphological characteristics on different agar media were assessed and compared to those of Gnomoniopsis castaneae, which can easily be confused with D. castaneae. Based on morphological and phylogenetic characteristics, we propose to reallocate D. castaneae to the genus Sirococcus, as S. castaneae comb. nov.

Chestnut blight, caused by Cryphonectria parasitica, is a devastating disease infecting American and European chestnut trees. The pathogen is native to East Asia and was spread to other continents via infected chestnut plants. This review summarizes the current state of research on this pathogen with a special emphasis on its interaction with a hyperparasitic mycovirus that acts as a biological control agent of chestnut blight. Taxonomy: Cryphonectria parasitica (Murr.) Barr. is a Sordariomycete (ascomycete) fungus in the family Cryphonetriaceae (Order Diaporthales). Closely related species that can also be found on chestnut include Cryphonectria radicalis, Cryphonectria naterciae, and Cryphonectria japonica. Host range: Major hosts are species in the genus Castanea (Fam. Fagaceae), particularly the American chestnut (C. dentata), the European chestnut (C. sativa), the Chinese chestnut (C. mollissima), and the Japanese chestnut (C. crenata). Minor, incidental hosts include oaks (Quercus spp.), maples (Acer spp.), European hornbeam (Carpinus betulus L.), and American chinkapin (Castanea pumila). Disease symptoms: C. parasitica causes perennial necrotic lesions (so-called cankers) on the bark of stems and branches of susceptible host trees, eventually leading to wilting of the plant part distal to the infection. Chestnut blight cankers are characterized by the presence of mycelial fans and fruiting bodies of the pathogen. Below the canker the tree may react by producing epicormic shoots. Non-lethal, superficial or callusing cankers on susceptible host trees are usually associated with mycovirus-induced hypovirulence. Disease control: After the introduction of C. parasitica into a new area, eradication efforts by cutting and burning the infected plants/trees have mostly failed. In Europe, the mycovirus Cryphonectria hypovirus 1 (CHV-1) acts as a successful biological control agent of chestnut blight by causing so-called hypovirulence. CHV-1 infects C. parasitica and reduces its parasitic growth and sporulation capacity. Individual cankers can be therapeutically treated with hypovirus-infected C. parasitica strains. The hypovirus may subsequently spread to untreated cankers and become established in the C. parasitica population. Hypovirulence is present in many chestnut growing regions of Europe, either resulting naturally or after biological control treatments. In North America, disease management of chestnut blight mainly focuses on breeding with the goal to backcross the Chinese chestnut's blight resistance into the American chestnut genome. This article is protected by copyright. All rights reserved.

Aim Early warning against potentially harmful organisms of woody plant species can be achieved by sampling sentinel plants in exporting countries. However, it is unclear where sentinel plants can best be located, and how many samples are required and when and how often sampling optimally should take place for the adequate assessment of the biodiversity associated with the target plant species. We aimed to review spatial and temporal factors affecting associate biodiversity of single woody plant species and to develop guidance for the design of global biodiversity sampling studies. Location Worldwide. Taxon Insects and Fungi. Methods Literature about factors affecting the diversity of insects and fungi in association with single plant species on global, regional, local and different temporal scales was reviewed. Case studies of insect and fungal diversity, primarily collected on single plant species, and the cost of collecting and analysing samples from locations around the world were analysed. Results The review of the literature illustrated various factors affecting diversity, and the case studies allowed quantification of the relative impact of some spatial, temporal and financial aspects on captured biodiversity and, thus, illustrate the need to consider all possible factors that may affect the result of the sampling when deciding on a sampling design. Main conclusions Our study illustrates the factors that should be considered when deciding on the location and timing of sampling for sentinel plants, which is important because of the trade‐off between the number of samples and sampling locations needed to detect many of the species which may be potential pests, and the cost of (repeated) sampling in many locations. Decisions about the sampling design must be based on the objective of the sampling, but our recommendations apply irrespective of the targeted plant species or country.

Intraspecific cryptic invasions may occur when new strains of an invasive species are introduced into an area where this species had already been introduced previously. In plant pathogens, such invasions are not well studied, even if, potentially, they can have severe consequences. Here, we investigated the effects of a potential intraspecific invasion in Europe of

International audience; The international seed trade is considered relatively safe from a phytosanitary point of view and is therefore less regulated than trade in other plants for planting. However, the pests carried by traded seeds are not well known. We assessed insects and fungi in 58 traded seed lots of 11 gymnosperm and angiosperm tree species from North America, Europe, and Asia. Insects were detected by X-raying and molecular methods. The fungal community was characterized using high-throughput sequencing (HTS) and by growing fungi on non-selective agar. About 30% of the seed lots contained insect larvae. Gymnosperms contained mostly hymenopteran (Megastigmus spp.) and dipteran (Cecidomyiidae) larvae, while angiosperms contained lepidopteran (Cydia latiferreana) and coleopteran (Curculio spp.) larvae. HTS indicated the presence of fungi in all seed lots and fungi grew on non-selective agar from 96% of the seed lots. Fungal abundance and diversity were much higher than insect diversity and abundance, especially in angiosperm seeds. Almost 50% of all fungal exact sequence variants (ESVs) found in angiosperms were potential pathogens, in comparison with around 30% of potentially pathogenic ESVs found in gymnosperms. The results of this study indicate that seeds may pose a greater risk of pest introduction than previously believed or accounted for. A rapid risk assessment suggests that only a small number of species identified in this study is of phytosanitary concern. However, more research is needed to enable better risk assessment, especially to increase knowledge about the potential for transmission of fungi to seedlings and the host range and impact of identified species.

International audience; The number of invasive alien pest and pathogen species affecting ecosystem functioning, human health and economies has increased dramatically over the last decades. Discoveries of invasive pests and pathogens previously unknown to science or with unknown host associations yet damaging on novel hosts highlights the necessity of developing novel tools to predict their appearance in hitherto naive environments. The use of sentinel plant systems is a promising tool to improve the detection of pests and pathogens before introduction and to provide valuable information for the development of preventative measures to minimize economic or environmental impacts. Though sentinel plantings have been established and studied during the last decade, there still remains a great need for guidance on which tools and protocols to put into practice in order to make assessments accurate and reliable. The sampling and diagnostic protocols chosen should enable as much information as possible about potential damaging agents and species identification. Consistency and comparison of results are based on the adoption of common procedures for sampling design and sample processing. In this paper, we suggest harmonized procedures that should be used in sentinel planting surveys for effective sampling and identification of potential pests and pathogens. We also review the benefits and limitations of various diagnostic methods for early detection in sentinel systems, and the feasibility of the results obtained supporting National Plant Protection Organizations in pest and commodity risk analysis.

Following its introduction from Asia in the 1990s, the ascomycete Hymenoscyphus fraxineus has caused a severe dieback of Fraxinus excelsior in Europe. In this study we assess and compare the virulence of 200 H. fraxineus isolates equally representing (i) two geographically distant populations with a different disease history (Switzerland, recently established populations at the epidemic front vs. Lithuania, old established populations), and (ii) two different types of host tissue (necrotic bark lesions as dead-end tissue vs. fallen leaf petioles as primary host tissue). Inoculations conducted on 3-year-old F. excelsior seedlings showed that the vast majority of the isolates (98%) were able to induce necrotic bark lesions after 10 months. Although a high variation in virulence was observed among isolates, no significant differences were detected between the older and the epidemic-front populations. Decline in virulence of populations of invasive organisms is generally assumed with increasing age of epidemics. This, however, does not appear to hold true for H. fraxineus. Either the Lithuanian population is still too young (15–20-years-old) to show a decline in virulence, or the size of the host population may still not be critical for pathogen survival. Given that bark lesions represent an epidemiological dead end and do not benefit the survival of H. fraxineus, a trend towards reduced “bark virulence” of isolates originating from leaf petioles compared to isolates from the bark lesions was expected. Such a trend, however, was observed neither in old, nor in recently established populations. This article is protected by copyright. All rights reserved.

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.