The Fungal Pathogen Gnomoniopsis castaneae Induces Damaging Cankers in Multiple Domestic Fagaceae Species.

Gnomoniopsis castaneae is internationally recognized as a destructive pathogen of chestnut species. Primarily associated with nut rot, it has also been associated with branch and stem cankers of chestnut and as an endophyte of multiple additional hardwood species. The present study evaluated implications of the recently reported United States presence of the pathogen for domestic Fagaceae species. Stem inoculation assays of Castanea dentata, C. mollissima, C. dentata × C. mollissima, and Quercus rubra (red oak) seedlings were utilized to examine the cankering ability of a regional isolate of the pathogen. The pathogen induced damaging cankers in all assessed species and significant stem girdling in all chestnut species. No previous study has associated the pathogen with damaging infection in Quercus species, and its presence in the United States has the potential to compound ongoing chestnut recovery programs and oak regeneration problems within forest systems.

Diversity of Monochaetia Species from Fagaceous Leaf Spots in China and Pathogenicity for Chinese Chestnut.

Pestalotioid fungi have been frequently studied with respect to their morphology, molecular phylogeny, and pathogenicity. Monochaetia is a pestalotioid genus that is morphologically characterized by 5-celled conidia with single apical and basal appendages. In the present study, fungal isolates were obtained from diseased leaves of Fagaceae hosts in China in 2016 to 2021 and identified based on morphology and phylogenetic analyses of the 5.8S nuclear ribosomal DNA gene with the two flanking internal transcribed spacer (ITS) regions, the nuclear ribosomal large subunit (LSU) region, the translation elongation factor 1-α (tef1) gene, and the ß-tubulin (tub2) gene. As a result, five new species are proposed here, namely, Monochaetia hanzhongensis, Monochaetia lithocarpi, Monochaetia lithocarpicola, Monochaetia quercicola, and Monochaetia shaanxiensis. In addition, pathogenicity tests for these five species and Monochaetia castaneae from Castanea mollissima were conducted with detached leaves of Chinese chestnut. Results demonstrated that only M. castaneae successfully infected the host C. mollissima and caused brown lesions. IMPORTANCE Monochaetia is a pestalotioid genus, with members that are commonly known as leaf pathogens or saprobes; some strains were isolated from air, in which case their natural substrate is so far unknown. Fagaceae represents an ecologically and economically important plant family that is widely distributed in the Northern Hemisphere, including an important tree crop species, Castanea mollissima, which is widely cultivated in China. In the present study, diseased leaves of Fagaceae in China were investigated, and five new Monochaetia species were introduced based on morphology and phylogeny of combined ITS, LSU, tef1, and tub2 loci. Additionally, six species of Monochaetia were inoculated onto healthy leaves of the crop host Castanea mollissima to test their pathogenicity. The present study provides significant data on the species diversity, taxonomy, and host range of Monochaetia and enhances our understanding of leaf diseases of Fagaceae hosts.

Morphology and Phylogeny of Pestalotiopsis (Sporocadaceae, Amphisphaeriales) from Fagaceae Leaves in China.

Fagaceae is a family of flowering plants widely distributed in the Northern Hemisphere, including deciduous and evergreen trees and shrubs. Species of Pestalotiopsis are well-known agents of leaf spot diseases, but targeted sampling on Fagaceae is still missing. To determine the diversity of Pestalotiopsis species associated with Fagaceae leaf spot in China, investigations were conducted in the main areas of Fagaceae distribution from 2016 to 2021. Diseased leaf tissues were collected, and fungal isolates were obtained from leaf spots. In the present study, 43 isolates of Pestalotiopsis were studied based on combined morphology and phylogeny. As a result, 10 new species were identified, viz., Pestalotiopsis anhuiensis, P. castanopsidis, P. changjiangensis, P. cyclobalanopsidis, P. foliicola, P. guangxiensis, P. guizhouensis, P. lithocarpi, P. shaanxiensis, and P. silvicola, and six new host records were recognized. IMPORTANCE Pestalotiopsis is a common fungal genus inhabiting plant tissues as endophytes, pathogens, and saprophytes. Fagaceae is a plant family including many important tree species, such as Castanea mollissima and Quercus spp. In this study, diseased leaves of Fagaceae in China were investigated, and 16 Pestalotiopsis species were identified based on morphology and phylogeny of combined loci of internal transcribed spacers (ITS), the translation elongation factor 1-α (tef1), and the beta-tubulin (tub2) genes. Among these, 10 new species were found, and six new host records were revealed. Our study significantly updates the taxonomy of Pestalotiopsis and enhances our understanding of leaf diseases of Fagaceae hosts.

Metabolites derived from fungi and bacteria suppress in vitro growth of Gnomoniopsis smithogilvyi, a major threat to the global chestnut industry.

INTRODUCTION: Chestnut rot caused by the fungus Gnomoniopsis smithogilvyi is a disease present in the world's major chestnut growing regions. The disease is considered a significant threat to the global production of nuts from the sweet chestnut (Castanea sativa). Conventional fungicides provide some control, but little is known about the potential of biological control agents (BCAs) as alternatives to manage the disease. OBJECTIVE: Evaluate whether formulated BCAs and their secreted metabolites inhibit the in vitro growth of G. smithogilvyi. METHODS: The antifungal potential of BCAs was assessed against the pathogen through an inverted plate assay for volatile compounds (VOCs), a diffusion assay for non-volatile compounds (nVOCs) and in dual culture. Methanolic extracts of nVOCs from the solid medium were further evaluated for their effect on conidia germination and were screened through an LC-MS-based approach for antifungal metabolites. RESULTS: Isolates of Trichoderma spp., derived from the BCAs, significantly suppressed the pathogen through the production of VOCs and nVOCs. The BCA from which Bacillus subtilis was isolated was more effective in growth inhibition through the production of nVOCs. The LC-MS based metabolomics on the nVOCs derived from the BCAs showed the presence of several antifungal compounds. CONCLUSION: The results show that G. smithogilvyi can be effectively controlled by the BCAs tested and that their use may provide a more ecological alternative for managing chestnut rot. The in vitro analysis should now be expanded to the field to assess the effectiveness of these alternatives for chestnut rot management.

The Use of qPCR to Detect Cryphonectria parasitica in Plants.

Cryphonectria parasitica is a fungal pathogen that causes lethal bark necrosis in chestnut. A duplex qPCR allowing detection of the pathogen and its host, Castanea sativa, is described. The method can be used for early detection of the pathogen in chestnut bark tissues with an internal control of false-negative results caused by PCR inhibitors and/or DNA extraction failure. A positive amplification control of qPCR that allows detection of any deviation from a normal qPCR run based on a control chart is also described. As C. parasitica is a regulated pathogen in Europe, the protocol also provides information on the way to collect and handle bark samples to fulfil biosecurity rules.

Frozen in time: Rangewide genomic diversity, structure, and demographic history of relict American chestnut populations.

American chestnut (Castanea dentata) was once the most economically and ecologically important hardwood species in the eastern United States. In the first half of the 20th century, an exotic fungal pathogen-Cryphonectria parasitica-decimated the species, killing billions of chestnut trees. Two approaches to developing blight-resistant American chestnut populations show promise, but both will require introduction of adaptive genomic diversity from wild germplasm to produce diverse, locally adapted restoration populations. Here we characterize population structure, demographic history, and genomic diversity in a range-wide sample of 384 wild American chestnuts to inform conservation and breeding with blight-resistant varieties. Population structure analyses suggest that the chestnut range can be roughly divided into northeast, central, and southwest populations. Within-population genomic diversity estimates revealed a clinal pattern with the highest diversity in the southwest, which likely reflects bottleneck events associated with Quaternary glaciation. Finally, we identified genomic regions under positive selection within each population, which suggests that defence against fungal pathogens is a common target of selection across all populations. Taken together, these results show that American chestnut underwent a postglacial expansion from the southern portion of its range leading to three extant genetic populations. These populations will serve as management units for breeding adaptive genetic variation into the blight-resistant tree populations for targeted reintroduction efforts.

Temporal changes in pathogen diversity in a perennial plant-pathogen-hyperparasite system.

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.

Impact of 'brown rot' caused by Gnomoniopsis castanea on chestnut fruits during the post-harvest process: critical phases and proposed solutions.

BACKGROUND: The brown rot fungus, Gnomoniopsis castanea, is the main organism responsible for the outbreak of chestnut postharvest decay that is threatening the sustainability of the chestnut market in Europe. Currently, no specific strategy is available to mitigate the impact and remediate the high losses of fruits in postharvest storage. In the present study, the different phases of chestnut handling in a standard facility plant were analyzed by evaluating the amount of fruit rot and infection by G. castanea at each phase. RESULTS: The warm bath (48 °C) was identified as the critical phase, requiring strict parametrization to effectively inactivate G. castanea in fruits. Laboratory tests indicated that maintaining fruits at 50 °C for a maximum of 45 min provided optimal conditions to completely inactivate G. castanea inoculum during postharvest handling. However, the warm bath at 50 °C and over was not effective in inactivating the complex of fungal taxa responsible for contamination and development of molds. Higher temperatures and extended treatment times caused significant losses in fruit quality, as indicated by taste panel evaluation. Upscaling of postharvest facilities is discussed and critically evaluated. CONCLUSION: The warm bath (50 °C for 45 min) is effective in completely inactivating G. castanea in fruits but did not reduce the impacts of the complex of molds responsible for external contamination and mycotoxin production. © 2021 The Authors. Journal of The Science of Food and Agriculture published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Pathogen-induced expression of a blight tolerance transgene in American chestnut.

American chestnut (Castanea dentata) is a susceptible host of the invasive necrotrophic fungus Cryphonectria parasitica, which causes chestnut blight disease. The fungal pathogen attacks chestnut stems by invading wounded tissue and secreting oxalate. This process leads to the death of infected host cells and the formation of cankers, eventually girdling stems and killing the tree above the infections. To reduce damage caused by fungal oxalate, American chestnut has been genetically engineered to express a wheat oxalate oxidase (OxO). This enzyme degrades the oxalate produced by the pathogen and confers elevated tolerance to Cryphonectria parasitica infection. We report new lines of transgenic American chestnut that have been developed with the win3.12 inducible promoter from poplar (Populus deltoides) driving OxO expression. This promoter is responsive to both wounding and pathogen infection, with a low level of baseline expression. Targeted expression of OxO to wounded and infected tissue is sought as an alternative to constitutive expression for potential metabolic resource conservation and transgene stability over the long lifetime of a tree and over successive generations of breeding. Transgenic Castanea dentata lines harbouring the win3.12-OxO construct were evaluated for transgene expression patterns and tolerance to chestnut blight infection. OxO transcript levels were low in uninfected plants, but robust infection-induced expression levels were observed, with one transgenic line reaching levels comparable to those of previously characterized CaMV35S-OxO lines. In chestnut blight infection bioassays, win3.12-OxO lines showed elevated disease tolerance similar to blight-resistant Chinese chestnut (Castanea mollissima) controls.

Emergence and diversification of a highly invasive chestnut pathogen lineage across southeastern Europe.

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. The chestnut blight fungus Cryphonectria parasitica first decimated the North American chestnut, and a more recent outbreak threatens European chestnut stands. To unravel the chestnut blight invasion of southeastern Europe, we sequenced 230 genomes of 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 or North America. The expansion across southeastern Europe was mostly clonal and is dominated by a single mating type, suggesting a fitness advantage of asexual reproduction. Our findings show how an intermediary, highly diverse bridgehead population gave rise to an invasive, largely clonally expanding pathogen.

Virome characterization of Cryphonectria parasitica isolates from Azerbaijan unveiled a new mymonavirus and a putative new RNA virus unrelated to described viral sequences.

Cryphonectria parasitica, the causal agent of chestnut blight is controlled in Europe through natural spread of Cryphonectria hypovirus 1 (CHV1), a mycovirus able to induce hypovirulence to the host. In recent years C. parasitica was reported infecting Azerbaijani population of chestnut, but the presence of CHV1 still needs to be confirmed. Aim of this work was to investigate fifty-five C. parasitica isolates collected in Azerbaijan to describe the associated viruses. Our work found i) the first negative-sense ssRNA virus known to infect C. parasitica naturally for which we propose the name Cryphonectria parasitica sclerotimonavirus 1 (CpSV1) and ii) an RNA sequence showing peculiar features suggesting a viral nature for which we propose the name Cryphonectria parasitica ambivirus 1 (CpaV1). The discovery of CpaV1 expands our knowledge of the RNA virosphere suggesting the existence of a new lineage that cannot presently be reliably associated to the monophyletic Riboviria.

In-Tree Behavior of Diverse Viruses Harbored in the Chestnut Blight Fungus, Cryphonectria parasitica.

The ascomycete Cryphonectria parasitica causes destructive chestnut blight. Biological control of the fungus by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe. To provide biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in chestnut trees. Field studies using living trees to date have focused on a selected family of viruses called hypoviruses, especially prototypic hypovirus CHV1, but there are now known to be many other viruses that infect C. parasitica Here, we tested seven different viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in trees. The test included cytosolically and mitochondrially replicating viruses with positive-sense single-stranded RNA or double-stranded RNA genomes. The seven viruses showed different in planta behaviors and were classified into four groups. Group I, including CHV1, had great biocontrol potential and could protect trees by efficiently spreading and converting virulent to hypovirulent cankers in the trees. Group II could induce high levels of hypovirulence but showed much smaller biocontrol potential, likely because of inefficient virus transmission. Group III showed poor performance in hypovirulence induction and biocontrol, while efficiently being transmitted in the infected trees. Group IV could induce hypovirulence and spread efficiently but showed poor biocontrol potential. Nuclear and mitochondrial genotyping of fungal isolates obtained from the treated cankers confirmed virus transmission between the two fungal strains in most isolates. These results are discussed in view of dynamic interactions in the tripartite pathosystem.IMPORTANCE The ascomycete Cryphonectria parasitica causes destructive chestnut blight, which is controllable by hypovirulence-conferring viruses infecting the fungus. The tripartite chestnut/C. parasitica/virus pathosystem involves the dynamic interactions of their genetic elements, i.e., virus transmission and lateral transfer of nuclear and mitochondrial genomes between fungal strains via anastomosis occurring in trees. Here, we tested diverse RNA viruses for their hypovirulence induction, biocontrol potential, and transmission properties between two vegetatively compatible but molecularly distinguishable fungal strains in live chestnut trees. The tested viruses, which are different in genome type (single-stranded or double-stranded RNA) and organization, replication site (cytosol or mitochondria), virus form (encapsidated or capsidless) and/or symptomatology, have been unexplored in the aforementioned aspects under controlled conditions. This study showed intriguing different in-tree behaviors of the seven viruses and suggested that to exert significant biocontrol effects, viruses must be able to induce hypovirulence and spread efficiently in the fungus infecting the chestnut trees.

Integrated Proteomic and Metabolomic Profiling of Phytophthora cinnamomi Attack on Sweet Chestnut (Castanea sativa) Reveals Distinct Molecular Reprogramming Proximal to the Infection Site and Away from It.

Phytophthora cinnamomi is one of the most invasive tree pathogens that devastates wild and cultivated forests. Due to its wide host range, knowledge of the infection process at the molecular level is lacking for most of its tree hosts. To expand the repertoire of studied Phytophthora-woody plant interactions and identify molecular mechanisms that can facilitate discovery of novel ways to control its spread and damaging effects, we focused on the interaction between P. cinnamomi and sweet chestnut (Castanea sativa), an economically important tree for the wood processing industry. By using a combination of proteomics, metabolomics, and targeted hormonal analysis, we mapped the effects of P. cinnamomi attack on stem tissues immediately bordering the infection site and away from it. P. cinnamomi led to a massive reprogramming of the chestnut proteome and accumulation of the stress-related hormones salicylic acid (SA) and jasmonic acid (JA), indicating that stem inoculation can be used as an easily accessible model system to identify novel molecular players in P. cinnamomi pathogenicity.

Development and characterization of an edible chitosan-whey protein nano composite film for chestnut (Castanea mollissima Bl.) preservation.

Chitosan (CHI) and whey protein are usually used to prepare edible films for food preservation. However, the composite film composed of the two components does not yield satisfactory properties for chestnut preservation. In this study, nano-cellulose and cinnamaldehyde (CMA) were added to CHI and whey protein, creating a new composite film with strong water retention, bacteriostatic, and mechanical properties. The water vapor permeability (WVP) of the film decreased by 21.61% with the addition of 0.5% (w/v) nano-cellulose, and 23.02% with the addition of 0.3% (w/v) CMA. Furthermore, water solubility (WS) decreased 22.05%, and the density of the film was significantly improved with the addition of 0.3% (w/v) CMA. The optimized formula of the film was CHI 2.5% (w/v), whey protein 3.0% (w/v), nano-cellulose 0.5% (w/v), CMA 0.3% (w/v), and pH 3.8, as determined by orthogonal testing L9(34 ), with fuzzy comprehensive assessment, of WVP, WS, tensile strength, and elongation at break. The film clearly inhibited the growth of E. coli, S. aureus, and Chinese chestnut fungus, destroying the mycelial structure of the fungus. In addition, coating effectively reduced the weight loss, mildew rate, and calcification index during 16 days of storage of chestnuts at 25 °C.

Growth Features of Castanea sativa Mill. in Forest Stands Infected by Cryphoneciria parasitica (Murrill) М. Е. Barr. in Abkhazia.

Currently, the forest plantations of Castanea sativa Mill. are largely damaged by phytopathogenic organisms, of which Cryphoneciria parasitica (Murrill) М. Е. Barr. has the most negative impact. The spread of this type of disease reduces the vital state and significantly reduces the life expectancy of C. sativa trees. For the first time, using dendrochronological analysis, the specificity of distribution and degree of phytopathogenic damage to C. sativa trunk tissues were assessed. It was revealed that in its lower part the dieback of the peripheral layers of wood occurs fairly evenly with a total degradation of cellular structures in the amount of 18-20%. In the middle segments of the trunk this figure increases almost twice. And the greatest degree of wood dieback is observed in the upper part of damaged trees. It was found that climatic changes of the last decades determine the strengthening of destructive processes in the woodland of C. sativa of the Caucasus.

Functional analysis of an essential Ran-binding protein gene, CpRbp1, from the chestnut blight fungus Cryphonectria parasitica using heterokaryon rescue.

A Ran binding protein (RanBP) homolog, CpRbp1, from Cryphonectria parasitica, has been identified as a protein that is affected by hypovirus infection or tannic acid supplementation. In this study, functional analyses of CpRbp1 were performed by constructing a knockout mutant and analyzing the resulting heterokaryon. Transformation-mediated gene replacement resulted in two putative CpRbp1-null mutants and genotype analyses identified these two mutants as heterokaryotic transformants consisting of two types of nuclei, one with the wild-type CpRbp1 allele and another with the CpRbp1-null mutant allele. Although stable mycelial growth of the heterokaryotic transformant was observed on selective medium containing hygromycin B, neither germination nor growth of the resulting conidia, which were single-cell monokaryotic progeny, was observed on the medium. In trans complementation of heterokaryons using a full-length wild-type allele of the CpRbp1 gene resulted in complemented transformants. These transformants sporulated single-cell monokaryotic conidia that were able to grow on media selective for replacing and/or complementing markers. These results clearly indicate that CpRbp1 is an essential gene, and heterokaryons allowed the fungus to maintain lethal CpRbp1-null mutant nuclei. Moreover, in trans complementation of heterokaryons using chimeric structures of the CpRbp1 gene allowed for analysis of its functional domains, which was previously hampered due to the lethality of the gene. In addition, in trans complementation using heterologous RanBP genes from Aspergillus nidulans was successful, suggesting that the function of RanBP is conserved during evolution. Furthermore, in trans complementation allowed for functional analyses of lethal orthologs. This study demonstrates that our fungal heterokaryon system can be applied effectively to determine whether a gene of interest is essential, perform functional analyses of a lethal gene, and analyze corresponding heterologous genes.

Genetic Diversity of Cryphonectria hypovirus 1, a Biocontrol Agent of Chestnut Blight, in Croatia and Slovenia.

Transmissible hypovirulence associated with Cryphonectria hypovirus 1 (CHV1) has been used for biological control of chestnut blight, devastating disease of chestnut caused by the fungus Cryphonectria parasitica. The main aims of this study were to provide molecular characterization of CHV1 from Croatia and Slovenia and to reveal its genetic variability, phylogeny, and diversification of populations. Fifty-one CHV1 haplotypes were detected among 54 partially sequenced CHV1 isolates, all belonging to Italian subtype (I). Diversity was mainly generated by point mutations while evidence of recombination was not found. The level of conservation over analyzed parts of ORF-A proteins p29 and p40 varied, but functional sites were highly conserved. Phylogenetic analysis revealed close relatedness and intermixing of Croatian and Slovenian CHV1 populations. Our CHV1 isolates were also related to Swiss and Bosnian hypoviruses supporting previously suggested course of CHV1 invasion in Europe. Overall, this study indicates that phylogeny of CHV1 subtype I in Europe is complex and characterized with frequent point mutations resulting in many closely related variants of the virus. Possible association between variations within CHV1 ORF-A and growth of the hypovirulent fungal isolates is tested and presented.

Hormone and secondary metabolite profiling in chestnut during susceptible and resistant interactions with Phytophthora cinnamomi.

Phytophthora cinnamomi (Pc) is a dangerous pathogen that causes root rot (ink disease) and threatens the production of chestnuts worldwide. Despite all the advances recently reported at molecular and physiological level, there are still gaps of knowledge that would help to unveil the defence mechanisms behind plant-Pc interactions. Bearing this in mind we quantified constitutive and Pc-induced stress-related signals (hormones and metabolites) complemented with changes in photosynthetic related parameters by exploring susceptible and resistant Castanea spp.-Pc interactions. In a greenhouse experiment, five days before and nine days after inoculation with Pc, leaves and fine roots from susceptible C. sativa and resistant C. sativa × C. crenata clonal 2-year-old plantlets were sampled (clones Cs14 and 111-1, respectively). In the resistant clone, stomatal conductance (gs) and net photosynthesis (A) decreased significantly and soluble sugars in leaves increased, while in the susceptible clone gs and A remained unchanged and proline levels in leaves increased. In the resistant clone, higher constitutive content of root SA and foliar ABA, JA and JA-Ile as compared to the susceptible clone were observed. Total phenolics and condensed tannins were highest in roots of the susceptible clone. In response to infection, a dynamic hormonal response in the resistant clone was observed, consisting of accumulation of JA, JA-Ile and ABA in roots and depletion of total phenolics in leaves. However, in the susceptible clone only JA diminished in leaves and increased in roots. Constitutive and Pc-induced levels of JA-Ile were only detectable in the resistant clone. From the hormonal profiles obtained in leaves and roots before and after infection, it is concluded that the lack of effective hormonal changes in C. sativa explains the lack of defence responses to Pc of this susceptible species.

Assessing the Phytosanitary Risk Posed by an Intraspecific Invasion of Cryphonectria parasitica in Europe.

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.