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Biological control of Cryphonectria parasitica fungus, the causal agent of chestnut blight, by virus infection (hypovirulence) is an effective control strategy against chestnut blight in Europe and some parts of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV1) type species of the Hypoviridae family. In this study, the CHV1 virus was studied within some highly infected British isolates of Cryphonectria parasitica, gained in the past through co-culture transmissions. The effects of six temperatures (5–30 °C, in 5 °C steps) on six infected isolates (three with viral strain E-5, and other three with viral strain L-18) and their respective negative non-infected controls, three isogenic virulent fungal isolates, were examined. Experiments were performed with the nine isolate types with three replicates on potato dextrose agar (PDA) with cellophane sheets per isolate and temperature. A recently developed rapid, specific, quantitative reverse transcription PCR (RT-qPCR) screening method was used. This enabled quantifying the concentration (nanograms per microliter or copy numbers) of the virus within each isolate repetition. The presence of the virus had a significant negative effect between 20 and 25 °C on the C. parasitica growth rate, which was anyway highly influenced by and positively correlated with the temperature. The temperature clearly determined the virus accumulation and its recovery from cold or heat, and the virus optimum temperature was estimated at 15–25 °C.

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.

Biological control of Cryphonectria parasitica fungus, causal agent of chestnut blight, by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe and some parts of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV-1) type species of the Hypoviridae family. To efficiently provide biocontrol, the virus must be able to induce hypovirulence in its fungal host in chestnut trees. Here, two different CHV-1 subtype I virus strains (E-5 and L-18), gained by transmissions, were tested for their hypovirulence induction, biocontrol potential, and transmission between vegetatively compatible (VCG) and incompatible fungal isolate groups in sweet chestnut seedlings and branches. Both strains of CHV-1 showed great biocontrol potential and could protect trees by efficiently transmitting CHV-1 by hyphal anastomosis between fungal isolates of the same VCG and converting virulent to hypovirulent cankers. The hypovirulent effect was positively correlated with the virus concentration, tested by four different reverse-transcription PCRs, two end-point and two real-time methods, one of which represents a newly developed real-time PCR for the detection and quantification of CHV-1.

Emerald ash borer (EAB; Agrilus planipennis ) represents a serious threat to North American and European ash species ( Fraxinus spp.). Spread of EAB westwards, from European Russia and Eastern Ukraine, could lead to dramatic consequences for native European ash populations. Early detection is essential for fast and successful eradication of new populations. In this study, we developed a new TaqMan qPCR assay allowing for sensitive and specific detection of EAB. We tested the specificity of the assay against 17 European Agrilus spp., eight buprestid species and nine species belonging to other wood-associated beetle taxa. The qPCR assay provided reliable amplification from samples with DNA concentrations as low as 0.5 picograms per reaction. Moreover, DNA could be amplified from different sample types, such as egg casings, leaves, faeces and bore dust from larval galleries. Robustness of the assay was verified by performing a blind test with four different laboratories. Here we provide a highly specific, robust and sensitive assay which can be used for enhanced surveillance of Agrilus planipennis on the European continent.

Abstract 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 Cryphonectria hypovirus 1 (CHV1). CHV1 is a biocontrol agent for the chestnut blight fungus (Cryphonectria parasitica), which co-invaded Europe alongside the fungus. We characterized the mutational and haplotypic intra-host virus diversity of thirty-eight natural CHV1 infections spread across four locations in Croatia and Switzerland. Intra-host CHV1 diversity values were shaped by purifying selection and accumulation of mutations over time as well as epistatic interactions within the host genome at defense loci. Geographical landscape features impacted CHV1 inter-host relationships through restricting dispersal and causing founder effects. Interestingly, a small number of intra-host viral haplotypes showed high sequence similarity across large geographical distances unlikely to be linked by dispersal.

Biological control of Cryphonectria parasitica fungus, the causal agent of chestnut blight, by virus infection (hypovirulence) is an effective control strategy against chestnut blight in Europe and some parts of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV1) type species of the Hypoviridae family. In this study, the CHV1 virus was studied within some highly infected British isolates of Cryphonectria parasitica, gained in the past through co-culture transmissions. The effects of six temperatures (5–30 ?, in 5 ? steps) on six infected isolates (three with viral strain E-5, and other three with viral strain L-18) and their respective negative non-infected controls, three isogenic virulent fungal isolates, were examined. Experiments were performed with the nine isolate types with three replicates on potato dextrose agar (PDA) with cellophane sheets per isolate and temperature. A recently developed rapid, specific, quantitative reverse transcription PCR (RT-qPCR) screening method was used. This enabled quantifying the concentration (nanograms per microliter or copy numbers) of the virus within each isolate repetition. The presence of the virus had a significant negative effect between 20 and 25 ? on the C. parasitica growth rate, which was anyway highly influenced by and positively correlated with the temperature. The temperature clearly determined the virus accumulation and its recovery from cold or heat, and the virus optimum temperature was estimated at 15–25 ?.

Biological control of Cryphonectria parasitica fungus, causal agent of chestnut blight, by virus infection (hypovirulence) has been shown to be an effective control strategy against chestnut blight in Europe and some parts of North America. The most studied mycovirus is the Cryphonectria hypovirus 1 (CHV-1) type species of the Hypoviridae family. To efficiently provide biocontrol, the virus must be able to induce hypovirulence in its fungal host in chestnut trees. Here, two different CHV-1 subtype I virus strains (E-5 and L-18), gained by transmissions, were tested for their hypovirulence induction, biocontrol potential, and transmission between vegetatively compatible (VCG) and incompatible fungal isolate groups in sweet chestnut seedlings and branches. Both strains of CHV-1 showed great biocontrol potential and could protect trees by efficiently transmitting CHV-1 by hyphal anastomosis between fungal isolates of the same VCG and converting virulent to hypovirulent cankers. The hypovirulent effect was positively correlated with the virus concentration, tested by four different reverse-transcription PCRs, two end-point and two real-time methods, one of which represents a newly developed real-time PCR for the detection and quantification of CHV-1.

Abstract The remarkable speed at which viral populations mutate allows them to evolve quickly, so that the viral diversity can change, especially when the virus is transmitted, i.e. its population goes through a bottleneck. Our experiments assessed the diversity of the intra-host populations of a mycovirus Cryphonectria hypovirus 1 (CHV1), a natural biocontrol agent of chestnut blight disease, using PacBio long-read HiFi sequencing. The intra-host viral population diversity before and after either vertical or horizontal transmission was estimated using two metrics—nucleotide (mutational) diversity measured as π, and viral variant diversity measured as Nei’s H. A significant bottleneck effect, demonstrated by the decline of the mutational diversity (π), was observed after vertical transmission of prototypical viral populations into conidia, in both investigated viral subtypes, French 1 (F1) and Italian (I). In contrast, the number of viral variants was significantly reduced after the vertical transmission of subtype I but increased for the subtype F1. In newly isolated fungal strains infected with CHV1 subtype I, fewer viral variants were vertically transferred into conidia, relative to the prototypical laboratory isolates, i.e. the average number of transmitted viral variants was smaller. In the horizontal viral transmission assays, the number of transmitted viral variants was closely linked to the genotype of the fungal host at the vegetative compatibility loci. Specifically, recipient viral populations’ diversity was greater when the alleles at loci vic2 and vic3 were the same in the donor and recipient fungal isolate, relative to when they were different. Heteroallelism at the vic4 locus had no impact on viral populations’ diversity. Despite the strong bottlenecks, purifying selection shaped the diversity of intra-host CHV1 populations. In both transmission experiments on average, synonymous mutational diversity was higher than non-synonymous, across all replicates. Signs of positive selection or mutation accumulations, inferred by a surplus of nonsynonymous mutations, were less common and mostly observed during vertical transmission experiments, i.e. in new viral populations arising from conidia.

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.