Possible Biological Control of Ash Dieback Using the Mycoparasite Hymenoscyphus Fraxineus Mitovirus 2.

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.

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.

Temporal and Spatial Genetic Population Structure of Cryphonectria parasitica and Its Associated Hypovirus Across an Invasive Range of Chestnut Blight in Europe.

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.

Chromosomal assembly and analyses of genome-wide recombination rates in the forest pathogenic fungus Armillaria ostoyae.

Recombination shapes the evolutionary trajectory of populations and plays an important role in the faithful transmission of chromosomes during meiosis. Levels of sexual reproduction and recombination are important properties of host-pathogen interactions because the speed of antagonistic co-evolution depends on the ability of hosts and pathogens to generate genetic variation. However, our understanding of the importance of recombination is limited because large taxonomic groups remain poorly investigated. Here, we analyze recombination rate variation in the basidiomycete fungus Armillaria ostoyae, which is an aggressive pathogen on a broad range of conifers and other trees. We analyzed a previously constructed, dense genetic map based on 198 single basidiospore progeny from a cross. Progeny were genotyped at a genome-wide set of single-nucleotide polymorphism (SNP) markers using double digest restriction site associated DNA sequencing. Based on a linkage map of on 11,700 SNPs spanning 1007.5 cM, we assembled genomic scaffolds into 11 putative chromosomes of a total genome size of 56.6 Mb. We identified 1984 crossover events among all progeny and found that recombination rates were highly variable along chromosomes. Recombination hotspots tended to be in regions close to the telomeres and were more gene-poor than the genomic background. Genes in proximity to recombination hotspots were encoding on average shorter proteins and were enriched for pectin degrading enzymes. Our analyses enable more powerful population and genome-scale studies of a major tree pathogen.

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.

Role of Fresh Dead Wood in the Epidemiology and the Biological Control of the Chestnut Blight Fungus.

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.

High-density genetic mapping identifies the genetic basis of a natural colony morphology mutant in the root rot pathogen Armillaria ostoyae.

Filamentous fungi exhibit a broad spectrum of heritable growth patterns and morphological variations reflecting the adaptation of the different species to distinct ecological niches. But also within species, isolates show considerable variation in growth rates and other morphological characteristics. The genetic basis of this intraspecific variation in mycelial growth and morphology is currently poorly understood. By chance, a growth mutant in the root rot pathogen Armillaria ostoyae was discovered. The mutant phenotype was characterized by extremely compact and slow growth, as well as shorter aerial hyphae and hyphal compartments in comparison to the wildtype phenotype. Genetic analysis revealed that the abnormal phenotype is caused by a recessive mutation, which segregates asa single locus in sexual crosses. In order to identify the genetic basis of the mutant phenotype, we performed a quantitative trait locus (QTL) analysis. A mapping population of 198 haploid progeny was genotyped at 11,700 genome-wide single nucleotide polymorphisms (SNPs) making use of double digest restriction site associated DNA sequencing (ddRADseq). In accordance with the genetic analysis, a single significant QTL was identified for the abnormal growth phenotype. The QTL confidence interval spans a narrow, gene dense region of 87kb in the A. ostoyae genome which contains 37 genes. Overall, our study reports the first high-density genetic map for an Armillaria species and shows its successful application in forward genetics by resolving the genetic basis of a mutant phenotype with a severe defect in hyphal growth.

Population genetic analysis of a parasitic mycovirus to infer the invasion history of its fungal host.

Hymenoscyphus fraxineus mitovirus 1 (HfMV1) occurs in the fungus Hymenoscyphus fraxineus, an introduced plant pathogen responsible for the devastating ash dieback epidemic in Europe. Here, we explored the prevalence and genetic structure of HfMV1 to elucidate the invasion history of both the virus and the fungal host. A total of 1298 H. fraxineus isolates (181 from Japan and 1117 from Europe) were screened for the presence of this RNA virus and 301 virus-positive isolates subjected to partial sequence analysis of the viral RNA polymerase gene. Our results indicate a high mean prevalence (78.7%) of HfMV1 across European H. fraxineus isolates, which is supported by the observed high transmission rate (average 83.8%) of the mitovirus into sexual spores of its host. In accordance with an expected founder effect in the introduced population in Europe, only 1.1% of the Japanese isolates were tested virus positive. In Europe, HfMV1 shows low nucleotide diversity but a high number of haplotypes, which seem to be subject to strong purifying selection. Phylogenetic and clustering analysis detected two genetically distinct HfMV1 groups, both present throughout Europe. This pattern supports the hypothesis that only two (mitovirus-carrying) H. fraxineus individuals were introduced into Europe as previously suggested from the bi-allelic nature of the fungus. Moreover, our data points to reciprocal mating events between the two introduced individuals, which presumably initiated the ash dieback epidemic in Europe.

Insights into the phylogeny of Northern Hemisphere Armillaria: Neighbor-net and Bayesian analyses of translation elongation factor 1-α gene sequences.

Armillaria possesses several intriguing characteristics that have inspired wide interest in understanding phylogenetic relationships within and among species of this genus. Nuclear ribosomal DNA sequence-based analyses of Armillaria provide only limited information for phylogenetic studies among widely divergent taxa. More recent studies have shown that translation elongation factor 1-α (tef1) sequences are highly informative for phylogenetic analysis of Armillaria species within diverse global regions. This study used Neighbor-net and coalescence-based Bayesian analyses to examine phylogenetic relationships of newly determined and existing tef1 sequences derived from diverse Armillaria species from across the Northern Hemisphere, with Southern Hemisphere Armillaria species included for reference. Based on the Bayesian analysis of tef1 sequences, Armillaria species from the Northern Hemisphere are generally contained within the following four superclades, which are named according to the specific epithet of the most frequently cited species within the superclade: (i) Socialis/Tabescens (exannulate) superclade including Eurasian A. ectypa, North American A. socialis (A. tabescens), and Eurasian A. socialis (A. tabescens) clades; (ii) Mellea superclade including undescribed annulate North American Armillaria sp. (Mexico) and four separate clades of A. mellea (Europe and Iran, eastern Asia, and two groups from North America); (iii) Gallica superclade including Armillaria Nag E (Japan), multiple clades of A. gallica (Asia and Europe), A. calvescens (eastern North America), A. cepistipes (North America), A. altimontana (western USA), A. nabsnona (North America and Japan), and at least two A. gallica clades (North America); and (iv) Solidipes/Ostoyae superclade including two A. solidipes/ostoyae clades (North America), A. gemina (eastern USA), A. solidipes/ostoyae (Eurasia), A. cepistipes (Europe and Japan), A. sinapina (North America and Japan), and A. borealis (Eurasia) clade 2. Of note is that A. borealis (Eurasia) clade 1 appears basal to the Solidipes/Ostoyae and Gallica superclades. The Neighbor-net analysis showed similar phylogenetic relationships. This study further demonstrates the utility of tef1 for global phylogenetic studies of Armillaria species and provides critical insights into multiple taxonomic issues that warrant further study.

Virulence and Stump Colonization Ability of Armillaria borealis on Norway Spruce Seedlings in Comparison to Sympatric Armillaria Species.

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.

Dynamics of Cryphonectria hypovirus infection in chestnut blight cankers.

Virulent strains of the chestnut blight fungus Cryphonectria parasitica cause lethal bark cankers on chestnut trees. Infection of C. parasitica with Cryphonectria hypovirus 1 in Europe biologically controls this disease, leading to nonlethal and inactive cankers. Unexpectedly, virus-free C. parasitica strains have been isolated from inactive cankers. In this study, we compared the virulence of virus-infected and virus-free C. parasitica strains isolated from either inactive or active cankers on chestnut seedlings and sprouts. In the seedling experiment, we assessed canker growth and seedling mortality. In the sprout experiment, we also assessed canker growth and made fungal reisolations to determine virus infection and immigration of foreign vegetative compatibility (vc) types over a period of 13 years in a coppice forest. Overall, the virulence of virus-free C. parasitica strains isolated from inactive versus active cankers did not differ. Significant differences were only attributed to virus infection. Virus infection and fungal strain composition in cankers changed over time. Foreign vc types immigrated into cankers and virus-free cankers became virus-infected within a few years. Most of the cankers were callused over time and became inactive. However, we observed that the virus did not always persist in these cankers. This study demonstrates that virus spread occurs effectively in European chestnut forests and that this biocontrol system is highly dynamic.

Commodity risk assessment of Ligustrum ovalifolium and Ligustrum vulgare plants from the UK.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of the evergreen Ligustrum ovalifolium and the semi-evergreen Ligustrum vulgare imported from the United Kingdom (UK) as: (a) bare root plants and (b) plants in pots, taking into account the available scientific information, including the technical information provided by the UK. The category (a) 'bare root plants' includes bundles of 1- to 3-year-old bare root whips or transplants and single 1- to 7-year-old bare root plants. The category (b) 'plants in pots' includes bundles of 1- to 2-year-old cell grown plants (only L. vulgare) and 1- to 5-year-old plants in pots. All pests associated with the commodities were evaluated against specific criteria for their relevance for this opinion. Two EU quarantine pests, Bemisia tabaci and Scirtothrips dorsalis, and one pest not regulated in the EU, Diaprepes abbreviatus, fulfilled all relevant criteria and were selected for further evaluation. For the selected pests, the risk mitigation measures proposed in the technical dossier from the UK were evaluated taking into account the possible limiting factors. For these pests, an expert judgement is given on the likelihood of pest freedom considering the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. In the assessment of risk, the age of the plants was considered, reasoning that older trees are more likely to be infested mainly due to longer exposure time and larger size. The degree of pest freedom varies among the pests evaluated, with B. tabaci being the pest most frequently expected on the imported plants. The Expert Knowledge Elicitation indicated with 95% certainty that between 9915 and 10,000 per 10,000 bare root plants and plants in pots will be free from B. tabaci.

Commodity risk assessment of Corylus avellana plants from the UK.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of Corylus avellana imported from the United Kingdom (UK) as: (a) bundles of 1- to 2-year old whips or transplants, (b) bundles of 1- to 2-year old cell grown plants, (c) 1- to 7-year old bare root single plants and (d) up to 15-year old single plants in pots, taking into account the available scientific information, including the technical information provided by the UK. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. Two EU quarantine pests, Phytophthora ramorum (non-EU isolates) and Thaumetopoea processionea fulfilled all relevant criteria and were selected for further evaluation. For the selected pests, the risk mitigation measures implemented in the technical dossier from the UK were evaluated taking into account the possible limiting factors. For these pests an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. In the assessment of risk, the age of the plants was considered, reasoning that older trees are more likely to be infested mainly due to longer exposure time and larger size. The degree of pest freedom varies among the pests evaluated, with P. ramorum being the pest most frequently expected on the imported plants. The expert knowledge elicitation indicated with 95% certainty that between 9939 and 10,000 of the single plants in pots up to 15-year old will be free from P. ramorum (non-EU isolates).

Commodity risk assessment of Tilia cordata and Tilia platyphyllos plants from the UK.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of Tilia cordata and T. platyphyllos imported from the United Kingdom (UK) as: (a) bundles of budwood/graftwood; (b) 1- to 2-year-old whips, seedlings or transplants; (c) bundles of 1- to 2-year-old cell grown plants; (d) 1- to 7-year-old bare root single plants; and (e) up to 25-year-old single plants in pots, taking into account the available scientific information provided by the UK. A list of pests potentially associated with the commodities was compiled. The relevance of any pest was assessed based on evidence following defined criteria. None of the pests on the list fulfilled all relevant criteria and therefore none were selected for further evaluation. As a result, risk mitigation measures proposed in the technical dossier from the UK were listed, but not further evaluated.

Commodity risk assessment of maple veneer sheets from Canada.

The European Commission requested the EFSA Panel on Plant Health to deliver a risk assessment on the likelihood of pest freedom from Union quarantine pests and pests subject to measures adopted pursuant to Article 30 of Regulation (EU) No 2016/2031 for the maple veneer sheets manufactured according to the process set out by Canada, with emphasis on the freedom from Davidsoniella virescens and Phytophthora ramorum (non-EU isolates). The assessment was conducted for veneer sheets of up to 0.7 mm and up to 6 mm thickness, taking into account the different phases in the veneer production in a systems approach. Some of those phases, taken alone, including the heat treatment of logs in a water bath, the cutting into thin veneer sheets and the final high heat drying of veneer sheets are expected to be effective against some of the pests, without uncertainties, making the system approach fully effective. The panel considers that no insects would survive cutting of logs into thin veneer sheets of 0.7 mm and that Xylella fastidiosa will not survive the temperatures in the water bath and final drying of veneers. The degree of pest freedom for the different groups of organisms is generally very high with slightly lower degree of pest freedom for veneer sheets of 6 mm thickness because of lower temperatures reached in the final drying of veneer sheets compared to thinner sheets. P. ramorum is not expected to survive the high heat drying of thin veneer sheets, but it may survive the lower temperatures inside thicker veneer sheets. The Expert Knowledge Elicitation (EKE) indicated, with 95% certainty, that between 9989 and 10,000 veneer sheets (thickness 6 mm) per 10,000 will be free from living P. ramorum. For D. virescens, the EKE indicated, with 95% certainty, that between 9984 and 10,000 veneer sheets (0.7 mm) per 10,000 and that between 9954 and 10,000 veneer sheets (6 mm) per 10,000 will be free from living inoculum. For other relevant groups of pests, the greatest likelihood of pest presence was observed for wood decay fungi. The EKE indicated, with 95% certainty, that between 9967 and 10,000 veneer sheets (0.7 mm) per 10,000 and that between 9911 and 10,000 veneer sheets (6 mm) per 10,000 will be free from living wood decay fungi.

A new multilocus approach for a reliable DNA-based identification of Armillaria species.

In this paper we highlight and critically discuss limitations to molecular methods for identification of fungi via the example of the basidiomycete genus Armillaria. We analyzed a total of 144 sequences of three DNA regions commonly used for identifying fungi (ribosomal IGS-1 and ITS regions, translation elongation factor-1 alpha gene) from 48 specimens of six Armillaria species occurring in Europe (A. cepistipes, A. ostoyae, A. gallica, A. borealis, A. mellea, A. tabescens). Species were identified by comparing newly obtained sequences with those from the NCBI database, phylogenetic analyses and PCR-RFLP analyses of the three regions considered. When analyzed separately, no single gene region could unambiguously identify all six Armillaria species because of low interspecific and high intrasequence variability. We therefore developed a multilocus approach, which involves the stepwise use of the three regions. Following this scheme, all six species could be clearly discriminated. Our study suggests that, to improve the reliability of DNA-based techniques for species identification, multiple genes or intergenic regions should be analyzed.

Temperature Effects on the Cryphonectria hypovirus 1 Accumulation and Recovery within Its Fungal Host, the Chestnut Blight Pathogen Cryphonectria parasitica.

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.

Physiological variations in hypovirus-infected wild and model long-term laboratory strains of Cryphonectria parasitica.

Introduction: Forest ecosystems are highly threatened by the simultaneous effects of climate change and invasive pathogens. Chestnut blight, caused by the invasive phytopathogenic fungus Cryphonectria parasitica, has caused severe damage to European chestnut groves and catastrophic dieback of American chestnut in North America. Within Europe, the impacts of the fungus are widely mitigated through biological control that utilizes the RNA mycovirus: Cryphonectria hypovirus 1 (CHV1). Viral infections, similarly to abiotic factors, can cause oxidative stress in their hosts leading to physiological attrition through stimulating ROS (reactive oxygen species) and NOx production. Methods: To fully understand the interactions leading to the biocontrol of chestnut blight, it is vital to determine oxidative stress damage arising during CHV1 infection, especially considering that other abiotic factors, like long-term cultivation of model fungal strains, can also impact oxidative stress. Our study compared CHV1-infected C. parasitica isolates from two Croatian wild populations with CHV1-infected model strains (EP713, Euro7 and CR23) that have experienced long-term laboratory cultivation. Results and Discussion: We determined the level of oxidative stress in the samples by measuring stress enzymes' activity and oxidative stress biomarkers. Furthermore, for the wild populations, we studied the activity of fungal laccases, expression of the laccase gene lac1, and a possible effect of CHV1 intra-host diversity on the observed biochemical responses. Relative to the wild isolates, the long-term model strains had lower enzymatic activities of superoxide dismutase (SOD) and glutathione S-transferase (GST), and higher content of malondialdehyde (MDA) and total non-protein thiols. This indicated generally higher oxidative stress, likely arising from their decades-long history of subculturing and freeze-thaw cycles. When comparing the two wild populations, differences between them in stress resilience and levels of oxidative stress were also observed, as evident from the different MDA content. The intra-host genetic diversity of the CHV1 had no discernible effect on the stress levels of the virus-infected fungal cultures. Our research indicated that an important determinant modulating both lac1 expression and laccase enzyme activity is intrinsic to the fungus itself, possibly related to the vc type of the fungus, i.e., vegetative incompatibility genotype.

Commodity risk assessment of Quercus petraea plants from the UK.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High-risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of Quercus petraea imported from the UK as: (a) bundles of 1- to 2-year-old whips and seedlings, (b) 1- to 7-year-old bare root plants for planting and (c) less than 1- to 15-year-old plants in pots, taking into account the available scientific information, including the technical information provided by the UK. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. Two EU quarantine pests, Cronartium quercuum and Phytophthora ramorum (non-EU isolates), two protected zone quarantine pests, Cryphonectria parasitica and Thaumetopoea processionea, and four pests not regulated in the EU, Coniella castaneicola, Meloidogyne mali, Phytophthora kernoviae and Trinophylum cribratum, fulfilled all relevant criteria and were selected for further evaluation. For the selected pests, the risk mitigation measures included in the technical dossier from the UK were evaluated taking into account the possible limiting factors. For these pests an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. In the assessment of risk, the age of the plants was considered, reasoning that older trees are more likely to be infested mainly due to longer exposure time and larger size. The degree of pest freedom varies among the pests evaluated, with C. castaneicola being the pests most frequently expected on the imported plants. The Expert knowledge elicitation indicated with 95% certainty that between 9,711 and 10,000 per 10,000 less than 1- to 15-year-old plants in pots will be free from C. castaneicola.

Commodity risk assessment of Quercus robur plants from the UK.

The European Commission requested the EFSA Panel on Plant Health to prepare and deliver risk assessments for commodities listed in Commission Implementing Regulation (EU) 2018/2019 as 'High-risk plants, plant products and other objects'. This Scientific Opinion covers plant health risks posed by plants of Quercus robur imported from the UK as: (a) bundles of 1- to 2-year-old whips and seedlings, (b) 1- to 7-year-old bare root plants for planting and (c) less than 1- to 15-year-old plants in pots, taking into account the available scientific information, including the technical information provided by the UK. All pests associated with the commodity were evaluated against specific criteria for their relevance for this opinion. Two EU quarantine pests, Cronartium quercuum and Phytophthora ramorum (non-EU isolates), two protected zone quarantine pests, Cryphonectria parasitica and Thaumetopoea processionea and four pests not regulated in the EU, Coniella castaneicola, Meloidogyne mali, Phytophthora kernoviae and Trinophylum cribratum, fulfilled all relevant criteria and were selected for further evaluation. For the selected pests, the risk mitigation measures included in the technical dossier from the UK were evaluated taking into account the possible limiting factors. For these pests an expert judgement is given on the likelihood of pest freedom taking into consideration the risk mitigation measures acting on the pest, including uncertainties associated with the assessment. In the assessment of risk, the age of the plants was considered, reasoning that older trees are more likely to be infested mainly due to longer exposure time and larger size. The degree of pest freedom varies among the pests evaluated, with C. castaneicola being the pest most frequently expected on the imported plants. The expert knowledge elicitation indicated with 95% certainty that between 9,711 and 10,000 per 10,000 less than 1- to 15-year-old plants in pots will be free from C. castaneicola.