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Characterization of the Cryphonectria parasitica population was initially done by a phenotypical assessment of 40 in vitro grown isolates obtained from 52 healing cankers collected from eight important chestnut-growing provinces of Turkey. The results of Bavendamm test, often correlated indirectly to hypovirulence, suggested 31 possibly hypovirulent and 9 virulent isolates. PCR tests amplified two regions of ORFs A and B of Cryphonectria hypovirus 1 (CHV-1) from 36 of 40 isolates. The PCR test confirmation was more sensitive than the Bavendamm test. Partial ORFA sequencing revealed 36 CHV-1 haplotypes belonging to Italian subtype (I), with all hypovirulent isolates being of EU-1 vc type. The CHV-1 from 10 native EU-1 isolates were first transferred to six European vc type testers, EU-2, EU-3, EU-5, EU-7, EU-26, and EU-44, having heteroallelism at one vic locus. The presence of the vic locus difference generally reduces virus transmission. The easiest and highest frequency virus transfer was obtained by vic4 and vic6 allelic differences, while the differences vic2 and vic7 made the transfer more challenging. Finally, in this study we successfully transferred CHV-1 to an EU-1 isolate obtained from the Bursa province to an EU-12 European tester isolate. [ABSTRACT FROM AUTHOR]

Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomy-cetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes (dcl1 and dcl2) and four argonaute-like genes (agl1 to agl4). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense. [ABSTRACT FROM AUTHOR]

The introduction in the same area of different invasive species can result in novel interactions, with unpredictable consequences. We carried out a study in Galicia (northwestern Iberian Peninsula) with the aim of clarifying the interactions between two invasive species Cryphonectria parasitica and Dryocosmus kuriphilus. In 2018, we selected five chestnut plots affected by both species. We compared trees affected only by the insect and trees affected by both the insect and the fungus with respect to attack level, gall characteristics, female size and fecundity, and concentrations of nitrogen, water and secondary metabolites. We also evaluated female preferences in a greenhouse assay. There were higher levels of attack in trees affected by both invaders. However, the greenhouse assay showed that ovipositing females do not preferentially choose trees attacked by the fungus. The presence of the fungus had no effect on the size, wall thickness, or hardness of D. kuriphilus galls, but larvae were smaller in trees also affected by the fungus. The fecundity of females was strongly related to the presence of chestnut blight; the number of eggs per female was almost double in trees affected by the fungus. There were no relations between blight and the nitrogen or water content in the galls, where the insects feed. There were also no effects of chestnut blight on the concentration of terpenes or phenols, but condensed tannins were higher in trees with chestnut blight. The higher tannins induced by chestnut blight may directly or indirectly benefit gall wasps. Positive relationships between condensed tannin concentration and reproductive performance of other gall makers were previously reported. Tannins can also improve the negative effects of environmental conditions inside the gall. Our results indicate that the presence of chestnut blight can increase the suitability of chestnut trees for the invasive insect, D. kuriphilus, through the increase in tannins due to the presence of the fungus. [ABSTRACT FROM AUTHOR]

Cryphonectria hypovirus 1 (CHV-1) has been widely studied and used as a biocontrol agent because of its ability to infect the chestnut blight fungus, Cryphonectria parasitica, and to reduce its virulence. Knowledge about the hypovirus, its presence, and diversity is completely lacking in South Tyrol (northern Italy), which may obstruct biocontrol measures for chestnut blight based on CHV-1. This work aimed to study the occurrence of CHV-1 infecting C. parasitica in South Tyrol and to perform a genetic characterization of the hypovirus. In South Tyrol, CHV-1 was found to occur in 29.2% of the fungal isolates investigated, varying in frequency between different regions and chestnut stands. Twenty-three haplotypes based on partial cDNA (complementary DNA) sequences of open reading frame (ORF)-A and 30 haplotypes based on partial cDNA sequences of ORF-B were identified among 47 and 56 hypovirulent fungal isolates, respectively. Phylogenetic analysis showed that all the haplotypes belonged to the Italian subtype of CHV-1 and that they were closely related to the populations of Italy, Switzerland, Croatia and Slovenia. Evidence of recombination was not found in the sequences and point mutations were the main source of diversity. Overall, this study indicated that the prevalence of CHV-1 in South Tyrol is low compared to many other central and western European populations and determined a need to actively impose biocontrol measures. Using sequence analysis, we identified some variants of interest of CHV-1 that should be studied in detail for their potential use in biocontrol. [ABSTRACT FROM AUTHOR]

Sweet chestnut, an Asiatic tree introduced in many parts of Europe including the United Kingdom, is planted for nut production, timber, and amenity. Its major threat is the disease called blight, caused by the fungus Cryphonectria parasitica, which infects through wounds by airborne spores. Field trapping using sticky rods rotating traps was performed in an infected area in Devon (between May 2021 and April 2023). An improved dual hydrolysis Taqman probes real-time PCR was used. The number of spores was calculated by comparing the cycle threshold to the Ct of standards with known amounts of conidia or known target fragment copies cloned into a plasmid. Weekly spore counts were in the range of around 60 to approximately 8.5 × 103, with fluctuations of peaks (mainly in late summer–autumn 2021) and troughs. The effects of weather parameters were modelled, finding correlations between spore numbers and temperature, humidity, dewpoint, rainfall, wind speed, and wind duration. Additionally, an insect trapping was performed to confirm the presence/absence and quantity of C. parasitica conidia potentially phoretic on some insects by using the same molecular approach. None of the ten collected insect species harboured spores of this fungus. [ABSTRACT FROM AUTHOR]

Sweet chestnut (Castanea sativa) is an ecologically and economically valuable tree species worldwide. Various pathogens, both endemic or introduced, cause severe damage or death to chestnut trees. Chestnut blight, caused by the fungus Cryphonectria parasitica, is one of the most devastating plant diseases known which influences chestnut trees in all major cultivation regions. The work presented here aimed to investigate how the incidence of chestnut blight varied between trees growing at two altitudes (200–400 m vs. 400–600 m) and to determine the impact of chestnut blight on tree‐ring width, morphology and anatomy of C. sativa trees. The incidence of C. parasitica infections was greater at 200–400 m than at 400–600 m. Greater stem diameters were found at 200–400 m than at 400–600 m. There were no differences in stem diameters and heights, however, between healthy and C. parasitica infected chestnut trees. The anatomical characteristics of wood were also compared between healthy and C. parasitica‐infected chestnut trees. Fibre cell traits (length/width, lumen width, lumen area) were higher in healthy trees than in C. parasitica‐diseased trees. Vessel diameters were greater in infected compared with healthy trees. Tree ring widths and wood anatomical characteristics therefore were significantly influenced by C. parasitica infections. [ABSTRACT FROM AUTHOR]

Cryphonectria hypovirus 1 (CHV-1) has been widely studied and used as a biocontrol agent because of its ability to infect the chestnut blight fungus, Cryphonectria parasitica, and to reduce its virulence. Knowledge about the hypovirus, its presence, and diversity is completely lacking in South Tyrol (northern Italy), which may obstruct biocontrol measures for chestnut blight based on CHV-1. This work aimed to study the occurrence of CHV-1 infecting C. parasitica in South Tyrol and to perform a genetic characterization of the hypovirus. In South Tyrol, CHV-1 was found to occur in 29.2% of the fungal isolates investigated, varying in frequency between different regions and chestnut stands. Twenty-three haplotypes based on partial cDNA (complementary DNA) sequences of open reading frame (ORF)-A and 30 haplotypes based on partial cDNA sequences of ORF-B were identified among 47 and 56 hypovirulent fungal isolates, respectively. Phylogenetic analysis showed that all the haplotypes belonged to the Italian subtype of CHV-1 and that they were closely related to the populations of Italy, Switzerland, Croatia and Slovenia. Evidence of recombination was not found in the sequences and point mutations were the main source of diversity. Overall, this study indicated that the prevalence of CHV-1 in South Tyrol is low compared to many other central and western European populations and determined a need to actively impose biocontrol measures. Using sequence analysis, we identified some variants of interest of CHV-1 that should be studied in detail for their potential use in biocontrol.

In England, Cryphonectria parasitica was detected for the first time in 2011 in a nursery and in 2016 in the wider environment. Surveys between 2017 and 2020 identified the disease at different sites in Berkshire, Buckinghamshire, Cornwall, Derbyshire, Devon, Dorset, London, West Sussex, and the island of Jersey, while the present study comprises the results of the 2020–2023 survey with findings in Derbyshire, Devon, Kent, Nottinghamshire, Herefordshire, Leicestershire, London, West Sussex, and the islands of Jersey and Guernsey. A total of 226 suspected samples were collected from 72 surveyed sites, as far north as Edinburgh and as far west as Plymouth (both of which were negative), and 112 samples tested positive by real-time PCR and isolation from 35 sites. The 112 isolates were tested for the vegetative compatibility group (VCG), mating type, and Cryphonectria hypovirus 1 (CHV1). Twelve VCGs were identified, with two of them (EU-5 and EU-22) being the first records in the UK. Both mating types were present (37% MAT-1 and 63% MAT-2), but only one mating type was present per site and VCG, and perithecia were never observed. Cryphonectria hypovirus 1 (CHV1), consistently subtype-I haplotype E-5, was detected in three isolates at a low concentration (5.9, 21.1, and 33.0 ng/µL) from locations in London, Nottinghamshire, and Devon. [ABSTRACT FROM AUTHOR]

Cryphonectria hypovirus 1 (CHV1), as a biological agent, has a great potential for controlling of chestnut blight. CHV1 infects Cryphonectria parasitica, the causal agent of chestnut blight, via horizontal transmission, subsequently weakening the pathogen's virulence. C. parasitica has a vegetative incompatibility system that restricts the transmission of CHV1 between different vc types, but not completely. This study aimed to determine the horizontal transmission of CHV1 between the vc types related to the C. parasitica population in Aydın Province. The two CHV1 infected isolates, Zgl709, and Fr22, which belong to EU-1 and EU-12 vc types, respectively, were used as the virus donor isolates in the horizontal transmission test. Thirty offspring single ascospore isolates, representing 16 vc types, which were obtained from the sexual cross between a local EU-1 and a local EU-12 isolate, served as the recipients. CHV1 was transmitted 100% if the donor and the recipient were from the same vc type. The average transmission rate between the donor EU-1 (Zgl709) and the recipients from different vc types was 13.6%. Fourteen recipient isolates from 8 vc types (EU-3, EU-4, EU-7, EU-8, EU-25, EU-26, EU-28, and EU-30) were successfully converted to hypovirulent by the donor EU-1. The average transmission rate between the donor EU-12 (Fr22) and the recipients from different vc types was 7.5%. Only 5 recipient isolates from 4 vc types (EU-4, EU8, EU-29, and EU-30) were converted to hypovirulent by the donor EU-12. There was no transmission of CHV1 between EU-1 and EU-12, which are the two dominant vc types present in Aydın Province. [ABSTRACT FROM AUTHOR]

Vegetative compatibility (vc) and mating types and genetic diversity of Cryphonectria parasitica isolates were determined using 183 isolates obtained from 215 infected chestnut trees growing in 13 provinces of Turkey. Based on the cultural aspects, 143 of these isolates were evaluated as virulent whereas the remaining 40 isolates were hypovirulent. When vc types of 183 isolates were classically differentiated, 135 of them matched to EU‐1 (82.3%), 29 of them to EU‐12 (17.6%) vc type, whereas 19 of them did not match to the two. When molecular vic markers were used, all the isolates were assigned to two EU vc types; 149 to EU‐1 (81.4%) and 34 (18.5%) to EU‐12. Of the majority of the isolates, 134 (73.2%) had mating‐type MAT‐1, while 44 (24%) isolates had MAT‐2 and 5 (2.8%) isolates had both mating types. The population analysis based on two DNA marker systems, Inter‐Primer Binding Site and Start Codon Targeted Polymorphism, showed no intraspecific genetic variation among the C. parasitica isolates. The prevalence of two dominant vc types revealed by this study shows that biological control with hypovirulent EU‐1 and EU‐12 isolates will be significant for the country. The results might be helpful to chestnut breeders carrying out resistance breeding studies to manage this disease based on hypovirulence attributed to Cryphonectria hypovirus 1. [ABSTRACT FROM AUTHOR]

Cryphonectria parasitica is the causal agent of chestnut blight, a fungal disease that almost entirely eliminated mature American chestnut from North America over a 50-year period. Here, we formally report the genome of C. parasitica EP155 using a Sanger shotgun sequencing approach. After finishing and integration with simple-sequence repeat markers, the assembly was 43.8 Mb in 26 scaffolds (L50 = 5; N50 = 4.0Mb). Eight chromosomes are predicted: five scaffolds have two telomeres and six scaffolds have one telomere sequence. In total, 11,609 gene models were predicted, of which 85% show similarities to other proteins. This genome resource has already increased the utility of a fundamental plant pathogen experimental system through new understanding of the fungal vegetative incompatibility system, with significant implications for enhancing mycovirus-based biological control.

Abstract Background Chestnut blight, one of the most serious branch diseases in Castanea caused by Cryphonectria parasitica, which has ravaged across American chestnut and most of European chestnut since the early twentieth century. Interestingly, the Chinese chestnut is strongly resistant to chestnut blight, shedding light on restoring the ecological status of Castanea plants severely affected by chestnut blight. To better explore the early defense of Chinese chestnut elicited in response to C. parasitica, the early stage of infection process of C. parasitica was observed and RNA sequencing-based transcriptomic profiling of responses of the chestnut blight-resistant wild resource ‘HBY-1’ at 0, 3 and 9 h after C. parasitica inoculation was performed. Results First, we found that 9 h was a critical period for Chinese chestnut infected by C. parasitica, which was the basis of further study on transcriptional activation of Chinese chestnut in response to chestnut blight in the early stage. In the transcriptome analysis, a total of 283 differentially expressed genes were identified between T9 h and Mock9 h, and these DEGs were mainly divided into two clusters, one of which was metabolism-related pathways including biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and photosynthesis; the other was related to plant-pathogen interaction and MAPK signal transduction. Meanwhile, the two clusters of pathways could be connected through junction among phosphatidylinositol signaling system, phytohormone signaling pathway and α-Linolenic acid metabolism pathway. It is worth noting that genes associated with JA biosynthesis and metabolic pathway were significantly up-regulated, revealing that the entire JA metabolic pathway was activated in Chinese chestnut at the early stage of chestnut blight infection. Conclusion We identified the important infection nodes of C. parasitica and observed the morphological changes of Chinese chestnut wounds at the early stage of infection. In response to chestnut blight, the plant hormone and MAPK signal transduction pathways, plant-pathogen interaction pathways and metabolism-related pathways were activated at the early stage. JA biosynthesis and metabolic pathway may be particularly involved in the Chinese chestnut resistance to chestnut blight. These results contributes to verifying the key genes involved in the resistance of Chinese chestnut to C. parasitica.

Sweet chestnut, an Asiatic tree introduced in many parts of Europe including the United Kingdom, is planted for nut production, timber, and amenity. Its major threat is the disease called blight, caused by the fungus Cryphonectria parasitica, which infects through wounds by airborne spores. Field trapping using sticky rods rotating traps was performed in an infected area in Devon (between May 2021 and April 2023). An improved dual hydrolysis Taqman probes real-time PCR was used. The number of spores was calculated by comparing the cycle threshold to the Ct of standards with known amounts of conidia or known target fragment copies cloned into a plasmid. Weekly spore counts were in the range of around 60 to approximately 8.5 × 103, with fluctuations of peaks (mainly in late summer–autumn 2021) and troughs. The effects of weather parameters were modelled, finding correlations between spore numbers and temperature, humidity, dewpoint, rainfall, wind speed, and wind duration. Additionally, an insect trapping was performed to confirm the presence/absence and quantity of C. parasitica conidia potentially phoretic on some insects by using the same molecular approach. None of the ten collected insect species harboured spores of this fungus.

Background: Chestnut blight, one of the most serious branch diseases in Castanea caused by Cryphonectria parasitica, which has ravaged across American chestnut and most of European chestnut since the early twentieth century. Interestingly, the Chinese chestnut is strongly resistant to chestnut blight, shedding light on restoring the ecological status of Castanea plants severely affected by chestnut blight. To better explore the early defense of Chinese chestnut elicited in response to C. parasitica, the early stage of infection process of C. parasitica was observed and RNA sequencing-based transcriptomic profiling of responses of the chestnut blight-resistant wild resource 'HBY-1' at 0, 3 and 9 h after C. parasitica inoculation was performed. Results: First, we found that 9 h was a critical period for Chinese chestnut infected by C. parasitica, which was the basis of further study on transcriptional activation of Chinese chestnut in response to chestnut blight in the early stage. In the transcriptome analysis, a total of 283 differentially expressed genes were identified between T9 h and Mock9 h, and these DEGs were mainly divided into two clusters, one of which was metabolism-related pathways including biosynthesis of secondary metabolites, phenylpropanoid biosynthesis, amino sugar and nucleotide sugar metabolism, and photosynthesis; the other was related to plant-pathogen interaction and MAPK signal transduction. Meanwhile, the two clusters of pathways could be connected through junction among phosphatidylinositol signaling system, phytohormone signaling pathway and α-Linolenic acid metabolism pathway. It is worth noting that genes associated with JA biosynthesis and metabolic pathway were significantly up-regulated, revealing that the entire JA metabolic pathway was activated in Chinese chestnut at the early stage of chestnut blight infection. Conclusion: We identified the important infection nodes of C. parasitica and observed the morphological changes of Chinese chestnut wounds at the early stage of infection. In response to chestnut blight, the plant hormone and MAPK signal transduction pathways, plant-pathogen interaction pathways and metabolism-related pathways were activated at the early stage. JA biosynthesis and metabolic pathway may be particularly involved in the Chinese chestnut resistance to chestnut blight. These results contributes to verifying the key genes involved in the resistance of Chinese chestnut to C. parasitica. [ABSTRACT FROM AUTHOR]

Cryphonectria parasitica is a microscopic fungus that attacks primarily tree species of the genus Castanea (chestnut) by penetrating their injured bark and colonizing the inner bark and cambium, disrupting nutrient and water flow and causing chestnut blight. Cr. parasitica severely impacts American chestnut (C. dentata) and European chestnut (C. sativa) causing major ecological damage and economic loss. Data from 2010 reveals that this pathogen partially or entirely defoliated and killed 40 % of Georgia's chestnut forests. In this study, we used generalized additive models (GAMs) to test whether environmental factors influence the severity of blight-induced defoliation of chestnut trees across Georgia. Mean annual precipitation emerged as the most impactful factor, followed in order of significance by slope, understory cover, mean annual temperature, and aspect. Our research contributes to a broader research agenda aimed at laying the groundwork for more informed conservation efforts. • High temperatures increase trees' susceptibility to chestnut blight infection. • Higher annual precipitation, steeper slopes, and greater understory cover significantly increase defoliation severity. • Drought may reduce disease spread but compromise trees' pathogen defenses. [ABSTRACT FROM AUTHOR]

Castanea species have enormous potential for broad applications in the biomedical sciences and chemical wood industries. Due to the occurrence of fungal diseases, especially chestnut blight, the survival status of Castanea plants is seriously threatened. Castanea plants in China have natural tolerance to chestnut blight. This study investigated the genetic diversity and chestnut blight resistance of Castanea plants in China and searched for germplasm with high tolerance resistance. Cluster and population structure analysis revealed that wild Castanea plants in China could be divided into three groups with significant interspecific boundary, C. henryi var. omeiensis is located between C. mollissima and C. henryi var. henryi. The ABBA-BBAA test showed that significant gene introgression occurred between C. mollissima and C. henryi in Mount Emei, supporting the conclusion that C. henryi var. omeiensis as a natural hybrid resource. The results of disease resistance evaluation showed that the tolerance of wild C. mollissima to chestnut blight was the highest and significantly higher than that of wild C. henryi and C. seguinii. In particular, there is a strong association between disease tolerance in C. henryi var. omeiensis and the level of genetic introgression from C. mollissima. Then, the rIBD fragments of C. henryi var. omeiensis derived from C. mollissima were analyzed to reveal the source of disease resistance using whole genome resequencing data. A total of 203 rIBD segments containing 435 genes were identified. Finally, 41 candidate genes for chestnut blight resistance were selected through multiple omics. In conclusion, this study investigated a type of interspecific disease-resistant hybrid resources, identified a set of candidate genes for chestnut blight resistance, and provided new strategies for disease-resistant breeding of chestnut resources in the future. • The tolerance of wild Castanea including three species to chestnut blight in China was evaluated. • It was confirmed that C. henryi var. omeiensis was an interspecific hybrid with high tolerance to chestnut blight. • A total of 203 rIBD segments and 41 candidate disease resistance genes were identified to enable next-generation precision breeding of chestnuts. [ABSTRACT FROM AUTHOR]

In England, Cryphonectria parasitica was detected for the first time in 2011 in a nursery and in 2016 in the wider environment. Surveys between 2017 and 2020 identified the disease at different sites in Berkshire, Buckinghamshire, Cornwall, Derbyshire, Devon, Dorset, London, West Sussex, and the island of Jersey, while the present study comprises the results of the 2020–2023 survey with findings in Derbyshire, Devon, Kent, Nottinghamshire, Herefordshire, Leicestershire, London, West Sussex, and the islands of Jersey and Guernsey. A total of 226 suspected samples were collected from 72 surveyed sites, as far north as Edinburgh and as far west as Plymouth (both of which were negative), and 112 samples tested positive by real-time PCR and isolation from 35 sites. The 112 isolates were tested for the vegetative compatibility group (VCG), mating type, and Cryphonectria hypovirus 1 (CHV1). Twelve VCGs were identified, with two of them (EU-5 and EU-22) being the first records in the UK. Both mating types were present (37% MAT-1 and 63% MAT-2), but only one mating type was present per site and VCG, and perithecia were never observed. Cryphonectria hypovirus 1 (CHV1), consistently subtype-I haplotype E-5, was detected in three isolates at a low concentration (5.9, 21.1, and 33.0 ng/µL) from locations in London, Nottinghamshire, and Devon.

Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) bark is a commonly used remedy to treat gastrointestinal disorders, throat and skin infections, hemorrhages, and dysentery. It has also been previously reported that its methanol extracts possess antibacterial activity, which can be related to the richness of Quercus spp. extracts in phenolic compounds, such as flavonoids and tannins. However, there is no information on the antifungal (including oomycete) properties of the bark from Q. ilex or its subspecies (ilex and ballota). In this work, we report the characterization of the aqueous ammonia extract of its bark by FTIR and GC-MS and the results of in vitro and ex situ inhibition tests against three phytopathogens. The main phytochemical components identified were inositols (19.5%), trans-squalene (13%), 4-butoxy-1-butanol (11.4%), gulopyranose (9.6%), lyxose (6.5%), 2,4-dimethyl-benzo[H]quinoline (5.1%), catechol (4.5%), and methoxyphenols (4.2%). The efficacy of the extract in controlling forest phytopathogens was tested in vitro against Fusarium circinatum (responsible for pitch canker of Pinus spp.), Cryphonectria parasitica (which causes chestnut blight), and Phytophthora cinnamomi (which causes 'root and crown rot' in a variety of hosts, including Castanea, conifers, Eucalyptus, Fagus, Juglans, Quercus, etc.), obtaining EC90 values of 322, 295, and 75 μg·mL−1, respectively, much lower than those attained for a commercial strobilurin fungicide (azoxystrobin). The extract was further tested ex situ against P. cinnamomi on artificially inoculated, excised stems of 'Garnem' almond rootstock, attaining complete protection at a dose of 782 μg·mL−1. The results suggest that holm oak bark extract may be a promising source of bioactive compounds against invasive forest pathogens, including the oomycete that is causing its decline, the so-called 'seca' in Spain. [ABSTRACT FROM AUTHOR]

Chestnuts are multipurpose trees significant for the economy and wildlife. These trees are currently found around the globe, demonstrating their genetic adaptation to different environmental conditions. Several biotic and abiotic stresses have challenged these species, contributing to the decline of European chestnut production and the functional extinction of the American chestnut. Several efforts started over the last century to understand the cellular, molecular, and genetic interactions behind all chestnut biotic and abiotic interactions. Most efforts have been toward breeding for the primary diseases, chestnut blight and ink disease caused by the pathogens, Cryphonectria parasitica and Phytophthora cinnamomi, respectively. In Europe and North America, researchers have been using the Asian chestnut species, which co-evolved with the pathogens, to introgress resistance genes into the susceptible species. Breeding woody trees has several limitations which can be mostly related to the long life cycles of these species and the big genome landscapes. Consequently, it takes decades to improve traits of interest, such as resistance to pathogens. Currently, the availability of genome sequences and next-generation sequencing techniques may provide new tools to help overcome most of the problems tree breeding is still facing. This review summarizes European and American chestnut’s main biotic stresses and discusses breeding and biotechnological efforts developed over the last decades, having ink disease and chestnut blight as the main focus. Climate change is a rising concern, and in this context, the adaptation of chestnuts to adverse environmental conditions is of extreme importance for chestnut production. Therefore, we also discuss the abiotic challenges on European chestnuts, where the response to abiotic stress at the genetic and molecular level has been explored. [ABSTRACT FROM AUTHOR]

Cryphonectria parasitica is an invasive fungal pathogen that causes blight disease on chestnut trees. Its destructive effect can be controlled with naturally occurring mycovirus Cryphonectria hypovirus 1 (CHV1). To date, the spread of C. parasitica and CHV1 in Europe is fairly well documented, but there are still several unexplored regions. Thus, we sampled blight cankers from four sweet chestnut populations in Bay of Kotor and Lake Skadar regions in Montenegro. We determined vegetative compatibility (vc) type and mating-type diversity using molecular vic and MAT1 genotyping, as well as confirming the presence of CHV1 by RT-PCR. We identified 11 vc types, with EU-12 being the dominant one represented by 58.2% of all fungal isolates. The Shannon diversity index ranged from 0.93 to 1.47. Both mating types of C. parasitica were found in all four populations. The prevalence of CHV1 ranged from 15% to 40%. All CHV1 isolates belonged to Italian subtype I of CHV1 and were closely related, with relatively recent common ancestors. Our results indicate a longer presence of C. parasitica and CHV1 in Montenegro than previously thought. Natural biocontrol with CHV1 seems to be well established. However, it has the potential for deterioration; thus, close monitoring is required. [ABSTRACT FROM AUTHOR]

Chestnuts are multipurpose trees significant for the economy and wildlife. These trees are currently found around the globe, demonstrating their genetic adaptation to different environmental conditions. Several biotic and abiotic stresses have challenged these species, contributing to the decline of European chestnut production and the functional extinction of the American chestnut. Several efforts started over the last century to understand the cellular, molecular, and genetic interactions behind all chestnut biotic and abiotic interactions. Most efforts have been toward breeding for the primary diseases, chestnut blight and ink disease caused by the pathogens, Cryphonectria parasitica and Phytophthora cinnamomi, respectively. In Europe and North America, researchers have been using the Asian chestnut species, which co-evolved with the pathogens, to introgress resistance genes into the susceptible species. Breeding woody trees has several limitations which can be mostly related to the long life cycles of these species and the big genome landscapes. Consequently, it takes decades to improve traits of interest, such as resistance to pathogens. Currently, the availability of genome sequences and next-generation sequencing techniques may provide new tools to help overcome most of the problems tree breeding is still facing. This review summarizes European and American chestnut’s main biotic stresses and discusses breeding and biotechnological efforts developed over the last decades, having ink disease and chestnut blight as the main focus. Climate change is a rising concern, and in this context, the adaptation of chestnuts to adverse environmental conditions is of extreme importance for chestnut production. Therefore, we also discuss the abiotic challenges on European chestnuts, where the response to abiotic stress at the genetic and molecular level has been explored.

Cryphonectria parasitica was detected for the first time in the United Kingdom in 2011. A 2017–2018 survey detected the disease at different sites in Berkshire, Derbyshire, Devon, Dorset and London, while the present study comprises the results of the 2019–2020 survey with new findings and additional sites in the United Kingdom (Berkshire, Buckinghamshire, Cornwall, Derbyshire, Devon, London, West Sussex) and in Jersey, reflecting the progressive detection of more infected trees. A total of 189 samples were collected from 52 sites, and 123 samples tested positive both by quantitative real‐time PCR and/or isolation from 43 sites. A total of 115 isolates were tested for mating type, vegetative compatibility group (VCG) and Cryphonectria hypovirus 1 (CHV‐1). Twelve VCGs were identified, with four of them being first records in United Kingdom. The highest diversity of VCGs was detected in Devon followed by West Sussex while London and Derbyshire presented the lowest. Both mating types were detected (41% MAT‐1 and 59% MAT‐2), and no heterokaryons were detected. Perithecia of C. parasitica were not observed at any site during this survey. CHV‐1 was detected in three isolates in very low concentration from three different locations in London and was always the unmutated subtype I haplotype E‐5. A greater diversity of VCGs at outbreak sites compared with previous surveys, combined with their scattered distribution and the slow spread of the pathogen, supports the hypothesis that this disease has been introduced through imports over time from Europe. [ABSTRACT FROM AUTHOR]

European chestnut (Castanea sativa) is threatened by the invasive fungus Cryphonectria parasitica, which causes chestnut blight. The virulence of the fungus can be reduced by a group of mycoviruses that can spread among vegetatively compatible strains through hyphal anastomosis. Hypovirulent isolates are used as biocontrol agents, but their efficiency can be diminished by restricted hyphal anastomosis if the variability of vegetative compatibility (vc) types in a population is high. Sexual reproduction could increase the vc type diversity and further complicate biocontrol in a region. Therefore, knowledge of genetic diversity of C. parasitica is important to assess the effectiveness of a biological control program. The present study was performed in the Autonomous Province of Bozen-Bolzano (South Tyrol) in northern Italy, where chestnut cultivation provides an additional income to farmers. The genetic characterization of C. parasitica isolates from 35 chestnut stands and one forest population in different districts of South Tyrol was performed based on the analysis of vegetative incompatibility loci, the mating type locus, and the internal transcribed spacer (ITS) region. In this study, a total of 23 different vc types were found all over South Tyrol with a Shannon diversity index of 1.86. EU-2, EU-1, and EU-13 were the most widespread vc types comprising 51%, 13%, and 9% of the fungal isolates, respectively. Both mating types were present in the region with a ratio close to 1:1. Three different haplotypes were identified based on ITS sequence analysis, which pointed to two introduction events of the fungus to the region and allowed placing C. parasitica from South Tyrol into a larger phylogeographic context. [ABSTRACT FROM AUTHOR]

Cryphonectria parasitica, the causal agent of chestnut blight, is a destructive Ascomycota fungal disease infecting European chestnut (Castanea sativa) and American chestnut (Castanea dentata) trees. On susceptible host trees, necrotic lesions (cancers) are caused by the disease on the bark of the trunk and branches. The disease leads to wilting and destruction of the chestnut trees. Protection against the pathogen is difficult. Biological control using hypovirulent strains of the pathogen is one of the best options, but its implementation requires a lot of preliminary testing, as well as compatibility between the virulent pathogen strain that infects the area and the hypovirulent strain used for treatment. Thus, this procedure is a rather complicated and slow process. Chemical control against the pathogen is not currently widespread, as its implementation is also difficult due to the large size of the trees and the nature of the forest-like plantations, and there are currently no available pesticides. At the same time, protection with chemical pesticides may be feasible in plantations in which the size of the trees allows for application (lowersized trees or young plantations). Therefore, it is necessary to find fungicides that can be used effectively, which is the purpose of this experiment. In vitro efficacy of four chemical pesticides [Pictor (dimoxystrobin+boscalid), Amistar Sun (azoxystrobin+difenoconazole), Score 250 EC (difenoconazole), Cuproxat FW (tribasic-cooper-sulphate)] has been tested against Cryphonectria parasitica. According to the results of this experiment Score 250 EC and Amistar Sun were the most effective fungicides, given that they inhibited fungal growth even at the lowest concentrations of the test solution. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Antiviral RNA interference (RNAi) is conserved from yeasts to mammals. Dicer recognizes and cleaves virus-derived double-stranded RNA (dsRNA) and/or structured single-stranded RNA (ssRNA) into small-interfering RNAs, which guide effector Argonaute to homologous viral RNAs for digestion and inhibit virus replication. Thus, Argonaute is believed to be essential for antiviral RNAi. Here, we show Argonaute-independent, Dicer-dependent antiviral defense against dsRNA viruses using Cryphonectria parasitica (chestnut blight fungus), which is a model filamentous ascomycetous fungus and hosts a variety of viruses. The fungus has two dicer-like genes ( dcl1 and dcl2 ) and four argonaute-like genes ( agl1 to agl4 ). We prepared a suite of single to quadruple agl knockout mutants with or without dcl disruption. We tested these mutants for antiviral activities against diverse dsRNA viruses and ssRNA viruses. Although both DCL2 and AGL2 worked as antiviral players against some RNA viruses, DCL2 without argonaute was sufficient to block the replication of other RNA viruses. Overall, these results indicate the existence of a Dicer-alone defense and different degrees of susceptibility to it among RNA viruses. We discuss what determines the great difference in susceptibility to the Dicer-only defense., Competing Interests: Competing interests statement:The authors declare no competing interest.

Laccase activity reduction in the chestnut blight fungus Cryphonectria parasitica usually accompanies the hypovirulence caused by the infection of fungus with Cryphonectria hypovirus 1 (CHV1). However, the different methods utilized for assessing this phenomenon has produced varied and often conflicting results. Furthermore, the majority of experimental setups included only one prototypic system, further confounding the results. Considering the diversity of fungal isolates, viral strains, and variability of their effects on the phytopathogenic process observed in nature, our goal was to ascertain if laccase activity variability is affected by (1) different C. parasitica isolates infected with several CHV1 strains, and (2) growth conditions. We have demonstrated that some CHV1 strains, contrary to previous assumptions, increase the activity of C. parasitica laccases. The specific fungal isolates used in the experiments and culture conditions also affected the results. Furthermore, we showed that two commonly used laccase substrates, 2,20-azino-bis(3-ethylbenzothiazoline-6- sulphonic acid) and 2,4-dimethoxyphenol, cannot be used interchangeably in C. parasitica laccase activity measurements. Our results illustrate the importance of conducting this type of study in experimental systems and culture conditions that resemble natural conditions as much as possible to be able to infer the most relevant conclusions applicable to natural populations. [ABSTRACT FROM AUTHOR]

Cryphonectria hypovirus 1 (CHV1) was detected by specific RT‐PCR in two strains of Cryphonectria parasitica from Azerbaijan in which reduction of laccase activity was also observed. This is the first report of a hypovirus in the chestnut blight fungus in Azerbaijan. Our work opens the way to further investigations devoted to characterizing the Azerbaijan CHV1 strain. [ABSTRACT FROM AUTHOR]

Holm oak (Quercus ilex subsp. ballota (Desf.) Samp.) bark is a commonly used remedy to treat gastrointestinal disorders, throat and skin infections, hemorrhages, and dysentery. It has also been previously reported that its methanol extracts possess antibacterial activity, which can be related to the richness of Quercus spp. extracts in phenolic compounds, such as flavonoids and tannins. However, there is no information on the antifungal (including oomycete) properties of the bark from Q. ilex or its subspecies (ilex and ballota). In this work, we report the characterization of the aqueous ammonia extract of its bark by FTIR and GC-MS and the results of in vitro and ex situ inhibition tests against three phytopathogens. The main phytochemical components identified were inositols (19.5%), trans-squalene (13%), 4-butoxy-1-butanol (11.4%), gulopyranose (9.6%), lyxose (6.5%), 2,4-dimethyl-benzo[H]quinoline (5.1%), catechol (4.5%), and methoxyphenols (4.2%). The efficacy of the extract in controlling forest phytopathogens was tested in vitro against Fusarium circinatum (responsible for pitch canker of Pinus spp.), Cryphonectria parasitica (which causes chestnut blight), and Phytophthora cinnamomi (which causes ‘root and crown rot’ in a variety of hosts, including Castanea, conifers, Eucalyptus, Fagus, Juglans, Quercus, etc.), obtaining EC90 values of 322, 295, and 75 μg·mL−1, respectively, much lower than those attained for a commercial strobilurin fungicide (azoxystrobin). The extract was further tested ex situ against P. cinnamomi on artificially inoculated, excised stems of ‘Garnem’ almond rootstock, attaining complete protection at a dose of 782 μg·mL−1. The results suggest that holm oak bark extract may be a promising source of bioactive compounds against invasive forest pathogens, including the oomycete that is causing its decline, the so-called ‘seca’ in Spain.

Xenorhabdus and Photorhabdus spp. are enteric bacterial symbionts of Steinernema and Heterorhabditis nematodes, respectively. These bacteria produce an extensive set of natural products (NPs) with antibacterial, antifungal, antiprotozoal, insecticidal, or other bioactivities when vectored into insect hemocoel by nematodes. We assessed the in vitro activity of different Xenorhabdus and Photorhabdus cell-free supernatants against important fungal phytopathogens, viz., Cryphonectria parasitica, Fusarium oxysporum, Rhizoctonia solani, and Sclerotinia sclerotiorum and identified the bioactive antifungal compound/s present in the most effective bacterial supernatant using the easyPACId (easy promoter–activated compound identification) approach against chestnut blight C. parasitica. Our data showed that supernatants from Xenorhabdus species were comparatively more effective than extracts from Photorhabdus in suppressing the fungal pathogens; among the bacteria assessed, Xenorhabdus szentirmaii was the most effective species against all tested phytopathogens especially against C. parasitica. Subsequent analysis revealed fabclavines as antifungal bioactive compounds in X. szentirmaii, generated by a polyketide synthase (PKS) and non-ribosomal peptide synthetase (NRPS) hybrid system. Fabclavines are broad-spectrum, heat-stable NPs that have great potential as biological control compounds against fungal plant pathogens. More studies are needed to assess the potential phytotoxicity of these compounds and their effects on non-target organisms before commercialization. Key points: • Chemical fungicides have toxic effects on humans and other non-target organisms. • Alternatives with novel modes of action to supplant current fungicide are needed. • A novel bioactive antifungal compound from Xenorhabdus szentirmaii was identified. [ABSTRACT FROM AUTHOR]

Ozark chinquapin (Castanea ozarkensis Ashe) is a forest tree, endemic to the Ozark Mountain region in Eastern United States. Its nutritious nuts were consumed by Native Americans, European settlers, livestock, and wild animals and its wood was an important rot-resistant construction material. Once a significant tree in regional forest communities, the species was nearly eradicated by a chestnut blight caused by Cryphonectria parasitca (Murill) Barr fungus. Some individuals have survived as sprouts from adventitious root buds, but they rarely reach reproductive maturity. While some in situ restoration efforts are underway, the development of a viable ex situ germplasm preservation method is critical to the conservation of this important food-bearing species. Our experiment aimed to develop a cryopreservation method for C. ozarkensis dormant winter buds subjected to eight experimental treatments before desiccation, slow cooling, and storage in liquid nitrogen vapor. The highest post cryogenic viability was 91.2 % for dormant buds pretreated with 0.3 M sucrose for 16 h followed by 0.75 M sucrose for 3 h; this treatment is suggested for cryopreservation of dormant winter buds of Ozark chinquapin germplasm. • A viable ex situ cryopreservation method for Castanea ozarkensis dormant buds was established. • Extended exposure to two different sucrose concentrations significantly enhanced post cryogenic viability. • Forcing dormant buds to sprout simplifies viability testing. [ABSTRACT FROM AUTHOR]

Twenty-two essential oil samples from Germany and Albania were assayed to test their inhibitory and fungicidal activity against Cryphonectria parasitica (Murr.) Barr., the major causal agent of chestnut blight on chestnut trees, using the fumigant macrodilution method. Test concentrations of the oils in air phase were: 0.02, 0.04, 0.08, 0.16 and 0.32 μl ml-1, and minimum inhibitory and fungicidal concentrations were determined. The strongest activity was shown by two samples of mint oil from Germany and Albania, and black pine at the concentration of 0.02 μl ml-1 of air for both test isolates (M1 and 4S). The tested plant essential oils also included: eucalyptus, sage, silver fir and cade. Only three samples of EOs (mint (2) and black pine) were lethal to both isolates. Sage and silver fir oils were more toxic to the M1 isolate, while cade and eucalyptus oils were more toxic to the 4S isolate. Fifteen oils: mint (2), black pine, cade, eucalyptus, silver fir, sage, pine (4), oregano, lavender, and rosemary (2), inhibited the growth of Cryphonectria parasitica, and seven oils (two samples of mint, eucaliptus, black pine, sage, silver fir and cade) showed fungicidal effects on the pathogen.

In contrast to many current applications of biotechnology, the intended consequence of the American Chestnut Research & Restoration Project is to produce trees that are well‐adapted to thrive not just in confined fields or orchards, but throughout their natural range. Our primary focus is on disease tolerance, but we believe it will also be critically important that optimal restoration trees should have robust genetic diversity and resilience, which can be supplied by a full complement of their wild‐type genes. Chestnut restoration offers a unique case study because many restoration or intervention options have been attempted: doing nothing, planting non‐native chestnut species, planting hybrids, mutagenesis (exposing seeds to high levels of radiation to induce random mutations), backcross breeding, and now genetic engineering. Any of these techniques may be advantageous independently or in combinations, depending on the specific goals of land managers or restoration practitioners, but genetic engineering offers a unique opportunity to enhance blight tolerance while minimizing other changes to the genome. [ABSTRACT FROM AUTHOR]

Chestnut blight, Cryphonectria parasitica (Murrill) Barr, was reported in Portugal in 1989. The disease rapidly spread within the chestnut region of the Trás-os-Montes (the Northeast of Portugal). Eradication and mechanical/burning exclusion failed to control the disease and natural hypovirulence is still absent or of very low incidence. The introduction of human-assisted therapeutic hypovirulence is therefore required to control the disease. We presente here the efficacy of field applications (short and long term) of a locally characterized hypovirulent strain (CHV1-RBB111), by the punch method in two chestnut orchards (Valpaços and Vinhais). The Valpaços disease incidence was 68% (65 out of 96 trees) and in Vinhais it was 46% (78 out of 169 trees). In both locations, diversity of vegetative incompatibility (vc type) was low and largely dominated (> 85%) by a single vc type (EU11). In Valpaços, all cankers of the orchard were treated (63 cankers) and in Vinhais 31 trees (cankers) were treated. One year after application, field efficacy was very high with 93.8% of healed cankers in Valpaços and 90.3% in Vinhais, these values increased to 100% and 93.5% (respectively) after four years. The persistence of the introduced CHV1-RBB 111 strain was high and present in almost all treated cankers and it was dominant both inwards and on the application site revealing an endophytic behavior. The non-C. parasitica microfungi were abundant with 14 different species. Biscogniauxia mediterranea (De Not.) Kuntze was the most abundant in both orchards. Treatment with the introduced hypovirulent strain (CHV1-RBB111) was found to be effective in controlling chestnut blight with regeneration of cortical tissues and recovery of treated trees. [ABSTRACT FROM AUTHOR]

Cryphonectria parasitica is an invasive fungal pathogen that causes blight disease on chestnut trees. Its destructive effect can be controlled with naturally occurring mycovirus Cryphonectria hypovirus 1 (CHV1). To date, the spread of C. parasitica and CHV1 in Europe is fairly well documented, but there are still several unexplored regions. Thus, we sampled blight cankers from four sweet chestnut populations in Bay of Kotor and Lake Skadar regions in Montenegro. We determined vegetative compatibility (vc) type and mating-type diversity using molecular vic and MAT1 genotyping, as well as confirming the presence of CHV1 by RT-PCR. We identified 11 vc types, with EU-12 being the dominant one represented by 58.2% of all fungal isolates. The Shannon diversity index ranged from 0.93 to 1.47. Both mating types of C. parasitica were found in all four populations. The prevalence of CHV1 ranged from 15% to 40%. All CHV1 isolates belonged to Italian subtype I of CHV1 and were closely related, with relatively recent common ancestors. Our results indicate a longer presence of C. parasitica and CHV1 in Montenegro than previously thought. Natural biocontrol with CHV1 seems to be well established. However, it has the potential for deterioration; thus, close monitoring is required.

The diversity of vegetative compatibility (vc) types of chestnut blight fungus, Cryphonectria parasitica, has been reported to be very low in all of Turkey, except in the eastern Black Sea Region. However, risk for increase in vc type diversity has been pointed out regarding the emergence of new vc types as recombinants during sexual reproduction. In this study, the potential increase in the diversity of vc types in Turkish populations of C. parasitica was demonstrated by experimental mating crosses between the two dominant vc types, EU‐1 and EU‐12. Two local isolates of C. parasitica, K5‐5 (EU‐1) and A‐2490 (EU‐12), were used setting crosses on autoclaved chestnut wood. Among the offspring from the crosses, two parental (EU‐1 and EU‐12) and 14 non‐parental vc types (EU‐2, EU‐3, EU‐4, EU‐7, EU‐8, EU‐11, EU‐14, EU‐17, EU‐22, EU‐25, EU‐26, EU‐28, EU‐29 and EU‐30) were detected by a routine test based on co‐culturing. Sixteen vc genotypes were confirmed by a PCR assay using specific primers targeting the alleles of six vic loci in C. parasitica. In field studies, no evidence of new vc types as recombinants was found in western Turkey whereas 14 different vc types were detected among single ascospore isolates isolated from field‐collected perithecia, which were sampled from the eastern Black Sea region. The results of this study indicate that the potential increase in vc type diversity in Turkish population of C. parasitica is high under the conditions where C. parasitica reproduces sexually. [ABSTRACT FROM AUTHOR]

Forest tree species are increasingly subject to severe mortalities from exotic pests, pathogens, and invasive organisms, accelerated by climate change. Such forest health issues are threatening multiple species and ecosystem sustainability globally. One of the most extreme examples of forest ecosystem disruption is the extirpation of the American chestnut (Castanea dentata) caused by the introduction of chestnut blight and root rot pathogens from Asia. Asian species of chestnut are being employed as donors of disease resistance genes to restore native chestnut species in North America and Europe. To aid in the restoration of threatened chestnut species, we present the assembly of a reference genome for Chinese chestnut (C. mollissima) “Vanuxem,” one of the donors of disease resistance for American chestnut restoration. From the de novo assembly of the complete genome (725.2 Mb in 14,110 contigs), over half of the sequences have been anchored to the 12 genetic linkage groups. The anchoring is validated by genetic maps and in situ hybridization to chromosomes. We demonstrate the value of the genome as a platform for research and species restoration, including signatures of selection differentiating American chestnut from Chinese chestnut to identify important candidate genes for disease resistance, comparisons of genome organization with other woody species, and a genome-wide examination of progress in backcross breeding for blight resistance. This reference assembly should prove of great value in the understanding, improvement, and restoration of chestnut species. [ABSTRACT FROM AUTHOR]

This study combined phytosanitary surveys, laboratory analyses, and mathematical modeling to show how hail-induced wounds can foster the infections of the blight pathogen Cryphonectria parasitica, locally associated with extensive dieback of chestnut (Castanea sativa). Orchards and coppices located within and outside the assessed dieback area in a single location in the North West of Italy were inspected to appraise the abundance of hail-induced wounds and C. parasitica infections. The incidence of C. parasitica was significantly higher within the dieback area compared with outside (92% versus 60%; P < 0.05). Hail-induced wounds were observed on small branches and shoots of all trees sampled within the dieback area, whereas they were less abundant outside (20% of trees), suggesting either that the dieback was directly associated with the injuries caused by the hailstorms or that those injuries may have facilitated infections of C. parasitica. Isolations conducted on 359 branches and shoots showed that hail-induced wounds served as infection courts for C. parasitica and that infections depended on the size rather than on the number of hail wounds. We fitted a logistic model showing that hail-induced wounds whose perimeter was larger than 66 mm were at particular risk of C. parasitica infection. A newly designed geometrical-based model is proposed to relate hailstones size, hail wound perimeter, and the risk of infection. We established that hail-induced wounds are entry points for virulent and hypovirulent strains of C. parasitica, since 6.5% of isolates were infected by Cryphonectria hypovirus-1. [ABSTRACT FROM AUTHOR]

Ambrosia beetles exhibit broad host ranges but a narrow preference based on the condition of the host. Tissues infected by pathogens or containing ethanol can facilitate attacks by ambrosia beetles, although it still remains unclear how these factors interact.The present study aimed to examine how (i) chestnut logs infected with the fungal pathogen Cryphonectria parasitica and treated with ethanol (i.e. baited with ethanol lure, soaked in ethanol or untreated) and (ii) hornbeam logs soaked in different ethanol concentrations (3–12.5%) affect host selection and colonization success of ambrosia beetles.Ethanol‐soaked logs were more attractive to Anisandrus dispar than ethanol‐baited logs or untreated logs, although this difference was more evident in uninfected than infected logs. Increasing ethanol concentration in host tissues was differentially attractive to Xyleborinus saxesenii and Xylosandrus germanus. A nonlinear relationship was also documented between ethanol concentration and emergence of X. germanus adults.Overall, the results obtained suggest that the presence of C. parasitica in chestnut logs can affect host selection in ambrosia beetles. In addition, the ethanol concentration in tree tissues affects host selection and colonization success, although the effect varies depending on the beetle species. This contrasting response could be a niche‐partitioning mechanism based on ethanol within host tissues. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cryphonectria parasitica (Murrill) M.E. Barr (Sordariomycetes, Valsaceae) is the causal agent of chestnut blight. This disease is a major concern for chestnut cultivation in Europe. The fungus colonizes vascular tissues and evolves generating cankers causing severe dieback and the death of the tree. Excised and debarked well-lignified shoots of 28 C. sativa genotypes (assay A) and of 10 progenies (assay B) were inoculated with C. parasitica strain FMT3bc2 (vcg: EU2). Fungal growth was measured along the longitudinal axis on the 3rd and 6th days after inoculation. Results indicated the inoculation methodology works and the results were clear after 6 days. Differences in susceptibility to chestnut blight among C. sativa trees of Montseny have been detected both at the individual genotype level and at the progeny level. Nineteen genotypes and four progenies showed a susceptibility to Blight not significantly different from C. mollissima. The methodology was easy to apply in extensive/preliminary selection screenings to assess the susceptibility of C. sativa materials to the Blight.

Laccase activity reduction in the chestnut blight fungus Cryphonectria parasitica usually accompanies the hypovirulence caused by the infection of fungus with Cryphonectria hypovirus 1 (CHV1). However, the different methods utilized for assessing this phenomenon has produced varied and often conflicting results. Furthermore, the majority of experimental setups included only one prototypic system, further confounding the results. Considering the diversity of fungal isolates, viral strains, and variability of their effects on the phytopathogenic process observed in nature, our goal was to ascertain if laccase activity variability is affected by (1) different C. parasitica isolates infected with several CHV1 strains, and (2) growth conditions. We have demonstrated that some CHV1 strains, contrary to previous assumptions, increase the activity of C. parasitica laccases. The specific fungal isolates used in the experiments and culture conditions also affected the results. Furthermore, we showed that two commonly used laccase substrates, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) and 2,4-dimethoxyphenol, cannot be used interchangeably in C. parasitica laccase activity measurements. Our results illustrate the importance of conducting this type of study in experimental systems and culture conditions that resemble natural conditions as much as possible to be able to infer the most relevant conclusions applicable to natural populations.

This paper showcases the development of plant virology in Croatia at the University of Zagreb, Faculty of Science, from its beginning in the 1950s until today, more than 70 years later. The main achievements of the previous and current group members are highlighted according to various research topics and fields. Expectedly, some of those accomplishments remained within the field of plant virology, but others make part of a much-extended research spectrum exploring subviral pathogens, prokaryotic plant pathogens, fungi and their viruses, as well as their interactions within ecosystems. Thus, the legacy of plant virology in Croatia continues to contribute to the state of the art of microbiology far beyond virology. Research problems pertinent for directing the future research endeavors are also proposed in this review.

Chestnut blight, a disease that has spread rampantly among American (Castanea dentata (Marsh.) Borkh.) and European chestnut (C. sativa Mill.) trees, results from infection by the fungal pathogen Cryphonectria parasitica (Murrill) M.E. Barr (C. parasitica). This fungus was introduced in the early 1900s and has almost functionally eliminated chestnut trees from the North American landscape. In 2017, we collected chestnut blight samples from two sites (Site B, (Fulton Co., IN) and Site C (Marshall Co., IN)). At the Fulton County planting, Site B, cankers had formed, healed over, and the trees were healthy. However, at the second site in Marshall County, (Site C), cankers continued to propagate until all of the chestnut trees had died back to the ground. Research evidence worldwide has indicated that these visual clues likely result from the presence of a hypovirus. Upon closer inspection and the subsequent isolation and reproduction of spores, no hypovirus has been identified from either site. Here, we present a curious coincidence where one site has completely succumbed to the disease, while the other has been able to spring back to health.

The objective of this study was to determine the factors causing the deterioration of the stand structures of chestnut forests in Kütahya-Simav and to reveal the relation between these factors and chestnut diseases. Chestnut forests in Kütahya Simav were chosen as research area in this study. The damages given to forests in the result of the combating against chestnut blight, Cryphonectria parasitica, and harvest of chestnut fruit were investigated in terms of silvicultural treatments. Three different sample plots were chosen from the research area. In each sample plot, the diameters, diseases and grafting status (exist or not) of the trees were determined. Stand structures of these forests were also investigated. As a result of the study, the disease state of the chestnut individuals in the sample areas was determined. It was found that on average 11% were completely dry, 6% were partially dry, and 46% were cancerous but capable of survival. Also, effectiveness in the combating against chestnut blight of silvicultural treatments was investigated. In the result of unconscious interventions made by the local people (grafting, harvesting, etc.) have observed that stand structure of chestnut forests are corrupted and disease increase.

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 İzmir and Kütahya 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 İzmir and Kütahya 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cryphonectriaceae species cause serious canker diseases on chestnut, oak and eucalypt trees. Recently, canker symptoms with typical orange fruiting bodies were observed on Chinese chestnut and oak trees in Hebei, Hubei, Shaanxi and Shandong Provinces in China. In the present study, isolates of these fungi were identified based on phylogenetic and morphological evidence, and their pathogenicities were tested on detached chestnut (Castanea mollissima) branches. DNA sequence comparisons of the internal transcribed spacer (ITS) regions and two regions of β‐tubulin (TUB1/TUB2) indicate that these isolates represent five species in the Cryphonectriaceae, viz. Cryphonectria japonica, Cryphonectria parasitica, Aurantiosacculus castaneae sp. nov., Cryphonectria neoparasitica sp. nov. and Endothia chinensis sp. nov. The sexual morph of Aurantiosacculus is discovered for the first time and can be distinguished from the other genera in Cryphonectriaceae by dark brown ascospores and tubiform appendages at both ends. Cryphonectria neoparasitica sp. nov. is different from the other Cryphonectria species by its aseptate ascospores. Endothia chinensis sp. nov. is the sole species in Endothia infecting the host genus Castanea. Additionally, it is much smaller than E. gyrosa and narrower than E. singularis in ascospores. The inoculation results showed that these five Cryphonectriaceae fungi isolated from chestnut or oak are all pathogenic to tested chestnut branches. Cryphonectria parasitica appears to be the most aggressive fungus, followed by C. neoparasitica sp. nov., C. japonica, E. chinensis sp. nov. and A. castaneae sp. nov. [ABSTRACT FROM AUTHOR]

Chestnut blight, caused by Cryphonectria parasitica, was identified in Devon, UK, in December 2016. Intensive surveys detected the disease at further sites in Devon (seven), Berkshire (one), Dorset (one), Derbyshire (four) and a cluster of eight sites in southeast London. Over 570 survey samples were tested, and 227 were positive for C. parasitica by isolation and real‐time PCR. A total of 227 isolates were tested for mating type, and 197 screened for vegetative compatibility group (VCG) and compared with VCGs known from mainland Europe. The same isolates were also screened for the presence of Cryphonectria hypovirus 1 (CHV‐1). Eleven VCGs were identified within the UK population. Five corresponded to already known European VCGs but six were unique. The European VCGs mainly came from the Devon, Dorset, Berkshire and Derbyshire disease outbreaks, whilst unique VCGs were almost exclusively from the southeast London cluster. Both mating types were detected, but only one mating type was present at each site, with the exception of a single Devon site. Perithecia of C. parasitica were never observed at any site. CHV‐1 was found in seven isolates from three different locations and was always subtype‐I, which has limited hypovirulence. Therefore, although CHV‐1 is associated with C. parasitica at some outbreaks, it probably has limited impact on virulence. The diversity of VCGs and their distribution at outbreak sites, together with findings of CHV‐1, suggests C. parasitica has been introduced to the UK multiple times over at least two decades through international plant trade. [ABSTRACT FROM AUTHOR]