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Cryphonectria hypovirus 1 (CHV1) is successful in controlling Cryphonectria parasitica, the causal agent of chestnut blight, but little is known regarding its transmission to other fungi, for example the European Cryphonectria radicalis. In this study, CHV1 was transmitted (circa 200,000–800,000 copies/microliter) to seven C. radicalis isolates from infected C. parasitica. Reverse transmission to virus-free C. parasitica (European 74 testers collection) was achieved, although it was less successful (250–55,000 copies/µL) and was dependent on the vegetative compatibility (VC) group. In C. radicalis, the virus infection led to colony colour change from pink to white and smaller colonies, dependent on the virus concentration. The virus was concentrated in the colony edges, and vertically transmitted to 77% of conidia. However, several in vitro experiments demonstrated that C. radicalis was always outcompeted by the blight fungus, only suppressing the pathogen between its 25–50% inoculum level. It presented good secondary capture only when acting as a pioneer. Two types of in planta assays (individual and challenge inoculations) were undertaken. Cryphonectria radicalis behaved as a saprotroph, while chestnut blight fungus behaved as an aggressive pathogen, and lesions after treatment with C. radicalis were no smaller in general, only when using cut branches. Overall, the results showed that infected C. radicalis was unable to control cankers. [ABSTRACT FROM AUTHOR]

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]

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]

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]

Various fungal species together with Cryphonectria parasitica (Murr.) Barr. have been isolated from chestnut tissues with blight symptoms. Microfungi remain in cankers during tissue healing, which occurs by transmitting hypovirulence by Cryphonectria hypovirus 1 (CHV1) compatible strains. However, studies focused on the diversity and ecology of the non-C. parasitica taxa on chestnut bark are lacking. This work evaluated the composition and richness of microfungi species associated with healthy chestnut trees, those with cankers healed by natural hypovirulence and those treated with hypovirulent strains (artificially introduced hypovirulence). Microfungi from diseased trees were isolated from six randomly selected points in the inner and external areas of the healed canker. In healthy trees, tissue samples were collected from 12 random locations on each tree's trunk. Fungal species were identified based on morphological characteristics and ITS region sequencing using the universal primers ITS1 and ITS4. Four hundred thirty-one fungal isolates were obtained from which 38 operational taxonomic units (OTUs) were identified. The fungal communities varied from tree to tree and did not display similar patterns. The endophyte Biscogniauxia mediterranea and epiphyte Cytospora eucalypticola fungi were detected in all study locations and tree health conditions. Notably, C. parasitica (virulent and hypovirulent strains) was dominant in the inner area of healed cankers, accounting for 64.3% of the isolates, and the saprobe fungi Penicillium glabrum was dominant among non-C. parasitica microfungi species. Dissimilarity analyses showed low similarity between the microfungi communities found in the inner and external areas of the healed cankers. The study reveals the long-life span of C. parasitica in healed cankers and the therapeutic effect of natural and introduced hypovirulence. [ABSTRACT FROM AUTHOR]

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. [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]

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. [ABSTRACT FROM AUTHOR]

We previously identified a protein spot that showed down‐regulation in the presence of Cryphonectria hypovirus 1 (CHV1) and tannic acid supplementation as a Hsp90 co‐chaperone p23 gene (CpCop23). The CpCop23‐null mutant strain showed retarded growth with less aerial mycelia and intense pigmentation. Conidia of the CpCop23‐null mutant were significantly decreased and their viability was dramatically diminished. The CpCop23‐null mutant showed hypersensitivity to Hsp90 inhibitors. However, no differences in responsiveness were observed after exposure to other stressors such as temperature, reactive oxygen species, and high osmosis, the exception being cell wall‐disturbing agents. A severe reduction in virulence was observed in the CpCop23‐null mutant. Interestingly, viral transfer to the CpCop23‐null mutant from CHV1‐infected strain via anastomosis was more inefficient than a comparable transfer with the wild type as a result of decreased hyphal branching of the CpCop23‐null mutant around the peripheral region, which resulted in less fusion of the hyphae. The CHV1‐infected CpCop23‐null mutant exhibited recovered mycelial growth with less pigmentation and sporulation. The CHV1‐transfected CpCop23‐null mutant demonstrated almost no virulence, that is, even less than that of the CHV1‐infected wild type (UEP1), a further indication that reduced virulence of the mutant is not attributable exclusively to the retarded growth but rather is a function of the CpCop23 gene. Thus, this study indicates that CpCop23 plays a role in ensuring appropriate mycelial growth and development, spore viability, responses to antifungal drugs, and fungal virulence. Moreover, the CpCop23 gene acts as a host factor that affects CHV1‐infected fungal growth and maintains viral symptom development. [ABSTRACT FROM AUTHOR]

Chestnut blight, caused by Cryphonectria parasitica, is a severe disease that may be biologically controlled by the use of hypovirulent strains, but the diversity of the pathogen population affects biocontrol feasibility. Villuercas–Ibores–Jara, the Jerte Valley and La Vera are the main chestnut production districts in Cáceres (central-western Spain). The purpose of this study is to determine the Vegetative Compatibility Groups (VCGs) and mating types in these districts and to identify hypovirulent isolates to be used in biological control. The VCGs were determined by the merging/barrage response; PCR tests were used for the mating type determination and CHV-1 hypovirus detection. In total, 40 sites were surveyed and 269 isolates were obtained, most of them (227) from the Villuercas–Ibores–Jara district, where EU11 was the predominant VCG (88.1%) with EU1 (6.6%) and EU12 (4.4%) being also present. In the Jerte Valley and La Vera, EU1 (61.9%), EU11 (11.9%) and EU12 (11.9%) were the predominant VCGs. Both mating types were detected (48% MAT-1; 50% MAT-2) and in general, only one mating type was found in each site. The presence of Cryphonectria hypovirus 1 (CHV-1), subtype I, was identified in only one isolate (VCG EU11) from Villuercas–Ibores–Jara. The characteristics of the C. parasitica population in this district and the occurrence of CHV-1 hypovirus support the potential of successful biological control in Villuercas–Ibores–Jara using hypovirulent strains, while in the Jerte Valley and La Vera only preventive measures are recommended. [ABSTRACT FROM AUTHOR]

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]

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

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]

Chestnut blight is the major disease of chestnuts (Castanea spp.) cultivated worldwide for the production of edible nuts. The disease is caused by the pathogenic fungus Cryphonectria parasitica, which infects trees by means of airborne propagules penetrating through fresh wounds on stems and branches. The aims of this study were to (I) assess the temporal propagule deposition patterns of C. parasitica in the Alpine district of North Western Italy, (II) test and model the effects of seasonality and climate on the above patterns, and (III) investigate the spatial distribution of propagule deposition at the within-site scale. A two-year-long spore trapping experiment was conducted in three chestnut orchards. Approximately 1300 samples were collected and processed with a species-specific qPCR assay to quantitatively assess the propagule deposition of C. parasitica. Results showed that C. parasitica can release propagules all over the year, though with significant seasonal peaks in the spring and fall (p < 0.05). Large propagule loads were significantly correlated (p < 0.05) with an increasing number of rainy days of the week (days providing 1–10 mm/day of water). Models predicting periods at high risk of infection based on climate and seasonality were fitted and successfully validated (p < 0.05). [ABSTRACT FROM AUTHOR]

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

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. [ABSTRACT FROM AUTHOR]

The article discusses the distribution and use of sweet chestnut (Castanea sativa Mill.) in Europe, and there is a focus on the Czech Republic. In this country, chestnut is a tree that can grow on various types of soil, and the main factor limiting its growing are late frosts. Th ere have been two main reasons for the introduction of sweet chestnut to our lands in history: (1) the tree ornamentality in the gardens of new castles and monasteries, and (2) the nutrition of people. In last few years, however, there has been taken into consideration a new reason for growing the sweet chestnut, namely the production of high-quality honey. On the other hand, there are some factors that pose a threat to the chestnut stands. Th e most serious and widespread is ink disease caused by fungi of genus Phytophtora, followed by the causative agent of the bark necrosis of chestnut (Cryphonectria parasitica Murr. Barr.). [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Invasive microbial species constitute a major threat to biodiversity, agricultural production and human health. Invasions are often dominated by one or a small number of genotypes, yet the underlying factors driving invasions are poorly understood. The chestnut blight fungus Cryphonectria parasitica first decimated the North American chestnut, and a more recent outbreak threatens European chestnut stands. To unravel the chestnut blight invasion of southeastern Europe, we sequenced 230 genomes of predominantly European strains. Genotypes outside of the invasion zone showed high levels of diversity with evidence for frequent and ongoing recombination. The invasive lineage emerged from the highly diverse European genotype pool rather than a secondary introduction from Asia or North America. The expansion across southeastern Europe was mostly clonal and is dominated by a single mating type, suggesting a fitness advantage of asexual reproduction. Our findings show how an intermediary, highly diverse bridgehead population gave rise to an invasive, largely clonally expanding pathogen. [ABSTRACT FROM AUTHOR]

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

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]

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]

Chestnut is an important crop in Portugal, representing an important source of revenue for mountain regions in this country. However, it has been strongly affected by the chestnut blight, a severe disease with negative consequences to fruit production, since there is still no definitive solution to this problem. Silicon is considered a nutrient for agricultural crops by conferring resistance to plants under biotic stress conditions through the combination of a physical and chemical defense system to fight against pathological fungi which attack leaves, roots and stems. Based on these factors, this study evaluates the influence of SiK® on chestnut plants infected with Cryphonectria parasitica. Three concentrations were studied: 5 mM, 7.5 mM and 10 mM SiK®. Results showed that Si fertilization can reduce the disease severity and the mortality rate of chestnut plants. The supply of 7.5 and 10 mM SiK® on PDA petri plates resulted in a total capacity of suppression (100% PI) of C. parasitica mycelium growth as opposed to the control petri plates (PDA). These concentrations also increased the antioxidant enzyme activity, catalase (CAT) and superoxide dismutase (SOD) measured 60 days after the inoculation with the fungus. In addition, the SiK® application increased the total phenolic compounds and soluble proteins content, which occurred over time. Also, increasing concentrations of SiK® increased the tolerance of seedlings to C. parasitica. The present study indicates that Si fertilization may be used in the future as a control method against chestnut blight. [ABSTRACT FROM AUTHOR]

Background: Chestnut blight, caused by Cryphonectria parasitica, is controlled in many European countries by the naturally occurring mycovirus Cryphonectria hypovirus 1 (CHV‐1). During surveys of recently identified chestnut blight outbreak in England, CHV‐1 was detected in several individuals of the pathogen isolated from affected trees. We investigated two of these CHV‐1‐infected isolates (L‐6 and Db‐1) as potential biocontrol agents for deployment in the UK comparing their virulence against virus‐free (M1275) and hypovirulent (M784) European isolates by inoculating sweet chestnut seedlings. Results: Both the European CHV‐1 M784 hypovirulent isolate and UK L‐6 isolate formed significantly smaller lesions in sweet chestnut seedling bark than the other three isolates (Db‐1, and virulent isolates FTC121 and M1275). The highest virus concentration was detected in isolate M784, followed by L‐6, with the lowest concentration in isolate Db‐1. White colony colouration indicative of hypovirulence was common in colonies re‐isolated from smaller lesions, and the same isolates also tended to be slower growing in culture, have a higher virus concentration, and caused less epicormic growth and fewer stromata to be present in plants. L‐6 and Db‐1 virus sequences, respectively, matched the virus haplotype E‐5 detected previously in Switzerland and a mutation of the same subtype I haplotype. Conclusion: Isolate L‐6 could potentially act as biocontrol for chestnut blight outbreaks in the UK but further laboratory and field experiments are needed. © 2019 Crown copyright. Pest Management Science © 2019 Society of Chemical Industry [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]

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

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 presence of Cryphonectria parasitica (Murrill) M.E. Barr was studied in six natural and planted stands of sweet chestnut in Serbia. The fungus was detected on the sweet chestnut in five localities and on the sessile oak in one locality. In total, 77 isolates from the sweet chestnut and five isolates from the sessile oak were obtained. Based on the culture morphology, all the obtained isolates were proven to be free from Cryphonectria (Saccardo) Saccardo & D. Saccardo hypovirus. The isolates of C. parasitica from the sweet chestnut were compatible with three different vegetative compatibility types, EU-12, EU-2, and EU-1, while the isolates from the sessile oak belonged to EU-12. After inoculation in laboratory conditions, the isolate from the sweet chestnut and sessile oak caused the decline in 88 and 76% of the sweet chestnut plants, respectively. In the case of the sessile oak, both isolates caused the decline in 52% of the plants. In field conditions, both isolates were aggressive to sessile oak trees after previous bark wounds and they were statistically significantly different compared to the control trees. The isolate from the sweet chestnut caused significantly larger cankers compared to both the isolate from the sessile oak and the control. [ABSTRACT FROM AUTHOR]

The recovery of the European chestnut from chestnut blight in Europe is attributed to hypovirulence, a viral disease that reduces the virulence of its host – Cryphonectria parasitica. Hypovirulence has been used in biocontrol programmes throughout Europe with the aim of not only treating individual chestnut blight cankers but causing the natural spread and establishment of viruses in C. parasitica populations. Almost 40 years after the first appearance of chestnut blight and more than 20 years after the first biocontrol treatment implementation in Slovakia the establishment and natural spread of hypovirulence were studied. The morphological characteristics of 179 chestnut blight cankers and 161 C. parasitica strains were evaluated for the presence of hypovirus from three chestnut cultivation sites in Slovakia. Nineteen cankers and isolates were considered hypovirulent based on visual assessment of their morphological characteristics. The hypovirus presence by RNA extraction was confirmed in 6 cankers/strains. [ABSTRACT FROM AUTHOR]

The vacuolar H+-ATPases (V-ATPases) are conserved ATP-dependent proton pumps that acidify intracellular compartments in eukaryotic cells. The role of Cpvmal, a V-ATPase catalytic subunit A of Cryphonectria parasitica, was investigated by generating c/wmai-overexpressing and cpvmal-silenced strains. The mutant strains were evaluated for phenotypic characteristics, V-ATPase activity, response to elevated pH and Ca2+ in the medium, virulence on chestnut, and accumulation of hypovirus RNA in the cells. Compared with the wild-type strain, cpvmuf-overexpressing strains showed no significant difference in phenotype; however, cpvmal-silenced strains exhibited a phenotype of reduced growth rate, lower level of sporulation, and a marked decrease in V-ATPase activity and virulence. In addition, silencing of cpvmal increased sensitivity to elevated pH and Ca2+, implicating an important role for Cpvmal in pH adaptation and Ca2+ homeostasis. Furthermore, silencing of cpvmal resulted in significantly decreased accumulation of hypoviral RNA. Taken together, our results indicate that Cpvmal plays an important role in the regulation of phenotypic traits and virulence and the accumulation of hypovirus RNA in C. parasitica. [ABSTRACT FROM AUTHOR]

The purpose of the paper is to briefly present the work of the 3rd meeting of REUFIS, which took place at Bursa, in Turkey, between November 19 and 20, 2019. The keynote lectures on invasive species affecting the nut trees in the REUFIS region and the other presentations are summarized. Also, the discussions that took place both in the working groups and in the plenary, the visit to the forestry museum in Bursa and to the sweet chestnut forest attacked by Cryphonectria parasitica in the Yeniköy forest area are presented. It is worth mentioning that, also on this occasion, the readiness of FAO to support the collaboration between specialists from different countries, both regarding the research of invasive forest species and on the actual implementation of the research results for solving practical problems, was reaffirmed. As a conclusion, the meeting is considered to be particularly useful and a complete success, both due to the organizers and participants, but also due to the hospitality and generosity of the hosts. The readers are encouraged to access the REUFIS website to find out more about the mission, objectives and activities of the network, but also to contact the national representative of Romania at REUFIS or persons from REUFIS management committee if they are interested in developing collaborations with colleagues from other countries included in the network. [ABSTRACT FROM AUTHOR]

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[Display omitted] Some mycoviruses such as Cryphonectria hypovirus 1 (CHV-1) have a potential application for biocontrol of Cryphonectria parasitica , the causal agent of chestnut blight, but little is known about the factors that regulate the fungus-virus interaction, especially when the concentration of the virus is low as it is usual in the wider environment in England. In this study, four CHV-1 infected isolate types of C. parasitica were tested for parameters relating to hypovirulence by inoculating onto potato dextrose agar (PDA), apples, and sweet chestnut branch fragments. Isolate L-6 (CHV-1 subtype I, haplotype E-5), together with the positive control isolate M784 (same haplotype), presented the highest hypovirulence values. The lowest hypovirulence was observed with isolate M784 previously kept in the cold at 8 °C, followed by isolate Db-1 (same haplotype but naturally mutated). The differences in hypovirulence were much more pronounced when inoculations took place using mycelial plugs taken from the edge of plate colonies as opposed to the centre of a colony. The same strains were used to further evaluate the effects of the growth medium (PDA or plant material), optimum and cold temperatures, and mycelium age (centre or edge plugs) on the respective virus concentration. Virus RNA was extracted and amplified by RT-PCR and the concentration of the CHV-1 DNA band (394 bp) measured. Virus concentration decreased when its host was growing on PDA when compared to its host growing on plant material. Virus concentration was reduced further if held in the cold temperature for 7 days, and the virus concentration decreased even further after an additional incubation period at 25 °C. Virus isolate L-6, sub-cultured from the colony centre, was the only one that recovered to its original concentration following incubation for 7 days on sweet chestnut wood fragments. The concentration of the virus was more stable in the centre of the plates than that found at the edge of a plate colony. Together with the expected 394 bp CHV-1 amplicon, another 90 bp band was occasionally simultaneously amplified using the same RT-PCR procedure. This band was quantified, and sequenced, finding no homologs. It is first time this satellite amplicon has been described and its concentration related with hypovirulence attributes of the virus infected C. parasitica , such as reduced fungal growth, and positive correlation with donor and recipient CHV-1 band load in donor-recipient transmission experiments. We discuss our findings in terms of previous parameters related to hypovirulence. [ABSTRACT FROM AUTHOR]

Summary Chestnut blight caused by Cryphonectria parasitica has recently been reported from Guilan province, the only region with natural chestnut ( Castanea sativa) stands in Iran. During the past few years, chestnut stands in Iran have been seriously threatened by the fungus, incidence of the disease is increasing and vast canopies are reduced to sprouts. As yet, there is no report on the disease distribution and severity in this region. Six sites from three main growing regions of chestnut in Guilan province were selected for investigation. We report occurrence and evaluation of the damage of the disease caused by C. parasitica. To evaluate the scale of damage, the investigated trees were classified into six categories based on the degree of crown damage, the number of canker wounds and the presence of the fungus. Index of health condition was calculated for all sites. During this study, a total of 250 isolates of Cryphonectria species were obtained, of which 232 isolates were C. parasitica and 18 were Cryphonectria radicalis. Castanea parasitica was observed in all regions. Index of health condition was scored from 0 to 6 with 0 being disease free and six being the most severe infection. Index results in the investigated sites varied between 0.69-5.45 and 0.93-5.55 for years 2006 and 2007, respectively. The highest damage was found in Doran ( I [ABSTRACT FROM AUTHOR]

Summary The occurrence of chestnut blight ( Cryphonectria parasitica) on oaks in mixed chestnut-oak forests was studied in 2003-2008 in Slovakia. Infections on living Quercus trees were found at four of seven localities. The disease incidence on oaks ranged from 1.3% to 15.8%. The symptoms on infected oaks were similar to those on chestnut, but less conspicuous. Cankers of C. parasitica were found only on Quercus robur and Q. petraea. A total of 22 isolates of C. parasitica, all virulent, were isolated. Each site yielded only a single vc type (EU12 or EU13). Field inoculation experiments on chestnuts with seven strains of C. parasitica from oaks and an isolate from Castanea sativa showed no differences in virulence. On Quercus robur stems, the cankered area was significantly smaller than on C. sativa and the cankers developed very slowly. [ABSTRACT FROM AUTHOR]

Cryphonectria parasitica, which causes chestnut blight, is one of the most important pathogens of forest trees. In Europe, mycovirus-mediated biocontrol is the most efficient method to control the disease but can be impeded by the lack of information about the population structure of the fungus within a region. In particular, sexual reproduction and the new introduction of the pathogen can complicate biocontrol strategies. For this reason, this study aimed to determine the population structure of C. parasitica, which causes chestnut blight, in the northern Italian region of South Tyrol, using eleven multilocus microsatellite markers. Fifty-one haplotypes were found across South Tyrol, belonging to three divergent clusters. Recombinant genotypes demonstrated that sexual reproduction occurs across the different clusters. The most dominant genotypes in the region were also the most dominant in neighboring areas, such as Switzerland, northern Italy and France. All of the clusters from South Tyrol were related to the Italian genotype pool and are thought to have been introduced from northern Italian and other European populations due to naturally occurring gene flow or human-mediated introduction. At least three separate introduction events of C. parasitica might have happened in South Tyrol that could be separated by time. This study demonstrated a high genetic diversity of C. parasitica in South Tyrol and helped to shed light on the sexual reproduction and introduction events in the local populations. [ABSTRACT FROM AUTHOR]

Abstract: We surveyed native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China, and C. nitschkei, a sympatric species on chestnut trees in Japan, to learn more about the diversity of hypoviruses and other double-stranded (ds) RNA viruses. In a sample of 472 isolates of C. parasitica and 45 isolates of C. nitschkei from six prefectures in Japan, we found 27 containing one or more dsRNAs. Twelve isolates of C. parasitica and two isolates of C. nitschkei were infected with Cryphonectria hypovirus 1 (CHV-1); four of these 12 C. parasitica isolates also contained other dsRNAs that did not hybridize to CHV-1. In China, only one of 85 C. parasitica isolates was CHV-1-infected; no dsRNAs were detected in the other isolates from China. No other known hypoviruses were found in this study. However, we found two previously undescribed dsRNAs in Japan approximately 9kb in size that did not hybridize to each other or to any known dsRNAs from C. parasitica. We also found three additional groups of dsRNAs, one of which represents the genome of a new member of the virus family Chrysoviridae and was found only in C. nitschkei; the other two dsRNAs were found previously in isolates of C. parasitica from Japan or China. The most significant result of this survey is the discovery of novel dsRNAs that can be characterized in future research. [Copyright &y& Elsevier]

Castanea sativa, one of the most precious forest trees in Greece, is affected by the spread of chestnut blight all over the country. Identification and mapping of the vegetative compatibility (vc) types of the fungus Cryphonectria parasitica was carried out and the occurrence of hypovirulence was checked. In 11 chestnut populations distributed all over Greece, 627 isolates were obtained and their vc type was determined. Four vc types were identified by means of tester isolates: EU-12 was found to be the dominant vc type reaching a percentage of 88%, with EU-2 (6%), EU-10 (3%) and EU-1 (2%) following. EU-12 was found in all 11 populations investigated whereas EU-2 was isolated from five populations and EU-10 and EU-1 were found only in one population each. Natural hypovirulence was also found in five populations of 11 examined. Perithecia were not found in either population, a finding supported by the fact that all Greek isolates examined belonged to a single mating type, MAT-1. The low diversity in vc types, the absence of the perithecial stage and the occurrence of natural hypovirulence in some areas support the feasibility of successful application of biological control on the national level in Greece. [ABSTRACT FROM AUTHOR]

Three indigenous field strains of the chestnut blight fungus, Cryphonectria parasitica collected in 1996 from a test site in the Meshomasic State Forest, Connecticut, USA, were engineered to contain a chromosomally integrated full-length infectious cDNA copy of the severe, virulence-attenuating hypovirus CHV1-EP713. These transgenic hypovirulent strains were introduced into the test site after it had been clear-cut, as sprayed conidia in multiple applications over 4 years, beginning in 1997. Evidence was obtained for cytoplasmic transmission of cDNA-derived hypovirus RNA and for recovery of transgenic strains from canker tissue of the spray-treated trees. However, there was no indication of spread of transgenic strains or cDNA-derived viral RNA to the control plot or outside the treatment plot. A single transgenic C. parasitica isolate was identified from a total of 156 isolates recovered from 1330 insects representing five insect orders. Only 2.6% of the 1082 C. parasitica bark isolates recovered during the course of this study were vegetatively compatible with the untransformed input strains. Transgenic ascospore progeny were produced by 40% of the perithecia collected in 1999 and by only 7.3% of the perithecia collected in 2000. A concurrent field release study performed in the Monongahela National Forest in West Virginia in 1998 and 1999 with transgenic hypovirulent strains constructed with the same CHV1-EP713 cDNA-containing plasmid identified seasonal application dates and delivery methods that resulted in very high levels of transgenic ascospore production. Results of the combined field release studies are discussed in terms of requirements for improved formulations and delivery methods and the importance of balancing ecological fitness and virulence attenuation. [ABSTRACT FROM AUTHOR]

Summary Seasonal variation in the development of chestnut blight, caused by Cryphonectria parasitica , was investigated by inoculating in situ chestnut trees and in vitro excised chestnut segments, at either monthly or 3-monthly intervals throughout 30 months. Inoculations were made with conidia and mycelium of a virulent isolate and with mycelium of a hypovirulent isolate. Conidial inoculations of living sprouts or excised segments between May and July resulted in the greatest incidence of infection whereas inoculations in autumn and winter, in vitro as well as in situ , did not reveal any visible disease. However, from these symptomless inoculated stems, C. parasitica was isolated 3 months after inoculation. Inoculations with the mycelium of the virulent isolate always resulted in lesions, except in January 1999, and the greatest rate of lesion development occurred for inoculations made in the spring and summer. There was a significant seasonal effect on lesion development. Lesions caused by the hypovirulent isolate, smaller than those caused by the virulent isolate, followed a similar seasonal pattern. The same seasonal variations were observed for inoculations in vitro of excised segments. Relative water content (RWC) of chestnut bark significantly varied with bark sampling date. The rate of lesion development in sprouts significantly correlated with average minimum (ATn) and maximum (ATx) temperatures and the sum of rainfall during inoculation period, with the rate of lesion development measured in excised segments 10 days after inoculation (R10d) and with RWC measured on the day of inoculation. In multiple regression models, variables ATx and R10d best explained variation in lesion development. [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. [ABSTRACT FROM AUTHOR]

The most important disease of European chestnut (Castanea sativa Mill.) is chestnut blight caused by the fungus Cryphonectria parasitica (Murrill) Barr which induces yield reduction in Europe and North America. This study aimed to investigate the impacts of C. parasitica infection on the physiological and biochemical characteristics of European chestnut at two different growth stages, 3 and 6 weeks after the infection. The amount of photosynthetic pigments (chlorophyll-a, chlorophyll-b, and carotenoids), the relative chlorophyll content, and the photochemical efficiency of the photosystem II (PSII) were measured in the leaves above and below the virulent and hypovirulent C. parasitica infections. The highest values were measured in the control leaves, the lowest values were in the leaves of the upper part of virulent necrosis. Antioxidant enzyme activities such as ascorbate peroxidase (APX), guaiacol peroxidase (POD), and superoxide dismutase (SOD), proline, and malondialdehyde concentrations were also investigated. In each of these measured values, the lowest level was measured in the control leaves, while the highest was in leaves infected with the virulent fungal strain. By measuring all of these stress indicator parameters the responses of chestnut to C. parasitica infection can be monitored and determined. The results of this study showed that the virulent strain caused more pronounced defense responses of chestnut's defense system. The measured parameter above the infection was more exposed to the blight fungus disease relative to the leaves below the infection. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Castanea sativa is one of the main multipurpose tree species valued for its timber and nuts. This species is susceptible to two major diseases, ink disease and chestnut blight, caused by Phytophthora spp. and Cryphonectria parasitica, respectively. The loss-of-function mutations of genes required for the onset of pathogenesis, referred to as plant susceptibility (S) genes, are one mechanism of plant resistance against pathogens. On the basis of sequence homology, functional domain identification, and phylogenetic analyses, we report for the first time on the identification of S-genes (mlo1, dmr6, dnd1, and pmr4) in the Castanea genus. The expression dynamics of S-genes were assessed in C. sativa and C. crenata plants inoculated with P. cinnamomi and C. parasitica. Our results highlighted the upregulation of pmr4 and dmr6 in response to pathogen infection. Pmr4 was strongly expressed at early infection phases of both pathogens in C. sativa, whereas in C. crenata, no significant upregulation was observed. The infection of P. cinnamomi led to a higher increase in the transcript level of dmr6 in C. sativa compared to C. crenata-infected samples. For a better understanding of plant responses, the transcript levels of defense genes gluB and chi3 were also analyzed. [ABSTRACT FROM AUTHOR]