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Some mycoviruses can be considered as effective biocontrol agents, mitigating the impact of phytopathogenic fungi and consequently reducing disease outbreaks while promoting plant health. Cryphonectria parasitica, the causal agent of chestnut blight and a highly destructive pathogen, experienced a notable decrease in its virulence with the identification of cryphonectria hypovirus 1 (CHV1), a naturally occurring biocontrol agent. In this study, two innovative diagnostic protocols designed for the accurate and efficient detection of CHV1 are introduced. The ORF A and ORF B regions of CHV1 are targeted by these techniques, which employ colorimetric loop-mediated isothermal amplification (LAMP) with 2 Colorimetric LAMP Master Mix and real-time quantitative PCR (qPCR) with SYBR Green chemistry, respectively. The LAMP assay presents a discernible color transition, changing from pink to yellow after a 35 min incubation period. Comparative analysis, when assessed against two established reverse transcription-PCR (RT-PCR) techniques, reveals a significant enhancement in sensitivity for both the LAMP approach, which offers a tenfold increase, and the qPCR method, which showcases a remarkable 100-fold sensitivity improvement. Throughout the comparison phase, it was evident that the RT-PCR, LAMP, and qPCR procedures displayed superior performance compared to the Bavendamm test, relying on phenol oxidase activity, effectively distinguishing hypovirulent strains. Consequently, this study introduces two pioneer diagnostic assays for highly sensitive CHV1 detection, representing a substantial advancement in the realm of CHV1 surveillance techniques. These methodologies hold significant promise for enhancing research endeavors in the domain of the biological control of 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.

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.

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.

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.

[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]

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.

Some mycoviruses can be considered as effective biocontrol agents, mitigating the impact of phytopathogenic fungi and consequently reducing disease outbreaks while promoting plant health. Cryphonectria parasitica , the causal agent of chestnut blight and a highly destructive pathogen, experienced a notable decrease in its virulence with the identification of cryphonectria hypovirus 1 (CHV1), a naturally occurring biocontrol agent. In this study, two innovative diagnostic protocols designed for the accurate and efficient detection of CHV1 are introduced. The ORF A and ORF B regions of CHV1 are targeted by these techniques, which employ colorimetric loop-mediated isothermal amplification (LAMP) with 2 Colorimetric LAMP Master Mix and real-time quantitative PCR (qPCR) with SYBR Green chemistry, respectively. The LAMP assay presents a discernible color transition, changing from pink to yellow after a 35 min incubation period. Comparative analysis, when assessed against two established reverse transcription-PCR (RT-PCR) techniques, reveals a significant enhancement in sensitivity for both the LAMP approach, which offers a tenfold increase, and the qPCR method, which showcases a remarkable 100-fold sensitivity improvement. Throughout the comparison phase, it was evident that the RT-PCR, LAMP, and qPCR procedures displayed superior performance compared to the Bavendamm test, relying on phenol oxidase activity, effectively distinguishing hypovirulent strains. Consequently, this study introduces two pioneer diagnostic assays for highly sensitive CHV1 detection, representing a substantial advancement in the realm of CHV1 surveillance techniques. These methodologies hold significant promise for enhancing research endeavors in the domain of the biological control of 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 × 10 3 , 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.

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.

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.

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., Competing Interests: The authors declare that they have no conflicts of interest.

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., (© 2019 Crown copyright. Pest Management Science © 2019 Society of Chemical Industry.)