Search Results (46)

Biotechnological tools have emerged as key alternatives for the protection, improvement, and sustainable use of forest species. This paper analyzes the main biotechnological strategies applied to the European chestnut, a species of significant ecological, economic, and cultural importance in many temperate regions. However, in recent decades, it has been seriously threatened by various factors, including devastating diseases such as chestnut blight and ink disease, as well as the impacts of climate change. First, classical and assisted breeding techniques are discussed, including controlled hybridization and the use of molecular markers to accelerate the selection of genotypes of interest. In the field of molecular biotechnology, studies related to the identification of key genes, the development of genetic markers (e.g., SSRs and SNPs), and the omics characterization of chestnut are reviewed. The use of micropropagation techniques for the clonal multiplication of elite individuals is also included. Furthermore, advances in genetic modifications are explored, highlighting the introduction of resistance genes through transgenic and cisgenic approaches, as well as emerging technologies such as CRISPR/Cas9. In the future, the integration of classical breeding with advanced genomics will enable the precise selection and accelerated development of European chestnut varieties, combining traditional trait improvement with genomic tools such as marker-assisted selection, genomic prediction, and gene editing to enhance disease resistance and climate resilience. Full article

Chestnut blight caused by Cryphonectria parasitica poses a major threat to chestnut plantations worldwide. Rapid, field-deployable diagnostic tools are essential for effective disease surveillance and management. Here, we developed and validated an enzyme-mediated duplex exponential amplification (EmDEA) assay for the specific and sensitive detection of C. parasitica. The assay successfully distinguished C. parasitica from non-target fungal species with a detection limit of 10 pg of genomic DNA per reaction. Application to naturally infected bark samples yielded results consistent with those of conventional qPCR. The complete workflow, including crude DNA extraction and fluorescence-based detection under isothermal conditions, was completed within 35 min. Our findings demonstrate that the EmDEA assay is a sensitive, robust, and field-adaptable tool that can be used for the early detection of chestnut blight, with significant potential for deployment in resource-limited environments. Full article

Hypovirus infection is known to reduce the pathogenicity of Cryphonectria parasitica, the causative agent of chestnut blight. Isoforms derived from a viral protein p48 have been discovered in host mitochondria and vesicles, which may contribute to virulence attenuation, as reported in earlier work using two-dimensional electrophoresis (2-DE). In this study, a total of 1739 fungal proteins were identified in fungal vesicles through Tandem Mass Tag (TMT)-based quantitative proteomics. The infection of CHV1-EP713 was associated with 75 up-regulated and 201 down-regulated proteins, predominantly involved in vesicular transport process and related cellular functions, including protein folding, membrane fusion, retrograde transport, autophagy, and ER stress responses. The down-regulation of calnexin, COPI, ArfGAP, importin-β, and Atg8 is consistent with impairments in protein folding, retrograde transport, and autophagy. Meanwhile, the up-regulation of clathrin, dynamin, Vps10p, HSP70, and t-SNAREs indicated enhanced trafficking to vacuoles and increased stress response activity. Overall, our findings indicate that hypoviral infection is associated with extensive alterations in the vesicular transport system of C. parasitica, likely mediated through changes in the abundance of multiple key protein regulators. These alterations may underlie attenuation of virulence by impacting crucial cellular processes. Full article

Chestnut blight, caused by Cryphonectria parasitica (Murrill) M.E. Bar, is a destructive fungal disease threatening chestnut cultivation and production. In response to the limitations of chemical control, biological control using antagonistic microbes has gained increasing attention. A rhizosphere-derived bacterium, strain D39, was isolated from healthy chestnut trees and identified as Bacillus amyloliquefaciens based on morphological characteristics and the phylogenetic analysis of 16S rRNA and gyrA genes. The antifungal activity of strain D39 against C. parasitica was evaluated using dual-culture and double-layer Oxford cup assays. The strain exhibited broad-spectrum and stable antagonistic effects and harbored five key genes associated with antimicrobial compound biosynthesis (srfAA, ituC, fenD, bmyB, and bacA), as confirmed by PCR. A 145 kDa extracellular protein with strong antifungal activity was extracted and purified by ammonium sulfate precipitation, DEAE ion-exchange chromatography, and Sephadex G-75 gel filtration. LC-MS analysis identified the protein as a serine peptidase belonging to the S8 family, and its structure was predicted using multiple bioinformatic tools. In pot experiments, treatment with the strain D39 significantly reduced disease severity, achieving control efficiencies of 66.07% and 70.89% at 10 and 20 days post-inoculation, respectively. These results demonstrate that B. amyloliquefaciens D39 has strong potential as a biocontrol agent against chestnut blight, offering an effective and environmentally friendly alternative for disease management. Full article

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. Full article

Since its introduction into Europe in the first half of the 20th century, Cryphonectria parasitica has been gradually spreading across the natural range of the sweet chestnut (Castanea sativa Mill.), infecting the trees and causing lethal bark cankers. Serendipitously, a hyperparasitic Cryphonectria hypovirus 1 (CHV1), which attenuates C. parasitica virulence in combination with more tolerant European chestnut species, was able to ward off the worst effect of the disease. In North America, unfortunately, the native Castanea dentata is now functionally extinct since it occurs only as root sprouts in eastern deciduous forests where it was once dominant. In our work, we investigated changes in C. parasitica populations over time and the regional variability in chestnut populations’ tolerance toward the blight disease. While vegetative compatibility (vc) type diversity and prevalence of hypovirulence remained similar as in previous studies, in the Buje population, unlike in previous studies, we were unable to find any hypovirulent fungal strains. The most common vegetative compatibility types (vc types) were EU-1, EU-2 and EU-12. However, several rare EU-types were found, including one previously unreported: EU-46. By inoculating several C. parasitica strains on tree stems from several chestnut populations, we observed that the induced lesion size was affected by the type of inoculum (CHV1-free or CHV1-infected), genotype-related individual chestnut stem and chestnut stem population of origin-related variability. The largest lesions were induced by CHV1-free fungal isolate DOB-G: 20.13 cm² (95% C.I. 18.10–22.15) and the smallest by CHV1-infected L14/EP713: 2.49 cm² (95% C.I. 1.59–3.39). Surprisingly, the size of the lesions induced by other CHV1-infected strains fell somewhere in between these extremes. The size of induced lesions was dependent on the population of origin as well and ranged from 11.60 cm² (95% C.I. 9.87–13.33) for stems from the Moslavačka gora population to 17.75 cm² (95% C.I. 15.63–19.87) for stems from Ozalj. Full article

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. Full article

The cross-hybridization of American chestnut (Castanea dentata (Marsh.) Borkh.) with Chinese chestnut (Castanea mollissima Bl.) is a promising strategy for restoring a blight-resistant strain of this keystone species to the Appalachian mountains. To assess the ecological impacts of hybridization on invertebrate communities, we conducted a study across chestnut plots with varying degrees of hybridization (75%, 94%, or 100% American chestnut). Our findings indicate American chestnut hybridization impacted invertebrate communities above- and belowground. Aboveground insect community composition, insect herbivory, gall infestation, and belowground invertebrate diversity were all altered. While some of these differences could be explained by different growth habits or environmental differences, stark differences in Asian chestnut gall wasp infestation (Dryocosmus kuriphilus Yasumatsu.) suggest a genetic component. These results suggest that chestnut hybridization, and particularly expanded restoration efforts using chestnut hybrids, could impact invertebrate communities above- and belowground in addition to pest dynamics. Understanding these effects is crucial for successful chestnut restoration and ecosystem management. Full article

Members of the fungal order Diaporthales are sac fungi that include plant pathogens (the notorious chestnut blight fungus), as well as saprobes and endophytes, and are capable of colonizing a wide variety of substrates in different ecosystems, habitats, and hosts worldwide. However, many Diaporthales species remain unidentified, and various inconsistencies within its taxonomic category remain to be resolved. Here, we aimed to identify and classify new species of Diaporthales by using combined morphological and molecular characterization and coupling this information to expand our current phylogenetic understanding of this order. Fungal samples were obtained from dead branches and diseasedleaves of Camellia (Theaceae) and Castanopsis (Fagaceae) in Fujian Province, China. Based on morphological characteristics and molecular phylogenetic analyses derived from the combined nucleotide sequences of loci of the internal transcribed spacer regions with the intervening 5.8S nrRNA gene (ITS), the 28S large subunit of nuclear ribosomal RNA gene (LSU), the translation elongation factor 1-α gene (tef1), the partial beta-tubulin gene (tub2), and partial RNA polymerase II second-largest subunit gene (rpb2), three new species of Diaporthales were identified and characterized. They are as follows: Chrysofolia camelliae sp. nov., Dendrostoma castanopsidis sp. nov., and Pseudoplagiostoma wuyishanense sp. nov. They are described and illustrated. This study extends our understanding of species diversity within the Diaporthales. Full article

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. Full article

Mites of the genus Thyreophagus (Acari: Acaridae) are distributed worldwide; they inhabit concealed habitats and include several beneficial and economically important species. However, species identification is difficult because many species are poorly described or delimited and their phoretic stages are unknown or uncorrelated. Furthermore, Thyreophagus is interesting because it includes entirely asexual (parthenogenetic) species. However, among the 34 described species of Thyreophagus, the asexual status is confirmed through laboratory rearing for only two species. Here, we provide detailed descriptions of five new species from North America (four) and Europe (one) based on adults and phoretic heteromorphic deutonymphs. Four of these species were asexual, while one was sexual. For most of these mites, the asexual status was confirmed and phoretic deutonymphs were obtained through rearing in the lab. We show that asexual mites retain seemingly functional copulatory and sperm storage systems, indicating that these lineages have relatively short evolutionary lifespans. One North American species, Thyreophagus ojibwe, was found in association with the native American chestnut Castanea dentata, suggesting a possibility that this mite can be used to control chestnut blight in North America. We also provide a diagnostic key to females, males, and heteromorphic deutonymphs of the Thyreophagus species in the world. Full article

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. Full article

Chestnuts are multipurpose trees that grow mainly in the Northern Hemisphere due to their aptitude for fruit and wood production. These trees are vastly significant for the economy and wildlife. The widespread distribution of these trees demonstrates their genetic adaptability to many environmental conditions. The main varieties of European chestnut (Castanea sativa Miller) cultivated in Portugal, their productive challenges and breeding and biotechnological efforts developed over the last decades are described. This paper highlights the efforts focused on the improvement of varieties and rootstocks through selection and hybridization of European chestnut with the Asian species Castanea crenata Siebold and Zuccarini and Castanea mollissima Blume, which are resistant to ink disease, which have been the foundation of the Portuguese chestnut breeding programs. Breeding and biotechnological efforts developed over the last decades, focused on ink disease and chestnut blight resistance, are described. The potentialities of this research to stimulate the competitiveness of bioeconomy-based knowledge and innovation in the productive chestnut sector is also discussed. Full article

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. Full article

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. Full article