Cryphonectria Parasitica Research Articles

Floristic changes following the chestnut blight may be delayed for decades.

A survey conducted in the 1920s, prior to the chestnut blight, indicated that chestnuts and oaks were codominant canopy species in White Oak Canyon, Shenandoah National Park, Virginia. A second survey in 1977 suggested that chestnuts were being replaced by tree species present before the blight, particularly oaks. In 2021, we resurveyed the 10 sites included in our 1977 survey and also recorded canopy and understory trees that grew above remnant chestnut sprouts. The canopy changed more substantially during the second interval (since 1977). Birch and maples were now more abundant. Hemlock declined, and oaks were less common in the canopy. In general, the trees considered as early to mid-successional have replaced oaks and hemlock. Chestnut sprouts have become much less common since 1977, presumably as repeated cycles of diebacks have weakened rootstocks. Those sites where chestnut sprouts have persisted until 2021 differed from neighboring sites without them. Chestnut sprouts were rare in sites with birch and hemlock; chestnut has persisted in locations with red oaks in the canopy and with few other understory competitors. This survey has been conducted over a longer time interval than previous studies that asked similar questions and our results suggest that changes to the forest composition following the loss of the American chestnut may be greater than previously recognized although the relative contribution of losing this codominant species is unclear.

Interactions between invasive pests and pathogens in a native chestnut forest

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

Early Thinning: A Promising Tool to Prevent Fistulina hepatica Heart Rot in Castanea sativa Coppice Stands

Castanea sativa is a high-value tree species whose stands have faced significant threats over the past century. The occurrence of one such threat, Chestnut Red Stain—a heartwood discoloration caused by the fungus Fistulina hepatica—has recently increased. This disease devalues the timber by up to 70% due to the rejection of stained wood by the industry. This study aimed to evaluate the effects of three management strategies on the occurrence of F. hepatica in chestnut coppice stands. Additionally, the co-occurrence of F. hepatica and Cryphonectria parasitica, both highly prevalent in the study area, was assessed. In 2013, three different silvicultural treatments were applied to five plots. Seven years later, the stand characteristics were measured, and the incidence of F. hepatica was evaluated using molecular tools. Data modeling revealed that the quadratic mean diameter before the interventions was related to the incidence of F. hepatica. Our results suggest that the thinning at earlier stand stages may be more critical than the type or intensity of the thinning in reducing the incidence of the fungus in the mid-term. This finding provides forest managers with new guidance to improve silvicultural treatments and prevent F. hepatica damage.

Applying palaeoecological analogues to contemporary challenges: community-level effects of canopy gaps caused by systematic decline of a prevalent tree species

Temperate woodland vegetation is initially determined by spatiotemporal and historical factors, mediated by complex biotic interactions. However, catastrophic events such as disease outbreaks (e.g., sweet chestnut blight, ash dieback), infestations of insect pests, and human-accelerated climate change can create canopy gaps due to systematic decline in, or loss of, tree species that was once an important part of the canopy. Resultant cascade effects have the potential to alter the composition of woodland ecosystems quickly and radically, but inherent lag times make primary research into these effects challenging. Here, we explore change in woodland vegetation at 10 sites in response to canopy opening using the Elm Decline, a rapid loss of Ulmus in woodlands across northwestern Europe ~ 5800 years ago that coexisted alongside other stressors such as increasing human activity, as a palaeoecological analogue. For arboreal taxa, community evenness significantly decreased, within-site turnover significantly increased, and richness remained unchanged. Changes in arboreal taxa were highly site-specific but there was a substantial decline in woody climbing taxa, especially Hedera (ivy), across the majority of sites. For shrub taxa, richness significantly increased but evenness and turnover remained consistent. Interestingly, however, there was a significant increase in abundance of shrubs at 70% of sites, including Calluna (heather), Ilex (holly) and Corylus (hazel), suggesting structural change. Surprisingly, there was no change in richness, evenness or turnover for herb taxa, possibly because change was highly variable spatially. However, there was a marked uptick in the disturbance indicator Plantago (plantain). Overall, these findings suggest that woodlands with sustained reduction in, or loss of, a tree species that once formed an important part of the canopy has profound, but often spatially idiosyncratic, impacts on vegetation diversity (richness), composition (evenness), stability (turnover), and on abundance of specific taxa, especially within the shrub layer. Use of this palaeoecological analogue, which was itself complicated by cooccurring changes in human activity, provides a valuable empirical insight into possible cascade effects of similar change in canopy opening in contemporary settings, including Ash Dieback.

Changes in VOCs from a chestnut blight fungus Cryphonectria parasitica by a hypovirus infection: mycoviral infection alters fungal smell for insect vectors

BackgroundThe chestnut blight fungus, Cryphonectria parasitica, and its Cryphonectria hypovirus 1 (CHV1) comprise a useful model system to study fungus–virus interactions. CHV1 infection results in various phenotypic changes in the fungal host, including hypovirulence and other associated symptoms. Many studies have investigated the effects of hypoviral infection and how this infection affects physiological and biochemical states: however, no studies have examined volatile organic compounds (VOCs).ResultsThis study characterized the global profile of VOCs released from C. parasitica, and by comparing CHV1-free with CHV1-infected isogenic strains, proved that CHV1 infection significantly affects the composition and levels of VOCs. We demonstrated that these VOC alterations were caused by viral regulation of the expression of fungal genes encoding enzymes responsible for the production of VOCs. The changed VOC profile resulting from CHV1 infection was more attractive to insects than the VOC profile from the virus-free strain, suggesting differences in the efficacy of fungal dissemination by the insect vector.ConclusionsWe characterized VOCs from C. parasitica and demonstrated that mycovirus (CHV1) infection affects fungal VOCs. We provided evidences that these alterations are caused by the modulation of the corresponding gene expression by CHV1 and resulted in changes in attractiveness to insects, suggesting the improved efficacy of hypovirulent C. parasitica for insect-borne dissemination.Graphical

Exploring the potential of chestnut (Castanea sativa Mill.): a comprehensive review and conceptual mapping

BackgroundCastanea sativa Mill. is important for ecosystems and societies. Its rich historical and cultural significance, remarkable ecological contributions, and diverse applications have inspired scientific research. This comprehensive review and conceptual mapping of chestnut research consolidates existing knowledge, identifies emerging trends, and highlights untapped potential to inform future investigations. First, the Scopus database was searched to retrieve all data-driven articles in English related to C. sativa published in English from 1951 to 2023. Second, the R language, Scopus Analytics, and VOSviewer were used to analyze the year of publication, authors, countries, affiliations, keywords, and citations. Finally, network analysis was performed to evaluate the hotspots and developmental trends of C. sativa. A total of 1889 research articles were recovered.ResultsThe articles showed an exponential progression, with a regression coefficient of 0.9435 (R2) and an annual growth rate of 6.28%. Italy (19.87%), Spain (15.56%), Portugal (13.39%), Turkey (6.92%), and Switzerland (5.17%) were the most prolific countries. C. sativa (798), sweet chestnut (54), cryphonectria parasitica (44), honey (38), phenolic compounds (35), chestnut blight (33) and antioxidant activity (31) are the keywords that occur the most frequently. The main research groups in the thematic map are "forest management," "Cryphonectria parasitica," "wood," "Spain" and "ethnobotany." Research on C. sativa has all the basic, motor, niche, and emerging or declining themes. Forest management, drought, Gnomoniopsis smithogilvyi, C. sativa shells, amino acids, honey, phenolic compounds, hydrolyzable tannins, antioxidant capacity, antioxidants, and extractives are trending topics.ConclusionsThis bibliometric analysis highlights the importance of C. sativa research, revealing its ecological contributions, cultural significance, and diverse applications. Future studies should focus on forest management, drought resistance, and bioactive properties to ensure sustainable utilization.

N6-methyladenosine RNA methyltransferase CpMTA1 mediates CpAphA mRNA stability through a YTHDF1-dependent m6A modification in the chestnut blight fungus.

In eukaryotic cells, N6-methyladenosine (m6A) is the most prevalent RNA epigenetic modification that plays crucial roles in multiple biological processes. Nevertheless, the functions and regulatory mechanisms of m6A in phytopathogenic fungi are poorly understood. Here, we showed that CpMTA1, an m6A methyltransferase in Cryphonectria parasitica, plays a crucial role in fungal phenotypic traits, virulence, and stress tolerance. Furthermore, the acid phosphatase gene CpAphA was implicated to be a target of CpMTA1 by integrated analysis of m6A-seq and RNA-seq, as in vivo RIP assay data confirmed that CpMTA1 directly interacts with CpAphA mRNA. Deletion of CpMTA1 drastically lowered the m6A level of CpAphA and reduced its mRNA expression. Moreover, we found that an m6A reader protein CpYTHDF1 recognizes CpAphA mRNA and increases its stability. Typically, the levels of CpAphA mRNA and protein exhibited a positive correlation with CpMTA1 and CpYTHDF1. Importantly, site-specific mutagenesis demonstrated that the m6A sites, A1306 and A1341, of CpAphA mRNA are important for fungal phenotypic traits and virulence in C. parasitica. Together, our findings demonstrate the essential role of the m6A methyltransferase CpMTA1 in C. parasitica, thereby advancing our understanding of fungal gene regulation through m6A modification.

New Detection Methods for Cryphonectria Hypovirus 1 (CHV1) through SYBR Green-Based Real-Time PCR and Loop-Mediated Isothermal Amplification (LAMP).

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.

Genome sequencing of Elaeocarpus spp. stem blight pathogen Pseudocryphonectria elaeocarpicola reveals potential adaptations to colonize woody bark

BackgroundElaeocarpus spp. stem blight, caused by Pseudocryphonectria elaeocarpicola, is a destructive disease, which will significantly reduce the productivity and longevity of Elaeocarpus spp. plants, especially in the Guangdong Province of China. However, few information is available for P. elaeocarpicola. To unravel the potential adaptation mechanism of stem adaptation, the whole genome of P. elaeocarpicola was sequenced by using the DNBSEQ and PacBio platforms.ResultsP. elaeocarpicola harbors 44.49 Mb genome with 10,894 predicted coding genes. Genome analysis revealed that the P. elaeocarpicola genome encodes a plethora of pathogenicity-related genes. Analysis of carbohydrate-active enzymes (CAZymes) revealed a rich variety of enzymes participated in plant cell wall degradation, which could effectively degrade cellulose, hemicellulose and xyloglucans in the plant cell wall and promote the invasion of the host plant. There are 213 CAZyme families found in P. elaeocarpicola, among which glycoside hydrolase (GH) family has the largest number, far exceeding other tested fungi by 53%. Besides, P. elaeocarpicola has twice as many genes encoding chitin and cellulose degradation as Cryphonectria parasitica, which belong to the same family. The predicted typical secreted proteins of P. elaeocarpicola are numerous and functional, including many known virulence effector factors, indicating that P. elaeocarpicola has great potential to secrete virulence effectors to promote pathogenicity on host plants. AntiSMASH revealed that the genome encoded 61 secondary metabolic gene clusters including 86 secondary metabolic core genes which was much higher than C. parasitica (49). Among them, two gene cluster of P. elaeocarpicola, cluster12 and cluster52 showed 100% similarity with the mycotoxins synthesis clusters from Aspergillus steynii and Alternaria alternata, respectively. In addition, we annotated cytochrome P450 related enzymes, transporters, and transcription factors in P. elaeocarpicola, which are important virulence determinants of pathogenic fungi.ConclusionsTaken together, our study represents the first genome assembly for P. elaeocarpicola and reveals the key virulence factors in the pathogenic process of P. elaeocarpicola, which will promote our understanding of its pathogenic mechanism. The acquired knowledge lays a foundation for further exploration of molecular interactions with the host and provide target for management strategies in future research.

A genome-guided strategy for climate resilience in American chestnut restoration populations

American chestnut (Castanea dentata) is a deciduous tree species of eastern North America that was decimated by the introduction of the chestnut blight fungus (Cryphonectria parasitica) in the early 20th century. Although millions of American chestnuts survive as root collar sprouts, these trees rarely reproduce. Thus, the species is considered functionally extinct. American chestnuts with improved blight resistance have been developed through interspecific hybridization followed by conspecific backcrossing, and by genetic engineering. Incorporating adaptive genomic diversity into these backcross families and transgenic lines is important for restoring the species across broad climatic gradients. To develop sampling recommendations for ex situ conservation of wild adaptive genetic variation, we coupled whole-genome resequencing of 384 stump sprouts with genotype-environment association analyses and found that the species range can be subdivided into three seed zones characterized by relatively homogeneous adaptive allele frequencies. We estimated that 21 to 29 trees per seed zone will need to be conserved to capture most extant adaptive diversity. We also resequenced the genomes of 269 backcross trees to understand the extent to which the breeding program has already captured wild adaptive diversity, and to estimate optimal reintroduction sites for specific families on the basis of their adaptive portfolio and future climate projections. Taken together, these results inform the development of an ex situ germplasm conservation and breeding plan to target blight-resistant breeding populations to specific environments and provides a blueprint for developing restoration plans for other imperiled tree species.

Characterisation of Cryphonectria hypovirus 1 strains in Turkey and their transmission to various vegetative compatibility types of Cryphonectria parasitica

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.

Argonaute-independent, Dicer-dependent antiviral defense against RNA viruses

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

The Disease Triangle of Chestnut: A review of host, pathogen, and environmental interactions of chestnuts cultivated in the eastern United States

Chestnuts, the edible seeds of the genus Castanea, are a perennial food crop closely tied to the global migration of humans throughout history and have recently been gaining popularity in agriculture and forest restoration in eastern North America. Cultivation of chestnuts yields nutritionally balanced food while fostering economic development, food security, and environmental health. However, diseases and insect pests threaten successful ecological restoration and food production. In this review we explore conditions affecting chestnuts in the eastern United States through the lens of the disease triangle. A “host” in the disease triangle is not merely a single tree, but a tree including its constituent population of fungal endophytes. Chestnut trees are rich with microbial life, and the sustainability of chestnuts in forest and cultivated settings may lie in understanding and manipulating microbial communities to improve plant health and control destructive diseases. To benefit from the ecological and economic potential of chestnuts on the landscape, it may be necessary to select locally adapted chestnut trees, regardless of pedigree, that are resilient against cosmopolitan pathogens. With transport of plants and pathogens throughout the globe, and with landscape level environmental changes over the last century, chestnut trees in the eastern United States (U.S.) are in a unique disease landscape compared to their ancestors. Diseases of economic concern from fungi and fungal-like organisms include chestnut blight (Cryphonectria parasitica) and ink disease (Phytophthora cinnamomi) on American and European chestnuts, oak wilt (Bretziella fagacearum) on all chestnut species, and the emerging diseases of brown rot (Gnomoniopsis smithogilvyi) and chestnut anthracnose (Colletotrichum henanense). The eastern U.S. has experienced profound environmental changes over the 20th century and into the early 21st century. These changes happen to coincide with the demise of the American chestnut in the eastern forest, efforts to re-establish chestnut as a forest species, and the rise in cultivation of multiple chestnut species and hybrids as a culinary crop. Chestnut trees growing in the early 21st century face different environmental circumstances than the American chestnuts of pre-colonial times, including changes in forest composition, rainfall changes and acidification, industrialized agriculture’s increased chemical inputs, rising global temperatures, and increased levels of carbon dioxide in the atmosphere. We conclude that chestnut tree species for both forestry and agriculture should be considered based on current fitness, adaptability, and economic and ecological value considering continued dynamics in host and pathogens distributions and anthropogenically driven climatic and edaphic conditions.

Phylogenetic and Morphological Evidence for Three New Species of Diaporthales (Ascomycota) from Fujian Province, China.

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.

The role of microbial coagulants on the physicochemical, proteolysis, microstructure and sensory properties of low-fat Edam cheese manufactured from ultrafiltered buffalo milk

This work investigates the influence of using microbial coagulants, including Rhizomucor miehei (MCR) protease and Cryphonectria parasitica (MCC) protease, on the quality characteristics of low-fat Edam cheese made from ultrafiltered buffalo milk (LFUE). Concurrently, a benchmark with calf rennet (CR) has been also performed. Throughout a 90-day ripening period, the cheeses were assessed for their physicochemical features, proteolysis, texture, free amino acid and free fatty acid content, microstructure, and sensory attributes. The study revealed that both microbial coagulants had no significant impact on the physicochemical composition and firmness of the cheeses while slightly affected the free fatty acids. Cheeses made with microbial coagulants displayed higher proteolysis, with MCR and MCC cheeses exhibiting greater levels of water-soluble nitrogen and 12% trichloroacetic acid-soluble nitrogen than CR cheese. MCR and MCC cheeses exhibited more extensive breakdown of αs- and β-caseins, as indicated by the SDS-PAGE electrophoretogram, compared to CR cheese during ripening. As for the proteolytic activity, the microbial coagulant contributed to shaping the free amino acid content, microstructure, and sensory qualities of the cheeses. Notably, MCC cheese outperformed MCR or CR cheeses in terms of free amino acid levels. MCR and MCC cheeses resulted in smooth microstructures with uniform protein networks as observed by microscopy, while CR cheese displayed rough, granular surfaces. With the highest scores for appearance, body, texture, and flavor, MCC cheese demonstrated superior sensory properties compared with MCR and CR cheeses.

Boscalid + Pyraclotrobin Resistance and Expression of Succinate Dehyrogenase Genes (sdhA and sdhB) in Chestnut Blight Fungus Cryphonectria parasitica

Sweet chestnut, Castanea sativa Mill., is a significant species in Türkiye. The tree has been encountered with numerous diseases. Chestnut blight, caused by Cryphonectria parasitica (Murrill) M. E. Barr, is one of the most common diseases having spread to every chestnut-growing region. In this study, the effects of Bellis® (25.2% Boscalid + 12.8% Pyraclotrobin), a fungicide commonly used in plant disease control, were evaluated at different doses (10 ppm and 50 ppm) against the chestnut blight agent C. parasitica, which had not been previously studied for its effects in vitro. The isolates included two virulent strains (Cp1_2023, Cp2_2023) and two hypovirulent strains (Cp7_2023, Cp9_2023). The effects of Boscalid + Pyraclotrobin on the mycelial growth of the pathogenic fungus were determined for the first time in vitro, and these effects were detected at the sdhA and sdhB genes expression level. The results showed that the mycelial growth and pathogenicity of C. parasitica isolates in the fungicide-treated medium were consistent with the gene expression profile. Specifically, the virulent Cp1_2023 isolate exhibited higher mycelial growth, pathogenicity, and gene expression levels compared to the other isolate. In conclusion, it is recommended to conduct more detailed studies before using fungicides and to employ them in controlled environments.

Molecular genetic analysis of natural introgression to enhance chestnut blight resistance of Castanea henryi var. omeiensis

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

CpSmt3, an ortholog of small ubiquitin-like modifier, is essential for growth, organelle function, virulence, and antiviral defense in Cryphonectria parasitica.

SUMOylation is an important post-translational modification that regulates the expression, localization, and activity of substrate proteins, thereby participating in various important cellular processes such as the cell cycle, cell metabolism, gene transcription, and antiviral activity. However, the function of SUMOylation in phytopathogenic fungi has not yet been adequately explored. A comprehensive analysis composed of proteomics, affinity pull-down, molecular and cellular approaches was performed to explore the roles of SUMOylation in Cryphonectria parasitica, the fungal pathogen responsible for chestnut blight. CpSmt3, the gene encoding the SUMO protein CpSmt3 in C. parasitica was identified and characterized. Deletion of the CpSmt3 gene resulted in defects in mycelial growth and hyphal morphology, suppression of sporulation, attenuation of virulence, weakening of stress tolerance, and elevated accumulation of hypovirus dsRNA. The ΔCpSmt3 deletion mutant exhibited an increase in mitochondrial ROS, swollen mitochondria, excess autophagy, and thickened cell walls. About 500 putative SUMO substrate proteins were identified by affinity pull-down, among which many were implicated in the cell cycle, ribosome, translation, and virulence. Proteomics and SUMO substrate analyses further revealed that deletion of CpSmt3 reduced the accumulation of CpRho1, an important protein that is involved in TOR signal transduction. Silencing of CpRho1 resulted in a phenotype similar to that of ΔCpSmt3, while overexpression of CpRho1 could partly rescue some of the prominent defects in ΔCpSmt3. Together, these findings demonstrate that SUMOylation by CpSmt3 is vitally important and provide new insights into the SUMOylation-related regulatory mechanisms in C. parasitica.

Mapping QTLs for blight resistance and morpho-phenological traits in inter-species hybrid families of chestnut (Castanea spp.).

Chestnut blight (caused by Cryphonectria parasitica), together with Phytophthora root rot (caused by Phytophthora cinnamomi), has nearly extirpated American chestnut (Castanea dentata) from its native range. In contrast to the susceptibility of American chestnut, many Chinese chestnut (C. mollissima) genotypes are resistant to blight. In this research, we performed a series of genome-wide association studies for blight resistance originating from three unrelated Chinese chestnut trees (Mahogany, Nanking and M16) and a Quantitative Trait Locus (QTL) study on a Mahogany-derived inter-species F2 family. We evaluated trees for resistance to blight after artificial inoculation with two fungal strains and scored nine morpho-phenological traits that are the hallmarks of species differentiation between American and Chinese chestnuts. Results support a moderately complex genetic architecture for blight resistance, as 31 QTLs were found on 12 chromosomes across all studies. Additionally, although most morpho-phenological trait QTLs overlap or are adjacent to blight resistance QTLs, they tend to aggregate in a few genomic regions. Finally, comparison between QTL intervals for blight resistance and those previously published for Phytophthora root rot resistance, revealed five common disease resistance regions on chromosomes 1, 5, and 11. Our results suggest that it will be difficult, but still possible to eliminate Chinese chestnut alleles for the morpho-phenological traits while achieving relatively high blight resistance in a backcross hybrid tree. We see potential for a breeding scheme that utilizes marker-assisted selection early for relatively large effect QTLs followed by genome selection in later generations for smaller effect genomic regions.

85 years counteracting an invasion: chestnut ecosystems and landscapes survival against ink disease

A retrospective examination of early pest and disease outbreaks, such as ink disease, offers new insights into their impact on ecosystems and landscapes. Ink disease, caused by Phytophthora spp., is one of the most destructive diseases affecting Castanea sativa Mill. It first appeared in Europe in the early 18th century, with the initial recorded case in Italy (Piedmont) dating back to 1845. However, its spread instilled significant concerns in several chestnut-growing regions primarily devoted to fruit production. In 1938, a comprehensive assessment of Phytophthora xcambivora outbreaks was conducted in the Bolognese Apennines (Italy), providing a detailed overview of chestnut cultivation status. Thirty-six disease foci were documented, and laboratory tests confirmed the presence of ink disease. To understand the disease’s impact on chestnut ecosystem and landscape over the past 85 years, the 1938 sites were revisited to assess chestnut persistence and phytosanitary status, with Phytophthora species detected using isolation and molecular techniques. Monitoring data revealed that C. sativa still thrives in all but one site, with its wood seemingly able to coexist in dynamic equilibrium with the disease. While P. xcambivora was still detected in several foci, the extent of damage appeared limited. The potential for natural control, likely influenced by the complexity of soil biota, along with the natural spread of hypovirulence in chestnut blight and biological control of the Asian chestnut gall wasp, could explain the continued presence of chestnut in the investigated area and potentially throughout the Italian chestnut range, despite nearly two centuries of ink disease prevalence. Nevertheless, ongoing monitoring is essential to dynamically comprehend the factors at play and their efficacy, particularly in the context of climate change and the possible spread of other Phytophthora species. The survival of chestnut ecosystems amidst ink disease underscores the preservation of the economic, ecological, and landscape values associated with these woodlands.