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Autophagy is an evolutionarily ancient process wherein cells are able to break down intracellular contents to support normal physiology and development. Autophagosome formation is regulated by several different proteins, including the key cysteine protease Atg4. The contribution of Atg4 protein in the pathogenic fungus Cryphonectria parasitica, which causes blight in chestnut plants, has not been completely understood. In this context, we aimed to investigate the role of Atg4 during autophagy formation and their contribution to nonautophagic events in C. parasitica. By complementation assay, we determined that the CpAtg4 gene from C. parasitica was able to functionally complement the deletion of yeast Atg4. Using a yeast two-hybrid assay system, we confirmed that CpAtg4 and CpAtg8 directly interact with one another, and amino acids 377 to 409 of CpAtg4 were identified as being responsible for its binding with CpAtg8. The deletion mutant of CpAtg4 did not demonstrate positive monodansylcadaverine staining, which indicated that CpAtg4 is required for autophagy in C. parasitica. Moreover, the ΔCpAtg4 strain exhibited a decrease in aerial hyphae formation and sporulation, and reduction in virulence on apple and chestnut stem. The ΔCpAtg4 strains were also more sensitive to H2O2 and Congo red-induced stress. We further determined that amino acids 377 to 409 of CpAtg4 were essential for the function of CpAtg4 in vivo. Together, our findings indicated that CpAtg4 is required for the autophagy formation, fungal phenotypic traits, stress tolerance, and virulence in C. parasitica.

In the current study the antifungal activity of inorganic reagents was tested against Cryphonectria parasitica in vitro in a mycelial growth inhibition test. Three reagents, each consisting of chloride silver (AgCl) in combination with (1) aluminum oxide − Al2O3, (2) zinc oxide − ZnO, and (3) Al2O3 and titanium dioxide – TiO2, were tested. Significant differences of the tested reagents on the growth of C. parasitica were recorded. The study demonstrated that silver in mixture with ZnO had an antifungal effect and significantly reduced the mycelial growth of C. parasitica in vitro. The mixture of AgCl with the other two combinations of inorganic metal oxides had no inhibition effect on the growth of the pathogen. It was confirmed that ZnO (applied in a single compound test) is responsible for inhibition of C. parasitica mycelium growth. A preliminary in planta assay was performed but statistically significant differences were not recorded in the average increment of canker length.

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.

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

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.

The American chestnut (Castanea dentata) was once a dominant tree species in the Appalachian Mountains and played a critical role in the ecological system. However, it was nearly eliminated by chestnut blight caused by the Ascomycetous fungus Cryphonectria parasitica. Identification of compounds specific to species and backcross hybrids may help further refine disease resistance breeding and testing. Phenolic compounds produced by plants are significant to their defense mechanisms against fungal pathogens. Therefore, an analytical platform has been developed to estimate the total phenolic content in leaf tissues of the American chestnut, Chinese chestnut (Castanea mollissima), and their backcross breeding generations (B3F2 and B3F3) using the Folin-Ciocalteu reagent assay with UV/Vis spectrophotometry which may be used to predict blight resistance. Adsorption (765 nm) results from leaf tissue extraction in methanol/water (95%:5% v/v) and pH 2, show that the variations among these four tree species are significant (ANOVA p = 2.3 × 10-7). The kinetics of phenolic compound solid-liquid extraction was elaborated using Peleg, second order, Elovich, and power law models. In addition, extensive analysis using headspace solid phase microextraction (SPME) gas chromatography and mass spectrometry was conducted to identify volatile organic compounds (VOCs) from the leaf of American chestnut, Chinese chestnut, and their backcross hybrids B3F2 and B3F3. A total of 67 VOCs were identified among all chestnut types. Many of the metabolites associated with the Chinese chestnut have been reported to have antifungal properties, whereas the native and hybrid American chestnut metabolites have not. Most of the antifungal metabolites showed the strongest efficacy towards the Ascomycota phylum. A partial least squares discriminant analysis (PLS-DA) model (R2X = 0.884, R2Y = 0.917, Q2 = 0.584) differentiated chestnut species and hybrids within the first five principal component (PCs).

The underlying molecular mechanisms of programmed cell death associated with fungal allorecognition, a form of innate immunity, remain largely unknown. In this study, transcriptome analysis was used to infer mechanisms activated during barrage formation in vic3-incompatible strains of Cryphonectria parasitica, the chestnut blight fungus. Pronounced differential expression occurred in barraging strains of genes involved in mating pheromone (mf2-1, mf2-2), secondary metabolite production, detoxification (including oxidative stress), apoptosis-related, RNA interference, and HET-domain genes. Evidence for secondary metabolite production and reactive oxygen species (ROS) accumulation is supported through UPLC-HRMS analysis and cytological staining, respectively. Differential expression of mating-related genes and HET-domain genes was further examined by RT-qPCR of incompatible interactions involving each of the six vegetative incompatibility (vic) loci in C. parasitica and revealed distinct recognition process networks. We infer that vegetative incompatibility in C. parasitica activates defence reactions that involve secondary metabolism, resulting in increased toxicity of the extra- and intracellular environment. Accumulation of ROS (and other potential toxins) may result in detoxification failure and activation of apoptosis, sporulation, and the expression of associated pheromone genes. The incompatible reaction leaves abundant traces of a process-specific metabolome as conidiation is initiated.

Forests in North America are threatened by a myriad of native and nonnative pests and pathogens, the latter of which are largely introduced via the international trade of raw wood products and live plants. The early twentieth-century arrival of white pine blister rust (WPBR) (Cronartium ribicola) and chestnut blight (Cryphonectria parasitica) on imported seedlings devastated forests across North America. Invasive species notably affect entire genera. For example, WPBR has now infected all North American five-needle pine (Pinus) species across the entire United States, which has affected species of commercial and noncommercial value and threatened sensitive, high-elevation ecosystems (Sniezko et al. 2011, 2014, Sniezko and Koch 2017).

Sonneratia apetala Francis Buchanan-Hamilton (Sonneratiaceae, Myrtales), is a woody species with high adaptability and seed production capacity. S. apetala is widely cultivated worldwide as the main species for mangrove construction. However, the study of diseases affecting S. apetala is limitted, with only a few fungal pathogens being recorded. Cryphonectriaceae (Diaporthales) species are the main pathogens of plants. They can cause canker diseases to several trees and thereby seriously threaten the health of the hosts. These pathogens include Cryphonectria parasitica (Cryphonectriaceae) causing chestnut blight on Castanea (Rigling and Prospero 2017) and Cytospora chrysosperma (Cytosporaceae) causing polar and willow canker to Populus and Salix (Wang et al. 2015) . Therefore, the timely detection of of Cryphonectriaceae canker pathogens on S. apetala is extremely important for protecting the mangrove forests. Two diaporthalean fungi, Celoporthe guangdongensis and Cytospora rhizophorae have been reported for the first time to cause canker on the branches of S. apetala. C. guangdongensis is significantly pathogenic and C. rhizophorae is saprophytic on S. apetala.

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.

Ecological interactions that cross domains of life have major impacts on ecosystems and human health. Although the coronavirus disease 2019 (COVID-19) pandemic makes this point with destructive clarity, it is clear that zoonotic pathogens pose a standing threat to our species as demonstrated by Ebola, Middle East respiratory syndrome (MERS), and severe acute respiratory syndrome (SARS). Other species experience similar pandemics and are both sources of, and sensitive to, shared pathogens. More than a century ago, Cryphonectria parasitica ( Left ), the fungus that causes Chestnut blight, devastated the American Chestnut tree. Until its eradication in 2011, Rinderpest morbillivirus ( Right ) decimated cattle and other ungulate populations. A biosurveillance system would help prevent future deadly and economically devastating pathogen outbreaks. Image credit: Paul Beales/UK Crown Copyright ( Left ); © Bioimaging at The Pirbright Institute ( Right ). Hence, there is an urgent need to establish a global, genomic-based biosurveillance platform, a development which would be of immense value to biosecurity, biodefense, and the economy. If implemented, this “pandemic interception system” would hugely advance our understanding of the natural world. Three major research programs are poised to support this effort: BIOSCAN, the Earth BioGenome Project (EBP), and the Global Virome Project (GVP). Each of these global programs is now working to develop approaches in comparative genomics that are needed to discover all species and to reveal their interactions. The diversity of infectious agents involved in host–pathogen interactions needs immediate clarification, especially with regard to those agents that transfect phylogenetically divergent lineages. Such information will enable a surveillance system that facilitates preemptive strikes and rapid responses to outbreaks as well as early development of diagnostic pipelines and vaccines. If pursued with a tiny fraction of the resources devoted toward the suppression of COVID-19, rapid progress could be made in identifying every pathogen hosted by birds and mammals … [↵][1]1To whom correspondence may be addressed. Email: kressj{at}si.edu. [1]: #xref-corresp-1-1

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

Chinese chestnut (Castanea mollissima) is an important crop tree species in China. However, branch canker and fruit rot are two kinds of severe diseases, which weaken the host and decrease chestnut production. During our investigations into chestnut diseases in China, several fungi have been confirmed as casual agents in previous studies, namely Aurantiosacculus castaneae, Cryphonectria neoparasitica, Cry. parasitica, Endothia chinensis and Gnomoniopsis daii. In this study, a new canker pathogen is introduced based on morphology, phylogeny and pathogenicity. Typical Gnomoniopsis canker sign of wide, orange tendrils emerging from hosts’ glaucous lenticels were obvious on the diseased trees in the field. Symptomatic branches or bark on stems from different chestnut plantations were sampled and isolated, then strains were identified by comparisons of DNA sequence data for the nuclear ribosomal internal transcribed spacer (ITS), partial translation elongation factor-1α (tef1) and β-tubulin (tub2) gene regions as well as morphological features. As a result, these strains appeared different from any known Gnomoniopsis species. Hence, we propose a novel species named Gnomoniopsis chinensis. Pathogenicity was further tested using the ex-type strain (CFCC 52286) and another strain (CFCC 52288) on both detached branches and 3-year-old chestnut seedlings. The inoculation results showed that Gnomoniopsis chinensis is mildly pathogenic to Chinese chestnut. However, further studies are required to confirm its pathogenicity to the other cultivated Castanea species in America, Europe and Japan.

In the past 25 to 30 years, a marked increase has occurred in numbers of invasive alien pests and pathogens recorded, damaging agriculture, horticulture and forest environments. The reasons for this upsurge are clear: all involve human-driven factors, with, arguably, global trade as the most important component in the complex. Climate change is another major factor. Each plant transported through trade could carry cryptic pests or pathogens, which may be introduced into previously naïve environments and lead to major health issues on previously unaffected plants. Global trade in plants is a complex system, driven by a desire to use large plants in landscapes and demand for varied species for gardens. Examples of invasive alien pathogens causing wide-spread problems on a global scale currently include many Phytophthora species, Dothistroma needle blight of pines, and Xylella fastidiosa on a wide range of trees. A striking example recently was the spread of the Dutch elm disease pathogen, Ophiostoma novo-ulmi, in North America and Western Europe. Many invasive pathogens cause problems in localized regions, including Ceratocystis platani, killing Platanus in some European countries. Other examples include the threat of pine wilt nematode (Bursaphelenchus xylophilus), damage to Cupressus in Mediterranean landscapes caused by Seiridium cardinale, dieback of sweet chestnut (Castanea) caused by the canker pathogen Cryphonectria parasitica, and many others. In addition to global trade, climate change appears to be exacerbating the problems, with major influences on establishment and spread of alien invasive species. Hope lies in the enormous genetic ‘power’ of plants: humans could establish selection and breeding programmes that will ultimately enable us to plant trees with greater tolerance of changing climate and pests and pathogens, whether native or invasive.

‘Mycoviruses’ is the term used for viruses infecting fungi and fungus-like organisms. Mycoviruses have been reported in Ascomycota, Basidiomycota, Chytridiomycota, Deuteromycota and Zygomycota (Ghabrial et al., 2015). Most reported mycoviruses consist of linear double-stranded RNA (dsRNA; ~70%), while a small number are composed of negative single-stranded RNA (− ssRNA) or positive single-stranded RNA (+ssRNA; ~30%) (Ghabrial et al, 2015; King et al, 2011). More rarely, mycoviruses with circular single-stranded DNA (ssDNA) have been found (Jiang et al., 2013; Li et al., 2020; Yu et al., 2010). Like animal and plant viruses, mycoviruses require the living cells of their host to replicate. The main differences between animal and plant viruses on the one hand, and mycoviruses on the other, lie in the fact that most mycoviruses lack an extracellular route for infection and are transmitted between cells through cell division, sporulation and occasionally cell fusion, as well as lacking movement proteins (Son et al., 2015) Many of these mycoviruses are considered to be cryptic, and supposedly have little to no effect on their host. However, some notable exceptions have been described, in which the viruses cause deleterious effects on their host, and where the host is a plant pathogen, may cause hypovirulence, reducing the infection, growth and reproduction of the fungus. Virocontrol, the biocontrol of pathogens via hypovirulence-causing mycoviruses, is a small field, but one which is now receiving more attention. This chapter discusses the characteristics of mycoviruses and the conditions that need to be met to make a successful virocontrol agent. The chapter describes one of the success stories so far, that of the virocontrol of Cryphonectria parasitica, the chestnut blight pathogen. The chapter ends by reviewing future trends and information on sources of more information on mycoviruses and virocontrol.

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

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

Laccase3 is an important virulence factor of the fungus Cryphonectria parasitica. Laccase3 gene (lac3) transcription is induced by tannic acid, a group of phenolic compounds found in chestnut trees, and its induction is regulated by the hypovirus CHV1 infection. CpHsp24, a small heat shock protein gene of C. parasitica, plays a determinative role in stress adaptation and pathogen virulence. Having uncovered in our previous study that transcriptional regulation of the CpHsp24 gene in response to tannic acid supplementation and CHV1 infection was similar to that of the lac3, and that conserved phenotypic changes of reduced virulence were observed in mutants of both genes, we inferred that both genes were implicated in a common pathway. Building on this finding, in this paper we examined whether the CpHsp24 protein (CpHSP24) was a molecular chaperone for the lac3 protein (LAC3). Our pull-down experiment indicated that the protein products of the two genes directly interacted with each other. Heterologous co-expression of CpHsp24 and lac3 genes using Saccharomyces cerevisiae resulted in more laccase activity in the cotransformant than in a parental lac3-expresssing yeast strain. These findings suggest that CpHSP24 is, in fact, a molecular chaperone for the LAC3, which is critical component of fungal pathogenesis.

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.

Cryphonectria is a fungal genus associated with economically significant disease of trees. Herein we characterized a novel double-stranded RNA virus from the fungal species Cryphonectria naterciae, a species unexplored as a virus host. De novo assembly of RNA-seq data and Sanger sequencing of RACE (rapid amplification of cDNA ends) clones gave the complete, non-segmented genome (10,164 bp) of the virus termed Cryphonectria naterciae fusagravirus (CnFGV1) that was phylogenetically placed within the previously proposed viral family Fusagraviridae. Of 31 field-collected strains of C. naterciae, 40% tested CnFGV1-positive. Cocultivation resulted in within-species transmission of CnFGV1 to virus-free strains of C. naterciae. Comparison of the mycelium phenotype and the growth rate of CnFGV1-infected and virus-free isogenic strains revealed frequent sectoring and growth reduction in C. naterciae upon virus infection. Co-culturing also led to cross-species transmission of CnFGV1 to Cryphonectria carpinicola and Cryphonectria radicalis, but not to Cryphonectria parasitica. The virus-infected C. naterciae and the experimentally infected Cryphonectria spp. readily transmitted CnFGV1 through asexual spores to the next generation. CnFGV1 strongly reduced conidiation and in some cases vegetative growth of C. carpinicola, which is involved in the European hornbeam disease. This study is the first report of a fusagravirus in the family Cryphonectriaceae and lays the groundwork for assessing a hypovirulence effect of CnFGV1 against the hornbeam decline in Europe.

The inadvertent introduction and rapid spread of chestnut blight (caused by Cryphonectria parasitica (Murr.) Barr) in the early 20th century resulted in the demise of American chestnut (Castanea dentata (Marsh.) Borkh.; Fagaceae) as a major component of forest canopies and had negative impacts on eastern forest communities. Research efforts over the last century have documented the persistence of occasional trees and root crown/stump sprouts throughout much of the species’ historic range providing the basis for ongoing breeding of blight-resistant stock and restoration efforts. Unfortunately, it remains unclear how much of the historic range remains climatically suitable for remnant trees that may harbor unique genetic variation for successful reintroduction efforts. Here we investigate whether the southwestern portion of the historical range remains environmentally suitable for undiscovered remnant populations of C. dentata using environmental niche modeling. We also use stage-structured matrix projection models to investigate the potential demographic future of C. dentata in W Tennessee, N Mississippi, SW Kentucky, and NW Alabama based upon observations of American chestnut in these areas over the last several decades. We found that suitable habitat associated with higher elevations and areas of high forest canopy cover occurs throughout much of the southwestern portion of the historical range and that populations of American chestnut in these areas are predicted to drastically decline over the next ~100-200 years without conservation interventions to mitigate the negative consequences of chestnut blight.

Knowledge of occurrence and severity of the damage caused by the fungus Cryphonectria parasitica (Murrill) M.E. Barr and forest fires is key to define a management plan of Castanea sativa forest masses in south-western Europe. The main goal of our study is to verify whether there is a concordance between field measurements and measures from orthophotographs acquired by an unmanned flight and to determine the influence of the number of severity levels on it. Accuracy of blight severity level estimate was computed used as ground truth a red-green-blue (RGB) orthophotograph of very high spatial resolution (8 cm) acquired by an Unmanned Aerial Vehicle (UAV). Starting from codes specifically defined for this study, the severity level of 823 chestnut trees was measured on the UAV orthophotograph. At same time, we ground measured the severity level on a sample of 182 chestnut trees using a standard methodology. From these measurements, overall accuracy and Kappa statistic were computed. Our results show that the concordance for 6 severity levels varies between moderate and good, whereas for 4 and 5 levels, varies between moderate and very good (kappa statistic>0.75). The study demonstrates the UAVs usefulness for studying forest damage in South-Western Europe.

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 Izmir and Kutahya 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 Izmir and Kutahya 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.

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.

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.

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.

Statistical experimental methods were used to optimize the medium for mass production of a novel laccase3 (Lac3) by recombinant Saccharomyces cerevisiae TYEGLAC3-1. The basic medium was composed of glucose, casamino acids, yeast nitrogen base without amino acids (YNB w/o AA), tryptophan, and adenine. A one-factor-at-a-time approach followed by the fractional factorial design identified galactose, glutamic acid, and ammonium sulfate, as significant carbon, nitrogen, and mineral sources, respectively. The steepest ascent method and response surface methodology (RSM) determined that the optimal medium was (g/L): galactose, 19.16; glutamic acid, 5.0; and YNB w/o AA, 10.46. In this medium, the Lac3 activity (277.04 mU/mL) was 13.5 times higher than that of the basic medium (20.50 mU/mL). The effect of temperature, pH, agitation (rpm), and aeration (vvm) was further examined in a batch fermenter. The best Lac3 activity was 1176.04 mU/mL at 25 °C, pH 3.5, 100 rpm, and 1 vvm in batch culture.

American chestnut (Castanea dentata) was a foundation species in the eastern United States until chestnut blight (Cryphonectria parasitica) infestation resulted in range-wide catastrophic reduction. Since 1983, The American Chestnut Foundation (TACF) has engaged in a breeding program aimed at restoring C. dentata to the wild. The primary goal has been to introduce blight resistance from Castanea mollissima while recovering a C. dentata phenotype via successive backcrosses. To diversify the genetic base, state chapters of TACF have been producing BC3 and BC4 (third and fourth backcross) lines using C. dentata from across its native range. This study focuses on morphology and chemistry of seeds, and morphology and early growth of seedlings derived from BC3 and BC4 trees selected for blight resistance in state chapters. Our primary comparisons were among backcross, pure C. mollissima, and pure C. dentata lines. Seed traits included a suite of morphological characters known to differ between C. dentata and C. mollissima, as well as dry matter, total carbohydrate, sugar, protein, lipid, and phenolic content. Seedling traits included variables such as stem basal diameter and height, leaf variables such as number and area, and relative growth in key parameters such as stem volume. C. mollissima lines tended to differ significantly from C. dentata and backcross lines in most parameters, while C. dentata and backcross types tended to overlap broadly in traits. These results suggest that seed and seedling characteristics of backcross hybrids studied here are likely to be sufficiently similar to C. dentata for use in restoration programs.

American chestnut (Castanea dentata [Marsh.] Borkh.) was once the dominant hardwood species in Eastern North America before an exotic fungal pathogen, Cryphonectria parasitica (Murrill) Barr, functionally eliminated it across its range. One promising approach toward restoring American chestnut to natural forests is development of blight-tolerant trees using genetic transformation. However, transformation and related processes can result in unexpected and unintended phenotypic changes, potentially altering ecological interactions. To assess unintended tritrophic impacts of transgenic American chestnut on plant-herbivore interactions, gypsy moth (Lymantria dispar L.) caterpillars were fed leaf disks excised from two transgenic events, Darling 54 and Darling 58, and four control American chestnut lines. Leaf disks were previously treated with an LD50 dose of either the species-specific Lymantria dispar multiple nucleopolyhedrovirus (LdMNPV) or the generalist pathogen Bacillus thuringiensis subsp. kurstaki (Btk). Mortality was quantified and compared to water blank controls. Tree genotype had a strong effect on the efficacies of both pathogens. Larval mortality from Btk-treated foliage from only one transgenic event, Darling 54, differed from its isogenic progenitor, Ellis 1, but was similar to an unrelated wild-type American chestnut control. LdMNPV efficacy was unaffected by genetic transformation. Results suggest that although genetic modification of trees may affect interactions with other nontarget organisms, this may be due to insertion effects, and variation among different genotypes (whether transgenic or wild-type) imparts a greater change in response than transgene presence.

The vacuolar H+-ATPases (V-ATPases) are conserved ATP-dependent proton pumps that acidify intracellular compartments in eukaryotic cells. The role of Cpvma1, a V-ATPase catalytic subunit A of Cryphonectria parasitica, was investigated by generating cpvma1-overexpressing and cpvma1-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, cpvma1-overexpressing strains showed no significant difference in phenotype; however, cpvma1-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 cpvma1 increased sensitivity to elevated pH and Ca2+, implicating an important role for Cpvma1 in pH adaptation and Ca2+ homeostasis. Furthermore, silencing of cpvma1 resulted in significantly decreased accumulation of hypoviral RNA. Taken together, our results indicate that Cpvma1 plays an important role in the regulation of phenotypic traits and virulence and the accumulation of hypovirus RNA in C. parasitica.

Epigenetic modifications may play an important role in invasion and adaptation of clonal and invasive populations to different environments. The aim of this study was to analyse epigenetic diversity and structure within and among populations of invasive pathogenic fungus Cryphonectria parasitica from south-eastern Europe, where one haplotype S12 dominates. The highest level of epigenetic diversity was found in haplotype S1, followed by S2, while the lowest level of epigenetic diversity was found in haplotype S12. Similar pattern of epigenetic diversity was detected in the control, genetically diverse Croatian population where S1 haplotype dominates. In four south-eastern European populations, the highest level of epigenetic diversity was observed in the Italian population, the oldest population in the studied area, while the lowest diversity was found in most recently established Bulgarian population. This relationship between epigenetic diversity and population age implies the important role of epigenetic modifications on the process of invasion. Our data suggest that epigenetic differences might affect the success of expansion of certain haplotype into new regions. Understanding the role of epigenetic processes in expansion and (pre)adaptation of fungal plant pathogens, besides fundamental knowledge, can contribute to development of strategies for control of fungal spread and pathogenesis.

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. Hypov...

Field-collected US strain C18 of Cryphonectria parasitica, the chestnut blight fungus, was earlier reported to be infected by a double-stranded RNA virus, mycoreovirus 2 (MyRV2). Next-generation sequencing has revealed co-infection of C18 by a positive-strand RNA virus, hypovirus 4 (CHV4). The current molecular and genetic analyses showed interesting commensal interactions between the two viruses. CHV4 facilitated the stable infection and enhanced vertical transmission of MyRV2, which was readily lost during subculturing and showed reduced vertical transmission in single infections. Deletion of a key antiviral RNA silencing gene, dcl2, in isolate C18 increased stability of MyRV2 in single infections. The ability of CHV4 to facilitate stable infection with MyRV2 appears to be associated with the inhibitory effect of CHV4 on RNA silencing via compromising the induction of transcriptional up-regulation of dcl2. These results suggest that natural infection of isolate C18 by MyRV2 in the field was facilitated by CHV4 co-infection.

Restoration of American chestnut (Castanea dentata) depends on combining resistance to both the chestnut blight fungus (Cryphonectria parasitica) and Phytophthora cinnamomi, which causes Phytophthora root rot, in a diverse population of C. dentata. Over a 14-year period (2004 to 2017), survival and root health of American chestnut backcross seedlings after inoculation with P. cinnamomi were compared among 28 BC3, 66 BC4, and 389 BC3F3 families that descended from two BC1 trees (Clapper and Graves) with different Chinese chestnut grandparents. The 5% most resistant Graves BC3F3 families survived P. cinnamomi infection at rates of 75 to 100% but had mean root health scores that were intermediate between resistant Chinese chestnut and susceptible American chestnut families. Within Graves BC3F3 families, seedling survival was greater than survival of Graves BC3 and BC4 families and was not genetically correlated with chestnut blight canker severity. Only low to intermediate resistance to P. cinnamomi was detected among backcross descendants from the Clapper tree. Results suggest that major-effect resistance alleles were inherited by descendants from the Graves tree, that intercrossing backcross trees enhances progeny resistance to P. cinnamomi, and that alleles for resistance to P. cinnamomi and C. parasitica are not linked. To combine resistance to both C. parasitica and P. cinnamomi, a diverse Graves backcross population will be screened for resistance to P. cinnamomi, survivors bred with trees selected for resistance to C. parasitica, and progeny selected for resistance to both pathogens will be intercrossed.

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.

Castanea sativa is a widespread and important multi-purpose tree in the Mediterranean area. Recently, intensive infestation of gall wasp decreased the production of chestnut, and makes the plants more susceptible to Cryphonectria parasitica, the causal agent of chestnut blight. Generally, biological control of chestnut blight is through natural spread or artificial release of hypovirulent strains of C. parasitica. However, this approach is not always successful. The aim here was to evaluate alternative approaches for control chestnut blight based on application of antagonistic microorganisms. In-vitro antagonistic activities of fungal species Trichoderma and Penicillium were tested after in vitro isolation from chestnut bark. In dual cultures, these antagonistic fungi consistently reduced growth of C. parasitica. Three commercial formulations based on Trichoderma spp., Glomus spp. and Bacillus subtilis were tested at three doses, both in vitro and directly applied to cuttings of chestnut stems as co-inoculations and as separated inoculations with C. parasitica mycelium plugs. With co-inoculation, B. subtilis showed the best performance against chestnut blight, with necrotic areas reduced by >70%. In separated inoculations, B. subtilis reduced the size of cankers on chestnut stems, by 29%–67%, as also for Trichoderma spp., by 36%–65%, and Glomus spp., by 31%–63%. Application of commercial formulations based on antagonistic microorganisms are effective tools in management of chestnut blight.

Cryphonectria hypovirus 1 strain CN280 (CHV1-CN280) was isolated from North China and exhibited typical hypovirulence-associated traits. We previously reported that CHV1-CN280 was more aggressive and had a higher horizontal transmission ability between Cryphonectria parasitica isolates belonging to different vegetative compatibility groups than two other CHV1 hypoviruses (namely, CHV1-EP713 and CHV1-Euro7), thus displaying greater potential for biological control of chestnut blight. The genome sequence of CHV1-CN280 shared approximately 70% identity with three other hypoviruses (CHV1-EP713, CHV1-Euro7, and CHV1-EP721). The coding region for p29, a papain-like protease encoded by CHV1-CN280 hypovirus, displayed an average of only approximately 60% amino acid identity among them, while the identity between the other three CHV1 isolates was higher than 89%. Protease p29 acted as a virus-encoded determinant responsible for altering fungal host phenotypes in other CHV1 isolates. In this study, the impacts of CHV1-CN280 p29 expression in virus-free C. parasitica were investigated. CHV1-CN280 p29 expression in C. parasitica resulted in significantly reduced sporulation, pigmentation, extracellular laccase activities, and pathogenicity, which is consistent with previous investigations. Subsequently, the potential of CHV1-CN280 p29 as a viral determinant responsible for suppression of host phenotypes in other phytopathogenic fungi such as Magnaporthe oryzae, the causal agent of rice blast disease, was discussed. However, heterologous expression of p29 in M. oryzae induced the opposite effect on sporulation, extracellular laccase activities, and pathogenicity; had no significant effect on pigmentation and mycelial growth; and contributed to extracellular peroxidase activities, suggesting that CHV1-CN280 p29 may disturb a unique regulatory pathway in C. parasitica, rather than a basic regulatory pathway conserved in diverse range of fungi. Alternatively, CHV1-CN280 p29-mediated modulation of fungal phenotypes may be facilitated by the specific interaction between p29 and a special fungal-host component, which exists only with C. parasitica but not M. oryzae.

Erworben im Rahmen der Schweizer Nationallizenzen (http://www.nationallizenzen.ch) The highly destructive chestnut blight disease can be successfully controlled by infecting the virulent strain of C. parasitica with the hyperparasitic mycovirus CHV-1. The artificial application of the virus-induced hypovirulence, however, requires that the vegetative compatibility (vc) diversity in the target C. parasitica population be determined beforehand. Conventional vc type determination by pairing the unknown isolate with tester strains of known vc types is time consuming and not always reliable. A genotyping assay was developed that uses DNA from C. parasitica isolates and discriminates the six known di-allelic vic loci and the two mating idiomorphs in two fluorescence-labeled multiplex PCRs. Validation was conducted by successfully genotyping the 74 known European vc tester strains and a conventionally characterized natural population of C. parasitica. Cross-species amplification tests with three Cryphonectria species (C. japonica, C. naterciae, and C. radicalis) revealed a high amplification specificity for C. parasitica, while the analytical sensitivity of the assay was established at 20 pg per reaction of genomic DNA. In conclusion, this fluorescent multiplex genotyping assay offers a simple and high-throughput tool for the characterization of the vegetative compatibility and mating type of C. parasitica at population level, which is particularly important for the application of mycovirus-mediated biological control of chestnut blight.

The invasive fungus Cryphonectria parasitica, the causal agent of chestnut blight, is able to survive and sporulate on the bark of fresh dead Castanea sativa wood for at least 2 years. Here, we experimentally investigated the role of fresh dead wood in the epidemiology of chestnut blight, specifically in the spread of the hyperparasitic virus Cryphonectria hypovirus 1, which acts as biocontrol agent of C. parasitica. A total of 152 artificially initiated, virulent bark cankers in four chestnut stands were treated with virus-infected asexual spores originating either from sporulating dead wood or from a spore suspension. Molecular markers for both the virus and the fungal carrier were used to examine the spread of the applied biocontrol virus. Fourteen months after treatment, 42 to 76% of the conidial spray-treated cankers and 50 to 60% of the cankers exposed to a sporulating dead stem had been virus infected by the applied hypovirulent conidia in all four study sites. Virus infection reduced canker expansion and promoted canker healing (callusing). Thus, fresh chestnut dead wood may play an important role in supporting the successful spread of natural hypovirulence in chestnut forests. Further, combined with the application of virus-infected conidial suspensions, it may help promote the establishment of artificially released hypoviruses in chestnut stands to control chestnut blight.

Horizontal transmission of virulence attenuating hypoviruses of Cryphonectria parasitica is restricted by an allorecognition system termed vegetative incompatibility (vic). A super donor formulation of two engineered C. parasitica strains (SD328/SD82) with gene disruptions at four of six vic loci transmitted hypovirus to strains in the laboratory independent of vic genotype. We now report the transmission of hypovirus by the SD328/82 formulation to a diverse, natural C. parasitica population infecting American chestnut in a forest setting. Hypovirulent (HV) isolates were recovered from 94% of cankers treated with the hypovirus-infected SD328/82 formulation compared to 51% of cankers treated with a hypovirus-infected EU5/6 formulation (strains having the same vic genotypes as SD strains but lacking vic gene disruptions). Overall, the SD328/82 formulation transmitted hypovirus into more divergent vic genotypes compared to the EU5/6 formulation. These results demonstrate the SD328/82 formulation can serve as an enhanced hypovirus vector for highly divergent C. parasitica populations.

The loss of American chestnut (Castanea dentata) in eastern North America to chestnut blight, a disease caused by the fungal pathogen (Cryphonectria parasitica), has devastated ecological and utilitarian processes and functions. A backcross breeding approach has been developed to confer disease resistance to hybrid seedlings, and forest reintroduction trials will provide important information on performance and durability of resistance in real-world forest conditions. Three plantings were established in 2009 in mesic, even-aged regeneration harvests (site index averaged 23 m for Quercus rubra) and were examined for eight-year blight resistance. These plantings are the first forest field trials to test blight resistance of the most advanced breeding generation currently available, the third generation of the third backcross (BC3F3), against less advanced breeding generations (BC1F3, BC2F3), disease-resistant Chinese chestnut (C. mollissima), and disease-susceptible American chestnut. We also examined if C. parasitica infection was related to tree size and growth. The pathogen infected 36 percent of trees across locations by year 8, but 31 percent of trees died prior to detection of infection. Non-pathogen related mortality was probably due to factors that are typical of hardwood plantings, including repeated deer browsing and native and non-native pest damage. The BC3F3 generation exhibited resistance more similar to the Chinese chestnut than the American chestnut, but exhibited significantly lower resistance than Chinese chestnut at the location with the highest blight incidence; genetic family differences among BC3F3 progeny were significant at this location. Interactions between planting location and breeding generation affected resistance rankings, suggesting additional or longer-term testing is needed to determine resistance of a particular breeding line across a variety of sites. Probability of disease incidence was positively related to ground-line diameter (GLD), but this relationship depended on location and breeding type. At two locations, American chestnut had 50 percent probability of C. parasitica infection when GLD was approximately 70 mm, and the BC3F3 had 50 percent probability when GLD was between 93 and 126 mm. The Chinese chestnut maintained low probability of disease incidence (

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

The mangrove is an ecosystem bounded by the line of the largest tide in size that occurs in climatic and subtropical regions. In this environment, microorganisms and their enzymes are involved in a series of transformations and nutrient cycling. To evaluate the biotechnological potential of fungi from a mangrove ecosystem, samples from mangrove trees were collected at the Paranagua Estuarine Complex in Brazil and 40 fungal isolates were obtained, cultivated, and screened for hydrolytic and ligninolytic enzymes production, adaptation to salinity and genetic diversity. The results showed a predominance of hydrolytic enzymes and fungal tolerance to ≤ 50 g L−1 sodium chloride (NaCl) concentration, a sign of adaptive halophilia. Through morphological and molecular analyses, the isolates were identified as: Trichoderma atroveride, Microsphaeropsis arundinis, Epicoccum sp., Trichoderma sp., Gliocladium sp., Geotrichum sp. and Cryphonectria sp. The ligninolytic enzymatic potential of the fungi was evaluated in liquid cultures in the presence and absence of seawater and the highest activity of laccase among isolates was observed in the presence of seawater with M. arundinis (LB07), which produced 1,037 U L−1. Enzymatic extracts of M. arundinis fixed at 100 U L−1 of laccase partially decolorized a real textile effluent in a reaction without pH adjustment and chemical mediators. Considering that mangrove fungi are still few explored, the results bring an important contribution to the knowledge about these microorganisms, as their ability to adapt to saline conditions, biodegradation of pollutants, and enzymatic potential, which make them promising candidates in biotechnological processes.

Mycovirus infection has been widely shown to attenuate the virulence of phytopathogenic fungi. Valsa mali is an agriculturally important fungus that causes Valsa canker disease in apple trees. In this study, two unrelated mycoviruses [Cryphonectria hypovirus 1 (CHV1, genus Hypovirus, and single-stranded RNA) and Mycoreovirus 1 (MyRV1, genus Mycoreovirus, double-stranded RNA)] that originated from Cryphonectria parasitica (chestnut blight fungus) were singly or doubly introduced into V. mali via protoplast fusion. CHV1 and MyRV1 stably infected V. mali and caused a reduction in fungal vegetative growth and virulence. Co-infection of both viruses further reduced the virulence of V. mali but compromised the stability of CHV1 infection and horizontal transmission through hyphal anastomosis. Infections of MyRV1 and, to a lesser extent, CHV1 up-regulated the transcript expression of RNA silencing-related genes in V. mali. The accumulation of CHV1 (but not MyRV1) was elevated by the knockdown of dcl2, a key gene of the RNA silencing pathway. Similarly, the accumulation of CHV1 and the efficiency of the horizontal transmission of CHV1 during co-infection was restored by the knockdown of dcl2. Thus, CHV1 and MyRV1 are potential biological control agents for apple Valsa canker disease, but co-infection of both viruses has a negative effect on CHV1 infection in V. mali due to the activation of antiviral RNA silencing by MyRV1 infection.

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.