Interaction between hypoviral-regulated fungal virulence factor laccase3 and small heat shock protein Hsp24 from the chestnut blight fungus Cryphonectria parasitica.

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.

HPLC-MS/MS Method for the Detection of Selected Toxic Metabolites Produced by Penicillium spp. in Nuts.

Penicillium spp. are emerging as producers of mycotoxins and other toxic metabolites in nuts. A HPLC-MS/MS method was developed to detect 19 metabolites produced by Penicillium spp. on chestnuts, hazelnuts, walnuts and almonds. Two extraction methods were developed, one for chestnuts and one for the other three nuts. The recovery, LOD, LOQ and matrix effect were determined for each analyte and matrix. Correlation coefficients were always >99.99%. In walnuts, a strong signal suppression was observed for most analytes and patulin could not be detected. Six strains: Penicillium bialowiezense, P. brevicompactum, P. crustosum, P. expansum, P. glabrum and P. solitum, isolated from chestnuts, were inoculated on four nuts. Chestnuts favored the production of the largest number of Penicillium toxic metabolites. The method was used for the analysis of 41 commercial samples: 71% showed to be contaminated by Penicillium-toxins. Cyclopenin and cyclopenol were the most frequently detected metabolites, with an incidence of 32% and 68%, respectively. Due to the risk of contamination of nuts with Penicillium-toxins, future studies and legislation should consider a larger number of mycotoxins.

Development of fermented chestnut with Bacillus natto: Functional and sensory properties.

Food fermentation can improve food nutritional value and sensory performance, it is considered as an ecofriendly bioprocessing technology. In this work, a fermented natto chestnut food was firstly developed and its active ingredients and functional properties were systematically studied. Through systematic experimental screening, including a single factor experiment and Box-Behnken design, the fermentation parameters of chestnut were optimized and selected. Under the optimal fermentation conditions, fermentation time 56 h, temperature 38 â„ƒ and 5% inoculum concentration, the fibrinolytic activity of the natto-chestnut reached 6479 IU/g. Meanwhile, higher antioxidant activity of the natto-chestnut was obtained due to the increased contents of total phenolic, total flavonoid and VC. In addition, α-glucosidase inhibition activity was also improved in the natto-chestnut. These results indicated that fermented chestnut could be a new dietary supplement with higher quality and better activities for people's health.

Isolation, Characterisation and Experimental Evolution of Phage that Infect the Horse Chestnut Tree Pathogen, Pseudomonas syringae pv. aesculi.

Bleeding canker of horse chestnut trees is a bacterial disease, caused by the bacterium Pseudomonas syringae pv. aesculi, estimated to be present in ~ 50% of UK horse chestnut trees. Currently, the disease has no cure and tree removal can be a common method of reducing inoculum and preventing spread. One potential method of control could be achieved using naturally occurring bacteriophages infective to the causative bacterium. Bacteriophages were isolated from symptomatic and asymptomatic horse chestnut trees in three locations in the South East of England. The phages were found to be belonging to both the Myoviridae and Podoviridae families by RAPD PCR and transmission electron microscopy. Experimental coevolution was carried out to understand the dynamics of bacterial resistance and phage infection and to determine whether new infective phage genotypes would emerge. The phages exhibited different coevolution patterns with their bacterial hosts across time. This approach could be used to generate novel phages for use in biocontrol cocktails in an effort to reduce the potential emergence of bacterial resistance.

Influence of leaf damage by the horse chestnut leafminer (Cameraria ohridella Deschka & Dimic) on mycorrhiza of Aesculus hippocastanum L.

In many parts of Europe, the white horse chestnut (Aesculus hippocastanum L.) has been attacked by the horse chestnut leafminer (Cameraria ohridella Deschka & Dimic), which causes premature leaf dieback. A. hippocastanum L. establishes mutualistic symbiosis with arbuscular mycorrhizal (AM) fungi. This study involved a comparison of mature A. hippocastanum individuals susceptible to C. ohridella and individuals resistant to this insect after a one-time treatment with a chemical preparation injected into the tree trunks 7 years before the investigation began. Concentration of macronutrients in soil and the activity of soil nonspecific dehydrogenase did not differ between soils under canopies of the treated and untreated trees. Concentrations of C and N were significantly higher in leaves of the treated than those of the untreated trees. The infestation by C. ohridella and defoliation of leaves of the untreated trees did not significantly influence the frequency and intensity of AM colonization compared to the chemically treated trees, although a tendency towards higher average AM colonization of roots of the untreated trees, infested by the herbivores, than roots of the non-infested trees was observed. The results also indicated a tendency for higher biomass of fine roots per soil volume under the trees treated against C. ohridella than under the trees invaded by the insect.

A signature of tree health? Shifts in the microbiome and the ecological drivers of horse chestnut bleeding canker disease.

Host susceptibility to pathogens can be shaped by genetic, ecological, and evolutionary factors. The ability to predict the spread of disease therefore requires an integrated understanding of these factors, including effects of pests on pathogen growth and competition between pathogens and commensal microbiota for host resources. We examined interactions between the leaf-mining moth Cameraria ohridella, the bacterial causal agent of bleeding canker disease Pseudomonas syringae pv aesculi, and the bark-associated microbiota of horse chestnut (Aesculus hippocastanum) trees. Through surveys of > 900 trees from 60 sites in the UK, we tested for ecological or life history predictors of leaf miner infestation, bleeding canker, or coinfection. Using culture-independent sequencing, we then compared the bark microbiomes from 46 trees to measure the association between microbiome composition and key ecological variables, including the severity of disease. Both pest and pathogen were found to respond to tree characteristics, but neither explained damage inflicted by the other. However, we found a clear loss of microbial diversity and associated shift in microbiome composition of trees as a function of disease. These results show a link between bark-associated microbiota and tree health that introduces the intriguing possibility that tree microbiota play key roles in the spread of disease.

Cryphonectria hypovirus 1-Induced Changes of Stress Enzyme Activity in Transfected Phytopathogenic Fungus Cryphonectria parasitica.

Cryphonectria parasitica is a phytopathogenic fungus introduced from Eastern Asia to North America and to Europe, where it causes chestnut blight, a devastating disease of chestnut trees. The disease can be biologically controlled utilising the mycovirus Cryphonectria hypovirus 1 (CHV1), which changes the physiology of the host, reducing its virulence towards chestnut. We measured fungal growth in vitro and activities of glutathione S-transferase, catalase and superoxide dismutase, enzymes involved in oxidative stress response, to elucidate the effects of CHV1 infection on the host. Six CHV1 strains of different subtypes and three fungal isolates were used in different combinations to better represent natural conditions, where higher genetic diversity of both fungus and virus is expected. The infection with different CHV1 strains decreased in vitro growth rate of infected fungal isolates and increased activity of their stress enzymes in most of the studied fungus/virus combinations, indicating increased oxidative stress following CHV1 infection. All our field CHV1 strains belong to the Italian subtype, but while strain M56-1 had equal or even stronger effect on its fungal host than prototypic strain EP713 of French subtype F1, strain B11 had no effect. Thus, the severity of the observed effects depended on a particular virus strain, fungal isolate, and the combination of the two, rather than solely on the virus subtype. Since previous research showed discordance between accumulation of mRNA and stress-related proteins in CHV1 infected C. parasitica, our results emphasise the importance of enzymes' activity measurements as an invaluable extension of transcriptomic and proteomic analyses.

Engineering super mycovirus donor strains of chestnut blight fungus by systematic disruption of multilocus vic genes.

Transmission of mycoviruses that attenuate virulence (hypovirulence) of pathogenic fungi is restricted by allorecognition systems operating in their fungal hosts. We report the use of systematic molecular gene disruption and classical genetics for engineering fungal hosts with superior virus transmission capabilities. Four of five diallelic virus-restricting allorecognition [vegetative incompatibility (vic)] loci were disrupted in the chestnut blight fungus Cryphonectria parasitica using an adapted Cre-loxP recombination system that allowed excision and recycling of selectable marker genes (SMGs). SMG-free, quadruple vic mutant strains representing both allelic backgrounds of the remaining vic locus were then produced through mating. In combination, these super donor strains were able to transmit hypoviruses to strains that were heteroallelic at one or all of the virus-restricting vic loci. These results demonstrate the feasibility of modulating allorecognition to engineer pathogenic fungi for more efficient transmission of virulence-attenuating mycoviruses and enhanced biological control potential.

The victorivirus Helminthosporium victoriae virus 190S is the primary cause of disease/hypovirulence in its natural host and a heterologous host.

A transmissible disease of the plant pathogenic fungus Helminthosporium victoriae, the causal agent of Victoria blight of oats, was reported more than 50 years ago. Diseased, but not normal, isolates, of H. victoriae contain two distinct viruses designated according to their sedimentation values as victorivirus Helminthosporium victoriae virus 190S (HvV190S) and chrysovirus Helminthosporium victoriae 145S (HvV145S). Although a viral etiology of the disease was previously proposed, conclusive evidence was lacking. Here we present unequivocal evidence based on transfecting virus-free H. victoriae protoplasts with purified virus particles showing that HvV190S is essential for disease development. Furthermore, we show an expansion of the host range of HvV190S to include Cryphonectria parasitica and we also show similarity in a subset of phenotypic traits between HvV190S-infected RNA silencing deficient mutant (Δdcl-2) of C. parasitica and a strain of H. victoriae. In virulence assays on detached American chestnut branches and Red Delicious apple fruits, HvV190S-infected C. parasitica strain Δdcl-2 was markedly less virulent than wild type and virus-free Δdcl-2 C. parasitica strains. Furthermore, the hypovirulent HvV190S-infected C. parasitica Δdcl-2 strain exhibited strong antifungal activity in dual culture with the plant pathogenic fungus Sclerotinia sclerotiorum. No such inhibitory activity was observed in comparable dual cultures with wild type and virus-free Δdcl-2 C. parasitica strains. The discovery that infection with HvV190S induced a hypovirulent phenotype in a heterologous plant pathogenic host is very significant since it might be possible to convert other economically important plant pathogenic fungi to hypovirulence using HvV190S.

A Permutation-Randomization Approach to Test the Spatial Distribution of Plant Diseases.

The analysis of the spatial distribution of plant diseases requires the availability of trustworthy geostatistical methods. The mean distance tests (MDT) are here proposed as a series of permutation and randomization tests to assess the spatial distribution of plant diseases when the variable of phytopathological interest is categorical. A user-friendly software to perform the tests is provided. Estimates of power and type I error, obtained with Monte Carlo simulations, showed the reliability of the MDT (power > 0.80; type I error < 0.05). A biological validation on the spatial distribution of spores of two fungal pathogens causing root rot on conifers was successfully performed by verifying the consistency between the MDT responses and previously published data. An application of the MDT was carried out to analyze the relation between the plantation density and the distribution of the infection of Gnomoniopsis castanea, an emerging fungal pathogen causing nut rot on sweet chestnut. Trees carrying nuts infected by the pathogen were randomly distributed in areas with different plantation densities, suggesting that the distribution of G. castanea was not related to the plantation density. The MDT could be used to analyze the spatial distribution of plant diseases both in agricultural and natural ecosystems.

The evolution of bacterial resistance against bacteriophages in the horse chestnut phyllosphere is general across both space and time.

Insight to the spatial and temporal scales of coevolution is key to predicting the outcome of host-parasite interactions and spread of disease. For bacteria infecting long-lived hosts, selection to overcome host defences is just one factor shaping the course of evolution; populations will also be competing with other microbial species and will themselves be facing infection by bacteriophage viruses. Here, we examine the temporal and spatial patterns of bacterial adaptation against natural phage populations from within leaves of horse chestnut trees. Using a time-shift experiment with both sympatric and allopatric phages from either contemporary or earlier points in the season, we demonstrate that bacterial resistance is higher against phages from the past, regardless of spatial sympatry or how much earlier in the season phages were collected. Similarly, we show that future bacterial hosts are more resistant to both sympatric and allopatric phages than contemporary bacterial hosts. Together, our results suggest the evolution of relatively general bacterial resistance against phages in nature and are contrasting to previously observed patterns of phage adaptation to bacteria from the same tree hosts over the same time frame, indicating a potential asymmetry in coevolutionary dynamics.

Biodegradation of chestnut shell and lignin-modifying enzymes production by the white-rot fungi Dichomitus squalens, Phlebia radiata.

As a discarded lignocellulosic biomass, chestnut shell is of great potential economic value, thus a sustainable strategy is needed and valuable for utilization of this resource. Herein, the feasibility of biological processes of chestnut shell with Dichomitus squalens, Phlebia radiata and their co-cultivation for lignin-modifying enzymes (LMEs) production and biodegradation of this lignocellulosic biomass was investigated under submerged cultivation. The treatment with D. squalens alone at 12 days gained the highest laccase activity (9.42 ± 0.73 U mg(-1)). Combined with the data of laccase and manganese peroxidase, oxalate and H2O2 were found to participate in chestnut shell degradation, accompanied by a rapid consumption of reducing sugar. Furthermore, specific surface area of chestnut shell was increased by 77.6-114.1 % with the selected fungi, and total pore volume was improved by 90.2 % with D. squalens. Meanwhile, the surface morphology was observably modified by this fungus. Overall, D. squalens was considered as a suitable fungus for degradation of chestnut shell and laccase production. The presence of LMEs, H2O2 and oxalate provided more understanding for decomposition of chestnut shell by the white-rot fungi.

[Micrococcus sp.--the pathogen of leaf necrosis of horse-chestnuts (Aesculus L.) in Kiev].

A group of phytopathogenic bacteria was isolated from patterns of drying horse-chestnuts (Aesculus L.), which grow in Kyiv. The properties of slowly growing, highly aggressive microorganisms have been described in the paper. They grow up on the 8-10th day after sowing. The investigated microorganisms form very small (0.5-1 mm in diameter) colonies on the potato agar. Bacteria are protuberant, shining, smooth with flat edges, they are pale yellow, yellow, or pink. The bacteria are Gram-positive, spherical, are disposed in smears singly, in pairs, as accumulations, or netting. They are aerobes, do not form spores, are not mobile. They are inert in respect of different sources of carbon. They reduce nitrates, do not dilute gelatin, do not hydrolyze starch, do not release hydrogen sulphide and indole. The bacteria are catalase-positive, oxidase-negative. They do not cause potato and carrot rot. They lose quickly their viability under the laboratory conditions. The saturated acids C 14:0; C 15:0; C16:0; C18:0 have been revealed in the composition of cellular fatty acids. Microorganisms are identified as Micrococcus sp. Under artificial inoculation this highly aggressive pathogen causes drying of the horse-chestnut buds and necrosis, which occupies 1/3-1/2 of the leaf plate. A wide zone of chlorosis, surrounding necrosis, may occupy the whole leaf surface. The infected leaves use to twist up from the top (apex) or along a midrib and to dry.

Hypovirus virulence and vegetative incompatibility in populations of the chestnut blight fungus.

Cryphonectria hypovirus 1 hyperparasitizes the chestnut blight fungus Cryphonectria parasitica and acts as a biocontrol agent for this serious tree disease. The virus is transmitted cytoplasmatically between fungal individuals. However, highly virulent viruses strongly debilitate their host and, thus, reduce their own transmission probability. Furthermore, vegetative incompatibility between fungi is an important transmission barrier. Therefore, virulent viruses are expected to be strongly selected against in fungal populations with high levels of vegetative incompatibility, eventually leading to the erosion of biocontrol. To test this prediction, we assessed the virulence of the virus in four European C. parasitica populations with high diversity of vegetative compatibility types and in four populations with low diversity. We expected the degree of virus virulence to be lower in fungal populations with high levels of vegetative incompatibility. However, our results did not reveal such a trend. No significant differences in virus virulence between populations with low versus high diversity of vegetative compatibility types were observed. There was no evidence for an erosion of disease control due to the presence of these transmission barriers. Thus, the findings of this study are promising for the sustainability of Cryphonectria hypovirus 1 as a biocontrol agent for chestnut blight in Europe.

Histological examination of horse chestnut infection by Pseudomonas syringae pv. aesculi and non-destructive heat treatment to stop disease progression.

Since its emergence in Northwest Europe as a pathogen that infects trunks and branches of Aesculus spp. (the horse chestnuts) approximately one decade ago, Pseudomonas syringae pv. aesculi has rapidly established itself as major threat to these trees. Infected trees exhibit extensive necrosis of phloem and cambium, which can ultimately lead to dieback. The events after host entry leading to extensive necrosis are not well documented. In this work, the histopathology of this interaction is investigated and heat-treatment is explored as method to eradicate bacteria associated with established infections. The early wound-repair responses of A. hippocastanum, both in absence and presence of P. s. pv. aesculi, included cell wall lignification by a distinct layer of phloem and cortex parenchyma cells. The same cells also deposited suberin lamellae later on, suggesting this layer functions in compartmentalizing healthy from disrupted tissues. However, monitoring bacterial ingress, its construction appeared inadequate to constrain pathogen spread. Microscopic evaluation of bacterial dispersal in situ using immunolabelling and GFP-tagging of P. s. pv. aesculi, revealed two discriminative types of bacterial colonization. The forefront of lesions was found to contain densely packed bacteria, while necrotic areas housed bacterial aggregates with scattered individuals embedded in an extracellular matrix of bacterial origin containing alginate. The endophytic localization and ability of P. s. pv aesculi to create a protective matrix render it poorly accessible for control agents. To circumvent this, a method based on selective bacterial lethality at 39 °C was conceived and successfully tested on A. hippocastanum saplings, providing proof of concept for controlling this disease by heat-treatment. This may be applicable for curing other tree cankers, caused by related phytopathogens.

Using experimental evolution to explore natural patterns between bacterial motility and resistance to bacteriophages.

Resistance of bacteria to phages may be gained by alteration of surface proteins to which phages bind, a mechanism that is likely to be costly as these molecules typically have critical functions such as movement or nutrient uptake. To address this potential trade-off, we combine a systematic study of natural bacteria and phage populations with an experimental evolution approach. We compare motility, growth rate and susceptibility to local phages for 80 bacteria isolated from horse chestnut leaves and, contrary to expectation, find no negative association between resistance to phages and bacterial motility or growth rate. However, because correlational patterns (and their absence) are open to numerous interpretations, we test for any causal association between resistance to phages and bacterial motility using experimental evolution of a subset of bacteria in both the presence and absence of naturally associated phages. Again, we find no clear link between the acquisition of resistance and bacterial motility, suggesting that for these natural bacterial populations, phage-mediated selection is unlikely to shape bacterial motility, a key fitness trait for many bacteria in the phyllosphere. The agreement between the observed natural pattern and the experimental evolution results presented here demonstrates the power of this combined approach for testing evolutionary trade-offs.

Local biotic environment shapes the spatial scale of bacteriophage adaptation to bacteria.

The ecological, epidemiological, and evolutionary consequences of host-parasite interactions are critically shaped by the spatial scale at which parasites adapt to hosts. The scale of interaction between hyperparasites and their parasites is likely to be influenced by the host of the parasite and potentially likely to differ among within-host environments. Here we examine the scale at which bacteriophages adapt to their host bacteria by studying natural isolates from the surface or interior of horse chestnut leaves. We find that phages are more infective to bacteria from the same tree relative to those from other trees but do not differ in infectivity to bacteria from different leaves within the same tree. The results suggest that phages target common bacterial species, including an important plant pathogen, within plant host tissues; this result has important implications for therapeutic phage epidemiology. Furthermore, we show that phages from the leaf interior are more infective to their local hosts than phages from the leaf surface are to theirs, suggesting either increased resistance of bacteria on the leaf surface or increased phage adaptation within the leaf. These results highlight that biotic environment can play a key role in shaping the spatial scale of parasite adaptation and influencing the outcome of coevolutionary interactions.

Cpkk1, MAPKK of Cryphonectria parasitica, is necessary for virulence on chestnut.

ABSTRACT The role of Cpkk1, a mitogen-activated protein kinase from Cryphonectria parasitica, was investigated by generating a number of mutant strains that overexpress, under the control of the cryparin promoter, both the wild-type protein and its allele with an extensive deletion in the catalytic domain. Furthermore, a hairpin construct was built and expressed to cause specific silencing of Cpkk1 mRNA transcripts. Specific mRNA silencing or overexpression was confirmed on both Northern and Western blot analysis. Selected C. parasitica strains with Cpkk1 either silenced or overexpressed were evaluated for their biological characteristics, including virulence on European chestnut, growth on different substrates, conidial sporulation, and resistance to cell-wall-degrading enzymes. Silencing of Cpkk1 and the overexpression of a defective Cpkk1 correlated with a marked reduction in virulence on 3-year-old chestnut trees, with no statistically significant effect on fungal growth in the various conditions tested.

Multiple introductions of divergent genetic lineages in an invasive fungal pathogen, Cryphonectria parasitica, in France.

The occurrence of multiple introductions may be a crucial factor in the successful establishment of invasive species, but few studies focus on the introduction of fungal pathogens, despite their significant effect on invaded habitats. Although Cryphonectria parasitica, the chestnut blight fungus introduced in North America and Europe from Asia during the 20th century, caused dramatic changes in its new range, the history of its introduction is not well retraced in Europe. Using 10 microsatellite loci, we investigated the genetic diversity of 583 isolates in France, where several introductions have been hypothesized. Our analyses showed that the seven most frequent multilocus genotypes belonged to three genetic lineages, which had a different and geographically limited distribution. These results suggest that different introduction events occurred in France. Genetic recombination was low among these lineages, despite the presence of the two mating types in each chestnut stand analysed. The spatial distribution of lineages suggests that the history of introductions in France associated with the slow expansion of the disease has contributed to the low observed rate of recombination among the divergent lineages. However, we discuss the possibility that environmental conditions or viral interactions could locally reduce recombination among genotypes.

Hypovirus-responsive transcription factor gene pro1 of the chestnut blight fungus Cryphonectria parasitica is required for female fertility, asexual spore development, and stable maintenance of hypovirus infection.

We report characterization of the gene encoding putative transcription factor PRO1, identified in transcriptional profiling studies as being downregulated in the chestnut blight fungus Cryphonectria parasitica in response to infection by virulence-attenuating hypoviruses. Sequence analysis confirmed that pro1 encodes a Zn(II)(2)Cys(6) binuclear cluster DNA binding protein with significant sequence similarity to the pro1 gene product that controls fruiting body development in Sordaria macrospora. Targeted disruption of the C. parasitica pro1 gene resulted in two phenotypic changes that also accompany hypovirus infection, a significant reduction in asexual sporulation that could be reversed by exposure to high light intensity, and loss of female fertility. The pro1 disruption mutant, however, retained full virulence. Although hypovirus CHV1-EP713 infection was established in the pro1 disruption mutant, infected colonies continually produced virus-free sectors, suggesting that PRO1 is required for stable maintenance of hypovirus infection. These results complement the recent characterization of the hypovirus-responsive homologue of the Saccharomyces cerevisiae Ste12 C(2)H(2) zinc finger transcription factor gene, cpst12, which was shown to be required for C. parasitica female fertility and virulence.