Fungus Cryphonectria Parasitica Research Articles

The role of production site isolation in the plant health situation of sweet chestnut (Castanea sativa)

The most destructive pathogen for the European chestnut is the blight fungus Cryphonectria parasitica (Murr.) Barr. The spread of the fungus was very fast in Europe within a few decades in the second half of the past century. During the tree-health checking in the chestnut andwalnut plantation in Romania, Hargita county, next to Homoródkarácsonyfalva village, we especially concentraded on the signs of blight disease occurrence. The grove is laying on a western slope, under a pine forest. This favourable geographical space protects it not only from pathogen attacts, but it has a special, mild microclimate for nut and chestnut trees. The European chestnut could be a valuable member of local forests, opening a new perspective under conditions of climate changes.

Occurrence of hypovirus-infected Cryphonectria parasitica isolates in northern Spain: an encouraging situation for biological control of chestnut blight in Asturian forests

The presence of hypovirulent (hypovirus-infected) isolates of the chestnut blight fungus Cryphonectria parasitica in Asturias, northern Spain, is first reported by using both morphological and molecular assessments. Hypovirulence was detected in 16 of the 69 councils where the fungus was previously observed. In total 30 of the 863 C. parasitica isolates analyzed were found to be hypovirus-infected. The isolates belonged to the two main vegetative compatibility (vc) types (EU-1 and EU-13) of the region. Asturian hypoviruses were genetically characterized by sequencing of ORFA and ORFB specific regions of the hypoviral RNA. The results show that 70% of the investigated hypovirulent isolates contained the Spanish subtype (subtype E) while 30% isolates the German subtype (D) of Cryphonectria hypovirus 1 (CHV-1). The presence of hypovirulent isolates compatible with the dominant vc types and the low vc diversity in this region favours the application of biological control. An initial disease management effort could be applied by using the hypovirus of subtype D, whereas further research related to the biocontrol properties of the subtype E hypovirus should be conducted.

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.

Factors influencing growth, sporulation and virus transfer in Cryphonectria parasitica isolates from Castilla and León (Spain)

Biological control with Cryphonectria hypovirus CHV1 of the chestnut blight, caused by the fungus Cryphonectria parasitica, has reduced the impact of the disease in Europe. The virus reduces the virulence of the fungus so that it causes non-lethal cankers, thus enabling the chestnut trees to overcome the disease. The virus can be transmitted horizontally by hyphal anastomosis or vertically to the conidia. In this study, we investigated growth and sporulation of the fungus as well as rates of horizontal transmission of the virus at different temperatures. We used fungal isolates of the vegetative compatibility types (vc types) that are most prevalent in Castilla and Leon (central northern Spain) to evaluate the effects of fungal strain on the parameters tested. In addition, we infected four isolates of C. parasitica with hypovirus subtypes CHV1-F1 and CHV1-I, to determine the influence of virus subtype on growth, sporulation and virus transfer. We assessed growth of fungal colonies and horizontal transmission of the virus at 15 °C and 25 °C. Colony growth was affected by an interaction between fungal isolates included in vc type EU1 or EU11 and virus at both 15 °C and 25 °C. However, horizontal transmission of the virus was only influenced by the fungal genotype of isolates included in vc type EU1 or EU11, and spore production was only affected by the virus subtype. Vertical transmission was also influenced by the fungal isolate and virus subtype. Growth of the fungal isolates varied depending on the virus subtype with which they were infected. This supports the theory that fungal host and virus subtype influence transmission and dissemination of hypovirulence. The fungal genotype affects colony growth and horizontal transmission of the virus. It is common to expect a good dissemination of the hypovirus with a low vc type diversity but the selection of the best combination of hypovirus and fungal isolate is crucial for the success of biological control not only for small areas but in larger chestnut populations as well.

Biological control of chestnut blight in Croatia: an interaction between host sweet chestnut, its pathogen Cryphonectria parasitica and the biocontrol agent Cryphonectria hypovirus 1.

Chestnut blight, caused by the fungus Cryphonectria parasitica, is a severe chestnut disease that can be controlled with naturally occurring hypoviruses in many areas of Europe. The aim of this research was to measure the effect of different Cryphonectria hypovirus 1 (CHV1) strains on the growth of the fungal host and select strains that could potentially be used for human-mediated biocontrol in forests and orchards, and to investigate whether and how chestnut-fungus-virus interactions affect the development and growth of the lesion area on cut stems. Two Croatian CHV1 strains (CR23 and M56/1) were selected as potential biocontrol agents. The sequencing of CHV1/ORF-A showed that both of these virus strains belonged to the Italian subtype of CHV1. In vitro transfection of selected virus strains from hypovirulent to genetically diverse virus-free fungal isolates and subsequent inoculation of all virus/fungus combinations on stems of genetically diverse sweet chestnut trees revealed that Croatian virus strain CR23 had an equally hypovirulent effect on the host as the strong French strain CHV1-EP713, while M56/1 had a weaker effect. Furthermore, it was shown that in some cases the same hypovirus/fungus combinations induced various degrees of canker development on different chestnut genotypes. Some CHV1 strains belonging to the Italian subtype have similar hypovirulent effects on C. parasitica to those belonging to the French subtype. Furthermore, chestnut susceptibility and recovery could be influenced by the response of chestnut trees to particular hypovirulent C. parasitica isolates, and virus-fungus-chestnut interactions could have significant implications for the success of chestnut blight biocontrol. © 2016 Society of Chemical Industry.

Comprehension of resistance to diseases in chestnut

Ink disease (caused by the pathogen Phytophthora cinnamomi) and chestnut blight (caused by the fungus Cryphonectria parasitica) are the two most destructive diseases affecting European chestnut (Castanea sativa) and American chestnut (C. dentata). Therefore, breeding for resistance to both pathogens is essential for the chestnut sustainability in Europe and the United States of America. Several genomic approaches have been implemented in order to map the resistance first to ink disease and then to blight chestnut using a hybrid population: C. sativa crossed with resistant Asian species (C. crenata and C. mollissima). The transcriptome of C. sativa and C. crenata inoculated and not with P. cinnamomi was recently sequenced and allowed the identification of genes putatively involved in ink disease resistance. Taking advantage of molecular markers (microsatellite and SNPs), developed from the referred tanscriptomes and from C. mollissima transcriptome, the first C. sativa x C. crenata genetic map was constructed. Additionally, a reliable phenotyping method was established to evaluate the level of P. cinnamomi resistance of each hybrid allowing the identification of two Quantitative Trait Loci (QTLs). These results are the first step for understanding the resistance to ink disease in chestnut.

A Mutant of the Bck1 Homolog from Cryphonectria parasitica Resulted in Sectorization with an Impaired Pathogenicity.

CpBck1, an ortholog of the cell-wall integrity mitogen-activated protein kinase kinase kinase of Saccharomyces cerevisiae, was cloned and characterized from the chestnut blight fungus Cryphonectria parasitica. The CpBck1-null mutant displayed cell wall integrity-related phenotypic changes such as abnormal cell morphology and wall formation and hypersensitivity to cell wall-disrupting agents. In addition, the mutant showed severely retarded growth without any sign of normal development, such as hyphal differentiation, conidiation, or pigmentation. As the culture proceeded, the mutant colony showed sporadic sectorization. Once sectored, the sectored phenotype of robust mycelial growth without differentiation was stably inherited. Compared with the wild type, both the parental CpBck1-null mutant and the sectored progeny exhibited marked impaired virulence. The present study revealed that a mutation in a signaling pathway component related to cell-wall integrity resulted in sporadic sectorization and these sectored phenotypes were stably inherited, suggesting that this signal transduction pathway is implicated in adaptive genetic changes for sectorization.

Chestnut tree: an appropriate choich for rural developement in Europe (review)

In chestnut plantations or in associations many pathogens and pests can be found. They are causing more than a hundred year problem in chestnut protection, for example the blight fungus Cryphonectria parasitica. Lot of breeders hybridized the Castanea sativa and Castanea dentata with the tolerant asian species. Nowadays the presence of Dryocosmus kuriphilus seems to be the main risk. However, there are more and more chestnut plantations in Europe because of the extensive growth of needs. The food industry produces many products from chestnut, but the wood industry also use it more and more. In most of European countries the chestnut stands represent landscape aesthetics and touristic value, but probably the number of farmers inquiring the plantation will be increase in Hungary.

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.

Heterokaryon analysis of a Cdc48-like gene, CpCdc48, from the chestnut blight fungus Cryphonectria parasitica demonstrates it is essential for cell division and growth

Functional analysis of a cell division cycle 48 (CDC48) ortholog, CpCdc48, from Cryphonectria parasitica was performed via construction of a CpCdc48-null mutant. Genotype analysis revealed that the putative CpCdc48-null mutant was a heterokaryotic transformant containing two different types of nuclei (i.e., one with the wild-type CpCdc48 allele and the other with the CpCdc48-null mutant allele). Although stable mycelial growth of the heterokaryotic transformant was observed on media containing hygromycin B, neither germination nor growth of the resulting spores was observed on selection media, suggesting that the CpCdc48 gene is essential. Microscopic analysis of germinated conidia from the heterokaryon demonstrated that although there were normal germinating spores due to the wild-type conidia, there were many residual conidia that did not germinate. However, with prolonged incubation, non-germinating conidia began to swell into gigantic globose spores. DAPI staining and FACS analysis of the gigantic spores revealed the presence of multiple nuclei. These gigantic conidia did not show any signs of polarized growth and underwent autolysis with further incubation. These findings indicate that the CpCdc48 gene is responsible for delayed cell cycle during spore germination, resulting in some karyokinesis, but not following spore cytokinesis. Thus, CpCdc48 is essential for cell division and polarized growth.

Using molecular markers to assess the establishment and spread of a mycovirus applied as a biological control agent against chestnut blight

Chestnut blight, caused by the invasive fungus Cryphonectria parasitica, is one of the few tree diseases in Europe for which biological control is possible. The fungus may be infected by a mycovirus that reduces its virulence and sporulation capacity. In this study, we used molecular markers to assess the establishment and dissemination of the Cryphonectria hypovirus (CHV1) after its release as a biological control agent. The study was conducted in two chestnut stands in northern Switzerland. In both stands, viruses were repeatedly introduced by conducting classical canker treatments. Sequence analysis of viral isolates revealed the presence of the released viruses in the majority of the treated cankers, confirming that therapeutic canker treatment is mostly effective. Analysis of the dissemination of the released viruses gave contradictory results. In both stands, more than 50 % of the sampled untreated cankers were found to be virus-infected. In one stand, all viral isolates from those cankers originated from the released viruses, which evidently had spontaneously spread in the local C. parasitica population. In the other stand, the released virus was outcompeted by a naturally occurring virus, which was dominant in untreated cankers. Our study emphasizes the usefulness of molecular markers to track biological control agents and to evaluate the success of their application.

Interaction between two invasive organisms on the European chestnut: does the chestnut blight fungus benefit from the presence of the gall wasp?

The impact of invasive fungal pathogens and pests on trees is often studied individually, thereby omitting possible interactions. In this study the ecological interaction between the chestnut blight fungus Cryphonectria parasitica and the chestnut gall wasp Dryocosmus kuriphilus was investigated. We determined if abandoned galls could be colonized by C. parasitica and thereby act as an entry point and a source of pathogen inoculum. Moreover we assessed the identity and diversity of other gall-colonizing fungal species. A total of 1973 galls were randomly sampled from 200 chestnut trees in eight Swiss stands. In a stand C. parasitica was isolated from 0.4-19.2% of the galls. The incidence of C. parasitica on the galls and the fungal diversity significantly increased with the residence time of D. kuriphilus in a stand. All but one C. parasitica cultures were virulent. The predominant fungus isolated from galls was Gnomoniopsis castanea whose abundance influenced negatively that of C. parasitica. This study shows that D. kuriphilus galls can be colonized by virulent strains of the chestnut blight fungus C. parasitica. This can have effects on the chestnut blight incidence even in chestnut stands where the disease is successfully controlled by hypovirulence. The gall wasp presence influences also the fungal species composition on chestnut trees.

Diversity of Cryphonectria parasitica populations from the Carpathian Basin.

The ascomycetous fungus Cryphonectria parasitica (Murr.) is one of the most important fungal pathogens of chestnut (Castanea sativa Mill.), causing chestnut blight. It is originated from Eastern Asia and was introduced into North America, and later into Europe. Almost 100 C. parasitica isolates were collected mainly from the Carpathian Basin to study their diversity and phylogenetic relationships. Three nuclear molecular markers were tested: (i) internal transcribed spacers (ITS), (ii) translation elongation factor 1 subunit alpha (tef1), and (iii) six microsatellite loci. The ITS region proved to be highly polymorphic but this kind of variability did not reflect the geographical regions of the isolates, while the tef1 sequences were identical in all isolates. Microsatellite haplotype diversity was relatively high (0.923-0.984) calculated for the fungal populations of the Carpathian Basin. This may have resulted from the multiple introductions from diverse origins of the populations, close to the first site of observation in Northern Italy. The BAPS analysis of the microsatellite markers occasionally grouped together samples from remote geographical origin, suggesting human assistance in the introduction of new C. parasitica haplotypes from distinct areas.

Cryphonectria parasitica isolates of the same vegetative compatibility type display different rates of transfer of CHV1 hypovirus

Hypovirulent strains of the chestnut blight fungus Cryphonectria parasitica have not been registered in the autonomous region of Castilla y Leon (Spain), except in the province of Leon. In this laboratory-based study, we analyzed the rates of horizontal transmission of hypovirus CHV1 subtype F1, isolated from chestnut stands in Leon. We tested the conversion capacity of the six vegetative compatibility (vc) types of C. parasitica isolates most commonly distributed in Castilla y Leon (EU1, EU11, EU12, EU66, CL5 and CL6). We investigated conversion rates of virulent isolates into hypovirulent isolates between pairings of isolates of the same vc type (EU1, EU11 and CL6) and also tested cross conversion rates between isolates of different vc types (EU1, EU11 and CL6 as donors and EU12, EU66, CL5 and CL6 as recipients). We carried out the hypovirus transmission assay with 1700 pairings, of which 700 had donor and recipient isolates of the same vc type and 1000 pairings of isolates had different vc types. Our results show that the conversion frequency to hypovirulent isolates was significantly affected by the vc type, the genotype of isolates with the same origin (province) and the interaction between both factors. In the conversion between isolates of the same vc type, the conversion rates were better with EU1 (ranging between 56 and 94 %) than with EU11 (varying from 4 to 58 %). In the cross conversion between donor and recipient isolates of different vc types, only CL5 and CL6 recipients were converted and the conversion of recipient isolates of EU12 and EU66 failed in all cases. For CL5 as recipient isolate, the conversion rates were similar with EU11 and CL6 as the donor isolates. Recipient isolates of CL6 had good conversion with donor isolates from the same vc type (CL6) and in cross conversion the results were better with donor EU1. Fungal isolates from chestnut stands in Leon displayed the best conversion rates, followed by those from Zamora, Salamanca and Avila when the donor and the recipient isolates were of the same vc type. The fungal isolates appear to have a strong influence on the transmission rate of the hypovirus, at least between strains isolated from chestnut stands in Castilla y Leon. EU1 isolates were more susceptible to conversion than the isolates from EU11. The results highlight the differences in hypovirus transmission, regarding vc types and the genotype of isolates from the same province, when donor and recipient isolates are of the same vc type. Further transmission assays would be useful to determine why hypovirus transmissions, when compared to the hypovirus transmission of other European assays, have different conversion rates with fungal isolates from Castilla y Leon.

Effect of the media on morphology of Cryphonectria parasitica (Murr.) Barr isolates and their Vegetative Compatibility Groups

The most dangerous pathogen for the European and American chestnuts is the blight fungus Cryphonectria parasitica (Murrill) Barr. Short after its introduction a big number of chestnut trees were destroyed on the infested area. The control could be really complicated, because of the numerous vegetative compatibility groups of the fungus. There is a type that carries a mycovirus viz. hypovirus in the cytoplasm. We are able to control effectively this pathogen by using mycovirus-carrying strains (called hypovirulent fungal strains also). In laboratory it is easy to multiply the virulent and the hypovirulent strains of the fungus but do not easy to differentiate colonies visually on simple PDA medium. During our research, we tested different types of media, based on potato and chestnut bark extract respectively. It was observed that on potato medium the virulent strains produce more orange pigments. So it is more easy to differentiate virulent or hypovirulent isolates of chestnut blight fungus based on colony colour and morphology.

Chestnut Blight Control by Agrochemicals in Castanea sativa under Managed Conditions

This research was conducted with the aim of evaluating the in vivo effectiveness of three agrochemical products to control chestnut blight disease in Castanea sativa infected by the fungus Cryphonectria parasitica under managed conditions. Three products (epoxiconazole, carbendazim and a mixture of carbendazim plus flutriafol) were tested using three different methods of application: i) preventive method applied by spraying, ii) curative method applied by spraying and iii) curative method applied directly over the lesion with a paint roller. Lesion length, sporulation and mortality parameters were evaluated presenting statistically significant differences. According to the methods, results showed that the curative method applied directly over the lesion is effective when the pathogen has not already spread, being the most effective application method in the short term. Regarding products, significant differences between control and treated chestnut plants were always observed, epoxiconazole being the most effective fungicide for chestnut blight control.

Controlo biológico do cancro do castanheiro: Deteção, identificação e caracterização do Hypovirus - CHV1

The chestnut blight caused by the fungus Cryphonectria parasitica is considered a major cause of the decline and disappearance of chestnut trees across Europe. C. parasitica is an ascomycete fungus (Diaporthales) that is native to eastern Asia. Infection of chestnut plants with this fungus is typically associated with extensive necrosis (cankers) of the bark on stems and branches, resulting in the subsequent death of the tree. Hypovirulence is a specific method for biological control of Chestnut Blight, it is an infection of C. parasitica with Cryphonectria hypovirus 1 (CHV1), a unencapsidate dsRNA virus of the genus Hypovirus. Biological control with hypovirulent strains is considered an efficient method to control the disease and improve the host plant recovery. The field application needs laboratorial production of compatible hypovirulent strain and introduction by inoculation at the margin of active cankers. In this work it was intended to identify and characterize the hypovirus with potential ability to integrate biological control programmes against Chestnut Blight. Using molecular techniques 11 hypovirus with hypovirulence potential were identified on white isolates of C. parasitica isolated on cankers from northeast of Portugal.

English

American chestnut (Castanea dentata Marsh. Borkh.) is a once- dominant hardwood species with the potential to be a valuable restoration tree for use on surface mined lands in the Appalachian region. Coupling soil ripping and plowing and disking with plantings of American and backcrossed chestnuts has resulted in high seedling survival on a reclaimed surface mine site in southeast Ohio. The objective of this study was to evaluate flowering, chestnut bur production (seed), and natural cankers caused by chestnut blight fungus (Cryphonectria parasitica) on three chestnut breeding lines, in three soil tillage treatments after seven field seasons. Pure American (Castanea dentata), and two types of C. dentata × C. mollissima hybrids (BC2F1 and BC3F1) were documented. Reproduction potential was measured via flower production in June. Canker incidence was recorded as necrotic bark lesions with the presence of orange spore bearing structures. When reproduction potential was compared among seed types, there were no differences; all chestnuts trees were flowering and producing chestnut burs after 7 seasons. Soil treatment had no impact on flowering incidence; however, hybrid breeding lines had a mild effect. Canker incidence and presence of flowers were not related statistically. When natural canker incidence was compared, pure American chestnut exhibited the most infection (P < 0.0001). There were also notable treatment effects, plots that applied the deep ripping had greater disease incidence on pure American chestnuts (P < 0.0001). Long-term survival and stand stability will depend on chestnut's tolerance to the blight at an age of fruiting and flowering. Results after seven years suggest that hybrids were exhibiting a decrease in blight incidence and were flowering and producing burs. Employing deep ripping methods to backcrossed American chestnut plantings provide a viable method for hardwood seedling establishment in soils impacted by surface mining.

Engineered Chestnut Withstands Blight but Spares Fungi at Its Roots

Chestnut trees that are genetically engineered to resist the pathogenic fungus Cryphonectria parasitica remain good hosts for symbiotic fungi that grow along their roots, according to William A. Powell of the SUNY College of Environmental Science and Forestry (ESF) in Syracuse, N.Y., and his collaborators. This sparing of benign fungi helps in overcoming a potentially important regulatory concern, keeping this transgenic variety of chestnut, Castanea dentate, on track for eventual widespread planting, he says. Details appeared 17 October 2014 in Applied and Environmental Microbiology (doi:10.1128/AEM.02169-14).

75 years of the chestnut blight fungus Cryphonectria parasitica (Murr.) Barr in Europe

The chestnut blight fungus Cryphonectria parasitica is a native pathogen in East Asia and has been introduced into North America and Europe. Historical records and population genetic studies revealed at least three major introduction events from Asia into Europe.&#x0D; Nowadays, chestnut blight is present in almost the entire distribution range of European chestnut, i.e. from the Iberian Peninsula to the Caucasus. The C. parasitica population in most countries has been studied in respect to the diversity of vegetative compatibility (vc) types and the occurrence of hypovirulence. The vc type diversity of the different populations varied considerably. Typically, a high diversity of vc types has been found in areas with a long history of chestnut blight and where sexual recombination between divergent genotypes commonly has occurred. On the other hand, newly established populations often showed a low diversity with only one, or a few vc types present.&#x0D; Hypovirulence, i.e. the occurrence of C. parasitica isolates infected by Cryphonectria hypovirus 1 has been found widespread in Europe. Natural dissemination and active biological control applications have lead to a high prevalence of the hypovirus and to the recovery of many chestnut stands. Virulent cankers became hypovirus-infected within a short time and ceased expansion. There is concern that the diversity of vegetative compatibility types could increase in Europe through sexual reproduction between C. parasitica genotypes originating from different introductions. A higher level of vegetative incompatibility would not only hamper hypovirus spread within a population but could also select for lower virulence in CHV-1 and subsequently lead to an erosion of biological control. Recent studies, however, indicate that the vc type barriers are not so restrictive than previously assumed and that so far no evidence for an erosion of biological control system in high diversity populations can be observed.