A real‐time PCR assay for improved rapid,specific detection of Cryphonectria parasitica

Cryphonectria parasitica, an ascomycete fungus, is the causal agent of chestnut blight. This highly destructive disease of chestnut trees causes significant losses, and is therefore a regulated pathogen in Europe. Existing methods for the detection of C. parasitica include morphological identification following culturing, or PCR; however, these are time‐consuming resulting in delays to diagnosis. To allow improved detection, a new specific real‐time PCR assay was designed to detect C. parasitica directly from plant material and fungal cultures, and was validated according to the European Plant Protection Organisation (EPPO) standard PM 7/98. The analytical specificity of the assay was tested extensively using a panel of species taxonomically closely related to Cryphonectria, fungal species associated with the hosts and healthy plant material. The assay was found to be specific to C. parasitica, whilst the analytical sensitivity of the assay was established as 2 pg µL^?1 of DNA. Comparative testing of 63 samples of naturally infected plant material by the newly developed assay and traditional morphological diagnosis demonstrated an increased diagnostic sensitivity when using the real‐time PCR assay. Furthermore the assay is able to detect both virulent and hypovirulent strains of C. parasitica. Therefore the new real‐time PCR assay can be used to provide reliable, rapid, specific detection of C. parasitica to prevent the accidental movement of the disease and to monitor its spread.     

Spread and population structure of <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> in a young chestnut orchard in Slovakia

The chestnut blight pathogen Cryphonectria parasitica was studied in a chestnut collection composed of both seedlings and grafts derived from selected Castanea sativa and C. sativa × C. crenata trees located in south-east Slovakia, near village Príbelce on an area of approximately 3.5 ha. The study was conducted during eight years (2003–2010). During this period 133 trees were infected, which represents 59.82% of chestnut trees of all chestnut accessions. Based on the phenotype of the fungus culture and the type of cankers in the field, all isolates were determined to be virulent. No hypovirulent strains were found. No vegetative compatibility (vc) type diversity was observed. More than 130 isolates were analyzed for vc and all were in single vc type, which was identical with EU 12. All isolates assayed for mating type were MAT-1. No perithecia were observed. No significant differences were found between the proportion of cankered and dead cankered trees in seedlings and grafts of hybrid origin (C. sativa × C. crenata) and of C. sativa origin. However, particular seedlings and grafts of hybrid origin seemed to exhibit certain resistance to chestnut blight.     

A membrane technique for producing protoplasts ofCryphonectria parasitica

A method for the formation and regeneration of protoplasts of several strains of the chestnut blight fungus,Cryphonectria parasitica, is presented. The procedure utillizes cellophane membranes for growth and employs centrifugation for separation of protoplasts from hyphal fragments. Yields averaged 8.04×10⁶ protoplasts per membrane. Regeneration frequencies were 40–50% with a soft-agar overlay. These protoplasts are suitable for use in experiments designed to determine the role of dsRNA in hypovirulence ofC. parasitica.     

Heterologous expression of a tannic acid-inducible laccase3 of <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Background  

A tannic acid-inducible and mycoviral-regulated laccase3 (lac 3) from the chestnut blight fungus Cryphonectria parasitica has recently been identified, but further characterization was hampered because of the precipitation of protein products by tannic acid supplementation. The present study investigated the heterologous expression of the functional laccase3 using a yeast Saccharomyces cerevisiae.     

Invasion history and demographic pattern of Cryphonectria hypovirus 1 across European populations of the chestnut blight fungus

We reconstructed the invasion history of the fungal virus Cryphonectria hypovirus 1 (CHV‐1) in Europe, which infects the chestnut blight fungus Cryphonectria parasitica. The pattern of virus evolution was inferred based on nucleotide sequence variation from isolates sampled across a wide area in Europe at different points in time. Phylogeny and time estimates suggested that CHV‐1 was introduced together with its fungal host to Europe and that it rapidly colonized the central range along the south facing slopes of the Alps and the north‐east facing slopes of the Dinaric Alps. These central populations were the source for two waves of simultaneous invasions toward the southern Balkans and Turkey, as indicated by migration rates. Our results showed that the evolutionary scenarios for CHV‐1 and C. parasitica were spatially congruent. As infection with CHV‐1 reduces the pathogenicity of C. parasitica toward the chestnut tree, CHV‐1 invasions of the newly established C. parasitica populations probably prevented the development of devastating chestnut blight epidemics in Europe. We propose that in this, and supposedly in other pathosystems, geographic, vegetation‐related, demographic, economic, and political factors may help explain the correlated invasion pattern of a parasite and its host.     

Differential Transfer and Dissemination of Hypovirus and Nuclear and Mitochondrial Genomes of a Hypovirus-Infected Cryphonectria parasitica Strain after Introduction into a Natural Population

Biological control of plant diseases generally requires release of living organisms into the environment. Cryphonectria hypoviruses function as biological control agents for the chestnut blight fungus, Cryphonectria parasitica, and hypovirus-infected C. parasitica strains can be used to treat infected trees. We used naturally occurring molecular marker polymorphisms to examine the persistence and dissemination of the three genomes of a hypovirus-infected C. parasitica strain, namely, the double-stranded RNA genome of Cryphonectria hypovirus 1 (CHV1) and the nuclear and mitochondrial genomes of its fungal host. The hypovirus-infected strain was experimentally introduced into a blight-infested chestnut coppice forest by treating 73 of 246 chestnut blight cankers. Two years after introduction, the hypovirus had disseminated to 36% of the untreated cankers and to 35% of the newly established cankers. Spread of the hypovirus was more frequent within treated sprout clusters than between sprout clusters. Mitochondrial DNA of the introduced fungus also was transferred into the resident C. parasitica population. Concomitant transfer of both the introduced hypovirus and mitochondrial DNA was detected in almost one-half of the treated cankers analyzed. The introduced mitochondrial DNA haplotype also was found in three resident isolates from newly established cankers. The nuclear genome of the introduced strain persisted in the treated cankers but did not spread beyond them.     

Polymorphic sequence‐characterized codominant loci in the chestnut blight fungus,Cryphonectria parasitica

Studies on the population biology of the chestnut blight fungus, Cryphonectria parasitica, have previously been carried out with dominant restriction fragment length polymorphism (RFLP) fingerprinting markers. In this study, we describe the development of 11 codominant markers from randomly amplified polymorphic DNAs (RAPDs). RAPD fragments were cloned and sequenced, and polymerase chain reaction (PCR) primers were designed flanking insertions/deletions. Primers labelled with fluorescent dyes were combined in multiplex reactions to assay five or six loci simultaneously in a capillary sequencing system. These codominant markers have the potential to complement RFLP methods for studying C. parasitica.     

Inadequate Cold Tolerance as a Possible Limitation to American Chestnut Restoration in the Northeastern United States

The American chestnut (Castanea dentata (Marshall) Borkh.), once a major component of eastern forests from Maine to Georgia, was functionally removed from the forest ecosystem by chestnut blight (an exotic fungal disease caused by Cryphonectria parasitica (Murr.) Barr), first identified at the beginning of the twentieth century. Hybrid‐backcross breeding programs that incorporate the blight resistance of Chinese chestnut (Castenea mollissima Blume) and Japanese chestnut (Castenea crenata Sieb. & Zuc.) into American chestnut stock show promise for achieving the blight resistance needed for species restoration. However, it is uncertain if limitations in tissue cold tolerance within current breeding programs might restrict the restoration of the species at the northern limits of American chestnut's historic range. Shoots of American chestnut and hybrid‐backcross chestnut (i.e., backcross chestnut) saplings growing in two plantings in Vermont were tested during November 2006, February 2007, and April 2007 to assess their cold tolerance relative to ambient low temperatures. Shoots of two potential native competitors, northern red oak (Quercus rubra L.) and sugar maple (Acer saccharum L.), were also sampled for comparison. During the winter, American and backcross chestnuts were approximately 5°C less cold tolerant than red oak and sugar maple, with a tendency for American chestnut to be more cold tolerant than the backcross chestnut. Terminal shoots of American and backcross chestnut also showed significantly more freezing damage in the field than nearby red oak and sugar maple shoots, which showed no visible injury.     

Novel insights into the emergence of pathogens: the case of chestnut blight

Exotic, invasive pathogens have emerged repeatedly and continue to emerge to threaten the world’s forests. Ecosystem structure and function can be permanently changed when keystone tree species such as the American chestnut (Castanea dentata) are eliminated from a whole range by disease. The fungal ascomycete pathogen Cryphonectria parasitica is responsible for causing chestnut blight. Once the pathogen was introduced into the Eastern US, where chestnuts were predominant, chestnuts were all but eliminated. This pathogen is currently causing extensive damage in Europe. A study in this issue of Molecular Ecology sheds new light on the pattern and process of emergence of this devastating plant pathogen ( Dutech et al. 2012 ). The authors used microsatellite markers to investigate the evolutionary history of C. parasitica populations introduced into North America and Europe. To infer sources of migrants and the migration events, the authors included putative source populations endemic to China and Japan, inferred potentially unsampled populations and conducted a multivariate population genetic and complex ABC analysis. Cryphonectria parasitica emerges as an example of an introduced pathogen with limited genotypic diversity and some admixture in the invaded ranges, yet repeated invasions into different areas of Europe and the United States. This work sheds new light on the emergence of C. parasitica providing compelling evidence that this pathogen emerged by repeated migration and occasional admixture.     

Functional characterization of the three mitogen‐activated protein kinase kinases (MAP2Ks) present in the Cryphonectria parasitica genome reveals the necessity of Cpkk1 and Cpkk2, but not Cpkk3, for pathogenesis on chestnut (Castanea spp.)

The biological function(s) of the cpkk1, cpkk2 and cpkk3 genes, encoding the three mitogen‐activated protein kinase kinases (MAP2Ks) of Cryphonectria parasitica, the causal agent of chestnut blight, were examined through knockout strains. Cpkk1, the Mkk1 orthologue, acts in a phosphorylation cascade essential for cell integrity; Cpkk2 is the Ste7 orthologue involved in the pheromone response pathway; Cpkk3 is the Pbs2 orthologue, the MAP2K activated during the high‐osmolarity response. Our analysis confirmed the role of each MAP2K in its respective signalling cascade with some peculiarities: abnormal hyphae with a reduced number of septa and thinner cell walls were observed in Δcpkk1 mutants, and a strong growth defect on solid media was evident in Δcpkk2 mutants, when compared with the controls. Virulence on chestnut was affected in both the Δcpkk1 and Δcpkk2 strains, which were also unable to complete the developmental steps essential for mating. No alterations were reported in Δcpkk3, except under hyperosmotic conditions and in the presence of fludioxonil. Δcpkk2 mutants, however, showed higher sensitivity during growth in medium containing the antibiotic G418 (Geneticin).     

Diversity of viruses in Cryphonectria parasitica and C. nitschkei in Japan and China, and partial characterization of a new chrysovirus species

We surveyed native populations of the chestnut blight fungus, Cryphonectria parasitica, in Japan and China, and C. nitschkei, a sympatric species on chestnut trees in Japan, to learn more about the diversity of hypoviruses and other double-stranded (ds) RNA viruses. In a sample of 472 isolates of C. parasitica and 45 isolates of C. nitschkei from six prefectures in Japan, we found 27 containing one or more dsRNAs. Twelve isolates of C. parasitica and two isolates of C. nitschkei were infected with Cryphonectria hypovirus 1 (CHV-1); four of these 12 C. parasitica isolates also contained other dsRNAs that did not hybridize to CHV-1. In China, only one of 85 C. parasitica isolates was CHV-1-infected; no dsRNAs were detected in the other isolates from China. No other known hypoviruses were found in this study. However, we found two previously undescribed dsRNAs in Japan approximately 9 kb in size that did not hybridize to each other or to any known dsRNAs from C. parasitica. We also found three additional groups of dsRNAs, one of which represents the genome of a new member of the virus family Chrysoviridae and was found only in C. nitschkei; the other two dsRNAs were found previously in isolates of C. parasitica from Japan or China. The most significant result of this survey is the discovery of novel dsRNAs that can be characterized in future research.     

Plant neighbour identity and invasive pathogen infection affect associational resistance to an invasive gall wasp

Theory predicts that mixed forests are more resistant to native pests than pure forests (i.e. associational resistance) because of reduced host accessibility and increased top-down control by natural enemies. Yet, whether the same mechanisms also apply to invasive pests remains to be verified. We tested the hypothesis of associational resistance against the invasive Asian chestnut gall wasp (ACGW, Dryocosmus kuriphilus) by comparing ACGW infestation rates on chestnuts (Castanea sativa) in stands varying in species composition (chestnut alone or associated with oaks, pines or ashes). We investigated the effects of reduced chestnut density and frequency in mixed stands, as well as the effect of biotic interactions between ACGW, its parasitoids and the chestnut blight disease (caused by Cryphonectria parasitica). ACGW infestation rates were significantly lower in chestnut–oak and chestnut–ash mixtures than in pure chestnut stands and chestnut–pine mixtures. Infestation rate decreased with decreasing chestnut relative proportion. The composition of native parasitoid communities emerged from galls significantly differed between pure and mixed chestnut stands, but not the species richness or abundance of parasitoids. The abundance of the introduced parasitoid Torymus sinensis was not correlated with ACGW infestation rates and was independent of stand composition. Blight symptoms modified ACGW infestation rates with taller trees being preferred when they were asymptomatic but avoided when they presented blight disease damage. Our results suggest that conservation biological control based on tree species mixtures could contribute to reducing the damage of invasive forest pests.     

Transformation of a filamentous fungus<Emphasis Type="Italic">Cryphonectria parasitica</Emphasis> using<Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

AsAgrobacterium tumefaciens, which has long been used to transform plants, is known to transfer T-DNA to budding yeast,Saccharomyces cerevisiae, a variety of fungi were subjected to theA. tumefaciens-mediated transformation to improve their transformation frequency and feasibility. TheA. tumefaciens-mediated transformation of chestnut blight fungus,Cryphonectria parasitica, is performed in this study as the first example of transformation of a hardwood fungal pathogen. The transfer of the binary vector pBIN9-Hg, containing the bacterial hygromycin B phosphotransferase gene under the control of theAspergillus nidulans trpC promoter and terminator, as a selectable marker, led to the selection of more than 1,000 stable, hygromycin B-resistant transformants per 1×10⁶ conidia ofC. parasitica. The putative transformants appeared to be mitotically stable. The transformation efficiency appears to depend on the bacterial strain, age of the bacteria cell culture and ratio of fungal spores to bacterial cells. PCR and Southern blot analysis indicated that the marker gene was inserted at different chromosomal sites. Moreover, three transformants out of ten showed more than two hybridizing bands, suggesting more than two copies of the inserted marker gene are not uncommon.     

Widespread Distribution of Fungivorus Aphelenchoides spp. in Blight Cankers on American Chestnut Trees

Previously we showed in laboratory studies that the fungivorus nematode, Aphelenchoides hylurgi, was attracted to and fed upon the chestnut blight fungus, Cryphonectria parasitica, from American chestnut bark cankers and was a carrier of biocontrol, white hypovirulent C. parasitica strains. In the present field study, we recovered Aphelenchoides spp. in almost all (97.0 %) of 133 blight canker tissue assays (three 5-g samples each) from four eastern states. High mean population densities (227 to 474 nematodes per 5 g tissue) of Aphelenchoides spp. were recovered from cankers in Virginia, West Virginia, and Tennessee but not from New Hampshire (mean = 75 nematodes per 5 g tissue). Overall, most canker assays yielded population densities less than 200 nematodes per 5 g tissue. All of 12 very small or young cankers yielded a few to many Aphelenchoides spp. Regression analysis indicated greatest recovery of Aphelenchoides spp. occurred in the month of May (r = 0.94). The results indicate that Aphelenchoides spp. appear to be widespread in blight cankers on American chestnut trees and could play a role in biocontrol of chestnut blight.     

Thyreophagus corticalis as a vector of hypovirulence in Cryphonectria parasitica in chestnut stands

The natural spread of hypovirulence in Cryphonectria parasitica (Murr.) Barr. occurs in chestnut (Castanea sativa Mill) stands and orchards in Italy and other European countries, leading to spontaneous recovery of the diseased trees. Little is known about how hypovirulence spreads in chestnut stands but various corticolous mite species frequently detected on chestnut cankers could be one of the many factors playing a role in the spread. Artificial virulent cankers created in inoculation field tests and treated with Thyreophagus corticalis (Acari, Sarcoptiformes, Acaridae) raised on hypovirulent cultures showed similar growth to those treated with mycelia of the hypovirulent strain over 18 months of inoculation. Cultures re-isolated from virulent cankers treated with mites were found to contain hypovirus like those derived from pairings of virulent and hypovirulent strains. Viral dsRNA could be carried externally and/or ingested by mites from the hypovirulent mycelia and then transmitted to the mycelia of virulent strains, causing their conversion. In a laboratory study, all fecal pellets collected from mites reared on hypovirulent and virulent strains grown on semi-selective media gave rise to colonies of C. parasitica with similar morphological characters and virulence to the original cultures. Field inoculation of stump sprouts with the resulting colonies revealed that mite digestive tract passage did not alter the virulence of the studied strains. These results are of interest for the biological control of chestnut blight.     

A methodology to evaluate disease severity: a case study of chestnut blight in El Bierzo region (northwestern Spain)

Correct knowledge of the incidence and severity of disease is essential for implementation of timely and effective management control strategies. In this article, multiple correspondence analysis (MCA) is applied to evaluate the severity of chestnut blight incited by the ascomyceteous fungus Cryphonectria parasitica. This economically important bark disease leads to the loss of an important part of the chestnut production and the progressive death of the tree. A total of 7240 living European chestnut (Castanea sativa) trees across 452 plots were surveyed in El Bierzo, NW Spain. For each tree, the main stem and canopy were visually assessed for signs of the pathogen and/or symptoms of the disease and the extent of the disease was classified on a qualitative ordinal scale consisting of six levels. The statistical procedure is useful because it quickly analyses measurable, discrete observations from assessed individuals to provide a disease severity measure related to tree features and disease extension inside the tree. The results indicated that the penetration of the pathogen is located in the lower part of the crown and spreads to the tips of the branches in the upper part of the crown. Thus, our results suggest that man‐made wounds, when the tree was grafted or pruned, are the main channel of pathogen penetration in El Bierzo region. Disease severity estimates and incidence data for C. parasitica across the region were compared. From the perspective of the management and control of the disease, a disease severity value provides a more accurate indication of the scenario of the disease in a region than presence or absence data alone.     

Comparisons of Ectomycorrhizal Colonization of Transgenic American Chestnut with Those of the Wild Type,a Conventionally Bred Hybrid,and Related Fagaceae Species

American chestnut (Castanea dentata [Marsh.] Borkh.) dominated the eastern forests of North America, serving as a keystone species both ecologically and economically until the introduction of the chestnut blight, Cryphonectria parasitica, functionally eradicated the species. Restoration efforts include genetic transformation utilizing genes such as oxalate oxidase to produce potentially blight-resistant chestnut trees that could be released back into the native range. However, before such a release can be undertaken, it is necessary to assess nontarget impacts. Since oxalate oxidase is meant to combat a fungal pathogen, we are particularly interested in potential impacts of this transgene on beneficial fungi. This study compares ectomycorrhizal fungal colonization on a transgenic American chestnut clone expressing enhanced blight resistance to a wild-type American chestnut, a conventionally bred American-Chinese hybrid chestnut, and other Fagaceae species. A greenhouse bioassay used soil from two field sites with different soil types and land use histories. The number of colonized root tips was counted, and fungal species were identified using morphology, restriction fragment length polymorphism (RFLP), and DNA sequencing. Results showed that total ectomycorrhizal colonization varied more by soil type than by tree species. Individual fungal species varied in their colonization rates, but there were no significant differences between colonization on transgenic and wild-type chestnuts. This study shows that the oxalate oxidase gene can increase resistance against Cryphonectria parasitica without changing the colonization rate for ectomycorrhizal species. These findings will be crucial for a potential deregulation of blight-resistant American chestnuts containing the oxalate oxidase gene.