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.     

Emerging new crown symptoms on Castanea sativa (Mill.): Attempting to model interactions among pests and fungal pathogens

In the 2015–2016 growing seasons, two novel symptoms were assessed on the crown of trees in orchards and coppices of chestnut groves in Central Italy. The first symptom was flagging of annual shoots with green leaves undergoing sudden wilt and turning brown later in the season. The second symptom consisted of leaves on annual shoots turning yellow before wilting in absence of flagging represented the second symptom. Samples were collected along transects in early summer, late summer and winter, and processed in the laboratory. The flagging symptom was associated in early summer with the presence of C. parasitica in cryptic dried buds on stems from the previous year's growth. The pathogen was also found in dormant buds in winter, suggesting that the infection could take place in summer during the Chinese gall wasp oviposition period. Cryphonectria parasitica was also isolated from abandoned galls in winter supporting the hypothesis that galls are a potential source of inoculum for crown infections. Aetiology of yellowing was not clarified and no fungal taxa were specifically associated with this symptom. Gnomoniopsis castanea, C. parasitica and, in early summer, Colletotrichum acutatum were the most abundant fungal taxa isolated from chestnut shoots and buds.     

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.     

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.     

Inhibition of virulent and hypovirulent Cryphonectria parasitica growth in dual culture by fungi commonly isolated from chestnut blight cankers

Chestnut blight cankers, caused by the fungus Cryphonectria parasitica, are prone to invasion by other microorganisms as the canker ages. This microbial community has the potential to alter canker expansion, which may influence the probability that the canker girdles the infected stem. Hypoviruses infect the pathogen mycelium directly and are known to decrease pathogen virulence (i.e. hypovirulent). These viral infections can slow pathogen growth, decreasing the rate of canker expansion and lowering the probability of girdling. Saprophytic fungi also invade the expanding canker and may antagonize C. parasitica leading to reduced pathogen growth. The combined effects of fungal antagonism and a hypovirulent pathogen could work in combination to reduce the probability of girdling the infected stem. We assessed the ability of different fungal taxa, isolated from low severity cankers, to inhibit the growth of virulent and hypovirulent forms of C. parasitica in dual culture tests on two cultural media. Percent growth inhibition of virulent C. parasitica by potentially antagonistic fungi ranged from 2 % to 34 %, while inhibition of hypovirulent C. parasitica ranged from 18 % to 54 %. Only one isolate, identified as Umbelopsis isabellina (UmbelopsisWS) inhibited the virulent form of the pathogen more than the hypovirulent form. All three Trichoderma isolates caused the greatest growth inhibition of virulent C. parasitica, but they, like all other fungal isolates tested, inhibited the hypovirulent form of the pathogen more than the virulent form. These results suggest that commonly occurring fungi in chestnut blight cankers, including Trichoderma, may inhibit the hypovirulent C. parasitica more than virulent C. parasitica. Thus, the presence of other fungi in cankers may not enhance the effect of hypovirulent C. parasitica to delay cankers from girdling a stem but instead intensify canker development.     

Ecological impacts of non-indigenous invasive fungi as forest pathogens

Non-indigenous pathogenic fungi increasingly threaten North American tree species. Ecosystems may be fundamentally changed when abundant tree species are functionally eliminated, as occurred with American chestnut (Castanea dentata (Marsh.) Borkh.). Conversely, changes may be more subtle but still significant and long lasting when populations are lost, or all trees in the larger size classes are killed. Proposed approaches for characterizing the magnitude of ecological impacts use characteristics of both the non-indigenous pathogen and the host species. Impacts are most significant when highly successful invading pathogens attack foundation species, setting in motion a long-lasting cascade of effects on the host and associated species. Such impacts have generally not been well documented at the ecosystem level. Several North American forest tree species have been functionally eliminated or severely reduced by non-indigenous pathogens. Historical invasions, such as that of chestnut blight (Cryphonectria parasitica (Murr.) Bar), caused very significant ecological impacts that will never be completely understood because of lack of quantitative data. Beech bark disease, caused by a combination of an introduced scale insect (Cryptococcus fagisuga Lindinger) and a fungus (Neonectria faginata (Lohman et al.) Castl. & Rossman), is still advancing and provides opportunities for studying ecosystem-level impacts when a major tree species is removed or markedly reduced in abundance from the overstorey. Butternut canker, caused by the fungus, Sirococcus clavigignenti-juglandacearum N.B. Nair, Kostichka & Kuntz, has spread throughout the host range, endangering species’ survival. Other non-indigenous invasive fungi such as Cronartium ribicola J.C. Fisch and Phytophthora lateralis Tucker & Milbrath continue to move into new populations, causing high mortality and associated restructuring of these ecosystems. Global trade and environmental change trends will ensure new challenges by non-indigenous fungal pathogens, presenting an urgent need for improved understanding of long-term impacts across ecological systems.     

Comparative efficacy of gypsy moth (Lepidoptera: Erebidae) entomopathogens on transgenic blight‐tolerant and wild‐type American,Chinese, and hybrid chestnuts (Fagales: Fagaceae)

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.     

Modeling climate impact on an emerging disease,the Phytophthora alni‐induced alder decline

Alder decline caused by Phytophthora alni is one of the most important emerging diseases in natural ecosystems in Europe, where it has threatened riparian ecosystems for the past 20 years. Environmental factors, such as mean site temperature and soil characteristics, play an important role in the occurrence of the disease. The objective of the present work was to model and forecast the effect of environment on the severity of alder Phytophthora outbreaks, and to determine whether recent climate change might explain the disease emergence. Two alder sites networks in NE and SW France were surveyed to assess the crown health of trees; the oomycete soil inoculum was also monitored in the NE network. The main factors explaining the temporal annual variation in alder crown decline or crown recovery were the mean previous winter and previous summer temperatures. Both low winter temperatures and high summer temperatures were unfavorable to the disease. Cold winters promoted tree recovery because of poor survival of the pathogen, while hot summer temperature limited the incidence of tree decline. An SIS model explaining the dynamics of the P. alni‐induced alder decline was developed using the data of the NE site network and validated using the SW site network. This model was then used to simulate the frequency of declining alder over time with historical climate data. The last 40 years' weather conditions have been generally favorable to the establishment of the disease, indicating that others factors may be implicated in its emergence. The model, however, showed that the climate of SW France was much more favorable for the disease than that of the Northeast, because it seldom limited the overwintering of the pathogen. Depending on the European area, climate change could either enhance or decrease the severity of the alder decline.     

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.     

Fungal pathogen and ethanol affect host selection and colonization success in ambrosia beetles

1. Ambrosia beetles exhibit broad host ranges but a narrow preference based on the condition of the host. Tissues infected by pathogens or containing ethanol can facilitate attacks by ambrosia beetles, although it still remains unclear how these factors interact.
2. The present study aimed to examine how (i) chestnut logs infected with the fungal pathogen Cryphonectria parasitica and treated with ethanol (i.e. baited with ethanol lure, soaked in ethanol or untreated) and (ii) hornbeam logs soaked in different ethanol concentrations (3–12.5%) affect host selection and colonization success of ambrosia beetles.
3. Ethanol‐soaked logs were more attractive to Anisandrus dispar than ethanol‐baited logs or untreated logs, although this difference was more evident in uninfected than infected logs. Increasing ethanol concentration in host tissues was differentially attractive to Xyleborinus saxesenii and Xylosandrus germanus. A nonlinear relationship was also documented between ethanol concentration and emergence of X. germanus adults.
4. Overall, the results obtained suggest that the presence of C. parasitica in chestnut logs can affect host selection in ambrosia beetles. In addition, the ethanol concentration in tree tissues affects host selection and colonization success, although the effect varies depending on the beetle species. This contrasting response could be a niche‐partitioning mechanism based on ethanol within host tissues.

     

Molecular genetic identification of the phytopathogenic fungus <Emphasis Type="Italic">Cryphonectria parasitica</Emphasis>

Mycological analysis of samples of sweet chestnut (Castanea sativa) collected in the northern Turkish zone of its habitat was carried out. A total of 300 strains of micromycetes belonging to 13 taxa were isolated in pure cultures. The phytopathogenic fungus Cryphonectria parasitica, the causal agent of chestnut blight, was identified among these strains. A technique for detection of C. parasitica in pure culture and host tissues using species-specific PCR markers was developed and tested. This technique can be used as an express method for detection of chestnut blight.     

The effect of disease induced by Albugo candida (white rust) and Peronospora parasitica (downy mildew) on the survival and reproduction of Capsella burna-pastoris (shepherd's purse)

Summary Disese induced by Albugo candida or Peronospora parasitica was found to have significant effects on the survival and reproductive output of affected individuals of Capsella bursa-pastoris. The time of infection by either pathogen was of considerable importance in determining the ultimate effect of disease. Systemic (primary) infection of seedling plants led to a high degree of mortality (88%) prior to reproductive maturity, however, localized or systemic secondary infection did not affect survival. Fruit production of surviving infected plants was negatively correlated with disease severity.     

Dynamics of twolined chestnut borer Agrilus bilineatus as influenced by defoliation and selection thinning

1 The twolined chestnut borer, Agrilus bilineatus (Coleoptera: Buprestidae), is a major mortality agent of stressed oak trees. However, patterns of abundance and population change are not well understood. 2 We studied the spatial and temporal variation in abundance of twolined chestnut borer adults during a gypsy moth, Lymantria dispar (Lepidoptera: Lymnatriidae), outbreak and examined the influence of both defoliation and thinning on twolined chestnut borer abundance. 3 In stands that were defoliated by gypsy moth, extensive defoliation occurred in one year, and major overstory tree mortality followed in the next. Most mortality occurred in the year preceding the peak year of twolined chestnut borer abundance and abundance of twolined chestnut borer was positively associated with defoliation and mortality in the previous year. 4 Twolined chestnut borers were more frequently associated with poor or fair crown condition trees than trees with good crown condition and were more abundant on members of the red oak group than the white oak group.     

Growth traits of juvenile American chestnut and red oak as adaptations to disturbance

American chestnut (Castanea dentata) was a dominant species in eastern North America prior to the importation of chestnut blight. In light of recent efforts to restore viable populations of chestnut in eastern forests, an increased understanding of its association with other co‐occurring, disturbance‐adapted oak species is necessary. We evaluated crown architecture and leaf morphology in juvenile chestnut and red oak (Quercus rubra) to assess potential differences in establishment strategies of both species. We also investigated differences in nonstructural carbohydrate reserves and whole tree biomass partitioning between species. Seedlings of both species were planted in forest stands treated either with midstory removal or small patch cuts, simulating potential restoration plantings. After 5–7 years, chestnut's allocation to its root system was lower than red oak's, with chestnut saplings instead diverting resources to branches and foliage. Chestnut had lower leaf area index, greater crown projection area, and higher specific leaf area than red oak, indicating the species may have an advantage in shaded understories. There were only minor differences in nonstructural root carbohydrate reserves, between red oak and American chestnut, indicating that chestnut may respond similarly to oak by resprouting after disturbances topkill young saplings. We suggest that American chestnut has morphological and physiological attributes that allow it to function as an opportunistic and plastic species that can utilize gaps to facilitate its canopy recruitment, yet still persist after occasional surface fire. This knowledge can guide restoration strategies for this iconic species of the eastern temperate forest region.     

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.     

Spatial analysis of nuclear and mitochondrial RFLP genotypes in populations of the chestnut blight fungus, Cryphonectria parasitica

Spatial structure of both nuclear and mitochondrial RFLPs were studied in several populations of the chestnut blight fungus, Cryphonectria parasitica, using a variety of spatial autocorrelation tests designed to detect nonrandom patterns. Fungal individuals were sampled from cankers on infected chestnut trees, and the location of each tree was mapped. Single-locus nuclear RFLPs, nuclear fingerprints, and mitochondrial DNA haplotypes were determined for each individual. Individuals with the same DNA fingerprint genotypes occurred closer together than would be expected at random in four of the five plots, while mitochondrial DNA haplotypes were aggregated in all five plots. Genetic distances between individuals, expressed as one minus the proportion of shared restriction fragment size classes for fingerprints and mitochondrial haplotypes, were significantly correlated with Euclidean distances between individuals in four of the five populations, but these correlations were very weak (r < 0.18). The same DNA fingerprint and single-copy nuclear RFLP alleles occurred on the same trees or immediately neighbouring trees more often than would be expected at random. Most of the aggregation for all three genetic markers occurred among individuals within the same cluster of chestnut stems or on neighbouring trees. Lack of spatial autocorrelation in one population was probably due to sampling on a larger scale that was too coarse to detect any patterns. Significant aggregation of genotypes in C. parasitica is most likely caused by some degree of restricted dispersal within populations. The implications of restricted dispersal are discussed in relation to the breeding system and isolation by distance in populations of. C. parasitica.     

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.     

Effects of transgenic American chestnut leaf litter on growth and survival of wood frog larvae

Biotechnology offers a new approach for the restoration of tree species affected by exotic pathogens; however, nontarget impacts of this novel strategy on other organisms have not been comprehensively assessed. We evaluated the effect of transgenic American chestnut (Castanea dentata) leaf litter on the growth and survival of larval wood frogs (Lithobates sylvaticus), a forest‐dwelling amphibian species widely sympatric with American chestnut, that forage almost entirely on periphyton and litter detritus that accumulate in temporary vernal pools in forests. We reared wood frog larvae on Castanea leaf litter (American chestnut genetically engineered for blight tolerance, nontransgenic American chestnut, Chinese chestnut [Castanea mollissima], and an American–Chinese chestnut hybrid) and litter from two non‐Castanea, nontransgenic “control” tree species, coupled with two levels of supplementary food. We observed no differences in growth or survival of wood frog larvae reared on transgenic versus nontransgenic American chestnut leaves. Without supplementary food, wood frog larvae provided leaves from American chestnut (both types) developed faster and grew larger than those exposed to other leaf litter treatments. Results of this study provide preliminary evidence that (1) American chestnut may have formerly been an important source of food for forest‐dwelling amphibians and (2) transgenic American chestnut litter generated as part of chestnut restoration efforts is unlikely to present direct novel risks to developing amphibian larvae in the forest environment.