Virulence of Hymenoscyphus albidus and H. fraxineus on Fraxinus excelsior and F. pennsylvanica.

European ash (Fraxinus excelsior) is currently battling an onslaught of ash dieback, a disease emerging in the greater part of its native area, brought about by the introduction of the ascomycete Hymenoscyphus fraxineus (= Hymenoscyphus pseudoalbidus). The closely-related fungus Hymenoscyphus albidus, which is indigenous to Europe, is non-pathogenic when in contact with F. excelsior, but could pose a potential risk to exotic Fraxinus species. The North American green ash (Fraxinus pennsylvanica) is planted widely throughout Europe and regenerates naturally within this environment but little is known about the susceptibility of this species to ash dieback. We performed wound inoculations with both fungi (nine strains of H. fraxineus and three strains of H. albidus) on rachises and stems of F. excelsior and F. pennsylvanica under field conditions in Southern Poland. Necrosis formation was evaluated after two months on the rachises and after 12 months on the stems. After inoculation of H. albidus, only small lesions (of up to 1.3 cm in length) developed on the F. excelsior and F. pennsylvanica rachises, but with no significant distinction from the controls. Hymenoscyphus albidus did not cause necrotic lesions on the stems of either Fraxinus species. In contrast, H. fraxineus induced necroses on all inoculated rachises of both ash species with mean lengths of 8.4 cm (F. excelsior) and 1.9 cm (F. pennsylvanica). Necroses also developed on all of the inoculated F. excelsior stems (mean length 18.0 cm), whereas on F. pennsylvanica such lesions only occurred on about 5% of the stems (mean length 1.9 cm). The differences between strains were negligible. No necroses were observed on the control plants. Reisolations of H. albidus were only successful in around 8-11% of the cases, while H. fraxineus was reisolated from 50-70% of the inoculated organs showing necrotic lesions. None of the Hymenoscyphus species were isolated from the control plants. Our data confirm H. fraxineus' high virulence with regards to F. excelsior and demonstrate a low virulence in relation to F. pennsylvanica under field conditions in Poland. Hymenoscyphus albidus did not express any perceivable pathogenicity on both host species.

Forest health in a changing world.

Forest pathology, the science of forest health and tree diseases, is operating in a rapidly developing environment. Most importantly, global trade and climate change are increasing the threat to forest ecosystems posed by new diseases. Various studies relevant to forest pathology in a changing world are accumulating, thus making it necessary to provide an update of recent literature. In this contribution, we summarize research at the interface between forest pathology and landscape ecology, biogeography, global change science and research on tree endophytes. Regional outbreaks of tree diseases are requiring interdisciplinary collaboration, e.g. between forest pathologists and landscape ecologists. When tree pathogens are widely distributed, the factors determining their broad-scale distribution can be studied using a biogeographic approach. Global change, the combination of climate and land use change, increased pollution, trade and urbanization, as well as invasive species, will influence the effects of forest disturbances such as wildfires, droughts, storms, diseases and insect outbreaks, thus affecting the health and resilience of forest ecosystems worldwide. Tree endophytes can contribute to biological control of infectious diseases, enhance tolerance to environmental stress or behave as opportunistic weak pathogens potentially competing with more harmful ones. New molecular techniques are available for studying the complete tree endobiome under the influence of global change stressors from the landscape to the intercontinental level. Given that exotic tree diseases have both ecologic and economic consequences, we call for increased interdisciplinary collaboration in the coming decades between forest pathologists and researchers studying endophytes with tree geneticists, evolutionary and landscape ecologists, biogeographers, conservation biologists and global change scientists and outline interdisciplinary research gaps.

Hymenoscyphus pseudoalbidus, the causal agent of European ash dieback.

The ascomycete Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea) causes a lethal disease known as ash dieback on Fraxinus excelsior and Fraxinus angustifolia in Europe. The pathogen was probably introduced from East Asia and the disease emerged in Poland in the early 1990s; the subsequent epidemic is spreading to the entire native distribution range of the host trees. This pathogen profile represents a comprehensive review of the state of research from the discovery of the pathogen and points out knowledge gaps and research needs. TAXONOMY: Members of the genus Hymenoscyphus (Helotiales, Leotiomycetidae, Leotiomycetes, Ascomycota) are small discomycetes which form their ascomata on dead plant material. A phylogeny based on the internal transcribed spacers (ITSs) of the rDNA indicated the avirulent Hymenoscyphus albidus, a species native to Europe, as the closest relative of H. pseudoalbidus. SYMPTOMS: Hymenoscyphus pseudoalbidus causes necrotic lesions on leaves, twigs and stems, eventually leading to wilting and dieback of girdled shoots. Bark lesions are characterized by a typical dark- to cinnamon-brown discoloration. LIFE CYCLE: Hymenoscyphus pseudoalbidus is heterothallic and reproduces sexually on ash petioles in the litter once a year. Ascospores are wind dispersed and infect ash leaves during the summer. The asexual spores only serve as spermatia. TOOLS AND TECHNIQUES: The most important techniques for fungal handling, such as detection, isolation, culturing, storage, crossing and ascocarp production, are briefly described. MANAGEMENT: Once the disease is established, management is hardly possible. The occurrence of a small fraction of partially tolerant trees constitutes hope for resistance breeding in the future. Healthy-looking trees should be preserved.

Networks in plant epidemiology: from genes to landscapes, countries, and continents.

There is increasing use of networks in ecology and epidemiology, but still relatively little application in phytopathology. Networks are sets of elements (nodes) connected in various ways by links (edges). Network analysis aims to understand system dynamics and outcomes in relation to network characteristics. Many existing natural, social, and technological networks have been shown to have small-world (local connectivity with short-cuts) and scale-free (presence of super-connected nodes) properties. In this review, we discuss how network concepts can be applied in plant pathology from the molecular to the landscape and global level. Wherever disease spread occurs not just because of passive/natural dispersion but also due to artificial movements, it makes sense to superimpose realistic models of the trade in plants on spatially explicit models of epidemic development. We provide an example of an emerging pathosystem (Phytophthora ramorum) where a theoretical network approach has proven particularly fruitful in analyzing the spread of disease in the UK plant trade. These studies can help in assessing the future threat posed by similar emerging pathogens. Networks have much potential in plant epidemiology and should become part of the standard curriculum.

Plant health and global change--some implications for landscape management.

Global change (climate change together with other worldwide anthropogenic processes such as increasing trade, air pollution and urbanization) will affect plant health at the genetic, individual, population and landscape level. Direct effects include ecosystem stress due to natural resources shortage or imbalance. Indirect effects include (i) an increased frequency of natural detrimental phenomena, (ii) an increased pressure due to already present pests and diseases, (iii) the introduction of new invasive species either as a result of an improved suitability of the climatic conditions or as a result of increased trade, and (iv) the human response to global change. In this review, we provide an overview of recent studies on terrestrial plant health in the presence of global change factors. We summarize the links between climate change and some key issues in plant health, including tree mortality, changes in wildfire regimes, biological invasions and the role of genetic diversity for ecosystem resilience. Prediction and management of global change effects are complicated by interactions between globalization, climate and invasive plants and/or pathogens. We summarize practical guidelines for landscape management and draw general conclusions from an expanding body of literature.

Cryptic speciation and community structure of Herpotrichia juniperi, the causal agent of brown felt blight of conifers.

Conifer twigs showing brown felt blight were collected along 100-m long transects at the timberline in the Swiss Alps and single-hyphal-tip cultures were prepared. Forty-seven of the sequenced 48 strains were Herpotrichia juniperi based on sequence comparisons of the internal transcribed spacers (ITS). A non-sporulating strain was tentatively identified as another, undescribed Herpotrichia species. Herpotrichia coulteri was not isolated. Most strains were from Juniperus communis var. saxatilis, the rest from Picea abies and Pinus mugo. Each twig was colonized by a different genotype as revealed by ISSR-PCR fingerprinting. More than one clone was present on some needles and twigs. Thus, importance of vegetative mycelial growth for dispersal seems to be limited to the spread of the disease to twigs of the same tree or of immediately adjacent trees, and, consequently, dispersal occurs mainly by ascospores. The H. juniperi strains could be assigned to five distinct groups based on the ISSR-PCR data. The strains from P. abies formed one of these groups but the other groups did not correlate with either host, transect or position along the transects. Multi-locus analysis based on beta-tubulin, elongation factor 1-alpha and ITS sequences confirmed the subdivision into five groups. Population differentiation among groups was distinct with N(ST) values varying between 0.545 and 0.895. H. juniperi seems to be composed of several cryptic species, one of them specific to P. abies.

Assignment of species rank to six reproductively isolated cryptic species of the Phialocephala fortinii s.1.-Acephala applanata species complex.

Phialocephala fortinii s.1. and Acephala applanata are the dominant dark septate endophytes (DSE) in roots of many trees and shrubs. Population genetic analysis led to the discovery of morphologically indistinguishable but reproductively isolated cryptic species (CSP) within Phialocephala fortinii s.1. In the present study we show that sequence data of two coding (beta-tubulin and translation elongation factor [EF-lalpha]) and three noncoding DNA loci confirm subdivision of P. fortinii s.1. and allow to differentiate seven CSP of P. fortinii. In addition we show that strains collected throughout Europe can be classified correctly based on these sequence markers. Statistically significant differences in growth response on different media were observed among CSP of P. fortinii and A. applanata. Growth inhibition on MEA amended with 100 mgl(-1) cycloheximide had the strongest differential effect of all physiological traits examined. In contrast exoenzyme production (laccase, proteinase, pectinase, phenol-oxidase, amylase, cytochrome oxidase and tyrosinase) rarely helped to differentiate CSP of P. fortinii. However A. applanata was a strong producer of amylases, laccases and proteinases. Based on these data we propose to assign species rank to six CSP of P. fortinii: P. turiciensis, P. letzii, P. europaea, P. helvetica, P. uotolensis, P. subalpina spp. nov. and P. fortinii s.s.

Community structure of Phialocephala fortinii s. lat. in European tree nurseries, and assessment of the potential of the seedlings as dissemination vehicles.

Patterns of colonization of conifer roots by dark septate endopyhtes of the Phialocephala fortinii s. lat. species complex in nurseries in Switzerland and Lithuania were studied. The potential for man-mediated genotype flow was estimated for two Swiss nurseries based on customers' addresses and the number of delivered plants. Two hundred and forty-nine strains from three Swiss and five Lithuanian nurseries and an afforestation site were characterized using a combination of inter-simple sequence repeat-anchored PCR (ISSR-PCR), single-copy RFLP analysis, and sequence analysis. P. fortinii s. lat. was abundant in nursery seedlings, but the frequency of seedlings colonized varied considerably among and within nurseries. Ten cryptic species (CSP) of P. fortinii s. lat. were identified, including four hitherto undiscovered CSP. P. helvetica was the dominant species in Swiss nurseries, whereas P. fortinii s. str. was the most abundant species in Lithuanian nurseries and the afforestation site. Swiss nurseries deliver plants over distances of more than 200km indicating the high potential for man-mediated genotype flow in P. fortinii s. lat.

Modelling disease spread and control in networks: implications for plant sciences.

Networks are ubiquitous in natural, technological and social systems. They are of increasing relevance for improved understanding and control of infectious diseases of plants, animals and humans, given the interconnectedness of today's world. Recent modelling work on disease development in complex networks shows: the relative rapidity of pathogen spread in scale-free compared with random networks, unless there is high local clustering; the theoretical absence of an epidemic threshold in scale-free networks of infinite size, which implies that diseases with low infection rates can spread in them, but the emergence of a threshold when realistic features are added to networks (e.g. finite size, household structure or deactivation of links); and the influence on epidemic dynamics of asymmetrical interactions. Models suggest that control of pathogens spreading in scale-free networks should focus on highly connected individuals rather than on mass random immunization. A growing number of empirical applications of network theory in human medicine and animal disease ecology confirm the potential of the approach, and suggest that network thinking could also benefit plant epidemiology and forest pathology, particularly in human-modified pathosystems linked by commercial transport of plant and disease propagules. Potential consequences for the study and management of plant and tree diseases are discussed.

Monitoring the spatial and temporal dynamics of a community of the tree-root endophyte Phialocephala fortinii s.l.

Phialocephala fortinii sensu lato was isolated from Picea abies roots that had been collected from the same 3 x 3-m forest plot in 2001 and 2004, to examine the spatial and temporal dynamics of this fungal community. RFLP analysis was used to define the multilocus haplotype (MLH) of each isolate. Pielou's measure of association and chi(2) tests of independence were employed to examine the randomness of patterns of spatial distribution of MLH observed in 2001 and 2004. Population differentiation between the two samplings was tested using the G(ST) statistic. In 2001, 144 strains of 28 MLH were isolated; in 2004, 139 strains of 29 MLH. Abundant MLH in 2001 also prevailed in 2004, and the same two cryptic species were dominant in both collections. The probability of being isolated in both years increased with increasing frequency of an MLH. The patterns of spatial distribution of most MLH did not differ between years. The G(ST) values indicated identity of the two collections. Communities of P. fortinii sensu lato remain spatially and genetically stable for at least 3 yr.

Evidence for subdivision of the root-endophyte Phialocephala fortinii into cryptic species and recombination within species.

The genetic structure of the root-endophyte Phialocephala fortinii was analyzed in three study sites using 11 single-copy RFLP probes. A total of 541 strains isolated from surface-sterilized, fine roots (diameter 0.5-3 mm) of Norway spruce (Picea abies) were examined. The average gene diversity (H) was high in all three study sites. Cluster analysis showed that up to four well-separated clusters of multi-locus haplotypes were present within the sites. Significant population subdivision was detected among these clusters, suggesting that groups of multi-locus haplotypes were reproductively isolated and that P. fortinii is a species complex composed of several cryptic species. This hypothesis was supported by ISSR-PCR which showed clusters consistent with those of the multi-locus haplotypes identified by RFLP analysis. In contrast, ITS sequence analysis did not allow to separate the species as clearly. The index of association (IA) did not deviate significantly from zero within any cryptic species, suggesting that recombination occurs within these species. Cryptic species occurred sympatrically. Thalli of two cryptic species were detected in the same 5-mm-long root segment in one instance. No significant differentiation was observed among populations of the same cryptic species in forest stands located approximately 5 km from each other. This finding is consistent with significant gene flow over this spatial scale. In addition, several isolates with both identical multi-locus haplotype and identical ISSR fingerprint were found at each study site indicating genotype flow or a recent common history between study sites.

Characterization of Guignardia mangiferae isolated from tropical plants based on morphology, ISSR-PCR amplifications and ITS1-5.8S-ITS2 sequences.

Phenotypic and genotypic variability of 18 endophytic Guignardia strains from different host plants (Anacardiaceae: Anacardium giganteum, Myracrodruon urundeuva, Spondias mombin; Apocynaceae: Aspidosperma polyneuron; Ericaceae: Rhododendron sp.; Fabaceae: Bowdichia nitida; Leguminosae: Cassia occidentalis; Rutaceae: Citrus aurantium) growing in different sites in Brazil was assessed by means of morphometric measurements and inter-single-sequence-repeat-anchored polymerase chain reaction (ISSR-PCR) amplifications of the DNA. Morphology of conidia and ascospores and growth rates of the Brazilian isolates corresponded well with those of G. mangiferae. Multiple correspondence analysis (MCA) of the ISSR-PCR data yielded three groups of strains, which did, however, not correspond either to the host or to the geographic origin. The same individual tree hosted genotypically different strains indicating multiple infections. Phylogeny based on ITS1-5.8S-ITS2 sequence analyses confirmed conspecificity of the Brazilian isolates with G. mangiferae (syn. G. endophyllicola).

Tree diseases and landscape processes: the challenge of landscape pathology.

Forest pathology inherently involves a landscape perspective, because tree pathogens propagate according to heterogeneous spatial patterns of flow and isolation. Landscape pathology is a field that is now emerging from the transdisciplinary cooperation of forest pathologists with landscape ecologists. Here, we review recent broad-scale assessments of tree disease risk, investigations of site and host preferences for several root rot pathogens, and regional historical analyses of pathogen outbreak in plantations. Crucial topics include fragmentation effects on pathogen spread and geophysical features that predispose forest patches to disease expression. Recent methodological developments facilitate the spatially explicit analysis of reciprocal coarse-scale relationships among hosts and pathogens. Landscape pathology studies fill a significant research gap in the context of our understanding of sustainable forest management, the introduction of exotic organisms and how climate change might affect the spread of disease.

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.

Primary resource capture in two sympatric Armillaria species in managed Norway spruce forests.

The ability of the preferentially saprotrophic fungus Armillaria cepistipes and the pathogenic A. ostoyae to capture fresh stump resources was investigated in managed Alpine Norway spruce (Picea abies) forests where both species occur sympatrically. The incidence of Armillaria species and genets as rhizomorphs in the soil as well as external and internal (heart rot) stump colonisation were determined in three comparable 1 ha plots. The results indicate that A. cepistipes and A. ostoyae have a very similar strategy to capture fresh stump resources. Both species produce dense networks of rhizomorphs in the soil enveloping living trees. After the felling of the trees, the stumps are rapidly captured through spread of mycelial fans in the cambial zone. Our study suggests that A. ostoyae is slightly more competitive than A. cepistipes in primary resource capture.

Cryphonectria radicalis: rediscovery of a lost fungus.

In an attempt to isolate the ascomycete Cryphonectria parasitica (Diaporthales, Valsaceae) from dead chestnut stems, we obtained three C. radicalis strains. All three strains were isolated in areas of Switzerland with high chestnut blight incidence. To confirm our species designation, we compared the three C. radicalis strains to hypovirus (hv)-free and hv-infected C. parasitica strains. The comparison revealed several distinctive characteristics. On potato dextrose agar in the dark, the C. radicalis strains produced a fluffy mycelium and small droplets of a purple exudate giving the mycelium a light pinkish appearance. On corn meal medium in the dark, the C. radicalis strains caused a color change of the medium to purple, whereas the C. parasitica strains did not cause any color change. Ascospores from C. radicalis were significantly smaller than C. parasitica ascospores and their dimensions fit within other published size ranges. Southern hybridization analysis of the two species using nuclear and mitochondrial probes support their taxonomic separation. This separation is further supported by the lack of successful interspecific crosses. In virulence tests on chestnut trees, the C. radicalis strains exhibited very low virulence, comparable to highly hypovirulent hv-infected C. parasitica strains. Our results suggest that C. radicalis still coexists with C. parasitica although at a low frequency.