Hymenoscyphus species associated with European ash1

The causative agent of dieback on European ash (Fraxinus excelsior) was first described as Chalara fraxinea based on cultural morphology because no sexual stage of the fungus was known. Later, based on culturing of ascospores of a candidate teleomorph, morphological comparison and nuclear ribosomal internal transcribed spacer sequencing, the sexual stage of C. fraxinea was assigned as Hymenoscyphus albidus, a native and widespread species in Europe. Recently, the morphological species concept of H. albidus was shown to cover two species that cannot be separated from each other based on teleomorph characters, but which can be distinguished by several DNA markers. As a result, the strains causing ash dieback were reassigned as H. pseudoalbidus. The closely related H. albidus is presumably a non‐pathogenic endophyte, but pathogenicity tests to confirm this hypothesis have not yet been performed. Genotyping of herbarium specimens has shown that H. pseudoalbidus was present in Switzerland for at least a decade prior to the epidemic outbreak in Europe. The origin of the ash dieback pathogen, and the general importance of correct pathogen identification to development of effective disease control, are discussed.     

Ash dieback: pathogen spread and diurnal patterns of ascospore dispersal,with special emphasis on Norway*

Dieback of European ash (Fraxinus excelsior), caused by the ascomycete Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea), started around 1992 in Poland and has since then spread over large geographical areas. By November 2010, the disease had been recorded in 22 European countries. The gradual expansion and high intensity of the ash dieback epidemic in Europe may suggest that H. pseudoalbidus is an invasive alien organism. In Norway, ash dieback was first reported in spring 2008, and a survey in early summer of the same year revealed that the disease had spread over large parts of the southern and eastern regions of the country. The distance from the southernmost to the northernmost infected stands was, at that time, about 400 km. Some old necrotic lesions were also observed, indicating that the ash dieback pathogen is likely to have been present in Norway since at least 2006. In 2009, a spore sampler was installed in a diseased ash stand at Ås, South‐Eastern Norway. Sampling started in late July and continued until late September. Large numbers of ascospores resembling those of H. pseudoalbidus were observed, with the maximum number of spores occurring from the end of July to mid‐August. The deposition of ascospores occurred mainly between 6 and 8 a.m. Ascospores are most likely to be the primary source initiating host infections and responsible for the rapid recent spread of H. pseudoalbidus in Europe.     

Occurrence of Hymenoscyphus pseudoalbidus on infected ash logs

Ash decline induced by Hymenoscyphus pseudoalbidus is an emerging disease that severely affects Fraxinus excelsior stands in Europe. There has been an invasive spread of the disease from east to west in Europe over the last decade. Wood discoloration on infected trunks has been reported, but few data are available on the involvement of H. pseudoalbidus in such symptoms. Transport and trade of ash logs could introduce the pathogen into disease‐free areas and therefore accelerate its dissemination. The aim of this study was to assess the prevalence and severity of H. pseudoalbidus in ash logs in infested areas located in the northeast of France and to clarify the role of secondary pathogens in ash decline. The results showed that prevalence of H. pseudoalbidus on collar lesions was high in the study area. The pathogen was able to produce conidia from infected wood. Thus, export of ash logs could represent a potential risk for spreading the disease. Involvement of Armillaria spp. in the decline process was confirmed, while no Phytophthora‐induced collar lesions were found. Studying both disease prevalence and the age of callus tissues surrounding collar lesions in 60 ash stands enabled the origin of the disease in the study area to be determined.     

Light and scanning electron microscopy studies of the early infection stages of Hymenoscyphus pseudoalbidus on Fraxinus excelsior

The ontogeny and morphology of infection structures associated with the early stages of infection of Hymenoscyphus pseudoalbidus on common ash (Fraxinus excelsior) leaves and leaf petioles were investigated using light and scanning electron microscopy. Ascospores were produced in mature ascocarps and infections on ash leaves were first observed 2 weeks later. Ascospores developed germ tubes, followed by appressorium formation and penetration of epidermal cells on ash leaves and petioles. Chalara fraxinea spores, the anamorph of H. pseudoalbidus, appeared and were arranged in chains, surrounded by a membranous sheath, and varied considerably in size and shape. Host invasion and colonization of all cell types of leaves and petioles were observed using light microscopy. The role of leaves and petioles as sites of infection in the life cycle of H. pseudoalbidus and the disease cycle of ash dieback is discussed.     

Variation in aggressiveness of Hymenoscyphus fraxineus genotypes amid the ash dieback epidemic

The ascomycete Hymenoscyphus fraxineus causes devastating damage to the European common ash (Fraxinus excelsior). The fungus originates from Asia, where it coexists with native ash species and completes its life cycle by sporulating on degrading ash leaf litter on the forest floor. Given this life cycle of the fungus, genotypes of H. fraxineus with varying degrees of aggressiveness may coexist in infected European ash forests. To test this hypothesis, we cultured 19 single-spore isolates from apothecia collected in a trial of heavily infected ash in Denmark and carried out stem inoculations on young ash seedlings. Microsatellite markers revealed that the 19 isolates were all genetically unique and did not show any genetic structure. High variation was observed among the 19 isolates in pathogenicity on young seedlings. The variation (assessed as necrosis development) was highly significant, but not correlated with the vigour of the fungal isolates when grown on culture media. The results support the hypothesis that aggressiveness of H. fraxineus may not be crucial for its fitness. In this sense, ash dieback disease may differ from other recent emerging infectious diseases on trees. We discuss the variation in aggressiveness of H. fraxineus in relation to durability of resistance and future management of the ash dieback epidemic. The findings of this study call for more research into natural variation in endophytic versus pathogenic behaviour of H. fraxineus on European ash.     

The ash dieback crisis: genetic variation in resistance can prove a long‐term solution

Over the last two decades, ash dieback has become a major problem in Europe, where the causative fungus has invaded the continent rapidly. The disease is caused by the invasive pathogenic fungus Hymenoscyphus pseudoalbidus (anamorph Chalara fraxinea), which causes severe symptoms and dieback in common ash, Fraxinus excelsior. It is becoming a significant threat to biodiversity in forest ecosystems and the economic and aesthetic impacts are immense. Despite the presence of the disease for at least 10 years in Scandinavia, a small fraction of F. excelsior trees have remained vigorous, and these trees exhibit no or low levels of symptoms even where neighbouring trees are very sick. This gives hope that a fraction of the ash trees will retain a sufficiently viable growth to survive. Following a period of high mortality in natural populations, selection and breeding of remaining viable ash trees could therefore provide a route for restoring the role of ash in the landscape. This paper reviews the available data on disease dissemination, and the consequences thereof in terms of symptom severity and mortality, and appraises studies that have tested the hypothesis that less‐affected trees have genetically based resistance. The implications of the results for the adaptive potential of common ash to respond to the disease through natural or assisted selection are discussed. The risks of adverse fitness effects of population fragmentation due to high mortality are considered. Finally, it is recommended that resistant trees (genotypes) should be selected to facilitate conservation of the species.     

Virulence of Hymenoscyphus albidus and native and introduced Hymenoscyphus fraxineus on Fraxinus excelsior and Fraxinus pennsylvanica

Ash dieback is caused by Hymenoscyphus fraxineus, a cryptic species of the putatively harmless Hymenoscyphus albidus. Recently, H. fraxineus was found to be native to East Asia. However, the virulence of Asian H. fraxineus strains on Fraxinus excelsior and the virulence of European H. albidus on hosts other than F. excelsior and Fraxinus mandshurica have not yet been assessed. In a wound inoculation study, the virulence of four H. albidus and four European and Japanese H. fraxineus strains was assessed on F. excelsior and Fraxinus pennsylvanica in a climate chamber. Lesion lengths were measured after approximately three and a half months. No lesions were observed on the negative control or on trees inoculated with H. albidus. In contrast, inoculation with H. fraxineus induced typical symptoms of ash dieback on both tree species. Japanese H. fraxineus strains induced significantly longer lesions compared to European strains. Fraxinus excelsior was highly susceptible and developed lesions averaging lengths of 1·7 and 8·4 cm for European and Japanese strains, respectively. Fraxinus pennsylvanica was less susceptible and developed average lesion lengths of 1·6 and 4·8 cm for European and Japanese strains, respectively. Most strains were successfully reisolated from necrotic lesions or inocula, fulfilling Koch's postulates. The data show that additional introductions of H. fraxineus strains from the native range to Europe could pose a threat to the conservation of F. excelsior. In addition, introduction of H. fraxineus to North America could potentially have a negative effect on the indigenous F. pennsylvanica.     

Pathogenicity of Hymenoscyphus fraxineus towards leaves of three European ash species: Fraxinus excelsior,F. angustifolia and F. ornus

This study aimed to demonstrate the association of the ash dieback pathogen Hymenoscyphus fraxineus with leaf symptoms on Fraxinus excelsior and to test its pathogenicity towards leaves of three European ash species, F. excelsior, F. angustifolia and F. ornus, in wound inoculation experiments. On F. excelsior, H. fraxineus was isolated from 94% of leaf rachises with necrotic lesions and from 74% of necrotic leaflet midribs. Following wound inoculation of leaf rachises, in two separate experiments performed in 2010 and 2011, the ash dieback pathogen caused symptoms (necrotic rachis lesions, leaf wilting and premature leaf shedding) on all three ash species, while control leaves remained symptomless. Hymenoscyphus fraxineus was consistently reisolated from fungus‐inoculated rachises. All 10 isolates tested were pathogenic to the three ash species and varied in virulence. Koch's postulates for H. fraxineus as causal agent of leaf symptoms on F. excelsior were fulfilled in this study. Complemented with the isolation of the fungus from naturally infected, symptomatic leaf rachises of F. angustifolia and F. ornus in previous investigations, H. fraxineus was confirmed to be a leaf pathogen of these ash species as well. The leaf inoculation experiments showed that F. excelsior was highly susceptible to H. fraxineus, F. angustifolia was equally or slightly less susceptible, whereas F. ornus was the least affected species; however, F. ornus should also be regarded as a host tree for the ash dieback pathogen. This susceptibility ranking corresponds well with field observations and previous stem inoculation experiments.     

Evaluation and validation of a loop-mediated isothermal amplification test kit for detection of <Emphasis Type="Italic">Hymenoscyphus fraxineus</Emphasis>

Hymenoscyphus fraxineus (anamorph Chalara fraxinea) is the ascomycete fungus which causes ash dieback, a potentially lethal disease of Fraxinus excelsior in Europe. Isolation and culturing of H. fraxineus is time consuming and there is a need for rapid, specific diagnostic tools to assist in the deployment of appropriate phytosanitary measures. Loop-mediated isothermal amplification (LAMP) is a nucleic acid amplification method which can be used for on-site diagnosis. OptiGene have recently released a kit for rapid in planta detection of H. fraxineus using LAMP. The performance of the kit was evaluated for use in the laboratory and in the field in a comparison with real-time PCR, and the kit was also validated according to EPPO standard 7/98. The analytical sensitivity of the kit was found to be 7 pg of DNA extracted from pure culture. The kit was rapid, with an average time to positive of 15.5 min for field samples. A comparison of real-time PCR and LAMP was carried out in the laboratory and in the field. The diagnostic sensitivity and specificity of the LAMP kit in the laboratory were 90.2% and 98.0% respectively, and 98.3% and 88.6% in the field. Positive and negative predictive values and likelihood ratios were also calculated and considered in relation to the prevalence of the disease. The kit was found to be a useful diagnostic tool which can be applied in the field.     

Pathogenicity of phylogenetic species in the Fusarium graminearum complex on soybean seedlings in Argentina

Soybean (Glycine max L.) is one of the main crops in Argentina. Most of the studies of pathogenicity in the Fusarium graminearum complex have focused on strains isolated from wheat and maize, and there is little information on strains isolated from soybean. Our objective in the present study was to compare the pathogenicity among soybean isolates of different phylogenetic species within the Fusarium graminearum complex on soybean seedlings under controlled conditions. Six strains representing three different phylogenetic species (F. graminearum, F. meridionale and F. cortaderiae) were identified by partial sequencing of the Translation Elongation Factor -1α gene (TEF-1) and evaluated for pathogenicity. All six strains reduced emergence, mainly by causing pre-emergence damping-off, seedling height and root dry weight and produced abnormal seedlings. The mean disease severity averaged across all isolates was approximately 3.0 in a 0–4 rating scale where 0?=?healthy seedling and 4?=?dead seedling. Significant differences in pathogenicity were observed among F. graminearum, F. meridionale and F. cortaderiae. These results are consistent with the hypothesis that different phylogenetic species within the Fusarium graminearum complex isolated from soybean are pathogenic under controlled conditions to soybean seedlings in Argentina. The present study demonstrates for the first time the pathogenic effect of F. meridionale on soybean in Argentina.     

昆虫病原线虫对小地老虎的致病力测定及防治效果

为探讨应用昆虫病原线虫防治小地老虎技术的可行性,采用培养皿滤纸法比较9个品系的昆虫病原线虫对小地老虎幼虫的致病力,同时通过盆栽试验验证了斯氏线虫属小卷蛾斯氏线虫Steinernema carpocapsae NC116品系、芫菁夜蛾斯氏线虫S.feltiae SF-SN品系和异小杆线虫属嗜菌异小杆线虫Heterorhabditis bacteriphora H06品系的保苗效果和防治效果,并测定了苦参碱与NC116品系混用对小地老虎3龄幼虫的联合作用效果。结果表明,NC116品系对小地老虎3龄幼虫致病力最高,其对3龄、4龄和5龄的LD50分别为2.4、7.5、和31.2条/头,表明随小地老虎幼虫龄期递增,其致病力下降。盆栽玉米苗上分别施用线虫100~200条/头时,对玉米保苗效果及对小地老虎3龄幼虫的控制效果依次为NC116H06SF-SN。0.8 mg/L苦参碱和NC116品系混用后,可使小地老虎3龄幼虫死亡率提高109.89%,二者表现增效作用。     

Influence of site and stand factors on Hymenoscyphus fraxineus‐induced basal lesions

Hymenoscyphus fraxineus is an invasive fungus in Europe and causes a severe decline affecting ash, which began in the late 1990s. One of the symptoms associated with the disease is lesions in the outer bark of the collar area. However, the aetiology of these basal lesions, and in particular the relative roles of H. fraxineus and Armillaria species, is still controversial; moreover, little is known about the influence of environmental factors on the disease epidemiology. This study therefore surveyed 42 plots located in northeastern France, in an area affected by ash decline since 2008, in order to determine which environmental factors condition the severity of lesions associated with H. fraxineus on ash collar. The spatial pattern that is a consequence of the invasive spread of the disease was taken into account in the analysis, using a spatial hierarchical Bayesian model fitted by integrated nested laplace approximation (INLA). Results show that while basal lesions are tightly associated with H. fraxineus, their severity is influenced by the Armillaria species present in the plot. Sites with vegetation indicating moist conditions, or more humid topographical positions, were associated with more developed basal lesions.     

The use of ascospores of the dieback fungus Hymenoscyphus fraxineus for infection assays reveals a significant period of biotrophic interaction in penetrated ash cells

Ascospores, discharged naturally from apothecia growing on rachis debris, were used as inoculum to examine the invasion of ash tissues by Hymenoscyphus fraxineus in order to understand the critical, but poorly understood, early interactions between host and pathogen. Methods were developed to collect ascospores for controlled infection assays on detached leaves, petioles and stem internode tissues. Light microscopy, using plasmolytic techniques, allowed the invasion of living plant cells to be observed. Ascospores were readily available from late May to September. On the plant surface, most spores differentiated directly to form appressoria without germ‐tube growth. Direct penetration was followed by a significant period of biotrophic fungal growth before lesions developed. Following the formation of a vesicle‐like structure after penetration, bulbous and elongated intracellular hyphae were produced in living plant cells. The use of ascospore inoculum, rather than mycelia, will allow natural and rapid screening of ash genotypes for resistance to the devastating dieback disease. The identification of the biotrophic phase of infection suggests that host range is controlled by effector‐triggered immunity.     

Ash dieback due to Hymenoscyphus fraxineus: what can be learnt from evolutionary ecology?

The future existence of common ash (Fraxinus excelsior), an important tree species throughout temperate Europe, is threatened. An invasive fungal disease (ash dieback) has spread through much of the distribution area of common ash. The causal agent of the disease is Hymenoscyphus fraxineus, a necrotrophic ascomycete, most probably introduced from Asia in the early 1990s. Hymenoscyphus fraxineus infects ash trees and saplings through their leaves, from which it grows into the stem. The fungus was studied intensively in recent years but there is still a need to address the topic from an evolutionary perspective. In this overview, some key evolutionary aspects of ash dieback are discussed, from the Red Queen dynamics of host–pathogen interactions to the probable consequences for virulence evolution of multiple infections. The progression of ash dieback in Europe does not show spatial differences, but studies show variation in susceptibility within host populations, a probable consequence of genetic differences, thus providing material for evolution of disease resistance or tolerance. Breeding programmes need to maintain the genetic diversity of Fraxinus, to enable it to withstand further threats such as climate change and the emerald ash borer. Because H. fraxineus reproduces exclusively sexually, the pathogen is likely to overcome a narrow genetic resistance. The introduction of further strains of H. fraxineus to Europe and the movement of infected plant material should be avoided. This case study shows that the integration of evolutionary ecology considerations would benefit plant disease management and biosecurity in general.     

<Emphasis Type="Italic">Fusarium temperatum</Emphasis> isolated from maize in France

The presence of Fusarium temperatum in France was investigated by analyzing 40 Fusarium strains, previously isolated from infected maize ears in 2011 and formerly identified as Fusarium subglutinans based on morphological characteristics. In this study, 26 strains out of the 40 were identified as F. temperatum and 14 as F. subglutinans based on sequencing of the translation elongation factor 1α gene. The phylogenetic analysis showed that the two species represented two clades strongly supported by bootstrap values. The pathogenicity of F. temperatum strains was confirmed on maize ears. This study provides new information about F. temperatum isolated from maize in France.     

Effect of mycoviruses on the virulence of Fusarium circinatum and laccase activity

Laccase enzymes (benzenediol: oxygen oxidoreductase, EC 1.10.3.2) play a major role in the degradation of phenolic compounds such as lignin. They are common in fungi and have been suggested to participate in host colonization by pathogenic fungi. Putative mycoviruses have recently been isolated from the causal agent of pine pitch canker disease, Fusarium circinatum Nirenberg & O'Donell. In this study, the effects of single and double mycoviral infections on laccase activity, growth rate and pathogenicity were investigated in fourteen F. circinatum strains. Extracellular laccase activity was analyzed by the Bavendamm test, image processing and a spectrophotometric method. Mycelial growth, in vivo pathogenicity and seedling survival probability were also determined in Monterrey pine (Pinus radiata D. Don) seedlings. The findings showed that (i) mycelial growth of isolates from the same fungal population was homogeneous, (ii) the presence of mycovirus appears to increase the virulence of fungal isolates, (iii) co-infection (with two mycoviruses) caused cryptic effects in fungal isolates, and (iv) laccases embody a possible auxiliary tool in fungal infection. The prospects for biocontrol, the adaptive role of F. circinatum mycoviruses and the importance of laccase enzymes in host colonization are discussed.     

Population structure of the ash dieback pathogen,Hymenoscyphus fraxineus,in relation to its mode of arrival in the UK

The ash dieback fungus, Hymenoscyphus fraxineus, a destructive, alien pathogen of common ash (Fraxinus excelsior), has spread across Europe over the past 25 years and was first observed in the UK in 2012. To investigate the relationship of the pathogen's population structure to its mode of arrival, isolates were obtained from locations in England and Wales, either where established natural populations of ash had been infected by wind‐dispersed ascospores or where the fungus had been introduced on imported planting stock. Population structure was determined by tests for vegetative compatibility (VC), mating type and single‐nucleotide polymorphisms (SNPs). VC heterogeneity was high at all locations, with 96% of isolate pairings being incompatible. Frequencies of the MAT1‐1‐1 and MAT1‐2‐1 idiomorphs were approximately equal, consistent with H. fraxineus being an obligate outbreeder. Most SNP variation occurred within study location and there was little genetic differentiation between the two types of location in the UK, or between pathogen populations in the UK and continental Europe. There was modest differentiation between UK subpopulations, consistent with genetic variation between source populations in continental Europe. However, there was no evidence of strong founder effects, indicating that numerous individuals of H. fraxineus initiated infection at each location, regardless of the route of pathogen transmission. The ssRNA virus HfMV1 was present at moderate to high frequencies in all UK subpopulations. The results imply that management of an introduced plant pathogen requires action against its spread at the continental level involving coordinated efforts by European countries.     

The mango sudden decline pathogen, Ceratocystis manginecans, is vectored by Hypocryphalus mangiferae (Coleoptera: Scolytinae) in Oman

In Oman, the bark beetle Hypocryphalus mangiferae is closely associated with trees affected by mango sudden decline disease caused by Ceratocystis manginecans. Although it has previously been assumed that this beetle plays a role in the dispersal of the pathogen, this has not been established experimentally. The aim of this study was to determine whether H. mangiferae vectors C. manginecans from infected to healthy mango trees. A survey conducted in northern Al Batinah region of Oman revealed that H. mangiferae was closely associated with mango sudden decline disease symptoms and it was found on trees in the early stages of the disease. Healthy, 2-year-old mango seedlings were exposed to H. mangiferae collected from diseased mango trees. Seedlings were infested by the bark beetles and after 6 weeks, typical mango sudden decline disease symptoms were observed. Ceratocystis manginecans was isolated from the wilted mango seedlings while uncolonized control seedlings remained healthy. The results show that H. mangiferae vectors C. manginecans in Oman and is, therefore, an important factor in the epidemiology of this disease.     

Genetic structure and pathogenicity of populations of Phytophthora infestans from organic potato crops in France, Norway, Switzerland and the United Kingdom

Genetic variation and pathogenicity of Phytophthora infestans isolates collected from organic potato crops of the susceptible cv. Bintje and the moderately resistant cv. Santé were assessed in France, Norway, and the United Kingdom in 2001 and in Switzerland in 2001 and 2002. Population structures differed considerably between the four P. infestans populations. Those from France, Switzerland and the UK were mainly clonal populations showing restricted levels of genetic diversity, whilst those from Norway were mixed A1 and A2 mating type populations with high levels of genetic diversity, suggesting periodical sexual reproduction. Isolates collected from cv. Bintje were on average more aggressive than or comparable to isolates from cv. Santé. Race complexity varied considerably between the regional P. infestans populations, with isolates from France and Switzerland showing the highest number of virulence factors. In all pathogen samples but the French, isolates collected from cv. Santé were more complex than isolates collected from cv. Bintje. No directional selection towards increased aggressiveness towards the more resistant cultivar Santé was observed. This suggests that there is no shift towards increased levels of pathogenicity in P. infestans populations following the large-scale introduction of more resistant potato varieties in organic production systems in Europe.     

Observation en Hollande de la forme parfaite du champignonGloeosporium perennans Zeller et Childs

Summary In October 1956 apothecia, belonging toPezicula malicorticis (Jackson)Nannfeldt (=Neofabraea malicorticis (Cordley) Jackson), the perfect stage of the parasite known in plant pathology literature asGloeosporium perennans, have been found on naturally infected appletrees in Druten and near Deest (Land van Maas en Waal) and on appletrees at Wageningen, which had been inoculated with mycelium ofG. perennans in October 1955. The apothecia occurred as well on the surface of the bark as in old conidial fructifications; they were sessile, clearly convex, bright-yellow under moist conditions and brownish when old or dry. The diameter of the apothecia was 0,4–1,6 mm. As many as 100 apothecia were found on a square cm. It is put forward that the formation of the apothecia has been favoured by the very wet weather of the preceding summer.

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Ingénieur agronome aux Stations fédérales d'essais agricoles, Lausanne (Suisse).