A molecular toolkit for population genetic investigations of the ash dieback pathogen Hymenoscyphus pseudoalbidus

The recently described ascomycete fungus Hymenoscyphus pseudoalbidus (anamorph: Chalara fraxinea) causes the current dieback of ash (Fraxinus excelsior) in large parts of Europe. The origin of this species and its relation to the native cryptic species Hymenoscyphus albidus are still enigmatic. The spatiotemporal pattern of the epidemic is typical for an introduced invasive species. However, the presence of two cryptic species indicates that hybridization or mutation might have been involved in driving speciation in this case. In this study, we present a set of 18 polymorphic microsatellite markers to study these processes in more detail on a population genetic level. Markers were designed such that they can be amplified in three individual multiplex PCRs and analysed in two fragment analysis runs. We thoroughly tested the marker set for pairwise linkage among loci, selective neutrality and Mendelian inheritance. Additionally, the markers were applied on two large collections of isolates derived from study sites in Germany. Population genetic calculations suggested a low yet significant level of differentiation, a large genotypic diversity and a limited genetic diversity within populations. Furthermore, we present additional data concerning the phylogenetic relation between H. albidus and H. pseudoalbidus, which seems to be more distantly related to each other than expected previously.     

Hymenoscyphus albidus is not associated with an anamorphic stage and displays slower growth than Hymenoscyphus pseudoalbidus on agar media

Examination of isolates of Hymenoscyphus albidus from France revealed that this fungus does not form an anamorphic stage in culture. The lack of an asexual stage in this fungus is a conspicuous morphological difference to the ash dieback pathogen Hymenoscyphus pseudoalbidus, which is associated with its Chalara fraxinea anamorphic state. In growth studies on malt extract agar (MEA) and MEA amended with ash leaflets (ash leaf malt extract agar, AMEA) at 20°C, isolates of H. albidus grew slower than those of H. pseudoalbidus. On AMEA, the growth of cultures of both species was greatly enhanced.     

The teleomorph of Chalara fraxinea,the causal agent of ash dieback

Ash dieback, caused by the pathogen Chalara fraxinea, is an emerging lethal disease of Fraxinus excelsior, threatening the host species in large parts of Europe. The ascomycete Hymenoscyphus albidus (Helotiaceae, Helotiales) was identified as the teleomorph of C. fraxinea by culturing from ascospores, morphological comparison and nuclear ribosomal internal transcribed spacer (ITS) sequencing.     

The development of a species‐specific test to detect Hymenoschyphus pseudoalbidus in ash tissues

Ash dieback, caused by the pathogen Hymenoscyphus pseudoalbidus, is an emerging lethal disease of Fraxinus excelsior in large parts of Europe. To develop a method for the early detection of H. pseudoalbidus, we designed primers for 46 microsatellites (simple sequence repeats, SSRs) of the pathogen. Seven pairs of primers (SSR38, SSR58, SSR114, SSR198, SSR206, SSR211 and SSR212) were found to bind only to the genome of H. pseudoalbidus, but not to the genome of H. albidus or to 52 different fungal endophytes isolated from F. excelsior and F. angustifolia. Using these seven primer pairs, H. pseudoalbidus was identified in fruiting bodies and different types of ash tissues including dead leaves, dead petioles and discoloured or non‐discoloured wood. Along one twig, H. pseudoalbidus was detected at different levels of intensity, which depended on the distance from symptomatic tissue. The detection limit was 0.9–1.8 pg of genomic DNA per PCR. Of 50 analysed commercially available seedlings, six were infected with H. pseudoalbidus. Two SSR loci (SSR198 and SSR211) showed fragment length polymorphism. Our results showed that the new primers not only provide an easy and inexpensive means of detecting H. pseudoalbidus in ash tissues, but can also provide information on the genetic heterogeneity of the species.     

Hymenoscyphus pseudoalbidus and Hymenoscyphus albidus: viridiol concentration and virulence do not correlate

Hymenoscyphus pseudoalbidus is the causal agent of ash dieback, a disease that is presently endangering Fraxinus spp. throughout most of Europe. The phytotoxin, viridiol, was previously isolated from culture extracts of H. pseudoalbidus and found to be toxic to leaves of F. excelsior. Thus, we were interested in learning to what extent viridiol is responsible for pathogenicity of H. pseudoalbidus and investigated this using twelve isolates of H. pseudoalbidus. We also included five isolates of the closely related avirulent species, Hymenoscyphus albidus, in our studies. Some, but not all, isolates of H. pseudoalbidus and H. albidus produced measurable quantities of viridiol in culture. Three tests were used to determine to what extent viridiol concentration correlates with virulence: culture extracts were tested for activity in leaf segment tests and for inhibition of germination of seedlings of Fraxinus excelsior; virulence of the isolates was tested following infection of axenically cultured ash seedlings. Activity of the culture extracts varied, as did virulence of the isolates following inoculation into seedlings. No correlations were found between viridiol concentration and activities of culture extracts in leaf segment tests or in the germination test, nor between viridiol concentration and disease symptoms when inoculated into seedlings. However, activities of culture extracts in leaf segment and in the germination test correlated, as did the results of each of these tests with virulence in the infection experiment. Apparently, as yet unidentified factors other than the concentration of viridiol play important roles in the virulence of H. pseudoalbidus.     

Detection of Chalara fraxinea from tissue of Fraxinus excelsior using species‐specific ITS primers

Chalara fraxinea (teleomorph: Hymenoscyphus albidus) is known as a serious pathogen of Fraxinus excelsior, causing massive dieback of trees in Europe. The fungus is able to cause latent infections, and has been previously detected as an endophyte in asymptomatic tissues. Chalara fraxinea is a slow grower in culture, and is thus likely to be overgrown by faster growing fungi whenever pure culture isolations are being attempted. This study reports species‐specific ITS primers allowing fast and reliable detection of the pathogen directly from infected tissues of F. excelsior.     

Pathogenicity of Hymenoscyphus fraxineus and Hymenoscyphus albidus towards Fraxinus mandshurica var. japonica

The ash dieback pathogen Hymenoscyphus fraxineus proved to be pathogenic in a stem wound inoculation experiment on Fraxinus mandshurica var. japonica. In contrast, Hymenoscyphus albidus was proven non‐pathogenic for this host species.     

Temporal development of ash dieback symptoms and spatial distribution of collar rots in a provenance trial of Fraxinus excelsior

European ash (Fraxinus excelsior) is threatened by Hymenoscyphus pseudoalbidus, the agent of ash dieback disease. Beside ordinary ash dieback symptoms, collar rots have been reported on declining ashes as an additional problem of increasing severity. Since 2009, ash dieback has been surveyed annually in a provenance trial on four different sites in southwest Germany (Metzler et al. in Ger J For Res 183:168–180, 2012). The trial was established in 2005. Data of tree growth, ash dieback symptoms and collar rot prevalence were collected by surveying the trial in summer 2012 and compared with previously published data of the trial. Evaluations revealed a continuous and considerable increase in dieback severity since 2009. The results suggest that the infection process has not come to a standstill yet. Up to 2012, 6 % of the trees remained symptomless, whereas mortality added up to 9 %. There were significant differences in ash dieback severity between the investigated provenances. Collar rot prevalence ranged from 19 to 59 % between study sites. Moreover, high spatial dependency of collar rot prevalence could be detected within sites. Collar rots were more abundant on trees of severe ash dieback intensity, but could also be detected on 15 % of otherwise healthy trees. Mycelium from collar rots could be identified by means of RFLP analyses and sequencing of the ITS region as most likely belonging to Armillaria gallica. The possible roles of Armillaria spp. and H. pseudoalbidus in collar rot formation are discussed.     

Genetic variation in dieback resistance in Fraxinus excelsior confirmed by progeny inoculation assay

Ash dieback caused by the pathogenic fungus Hymenoscyphus fraxineus [previously known as H. pseudoalbidus (sexual stage) and Chalara fraxinea (asexual stage)] is a widespread problem in Europe. Here, we assess crown damage from natural infection and necrosis development following artificial controlled inoculations on full‐sib and half‐sib progeny from Danish Fraxinus excelsior clones with contrasting and well‐characterized levels of susceptibility to the disease. The inoculation assay was performed on a total of 123 offspring, and necrosis development monitored over two years. The offspring from low susceptible mother clones developed smaller necroses when compared to offspring from susceptible clones. Their crown damage due to natural infections was also significantly less. The correlation coefficient between average crown damages of mother clones and the average of their progeny was 0.85 (natural infections), while the correlation between crown damage of mother clones and the average necrosis development in their progeny after controlled inoculation was 0.73. The correlation between resistance of parent trees and crown damage/necrosis development on their offspring confirms the presence of heritable resistance and indicates that a bioassay based on controlled inoculations has the potential of becoming a fast and cost‐effective tool for estimation of dieback susceptibility in breeding programmes for resistance in ash trees.     

Fungi in foliage and shoots of Fraxinus excelsior in eastern Ukraine: a first report on Hymenoscyphus pseudoalbidus

In eastern Ukraine, the first symptoms of dieback on common ash (Fraxinus excelsior) were observed in 2010, as sparse flushing of leaves, bark necrosis and wood discoloration of shoots. The aim of this study was to assess possible causal agents of the damage by studying fungal communities in both symptomatic and healthy‐looking shoots, and leaf petioles. Field sampling was carried out in 2010 in Kharkiv and Sumy regions in eastern Ukraine and included 68 segments of symptomatic shoots, 68 segments of healthy‐looking shoots and 240 segments of petioles. DNA isolation from individual segments and direct sequencing of fungal ITS rRNA resulted in 430 fungal sequences representing 29 distinct taxa. Results showed that Hymenoscyphus pseudoalbidus – the primary causal agent of ash dieback in Europe – was present at low proportion (5.6%) in symptomatic shoots. Other more frequently detected fungi were Epicoccum nigrum, Venturia fraxini, Colletotrichum truncatum, Aureobasidium pullulans, Alternaria alternata, Alternaria sp. and Lophiostoma corticola. In conclusion, the study reports on the first incidence of ash decline in the Ukraine and other possible causal agents of damage which may help to evaluate and forecast the future situation with F. excelsior stands in the region.     

Forest self-regeneration following clear-felling of dieback-affected Fraxinus excelsior: focus on ash

Due to the dieback caused by invasive fungus Hymenoscyphus pseudoalbidus, ash is threatened in many regions of Europe. In order to predict the situation with ash ecosystems, it is important to examine forest regeneration in areas formerly dominated by ash with long disease history and known management. The main aim of the present study was, therefore, to evaluate forest self-regeneration following clear-felling of ash dieback-devastated stands, focusing on ash. In Lithuania, during 2011, a forest regeneration inventory was performed on twenty clear-cuts, initially dominated by stands of ash, but severely damaged by the dieback caused by H. pseudoalbidus and clear-felled 1–10 years previously. In subsequent forest regeneration, grey alder and birch dominated. Ash regeneration was scarce, and its proportion decreased from 40–100 % in pre-dieback stands to 0–21 % in current stands. Of all observed ash trees (n = 775), 53.9 % were diseased, 16.8 % were dead, and only 29.3 % were visually healthy. Moreover, ash was among the slowest growing species, while grey alder, aspen and birch were the fastest. The regeneration and health condition of ash in forests, where previous to the dieback ash was dominant, demonstrated a sharp decrease in occurrence of this species and a clear shift in species composition towards grey alder and birch.     

First definite report of natural infection of Fraxinus ornus by Hymenoscyphus fraxineus

Hymenoscyphus fraxineus was isolated from four leaf rachises with necrotic lesions of flowering ash (Fraxinus ornus) seedlings, which had been planted on a forest site in Austria where they were exposed to a massive natural infection pressure. This represents the first definite report of natural infection of this ash species by the ash dieback pathogen.     

First record of Chalara fraxinea in Finland and genetic variation among isolates sampled from Åland,mainland Finland,Estonia and Latvia

We have isolated and confirmed the identification of ash dieback fungus (Chalara fraxinea, teleomorph Hymenoscyphus albidus) for the first time in Finland. In a preliminary analysis, considerable amount of genetic variation was detected among 20 Finnish (Åland and mainland Finland), one Latvian and 11 Estonian isolates, analysed by random amplified microsatellite (RAMS) markers.     

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

Hymenoscyphus fraxineus, the causal agent of ash dieback, was inoculated onto intact, unwounded current‐year shoots and leaf scars of 4‐year‐old, potted Fraxinus excelsior seedlings. Pieces of ash wood colonized by the fungus were used as inoculum. Three of 25 (12%) of the inoculated intact shoots and nine of 25 (36%) of the inoculated leaf scars were infected by H. fraxineus and developed typical symptoms of ash dieback, including necrotic lesions on the shoot surface and wood discoloration as well as shoot and leaf wilting distal to the inoculation site. No symptoms occurred on control seedlings, which had been inoculated in the same way but with sterile wood pieces. Visible necrotic lesions on shoots and wood discoloration were statistically significantly longer in proximal than in distal direction from the inoculation site, a pattern which resembles symptoms after natural infection. The ash dieback pathogen was re‐isolated from nine of 12 (75%) of the symptomatic seedlings. These results provide indirect supportive evidence that the fungus infects shoots via leaves and shows that it is able, under experimental conditions using a massive mycelial inoculum, to directly infect intact, unwounded current‐year shoots of its main host in Europe.     

Introduction of Mandshurian ash (Fraxinus mandshurica Rupr.) to Estonia: Is it related to the current epidemic on European ash (F. excelsior L.)?

Recent investigations in Japan have suggested that the causal organism of the ongoing epidemic affecting European ash (Fraxinus excelsior) in Europe, Hymenoscyphus pseudoalbidus, may originate in East Asia. The fungus may have been unintentionally carried to Europe during the introduction of Mandshurian ash (F. mandshurica), the host tree of the fungus in East Asia. Still unicentric emergence hypothesis is in force: An area in the eastern Poland or Baltic has been shown to be the presumed epicentre of the epidemic. Really, during the Soviet occupation, several consignments of F. mandshurica seeds and plants, originating directly from the natural range of F. mandshurica in East Asia (Russian Far East), reached Baltic areas. In this paper, an overview about the Mandshurian ash is presented, the history of introduction of F. mandshurica to Estonia is reviewed and colonization of F. excelsior in this country by H. pseudoalbidus is briefly discussed. At present, we could not find any evidence, spatial or temporal, for a direct connection of the disease emergence on native F. excelsior with the introduction of F. mandshurica. The pathogen first colonized northwest Estonia and moved southeast and not from south to north as would be expected according to the hitherto existing unicentric hypothesis. However, more information is needed from different regions before to pose a multicentric emergence hypothesis and to deepen more into the investigations of the environmental factors that affected the host and supported to the epidemic in different areas.     

Genetic resistance to Hymenoscyphus pseudoalbidus limits fungal growth and symptom occurrence in Fraxinus excelsior

The European common ash (Fraxinus excelsior) is currently threatened by a pathogenic fungus, Hymenoscyphus pseudoalbidus, which seems to enter the trees through the leaves. Continuous assessments of 39 clones in Danish field trials have shown that there are significant differences in the susceptibility of clones to the new disease. Interestingly, clones that showed early leaf senescence in the autumn were in general less susceptible to the disease than late‐senescing clones. Thus, variation in susceptibility could be owing to phenological differences associated with the infection biology. To test whether differences in susceptibility are driven by genetically based factors other than phenology, we compared inoculations with H. pseudoalbidus on four highly susceptible clones with those of four less susceptible clones. Development of necrosis was hereafter followed regularly. The growth of the fungus in the inner bark was further detected with species‐specific PCR primers. The severity of the response to infection shows significant differentiation among clones and significant correlation with clone susceptibility, as assessed from natural infections in field trials. The fungus was detected in tissues immediately surrounding the necrosis but showed some signs of endophytic growth. The results suggest that healthier clones are able to limit the growth and spread of the fungus and thereby minimize the occurrence of symptoms. This gives hope for the future preservation of F. excelsior in Europe through selection and breeding.     

First report of Hymenoscyphus fraxineus on Fraxinus excelsior in Montenegro

During the monitoring of the mycological complex on different forest tree species in the Biogradska Gora National Park in north‐east Montenegro, symptoms indicative of ash dieback caused by Hymenoscyphus fraxineus were observed on young Fraxinus excelsior trees in the protected virgin forest, including dieback of plants and branches, wilting of leaves and shoots leading to a “flag‐like” habitus, premature shedding of leaves and longitudinal bark necroses. Using standard isolation methods, slow‐growing cultures with numerous phialides, typical of the asexual phase of the ash dieback fungus, were obtained. In addition, petioles with numerous characteristic apothecia were also recorded. This is the first report of H. fraxineus on common ash in Montenegro. Possible pathways of introduction and implications of the findings are discussed.     

Temporal evolution of collar lesions associated with ash dieback and the occurrence of Armillaria in Belgian forests

Ash dieback, caused by the fungus Hymenoscyphus fraxineus, has been observed in Europe for several years. In Belgium, the disease was first reported in 2010. Besides crown defoliation and dieback, collar lesions have sometimes been reported. To evaluate the prevalence and the progression of collar lesions and crown defoliation in ash dieback‐affected stands of various ages, a survey was conducted in 2013 and 2014 on 268 ash trees (Fraxinus excelsior) originating from 17 Walloon forest stands. The results showed that the proportion of trees with collar lesions greatly increased between June 2013 and September 2014 and that there appeared to be no significant link between a tree's diameter‐at‐breast height (DBH) and collar lesion occurrence. The mean percentage of defoliation increased in each forest stand across time, with observations conducted in September 2013 and 2014 showing a positive correlation with the mean percentage of trees with collar lesions. Molecular tests were carried out on 103 additional trees originating from 12 of the 17 stands to evaluate the occurrence of H. fraxineus and Armillaria spp. at the collar level. Most of the trees (98%) were infected by H. fraxineus. In contrast, only 41% of the samples were infected with Armillaria spp., most commonly A. gallica and A. cepistipes. This study discusses the role of Armillaria spp. and the rapid increase in the number of trees with collar lesions within the context of the evolution of ash dieback in Europe.     

Genetic variation in dieback resistance: growth and survival of Fraxinus excelsior under the influence of Hymenoscyphus pseudoalbidus

The invasive fungal pathogen, Hymenoscyphus pseudoalbidus V. Queloz, has decimated stands of Fraxinus excelsior L. over most of the species' natural distribution area. We assessed crown damage from 2009 to 2014 (at ages 8, 11 and 13) in 43 open-pollinated ash families planted in north-eastern Zealand, Denmark, and confirmed the presence of substantial genetic variation in ash dieback susceptibility. The average crown damage increased in the trial from 61% in 2009 to 66% in 2012 and 72% in 2014, while the estimated heritability was 0.42 in both 2009 and 2012 but increased to 0.53 in 2014. Genetic correlation between assessments was 0.88 between 2009 and 2012 and 0.91 between 2009 and 2014, suggesting fairly good possibilities for early selection of superior genotypes in the presence of high infection levels in the trial. The level of crown damage had strong negative effect on growth and survival. Only 34% of the trees with high levels of damage in 2009 were still alive in 2014, emphasising that high susceptibility is associated with low fitness.