Differences in susceptibility to ash dieback in Czech provenances of Fraxinus excelsior

The impact of ash dieback caused by Hymenoscyphus fraxineus on 17 provenances of Fraxinus excelsior and one provenance of Fraxinus angustifolia was studied in an extensive field trial established in the Czech Republic prior to the H. fraxineus invasion in 1999. A difference in the level of resistance to ash dieback between the species was found: F. angustifolia was significantly less affected by the disease than F. excelsior. Moreover, particular provenances of F. excelsior showed important differences in the level of resistance to H. fraxineus. A relationship between the impact of ash dieback and altitude was also discovered – the provenances from altitudes above 600 m a.s.l. were less affected by the pathogen than were the provenances from lower areas. No difference in the impact of the disease among provenances of F. excelsior from different ecotopes (ravine, calcareous ravine and alluvial) was found. Substantial among‐tree variability in resistance to H. fraxineus was observed throughout the trial – promising genotypes (with crown defoliation up to 5%) were identified in all 18 tested provenances. In regard to this finding, it appears that the main source of resistance to the pathogen is probably at the individual genotype level in the trial. A secondary but massive attack by Hylesinus fraxini was identified in the trees that had been greatly damaged by ash dieback, and the beetle caused their health to deteriorate significantly. A significant negative effect of the presence of collar necroses caused by H. fraxineus and browse damage was also identified.     

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 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.     

Hymenoscyphus fraxineus mycelial growth on media containing leaf extracts of different Oleaceae

We assessed the mycelial growth rate of Hymenoscyphus fraxineus, the causal agent of ash dieback, on agar media containing leaf extracts of seven common Mediterranean species of the Oleaceae (Fraxinus excelsior, F. angustifolia, F. ornus, Ligustrum vulgare, Olea europaea, Phyllirea latifolia and Syringa vulgaris). The pathogen grew on all media, but growth rates showed significant differences among media and H. fraxineus isolates. Growth rates were highest on media containing F. excelsior and F. angustifolia, intermediate on media containing O. europaea and P. latifolia and lowest on those containing F. ornus and L. vulgare.     

Identifying Fraxinus excelsior tolerant to ash dieback: Visual field monitoring versus a molecular marker

Ash dieback (ADB) caused by the pathogen Hymenoscyphus fraxineus is the cause of massive mortality of Fraxinus spp. in Europe. The aim of this work was to check for the presence of the molecular marker for ADB tolerance in mapped healthy‐looking F. excelsior trees, and to compare its occurrence in trees exhibiting severe ADB symptoms. Monitoring of 135 healthy‐looking F. excelsior on the island of Gotland, Sweden, showed that after 3–4 years 99.3% of these trees had 0%–10% crown damage, thus remaining in a similar health condition as when first mapped. After 5–6 years, 94.7% of these trees had 0%–10% crown damage. Molecular analysis of leaf tissues from 40 of those showed the presence of the molecular marker in 34 (85.0%) trees, while it was absent in 6 (15.0%) trees. Analysis of leaf tissues from 40 severely ADB‐diseased trees showed the presence of the molecular marker in 17 (42.5%) trees, but its absence in 23 (57.5%) trees (p < .0001). The results demonstrated that monitoring of healthy‐looking F. excelsior is a simple and straightforward approach for the selection of presumably ADB‐tolerant ash for future breeding. The cDNA‐based molecular marker revealed moderate capacity on its own to discriminate between presumably ADB‐tolerant and susceptible F. excelsior genotypes.     

Ascocarp formation of Hymenoscyphus fraxineus on several‐year‐old pseudosclerotial leaf rachises of Fraxinus excelsior

The ash dieback pathogen Hymenosycphus fraxineus can form ascocarps on pseudosclerotial leaf rachises of Fraxinus excelsior not only in the year after leaf fall but also on older rachises, at least up to five growing seasons after the leaves have been shed. The significance of this finding for the epidemiology of ash dieback is discussed.     

Fungal colonization patterns in necrotic rootstocks and stem bases of dieback‐affected Fraxinus excelsior L.

European ash (Fraxinus excelsior) trees currently face the major threat of ash dieback caused by an invasive fungus, Hymenoscyphus fraxineus. Collar rots in F. excelsior have been increasingly associated with infections by this pathogen. However, the aetiology of the collar rots is still unclear and remains heavily debated. In contrast to most studies of this kind, entire rootstocks of four diseased ash trees were dug out to examine necrotic tissues in these rootstocks and stem bases in detail and to sample necrotic wood for fungal isolation. With the aid of morphological and molecular identification techniques, five to twelve fungal taxa were detected per tree. Members of the Nectriaceae family and Botryosphaeria stevensii, the causal agent of stem and branch cankers on many tree species, were frequently isolated from outer xylem. In contrast, H. fraxineus was the dominating species in interior wood layers. Microsatellite genotyping of 77 H. fraxineus isolates helped to identify up to six different genotypes per tree. The role of H. fraxineus and other isolated fungi in the aetiology of ash collar rots are discussed.     

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.     

Aqueous leaf extract of Ligustrum vulgare inhibits ascospore germination and mycelial growth of Hymenoscyphus fraxineus

Hymenoscyphus fraxineus, the causal agent of common ash dieback, possesses a low level of genetic diversity in Europe. The introduction of novel strains of this fungus must be prevented, due to the possible emergence of new virulence alleles, which could result in the infestation of the small proportion of hitherto resistant or tolerant ash trees. More comprehensive knowledge of the host spectrum of H. fraxineus is necessary for preventing further introductions. It is possible that H. fraxineus manifests itself in hosts beyond the genus Fraxinus, though this proposition has received little attention thus far. Two in vitro experiments were set up to investigate whether privet (Ligustrum vulgare) could serve as a host: germination rate of fresh H. fraxineus ascospores and colony growth of H. fraxineus were tested on agar media containing leaf extracts of privet, common ash (Fraxinus excelsior) and manna ash (Fraxinus ornus). Two different media with leaf extracts were tested, one with high (60%) and one with low (12%) extract content. Barely any significant differences were recorded in the case of the media with low extract content. Significant effects occurred only at the higher extract content level: germination was completely inhibited on the privet medium. Mycelial growth on the privet medium was slower than on both the common ash and manna ash media and, in addition, one of the three H. fraxineus strains was completely inhibited. These observations indicate the presence of inhibitors in privet. It is therefore unlikely to be a suitable host for H. fraxineus.     

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.     

Direct evidence of Hymenoscyphus fraxineus infection pathway through the petiole‐shoot junction

The symptoms of ash dieback caused by the fungus Hymenoscyphus fraxineus include wilting of the foliage followed by dieback of shoots, twigs and branches. Necroses in shoots are assumed to develop after infection through leaf petioles; however, clear evidence of this infection pathway has not yet been provided. Considering the multiple pathogen genotypes in dead ash petioles, we aimed to obtain a spatial overview of all H. fraxineus genotypes colonizing individual shoots and their corresponding petioles before leaf shedding to acquire precise information about the infection biology of H. fraxineus and its ability to cross the petiole‐shoot junction. Individual genotypes of H. fraxineus were characterized by the analysis of microsatellites using DNA extracted directly from petiole segments or cultures isolated from the segments. We detected 150 different multilocus genotypes in 10 analysed shoots and their respective petioles; the highest number of genotypes was eight for a single petiole and three for a single shoot. The genotypes of most shoot lesions were identical to particular genotypes from the proximal segments of petioles, implicating the main pathway of shoot infections. To test whether the amount of colonized substrate or intraspecific competition have an effect on successful infection, genotypes that reached the most proximal end of the petioles were scored for the number of invaded petiole segments and for the number of other H. fraxineus genotypes co‐occurring in the segments. However, the extent of colonization of the scored genotypes and intraspecific competition with other H. fraxineus strains did not influence pathogen success in entering the shoot. This study confirms that the majority of ash shoot infections are caused by genotypes of H. fraxineus originating from petioles. Compared to petioles, the frequency of shoot colonization as well as number of H. fraxineus genotypes in shoots was much lower.     

The introduction of Hymenoscyphus fraxineus to Northern Ireland and the subsequent development of ash dieback

Ash dieback caused by Hymenoscyphus fraxineus was first recorded in Northern Ireland (NI) in November 2012. The disease was observed only on recently (<6 years) planted trees. An in-depth case study in 2015 of an ash plantation with severe symptoms indicated that many of the trees were infected at the time of planting. Apothecia were observed developing from pseudosclerotia beneath the epidermis of dead branches still attached to the tree, suggesting a possible mechanism whereby H. fraxineus could be disseminated without leaf or rachises infection. Apothecia also formed on roots, indicating that infections may also occur in the soil. Often young trees were killed by the formation of large basal lesions which did not arise from stem infections higher up. On first detecting the disease on the island of Ireland the Governments of NI and the Republic of Ireland published an “All-Ireland Chalara Control Strategy.” Part of that strategy was a ban on the importation of ash plants from regions where the disease was known to be present, to prevent the introduction of further inoculum, and the implementation of an ‘eradication and containment’ policy with the aim of preventing the establishment and spread of the disease. While these measures may have slowed disease establishment, they were ultimately unsuccessful and by 2018 ash dieback was widespread and established throughout the whole of NI in plantations and in the wider environment.     

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.     

Phytophthora dieback on narrow leaved ash in the Black Sea region of Turkey

Severe dieback symptoms were observed in a 490‐ha moist ash (Fraxinus angustifolia) lowland forest stand, comprising trees over 100 years old and in 100 ha of newly planted F. angustifolia near Sinop, in Turkey. Five of the 10 soil samples collected around stem bases of the diseased trees were baited using ash leaves and yielded a Phytophthora sp. This heterothallic species produced non‐caducous, non‐papillate sporangia in non‐sterile soil extract, and fluffy, even growth on corn meal and potato dextrose agars, and suppressed, even growth on grated carrot agar. Isolates were identified as Phytophthora taxon salixsoil based on internal transcribed spacer DNA sequences. This species has been recently redesignated as P. lacustris. Three isolates were found to be pathogenic when inoculated on the stem bases of three‐year‐old F. angustifolia saplings.     

Fraxinus nigra (black ash) dieback in Minnesota: Regional variation and potential contributing factors

Extensive tree dieback is a recurrent issue in many regions. Crown dieback of Fraxinus nigra Marsh. (black ash; brown ash) in the northeastern and north central United States is an example. F. nigra is a widely distributed hardwood that is often the dominant species in wetland forests from Manitoba to Newfoundland and West Virginia to Indiana. Widespread crown dieback of F. nigra has been noted in many regions, but there are few quantitative assessments of dieback extent or relationship to potential causes. Most F. nigra dieback episodes are not associated with specific disease or pest agents. Drought, excessive soil moisture, cohort senescense, and road influences, have all been suggested as potential contributing factors. Our objectives were to (1) quantify variable dieback across northern Minnesota, a region described as having extensive dieback, (2) determine the relationship between dieback and site moisture, (3) relate dieback to tree age/size distributions, and (4) assess whether dieback was related to road proximity. Given the increasing threat of Agrilus planipennis (emerald ash borer) in the region, it is important to know the current health status of F. nigra populations before widespread infestation occurs. Many stands in our study exhibited high incidences of crown dieback. However, the incidence of dieback was variable across the region. Spatial variability in dieback was associated with site wetland characteristics; more dieback occurred on jurisdictional wetlands and on sites with a higher wetness index and a deeper depth to a perching layer. Dieback was also positively correlated with mean stand diameter, and tree diameters were generally correlated with age, suggesting that stands with larger and older individuals experienced more dieback. Cohort senescence is a possible explanation for this trend. Finally, dieback occurred with higher frequency nearer to roads. The road influence could be related to hydrological alterations or perhaps toxicity from road deicing salt. The fact that dieback is more severe close to roads may contribute to a general perception that black ash dieback is more severe throughout the region than our study suggests. Collectively, our results indicate that the healthiest F. nigra stands in our study region are likely to be younger and located on relatively drier sites and farther from roads, compared to stands with significant crown dieback.     

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.     

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.     

A versatile method for assessing pathogenicity of Hymenoscyphus fraxineus to ash foliage

We describe a method for inoculating rachises of Fraxinus excelsior (European or common ash) with Hymenoscyphus fraxineus, which is faster than previous methods and allows associated foliar symptoms to be assessed on replicate leaves. A total of ten ash seedlings were inoculated with five isolates of H. fraxineus and lesion development assessed over four weeks. A five‐point disease progress scale of symptom development was developed from no lesion (0), lesion on rachis (1), “pre‐top dead,” with curling of distal leaflets and bending of the rachis (2), top dead, with wilting and death of distal leaflets (3) to leaf abscission (4). The method revealed variation in aggressiveness of H. fraxinus isolates and may be suitable for assessing the resistance of F. excelsior and other Fraxinus species to dieback. The in vitro growth rate of H. fraxineus isolates was highly correlated with both disease progress and the length of rachis lesions on susceptible plants, indicating that it can be used as a preliminary step in selecting isolates with high aggressiveness for use in resistance screening.     

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.