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.     

Fraxinus excelsior seed is not a probable introduction pathway for Hymenoscyphus fraxineus

We investigated the transmission of Hymenoscyphus fraxineus from infested seed to germinating seedlings of common ash (Fraxinus excelsior) in order to determine the potential risk associated with intra‐ and intercontinental movement of seed. Neither fungal isolations from necrotic or healthy embryos nor PCR testing with H. fraxineus‐specific primers detected the pathogen. Similarly, H. fraxineus was not detected in axenically grown seedlings generated from infested seed lots. The results help clear up prior confusion of the pathogen being seed‐borne. Any remaining surface contamination by pathogen spores could be washed off seeds as a quarantine measure.     

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.     

Individual resistance of Fraxinus angustifolia clones to ash dieback

The presumed resistance of individual ash trees to ash dieback caused by invasive pathogen Hymenoscyphus fraxineus is an important issue for the maintenance of ash in European forests. All known studies regarding the resistance of ash trees to ash dieback were conducted in plantations and stands of F. excelsior; however, no such data exist for F. angustifolia. Crown damage assessments were performed over four consecutive years between 2009 and 2012 at a F. angustifolia clonal plantation in Hra??ica, Slovenia. Inoculation of H. fraxineus into the branches of the most and least damaged clones of F. angustifolia and leaf phenology assessments was performed to verify the presence of defence mechanisms that limit fungal growth or promote disease escape. Additionally, root collars of selected clones were inspected for fungal infections. The crown damage assessments showed considerable differences among F. angustifolia clones, indicating genetic variability in susceptibility to ash dieback. Crown dieback progressed significantly over the 4‐year time period; the mean crown damage of individual clones in 2012 varied between 16.7% and 83.8%. Significant differences among F. angustifolia clones were found in the inoculation trials and leaf phenology assessments. However, defence mechanisms such as early leaf flushing, early leaf shedding and the ability to inhibit pathogen growth in host tissues were not confirmed. High frequency of Armillaria spp. and H. fraxineus root collar infection demonstrated the need for whole tree inspection to determine causal agent of damages on individual ash trees. Armillaria spp. may be highly associated with ash decline epidemiology.     

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.     

Pathogenicity of fungi associated with ash dieback symptoms of one‐year‐old Fraxinus excelsior in Montenegro

In addition to Hymenoscyphus fraxineus, two fungi identified as Diaporthe eres aff. and Fusarium sambucinum aff. were also isolated from necrotic bark lesions on declining one‐year‐old Fraxinus excelsior in a forest stand in Montenegro. To examine their involvement in ash decline, a pathogenicity test was performed using under bark inoculations on one‐year‐old Fraxinus excelsior. Hymenoscyphus fraxineus was included as comparison. All three fungal species proved highly pathogenic towards one‐year‐old seedlings although lesion sizes differed significantly between the different species. Hymenoscyphus fraxineus was most aggressive, followed by F. sambucinum aff., while D. eres aff. caused the smallest lesions. This study demonstrates for the first time the ability of isolates in the D. eres and F. sambucinum species complexes to cause decline on one‐year‐old common ash seedlings.     

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.     

Tissue‐specific DNA levels and hyphal growth patterns of Hymenoscyphus fraxineus in stems of naturally infected Fraxinus excelsior saplings

Ten saplings of European ash (Fraxinus excelsior L.) naturally infected by the invasive ash dieback pathogen Hymenoscyphus fraxineus were collected in Ukraine and Norway and examined for bark necrosis and extension of discoloration in sapwood and pith in a stem region. Tissue‐specific colonization profiles were determined by spatial analyses of symptomatic and visually healthy stem tissues using a H. fraxineus‐specific qPCR assay and light microscopy. Our data suggest that hyphal growth in the starch‐rich perimedullary pith is of particular importance for both axial and radial spread of H. fraxineus, but that most of its biomass accumulates in sapwood parenchyma. The study confirms the results from earlier work and presents new information that refines the current stem invasion model.     

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.     

Phytophthora root and collar rot of mature Fraxinus excelsior in forest stands in Poland and Denmark

In recent years, Common ash (Fraxinus excelsior) throughout Europe has been severely impacted by a leaf and twig dieback caused by the hyphomycete Chalara fraxinea. The reasons for its current devastating outbreak, however, still remain unclear. Here, we report the presence of four Phytophthora taxa in declining ash stands in Poland and Denmark. Phytophthora cactorum, Phytophthora plurivora, Phytophthora taxon salixsoil and Phytophthora gonapodyides were isolated from rhizosphere soil samples and necrotic bark lesions on stems and roots of mature declining ash trees in four stands. The first three species proved to be aggressive to abscised roots, twigs and leaves of F. excelsior in inoculation experiments. Soil infestation tests also confirmed their pathogenicity towards fine and feeder roots of ash seedlings. Our results provide first evidence for an involvement of Phytophthora species as a contributing factor in current decline phenomena of F. excelsior across Europe. Specifically, they may act as a predisposing factor for trees subsequently infected by C. fraxinea. Phytophthora species from ash stands also proved to be aggressive towards a wide range of tree and shrub species commonly associated with F. excelsior in mixed stands. Although damage varied considerably depending on the Phytophthora species/isolate–host plant combination, these results show that many woody species may be a potential source for survival and inoculum build‐up of soilborne Phytophthora spp. in ash stands and forest ecosystems in general.     

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.     

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.     

Can Hymenoscyphus fraxineus infect hardy members of the Oleaceae other than ash species?

The interaction between the ash dieback fungus, Hymenoscyphus fraxineus, and two commonly grown members of the Oleaceae (Forsythia × intermedia ‘Lynwood’ and Ligustrum vulgare) was investigated. Shoots were inoculated with three isolates of H. fraxineus and harvested after 2 and 10 weeks. DNA was extracted from tissue collected at the point of inoculation, as well as 15 mm below and above (distal), and analysed using fungal‐specific ITS primers and specific primers for H. fraxineus. The fungus was frequently detected in tissue at the point of inoculation, but rarely in distal samples. There was no evidence that H. fraxineus was pathogenic on either of the two oleaceous species over the duration of the experiment.     

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.     

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.     

Control of Hymenoscyphus fraxineus,the causal agent of ash dieback,using composting

Viability of Hymenoscyphus fraxineus inocula following temperature treatments for different exposure times was examined in vitro and in aerated flask‐ and large‐scale composting tests using green waste. After an exposure for up to 10 days at 20°C, 97.3% of H. fraxineus mycelium and pseudosclerotia plate cultures remained viable. No viability was detected following a 3‐day exposure to 40°C or a 1‐day exposure to 45°C although pseudosclerotia were more tolerant than mycelium to an exposure to 35°C. Primordial apothecia of H. fraxineus emerged from 62%–100% of infected ash rachises collected from two infected sites and stored at 4°C for 0–5 months; exposure to compost for up to 10 days at 20°C did not affect this emergence. No emergence of H. fraxineus apothecia was observed from ash rachises that were exposed to compost at 45°C for 1 day or at 35°C or 40°C for 3 days in flasks or at 40°C for 1 day or at 30°C for 5 days in a large‐scale composting system. Based on a fitted model, estimates of the survival of H. fraxineus inoculum in infected ash rachises exposed to compost at 50°C for 1 day were 0.081% of that in the untreated H. fraxineus ash rachis inoculum. Increasing loss in viability of H. fraxineus inoculum in infected ash rachises during longer and warmer exposures to compost at 35°C–45°C corresponded with a reduced concentration of pathogen DNA detected in the rachises using real‐time PCR. However, exposure of rachises to compost at >53°C resulted in a smaller reduction in pathogen DNA detected than exposure to compost at lower temperatures, possibly due to the inhibition of enzymatic degradation of DNA at elevated temperatures.     

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.