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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Isolation,identification and necrotic activity of viridiol from Chalara fraxinea,the fungus responsible for dieback of ash

Investigation of secondary metabolite production of Chalara fraxinea, the fungus responsible for dieback of ash, led to the isolation of the phytotoxin viridiol and the fungistatic compound viridin. Viridiol showed necrotic activity towards ash‐seedlings.     

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.     

Infection of Norway spruce container seedlings by Gremmeniella abietina

First‐ and second‐year containerized Norway spruce seedlings were inoculated with conidia of type A (large tree type) and type B (small tree type) of Gremmeniella abietina var. abietina at different times during the summer. The appearance of symptoms after artificial inoculation and natural infection on spruce seedlings were recorded the following spring and compared with the disease symptoms on Scots pine seedlings. The proportion of diseased seedlings after inoculation reached as high as 80%. The susceptible period during the summer began later on the first‐year seedlings than on the second‐year seedlings, and was similar for the pine seedlings. Susceptibility of first‐year seedlings was highest in August and on second‐year seedlings in July. The accumulated temperature sum, relative humidity and height growth for first‐ and second‐year seedlings was assessed. Natural infection in 2002 caused more disease on pine than on spruce seedlings. Experimental thinning of seedlings had no effect on disease incidence. In a preliminary comparison between the ability of A and B types to cause disease in Norway spruce seedlings, type B caused more damage than type A after inoculation. However, type A caused a high disease frequency in other experiments in this study. Symptoms on Norway spruce seedlings often first occurred in the mid‐section of the shoot, and were similar to those observed on pine seedlings: needles turned brown, starting at the needle base, in the spring following inoculation. On first‐year spruce, diseased needles were shed rapidly, in contrast to a slower rate of shedding on first‐year pine seedlings. Pycnidia developed about 2 years after inoculation (on pine 1 year after inoculation). On Norway spruce seedlings the lower part of the shoot, including the lateral shoots, often remained alive. The experiments show that G. abietina can cause disease on containerized Norway spruce seedlings under nursery conditions in Finland. The coincidence of spore dispersal, seedling susceptibility and predisposing factors are important in disease development.     

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.     

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.     

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.     

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.     

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.     

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.     

Effect of Betula pendula clone and leaf age on Melampsoridium betulinum rust infection in a field trial

Ten 5‐year‐old Betula pendula clones were studied for their rust resistance in the field. The trees were treated by inoculating 10 leaves on a shoot with Melampsoridium betulinum urediniospore suspension or spraying the control leaves with water. The birch clones differed significantly in their resistance to M. betulinum leaf rust fungus and the clones also varied in their responses to the local rust strain and the inoculated rust strains. However, natural rust infections and inoculation treatment were positively correlated. The older leaves had fewer infections than the younger ones on the tip of the shoot in the control trees, but in the inoculation treatment no significant correlation was found between the leaf ages and rust infection. The factors behind the different leaf susceptibilities 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.     

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.