Natural infection of Fraxinus excelsior seeds by Chalara fraxinea

The occurrence of Chalara fraxinea, the fungus responsible for dieback of European ash (Fraxinus excelsior), was investigated in the current and previous seed years collected from symptomatic trees in Latvia and Sweden using molecular techniques (DNA extraction, ITS‐PCR, Sanger sequencing). Molecular analysis of seeds revealed the presence of 30 different fungal taxa. Chalara fraxinea was detected in 8.3% of seeds tested from the current year originating from Latvia. The presence of C. fraxinea in seeds of F. excelsior is of great concern to phytosanitary protection authorities in countries outside the current zone of infestation.     

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.     

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.     

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.     

Pathogenicity of Chalara fraxinea

The mitosporic fungus Chalara fraxinea (Ascomycota) is associated with dieback of common ash, an emerging disease of Fraxinus excelsior (Oleaceae) in Europe. The pathogenicity of C. fraxinea was demonstrated by field inoculations on young trees.     

Mycological and histological investigations of Fraxinus excelsior nursery saplings naturally infected by Chalara fraxinea

Ash dieback is an emerging disease of Fraxinus excelsior in Germany. To date, economical damage is significant in nurseries, which also contribute towards spread of the disease, but damage to forests is increasing. The study presents the results of mycological and histological investigations on three hundred 3‐year‐old nursery ash saplings. The infection rate by the causative pathogen was determined for bark, outer and inner xylem, the pith and also separately for the above‐ground portion and root system of the plants. The invasion and colonization strategy of the fungus in the woody stem was examined. In addition, the presence of soil‐borne Oomycetes as possible primary or accompanying causal organisms was investigated. The results verify the dominant role of Chalara fraxinea as a causal agent of ash dieback and rule out the role of Oomycetes in the disease process. We conclude that C. fraxinea is not primarily endophytic in nature and spreads very effectively in the central stem tissues, which enables colonization of the woody stem in all three dimensions. Infections arising in the upper part of plants can thus spread extensively to lower parts.     

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.     

Occurrence and pathogenicity of fungi in necrotic and non-symptomatic shoots of declining common ash (<Emphasis Type="Italic">Fraxinus excelsior</Emphasis>) in Sweden

Currently, massive dieback of Fraxinus excelsior is observed in countries of eastern, northern and central Europe, and the reasons for it are unclear. The aims of the present work were (a) to study fungal communities in declining F. excelsior crowns; (b) to clarify role of fungi in the decline. Shoots from symptomatic crowns were collected in four localities in central Sweden, and distributed into the following categories: (a) visually healthy; (b) initial necroses; (c) advanced necroses; (c) dead tops. The most frequently isolated fungi were Gibberella avenacea, Alternaria alternata, Epicoccum nigrum, Botryosphaeria stevensii, Valsa sp., Lewia sp., Aureobasidium pullulans and Phomopsis sp., and these taxa were consistently found in shoots of all four symptomatic categories. Forty-eight taxa of other fungi were isolated, and fungal diversity was not exhausted by the sampling effort. The same taxa of fungi were dominant in F. excelsior shoots of different symptomatic categories, and moderate to high similarity of fungal communities was observed in shoots despite the symptoms. Forty-four isolates from 24 fungal taxa were used for artificial inoculations of 277 1-year-old F. excelsior seedlings in bare root nursery. After 2 years, only four fungi caused symptomatic necroses of bark and cambium: A. alternata, E. nigrum, Chalara fraxinea and Phomopsis sp. The most pathogenic was C. fraxinea, inducing symptoms on 50% of inoculated trees, while three other fungi caused necroses on 3–17% of inoculated trees. Infection biology of C. fraxinea and environmental factors determining susceptibility of F. excelsior to decline deserve future investigations.     

Effect of long-phase stratification treatments on seed germination in ash

• Introduction   

Ash (Fraxinus excelsior L.) seeds require warm treatment followed by cold treatment to release dormancy and this is most effective if carried out in a medium. The objective of this study was to determine if long periods of warm treatment would enhance germination at 15 (8-h light)/5°C (dark) and 15°C (same lighting conditions) in ash seeds of north-western European origin.     

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.     

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.     

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.     

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.     

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.     

First detection of Neoscytalidium dimidiatum associated with canker disease in Egyptian Ficus trees

In September 2013, a canker disease of Ficus nitida and F. benjamina was reported for the first time in Assiut governorate, Egypt. Infected samples were collected from various locations. Pure cultures of a fungus were isolated on potato dextrose agar at 25°C from diseased plants. Morphological investigation and DNA sequencing showed that the causal agent was Neoscytalidium dimidiatum. A pathogenicity test conducted using 2‐year‐old plants of each host gave 70%–80% infection, and the pathogen was reisolated from the inoculated plants. A pure culture of N. dimidiatum was deposited in the culture collection of the Assiut University Mycological Centre (AUMC) under the code AUMC 9293 and the ITS sequence was placed in NCBI under accession number KX985929 .     

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.     

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.     

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.     

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.     

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.