Invasive Fungus Hymenoscyphus Fraxineus Research Articles

Detection of Viruses in Special Stands of Common Ash Reveals Insights into the Virome of Fraxinus excelsior

Plant diseases are mostly multicausal with several factors influencing the health status of affected hosts. Common ash (Fraxinus excelsior), a significant tree species of European forests, is currently mostly endangered by ash dieback, caused by the invasive fungus Hymenoscyphus fraxineus. However, contributing factors, including pathogenic viruses, are poorly understood. Here, we report the results of a virus screening conducted on selected special stands of F. excelsior. Over three consecutive years, ash trees from different origins were tested, including leaf material from mature seed trees, young trees and ash seedlings from the natural regeneration. Using RT-PCR, we screened for five viruses, including the generalist species ArMV (Nepovirus arabis) and CLRV (Nepovirus avii), as well as newly discovered viruses in ash, including the emaravirus ASaV (Emaravirus fraxini), the idaeovirus PrLBaV (Idaeovirus ligustri), and cytorhabdoviruses. The results revealed a high virus diversity in common ash. An association of ASaV detection with specific leaf symptoms, including shoestring, chlorotic ringspots, and vein yellowing, was documented. An analyses of relevant gene products of cytorhabdoviruses obtained from ashes of different sites revealed sequence diversities and two distinct phylogenetic groups present in ash populations. Signatures of novel viruses from different families have been identified by high-throughput sequencing. Together, our results provide insights into the virus diversity and distribution of viruses in ash and expand our knowledge about the virome of this endangered tree species.

Ash dieback assessments on intensive monitoring plots in Germany: influence of stand, site and time on disease progression

AbstractSince 2002, ash dieback caused by the invasive fungus Hymenoscyphus fraxineus has been observed in Germany. The pathogen and its associated symptoms have fatal consequences for the vitality and survival of European ash (Fraxinus excelsior L.), an economically and ecologically important tree species. This study analyses the ash monitoring results of eleven intensive monitoring plots of the FraxForFuture research network distributed across Germany and focuses on within-stand differences of symptoms in dependence of small-scale site and tree properties. A cohort of 1365 ash trees was surveyed six times over three years, testing and applying a summer and a winter version of a nationally standardised ash dieback assessment key. The main disease symptoms (crown dieback and basal lesions) were more pronounced in areas with higher ash density, in edaphically moist areas (hydromorphic soils), on younger/smaller ash trees, and generally increased over time. However, the trend over time differed between single plots. In case of considering only the surviving part of the ash populations, crown condition even improved in 6/11 plots, indicating a selection process. Large basal lesions at the beginning of the observation period were a very good predictor for deadfall probability, especially on trees with lower stem diameter. Generally, ash dieback related symptoms at stem and crown were highly correlated. Silvicultural management practice in the past that actively pushed ash towards the moister end of its water demand spectrum has to be questioned in the light of ash dieback. Cost-intensive ash re-cultivation in the future—possibly with less dieback-susceptible progenies—should avoid pure ash stands and hydromorphic soil conditions.

Potential Westward Spread of Emerald Ash Borer, Agrilus planipennis Fairmaire, 1888 (Coleoptera: Buprestidae) from Eastern Ukraine

Emerald ash borer (EAB), Agrilus planipennis, is a phloem-boring beetle, native to East Asia that has become a serious invasive pest of ash (Fraxinus spp.) trees in North America and European Russia since the early 2000s. In 2019, EAB was detected in Ukraine. It had spread over 300 km from the entry point over two years and killed hundreds of Fraxinus excelsior and F. pennsylvanica trees. EAB poses a threat to the ash forests of neighboring European countries, which have already been damaged by the invasive fungus Hymenoscyphus fraxineus. The purpose of this research was (i) to reveal the traits of EAB and the climatic variables that affect its survival; (ii) to predict the EAB expansion range in Ukraine and westward; and (iii) to compare the most significant bioclimatic variables in the native, invasive ranges of EAB, as well as outside these ranges. The results demonstrated the following: (i) in all ranges, EAB has adapted to the seasonal temperature variations; (ii) the MaxEnt model predicted the potential distribution of EAB with high accuracy (AUC = 0.988); the predicted area of EAB invasion covered 87%, 48%, and 32% in Luhansk, Kharkiv, and Donetsk regions, respectively; and (iii) the ranges of climatic variables in EAB-inhabited regions demonstrated the high ecological plasticity of this pest. However, the predictions could be improved by considering forest structure, as well as the localization of roads.

Monitoring Ash Dieback in Europe—An Unrevealed Perspective for Remote Sensing?

The ash dieback pandemic, caused by the invasive fungus Hymenoscyphus fraxineus, represents one of Europe’s biggest threats to preserving natural biodiversity. To ensure the suppression of forest damage caused by fungi, timely recognition of the symptoms of ash dieback and further continuous monitoring on an adequate spatial scale are essential. Visual crown damage assessment is currently the most common method used for identifying ash dieback, but it lacks the spatial and temporal coverage required for effective disease suppression. Remote sensing technologies, with the capabilities of fast and repetitive retrieval of information over a large spatial scale, could present efficient supplementary methods for ash damage detection and disease monitoring. In this study, we provided a synthesis of the existing remote sensing methods and applications that considers ash dieback disease, and we described the lifecycle of the disease using the major symptoms that remote sensing technologies can identify. Unfortunately, although effective methods of monitoring biotic damage through remote sensing have been developed, ash dieback has only been addressed in two research studies in the United Kingdom and Germany. These studies were based on single-date hyperspectral and very-high-resolution imagery in combination with machine learning, using previously specified ground-truth information regarding crown damage status. However, no study exists using high-resolution imagery such as Sentinel-2 or radar Sentinel-1, although some preliminary project results show that these coarser sources of information could be applicable for ash dieback detection and monitoring in cases of Fraxinus angustifolia, which forms pure, more homogenous stands in Southern Europe.

Assessment of lesion development in Fraxinus excelsior cultivars Altena, Atlas and Westhof's Glorie inoculated with different isolates of Hymenoscyphus fraxineus

AbstractAsh dieback caused by the invasive fungus Hymenoscyphus fraxineus leads to massive mortality among common ash (Fraxinus excelsior) in Europe. To select tolerant genotypes, inoculation experiments are frequently conducted with isolates of the fungus. The aim of this study was to improve the inoculation methodology for evaluating susceptibility of ash genotypes to ash dieback through (i) testing the virulence of different isolates of Hymenoscyphus fraxineus and (ii) investigating temporal lesion development using three widely applied cultivars: Altena, Atlas and Westhof's Glorie. First, an experiment was conducted with a repeated measure design in which virulence of five different isolates was tested. Second, a subset of two isolates that induced the longest downward lesion length in experiment one was used in combination with a slightly adjusted inoculum protocol. Significant differences were found between isolates for downward lesion length, but a significant interaction effect of isolates and cultivars was absent. Also, the inoculation position within the stem affected lesion length; the largest lesions were found on the highest inoculation position within the stem. Furthermore, we found that cryopreserved isolates can remain virulent over years. The timing of inoculation at the end of the growing season was effective as large lesions already occurred during winter dormancy. For future inoculation studies, we propose to use: (i) isolates that induce large lesions, as less virulent isolates induced not only shorter but also fewer lesions; (ii) a similar inoculation position to better compare inoculations within and between experiments; (iii) cryopreserved isolates for testing over years; (iv) mycelial suspensions for inoculum preparation to cover wood chips more evenly; (v) reference clone—like the studied cultivars—to standardize research outcomes between years and research groups.

Comparative analyses of the Hymenoscyphus fraxineus and Hymenoscyphus albidus genomes reveals potentially adaptive differences in secondary metabolite and transposable element repertoires

BackgroundThe dieback epidemic decimating common ash (Fraxinus excelsior) in Europe is caused by the invasive fungus Hymenoscyphus fraxineus. In this study we analyzed the genomes of H. fraxineus and H. albidus, its native but, now essentially displaced, non-pathogenic sister species, and compared them with several other members of Helotiales. The focus of the analyses was to identify signals in the genome that may explain the rapid establishment of H. fraxineus and displacement of H. albidus.ResultsThe genomes of H. fraxineus and H. albidus showed a high level of synteny and identity. The assembly of H. fraxineus is 13 Mb longer than that of H. albidus’, most of this difference can be attributed to higher dispersed repeat content (i.e. transposable elements [TEs]) in H. fraxineus. In general, TE families in H. fraxineus showed more signals of repeat-induced point mutations (RIP) than in H. albidus, especially in Long-terminal repeat (LTR)/Copia and LTR/Gypsy elements.Comparing gene family expansions and 1:1 orthologs, relatively few genes show signs of positive selection between species. However, several of those did appeared to be associated with secondary metabolite genes families, including gene families containing two of the genes in the H. fraxineus-specific, hymenosetin biosynthetic gene cluster (BGC).ConclusionThe genomes of H. fraxineus and H. albidus show a high degree of synteny, and are rich in both TEs and BGCs, but the genomic signatures also indicated that H. albidus may be less well equipped to adapt and maintain its ecological niche in a rapidly changing environment.

Spread and Severity of Ash Dieback in Switzerland – Tree Characteristics and Landscape Features Explain Varying Mortality Probability

Since the 1990s the invasive fungus Hymenoscyphus fraxineus has been threatening European ash (Fraxinus excelsior), a tree species with high ecological and economic importance. This pathogen is causing severe crown dieback, leading to high mortality rates across Europe and is present in Switzerland since 2008. In this study, we provide a comprehensive overview of the temporal evolution of crown damage and mortality rates in Switzerland over the 2009–2019 period. Harnessing the power of the annualized design of the Swiss national forest inventory (NFI), we show that annual mortality rates (AMRs) of ash increased significantly since the arrival of the fungus, with stronger effects in small trees [<26 cm diameter at breast height (DBH)]. Mortality modeling confirmed a size and growth-rate dependent mortality probability (MP). It also revealed that stands with higher humidity – either through higher mean annual precipitation or more humid soil conditions – showed also increased MP. Decreasing host abundance with increasing elevation was also associated with lower MP. Special ash surveys performed over the last 10 years still show a large percentage of ash trees with very low defoliation. This gives hope to finding possible tolerant or resistant trees for (inter-)national breeding programs. In the mean-time our results reinforce previously published management guidelines to promote not only healthy big trees, but also healthy and fast-growing young trees in more open stands for long-term conservation of ash in Europe.

Marker-Trait Associations for Tolerance to Ash Dieback in Common Ash (Fraxinus excelsior L.)

Common ash (Fraxinus excelsior L.) is a tree species of significant ecological and economic importance that has suffered a devastating decline since the 1990s in Europe. Native ash species are being threatened by the alien invasive fungus Hymenoscyphus fraxineus, which causes ash dieback. The main goal of the study was to develop markers for traits related to tolerance to ash dieback and to investigate whether genotypes selected for tolerance were genetically different from susceptible wild populations. We phenotyped 326 ash trees from Sweden for disease severity and genotyped them using 63 amplicon-derived single-nucleotide polymorphism (SNP) markers derived from genes in 40 scaffolds spanning 8 MB in total, which represents approximately 1% of the ash genome. We used a mixed linear model to test for an association between genotypic variation at these loci and disease severity of ash. In total, two SNPs were found to have significant associations. One non-synonymous SNP associated with the disease severity of ash was found in a gene predicted to encode a subtilisin-related peptidase S8/S53 domain. A second marginally significant marker was associated with an LRR gene. Our results demonstrate an inexpensive time-effective method for generating genomic data that could have potential for use in future tree breeding programs and provide information for marker-assisted selection. Our study also showed a low differentiation between genotypes selected for disease tolerance and the wild population of ash representing a range of susceptibilities to ash dieback, indicating opportunities for further selection without significantly losing genetic diversity in the ash population.

Amplifying feedback loop between growth and wood anatomical characteristics of Fraxinus excelsior explains size-related susceptibility to ash dieback.

Since the 1990s the invasive fungus Hymenoscyphus fraxineus has caused severe crown dieback and high mortality rates in Fraxinus excelsior in Europe. In addition to a strong genetic control of tolerance to the fungus, previous studies have found landscape heterogeneity to be an additional driver of variability in the severity of dieback symptoms. However, apart from climatic conditions related to heat and humidity influencing fungal infection success, the mechanistic understanding of why smaller or slower-growing trees are more susceptible to dieback remains less well understood. Here, we analyzed three stands in Switzerland with a unique setting of 8years of data availability of intra-annual diameter growth and annual crown health assessments. We complemented this by ring width and quantitative wood anatomical measurements extending back before the monitoring started to investigate if wood anatomical adjustments can help better explain the size-related dieback phenomenon. We found that slower-growing trees or trees with smaller crowns already before the arrival of the fungus were more susceptible to dieback and mortality. Defoliation directly reduced growth as well as maximum earlywood vessel size, and the positive relationship between vessel size and growth rate caused a positive feedback amplifying and accelerating crown dieback. Measured non-structural carbohydrate (NSC) concentrations in the outermost five rings did not significantly vary between healthy and weakened trees, which translate into large differences in absolute available amount of NSCs. Thus, we hypothesize that a lack of NSCs (mainly sugars) leads to lower turgor pressure and smaller earlywood vessels in the following year. This might impede efficient water transport and photosynthesis, and be responsible for stronger symptoms of dieback and higher mortality rates in smaller and slower-growing trees.

Canditate metabolites for ash dieback tolerance in Fraxinus excelsior.

Ash dieback, a forest epidemic caused by the invasive fungus Hymenoscyphus fraxineus, threatens ash trees throughout Europe. Within Fraxinus excelsior populations, a small proportion of genotypes show a low susceptibility to the pathogen. We compared the metabolomes from a cohort of low-susceptibility ash genotypes with a cohort of high-susceptibility ash genotypes. This revealed two significantly different chemotypes. A total of 64 candidate metabolites associated with reduced or increased susceptibility in the chemical families secoiridoids, coumarins, flavonoids, phenylethanoids, and lignans. Increased levels of two coumarins, fraxetin and esculetin, were strongly associated with reduced susceptibility to ash dieback. Both coumarins inhibited the growth of H. fraxineus in vitro when supplied at physiological concentrations, thereby validating their role as markers for low susceptibility to ash dieback. Similarly, fungal growth inhibition was observed when the methanolic bark extract of low-susceptibility ash genotypes was supplied. Our findings indicate the presence of constitutive chemical defense barriers against ash dieback in ash.

Ash dieback risks an extinction cascade

Large-scale decline in populations of European ash (Fraxinus excelsior) are occurring throughout Europe due to the invasive fungus Hymenoscyphus fraxineus. This has grave ecological implications not only for ash trees, but also for the biodiversity supported by, and in some cases solely dependent on ash. Here we used data on the tree-species associations of biodiversity in Sweden, to predict extinction risks for ash-associated organisms, and the potential for combinations of other tree species to sustain ash-associated biodiversity. Of the 483 ash-associated species identified, 11% are exclusive to ash, and a further 23% prefer mainly ash. Notably, many ash-associated species are shared with wych elm (Ulmus glabra) which is similarly threatened by an invasive fungus. Considering the level of host association and the species' conservation status, 115 species were deemed at high risk of regional extinction. Using a mathematical optimization model we found that up to nine additional tree species would be needed to sustain all non-obligate ash dependent/preferring species in the absence of ash and elm. We discuss mitigation and adaption options to reduce the potential for an extinction cascade and conserve ash-associated biodiversity, but all pose unique challenges.

Fungal endophytes for biocontrol of ash dieback: The antagonistic potential of Hypoxylon rubiginosum

Abstract Our goal is to develop a biocontrol agent using endophytic fungi to control ash dieback, a disease that endangers the very existence of the European ash, Fraxinus excelsior. Such a protective endophyte should produce metabolites toxic to the invasive fungus Hymenoscyphus fraxineus, the causal agent of ash dieback. From a total of 340 fungal endophytes from F. excelsior, the interactions with 13 exemplary isolates are presented here. In co-culture between H. fraxineus and endophyte, there was almost always reciprocal inhibition between pathogen and endophyte. Endophytes that inhibited H. fraxineus more than they were inhibited by the pathogen were inoculated into axenically cultured F. excelsior to select those that grew asymptomatically in the host. Most of the endophytes caused disease symptoms in axenically cultured F. excelsior seedlings, highlighting the importance of not only relying on co-culture experiments, but also using in planta experiments. By means of a multi-locus genealogy in conjunction with morphological studies and a comparison with the ex-epitype culture, one of the most promising isolates was identified as Hypoxylon rubiginosum, an ascomycete fungus that is closely associated with Fraxinus and frequently produces its sexual morph on dead wood of the host. Using preparative HPLC, metabolites were isolated from mono- and co-cultures of H. fraxineus and H. rubiginosum and their structures elucidated by NMR spectroscopy and HR mass spectrometry. The known phytotoxin viridiol was detected as a major metabolite of the ash pathogen in co-culture and the previously known antifungal metabolite, phomopsidin, as the major secondary metabolite of H. rubiginosum. In addition, the new metabolite, 10-hydroxyphomopsidin, was obtained from H. rubiginosum. Phomopsidin, a known inhibitor of beta-tubulin that targets the eukaryotic cytoskeleton, inhibited the pathogen, but also showed mild cytotoxic effects. The fact that H. rubiginosum grows asymptomatically in planta and that it produces the antifungal metabolite phomopsidin qualifies it as a promising candidate for further development as a biocontrol agent for ash dieback.

Fungi isolated from shoots showing ash dieback in the Wolica Nature Reserve in Poland and artificially inoculated seedlings with Hymenoscyphus fraxineus

Abstract Ash dieback caused by an alien, invasive fungus Hymenoscyphus fraxineus is a serious disease of European ash species in many parts in Europe. In Poland, the disease was recorded in the beginning of the 1990s. This study was performed in 2016–2017 with the aim to identify fungi isolated from ash shoots showing dieback symptoms in the Wolica Nature Reserve in Poland, as well as from shoots of two years-old ash seedlings inoculated with H. fraxineus in the greenhouse. The most frequently isolated fungi from shoots of common ash (associated with the pathogenic fungus H. fraxineus) were identified on the basis of sequencing of the internal transcribed spacer region (ITS1) of fungal rDNA. In total, 19 fungal taxa were identified for ash shoots as follows: H. fraxineus, Fusarium avenaceum, Alternaria spp., Phomopsis oblonga, Diplodia mutila and other Phomopsis spp. The pathogen H. fraxineus was not found for all the shoots samples; one year after inoculation the aforementioned fungi and other species as: Alternaria alternata, Bionectria ochroleuca, Epicoccum nigrum, F. acuminatum, F. avenaceum, and Paraphaesphaeria neglecta were identified in inoculation point, as well as H. fraxineus. The same quantitative and qualitative changes of organisms were observed in the case of artificially colonised ash seedlings in the greenhouse, as well as in the shoots of adult ash trees in the forest.

Advanced spectroscopy-based phenotyping offers a potential solution to the ash dieback epidemic

Natural and urban forests worldwide are increasingly threatened by global change resulting from human-mediated factors, including invasions by lethal exotic pathogens. Ash dieback (ADB), incited by the alien invasive fungus Hymenoscyphus fraxineus, has caused large-scale population decline of European ash (Fraxinus excelsior) across Europe, and is threatening to functionally extirpate this tree species. Genetically controlled host resistance is a key element to ensure European ash survival and to restore this keystone species where it has been decimated. We know that a low proportion of the natural population of European ash expresses heritable, quantitative resistance that is stable across environments. To exploit this resource for breeding and restoration efforts, tools that allow for effective and efficient, rapid identification and deployment of superior genotypes are now sorely needed. Here we show that Fourier-transform infrared (FT-IR) spectroscopy of phenolic extracts from uninfected bark tissue, coupled with a model based on soft independent modelling of class analogy (SIMCA), can robustly discriminate between ADB-resistant and susceptible European ash. The model was validated with populations of European ash grown across six European countries. Our work demonstrates that this approach can efficiently advance the effort to save such fundamental forest resource in Europe and elsewhere.

The susceptibility of Asian, European and North American Fraxinus species to the ash dieback pathogen Hymenoscyphus fraxineus reflects their phylogenetic history

In Europe, common ash (Fraxinus excelsior) is being decimated because of the invasive fungus Hymenoscyphus fraxineus. In its native range in Asia this ascomycete is considered a harmless leaf associate of F. mandshurica and F. chinensis subsp. rhynchophylla. Field observations from Europe suggest that there is species-specific variation in disease susceptibility among European and North American Fraxinus species, but a wider comparison at the genus level has been missing so far. We assessed disease symptoms and pathogen apothecium development in 17 Fraxinus species from Asia, Europe and North America exposed to high infection pressure in a Danish arboretum. We also tested their susceptibility to pathogen infection through controlled stem and leaf inoculations and subsequently assessed the level of pathogen DNA by a qPCR assay. The results suggested the presence of a phylogenetic signal in disease susceptibility where closely related Asian, European and North American species in section Fraxinus had relatively high levels of H. fraxineus DNA in the leaves and supported high production of apothecia. Leaves from some North American species also contained relatively high levels of H. fraxineus DNA, supported moderate production of apothecia and developed lesions—stating the need to avoid introduction of H. fraxineus to North America.