Causal Agent Of Ash Dieback Research Articles

Drivers of Hymenoscyphus fraxineus Infections in the Inner-Alpine Valleys of Northwestern Italy

Fraxinus excelsior L. (ash) is a key forest tree species challenged by Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz, Hosoya, the causal agent of ash dieback. The goals of this study were (I) to assess the presence, spatial distribution, and incidence of H. fraxineus in the inner-alpine valleys of northwestern Italy, along with the severity of ash dieback; (II) to model the probability of infection by H. fraxineus based on environmental variables; (III) to reconstruct the direction of provenance of the front of invasion of the pathogen; and (IV) to test whether H. fraxineus has replaced the native relative Hymenoscyphus albidus (Gillet) W. Phillips, a saprobe of ash litter. By combining phytosanitary monitoring and samplings in 20 forest stands, laboratory analyses, and statistical modelling, this study showed that H. fraxineus was present in 65% of stands with an average incidence of 27%, reaching peaks of 80%. Rainfalls were the most relevant drivers of the probability of infection by H. fraxineus, rising up to 80% with the increased precipitation in April and July. Other drivers included elevation, maximal temperatures, latitude, and longitude. The front of invasion likely moved from Italy and/or Switzerland, rather than from France, while the replacement of H. albidus is uncertain.

Novel RNA viruses from the native range of Hymenoscyphus fraxineus, the causal fungal agent of ash dieback

The native Japanese population of the fungus Hymenoscyphus fraxineus, the causal agent of ash dieback in Europe, was screened for viruses using a high-throughput sequencing method. Five RNA viruses were detected in 116 fungal isolates sequenced via Illumina RNA-seq platform, with an overall virus prevalence of 11.2%. The viruses were completely sequenced by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) followed by Sanger sequencing. The sequences appear to represent new species from three established families (Mito-, Endorna- and Partitiviridae), one recognized genus (Botybirnavirus) and a negative-sense single-stranded RNA virus in the order Bunyavirales from the proposed family “Mybuviridae”. The highest prevalence was found for the mitovirus (7.8%), that had two genomic forms (linear and circular), while the other viruses were detected each in one isolate. Co-infection of a mitovirus and an endornavirus was also observed in one of the infected isolates. Here we describe the molecular characterization of the identified viruses. This study expands the diversity of viruses in H. fraxineus and provides the basis for investigating the virus-mediated control of ash dieback in Europe.

Physiological and genomic characterisation of Luteimonas fraxinea sp. nov., a bacterial species associated with trees tolerant to ash dieback

A group of isolates of the genus Luteimonas was characterised, which represented a specific component of the healthy core microbiome of Fraxinus excelsior in forest districts with a high infection rate of H. fraxineus, the causal agent of ash dieback. Based on phylogenomic and phenotypic analyses, a clear differentiation from related Luteimonas species was shown. Comparisons of the overall genome relatedness indices with the closest phylogenetic neighbours resulted in values below the recommended species cut-off levels. In addition, differences in several physiological and chemotaxonomic traits allowed a clear demarcation from the type strains of closely related species. Conclusively, the strain group was considered to represent a novel species in the genus Luteimonas, for which the name Luteimonas fraxinea sp. nov. is proposed, with strain D4P002T (=DSM 113273T = LMG 32455T) as the type strain. A functional analysis of the genome revealed features particularly associated with attachment, biofilm production and motility, indicating the ability of D4P002T to effectively colonise ash leaves. In nursery trials, ash seedlings inoculated with this strain showed suppression of the pathogen over a period of three years. This effect was accompanied by a significant shift in the bacterial microbiome of the plants. Altogether, the exclusive occurrence in the microbiome of tolerant ash trees, the genetic background and the results of the inoculation experiment suggest that strain D4P002T may suppress the penetration and spreading of H. fraxineus in or on ash leaves via colonisation resistance or trigger a priming effect of plant defences against the pathogen.

Expansion of Ash Dieback towards the scattered Fraxinus excelsior range of the Italian peninsula

Hymenoscyphus fraxineus, causal agent of Ash Dieback, has posed a threat to Fraxinus excelsior (common ash) in Europe since the 1990s. In south-western Europe, optimal climatic conditions for H. fraxineus become scattered and host density decreases, reducing disease spread rates. To date, the Ash Dieback agent has not been reported from southern and most of central Italy, where native F. excelsior is present as small fragmented populations. This study examines the expansion of Ash Dieback into central Italy, and it considers the consequences of further local spread with regards to the loss of F. excelsior genetic resource. Symptomatic F. excelsior were sampled from sixteen sites in northern and central Italy during 2020. Specimens were analyzed with a culturomics and a quantitative PCR approach. A bibliographic search of F. excelsior floristic reports was conducted for the creation of a detailed range map. The combined use of both techniques confirmed the presence of H. fraxineus in all the sites of central Italy where host plants were symptomatic. These new records represent the southern limit of the current known distribution of this pathogen in Italy, and together with Montenegro, in Europe. The characterization of the F. excelsior scattered range suggests that further spread of Ash Dieback across southern Italy is a realistic scenario. This presents a threat not just to the southern European proveniences of F. excelsior, but to the species as a whole, should Ash Dieback lead to the loss of warm climate adapted genetic material, which may become increasingly valuable under climate change.

A Comparative Analysis of Ash Leaf-Colonizing Bacterial Communities Identifies Putative Antagonists of Hymenoscyphus fraxineus.

In the last few years, the alarming spread of Hymenoscyphus fraxineus, the causal agent of ash dieback, has resulted in a substantial threat to native ash stands in central and northern Europe. Since leaves and leaf petioles are the primary infection sites, phyllosphere microorganisms are presumed to interact with the pathogen and are discussed as a source of biocontrol agents. We studied compound leaves from susceptible and visible infection-free trees in four ash stands with a high likelihood of infection to assess a possible variation in the bacterial microbiota, depending on the health status of the trees. The bacterial community was analyzed by culture-independent 16S rRNA gene amplicon sequencing and through the isolation and taxonomic classification of 2,589 isolates using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The bacterial community structure did not show significant differences. However, a set of amplicon sequence variants (ASVs) and MALDI groups belonging to Luteimonas, Aureimonas, Pseudomonas, Bacillus, and Paenibacillus were distinctly increased in tolerant trees, which may be associated with the ability of the tree to resist the pathogen. The most obvious differences were observed for Luteimonas, a genus that is also exclusively present in the healthy core microbiome. In a first in vitro screen of antagonists, approximately 11% of total isolates suppressed the growth of H. fraxineus, but a statistical test with two different H. fraxineus strains confirmed only the antagonistic activity of 8% of these isolates. The antagonistic isolates were assigned to Bacillus velezensis, Pantoea vagans, and Pseudomonas caspiana. Overall, our study provides a set of isolates or phylogenetic groups that might be involved in the process that prevents the penetration and spread of H. fraxineus. In the next step, in planta experiments are required with a longer period of exposure to H. fraxineus to evaluate effective isolates or consortia of isolates acting through direct antagonism or competition or indirectly by inducing resistance.

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.

The Endophytic Mycobiome of European Ash and Sycamore Maple Leaves - Geographic Patterns, Host Specificity and Influence of Ash Dieback.

The European ash (Fraxinus excelsior) is threatened by the introduced ascomycete Hymenoscyphus fraxineus, the causal agent of ash dieback. Endophytic fungi are known to modulate their host’s resistance against pathogens. To understand possible consequences of ash dieback on the endophytic mycobiome, F. excelsior leaves were collected in naturally regenerated forests and the fungal communities analyzed by classic culture and Illumina amplicon sequencing using a newly developed and validated fungal-specific primer. Collections were done in the area infested by ash dieback north of the Alps, and in the disease free area on the south side. Sycamore maple (Acer pseudoplatanus) was additionally collected, as well as the flowering ash (F. ornus), which occurs naturally in the south and shows tolerance to ash dieback. Both cultivation and amplicon sequencing revealed characteristic endophytic fungal communities dominated by several strictly host specific Venturia species. On A. pseudoplatanus, a hitherto undescribed Venturia species was identified. Due to its dominance on F. excelsior, V. fraxini is unlikely to go extinct in case of reduced host densities. A majority of species was not strictly host specific and is therefore likely less affected by ash dieback in the future. Still, shifts in community structure and loss of genetic diversity cannot be excluded. The potentially endangered endophyte Hymenoscyphus albidus was rarely found. In addition to host specificity, species with preferences for leaf laminae or petioles were found. We also detected considerable geographical variation between sampling sites and clear differences between the two sides of the Alps for endophytes of F. excelsior, but not A. pseudoplatanus. Since sycamore maple is not affected by an epidemic, this could point toward an influence of ash dieback on ash communities, although firm conclusions are not possible because of host preferences and climatic differences. Furthermore, the mycobiota of F. excelsior trees with or without dieback symptoms were compared, but no clear differences were detected. Besides methodical refinement, our study provides comprehensive data on the ash mycobiome that we expect to be subject to changes caused by an emerging disease of the host tree.

The ash dieback invasion of Europe was founded by two genetically divergent individuals

Accelerating international trade and climate change make pathogen spread an increasing concern. Hymenoscyphus fraxineus, the causal agent of ash dieback, is a fungal pathogen that has been moving across continents and hosts from Asian to European ash. Most European common ash trees (Fraxinus excelsior) are highly susceptible to H. fraxineus, although a minority (~5%) have partial resistance to dieback. Here, we assemble and annotate a H. fraxineus draft genome, which approaches chromosome scale. Pathogen genetic diversity across Europe and in Japan, reveals a strong bottleneck in Europe, though a signal of adaptive diversity remains in key host interaction genes. We find that the European population was founded by two divergent haploid individuals. Divergence between these haplotypes represents the ancestral polymorphism within a large source population. Subsequent introduction from this source would greatly increase adaptive potential of the pathogen. Thus, further introgression of H. fraxineus into Europe represents a potential threat and Europe-wide biological security measures are needed to manage this disease.

Hyfraxins A and B, cytotoxic ergostane-type steroid and lanostane triterpenoid glycosides from the invasive ash dieback ascomycete Hymenoscyphus fraxineus

A virulent culture of Hymenoscyphus fraxineus, the causal agent of ash dieback, was investigated for its production of secondary metabolites in a 70 L batch fermentation. Chemical analysis of the mycelial extract by means of flash chromatography and preparative HPLC led to the isolation of a new ergostane-type steroid (1) and a new related lanostane triterpenoid (2), both revealing the same glycosylation pattern. While their planar structures were elucidated by HR-ESIMS and NMR data, relative stereochemistry was assigned by ROESY correlations in conjunction with H,H and C,H coupling constants. Absolute configuration was determined based on ROESY correlations between the aglycons and the sugar moieties, which were identified in both cases as d-mannose by GC/MS analysis of the trimethylsilylated derivatives. The isolated compounds, for which we propose the trivial names hyfraxins A (1) and B (2), were found to be cytotoxic against the mouse fibroblast cell line L929 and exhibited moderate to weak activity against Gram-positive bacteria.

Genetic evaluation of damage caused by ash dieback with emphasis on selection stability over time

Abstract Damage caused by Hymenoscyphus fraxineus, the causal agent of ash dieback, was genetically evaluated based on scorings made on five occasions during the years 2006–2016 at two seed orchards of ash (Fraxinus excelsior L.) established in southern Sweden in 1992 and 1995, respectively. The studied population included grafts originating from 106 plus-tree clones selected from 27 stands in southern Sweden. The study verified previous results of (1) high genetic control of damage traits, (2) high genotypic variation in disease susceptibility and (3) that only a small proportion of the natural population are at least partly resistant. The clonal response for dieback damage was stable over the 10-year period, i.e. the least susceptible clones selected in 2006 still belong to the healthiest ones after several years of heavy infection pressure. There were no clear trends in the development of damage over time for trees being alive at each assessment. However, the proportion of trees that died was substantial (7–8% per year), but this estimation was influenced by other factors than the disease, such as wild game and sanitary cuttings. Altogether, the results are promising for initiating breeding programs to improve the dieback resistance. However, this is a heavy and costly task, since only a very small proportion of the ash trees are partially resistant to the dieback disease. Thus, a joint breeding program involving all the northern European countries is justified.

Hymenoscyphus fraxineusmycelial growth on media containing leaf extracts of different Oleaceae

Summary 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.

Identification of Hymenoscyphus fraxineus, the causal agent of ash dieback in Slovakia

AbstractSymptoms of ash dieback have been recorded in Slovakia since 2004. The field sampling was carried out in 2013, included 59 segments of shoots and 10 and more petioles per locality from four localities. The causal agent of ash dieback, the hyphomycete Chalara fraxinea T. Kowalski, was isolated from Fraxinus excelsior L. from seven localities in Slovakia. The morphology of C. fraxinea isolates and the teleomorph Hymenoscyphus fraxineus (T. Kowalski) Baral, Queloz, Hosoya are described and ITS sequences are provided.

Local population structure of Hymenoscyphus fraxineus surveyed by an enlarged set of microsatellite markers

SummaryThe population structure of Hymenoscyphus fraxineus, the causal agent of ash dieback, was assessed at four closely located sites in the Czech Republic. To analyse the genetic variation, one Swiss and one Norwegian population with known population structures were selected as reference points. The analysis was performed using 16 previously published and five newly developed microsatellite markers. The quality of the new markers was assessed by sequencing the flanking region, identifying the type of the mutation and analysing the inheritance and linkage between all pairs of loci. In addition, markers were tested on 45 Japanese strains to confirm their usability on native H. fraxineus populations in Asia. One of the new markers was monomorphic in all European populations and one marker exhibited a high percentage of null alleles in the Japanese samples. Twenty markers in the four Czech populations showed lower average gene diversity than in the other two European populations. One Czech population significantly differed from all the others, with a pairwise GST of approximately 0.2, the lowest allelic richness and very low average gene diversity, likely resulting from a founder effect. The amount of genetic differentiation between the four neighbouring Czech populations exceeds the expected value from previous Europe‐wide population studies and suggests that local population dynamics can affect the population structure of H. fraxineus.

Hymenoscyphus fraxineus can directly infect intact current‐year shoots of Fraxinus excelsior and artificially exposed leaf scars

SummaryHymenoscyphus 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.

Real-Time LAMP for Chalara fraxinea Diagnosis.

Chalara fraxinea is the causal agent of ash dieback, a disease affecting Fraxinus excelsior and F. angustifolia across Europe. Loop-mediated isothermal amplification (LAMP) is a rapid, DNA-based method which can be used for specific detection of plant pathogens in infected material. The combination of a rapid LAMP assay for C. fraxinea with a simple sample preparation method in a user-friendly kit format raises the potential for testing to be carried out away from conventional laboratory facilities, to expedite measure to manage this damaging disease.

Detection and genetic characterisation of a novel mycovirus in Hymenoscyphus fraxineus, the causal agent of ash dieback

The ascomycete fungus Hymenoscyphus fraxineus (synonym: Hymenoscyphuspseudoalbidus, basionym: Chalara fraxinea) is a new invasive pathogen causing severe dieback of ash trees (Fraxinus spp.) in Europe. The disease was first recorded in the 1990s in Eastern Poland and Lithuania and has meanwhile spread across the continent and to Great Britain. Mycoviruses are commonly found in all major groups of plant pathogenic fungi. Some of these viruses can cause debilitating disease or reduce virulence in their fungal host and thereby can act as biological control agents (e.g. hypoviruses in the chestnut blight fungus). The main aim of our study is to identify mycoviruses in the ash dieback pathogen and to describe their phylogenetic position. Using RNAseq we could identify a viral sequence in two fungal isolates. Amino acid sequence comparison suggests that the detected mycovirus is a putative new member of the genus Mitovirus (family Narnaviridae), and we suggest naming it Hymenoscyphus fraxineus mitovirus 1 (HfMV1). The novel mitovirus is highly variable amongst European isolates and seems to diverge rapidly. This is very promising for the future search of a strain adequate for biological control measures.

Hymenoscyphus pseudoalbidus and Hymenoscyphus albidus: viridiol concentration and virulence do not correlate

SummaryHymenoscyphus pseudoalbidus is the causal agent of ash dieback, a disease that is presently endangering Fraxinus spp. throughout most of Europe. The phytotoxin, viridiol, was previously isolated from culture extracts of H. pseudoalbidus and found to be toxic to leaves of F. excelsior. Thus, we were interested in learning to what extent viridiol is responsible for pathogenicity of H. pseudoalbidus and investigated this using twelve isolates of H. pseudoalbidus. We also included five isolates of the closely related avirulent species, Hymenoscyphus albidus, in our studies. Some, but not all, isolates of H. pseudoalbidus and H. albidus produced measurable quantities of viridiol in culture. Three tests were used to determine to what extent viridiol concentration correlates with virulence: culture extracts were tested for activity in leaf segment tests and for inhibition of germination of seedlings of Fraxinus excelsior; virulence of the isolates was tested following infection of axenically cultured ash seedlings. Activity of the culture extracts varied, as did virulence of the isolates following inoculation into seedlings. No correlations were found between viridiol concentration and activities of culture extracts in leaf segment tests or in the germination test, nor between viridiol concentration and disease symptoms when inoculated into seedlings. However, activities of culture extracts in leaf segment and in the germination test correlated, as did the results of each of these tests with virulence in the infection experiment. Apparently, as yet unidentified factors other than the concentration of viridiol play important roles in the virulence of H. pseudoalbidus.

Fungi in foliage and shoots of Fraxinus excelsior in eastern Ukraine: a first report on Hymenoscyphus pseudoalbidus

SummaryIn 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.

Hymenoscyphus pseudoalbidus, the correct name for Lambertella albida reported from Japan

Recent molecular analyses separate Hymenoscyphus pseudoalbidus (causal agent of ash dieback in Europe) from the morphologically scarcely distinguishable H. albidus. Hymenoscyphus albidus was reported (as "Lambertella albida") on petioles of Fraxinus mandshurica in Japan. Phylogenetic analysis in the present study shows Japanese "L. albida" to be conspecific with H. pseudoalbidus but with a higher genetic variability compared to European isolates. The presence of croziers at the ascus base was found to be a clear distinguishing character of H. pseudoalbidus. Our phylogenetic analysis of the combined ITS and LSU-D1/D2 dataset supports Hymenoscyphus as more appropriate than Lambertella for H. pseudoalbidus. As the Hymenoscyphus clade includes members with two major characters (presence of substratal stroma and brown ascospores) currently used to circumscribe Lambertella, the generic delimitation of Lambertella requires redefinition.

Neue Erkenntnisse zur Pathogenese des Triebsterbens an Esche (Fraxinus excelsior)

Ash dieback is currently one of the most important tree diseases in Europe. It is already widespread in Northern and Central Europe as well as in some parts of Western and Southern Europe. Trees of the species Fraxinus excelsior an F. angustifolia of all ages on various site types are affected in forests, in the landscape as well as in nurseries and urban plantings. Symptoms include wilting of leaves and young shoots, shoot dieback, premature leaf fall and variously colored bark necroses of different sizes, always lacking exudates. In young trees the disease leads to mortality within a few years, whereas a chronic process can often be observed in older trees. In Germany the impact of the disease varies greatly in different regions, but a countrywide distribution is assumed. Significant damage was especially established in nurseries and forests of Northern Germany. The paper presents a recent investigation of a large number of naturally infested nursery saplings from Northern Germany. The real infection rates, invasion and spreading strategies of the fungus inside of plant tissues and the role of soil-borne Oomycetes as possible primary or accompanying causal organisms of the disease were examined. The results confirm the dominant role of Chalara fraxinea (teleomorph: Hymenoscyphus albidus) as the causal agent of ash dieback and rule out the role of pathogenic Oomycetes in the disease process. The authors conclude that the fungus is able to spread very effectively in woody tissue as a parasite and does not originate from the root system.