First report of Diplodia fraxini and Diplodia subglobosa causing canker and dieback of Fraxinus excelsior in Slovenia

Over the last decades the vitality and productivity of European ash trees in Slovenia has been seriously impacted by the onset of canker and dieback disease symptoms on young and old trees, primarily identified as ash dieback caused by Hymenoscyphus fraxineus. Given the limited information available about the aetiology of this emerging disease, a study was carried out to isolate, identify and characterize the fungal species involved in the observed ash symptoms. Field surveys were conducted in five forest sites where 50 symptomatic branch samples were collected. All samples were inspected and used for fungal isolation. Based on morphology, colony appearance and DNA sequence data of the internal transcribed spacer region (ITS), 125 fungal colonies belonging to five species were isolated and identified. Only a few symptomatic ash samples yielded colonies of H. fraxineus, whereas Botryosphaeriaceae species were isolated with a high frequency, with Diplodia fraxini as the dominant species. A pathogenicity test proved that all isolated species were pathogenic on European ash, causing bark lesions and wood discoloration. All Botryosphaeriaceae species isolated in this study are reported for the first time on European ash in Slovenia.

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Diplodia fraxini and Diplodia subglobosa: The Main Species Associated with Cankers and Dieback of Fraxinus excelsior in North-Eastern Italy

Forests ◽

10.3390/f11080883 ◽

2020 ◽

Vol 11 (8) ◽

pp. 883

Author(s):

Benedetto T. Linaldeddu ◽

Francesco Bottecchia ◽

Carlo Bregant ◽

Lucia Maddau ◽

Lucio Montecchio

Keyword(s):

Dna Sequences ◽

Fungal Pathogens ◽

Fungal Species ◽

Fraxinus Excelsior ◽

Limited Information ◽

Ash Dieback ◽

Alpine Regions ◽

Hymenoscyphus Fraxineus ◽

North Eastern ◽

Friuli Venezia Giulia

In Italy, after the first report in Friuli-Venezia Giulia along the border with Slovenia in 2009, ash dieback has successively been reported in Veneto, Tuscany and Trentino-Alto Adige. Given its alarming expansion in European ash formations along the sub-montane belt of north-eastern Italy and the limited information about the associated fungal microorganisms; since 2017, a study has been conducted in order to isolate and characterize the fungal species involved in the aetiology of the disease. The surveys were conducted in six ash–maple forests distributed along the Veneto and Friuli-Venezia Giulia pre-Alpine regions (Italy). In each site, the health status of ash trees was assessed and a sample (shoot or branch) with the typical symptoms of the disease was taken from ten trees to isolate the associated pathogens. The fungal colonies developed were identified using morphological features and DNA sequences. The 60 samples processed yielded a total of 109 fungal isolates belonging to 9 families including: Botryosphaeriaceae (62 isolates), Diaporthaceae (18), Nectriaceae (10), Didymellaceae (9), Helotiaceae (5), Diatrypaceae (2), Didymosphaeriaceae (1), Phaeosphaeriaceae (1) and Valsaceae (1). In particular, three species—Diplodia subglobosa, Diplodia fraxini and Diaporthe eres—were isolated with high frequency, while Hymenoscyphus fraxineus was isolated from only five plants distributed in four sites. The pathogenicity tests, conducted on 3-year-old seedlings, detached branches (3–4 cm diameter), and leaves of Fraxinus excelsior, showed that Diplodia fraxini is the most virulent species and the only one able to reproduce the symptoms observed in nature. Overall, the results obtained emphasize that several fungal pathogens are involved in the aetiology of the disease, many of which belong to the Botryosphaeriaceae family.

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Mycobiome of Fraxinus excelsior With Different Phenotypic Susceptibility to Ash Dieback

Frontiers in Forests and Global Change ◽

10.3389/ffgc.2021.580514 ◽

2021 ◽

Vol 4 ◽

Author(s):

Marta Agostinelli ◽

Diem Nguyen ◽

Johanna Witzell ◽

Michelle Cleary

Keyword(s):

Large Scale ◽

Population Decline ◽

Keystone Species ◽

Fungal Species ◽

Fraxinus Excelsior ◽

Fungal Communities ◽

Ash Dieback ◽

Hymenoscyphus Fraxineus ◽

Ash Trees ◽

European Ash

For the last two decades, large-scale population decline of European ash (Fraxinus excelsior) has occurred in Europe because of the introduction of the alien fungal pathogen, Hymenoscyphus fraxineus, from East Asia. Since European ash is a keystone species having critical importance for biodiversity, and only a small percentage of the ash population appears to show some tolerance against the pathogen, the loss of ash trees means that other associated organisms, especially those with high or obligate associations to ash, are at risk of further species declines. In this study, we used high throughput DNA sequencing and multivariate analysis to characterize: (i) the mycobiome in aerial tissues (i.e., leaf, bark, and xylem) of ash trees showing different phenotypic response to ash dieback, (ii) the temporal variation in fungal communities across the growing season, and (iii) the similarity in fungal community structure between ash and other common trees species that may serve as an ecological niche substitute for ash microfungi. Results showed that fungal communities differed among the three tissue types, susceptibility classes, in time and between sites. Trophic analysis of functional groups using the FUNGuild tool indicated a higher presence of pathotrophic fungi in leaves than in bark and xylem. The share of pathotrophic fungi increased along a gradient of low to high disease susceptibility in both bark and xylem tissue, while the proportion of symbiotrophic fungi correspondingly decreased in both tissue types. Neighboring, alternative host trees did not share all the fungal species found in ash, however, most microfungi uniquely associated to ash in this study are generalists and not strictly host specific. The progressive disappearance of ash trees on the landscape imposes a high risk for extinction of Red-listed macrofungal species, and breeding for resistance against ash dieback should help sustain important biodiversity associated to ash. Microfungal diversity though may be less prone to such demise since most ash-associated endophytes appear to occur on a broad range of host species.

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Genomic basis of European ash tree resistance to ash dieback fungus

10.1101/626234 ◽

2019 ◽

Author(s):

Jonathan J. Stocks ◽

Carey L. Metheringham ◽

William Plumb ◽

Steve J. Lee ◽

Laura J. Kelly ◽

...

Keyword(s):

Sequence Data ◽

Seed Source ◽

Source Zone ◽

Whole Genome Sequence ◽

Ash Dieback ◽

Hymenoscyphus Fraxineus ◽

A Genome ◽

Ash Trees ◽

Gp Model ◽

European Ash

SummaryPopulations of European ash trees (Fraxinus excelsior) are being devastated by the invasive alien fungus Hymenoscyphus fraxineus, which causes ash dieback (ADB). We sequenced whole genomic DNA from 1250 ash trees in 31 DNA pools, each pool containing trees with the same ADB damage status in a screening trial and from the same seed-source zone. A genome-wide association study (GWAS) identified 3,149 single nucleotide polymorphisms (SNPs) associated with low versus high ADB damage. Sixty-one of the 203 most significant SNPs were in, or close to, genes with putative homologs already known to be involved in pathogen responses in other plant species. We also used the pooled sequence data to train a genomic prediction (GP) model, cross-validated using individual whole genome sequence data generated for 75 healthy and 75 damaged trees from a single seed source. Using the top 30% of our genomic estimated breeding values from 200 SNPs, we could predict tree health with over 90% accuracy. We infer that ash dieback resistance in F. excelsior is a polygenic trait that should respond well to both natural selection and breeding, which could be accelerated using GP.

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Morphological and Molecular Characteristics of Fungal Species Associated with Crown Rot of Strawberry in South Korea

10.21203/rs.3.rs-713894/v1 ◽

2021 ◽

Author(s):

Oliul Hassan ◽

Taehyun Chang

Keyword(s):

South Korea ◽

Root Rot ◽

Species Complex ◽

Sequence Data ◽

Morphological Characteristics ◽

Fungal Species ◽

Crown Rot ◽

Molecular Characteristics ◽

Pathogenicity Test ◽

Crown And Root Rot

Abstract Crown and root rot is the most important and destructive strawberry diseases in Korea as it causessubstantial economic loss. In August 2020, a severe outbreak of crown and root rot on strawberries (Fragaria×ananassa Duch.) was observed in the greenhouse at Sangju, South Korea. Infected plantlets displayed browning rot within the crown and root, stunted growth, and poor rooting. Thirty fungal isolates were procured from the affected plantlet. Isolates were identified based on morphological characteristics and pathogenicity test as well as sequence data obtained from internal transcribed spacer, large subunit ribosomal ribonucleic acid, translation elongation factor,and RNA polymerase Ⅱ-second largest subunit. Results showed that thecrown and root rot of strawberry in Korea was caused by three distinct fungal species:Fusarium oxysporum species complex, F. solani species complex, andPlectosphaerella cucumerina. To the best of our knowledge,F. solani species complex andP. cucumerinaare reported for the first time as the causal agents of the crown and root rot of strawberryin South Korea.Pathogenicity tests confirmed that these isolates are pathogenic to strawberry.Understanding the composition and biology of the pathogen population will be helpful toprovide effectivecontrol strategies for the disease.

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Plant Pathogenic Fungi Associated with Coraebus florentinus (Coleoptera: Buprestidae) Attacks in Declining Oak Forests

Forests ◽

10.3390/f10060488 ◽

2019 ◽

Vol 10 (6) ◽

pp. 488 ◽

Cited By ~ 2

Author(s):

Claudia Pinna ◽

Benedetto T. Linaldeddu ◽

Vitale Deiana ◽

Lucia Maddau ◽

Lucio Montecchio ◽

...

Keyword(s):

Sequence Data ◽

Management Strategies ◽

Pathogenic Fungi ◽

Fungal Species ◽

Western Mediterranean ◽

Plant Pathogenic Fungi ◽

Limited Information ◽

Natural Ecosystems ◽

Oak Trees ◽

Coraebus Florentinus

The black-banded oak borer, Coraebus florentinus, is an emerging pest of oak trees in the western Mediterranean region. Larvae of the insect are xylophagous and progressively excavate an annular gallery that interrupts sap flow, resulting in the death of the attacked branches. Until now, limited information has been available regarding the ecological interactions between C. florentinus and the main plant pathogenic fungi involved in the etiology of oak decline. Knowledge of these interactions is important in understanding their impact in natural ecosystems and developing appropriate management strategies. Therefore, in this study, we characterized the fungal communities occurring in the exoskeleton of adults and larvae of C. florentinus and associated with the necrotic wood tissues surrounding the branch galleries of declining oak trees. A total of 29 fungal species were identified based on DNA sequence data and morphological features, of which 14 were from symptomatic woody tissues, six from insect exoskeleton, and nine from both insects and symptomatic wood tissues. The most frequent fungal species, Cryphonectria naterciae (15.9% of isolates), Dothiorella iberica (11.3%), and Diplodia corticola (9.9%), were isolated from both insect and gallery systems. All three species are well-known oak pathogens and are reported here, for the first time, to be associated with C. florentinus. At the same time, 89.6% of the fungal taxa were isolated from one or two sites, highlighting the site-dependence of fungal community assemblages.

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Estimating mortality rates of European ash ( Fraxinus excelsior ) under the ash dieback ( Hymenoscyphus fraxineus ) epidemic

Plants People Planet ◽

10.1002/ppp3.11 ◽

2018 ◽

Vol 1 (1) ◽

pp. 48-58 ◽

Cited By ~ 27

Author(s):

Tim L. R. Coker ◽

Jiří Rozsypálek ◽

Anne Edwards ◽

Tony P. Harwood ◽

Louise Butfoy ◽

...

Keyword(s):

Mortality Rates ◽

Fraxinus Excelsior ◽

Ash Dieback ◽

Hymenoscyphus Fraxineus ◽

European Ash

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Occurrence and Diversity of Black-Foot Disease Fungi in Symptomless Grapevine Nursery Stock in Spain

Plant Disease ◽

10.1094/pdis-03-19-0484-re ◽

2020 ◽

Vol 104 (1) ◽

pp. 94-104 ◽

Cited By ~ 8

Author(s):

Carmen Berlanas ◽

Sonia Ojeda ◽

Beatriz López-Manzanares ◽

Marcos Andrés-Sodupe ◽

Rebeca Bujanda ◽

...

Keyword(s):

Sequence Data ◽

Elongation Factor ◽

Fungal Species ◽

Nuclear Ribosomal Dna ◽

Northern Spain ◽

Internal Transcribed Spacer Region ◽

Tubulin Genes ◽

Foot Disease ◽

Black Foot ◽

Black Foot Disease

In this study, 3,426 grafted grapevines ready to be planted from 15 grapevine nursery fields in Northern Spain were inspected from 2016 to 2018 for black-foot causing pathogens. In all, 1,427 isolates of black-foot pathogens were collected from the asymptomatic inner tissues of surface sterilized secondary roots and characterized based on morphological features and DNA sequence data of the nuclear ribosomal DNA-internal transcribed spacer region, histone H3, translation elongation factor 1-alpha and β-tubulin genes. Eleven species belonging to the genera Dactylonectria, Ilyonectria, Neonectria, and Thelonectria were identified, including Dactylonectria alcacerensis, D. macrodidyma, D. novozelandica, D. pauciseptata, D. torresensis, Ilyonectria liriodendri, I. pseudodestructans, I. robusta, Neonectria quercicola, Neonectria sp. 1, and Thelonectria olida. In addition, two species are newly described, namely D. riojana and I. vivaria. Twenty-four isolates representing 13 black-foot species were inoculated onto grapevine seedlings cultivar ‘Tempranillo’. The pathogenicity tests detected diversity in virulence among fungal species and between isolates within each species. The most virulent species was D. novozelandica isolate BV-0760, followed by D. alcacerensis isolate BV-1240 and I. vivaria sp. nov. isolate BV-2305. This study improves our knowledge on the etiology and virulence of black-foot disease pathogens, and opens up new perspectives in the study of the endophytic phase of these pathogens in grapevines.

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Decline of Fraxinus excelsior L. in parks of Saint Petersburg: Who is to blame – Hymenoscyphus fraxineus or Diplodia spp.?

Forestry Studies / Metsanduslikud Uurimused ◽

10.2478/fsmu-2020-0013 ◽

2020 ◽

Vol 73 (1) ◽

pp. 43-51

Author(s):

Dmitrii A. Shabunin ◽

Andrey V. Selikhovkin ◽

Elena Yu. Varentsova ◽

Dmitry L. Musolin

Keyword(s):

Light Microscopy ◽

Fraxinus Excelsior ◽

Ash Dieback ◽

Saint Petersburg ◽

Hymenoscyphus Fraxineus ◽

Ash Trees ◽

Fallen Leaves ◽

European Ash

Abstract The weakening and decline of European ash Fraxinus excelsior L. and other ash species have been recorded at different locations in the suburbs of Saint Petersburg, Russia. During the summer of 2019 and spring of 2020, samples from leaves, petioles, and shoots were collected from the weakened and declining ash trees in three parks in Pushkin and Gatchina and maintained in humid chambers to induce the fructification of fungi. In total, 30 taxa of micromycetes belonging to 23 genera were identified using methods of light microscopy. Hymenoscyphus fraxineus, a putative agent of ash dieback, was not recorded in the samples collected in the crowns of trees, but only on the petioles of the fallen leaves in spring. Out of all the micromycetes recorded, only coelomycetes from the genus Diplodia Fr. (in particular, D. mutila) can damage the branches of ash trees and, thus, be considered pathogenic. It is likely that H. fraxineus opens “the entry of infection” and Diplodia spp. cause the major weakening and decline of branches. The data obtained can significantly change our understanding of the causes of ash dieback and possible methods of ash stand preservation. The reason for the low pathogenicity and activity of H. fraxineus, as well as the possible role of ascomycetes Diplodia spp. in the dieback of ash stands requires further research.

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The Relationship between Fungal Diversity and Invasibility of a Foliar Niche—The Case of Ash Dieback

Journal of Fungi ◽

10.3390/jof6030150 ◽

2020 ◽

Vol 6 (3) ◽

pp. 150

Author(s):

Ahto Agan ◽

Rein Drenkhan ◽

Kalev Adamson ◽

Leho Tedersoo ◽

Halvor Solheim ◽

...

Keyword(s):

Niche Partitioning ◽

Carbon Utilization ◽

Dimorphic Fungus ◽

Dna Amount ◽

Ash Dieback ◽

Hymenoscyphus Fraxineus ◽

Leaf Tissues ◽

Ecological Success ◽

Carbon Utilization Profiles ◽

European Ash

European ash (Fraxinus excelsior) is threatened by the invasive ascomycete Hymenoscyphus fraxineus originating from Asia. Ash leaf tissues serve as a route for shoot infection but also as a sporulation substrate for this pathogen. Knowledge of the leaf niche partitioning by indigenous fungi and H. fraxineus is needed to understand the fungal community receptiveness to the invasion. We subjected DNA extracted from unwashed and washed leaflets of healthy and diseased European ash to PacBio sequencing of the fungal ITS1-5.8S-ITS2 rDNA region. Leaflets from co-inhabiting rowan trees (Sorbus aucuparia) served as a reference. The overlap in leaflet mycobiomes between ash and rowan was remarkably high, but unlike in rowan, in ash leaflets the sequence read proportion, and the qPCR-based DNA amount estimates of H. fraxineus increased vigorously towards autumn, concomitant with a significant decline in overall fungal richness. The niche of ash and rowan leaves was dominated by epiphytic propagules (Vishniacozyma yeasts, the dimorphic fungus Aureobasidion pullulans and the dematiaceous hyphomycete Cladosporium ramotenellum and H. fraxineus), and endophytic thalli of biotrophs (Phyllactinia and Taphrina species), the indigenous necrotroph Venturia fraxini and H. fraxineus. Mycobiome comparison between healthy and symptomatic European ash leaflets revealed no significant differences in relative abundance of H. fraxineus, but A. pullulans was more prevalent in symptomatic trees. The impacts of host specificity, spatiotemporal niche partitioning, species carbon utilization profiles and life cycle traits are discussed to understand the ecological success of H. fraxineus in Europe. Further, the inherent limitations of different experimental approaches in the profiling of foliicolous fungi are addressed.

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First Report of the Ash Dieback Pathogen Hymenoscyphus pseudoalbidus (Anamorph Chalara fraxinea) on Fraxinus excelsior in Belgium

Plant Disease ◽

10.1094/pdis-07-10-0540 ◽

2011 ◽

Vol 95 (2) ◽

pp. 220-220 ◽

Cited By ~ 11

Author(s):

A. Chandelier ◽

N. Delhaye ◽

M. Helson

Keyword(s):

Fraxinus Excelsior ◽

Malt Extract ◽

Internal Transcribed Spacer Region ◽

Ash Dieback ◽

Fungal Isolation ◽

First Report ◽

Chalara Fraxinea ◽

Fungal Biology ◽

Hymenoscyphus Pseudoalbidus ◽

Branch Dieback

Since the early 1990s, European ash (Fraxinus excelsior L.) has been affected by a lethal disease caused by the ascomycete fungus, Hymenoscyphus pseudoalbidus, originally known under the name of its anamorph, Chalara fraxinea (2,4). Pathogenicity of H. pseudoalbidus was demonstrated by inoculations on young trees (3). This emerging pathogen induces necrosis of leaf rachises, leaf wilting and shedding, bark necrosis, and wood discoloration as well as shoot, twig, and branch dieback. First observed in Poland, ash dieback now occurs in many parts of Europe. Since 2009, a survey of ash dieback caused by H. pseudoalbidus has been conducted in Wallonia (southern Belgium). Sampling units were selected to take the occurrence of ash stands and the potential points of entry of the pathogen into the country (nurseries, sawmills, rivers, and roads) into account. While the disease was not detected in 2009, young, naturally regenerated trees displaying typical symptoms of ash dieback were found in June 2010 in Silly, a village in the province of Hainaut. Symptomatic trees were located along a road in front of a large ash stand. Examination of shoots with bark necrosis from three symptomatic trees yielded positive results on the basis of a real time PCR test developed in our laboratory for the detection of H. pseudoalbidus (1). To confirm the molecular identification, fungal isolation from discolored wood onto malt extract agar supplemented with 100 mg liter–1 of streptomycin sulfate was attempted. After 18 days at 20 to 22°C in the dark, slow-growing, dull white colonies with gray patches, resembling those of C. fraxinea, had formed. The nuclear ribosomal internal transcribed spacer region (ITS) was amplified with primers ITS1 and ITS4 (4) and partly sequenced (GenBank Accession No. FR667687). A BLASTn search in GenBank revealed that the sequence of the Belgian isolate (452 bp) displayed 100% identity with sequences of a H. pseudoalbidus isolate from Switzerland (GenBank Accession No GU586932). In contrast, the sequence showed some mismatches with that of the closely related and probably strictly saprotrophic fungus, Hymenoscyphus albidus (GenBank Accession No GU586891.1). The strain was deposited as reference material in the Fungal Biology collection (CBS 128012). To our knowledge, this is the first report of ash dieback caused by H. pseudoalbidus in Belgium. The discovery of this aggressive tree pathogen in Wallonia documents its further westward spread in Europe. In the future, we expect that H. pseudoalbidus will continue its range expansion into areas that have so far not been affected by ash dieback. References: (1) A. Chandelier et al. For. Pathol. 40:87, 2010. (2) T. Kowalski. For. Pathol. 36:264, 2006. (3) T. Kowalski and O. Holdenrieder. For. Pathol. 39:1, 2009. (4) V. Queloz et al. For. Pathol. Online publication. doi:10.1111/j.1439-0329.2010.00645.x, 2010.

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