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.

Transformation of European Ash (Fraxinus excelsior L.) Callus as a Starting Point for Understanding the Molecular Basis of Ash Dieback

The population of European ash (Fraxinus excelsior L.) is currently facing the risk of collapse, mainly due to ash dieback, a disease caused by a pathogenic fungus, Hymenoscyphus fraxineus. To facilitate studies into the molecular basis of ash dieback and design breeding strategies for a generation of resistant trees, it is necessary to develop tools enabling the study of gene function in F. excelsior. Despite this, a method for the genetic engineering of F. excelsior is still missing. Here, we report the first successful genetic transformation of F. excelsior callus and a selection process enabling the formation of stable transgenic callus lines. The protocol relies on the use of Agrobacterium tumefaciens to transform callus tissue derived from embryos of F. excelsior. In our experiments, we used the β-glucuronidase (GUS) reporter system to demonstrate the transformation of callus cells and performed RT-PCR experiments to confirm the stable expression of the transgene. Since ash dieback threatens the long-term stability of many native F. excelsior populations, we hope that the transformation techniques described in this manuscript will facilitate rapid progress in uncovering the molecular basis of the disease and the validation of gene targets previously proposed to be linked to the resistance of trees to H. fraxineus pathogenicity.

Advances in the Detection of Emerging Tree Diseases by Measurements of VOCs and HSPs Gene Expression, Application to Ash Dieback Caused by Hymenoscyphus fraxineus

Ash shoot dieback has now spread throughout Europe. It is caused by an interaction between fungi that attack shoots (Hymenoscyphus fraxineus) and roots (Armillaria spp., in our case Armillaria gallica). While detection of the pathogen is relatively easy when disease symptoms are present, it is virtually impossible when the infestation is latent. Such situations occur in nurseries when seedlings become infected (the spores are carried by the wind several dozen miles). The diseases are masked by pesticides, fertilisers, and adequate irrigation to protect the plants. Root rot that develops in the soil is also difficult to detect. Currently, there is a lack of equipment that can detect root rot pathogens without digging up root systems, which risks damaging trees. For this reason, the use of an electronic nose to detect pathogens in infected tissue of ash trees grown in pots and inoculated with the above fungi was attempted. Disease symptoms were detected in all ash trees exposed to natural infection (via spores) in the forest. The electronic nose was able to detect the pathogens (compared to the control). Detection of the pathogens in seedlings will enable foresters to remove diseased trees and prevent the path from nursery to forest plantations by such selection.

Fungi Detected in the Previous Year’s Leaf Petioles of Fraxinus excelsior and Their Antagonistic Potential against Hymenoscyphus fraxineus

Studies on fungal communities in the previous year’s leaf petioles of Fraxinus excelsior found in litter in five ash stands in southern Poland were made in 2017. Fungi were identified on the basis of isolation from 300 surface sterilized leaf petioles and by in situ inventory of fruit bodies (on 600 petioles, in spring and autumn). Identification was based on morphology of colonies and fruit bodies, and sequencing of ITS region of the rRNA gene cluster. In total, 2832 isolates from 117 taxa (Ascomycota—100; Basidiomycota—15; Mucoromycota—2 taxa) were obtained with the isolation method. The most frequent taxa (with frequency >10%) were: Nemania serpens, Hymenoscyphus fraxineus, Alternaria sp. 1, Boeremia sp., Helotiales sp. 1, Epicoccum nigrum, Venturia fraxini, Fusarium sp., Fusarium lateritium, Nemania diffusa, Typhula sp. 2 (in descending order). In total, 45 taxa were detected with the in situ inventory method. Eleven taxa were classified as dominant: Hymenoscyphus fraxineus, Venturia fraxini, Leptosphaeria sp. 2, Cyathicula fraxinophila, Typhula sp. 2, Hypoderma rubi, Pyrenopeziza petiolaris, Cyathicula coronata, Hymenoscyphus scutula, Leptosphaeria sclerotioides and Hymenoscyphus caudatus. Among 202 leaf petioles colonized by H. fraxineus, 177 petioles also showed fructification of 26 other fungi. All the isolated saprotrophs were tested in dual-culture assay for antagonism to two strains of H. fraxineus. Three interaction types were observed: type A, mutual direct contact, when the two fungi meet along the contact line (occurred with 43.3% of test fungi); type B, with inhibition zone between colonies (with 46.9% of test fungi); type C, when the test fungus overgrows the colony of H. fraxineus (with 9.8% of test fungi). The possible contribution of the fungal saprotrophs in limiting of the expansion of H. fraxineus in ash leaf petioles, which may result in reduction in the inoculum of ash dieback causal agent, is discussed.

Invasive Populations of Emerald Ash Borer Agrilus planipennis Fairmaire, 1888 (Coleoptera: Buprestidae) in Saint Petersburg, Russia: a “Hitchhiker”?

Emerald ash borer (EAB), Agrilus planipennis, is an invasive beetle of East Asian origin that in North America and Russia killed millions of ash trees (Fraxinus spp.). In September 2020, EAB was de-tected in Saint Petersburg, becoming resonant event for the metropolitan city. The aim of the present study was to investigate occurrence and ecology of EAB in Saint Petersburg. The presence of two distinct enclave populations of EAB was revealed, each of which has (very likely) been established by separate events of “hitchhiking” transport vehicles. Following the invasion, further spread of EAB in Saint Petersburg was slow and locally restricted, main explanation for which is climatic factor. Due to spread by “hitchhiking”, the possibility of EAB further long-distance ge-ographic spread of EAB in the Baltic Sea region (EU) is high, and not only by ground transport (120–130 km distance from EU borders), but also by ferries transporting cars (traditional means of transportation across the Baltic Sea). In certain cases, development of EAB on F. excelsior was more successful (stem portion colonized, larval densities, number of galleries, exit holes, viable larvae, emerged beetles) than in (adjacent) F. pennsylvanica trees. Observed relatively high EAB-sensitivity of F. excelsior therefore questions the efficacy and benefits of the currently ongoing selection and breeding projects against ash dieback (ADB), caused by Hymenoscyphus fraxineus. Inventory, mapping, and monitoring of surviving F. excelsior trees in areas infested by both ADB and EAB are necessary to acquire genetic resource for work on strategic long-term restoration of F. excelsior, tackling (inevitable) invasion of EAB to the EU.

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.

European-wide forest monitoring substantiate the neccessity for a joint conservation strategy to rescue European ash species (Fraxinus spp.)

European ash (Fraxinus excelsior) and narrow-leafed ash (F. angustifolia) are keystone forest tree species in Europe with a broad ecological amplitude and significant economic importance. Besides global warming both species are currently under significant thread by an invasive fungal pathogen that has been progressively spreading throughout the continent for almost three decades. Ash dieback caused by the invasive ascomycete Hymenoscyphus fraxineus is capable of damaging ash trees of all age classes and often leads to the ultimate death of a tree after years of progressively developing crown defoliation. While studies at national and regional level already suggested rapid decline of ash populations as a result of ash dieback, a comprehensive survey at European level with harmonized crown assessment data across countries could shed more light into the population decline from a pan-European perspective and could also pave the way for a new conservation strategy beyond national boarders. Here, we present data from the ICP Forests Level I crown condition monitoring including 27 countries, covering the timespan from 1987-2020. In total, 407 survey plots randomly distributed across these countries were analyzed resulting in >36,000 individual observations. We found a substantial increase in defoliation and mortality over time indicating that crown defoliation has almost doubled during the last three decades. Hotspots of mortality are currently situated in southern Scandinavia and north-eastern Europe, well corresponding to the fact that the disease spread fast from north-east to north-west. Overall survival probability after nearly 30 years of infection has already reached a critical value of 0.51, but with large differences among regions (0.00-0.907). Both a Cox proportional hazard model as well as an Aalen additive regression model strongly suggest that survival of ash is significantly lower in locations with excessive water regime and which experienced more extreme precipitation events during the last two decades. Our results underpin the necessity for fast governmental acting and joint rescue efforts beyond national boarders since overall mean defoliation will likely reach 50% as early as 2030 as suggested by time series forecasting . We strongly recommend to develop a pan-European conservation strategy before the decline will reach its tipping point resulting into non-reversible loss of diversity in the European forest landscape.

Assessing Genotypic and Environmental Effects on Endophyte Communities of Fraxinus (Ash) Using Culture Dependent and Independent DNA Sequencing

Fraxinus excelsior populations are in decline due to the ash dieback disease Hymenoscyphus fraxineus. It is important to understand genotypic and environmental effects on its fungal microbiome to develop disease management strategies. To do this, we used culture dependent and culture independent approaches to characterize endophyte material from contrasting ash provenances, environments, and tissues (leaves, roots, seeds). Endophytes were isolated and identified using nrITS, LSU, or tef DNA loci in the culture dependent assessments, which were mostly Ascomycota and assigned to 37 families. Few taxa were shared between roots and leaves. The culture independent approach used high throughput sequencing (HTS) of nrITS amplicons directly from plant DNA and detected 35 families. Large differences were found in OTU diversity and community composition estimated by the contrasting approaches and these data need to be combined for estimations of the core endophyte communities. Species richness and Shannon index values were highest for the leaf material and the French population. Few species were shared between seed and leaf tissue. PCoA and NMDS of the HTS data showed that seed and leaf microbiome communities were highly distinct and that there was a strong influence of Fraxinus species identity on their fungal community composition. The results will facilitate a better understanding of ash fungal ecology and are a step toward identifying microbial biocontrol systems to minimize the impact of the disease.