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.

Investigation of the Biocontrol Potential of Two Ash Endophytes against Hymenoscyphus fraxineus Using In Vitro Plant–Fungus Dual Cultures

Development of effective biocontrol procedures using ash endophytes to combat an ash pathogen Hymenoscyphus fraxineus would be an appropriate contribution to the ongoing effort to protect European ash stands against ash decline. In this study we investigated the biocontrol potential of two ash endophytes, Thielavia basicola and Minimidochium sp., against H. fraxineus using in vitro plant-fungus and fungus-fungus dual cultures approach in three biocontrol models. The tests aimed to determine whether the endophytes show antagonism toward Fraxinus excelsior and F. pennsylvanica, to assess the level of antagonism of the endophytes toward H. fraxineus and to identify potential secondary metabolites induced by the presence of H. fraxineus. The results that dual culture experiments modeled according to our design may be a very useful tool to precisely study biocontrol potential of fungi, i.e., without the impact of environmental factors. Such experiments also enable the selection of most resistant ash genotypes and rapid propagation, producing large numbers of pathogen-free seedlings. It should be noted, however, that both of the endophytes tested in the dual cultures strongly inhibited the growth of H. fraxineus. Their growth under the influence of callus/seedlings was also inhibited. Comparison of HPLC profiles showed that the presence of H. fraxineus in the post-culture medium induced the production of an unknown secondary metabolite in this species. Such results suggest that some of the plant–fungus combinations examined in this study may have potential to be developed as biocontrol methods, thus increasing the survivability of ash stands under natural conditions.

Priming of Resistance-Related Phenolics: A Study of Plant-Associated Bacteria and Hymenoscyphus fraxineus

European ash (Fraxinus excelsior) is highly affected by the pathogenic fungus Hymenoscyphus fraxineus in all of Europe. Increases in plant’s secondary metabolite (SM) production is often linked tol enhanced resistance to stress, both biotic and abiotic. Moreover, plant-associated bacteria have been shown to enhance SM production in inoculated plants. Thus, our hypothesis is that bacteria may boost ash SM production, hence priming the tree’s metabolism and facilitating higher levels of resilience to H. fraxineus. We tested three different ash genotypes and used Paenibacillus sp. and Pseudomonas sp. for inoculation in vitro. Total phenol (TPC), total flavonoid (TFC) and carotenoid contents were measured, as well as the chlorophyll a/b ratio and morphometric growth parameters, in a two-stage trial, whereby seedlings were inoculated with the bacteria during the first stage and with H. fraxineus during the second stage. While the tested bacteria did not positively affect the morphometric growth parameters of ash seedlings, they had a statistically significant effect on TPC, TFC, the chlorophyll a/b ratio and carotenoid content in both stages, thus confirming our hypothesis. Specifically, in ash genotype 64, both bacteria elicited an increase in carotenoid content, TPC and TFC during both stages. Additionally, Pseudomonas sp. inoculated seedlings demonstrated an increase in phenolics after infection with the fungus in both genotypes 64 and 87. Our results indicate that next to genetic selection of the most resilient planting material for ash reforestation, plant-associated bacteria could also be used to boost ash SM production.

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.

Meteorological factors associated with the timing and abundance of Hymenoscyphus fraxineus spore release

The ascomycete Hymenoscyphus fraxineus has spread across most of the host range of European ash with a high level of mortality, causing important economic, cultural and environmental effects. We present a novel method combining a Monte-Carlo approach with a generalised additive model that confirms the importance of meteorology to the magnitude and timing of H. fraxineus spore emissions. The variability in model selection and the relative degree to which our models are over- or under-fitting the data has been quantified. We find that both the daily magnitude and timing of spore emissions are affected by meteorology during and prior to the spore emission diurnal peak. We found the daily emission magnitude has the strongest associations to weekly average net radiation and leaf moisture before the emission, soil temperature during the day before emission and net radiation during the spore emission. The timing of the daily peak in spore emissions has the strongest associations to net radiation both during spore emission and in the day preceding the emission. The seasonal peak in spore emissions has a near-exponential increase/decrease, and the mean daily emission peak is approximately Gaussian.

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.