First report of Hymenoscyphus fraxineus causing ash dieback in Spain

In August 2021, mature Fraxinus excelsior trees and associated regeneration with typical symptoms of ash dieback (shoot dieback, wilting of leaves, necrotic rachises and lenticels, and fungal fruiting bodies) were observed in the localities of Oviedo (43.3804, -5.8679) and Bulnes (43.2352, -4.8241) in the principality of Asturias in north-western Spain (Figs. 1–2). The lethal disease is caused by the ascomycete fungus Hymenoscyphus fraxineus (anamorph Chalara fraxinea), which was introduced into Europe from Asia. Since its introduction in the mid1990s in north-east Poland,H. fraxineushas spread across Europe causing severe decline and increased mortality of European ash (Fraxinus excelsior) (Kowalski, 2006; Queloz et al., 2011) and narrow-leaved ash (Fraxinus angustifolia). In both Oviedo and Bulnes, 10–15 necrotic rachises and fruiting bodies were collected in August 2021 (Fig. 3). The rachises were dried and fresh fruiting bodies stored in 99% (v/v) ethanol until further processing. For fungal isolation, five rachises per site were wiped clean with 70% (v/v) ethanol, then small wedges were cut out with a scalpel and laid on ash malt extract agar (five wedges per rachis, i.e. 25 isolates per site; Gross et al., 2014). Fungal cultures were left to grow at 20C (daylight) for two weeks. Forty-two cultures showed the typical mycelial morphology of H. fraxineus on ash malt agar, that is dark discolouration of the agar at the edge of the culture and creamy-white mycelium (Fig. 4), five cultures were contaminated with Penicillium and three wedges showed no fungal growth. Phialophores and conidia of the Chalara anamorph were observed under the microscope from cultures considered to beH. fraxineus (Fig. 5). FourH. fraxineus-like isolates and one fruiting body per site were chosen for molecular analysis.


European ash, Fraxinus excelsior
In August 2021, mature Fraxinus excelsior trees and associated regeneration with typical symptoms of ash dieback (shoot dieback, wilting of leaves, necrotic rachises and lenticels, and fungal fruiting bodies) were observed in the localities of Oviedo (43.3804,) and Bulnes (43.2352, -4.8241) in the principality of Asturias in north-western . The lethal disease is caused by the ascomycete fungus Hymenoscyphus fraxineus (anamorph Chalara fraxinea), which was introduced into Europe from Asia. Since its introduction in the mid-1990s in north-east Poland, H. fraxineus has spread across Europe causing severe decline and increased mortality of European ash (Fraxinus excelsior) (Kowalski, 2006;Queloz et al., 2011) and narrow-leaved ash (Fraxinus angustifolia).
In both Oviedo and Bulnes, 10-15 necrotic rachises and fruiting bodies were collected in August 2021 (Fig. 3). The rachises were dried and fresh fruiting bodies stored in 99% (v/v) ethanol until further processing. For fungal isolation, five rachises per site were wiped clean with 70% (v/v) ethanol, then small wedges were cut out with a scalpel and laid on ash malt extract agar (five wedges per rachis, i.e. 25 isolates per site; Gross et al., 2014). Fungal cultures were left to grow at 20 • C (daylight) for two weeks. Forty-two cultures showed the typical mycelial morphology of H. fraxineus on ash malt agar, that is dark discolouration of the agar at the edge of the culture and creamy-white mycelium ( Fig. 4), five cultures were contaminated with Penicillium and three wedges showed no fungal growth. Phialophores and conidia of the Chalara anamorph were observed under the microscope from cultures considered to be H. fraxineus (Fig. 5). Four H. fraxineus-like isolates and one fruiting body per site were chosen for molecular analysis.
This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.  In France, H. fraxineus was first reported in 2008 in the north-east of the country (Anonymous, 2008). The pathogen has since spread south and west, reaching the French Pyrenees in 2020 (https://draaf.paca. agriculture.gouv.fr/Carte-nationale-des-premiers). Based on its progression it is likely to be detected in the Spanish Pyrenees in the next few years. Both F. excelsior and F. angustifolia are native to Spain and susceptible to ash dieback. Based on previous studies, the epidemic is F I G U R E 3 Necrotic rachis with fruiting bodies F I G U R E 4 Mycelial growth of Hymenoscyphus fraxineus isolates on ash malt agar F I G U R E 5 Phialophores and conidia of the Chalara anamorph under the light microscope certain to cause severe damage to ash populations (Coker et al., 2019).
High summer temperatures and differences in climate in the different regions of Spain make it difficult to predict the spread of the pathogen.
A monitoring system should be implemented to track the progression of the disease and to evaluate its impact. Breeding efforts are required to develop gene banks that can act as a source of tolerant plant material in the future.