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

Abstract

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.