First report of <i>Diplocarpon coronariae</i> causing apple blotch disease of apple tree in Belgium

In October 2023, apple tree leaves presenting typical symptoms caused by the fungus Diplocarpon coronariae (Crous et al., 2020) were observed in an unsprayed genetic resources evaluation orchard at the Walloon Agricultural Research Centre in Gembloux, Belgium. Apple blotch disease causes severe premature defoliation leading to reduce fruit load and quality, and reduced tree vigour (Wöhner & Emeriewen, 2019). Symptoms (Fig. 1) were visible on a few leaves located at the centre of the tree crowns. Leaves presenting typical symptoms of necrosis, chlorosis and star-shaped structures with dark brown acervuli were collected from three different apple tree accessions (cvs. Sabot d'Eijsden Rouge, Madame Galopin and an unknown cultivar) planted close to each other. Areas of the leaves showing clear symptoms and acervuli were surface disinfected (30 seconds in 70% v/v ethanol, three minutes in sodium hypochlorite solution [1.5% active chlorine], 30 seconds in 70% v/v ethanol and finally rinsed twice for 30 seconds in sterile demineralised water). The content of acervuli was then scraped and stabbed into potato dextrose agar (PDA) medium (Difco, USA). Plates were incubated in the dark at 22°C for one month. Typical colonies of Diplocarpon were observed for material collected from the three apple cultivars. Monoconidial cultures were then prepared for an isolate sampled from each cultivar. The colonies were first creamy then turned brownish and finally became dark brown to black with invaginated structures both on PDA and peptone potato dextrose agar media (Fig. 2). Genomic DNA was extracted from each of the three pure cultures. PCR amplification and sequencing of the ITS region with the primers ITS5 and ITS4 (White et al., 1990) confirmed the identity of the pathogen (GenBank Accession No. PQ471560). To fulfil Koch's postulates, seedlings of apple cv. Topaz, grown in a greenhouse, were sprayed at the five-leaf stage with a conidial suspension (2.44×105 conidia/ml) of the isolate collected from apple cv. Madame Galopin. The inoculated seedlings were incubated in 100% relative humidity at 21°C for 72 hours. The plants were then maintained in a phytotron (light 16h/24h at 21°C) and regularly observed for the appearance of symptoms. Necrosis and acervuli were clearly visible three to four weeks post-inoculation (Fig. 3). The fungus was consistently reisolated on PDA medium from the lesions present on the infected leaves. DNA extraction and sequencing of the ITS region from these isolates confirmed the identity of the pathogen. To our knowledge, this is the first report of D. coronariae in Belgium. The threat it could represent for organic apple orchards in Belgium, and particularly for untreated orchard meadows, is unknown but the pathogen is responsible for significant economic losses in several countries (Khodadadi et al., 2022; Wöhner & Emeriewen, 2019). Breeding efforts to select for resistant cultivars are urgently needed. This work received funding from Horizon Europe Framework Program of the EU (grant agreement no. 101061028), InnOBreed project.