First report of apple bitter rot caused by colletotrichum fioriniae in brittany, France

France is the third largest European producer of apples (Malus domestica), after Poland and Italy, with 2.1 million tons yearly (Eurostat 2015), and the largest producer of European cider apples, with 8,500 ha and 250,000 tons of fruit. Cider apples from Brittany represent over a quarter of the national tonnage (Anonymous 2012). Apple bitter rot symptoms have been observed on ‘Galeuse’ apples (a local variety in Brittany) for nearly 5 to 7 years and again in September 2015, during harvest at one apple orchard near Brest, France. Lesions were round, 1 to 4 cm in diameter, brown and dry with acervuli, producing orange spore masses in concentric rings. Fungal isolates were obtained from symptomatic apples by culturing necrotic tissue pieces on potato dextrose agar at 25°C in the dark. Cultures were light gray, with cottony aerial mycelium becoming darker with age and with reverse colors ranging from brownish pink to dark gray with black spots. Cultures had dark melanized structures similar to acervuli that oozed orange-colored conidia. Conidia were cylindrical to fusiform, pointed at one or both ends, and 10.0 to 14.0 μm × 3.0 to 3.5 μm. Both cultural and morphological characteristics were similar to those described for Colletotrichum acutatum sensu lato (Damm et al. 2012). Total genomic DNA was extracted and the ITS region of rDNA was amplified using the universal primers ITS4 and ITS5, then sequenced. The resulting sequence was 100% identical to C. acutatum species complex sequences obtained by a BLAST search in GenBank. Based on Damm et al. (2012), five other loci were used to further characterize the isolate: partial GAPDH, CHS-1, HIS3, ACT, and TUB2 genes sequences were amplified and sequenced. Sequences were deposited in GenBank (Accession Nos. KT887552 for ITS, KT887551 for TUB, KT887555 for CHS, KT887553 for HIS3, KT887556 for ACT, and KT887554 for GAPDH). The multilocus phylogenetic analysis carried out with the obtained sequences and reference sequences (Damm et al. 2012) revealed that the isolate clustered within C. fioriniae, as also suggested by the BLAST results, and consistent with the initial identification as C. acutatum. Koch’s postulates were performed on 10 ‘Golden Delicious’ apples. Surface sterilized fruits were wound-inoculated with 20 μl of a conidial suspension (105 conidia/ml). After 10 days incubating at 20°C, symptoms identical to those initially observed developed around the inoculation point, while controls inoculated with water remained symptomless. Fungal colonies reisolated from the lesions were morphologically similar to the original isolate. Previous studies have demonstrated that this pathogen is present worldwide on Malus spp., and apple bitter rot is considered one of the most important diseases in the United States (Ivic et al. 2013). In Europe, there are increasing numbers of reports ofColletotrichum species on apples (Ivic et al. 2013; Baroncelli et al. 2014), but to our knowledge, this is the first report in France of anthracnose on apples caused by C. fioriniae. In Europe, bitter rot caused by C. acutatum species may become a major problem and require additional investigation.