Apple (Malus × domestica Borkh.) is economically the most important fruit crop in South Tyrol (Italy). At the end of the growing season 2020, necrotic lesions and chlorosis developed on leaves and premature leaf dropping was observed on the cultivars Gala, Granny Smith and Cripps Pink(cov)/Rosy Glow(cov) in the Etsch/Adige valley. After the appearance of these symptoms, small circular brownish spots were observed on above 90 % of apples in the respective orchards. Fungal isolates were obtained from symptomatic apples by culturing small portions of fruit flesh from the lesion margin on potato dextrose agar (PDA) at 25°C in the dark. The colonies showed a white to peachy color on the upper surface and were greyish on the reverse side. Conidia were cylindrical, predominantly rounded and averaged 16.39 ± 1.37 µm and 5.75 ± 0.81 µm (n = 33), consistent morphological characteristics as described recently in Fuentes-Aragón et al. (2021). A multi-locus sequence analysis according to Astolfi et al. (2022) and Weir et al. (2022) was conducted based on the internal transcribed spacer (ITS) region and on fragments of actin (ACT), DNA-(apurinic or apyrimidinic site) lyase (APN2), calmodulin (CAL), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), glutamine synthetase (GS), beta-tubulin (TUB2), and intergenic spacer and partial mating type (Mat1-2) genes. MegaBlast analysis revealed 100 % identity to the reference genome of C. chrysophilum for ITS (NR_160821), for ACT (KX093982), APN2 (KX094018), CAL (KX094063), GAPDH (KX094183), 99.29 % for TUB2 (KX094285), 99.85 % for Mat1-2 (KX094325) and 99.87 % for GS (KX094204). Sequences were deposited in GenBank (ITS: OK485032; ACT: OK539650; APN2: ON624100; CAL: ON624104; GAPDH: OK539654; GS: ON624108; TUB2: OK539658; Mat1-2: ON645973). Pathogenicity assays were performed on wounded and unwounded apples of cvs. Gala and Rosy Glow(cov). Apples of both cvs. were wounded with a sterile needle. For each variant, 8 fruits were inoculated with 10 µL spore suspension (1 × 106 spores mL-1) in 0.05 % Tween®20 or with 10 µL 0.05 % Tween®20 as control. The apples were put into plastic boxes containing moist tissue and incubated at 25°C, 100 % relative humidity and 12 h photoperiod. First lesions appeared on inoculated wounded fruits after 5 days, whereas unwounded and control fruits remained asymptomatic. After 15 days, symptoms could be observed only on inoculated wounded apples with spore suspension: on 95 % of individual wounds of Rosy Glow(cov) and on 77.5 % of Gala. Koch's postulates were fulfilled by re-isolating the fungus and by identifying the re-isolates as C. chrysophilum (ITS: OK485033-OK485035; ACT: OK539651-OK539653; APN2: ON624101-ON624103; CAL: ON624105-ON624107; GAPDH: OK539655-OK539657; GS: ON624109; TUB2: OK539659-OK539661; Mat1-2: ON645974-ON645976). To date, only one study confirmed C. chrysophilum as causal agent of apple bitter rot in Europe (Cabrefiga et al. 2022). Recently, Astolfi et al. (2022) reclassified C. chrysophilum as the main causal agent of GLS on apples in Southern Brazil and Uruguay. The authors stressed that C. chrysophilum might also be the potential agent of GLS in Europe (Astolfi et al. 2022). This confirms the observations made in 2020 in South Tyrol, where massive leaf spots preceded the symptoms on fruit. To the best of our knowledge, this is the first report of a preharvest decay on apples caused by C. chrysophilum in Italy (South Tyrol).
Keywords: Causal Agent; Crop Type; Fruit; Fungi; Glomerella leaf spot (GLS); Pathogenicity assay; fungal isolates; multi-locus sequence analysis; tree fruits; wounded and unwounded.