First report of Colletotrichum nymphaeae and C. alienum causing apple bitter rot in Italy

Apple bitter rot (ABR) affects apples (Malus × domestica Borkh.) in pre- and post-harvest conditions and has become an increasing concern in European orchards. The disease is caused by Colletotrichum species, mainly included in the C. gloeosporioides and C. acutatum species complexes (Calì et al. 2025; McLaughlin et al. 2024). In 2024, symptoms of ABR, with an estimated disease incidence of approximately 15%, including small, circular, light-brown lesions that expanded in concentric rings, forming sunken depressions bearing acervuli, were observed in late August on apple fruits cv Opal in Chivasso (TO, 45°12'44.1"N 7°54'35.9"E) and in mid-September on cv Inored Story® in Savigliano (CN, 44°38'58.6"N 7°35'37.0"E), Piedmont, Northern Italy. Small fragments (0.2–0.5 cm) were cut from the lesion edge of sampled fruits, surface-sterilized in 1% sodium hypochlorite for one minute, rinsed in sterile distilled water, dried on sterile paper, transferred onto Potato Dextrose Agar (PDA) with streptomycin sulphate (25 mg/L), and incubated at 25 ± 1 °C in the dark. Single-spore Colletotrichum-like colonies were obtained, and two isolates were selected as representative based on distinct colony aspect. Isolate CVG2470, from cv Opal, produced colonies characterized by a dense, olivaceous grey, velvety mycelium with abundant pale salmon masses producing conidia measuring 11.02 ± 0.91 × 3.34 ± 0.30 µm (n = 30). Isolate CVG2563, from cv Inored Story®, showed colonies with a dense, white, cottony to felted mycelium with conidia measuring 10.37 ± 0.87 × 4.02 ± 0.39 µm (n = 30). For species identification, the genomic DNA was extracted and the following loci were amplified: glyceraldehyde-3-phosphate dehydrogenase (gapdh), chitin synthase 1 (chs-1), β-tubulin (tub2), actin (act), histone H3 (his3), calmodulin (cal), glutamine synthetase (gs) and Apn2–Mat1-2 intergenic spacer and partial mating-type gene (ApMat) (Damm et al. 2012; Talhinhas and Baroncelli 2021; Weir et al. 2012). BlastN analyses of the obtained sequences for CVG2470 (Genbak accession numbers: PX966954 to PX966958) resulted in 100% homology to the epitype CBS 515.78 of C. nymphaeae for gapdh, act and tub2, 99.64% for chs-1 and 98.95% for his3. The sequences of CVG2563 (Genbank accession numbers: PX966959 to PX966965) showed 100% homology to the epitype ICMP 12071 of C. alienum for gapdh, act, tub2 and ApMat, 99.59% for chs-1, 99.86% for cal and 99.87% for gs. Pathogenicity tests were performed on surface sterilized apples cv Opal (isolate CVG2470) and Inored Story® (isolate CVG2563): 10 were wounded and inoculated with 20 µl of a conidia suspension (104conidia/ml), 10 were sprayed with 8 ml of the same suspension, and 10 (five wounded and five unwounded) were treated with sterile water as controls. Fruits were placed in humid chambers, maintained at 25 ± 1°C with 12 h photoperiod and monitored daily. Wounded fruits showed 5.22 ± 0.82 × 5.04 ± 0.67 mm and 3.89 ± 0.73 × 3.90 ± 1.04 mm lesions 14 days post-inoculation (dpi), for CVG2470 and CVG2563, respectively. Non-wounded fruits showed necrotic spots at 12-14 dpi. The controls remained asymptomatic. Identity of reisolated fungi was confirmed by sequencing his3 and gs regions. Colletotrichum nymphaeae has been previously reported on apple in Brazil, Japan, South Korea, USA and Uruguay, whilst C. alienum in New Zealand (Farr et al. 2026). To the best of our knowledge, this is the first report of these species on apple in Italy, as well as in Europe, as agents of ABR.