First report of anthracnose caused by Colletotrichum grevilleae on apple in Korea.

In October 2022, typical symptoms of anthracnose were observed on apple (Malus ⅹ domestica cv. Fuji) fruits collected from Pocheon in Gyeonggi province, South Korea (N37.98074°, E127.33995°). In the surveyed orchard, the incidence rate of apple anthracnose was less than 1%. The initial symptoms were brown-to-dark brown lesions, and with disease progression, they enlarged and the pulp became soft, forming a brown band. In total 29 apple fruits were collected, and the causal agent was isolated by removing the peel, and the diseased tissues were directly transferred onto potato dextrose agar (PDA), followed by incubation for 7 days at 25°C. As the results, two isolates (GgPc22-1-11 and GgPc22-1-13) were obtained. For describing morphological and cultural characteristics, isolate GgPc22-1-11 was cultured on PDA and synthetic nutrient-poor agar (SNA) at 25°C under near-UV light with a 12-h photoperiod for 10 days. The colonies of GgPc22-1-11 on PDA were initially white and subsequently appeared light gray to olivaceous with white margins. The reverse side of the plates were dark brown and slate blue (Supplementary Fig. S1). Colonies on SNA were flat with an entire margin and short sparse white aerial mycelium. No setae were observed. Conidia on PDA were hyaline, straight, aseptate with a rounded apex, clavate to cylindrical, and measured 16.4 ± 2.4 (10.8-23.8) × 5.5 ± 0.7 (3.6-7.7) μm (n = 200). Appressoria were medium-to-dark brown, aseptate, solitary or in groups with irregular outlines, and lobate or having undulate margins (Supplementary Fig. S1). These morphological and cultural characteristics of GgPc22-1-11 were consistent with those of Colletotrichum grevilleae F. Liu, Damm, L. Cai & Crous, pathogens of Proteaceae and Punica granatum (Liu et al. 2013; Huang et al. 2023). DNA was extracted from GgPc22-1-11, PCR was performed and Phylogenetic analysis of concatenated partial sequences of the internal transcribed spacer (ITS) of rDNA, β-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), chitin synthase 1 (CHS-1), and actin (ACT) genes was conducted (Weir et al. 2012). The resulting sequences were deposited in GenBank under the accession numbers LC773710-LC773714. A nucleotide BLAST search revealed that the ITS sequences of the isolates were 98.95% identical to those of C. grossum CAUG7 (KP890165.1). The TUB2, GAPDH, CHS-1, and ACT sequences of the isolates were 99.79%, 99.24%, 100%, and 100%, respectively, identical to those of C. grevilleae WP4. GgPc22-1-11 was clustered with C. grevilleae WP4 using neighbor joining analysis conducted with MEGA X software (Kumar et al. 2018) (Supplementary Fig. S2). Pathogenicity tests were conducted using GgPc22-1-11 and repeated three times. A total of 12 symptomless apples of each variety were selected, including Fuji, Hongro, Tsugaru, and RubyS. The apples were surface-sterilized with 70% ethanol and wounded using a sterile needle. Both wounded and unwounded apples were inoculated with mycelium plugs and paper disks containing a conidial suspension (1 × 106 conidia/ml) and placed in a plastic box with moist paper towels (>90% relative humidity) at 25°C in dark. At 5 days after inoculation, all artificially wounded fruits exhibited symptoms and 30% (4 out of 12) of unwounded inoculated fruits showed symptoms in each apple variety while control fruits were asymptomatic both the unwounded and wounded inoculations (Supplementary Fig. S1). To fulfill Koch's postulates, the fungi were reisolated from symptomatic tissues and were identical to GgPc22-1-11 confirmed by morphological and molecular analysis. To the best of our knowledge, C. grevilleae has been reported in Protea sp. and pomegranate (Liu et al. 2013; Huang et al. 2023) but not in apples to date, and this is the first report of C. grevilleae causing anthracnose in apple fruits. This research of the newly emerged unreported Colletotrichum species can offer valuable information for development of an effective fungicide spray program to control apple anthracnose.