In 2020 and 2021, we surveyed disease occurrence on fruit of papaya trees growing in greenhouses located in Taean and Gokseong, Korea. During the disease surveys in a greenhouse in each location, we found anthracnose symptoms on the fruits in the harvest season. Three sites were observed in each greenhouse, and 100 fruits at each site were investigated for the disease incidence. The diseased fruits were collected from the investigated greenhouses, and fungi were isolated from the fruits with anthracnose symptoms. Conidial mass of fungi formed on the diseased fruits was diluted in sterile distilled water to prepare a conidial suspension. The 50 μl of conidial suspension from the conidial mass was poured into 2% water agar (WA) plates and streaked using a sterile loop. After incubating the WA plates at 25°C for one day, germinated conidia on WA were observed under a stereo microscope (SMZ 1780; Nikon, Tokyo, Japan) and transferred to new WA plates. The single-conidium isolates grown in the WA plates at 25°C for 3 days were transferred to potato dextrose agar (PDA) slants and cultured at 25°C. Among the 21 isolates obtained from the diseased fruits, seven were selected in consideration of the location and the timing of the disease outbreak (Table 1), and used for identification.

The seven isolates were cultured on PDA in 9-cm-diameter Petri dishes at 25°C in the dark for 30 days, and conidia produced in the PDA cultures were investigated for their morphological characteristics using a light microscope (Eclipse Ci-L; Nikon). The 50 conidia of each isolate produced in the PDA cultures were examined for their shape and size by using a light microscope. A conidial suspension (1-3×10⁶ conidia/ml) of each isolate was prepared from the PDA culture. The 20 µl conidial suspension was dropped onto WA, and a sterile cover glass (CG) was placed on it. The WA-CG-culture plates were incubated at 25°C for 2 days, and then the morphology of 50 appressoria from each isolate was examined using a light microscope.

The seven isolates were used for phylogenetic analysis. Genomic DNA of the isolates was extracted using the methods in a previous study (Kim et al., 2024). Amplification of the ribosomal internal transcribed spacer (ITS), actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2), and Apn2-Mat1-2 intergenic spacer and partial mating type (ApMat) gene regions of the isolates was conducted using the respective primer pairs (Table 2). The polymerase chain reaction (PCR) products were prepared with DNA Free-Multiplex Master Mix (Cellsafe, Yongin, Korea) and the Universal DNA Purification Kit (Tiangen, Beijing, China), following the manufacturers’ protocols. The PCR products were sequenced at Bionics Co. (Seoul, Korea) using the same primers.

Sequences of the isolates from papaya fruit and other relevant Colletotrichum species from Genbank were refined using MEGA version 7 (Kumar et al., 2016). Maximum-likelihood analysis for the concatenated alignments of the ITS, ACT, GAPDH, TUB2, and ApMat genes was conducted using a general time-reversible+GAMMA model with 1,000 bootstrap replicates, performed by MEGA version 7 software. Colletotrichum boninense CBS 123755 and Colletotrichum acutatum CBS 112996 were used as outgroup taxa.

Two isolates of each Colletotrichum species identified through phylogenetic analysis were tested for their pathogenicity on papaya fruit. Healthy mature papaya fruits in length of 14-19 cm were surface-sterilized using 1% sodium hypochlorite for 5 min and then washed with sterile distilled water. The papaya fruits were drained with paper towels and placed in plastic boxes (50×36×25 cm). One fruit per isolate was used for inoculation test. Conidial suspension (1-3×10⁶ conidia/ml) prepared from the 30-day-old PDA cultures was used for inoculation tests. The 30 μl of each conidial suspension was dropped in three points 4 cm apart on a fruit for non-wound inoculation. For wound inoculation, the conidial suspension was dropped in the same way as the non-wound inoculation on the fruit stabbed to a depth of 2-3 mm with a sterile needle. Control fruits were treated with the same amount of sterile water by the same inoculation methods. The plastic boxes containing the inoculated fruits were covered and placed in a culture room under 60-80% relative humidity at 24-26°C. The result of inoculation tests was investigated based on formation of lesions on the fruits at 7 days after inoculation. The inoculation test was conducted thrice.

In 2020 and 2021, we encountered outbreaks of anthracnose symptoms on fruit of papaya trees cultivated in greenhouses at locations Taean and Gokseong, Korea, during surveys of crop diseases. The initial symptoms on the fruit appeared as small, water-soaked, slightly depressed, circular or oval dark brown spots. As the disease progressed, the symptoms turned sunken, dark brown to black, and enlarged (Fig. 1A-C). In the later stages, yellowish conidial masses formed on the lesions. The disease on the fruits occurred up to 5% in the investigated greenhouses at the two locations (Table 3).

The seven isolates from papaya fruits were identified by phylogenetic analysis (Fig. 2). The concatenated alignments of the isolates and relevant Colletotrichum spp. contained a total of 2,799 characters (563, 255, 295, 503, and 1,183 characters for ITS, ACT, GAPDH, TUB2, and ApMat, respectively). The first group comprising two isolates (CPCO-2001 and CPCO-2004) clustered with Colletotrichum aenigma ICMP 18608. The second group comprising five isolates (CPCO-2007, CPCO-2011, CPCO-2013, CPCO-2101, and CPCO-2103) clustered with the strains ICMP 18581 and ICMP 18646 of Colletotrichum fructicola. Therefore, the two groups of isolates were identified as C. aenigma and C. fructicola, respectively, with significant bootstrap values of more than 95%. The sequence data of ITS, ACT, GAPDH, TUB2, and ApMat genes obtained from the isolates of C. aenigma and C. fructicola were deposited in GenBank with accession numbers (Table 4).

The morphological characteristics of the two Colletotrichum spp. isolates were generally consistent with those reported in previous studies (Choi and Park, 2021; Lee et al., 2021; Prihastuti, et al., 2009; Weir et al., 2012), although they differed slightly in conidial size (Table 5). The conidia of C. aenigma isolates were cylindrical with round ends, sometimes slightly curved (Fig. 3A) and measured 10.8-20.7×3.0-6.8 μm (average 15.5×5.3 μm). The appressoria of C. aenigma isolates were subglobose and lobate, brown to dark brown (Fig. 3B) and measured 7.0-17.2×4.4-9.8 μm (average 11.0×7.5 μm). The conidia of C. fructicola isolates were cylindrical with obtuse or slightly round ends, sometimes slightly curved (Fig. 3C) and measured 13.3-21.0×4.6-6.3 μm (average 16.0×5.4 μm). The appressoria of C. fructicola isolates were ovoid, clavate and slightly irregular, brown to dark brown (Fig. 3D) and measured 6.0-13.0×5.0-9.0 μm (average 9.5×6.6 μm).

All tested isolates of C. aenigma and C. fructicola induced anthracnose symptoms on papaya fruits by non-wound inoculation as well as wound inoculation (Table 6). In general, the anthracnose symptoms were more severely induced by wound inoculation than by non-wound inoculation. The symptoms on the fruits in non-wound inoculation tests with the two Colletotrichum spp. were similar to those observed on the fruits in the investigated greenhouses (Fig. 1D, E). No symptoms were observed on the control fruits (Fig. 1F). Inoculated isolates of the two Colletotrichum spp. were re-isolated from the lesions induced by the inoculation tests and confirmed as morphologically identical to the original isolates.

Colletotrichum spp. are known to cause anthracnose in numerous plants (Sutton, 1992; Von Arx, 1970). It has been reported that Colletotrichum gloeosporioides causes anthracnose in papaya fruit (Cooke et al., 2009; Persley and Ploetz, 2003; Ploetz et al., 2008; Tan et al., 2023). Colletotrichum chrysophilum has also been reported as a pathogen causing papaya anthracnose in Mexico (Pacheco-Esteva et al., 2022). C. gloeosporioides was recognized as a species complex and divided into many species on the basis of multi-gene phylogenies (Weir et al., 2012).

In the present study, Colletotrichum sp. isolates causing anthracnose in papaya fruit were identified as C. aenigma and C. fructicola through phylogenetic analysis and morphological characteristics. C. aenigma and C. fructicola were newly named based on phylogenetic analysis and have been reported to cause anthracnose in various crops worldwide (Prihastuti et al., 2009; Weir et al., 2012). However, there have been no reports on occurrence of papaya anthracnose caused by the two Colletotrichum spp. In Korea, it has been reported that C. aenigma causes anthracnose in apple, grape, and orange stonecrop, and C. fructicola in apple, grape, mango, peach, pear, and spotted laurel (Korean Society of Plant Pathology, 2026). This is the first report of C. aenigma and C. fructicola causing papaya anthracnose in Korea.