Papaya (Carica papaya) is a tree belonging to the family Caricaceae and grows primarily in the wet tropical biome. The native range of the tree is South Mexico to Venezuela (Plants of the World Online, 2024). The tree fruit is mainly used for food, medicine, and cooking in the production area. In the southern regions of Korea, farmers cultivate it as a fruit tree in vinyl greenhouses.

Fruit rot symptoms were observed in papaya trees grown in a greenhouse in Gokseong, Korea, during disease surveys in 2021 and 2022. The rot symptoms initially occurred at the tip of fruit and later developed into the inside of fruit, with white hyphae growing in the lesions (Fig. 1A, B). In the later stages, yellowish conidial masses were formed on the hyphae in the lesions (Fig. 1C). The incidence of diseased papaya fruits in the investigated greenhouse was 0.5-1.0%.

Papaya fruits with rot symptoms were collected during the disease surveys to isolate fungal pathogens. A conidial suspension was prepared from conidial mass on the diseased fruit using sterile distilled water and streaked on 2% water agar (WA) plates with a sterile loop. The WA plates were incubated at 25^°C for 1 day. Germinated conidia on WA observed under a dissecting microscope (Nikon SMZ 1780; Nikon, Tokyo, Japan) were transferred to new WA plates and incubated at 25^°C for 4 days. Six single-conidium isolates were obtained from the WA cultures and used for identification and pathogenicity tests. The morphological characteristics of the isolates cultured on potato dextrose agar (PDA) in 9 cm diameter Petri dishes for 20-25 days were investigated using a compound microscope (Nikon Eclipse Ci-L; Nikon). The investigation results revealed that the isolates belonged to the genus Fusarium (Booth, 1971).

Phylogenetic analysis was performed to identify clades of the Fusarium sp. isolates. Genomic DNA of the isolates was extracted using a slight modified method from a previous study (Lee et al., 2023). Amplification of translation elongation factor 1-α (TEF1) and RNA polymerase II second largest subunit (RPB2) gene regions of the isolates was conducted using primer sets of EF-1 and EF-2 for TEF1 (O'Donnell et al., 1998), and 5F2-7cR and 7cF-11aR for RPB2 (O'Donnell et al., 2007), along with DNA Free-Multiplex Master Mix (Cellsafe, Yongin, Korea), following amplification protocols from previous studies (O'Donnell et al., 2004, 2007). The polymerase chain reaction products were purified using ExoSAP-IT™ (Applied Biosystems, Waltham, MA, USA) and sequenced at Bionics (Seoul, Korea) using the same primers. Adjustment of the sequences was carried out as needed using SeqMan II (DNASTAR, Madison, WI, USA). Sequence alignment of the isolates and other relevant Fusarium species from Genbank was performed and refined with MEGA version 7 (Kumar et al., 2016), if necessary. Maximum likelihood estimation for the concatenated alignments was conducted using a general time-reversible model with 1,000 bootstrap replicates, performed by MEGA version 7 software (Kumar et al., 2016). Geejayessia atrofusca (NRRL 22316) was set as an outgroup taxon. The phylogenetic analysis showed that the isolates were divided into two groups of Fusarium spp. The first group consisting of two isolates (CPFU-01 and CPFU-07) clustered with a reference strain F. bostrycoides NRRL 31169 (Fig. 2). The second group consisting of four isolates (CPFU-04, CPFU-10, CPFU-13, and CPFU-15) clustered with each other but not with any reported Fusarium spp., suggesting that they might be a novel species. The sequence data of TEF1 and RPB2 obtained from the two isolates of F. bostrycoides in the first group were deposited in GenBank under the accession numbers PQ303562-PQ303563 and PQ368281-PQ368282, respectively. The sequence data of TEF1 and RPB2 obtained from the four isolates of Fusarium sp. in the second group were deposited in GenBank under the accession numbers PQ368283-PQ368286 and PQ303564-PQ303567, respectively.

The six isolates of Fusarium spp. were used for pathogenicity tests to papaya fruits. Each isolate was cultured on PDA at 25^°C in the dark for 20-30 days, and a conidial suspension (3-5×10⁶ conidia/ml) was prepared from the PDA cultures using sterile distilled water. For inoculation tests, immature papaya fruits in length of 7-12 cm were immersed in 1% sodium hypochlorite solution for 5 min for surface sterilization and then rinsed with sterile distilled water. The papaya fruits were placed in humid plastic boxes (29.0×22.5×12.0 cm) after removing moisture, and 10 μl of each conidial suspension was dropped on a fruit tip. For wound inoculation, the central portion of the conidial suspension droplet on the fruit tip was stabbed to a depth of 2-3 mm using a needle. The same amount of sterile distilled water was used to control papaya fruits. The plastic boxes containing the inoculated papaya fruits were placed in a room at 24-26°C. After 10 days, lesion formation on the fruits was investigated. The inoculation test was repeated three times.

The pathogenicity tests revealed that all tested isolates of Fusarium spp. caused fruit rot symptoms on papaya fruits in wound or non-wound inoculation (Table 1). The symptoms on the fruits induced by pathogenicity tests with the isolates were similar to those observed on fruits from the investigated greenhouse (Fig. 1D,E). No symptoms were observed on the control fruits (Fig. 1F). Fusarium spp. were re-isolated from the lesions formed in the inoculation tests, and the isolated Fusarium spp. were found to be morphologically identical to the inoculated species. The morphology of 25 conidia and 20 phialides of each isolate produced on the lesions were examined. The morphological characteristics of the two isolates (CPFU-01 and CPFU-07) belonging to F. bostrycoides and four isolates (CPFU-04, CPFU-10, CPFU-13, and CPFU-15) of Fuarium sp. in phylogenetic analysis were shown in Table 2 and Fig. 3. The morphological features of the F. bostrycoides isolates from papaya fruits were similar to those of F. bostrycoides described in a previous study (Sandoval-Denis et al., 2019). However, the morphological features of Fusarium sp. isolates from papaya fruits were inconsistent with those of other reported Fusarium spp. Therefore, further taxonomic studies of this unknown species is needed to be carried out.

It has been reported that Fusarium solani, Fusarium moniliforme, and Fusarium equiseti cause papaya fruit rot in foreign countries (Persley and Ploetz, 2003). In Korea, no disease occurrence has been reported except for powdery mildew in papaya (Korean Society of Plant Pathology, 2024). F. bostrycoides has been reported to cause wilt in yellow passion fruit (Ninos et al., 2021). However, there have been no reports on disease occurrence caused by the pathogen in papaya. This is the first report of F. bostrycoides and a new type of Fusarium sp. causing fruit rot in papaya.