First Report of Papaya Fruit Anthracnose Caused by Colletotrichum okinawense in Brazil

Papaya anthracnose caused by Colletotrichum spp. is an important disease worldwide, affecting yield and production of papaya fruits (Carica papaya L.). In Brazil, Colletotrichum sp. is mainly a postharvest pathogen, which severely affects the marketability of several fruits. Five papaya fruits with sunken, dark brown lesions covered with salmon-colored spore masses, typical of anthracnose symptoms, were collected from a fresh produce market in the city of São Luís/Maranhão from January to February 2018. The disease incidence was estimated at around 60%. These fruits were surface disinfected with 1.5% NaOCl and allowed to dry on a filter paper for 30 min. The symptomatic tissues were excised into small pieces (4 to 5 mm) and placed on potato dextrose agar (PDA) medium. Six fungal strains from six diseased papaya fruits were cultured. Fungal strains were grown on PDA at 25 ± 2°C with a 12-h photoperiod, and six single-spore strains were obtained. All isolates showed similar morphological characteristics, and the representative strain SB-8 was further investigated. On oatmeal agar medium, 10-day-old colonies were flat with entire margin, surface saffron to salmon, aerial mycelium lacking, reverse salmon; 29 to 30.2 mm of mycelial growth in 7 days (≥40 mm in 10 days); and conidial mass were saffron. On Spezieller Nahrstoffarmer agar medium, the conidia were hyaline, smooth-walled, septate, straight, clavate to cylindrical, the apex rounded, the base round or truncate. The 95% confidence intervals for conidia dimensions were (12.1) 13.9 to 14.7 (16.1) µm (extremes given in parentheses). The mean ± SD were 14.3 ± 1.1 × 4.3 ± 0.2 µm, length/width ratio = 3.3; n = 30. DNA was extracted from SB-8 strain and used for amplification of partial sequences of the internal transcribed spacer (ITS), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and histone 3 (HIS3) genes (GenBank accession nos. MK830706 [ITS], MK820658 [GAPDH], MK820659 [ACT], and MK820660 [HIS3]). A BLAST search of the NCBI database revealed that SB-8 gene sequences displayed 100% nucleotide identity to the respective gene sequences of Colletotrichum okinawense Damm & Toy. Sato MAFF240517 (MG600971, MG600827, and MG600971). Maximum parsimony analysis was done using recently published datasets (Damm et al. 2019), and the strain clustered with the type strain sequence of C. okinawense. Based on morphological characteristics and phylogenetic analysis, the SB-8 strain was identified as C. okinawense. Koch’s postulates were completed using five healthy papaya fruits (cv. Golden), which were disinfected with 300 ppm NaOCl (30 min). Two points were selected for each fruit, and wounds were made at the points with a sterile needle to a depth of 3 mm. Mycelial plugs taken from the margin of actively growing colonies (PDA) of each strain were placed in shallow wounds. PDA discs without fungal growth were used as control. Inoculated fruits were maintained in a humid chamber for 48 h at 24 ± 2°C with a 12-h photoperiod. The first symptoms appeared 2 days after inoculation, and at the end of the test, the symptoms were similar to those observed initially in the fresh produce market. No symptom was observed on fruit inoculated with PDA discs without fungal growth. C. okinawense was successfully reisolated from symptomatic fruits and identified as previously described, thus fulfilling Koch’s postulates. The experiment was repeated once showing similar results. Recently, Damm et al. (2019) mentioned several strains previously identified as C. brevisporum causing anthracnose in papaya in Brazil probably belong to C. okinawense based on high identity for ACT gene. Based on multilocus phylogeny concatenated four genes (ITS, GAPDH, ACT, and HIS3) we confirm the presence of C. okinawense causing papaya fruit anthracnose in Brazil.