Watermelon (Citrullus lanatus), it's an important fruit in Brazil, producing 1.9 million ton/year, occupies the fifth place in the world, (FAO, 2022), but post-harvest diseases are a major limitation, leading to losses of up to 15% (Balasubramaniam et al. 2023). In 2022, 65 fresh and healthy watermelons cv. Harmonium (Basf®) were collected from a field in Rio Grande do Norte state (4°54'25.0"S 37°24'03.0"W), Brazil. The fruits were sanitized with 2.0% sodium hypochlorite for 3 min, rinsed with water and then, placed in a humid chamber to induce fungal growth. After one week, 51% showed lesions and mycelial growth, mainly in the peduncular area. Subsequently, about 132 small pieces (5 × 5 mm) of diseased tissues were placed on potato dextrose agar (PDA) and incubated at 25 ± 1 °C for 7 days. 19 single-spores isolates were obtained, which presented abundant colonies of aerial mycelium, with coloration ranging from white to dark orange, with the presence of cylindrical, slightly curved macroconidia and rounded, aseptate apices, with average length and width of 12.40 ± 1.99 μm (8.29 to 15.73 μm) and 3,11 ± 0.68 μm (2.26 to 4.00 μm), respectively (n = 50). Microconidia were oval to fusiform, aseptate with 2 septa, with average length and width of 3.90 ± 1.03 μm (1.94 to 7.08 μm) and 1.47 ± 0.35 μm (0.64 to 2.31 μm), respectively (n = 50). The presence of monophialides and polyphialides was observed, with the presence of terminal and intercalary chlamydospores. Based on these morphological features, the isolates were identified as Fusarium spp. (O'Donnell et al., 2020). Three isolates (CFC1756, CFC1759 and CFC1761) were selected for further molecular analysis. Partial gene sequences for the translation elongation factor 1- (TEF-1) and the second-largest subunit of RNA polymerase II (RPB2) were amplified using the primer pairs EF1/EF2 (O'Donnell et al. 1998) and RPB2-5F/RPB2-7cR (Sung et al. 2007; Liu et al., 1999), respectively. A BLAST search showed that TEF-1 sequences had 99.54% similarity to Fusarium isolate, accession number MK752504, and the RPB2 sequences matched 100% similarity to accession number KC808359, identified as F. falciforme. Maximum likelihood analysis based on the combined gene sequences was performed using IQ-TREE (Nguyen et al. 2015), with simultaneous inference of the best substitution models for each gene (TIMe+G4 for TEF-1α and TNe+I for RPB2), according to the Bayesian Information Criterion (BIC). Nodes with support values ≥80% were considered well supported (Minh et al. 2013). The phylogenetic tree showed that isolates CFC1756, CFC1759, and CFC1761 clustered with F. falciforme isolate CBS 475.67 (O'Donnell et al. 2020), in a clade with 89.3% support. The sequences were deposited in GenBank (for TEF: PQ217626, PQ217627, PQ217628; and for RPB2:PQ200854, PQ200855, PQ200856) for CFC1756, CFC1759 and CFC1761, respectively. Koch's postulate were assessed on healthy watermelons cv. Harmonium. The fruits were sanitized with 2.0% sodium hypochlorite for 3 min, rinsed with water and then, wounded with 2 mm depth the watermelons with a sterile awl, mycelium disks from 7-day-old plates of similar diameter were placed face down on the 8 watermelons (8 replicates for each isolate). In control watermelons, the fruits were inoculated using only disks containing PDA. After 7 days of inoculation, at room temperature of 25 ± 2 °C and in a humid chamber, white mycelium was produced from the inoculation points, followed by soft and sunken rot. The pathogens were reisolated and presented the same morphology as the inoculated isolates; the control fruits did not present symptoms. This experiment was conducted twice, and similar results were obtained. Fusarium falciforme has been reported as causing various diseases on different hosts in several countries, including Korea (Kang et al., 2024), Malaysia (Balasubramaniam et al. 2023), and Mexico (Payán-Arzapalo et al., 2024). To our knowledge, this is the first report of F. falciforme causing postharvest rot in watermelons in Brazil and this information is important for the management of strategies to control the disease.
Keywords: fruits; pathogenicity; postharvest disease.