First Report of <i>Fusarium triseptatum</i> Causing Fruit Rot Disease in Dragon Fruit in Ecuador

Dragon Fruit (Selenicereus undatus) production has recently emerged along Ecuador's coastal region. In 2022, in Manabí province (Montecristi-Manabi, 1°1'38.46"S, 80°42'2.65"W), a postharvest disease was observed, causing sunken, light brown circular lesions at the edges that darken to brown in the center, leading to tissue softening and fruit rot. The causal pathogen was not identified despite the disease's incidence of 25% among 1000 fruit samples. Ten symptomatic fruits were collected for pathogen identification and stored for 21 days at 8 to 12 °C (Fig S1). Peel sections exhibiting disease symptoms were surface-sterilized with 2% sodium hypochlorite and 70% ethanol, rinsed with sterile water, and cultured on potato dextrose agar (PDA) supplemented with gentamicin (160 mg/L). Petri dishes were incubated at 25°C for 5 days. A single hyphal-tip isolation method was employed to isolate five pure isolates after 14 days. These isolates were incubated at 28°C under white light to induce fungal sporulation for two days. Initial fungal colonies displayed fluffy white to pinkish mycelium that later turned purple (Rafique et al. 2015). Microscopic analysis revealed straight primary conidia and hyaline microconidia with oval to reniform (5-10.7 µm length, 1.4-3 µm width), on PDA+G. Based on morphological characteristics, all isolates were preliminarily identified as Fusarium sp. (Dubey et al. 2010; Mahmud et al. 2020; Fig S1). Pathogenicity tests were performed using five isolates. Healthy dragon fruits were surface disinfected with 2% hypochlorite and 70% ethanol, rinsed, inoculated with 10 µl of a conidial suspension (1×10⁶ conidia per ml) and placed on unwounded peels. Controls received sterile water. Fruits were incubated in a humid chamber (90% relative humidity). Within 10 days, inoculated fruits developed brown circular lesions, whereas controls remained symptom-free. The pathogen was successfully reisolated and showed identical morphology to the original isolates. Pathogenicity assays were repeated three times with consistent results. Infection tests of these isolates were performed in stems, obtaining no infection. All isolates were selected for molecular identification. DNA was extracted, and the translation elongation factor 1-alpha (TEF1-α) and RNA polymerase II second largest subunit (RPB2) gene regions were amplified with the primers EF-1/EF-2 and fRPB2-5F/fRPB2-7cR accordingly (Liu et al. 1999; O´Donnell et al. 1998). Sequences were aligned for both markers using Clustal X in MEGA X (Kumar et al. 2018), revealing 100% identity among sequenced isolates. Consensus sequences of TEF1-α and RPB2 (GenBank TEF1-α : PQ434664, RPB2: PQ434663) were more than 99,0 % identical to isolate PQ260732 and PP682396, both showing Fusarium identity. A Bayesian phylogenetic tree of concatenated TEF1-α and RPB2 was built using BEASTv1.10.4 (Drummond et al. 2012), and the analysis showed that the consensus sequence clustered with Fusarium triseptatum (Fig S2). This study represents the first report of F. triseptatum as the causal agent of postharvest fruit rot in S. undatus in Ecuador. These findings provide critical information for disease management and emphasize the need for further research to develop postharvest control strategies.