First report of Cercospora citrullina causing leaf spot on Momordica charantia in Mexico
TL;DR
To determine the species, the four isolates were molecularly identified by amplifying and sequencing the internal transcribed spacer (ITS) region and the translation elongation factor 1‑α (EF‑1α) using primers ITS1/ITS4 and EF1‑728F/EF1‑986R, respectively (White et al. 1990).
Abstract
In March 2025, leaf spot symptoms were detected in approximately 50% of five‑month‑old Momordica charantia plants in a 1.5‑ha commercial field located in Culiacán, Sinaloa, Mexico (24°14'26.8"N, 107°11'43.2"W). Affected plants exhibited circular to irregular leaf spots lesions with grayish centers and brown to reddish margins, along with chlorosis and in advanced stages, mild defoliation. Forty symptomatic leaf samples were randomly collected for pathogen isolation. Samples were surface‑disinfected with 1% NaClO and rinsed with sterile distilled water. After drying, five tissue fragments were placed per Petri dish containing PDA supplemented with streptomycin sulfate (0.3 g L⁻¹) and incubated at 25 °C for 7 days. Among eight isolates obtained, four isolates were selected for identification: CERCCH1CULSIN, CERCCH2CULSIN, CERCCH3CULSIN, and CERCCH4CULSIN. Microscopic examination showed smooth, branched, brown and hyphae. Conidiophores were fascicles, brown, straight and unbranched, measuring 18.45–295.60 × 3.20–6.55 μm (n = 10). Conidia were hyaline, straight or slightly curved, and acicular to clavate, measuring 20.10–228.75 × 3.05–6.90 μm (n = 10). Based on these traits, the isolates were preliminarily identified as Cercospora spp. (Chupp 1954). To determine the species, the four isolates were molecularly identified by amplifying and sequencing the internal transcribed spacer (ITS) region and the translation elongation factor 1‑α (EF‑1α) using primers ITS1/ITS4 and EF1‑728F/EF1‑986R, respectively (White et al. 1990; Carbone and Kohn 1999). Sequences were deposited in GenBank under codes PQ670974, PV628715, PV628716, and PV628717 for ITS, and PQ671957, PV651779, PV651780, and PV651781 for EF‑1α, corresponding to isolates CERCCH1CULSIN, CERCCH2CULSIN, CERCCH3CULSIN, and CERCCH4CULSIN. BLAST analysis showed that ITS sequences shared about 99.8% identity with the reference C. citrullina sequence ON849061.1 (PQ670974.1, 476/477 pb; PV628715.1, 480/481 pb; PV628716.1, 479/481 pb; PV628717.1, 472/473 pb), while EF‑1α sequences showed 100% with ON890306.1 (PQ671957.1, 246/246 pb; PV651779.1, 249/249 pb; PV651780.1, 253/253 pb; PV651781.1. 253/253 pb). A Phylogenetic analysis using the Neighbor‑Joining method with 1000 bootstrap replicates grouped the isolates within C. citrullina clade. Pathogenicity of each of the four isolates was confirmed through foliar spray inoculation. Spore suspensions (1.0 × 10⁴ conidia mL⁻¹) of each isolate were applied to 15 healthy, 30‑day‑old M. charantia plants. An additional set of 15 plants was sprayed with sterile water to serve as the control. All plants were maintained at 25 ± 2 °C, 90% RH, and a 12:12 h photoperiod. Eight to ten days after inoculation, leaf spot symptoms developed and were like those observed in the field for all four isolates, whereas no symptoms were observed in the control plants. Cercospora citrullina was successfully reisolated and identified from symptomatic foliar tissue of each isolate, thereby fulfilling Koch’s postulates. This fungus has previously been reported as a causal agent of foliar disease in M. charantia (Hsieh and Goh 1990; Gautam et al. 2020); however, no prior reports exist for this pathogen on this host in Mexico. Therefore, this study constitutes the first report of C. citrullina causing leaf spot on M. charantia in Mexico, and this finding will support future research aimed at developing integrated disease management strategies for this disease.