First Report of Chili Pepper Charcoal Rot Caused by Macrophomina phaseolina in Korea

Chili pepper (Capsicum annuum L.) is a major vegetable crop in Korea. In August 2018 and July 2025, wilting was observed in chili pepper fields in Jeongeup (35.56°N, 126.86°E) and Suncheon (35.00°N, 127.51°E), Republic of Korea. The areas of the observed fields were 0.2 ha and 0.3 ha, respectively, and disease incidence ranged from approximately 5% and reached up to 30% in affected fields. To identify the causal agent, symptomatic plants were randomly selected from each field. Basal stem tissues from the symptomatic plants were surface-disinfested in 1% sodium hypochlorite for 1 min, rinsed three times with sterile distilled water, and a total of 21 segments (seven per plant) were plated onto water agar (WA). Plates were incubated at 25°C for 5 days, and single-hyphal-tip cultures were obtained. Fungal isolates were obtained from all 21 segments, and three representative isolates (NC18-688, NC18-689, and P017) were selected for further characterization. For cultural characterization, 5-mm-diam. mycelial plugs were placed on potato dextrose agar (PDA) and incubated at 25°C for 3 days. Colonies were dark brown to dark olive and darkened with age, reaching 77 ± 7 mm in diameter. Although the overall colony morphology was consistent with M. phaseolina, variations among the three isolates were observed. Isolates NC18-688 and P017 exhibited rapid and abundant microsclerotia formation with minimal mycelial growth. In contrast, isolate NC18-689 produced relatively abundant mycelium but showed the slowest rate of microsclerotia formation. For morphological characterization, 5-mm-diam. plugs were transferred to oatmeal agar (OMA) and incubated at 28°C in the dark for 24 h, followed by incubation under UV light at 28°C for 5 days (Zhao et al. 2020). Microsclerotia were black, smooth, round to oblong, and 38.6–79.6 µm in diameter (n = 50). Pycnidia formed on OMA but not on PDA or WA; they were black, thin-walled, globose, with 0–1 ostiole, and 139.7–234.2 µm in diameter. Conidiogenous cells and conidia were observed from ruptured pycnidia. Conidiogenous cells were hyaline, thin-walled, cylindrical to lageniform, producing conidia at the apex. Conidia were hyaline, smooth-walled, aseptate, granular, ellipsoid to obovoid, and 16.5–22.1 × 6.1–9.7 µm. These characteristics were consistent with descriptions of Macrophomina phaseolina (Huda-Shakirah et al. 2019). Molecular identification was performed by PCR amplification and sequencing of the ITS, EF1-α, and β-tubulin (TUB) regions using primers ITS1/ITS4 (White et al. 1990), EF-728F/EF-986R (Carbone and Kohn 1999), and Bt2a/Bt2b (Glass and Donaldson 1995), respectively. Sequences were deposited in GenBank (accession nos. PX775556–PX775558 [ITS], PX833991–PX833993 [EF1-α], and PX833994–PX833996 [TUB]). Concatenated ITS, EF1-α, and TUB sequences were used for phylogenetic analysis. Bayesian inference (BI) analysis was performed using MrBayes v3.2.7. The GTR+I+G (General Time Reversible with proportion of invariable sites and gamma-distributed rate variation) model was selected as the best-fit nucleotide substitution model, with parameters set to Nst = 6 and Rates = Invgamma. The Metropolis-coupled Markov chain Monte Carlo (MCMCMC) algorithm was run with four chains for 100,000 generations, and trees were sampled every 1,000 generations. The first 25% of the sampled trees was discarded as burn-in, and the remaining trees were used to construct a consensus tree. Although the three isolates shared 100% sequence identity in both the ITS (377 bp) and TUB (405 bp) regions, minor nucleotide variations were detected in the EF1-α region (9 polymorphic sites out of 245 bp; 96.33% identity). Overall, the isolates displayed a high sequence similarity of 99.12% across the concatenated regions (1,018/1,027 bp), reflecting subtle intraspecific genetic diversity. Bayesian inference placed the three isolates in a clade with M. phaseolina reference isolates CBS 162.25, CBS 205.47, and CBS 227.33. Pathogenicity was tested by soil infestation using sterilized sorghum grain inoculum. Sterilized grains were inoculated with ten 5-mm-diam. PDA plugs from actively growing cultures; control grains received ten sterile PDA plugs. After incubation at 25°C in the dark for 14 days, colonized sorghum was mixed with potting soil at 5% (v/v). Three-week-old chili pepper seedlings were transplanted into infested soil (five replicate experiments). Wilting developed approximately 2 weeks after transplanting in inoculated plants but not in controls. Among the three isolates, NC18-688 showed the highest virulence in pathogenicity assays. The pathogen was re-isolated from symptomatic tissues and identified as M. phaseolina, fulfilling Koch’s postulates. However, the pathogen was not recovered from any control plants. To our knowledge, this is the first report of charcoal rot of chili pepper caused by M. phaseolina in Korea. Given that warm and dry conditions favor charcoal rot, continued monitoring is needed as climate change may increase disease risk in chili pepper–growing regions of Korea.