Shifting Populations and Increasing Diversity of Colletotrichum Species Causing Pepper Anthracnose Under Rising Temperatures and Fungicide Selection Pressure
Article information
Abstract
Pepper anthracnose, a devastating and economically important disease in Korea, is caused by diverse Colletotrichum species that differ significantly in their pathogenicity and environmental adaptation. In a three-year study from 2022 to 2024, pathogens were isolated and species characteristics were investigated. Using multi-locus phylogenetic analysis and species-specific PCR, seven species were identified: C. scovillei, C. nymphaeae, and C. fioriniae in the C. acutatum species complex; C. fructicola, C. aenigma, and C. gloeosporioides in the C. gloeosporioides species complex; and C. truncatum. C. scovillei remained dominant, but species diversity increased in 2024, with non-dominant species such as C. fructicola and C. truncatum increasing in specific regions. Temperature-dependent assays revealed distinct ecological niches, showing that while C. scovillei prefers a moderate 25°C, high temperature-adapted species like C. fructicola and C. truncatum exhibit enhanced growth and pathogenicity at 30°C. Fungicide sensitivity tests showed that tebuconazole and fluazinam remained effective against all species of Colletotrichum, whereas pyraclostrobin (QoI) resistance was widespread. The mycelial growth inhibition of C. scovillei by pyraclostrobin ranged from 29.5% to 43.3% during 2022–2024, indicating reduced sensitivity. All C. truncatum isolates were resistant, consistently associated with the G143A mutation in the cytb gene. These findings indicate that climate warming and fungicide selection pressure are driving shifts toward more resilient populations, highlighting the need for climate-adaptive disease management through monitoring and fungicide rotation.
Pepper anthracnose is a major pepper disease caused by fungi of the genus Colletotrichum and occurs annually in pepper-growing regions in Korea, resulting in significant losses in yield and fruit quality (Cannon et al., 2012; Dean et al., 2012; Kim et al., 2015; Sawant et al., 2023; Than et al., 2008b). Disease incidence generally increases under warm and humid conditions, and disease development often coincides with the rainy season characterized by frequent rainfall and strong winds, which makes effective disease control difficult. In addition, the species composition of the causal pathogens may vary even within the same field (Shin et al., 2025), leading to variation in disease development and control efficacy.
The pathogens causing pepper anthracnose consist of multiple Colletotrichum species that differ in host range, pathogenicity, and physiological characteristics, and the coexistence of multiple species within a single crop field has been widely reported (De Silva et al., 2017; Mongkolporn and Taylor, 2018; Noor and Zakaria, 2018; Oo et al., 2017). Such interspecific differences may result in variation in fungicide sensitivity and resistance development, as well as differential adaptation to environmental conditions, ultimately leading to unstable disease control in the field.
Historically, C. gloeosporioides was regarded as the primary causal agent of pepper anthracnose in Korea based on morphological characteristics (Park and Kim, 1992). However, morphology-based identification is often unreliable due to overlapping characteristics among species and substantial intraspecific variation (De Silva et al., 2017; Kim et al., 2008). With the introduction of molecular approaches, multi-locus phylogenetic analysis has enabled the reclassification of species previously regarded as single taxa. As a result, the C. acutatum species complex and the C. gloeosporioides species complex were resolved into multiple distinct species (Cannon et al., 2012; Damm et al., 2012). In particular, the C. acutatum species complex was subdivided into C. scovillei, C. nymphaeae, and C. fioriniae (Damm et al., 2012; Oo et al., 2017), while the C. gloeosporioides species complex was re-identified as C. fructicola, C. aenigma, C. siamense, C. gloeosporioides, and related species (Damm et al., 2012; Oliveira et al., 2022; Zakaria, 2021). These advances in classification have enabled accurate discrimination among morphologically similar species and provided a basis for comparative studies of species-specific pathogenicity and physiological traits. In Korea, molecular marker-based studies have been actively conducted since the 2010s, leading to the re-identification of C. scovillei as the predominant pathogen of pepper anthracnose in the field (Oo et al., 2017). However, most previous studies focused on species identification within a single year or limited geographic regions, and long-term, nationwide analyses linking changes in species composition to recent environmental conditions remain limited.
Disease development of Colletotrichum species is strongly influenced by environmental factors such as temperature and humidity (Saxena et al., 2016). Most Colletotrichum species exhibit optimal mycelial growth, conidial germination, and pathogenicity at temperatures ranging from 25 to 28°C. In contrast, C. truncatum has been reported as a heat-adapted species capable of maintaining high growth rates and pathogenicity even at 35°C, whereas C. scovillei, C. nymphaeae, and C. fioriniae show a sharp decline in pathogenicity at temperatures above 30°C (Salotti et al., 2022). These differences in temperature adaptation may drive shifts in species composition under changing environmental conditions and contribute to instability in disease control.
Control of pepper anthracnose primarily relies on chemical methods due to their high cost-effectiveness and field applicability (Saxena et al., 2016). In particular, quinone outside inhibitor (QoI) fungicides are single-site action fungicides that block electron transport by targeting cytochrome b at complex III of the mitochondrial electron transport chain, exhibiting high control efficacy against various crop diseases (Baumler et al., 2003; Ishii et al., 2001; Kim et al., 2003; Ma et al., 2003; Sierotzki et al., 2000). However, this single-site mechanism of action is limited by a high risk of resistance development. Indeed, numerous QoI-resistant isolates have been reported within the genus Colletotrichum (Adaskaveg et al., 2025; Kongtragoul et al., 2020), and pyraclostrobin-resistant isolates have also been identified in South Korea (Kim et al., 2019). Molecular biological methods are mainly utilized for the diagnosis of QoI resistance, where the detection of point mutations (such as G143A, F129L, and G137R) occurring at the Qo binding site of the cytochrome b (cytb) gene serves as a key indicator (Gisi et al., 2002; Grasso et al., 2006). These findings suggest that the repeated use of fungicides within the same class can accelerate the spread of resistant populations, emphasizing the importance of resistance management through systematic sensitivity monitoring.
In this study, we investigated changes in the species composition of Colletotrichum populations isolated from major pepper-growing regions in Korea from 2022 to 2024. We compared the pathogenicity and physiological characteristics of major species. Using multi-locus phylogenetic analysis and species-specific PCR, we re-identified the distribution of Colletotrichum species in anthracnose-infected pepper fields and compared mycelial growth and pathogenicity on pepper fruits at 25°C and 30°C. In addition, sensitivity to QoI fungicides and the occurrence of QoI resistance were analyzed to elucidate the ecological and pathological factors underlying the recently observed increase in species diversity.
Materials and Methods
Sample collection and fungal isolation
To collect anthracnose pathogens, pepper fruits with typical symptoms of anthracnose and black spots were collected after August in major pepper growing regions in Korea, including Gyeonggi-do, Gangwon-do, Chungcheongbuk-do, Chungcheongnam-do, Gyeongsangbuk-do, and Jeollabuk-do, from 2022 to 2024. Conidia were collected from sporulating lesions using sterile toothpicks and suspended in sterile distilled water supplemented with streptomycin (300 μg/mL). The concentration of conidial suspensions was adjusted to 1 × 104 – 105 conidia/mL using a hemocytometer (Marienfeld Superior, Lauda-Königshofen, Germany). Aliquots (20 μL) of the suspensions were spread onto potato dextrose agar (PDA; Difco™, Becton, Dickinson and Company, Franklin Lakes, NJ, USA) containing streptomycin (300 μg/mL). Plates were incubated at 25°C in the dark for 3 days, and single-spore colonies were isolated. Pure isolates were subcultured on PDA, and 3-mm-diameter mycelial plugs were excised from the actively growing colony margins. For long-term preservation, twelve mycelial plugs were transferred into 2-mL Cryovial® tubes (Simport® Scientific Inc., Saint-Mathieu-de-Beloeil, QC, Canada) containing 1.7 mL of 20% (v/v) glycerol (Merck, Darmstadt, Germany) and stored at −70°C. In addition, PDA slant cultures were prepared in 2-mL Cryovial® tubes, inoculated with 3-mm mycelial plugs, and incubated at 25°C for 3 days. Sterile liquid paraffin was then overlaid onto the colonies, and cultures were stored at room temperature until use.
Isolates used in this study
A total of 1,975 isolates were collected between 2022 and 2024. Among these, 32 representative isolates were selected based on the year of isolation, geographic origin, and colony morphology for re-identification through multi-locus phylogenetic analysis. As detailed in Supplementary Table 1, an expanded set of 69 isolates was utilized to characterize mycological properties, temperature-dependent mycelial growth, and pathogenicity. Furthermore, 168 isolates were selected from the total collection based on their response to pyraclostrobin; these were used for EC50 determination via the agar dilution assay and for detecting point mutations in the cytb gene.
Extraction of gDNA and total RNA, and synthesis of cDNA
To extract gDNA and total RNA, isolates were cultured on PDA at 25°C in the dark for 7 days. Mycelia were harvested and transferred to 2-mL microtubes co ntaining 0.1-mm glass beads, then frozen at −70°C for at least 24 h. Frozen samples were lyophilized using a freeze dryer (FD8508, IlshinBioBase, Dongducheon, Korea) and ground using a Bullet Blender homogenizer (BBY24M, Next Advance, Troy, NY, USA). Genomic DNA was extracted using the ExgeneTM Plant SV Mini Kit (GeneAll Biotechnology, Seoul, Korea), and total RNA was extracted using the Hybrid-RTM kit (GeneAll Biotechnology), according to the manufacturer’s instructions. The concentration and purity of gDNA and total RNA were measured using a microvolume spectrophotometer (OPTIZEN Nano Q, K Lab, Daejeon, Korea), and they were adjusted to a final concentration of 50 ng/μL for subsequent PCR analyses. Total RNA was used for the synthesis of cDNA for isolates belonging to the C. gloeosporioides species complex, because introns in the cyt b gene interfered with direct amplification from genomic DNA. For cDNA synthesis, 20 μL reaction mixtures containing 4 μL Reverse Transcription 5× Master Mix (Elpis Biotech, Daejeon, Korea), 5 μL total RNA, and 11 μL dH2O were prepared. Reverse transcription was performed at 37°C for 1 h, followed by incubation at 94°C for 5 min to synthesize cDNA.
Multi-locus phylogenetic analysis
Multi-locus phylogenetic analysis was conducted to identify Colletotrichum species. A total of 32 representative isolates were selected based on year of isolation, geographic origin, and colony morphology to represent the diversity of Colletotrichum populations collected during the survey period (Supplementary Table 1). The internal transcribed spacer (ITS), chitin synthase 1 (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), actin (ACT), and β-tubulin (TUB2) gene regions were amplified using the primer pairs ITS1/ITS4 (White et al., 1990), CHS79F/CHS354R (Carbone and Kohn, 1999), GDF1/GDR1 (Guerber et al., 2003), ACT512F/ACT783R (Carbone and Kohn, 1999), and T1/Bt2b (Glass and Donaldson, 1995; O’Donnell and Cigelnik, 1997), respectively (Table 1). PCR amplification was performed in 20 μL reaction volumes containing 12 μL dH2O, 4 μL EzPCR HS 5× Master Mix (Elpis Biotech), 1 μL of each primer (10 pmol), and 2 μL of template DNA. Amplification was performed with an initial denaturation at 95°C for 5 min, followed by 30 cycles of denaturation at 95°C for 1 min, annealing at gene-specific temperatures (ITS, 55°C; CHS-1, 58°C; GAPDH, 56°C; ACT and TUB2, 61°C) for 30 s, and extension at 72°C for 1 min, followed by a final extension at 72°C for 10 min. PCR products were separated by electrophoresis on 1% agarose gels in 0.5× Tris-borate-EDTA (TBE) buffer at 100 V for 40 min. Amplified fragments were purified using the ExpinTM PCR SV Mini Kit (GeneAll Biotechnology) and sequenced by Macrogen (Daejeon, Korea). Obtained sequences were edited using MEGA 11 (version 11.0.11), and forward and reverse sequences were assembled to correct ambiguous bases. All sequences generated in this study were deposited in the NCBI GenBank database, and reference sequences were retrieved from GenBank (Supplementary Table 1). Concatenated sequences were used to construct phylogenetic trees using the Maximum Likelihood method implemented in MEGA 11.
Primer used for amplification of ITS, CHS-1, GAPDH, ACT, and TUB2 gene regions
Species identification using species-specific primers
To identify Colletotrichum species isolated from pepper anthracnose lesions, species-specific PCR assays were conducted. Isolates belonging to the C. acutatum species complex and the C. gloeosporioides species complex were initially distinguished using the primer pairs CaSc-Bt498F/CSc-Bt810R and CgSc-Bt675F/CSc-Bt810R, respectively (Abdulkareem, 2025). For species-level identification within the C. acutatum species complex, the primer sets CSC_BT293/T2, CNY_BT270_1/T2, and CFI_BT302(R)/T2 were used to identify C. scovillei, C. nymphaeae, and C. fioriniae, respectively (Seo, 2024). Similarly, the primer pairs Cfr-Bt492F/CgSc-Bt691R, Cae-Bt462F/CgSc-Bt691R, and Cgl-Bt591F/CgSc-Bt691R were used to identify C. fructicola, C. aenigma, and C. gloeosporioides, respectively (Abdullahi and Kim, 2024). In addition, C. truncatum was identified using the primer pair CcapF/CcapR (Torres-Calzada et al., 2011). Primer sequences are listed in Table 2. PCR reactions were performed using the same reaction mixture described above and amplified under the conditions listed in Table 3. Amplified products were separated on 1% agarose gels in 0.5× TBE buffer at 100 V for 40 min and visualized under UV illumination using a gel documentation system (NaBI set, NeoScience, Korea).
Species-specific primers used for identification of Colletotrichum species isolated from pepper
PCR conditions for species-specific identification of Colletotrichum spp.
Cyt b gene amplification
PCR amplification of the cyt b gene was performed in 20 μL reactions containing 12 μL dH2O, 4 μL EzPCR HS 5× Master Mix, 1 μL of each primer (forward: RF-CYT1A, 5′-AYA GAG CTC CWA GAA CWT TAG-3′; reverse: RF-CYT2B, 5′-GAA ACA CCT AAW GGG TTA CTT GA-3′), and 2 μL template DNA. The PCR program consisted of initial denaturation at 94°C for 4 min; 30 cycles of 98°C for 10 s, 37°C for 30 s, and 72°C for 1 min; followed by a final extension at 72°C for 7 min. PCR products were separated on 1% agarose gels in 0.5× TBE buffer at 100 V for 40 min and visualized under UV light. Amplicons were purified using the ExpinTM PCR SV Mini Kit and sequenced by Macrogen.
Morphological characteristics of colonies and conidia
A total of 69 representative Colletotrichum isolates, which were previously identified by multi-locus phylogenetic analysis and PCR with species-specific primers, were selected for the observation of morphological characteristics based on year of isolation and geographic origin. To examine colony morphology and conidial characteristics, 3-mm mycelial plugs from selected isolates were inoculated onto PDA plates and incubated at 25°C in the dark for 10 days. Colony morphology was recorded, and aerial mycelia were gently removed from the colony surface using sterile glass slides. Conidiation was induced by exposing cultures to near-ultraviolet light at 20°C for 2 days at 12-h intervals. Conidia were harvested by adding 10 mL of sterile distilled water to the PDA plates and gently scraping the colony surface with sterile glass slides. The conidial suspension was filtered through four layers of sterile gauze to remove mycelial fragments and debris, followed by centrifugation at 3,000 rpm for 10 min twice for washing. Conidial morphology was observed using a light microscope (KB-300; Korea Lab Tech, Seongnam, Korea), and conidial length was measured using ToupView software (ToupTek Photonics, Hangzhou, China), with 50 conidia measured per replicate.
Mycelial growth at different temperatures
The same 69 Colletotrichum isolates used for morphological characterization were used to evaluate mycelial growth at 25°C and 30°C. Mycelial plugs (3 mm in diameter) were placed at the center of PDA plates and incubated at either 25°C or 30°C in the dark. Colony diameter was measured at 2-day intervals using a digital caliper (BD-DC150P, BLUETEC). Each isolate was tested with three replicates, and mean colony diameters of isolates belonging to the same species were used for interspecific comparisons.
Pathogenicity on pepper fruits at different temperatures
To compare temperature-dependent pathogenicity, the same 69 Colletotrichum isolates used for morphological characterization were inoculated onto pepper fruits and incubated at 25°C and 30°C. Conidia were harvested from 10-day-old PDA cultures, and conidial suspensions were adjusted to 5 × 105 conidia/mL. Pepper fruits were surface-sterilized with 1% sodium hypochlorite, rinsed with sterile distilled water, and wounded using sterile lancets (SD BIOSENSOR, Suwon, Korea). A 20-μL aliquot of conidial suspension was deposited onto each wound site. Inoculated fruits were placed in plastic containers and maintained under high-humidity conditions at either 25°C or 30°C for 5 days. After the humid incubation period, container lids were removed, and fruits were maintained at 25°C to promote lesion development. Disease severity was assessed by measuring lesion diameter using a digital caliper.
Assay for fungicidal response using the agar dilution method
The mycelial growth inhibition effects of the fungicides pyraclostrobin (a.i. 20%, WG), tebuconazole (a.i. 20%, SC), and fluazinam (a.i. 50%, WP) against the pathogens were evaluated using a large-scale agar dilution method (Isa et al., 2026). Each fungicide was added to PDA to achieve final concentrations of 10.0, 5.0, and 10.0 μg/mL, respectively. For the large-scale agar dilution, 15 cm diameter Petri dishes (cat. no. 10150; SPL Life Sciences Co., Ltd., Pocheon, Korea) were utilized, with ten isolates inoculated at uniform intervals per dish. The inoculated plates were incubated at 25°C for 5 days, after which the colony diameters were measured. The mycelial growth inhibition rate was calculated relative to the untreated control using the formula below. For pyraclostrobin, all isolates collected annually from 2022 to 2024 (651, 711, and 613 isolates, respectively) were tested, while only the 613 isolates collected in 2024 were used for tebuconazole and fluazinam.
T: the diameter of pathogen colonies on media amended with fungicides
C: the diameter of pathogen colonies on media without fungicide
To determine the EC50 values of pyraclostrobin against the pathogens, 168 isolates were selected from the total collection based on mycelial growth inhibition rates (%) obtained from the large-scale agar dilution assay at 10 μg/mL pyraclostrobin (Supplementary Table 2). The isolates were classified into three inhibition groups (0.0 ≤ x < 50.0%, 50.0 ≤ x < 70.0%, and 70.0 ≤ x ≤ 100.0%), and representative isolates from each group were selected for each Colletotrichum species. The final concentrations of pyraclostrobin in the PDA medium were adjusted to 0.016, 0.08, 0.4, 2.0, and 10.0 μg/mL. The inoculated pathogens were incubated at 25°C for 6 days, followed by the measurement of colony diameters. The mycelial growth inhibition rate of pyraclostrobin at each concentration was calculated using the aforementioned formula, and the EC50 values were subsequently determined using Microsoft Excel. All experimental treatments were performed in triplicate.
Statistical analysis
All experimental data were analyzed using IBM SPSS Statistics version 26 (IBM Corp., Armonk, NY, USA). Differences in mycelial growth and pathogenicity between temperatures for each species were compared using the t-test. Conidial size comparisons and fungicide sensitivity assays comparing sensitivities among species were analyzed using analysis of variance (ANOVA), and mean comparisons were performed using Duncan’s multiple range test (DMRT). A significance level of P < 0.05 was applied throughout the analyses.
Results
Species identification based on multi-locus phylogenetic analysis
Multi-locus phylogenetic analysis clearly resolved the Colletotrichum isolates into three major clades corresponding to species complexes (Fig. 1). The first clade comprised members of the C. acutatum species complex, in which C. scovillei, C. nymphaeae, and C. fioriniae formed distinct and well-supported subclades. The second clade consisted of isolates belonging to the C. gloeosporioides species complex, including C. fructicola, C. aenigma, and C. gloeosporioides, each clustering independently. The third clade represented the C. truncatum species complex, in which all C. truncatum isolates grouped together as a single monophyletic lineage. The topology of the phylogenetic tree was consistent with previously reported Colletotrichum species delimitations based on multi-locus sequence data, confirming the reliability of the species identification in this study.
Multi-gene phylogenetic analysis of Colletotrichum species isolated from pepper based on ITS, CHS-1, GAPDH, ACT, and TUB2 sequences. The phylogenetic tree was constructed using the Maximum Likelihood method in MEGA 11 with 1,000 bootstrap replications. Thirty-two representative isolates covering all seven identified species (C. scovillei, C. nymphaeae, C. fioriniae, C. fructicola, C. aenigma, C. gloeosporioides, and C. truncatum) were used to confirm the phylogenetic accuracy of our species identification.
Species identification using species-specific PCR
Species-specific PCR assays were conducted to validate the results of the phylogenetic analysis. All isolates were initially classified into either the C. acutatum species complex or the C. gloeosporioides species complex using complex-specific primer sets (Fig. 2A, 2B). Subsequent amplification with species-specific primers enabled unambiguous identification of individual species within each complex. Within the C. acutatum species complex, C. scovillei, C. nymphaeae, and C. fioriniae produced PCR amplicons of 330, 352, and 320 bp, respectively (Fig. 2C–2E). Within the C. gloeosporioides species complex, C. fructicola, C. aenigma, and C. gloeosporioides yielded products of 200, 230, and 101 bp, respectively (Fig. 2F–2H). In addition, C. truncatum isolates produced a distinct amplicon of 394 bp using the primer pair CcapF/CcapR (Fig. 2I). The results of species-specific PCR assays were fully consistent with those obtained by multi-locus phylogenetic analysis.
Gel electrophoresis showing specific PCR amplicons of Colletotrichum species complexes and individual species using species-specific primers. (A) C. acutatum species complex amplified with primers CaSc-Bt498F/CSc-Bt810R. (B) C. gloeosporioides species complex amplified with primers CgSc-Bt675F/CSc Bt810R. (C–I) Species-specific amplification of seven Colletotrichum species: (C) C. scovillei (CSC Bt293/T2), (D) C. nymphaeae (CNY-Bt270_1/T2), (E) C. fioriniae (CFI-Bt302(R)/T2), (F) C. fructicola (Cfr Bt492F/CgSc-Bt691R), (G) C. aenigma (Cae-Bt462F/CgSc-Bt691R), (H) C. gloeosporioides (Cgl Bt591F/CgSc-Bt691R), and (I) C. truncatum (CcapF/CcapR). M, DNA size marker (100 bp plus DNA ladder); P, positive control; N, negative control.
Distribution of Colletotrichum species
Analysis of species composition revealed that C. scovillei was the predominant pathogen in all survey years (Fig. 3A). In 2022 and 2023, C. scovillei accounted for more than 90% of the total isolates, with particularly high dominance in 2023. In contrast, although C. scovillei remained dominant in 2024, the relative proportions of non-dominant species increased, including C. fructicola (6.4%), C. fioriniae (3.8%), C. nymphaeae (1.6%), and C. truncatum (1.1%), as well as C. aenigma and C. gloeosporioides, indicating a shift toward greater species diversity. Regional analysis in 2024 revealed marked differences among regions (Fig. 3B). In Gyeongsangbuk-do, C. scovillei remained predominant at 64.3%, while C. nymphaeae (12.0%) and C. fioriniae (15.0%) were detected at relatively high frequencies. In Jeollabuk-do, C. fructicola (37.3%), C. nymphaeae (14.5%) and C. truncatum (14.1%) accounted for a substantial proportion of the isolates. These results show an increase in species diversity in certain regions in 2024.
Proportions of Colletotrichum species isolated from anthracnose-infected pepper fruits in Korea. (A) Annual distribution of Colletotrichum species from 2022 to 2024. A total of 651 isolates in 2022, 711 in 2023, and 613 in 2024 were utilized for the analysis. (B) Regional distribution of Colletotrichum species across major pepper cultivation areas in 2024. Numbers in parentheses indicate the total number of isolates analyzed in each province.
Mycological characteristics of re-identified Colletotrichum species
Distinct differences in colony morphology and conidial characteristics were observed among the re-identified Colletotrichum species (Fig. 4). Species belonging to the C. acutatum species complex generally produced white to grayish colonies on PDA, with reverse colony colors ranging from ivory to pale yellow or pinkish tones. Conidia of C. scovillei, C. nymphaeae, and C. fioriniae were hyaline and cylindrical, with mean lengths of 12.0 ± 3.0 μm, 14.3 ± 2.7 μm, and 15.3 ± 2.2 μm, respectively. Isolates of the C. gloeosporioides species complex exhibited colonies that gradually darkened toward the center during incubation. Their conidia were also cylindrical and hyaline, with relatively small differences in size among species. The mean conidial lengths of C. fructicola, C. aenigma, and C. gloeosporioides were 16.8 ± 3.9 μm, 16.6 ± 3.3 μm, and 16.6 ± 3.6 μm, respectively, while widths ranged from 7.1 to 7.8 μm. In contrast, C. truncatum formed dark green to black colonies and produced falcate (crescent-shaped) conidia. The mean conidial size of C. truncatum was 27.2 ± 3.5 × 5.0 ± 0.1 μm, which was the largest among the seven species examined.
Colony morphology and conidial characteristics of Colletotrichum species isolated from pepper. Representative colony morphology showing reverse (left) and surface (right) views on PDA is presented in (A–C) and (G–J). Microscopic observations of conidia are shown in (D–F) and (K–N). (A, D) C. scovillei JBWJ1-5; (B, E) C. nymphaeae JBJJ1-6; (C, F) C. fioriniae JBWJ2-1; (G, K) C. fructicola JBIS1-6; (H, L) C. aenigma JBIS1-4; (I, M) C. gloeosporioides JBIS1-3; and (J, N) C. truncatum JBJS2-4. Conidia were obtained from 7-day-old cultures grown on PDA. Scale bars = 50 μm.
Mycelial growth at different temperatures
Mycelial growth of the seven Colletotrichum species differed significantly depending on temperature (Table 4). Species belonging to the C. acutatum species complex exhibited faster mycelial growth and larger colony diameters at 25°C, whereas growth was significantly reduced at 30°C. In contrast, species within the C. gloeosporioides species complex showed comparable growth rates and colony diameters at both temperatures. C. truncatum displayed enhanced mycelial growth at 30°C compared with 25°C, indicating a higher tolerance to elevated temperatures. These results demonstrate that temperature-dependent growth responses vary among species complexes.
Comparison of mycelial growth rate and colony diameter at 25°C and 30°C in Colletotrichum species isolated from diseased peppers
Temperature-dependent pathogenicity on pepper fruits
Pathogenicity assays on pepper fruits revealed significant differences in lesion development among species and temperature conditions (Table 5, Fig. 5). Species of the C. acutatum species complex produced larger lesions at 25°C than at 30°C, with C. scovillei showing the highest pathogenicity within the complex. In contrast, C. fructicola and C. aenigma produced significantly larger lesions at 30°C than at 25°C, indicating enhanced pathogenicity under higher temperature conditions. Similarly, C. truncatum exhibited significantly increased lesion sizes at 30°C, confirming its strong pathogenicity at elevated temperatures.
Comparison of pathogenicity based on lesion diameter at 25°C and 30°C on pepper fruits among Colletotrichum species isolated from diseased peppers
Comparison of pathogenicity of Colletotrichum species on pepper fruits under two different incubation temperatures. The upper and lower rows represent symptoms induced at 25°C and 30°C, respectively. (A) C. scovillei (JBWJ1-5 and CBES1-5), (B) C. nymphaeae (JBJJ1-6 and JBJJ1-8), (C) C. fioriniae (JBWJ2-1 and GBYY3-6), (D) C. fructicola (JBIS1-6 and GBYY3-13), (E) C. aenigma (JBIS1-4 and GBYY3-3), (F) C. gloeosporioides (JBIS1-3 and 22UGM9), and (G) C. truncatum (JBWJ1-2 and JBJS2-2).
Fungicidal responses of Colletotrichum species
Using a large-scale agar dilution method, the fungicidal responses of various Colletotrichum species to pyraclostrobin were evaluated by measuring mycelial growth inhibition (%) at a concentration of 10 μg/mL over a three-year period from 2022 to 2024 (Table 6, Fig. 6A). In 2022, species within the C. gloeosporioides species complex showed relatively high inhibition rates, with C. aenigma (72.8%) and C. gloeosporioides (77.7%) exhibiting the highest sensitivity, followed by C. fructicola (58.7%). In contrast, C. scovillei (43.3%) and C. truncatum (21.9%) showed significantly lower inhibition levels. In 2023, only C. scovillei and C. fructicola were analyzed. While C. fructicola showed a higher inhibition (56.8%) than C. scovillei (29.5%), the difference between the two species was not statistically significant (P > 0.05). The results from 2024 revealed distinct levels of sensitivity among the species. Within the C. acutatum species complex, C. nymphaeae (97.8%) and C. fioriniae (98.0%) were highly sensitive to pyraclostrobin, whereas C. scovillei (31.7%) showed significantly lower inhibition. The species in the C. gloeosporioides species complex (C. fructicola, C. aenigma, and C. gloeosporioides) exhibited moderate to high inhibition rates ranging from 66.0% to 78.4%. Similar to the trends observed in 2022, C. truncatum (34.9%) and C. scovillei (31.7%) showed the lowest inhibition rates in 2024, with no significant statistical difference between them.
Evaluation of mycelial growth inhibition by fungicides using the large-scale agar dilution method. The assay included (A) pyraclostrobin, (B) tebuconazole, and (C) fluazinam. Each fungicide was incorporated into PDA at final concentrations of 10.0, 5.0, and 10.0 μg/mL, respectively. Abbreviations for Colletotrichum species are as follows: cas, C. scovillei; can, C. nymphaeae; caf, C. fioriniae; cgf, C. fructicola; cga, C. aenigma; cgg, C. gloeosporioides; and ct, C. truncatum. The same letters on the boxes indicate no significant difference according to DMRT test (P < 0.05).
The mycelial growth inhibition of 613 Colletotrichum isolates collected in 2024 was evaluated at 5.0 μg/mL of tebuconazole, and 10.0 μg/mL of fluazinam, respectively (Fig. 6B and C). Tebuconazole showed lower inhibition in the thermophilic groups, including the C. gloeosporioides species complex and C. truncatum, compared to the C. acutatum species complex. While C. scovillei, C. nymphaeae, and C. fioriniae generally showed higher sensitivity, the thermophilic groups exhibited a wider range of responses and lower median inhibition values. Fluazinam was the most effective across all 613 isolates, showing high mycelial growth inhibition regardless of the species. Although the inhibition rate for C. truncatum was relatively lower than that of other species, it still maintained a substantial level of suppression, and the overall inhibitory effect remained consistently high across the C. acutatum and C. gloeosporioides species complexes.
The EC50 values of the 168 selected isolates for pyraclostrobin are presented in Supplementary Table 2 and Fig. 7. Isolates with EC50 values exceeding 2 μg/mL exhibited a mycelial growth inhibition rate of 55.0% or less at a concentration of 10.0 μg/mL.
Distribution of EC50 values for pyraclostrobin among various Colletotrichum species. EC50 values were determined through mycelial growth inhibition assays at concentrations of 0.016, 0.08, 0.4, 2.0, 10.0 μg/mL. (A) C. scovillei (n = 46), (B) C. nymphaeae (n = 10), (C) C. fioriniae (n = 23), (D) C. fructicola (n = 54), (E) C. aenigma (n = 4), (F) C. gloeosporioides (n = 2), and (G) C. truncatum (n = 29). Blue dots represent sensitive isolates with EC50 ≤ 2.0 μg/mL, while red dots indicate resistant isolates with EC50 > 2.0 μg/mL.
Molecular verification of pyraclostrobin resistance via cytb gene analysis
The amino acid sequences of the cytb gene were analyzed to identify resistance-associated mutations (Fig. 8). All isolates showing high inhibition levels, regardless of species, possessed the wild-type amino acids phenylalanine, glycine, and glycine at positions 129, 137, and 143, respectively. In contrast, all isolates with low inhibition levels consistently harbored a point mutation resulting in the substitution of glycine with alanine at position 143 (G143A). This association was further supported by the analysis of 168 isolates (Supplementary Table 2), where all isolates with EC50 ≥ 2.0 μg/mL or inhibition <55.0% carried the G143A mutation. No amino acid substitutions were detected at positions 129 or 137 in any isolate. These results demonstrate a complete association between the G143A mutation and reduced inhibition, indicating that G143A is a reliable molecular marker for pyraclostrobin resistance in Colletotrichum spp.
Amino acid alignment of partial cytochrome b (cytb) sequences from Colletotrichum species. The alignment highlights conserved and substituted residues at positions 129, 137, and 143. (A) Sequences from pyraclostrobin-sensitive isolates showing the wild-type glycine (G) at position 143. (B) Sequences from pyraclostrobin-resistant isolates showing the G143A mutation, where glycine is substituted with alanine (highlighted in red). Positions 129 and 137 remained conserved across all tested isolates.
Discussion
The accurate identification and classification of Colletotrichum species causing pepper anthracnose are fundamental for developing effective disease management strategies. Until now, the identification and classification of Colletotrichum species causing pepper anthracnose have undergone significant revisions with the advancement of molecular biology. Historically, based on morphological characteristics, C. gloeosporioides was considered the primary causal agent of the disease in Korea (Park and Kim, 1992). However, reliance on morphology alone is often misleading due to the overlapping dimensions of conidia and high intraspecific variation in colony appearance among different species (De Silva et al., 2017). The implementation of multi-locus phylogenetic analysis using genes such as ITS, CHS-1, GAPDH, ACT, and TUB2 has allowed for a much more precise resolution of species complexes (Cannon et al., 2012; Damm et al., 2012). According to Shin et al. (2025), C. scovillei, C. nymphaeae, C. fructicola, C. gloeosporioides, C. cigarro, and C. truncatum were reported from pepper and bell pepper in Gyeongsangbuk-do, Korea. In this study, multi-locus phylogenetic analysis and species-specific PCR confirmed the presence of seven distinct species: C. scovillei, C. nymphaeae, and C. fioriniae within the C. acutatum species complex; C. fructicola, C. aenigma, and C. gloeosporioides within the C. gloeosporioides species complex; and C. truncatum.
Between 2022 and 2024, C. scovillei remained the dominant species, accounting for 86.4–99.5% of isolates. This dominance is consistent with studies conducted since the 2010s which identified C. scovillei as the major driver of anthracnose outbreaks (Oo et al., 2017). However, in 2024, non-dominant species increased in specific regions, with C. nymphaeae, C. fructicola, and C. truncatum in Jeollabuk-do, and C. nymphaeae and C. fioriniae in Gyeongsangbuk-do. Such regional variation suggests that local environmental conditions and selection pressures, including fungicide use patterns, cultivated varieties, and microclimatic differences, may drive the increase of non-dominant species (Burdon and Thrall, 2014).
According to the Korea Meteorological Administration, average summer temperatures and the frequency of heatwave events in Korea have increased continuously in recent decades. In particular, the average temperatures during July and August, when pepper anthracnose outbreaks are most severe, increased from 24.1°C in 2022 to 26.3°C in 2024 (KMA, 2025). In addition, the maximum temperatures recorded during this period reached 37.9°C in 2022, 38.4°C in 2023, and 39.3°C in 2024, indicating a sustained trend toward elevated temperature conditions. These environmental conditions may provide selective advantages to thermotolerant Colletotrichum species and contribute to shifts in species composition in the field. To better understand the ecological implications of these elevated temperature conditions, temperature-dependent assays were conducted for Colletotrichum species. These assays provided crucial ecological insights into the observed population shifts. Colletotrichum species exhibit varying thermal requirements that directly influence their survival and virulence in the field (Ji et al., 2023; Salotti et al., 2022). Members of the C. acutatum species complex, including the dominant C. scovillei, showed optimal mycelial growth and pathogenicity at 25°C, with a significant decline in activity when temperatures reached 30°C. In contrast, heat-adapted species such as C. fructicola, C. aenigma, and C. truncatum maintained or even enhanced their growth and pathogenicity at 30°C. These findings align with Ji et al. (2023), who reported that members of the C. gloeosporioides complex exhibit high-temperature adaptability. This trait allows them to maintain a competitive advantage under elevated temperatures, increasing their potential to become dominant as climate warming progresses. Notably, C. truncatum also maintained high growth rates under elevated temperatures, further supporting its potential as an emerging threat (Salotti et al., 2022). In the present study, C. truncatum isolates from pepper exhibited enhanced mycelial growth and pathogenicity at 30°C compared to 25°C. In contrast, previous research on C. truncatum causing lentil anthracnose reported that the optimal conditions for disease incidence, lesion size, and sporulation were 24/20°C (day/night), with a sharp decline observed at 28/24°C (Chongo and Bernier, 2000). While those lentil-derived isolates showed increased lesion size and sporulation rates as temperatures rose from 16/12°C to 24/20°C, their thermal optimum remained significantly lower than that of the pepper-derived isolates in our study. These results suggest that C. truncatum populations originating from infected pepper fruits possess a higher thermal optimum for growth than those from lentils. Furthermore, this discrepancy serves as a compelling example of how shifts in climatic conditions can drive the adaptation of fungal pathogens, potentially altering their optimal growth temperatures and subsequent disease dynamics in the field.
The increasing prevalence of thermotolerant species poses important challenges for disease management. Traditionally, anthracnose outbreaks in Korea were expected to decline during extreme mid-summer heat (above 30°C). However, the increasing prevalence of thermotolerant species such as C. fructicola and C. truncatum suggests that the disease may persist even under high-temperature conditions (Ji et al., 2023; Salotti et al., 2022; Shin et al., 2025). This shift is particularly concerning as these species maintain high virulence at temperatures where traditional dominant species typically show reduced activity (Than et al., 2008a). Moreover, the synergistic effects of elevated temperatures and heavy rainfall during the monsoon season may further accelerate pathogen proliferation and spread, potentially leading to more frequent and severe yield losses. Consequently, the expansion of the pathogen’s active period will complicate the timing and efficacy of fungicide applications.
Fungicide sensitivity tests highlighted the complexity of controlling diverse Colletotrichum populations. QoI fungicide resistance has been widely reported as a major issue in Colletotrichum spp. (Abdulkareem et al., 2024; Kim et al., 2019; Kongtragoul et al., 2020), and our results are consistent with previous reports. Pyraclostrobin and tebuconazole showed clear species-dependent responses, whereas fluazinam remained effective across all species. The dominant species, C. scovillei, consistently showed low inhibition levels throughout the three-year survey, and all C. truncatum isolates were resistant, indicating a substantial risk of control failure under intensive QoI fungicide use. In contrast, C. nymphaeae and C. fioriniae remained highly sensitive, suggesting that fungicide efficacy may vary considerably depending on regional shifts in species composition. Tebuconazole also showed reduced efficacy against members of the C. gloeosporioides species complex and C. truncatum compared with species in the C. acutatum species complex. Because fungicide responses differ substantially among Colletotrichum species, shifts in population structure may reduce the effectiveness of conventional disease management programs. In particular, C. fructicola, C. aenigma, and C. truncatum, which maintain high pathogenicity under elevated temperatures, may become increasingly prevalent under climate warming conditions, thereby increasing disease pressure in pepper-growing regions. Among these species, C. truncatum is of particular concern because of its combined thermotolerance and reduced sensitivity to multiple fungicides, highlighting its potential as a major threat to sustainable anthracnose management under future climate conditions.
A strong and consistent association among the mycelial growth inhibition rate at 10.0 μg/mL, EC50 values, and the G143A mutation of the cyt b gene validated the resistance criteria established in this study. Isolates with a mycelial growth inhibition rate ≤55.5% consistently exhibited EC50 values > 2 μg/mL and carried the G143A mutation, whereas isolates with >55.5% inhibition showed lower EC50 values and retained the wild-type genotype. These findings demonstrate that the agar dilution method provides a reliable and practical approach for the primary screening of pyraclostrobin resistance in large-scale monitoring programs.
The coexistence of multiple Colletotrichum species with differing physiological traits and fungicide sensitivities within a single field complicates disease control. The application of specific fungicides may suppress sensitive species while allowing resistant, heat-adapted species to flourish under double selection pressure. Therefore, uniform management strategies may become less effective. Instead, integrated management should incorporate real-time monitoring of species composition and environmental factors to optimize control efficiency. In conclusion, the population structure of Colletotrichum in Korean pepper fields is becoming more dynamic under the combined influence of climate conditions and fungicide use, with an increasing presence of heat-adapted species. In addition, resistance to pyraclostrobin has become more pronounced, highlighting the need for continuous monitoring to support appropriate fungicide use. Under these conditions, effective disease management will require integrated strategies, including fungicide rotation and ongoing monitoring of species composition and resistance profiles.
Notes
Conflicts of Interest
No potential conflict of interest relevant to this article was reported.
Acknowledgments
This work was carried out with the support of the Cooperative Research Program for Agriculture Science & Technology Development (Project No. RS-2024-00398990).
Electronic Supplementary Material
Supplementary materials are available at The Plant Pathology Journal website (http://www.ppjonline.org/).