Characterization and Fungicide Resistance Profiling of Colletotrichum Species from Soybean in Korea

Abstract

Multi-locus phylogenetic analysis (ITS, gapdh, act, chs-1, and tub2) of 36 Colletotrichum isolates collected from major soybean-growing areas in Korea resolved them into nine species across six species complexes: Colletotrichum truncatum, C. sojae, C. plurivorum, C. gloeosporioides, C. fructicola, C. nymphaeae, C. tabaci, C. chlorophyti, and C. reniforme. Notably, C. chlorophyti, C. tabaci, and C. reniforme were identified as newly reported pathogens of soybean anthracnose in Korea. Representative isolates exhibited optimal mycelial growth at 25–27.5oC, whereas C. truncatum and C. chlorophyti adapted better to higher temperatures (27.5–30oC). Pathogenicity tests demonstrated that all isolates induced disease symptoms on soybean seeds, stems, and pods, with significant variations in virulence depending on the host organ and wounding status. Fungicide sensitivity assays revealed that eight isolates of C. truncatum were resistant to benomyl (EC50 ≥2.0 μg/ml), and ten isolates (C. truncatum, C. sojae, and C. reniforme) were resistant to pyraclostrobin (EC50 ≥10.0 μg/ml). Seven C. truncatum isolates exhibited dual resistance to both fungicides. All isolates remained sensitive to prochloraz, fluazinam, and fludioxonil. Sequence analysis of target genes confirmed that benomyl resistance was driven by the E198A mutation in the tub2 gene, while pyraclostrobin resistance was mediated by the G143A mutation in the cytb gene. Cross-resistance was observed within the benzimidazole (thiophanate-methyl) and strobilurin (azoxystrobin, kresoxim-methyl, trifloxystrobin) fungicide classes. Seed treatment with prochloraz effectively controlled anthracnose caused by both sensitive and resistant isolates. Furthermore, allele-specific polymerase chain reaction assays successfully identified the E198A and G143A point mutations, offering a rapid molecular diagnostic tool for resistance monitoring.