First Report of Colletotrichum karstii Causing Anthracnose on Soybean in China

Soybean (Glycine max) is a significant grain and oil crop. Among the various challenges faced by soybean cultivation, anthracnose stands out as one of the most prevalent diseases. In June 2023, anthracnose symptoms on leaves characterized by irregular disease spots featuring gray-white centers and brown edges, along with many small black dots on their surface, were observed in a 20-hectare soybean (variety "Liu Yuehuang") field located in Luodian County (25°40'20″ N, 106°53'50″ E, 575 m), Guizhou Province, China. Around 30% of the 300 soybean plants examined were symptomatic, and a total of ten leaves were collected. Fragments (5×5 mm) from the edge of disease spots were sheared and surface-sterilized with 3% sodium hypochlorite and 75% ethanol for 60 s and 30 s, respectively. They were then flushed twice with sterile water, dried using sterile filter papers, finally placed on potato dextrose agar (PDA) and incubated at 28°C for two days. In total, 11 isolates with identical morphological characteristics were obtained. The colonies grown with white aerial mycelia on their surface; conidia were cylindrical, both ends are rounded, aseptate, hyaline, 11.0-14.0 (12.5) × 4.5-6.0 (5.0) μm (n = 30); appressoria were nearly ovoid, brown to black, 8.5-10.5 (9.5) × 5.5-7.5 (6.0) μm (n = 30). The morphological characteristics closely resembled the description of C. karstii (Damm et al., 2012). To further identify the isolates, chitin synthase (CHS-1), actin (ACT), beta-tubulin (TUB2), glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and the internal transcribed spacer (ITS) loci were amplified by using CHS-79F/CHS-345R, ACT-512F/ACT-783R (Carbone and Kohn, 1999), Bt2F/Bt2R (Woudenberg et al., 2009), GDF/GDR (Guerber et al., 2003) and ITS1/ITS4 (White et al., 1990) PCR primers, respectively. The BLAST results showed that the sequences of two representative strains, LD 2023048-1 and LD 2023048-2, were highly similar to those of strain C. karstii CGMCC3.14194 (ITS: OR342620 (99%) and OR342621 (99%) with HM585409, ACT: OR412337 (97%) and OR423341 (100%) with HM581995, CHS-1: OR423344 (99%,) and OR423345 (100%) with HM582023, GAPDH: OR423348 (98%) and OR423349 (98%) with HM585391, and TUB: OR423352 (99%) and OR423353 (99%) with HM585428). The phylogenetic tree combined five sequences showed that the two strains clustered into a branch of C. karstii CGMCC3.14194 with high support values. Thirty-day-old soybean plants (n = 10) (variety Liu Yuehuang) were separately sprayed with 1 × 105 spore suspensions/mL of the two strains by spray method, and plants sprayed with sterile distilled water were used as the negative control (n = 5). All the plants were then covered with plastic bags and cultured in the greenhouse (28℃, 80% humidity, 12 h light dark cycle). After ten days of inoculation, plants inoculated with C. karstii began to produce typical anthracnose symptoms, while the control remained asymptomatic. The confirmation of the reisolated pathogen as C. karstii was established through a comprehensive analysis of morphology and five sequencing loci. Pathogenicity tests were repeated three times. Anthracnose on soybean is caused by Colletotrichum spp. reported in China including C. truncatum (Hu et al., 2015), C. brevisporum (Shi et al., 2021) and C. fructicola (Xu et al., 2023). As far as we know, this study is the initial report of C. karstii inducing anthracnose on soybean to date, which establishes a fundamental reference for preventing and controlling this disease.