First Report of Colletotrichum aenigma causing Leaf Anthracnose of Maize in Shanghai, China

China is the world’s second-largest maize producer, contributing 23% to global production (Luo et al. 2023). During a field survey in July 2025, about 15 to 20% of maize leaves showed symptoms on maize cv. Zhengdan958 grown in a farm field in Zhuanghang Town, Fengxian District, Shanghai, China. (121.23°E, 30.95°N). Initial symptoms consisted of small dark-brown spots on leaves that enlarged into irregular leaf blight lesions. Samples showing leaf spot symptoms were collected randomly from five plants and the infected leaf sections (5 × 5 mm) were cut from the edge of the necrotic/healthy tissues, surface-sterilized with 70% ethanol for 30 s and 2% sodium hypochlorite for 90 s before being rinsed twice with sterilized distilled water and placed on potato dextrose agar (PDA). After three days of incubation at 25°C in the dark, the newly grown colonies were transferred to fresh medium and purified using the single-spore isolation method. In total, five fungal isolates were obtained. Fungal colonies on PDA produced whitish mycelium after 7 to 14 days at 25 °C with a 12-h photoperiod. The upper side of colonies on the PDA plates was predominantly white, with a dense, cottony texture, and the reverse side was dark brown to yellow at the center and pale white at the margin. Light microscopic observations showed that conidia were smooth-walled, hyaline, aseptate, guttulate, cylindrical, and straight with rounded ends, measuring 15.5-21.5 × 5.5-7.5 μm (mean 18.6 × 6.4 μm, n = 30). Additionally, some conidia were slightly distorted and had one slightly acute end. The morphological characteristics of the isolates matched those of Colletotrichum species within the C. gloeosporioides complex (Weir et al., 2012). To further identify isolates, actin (ACT), calmodulin (CAL), chitin synthase (CHS-1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin 2 (TUB2), manganese-superoxide dismutase (SOD2), and the ribosomal internal transcribed spacers (ITS) loci of representative isolates were amplified using ACT-512F+ACT-783R, ICL1/CL2, CHS-79F/CHS-345R, GDF1/GDR1, Bt2a/Bt2b, SODglo2-F/SODglo2-R, and ITS1/ITS4 primers (Weir et al. 2012). Sequences of representative isolate Sh01 were submitted to GenBank (ACT, PX883930; CAL, PX883932; CHS-1, PX883931; GAPDH, PX883935; TUB2, PX883933; SOD2, PX883934; ITS, PX875895). Maximum likelihood analysis of the representative isolate and reference sequences of Colletotrichum spp. from GenBank showed that the representative isolate clustered with the ex-type culture of C. aenigma (ICMP18686, ICMP18608) with 100% bootstrap support. Based on morphological and molecular studies, the pathogen was identified as C. aenigma. The pathogenicity of isolate Sh01 was tested on maize plants (growth stage V3). For inoculation, plants were laid horizontally in trays, and the third leaf of each plant was treated with 7.5 μl droplets containing 1×10⁶ conidia/mL without wounding. Negative controls consisted of maize leaves inoculated with sterile distilled water droplets. Following inoculation, all plants were placed vertically and incubated in a growth chamber under 23°C, 90% relative humidity, and a 12-h photoperiod (Vargas et al., 2012). Five days post-inoculation, brown necrotic lesions characteristic of C. aenigma infection appeared on the inoculated leaves, while the control leaves remained asymptomatic. The pathogen was successfully reisolated from these lesions and morphologically identified, providing further evidence to support the pathogenicity of C. aenigma on maize. To our knowledge, this is the first report of C. aenigma causing leaf anthracnose on maize in China. This finding will facilitate the development of effective management strategies for the anthracnose of maize.