First Report of Anthracnose Caused by Colletotrichum siamense on Hydrangea macrophylla in China

Hydrangea macrophylla (Thunb.) Ser. (Hydrangeaceae), a shrubby perennial plant, is widely used as an ornamental flowering plant because of its showy inflorescences and colorful sepals. In October 2022, leaf spot symptom was observed on H. macrophylla in Meiling Scenic Spot, which covers an area of about 143.58 km2 in Nanchang, Jiangxi Province, China (28.78°N, 115.83°E). An investigation was carried out in a 500 m2 mountain area with 60 H. macrophylla plants in a residential garden, the incidence of disease observed was 28~35%. The symptoms were visible as nearly round dark brown spots on the leaves in the early stages of infection. At later stages, the spots gradually developed grayish white center with dark brown margins. To isolate the pathogen, seven leaves randomly selected from 30 infected leaves were cut into 4-mm2 pieces, surface disinfected with 75% ethanol for 30s followed by 5% NaClO for 1 min, rinsed in sterile water three times, placed on potato dextrose agar (PDA), and cultured at 25 °C in the dark for 7 days, and four strains with similar morphological characteristics were obtained from 7 diseased samples. Conidia were aseptate, cylindrical, hyaline, obtuse at both ends, and measured 13.31 to 17.53 × 4.43 to 7.45 µm (15.47 ± 0.83 × 5.91 ± 0.62 µm, n = 60). Morphological characteristics matched Colletotrichum siamense (Weir et al. 2012; Sharma et al. 2013). For molecular identification, two representative isolates (HJAUP CH003 and HJAUP CH004) were used for genomic DNA extraction, and the internal transcribed spacer (ITS), partial sequences of actin (ACT), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-tubulin (TUB2) and partial calmodulin (CAL) were amplified, using primer pairs ITS4/ITS5 (White et al. 1990), ACT-512F/ACT-783R, GDF1/GDR1, Bt2a/Bt2b and CL1C/CL2C (Weir et al. 2012), respectively. The sequences were deposited in GenBank (accessions nos. ITS: OQ449415, OQ449416; ACT: OQ455197, OQ455198; GAPDH: OQ455203, OQ455204; TUB2: OQ455199, OQ455200; CAL: OQ455201, OQ455202). Concatenated sequences of the five genes were used to conduct phylogenetic analyses using the maximum-likelihood method in MEGA7.0 (Sudhir et al. 2016) and Bayesian inference analysis in MrBayes 3.2 (Ronquist et al. 2012). Our two isolates cluster together with four strains of C. siamense with 93%ML/1.00BI bootstrap support. The isolates were identified as C. siamense based on the morpho-molecular approach. Pathogenicity of HJAUP CH003 was tested indoors by inocu