Occurrence of Anthracnose Caused by <i>Colletotrichum fructicola</i> on Mulberry in Zhejiang, China

Mulberry trees (Morus L.) are economically crucial crops in China, highly valued for their roles in silk production, livestock feeding, and as sources of food and medicinal products. From 2021 to 2023, leaf spot symptoms emerged in mulberry orchard in Qiandao Lake Town, Hangzhou, Zhejiang, China (29.61° N, 118.91° E). A survey of 60 trees over 200 m2 area revealed 45% of the foliage was infected with leaf spot disease, significantly reducing leaf quality. Infection typically began at the leaf tips or edges, initially as small dark brown spots (0.6 to 1.4 mm) that expanded into irregular lesions with grayish white centers and brown margins (2.5 to 3.6 mm). The infected leaves eventually wither and decay, and in severe cases, they may drop. Twelve freshly infected leaves from ten different mulberry trees were collected, cut into 5 × 5 mm sections, disinfected with 75% ethanol for 45 s and 0.5% NaClO for 2 min, rinsed three times with sterile water, the sections were plated on potato dextrose agar (PDA) and incubated at 28°C in the dark for 5 days. Six morphologically similar isolates were obtained by transferring hyphal tips to pure cultures. After 7 days on PDA, colonies were dense with white cottony aerial mycelia and light gray-black hyphae. The average mycelial growth rate was 8.2 mm/day, ranging from 5.8 to 11.2 mm/day. Conidia were cylindrical, hyaline, aseptate, with rounded ends and an inconspicuous hilum, measuring 12.4 to 16.4 × 3.6 to 6.8 μm. The appressoria were brown to dark brown, nearly ovoid to cylindrical, and slightly irregular, measuring 6.32 to 13.65 ×5.25 to 7.68 µm. These features were consistent with the description of Colletotrichum spp. (Fuentes-Aragón et al. 2018). Genomic DNA was extracted, and the rDNA internal transcribed spacer (ITS), partial actin (ACT), chitin synthase (CHS1), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and β- tubulin 2 (TUB2) genes were amplified using the primers ITS1/ITS4, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GDF/GDR, T1/Bt2b (Weir et al. 2012). The sequences were submitted to GenBank with accession numbers ON042364.1 (ITS), PQ567195 (ACT), PQ56196 (CHS1), PQ567197 (GAPDH), and PQ567198 (TUB2). BLASTN analysis revealed high similarity to Colletotrichum fructicola strain ICMP 18581, with sequence identities of 99.62% (ITS), 96.20% (ACT), 98.89% (CHS1), 98.79% (GAPDH), and 99.56% (TUB2) (Weir et al., 2012). A phylogenetic tree was constructed using the Bayesian inference (BI) method in MrBayes v.3.2.7 based on concatenated sequences of five genes (ITS-ACT-CHS1-GAPDH-TUB2). Based on morphological characteristics and phylogenetic analysis, the isolates were identified as C. fructicola. To confirm pathogenicity, ten 6-month-old potted plants were used for inoculation with each representative isolate. Tested plants were sprayed with 5 ml of a conidial suspension (1 × 106 conidia/ml), and the controls plants were sprayed with sterile water. All the plants were incubated in a growth chamber at 25 ± 2°C with 85% relative humidity. After 15 days, typical lesions like those observed on the field plants appeared on all inoculated plants, while the control remained healthy. The same fungal pathogen was reisolated and the identity was confirmed by morphological characterization and molecular analysis, confirming Koch's postulates. The pathogen has been reported as the causal agent of anthracnose on a wide range of plant hosts worldwide (Dos Santos et al. 2023; Ma et al. 2023). To our knowledge, this is the first report of anthracnose on morus caused by C. fructicola in Zhejiang, China.