First Report of Causing Anthracnose on Mulberry Leaves in China.

Mulberry ( L.) has been grown worldwide as a crop for silkworm rearing for over five thousand years (Jiao et al. 2020). In July 2021, a leaf spot disease was observed on mulberry leaves in Wuhan city (114°33'E, 30°48'N), Hubei province, China, with approximately 40% of leaves (about 300 trees) affected. Early symptoms were light brown, with small lesions subsequently expanding to larger sometimes irregular dark brown or black spots surrounded by yellow-brown margins, with easily perforated necrotic lesions. Leaf tissues (5 mm×5 mm) were excised from the border between diseased and healthy tissues, surface sterilized with 75% ethanol solution for 30 s and 2.5% sodium hypochlorite for 2 min, washed thrice in sterile distilled water, and then placed on potato dextrose agar (PDA), and incubated at 25°C in darkness. Four isolates (C1, C9, CHS2, and CHS6) were subcultured using the single-spore method. On PDA, colonies were cottony, pale white from above, and white to grayish-green on the reverse side. Conidia were aseptate, hyaline, subcylindrical with broadly rounded ends, 8.4 to 18.3×4.1 to 7.7 μm (mean = 13.9×5.5 μm, n = 30). Appressoria were typically elliptic or irregular with a few lobes, dark brown, 5.9 to 9.6×4.2 to 8.1 μm (mean = 7.9 ×5.7 μm, n = 30). The morphological characteristics of the isolates matched the descriptions of species complex (Weir et al. 2012). The isolates were further identified by analysis of the ribosomal internal transcribed spacers (ITS), glyceraldehyde-3-phosphate dehydrogenase (), calmodulin (), actin (), chitin synthase (), glutamine synthetase (), and β-tubulin 2 () genes, amplified respectively with ITS1/ITS4, GDF/GDR, CL1C/CL2C, ACT-512F/ACT-783R, CHS-79F/CHS-345R, GSF1/GSR, and Bt2a/Bt2b (Glass and Donaldson 1995; Weir et al. 2012; White et al. 1990). The sequences were deposited in GenBank (ON492187-ON492214). Concatenated sequences of the seven genes in addition to species sequences from GenBank were used to conduct a phylogenetic analysis using Maximum-Likelihood (ML) method in MEGA7. The four isolates were grouped into a clade with supported by a high bootstrap value (89%), and hence, they were identified as based on the morphological and molecular analyses. To confirm Koch's postulates, wounded leaves of six healthy 2-month-old seedlings made by a sterile needle were inoculated with each isolate by spraying 10 ml of conidial suspensions (10 conidia/ml) on each plant, and the control plants were treated with sterile distilled water. All the treated plants were kept in a plastic box containing sterile water and incubated at 28°C in a 12 h/12 h light/dark cycle. The test was performed three times. After 7 days, typical anthracnose lesions appeared on all inoculated leaves, whereas control plants remained asymptotic. Furthermore, was only reisolated from the symptomatic leaves. Previous studies reported five species (, , , , and subsp. ) to cause this disease on mulberry in China (Tian, 1981; Xue et al. 2019). To our knowledge, this is the first report of causing anthracnose on mulberry in China. The finding will facilitate epidemiological studies and the development of effective control strategies for the disease.