First report of Corynespora cassicola causing black spot on Sarcandra glabra in China

Sarcandra glabra is an important Chinese medicinal plant, which was widely cultivated under forest in south China. Guangxi province is the main producing areas of this herb. In June 2019, a serious leaf disease was found causing severe defoliation in the S. glabra plantation under bamboo forest in Rongan country, Guangxi province (109°13'N''E). About 70% of the plants in the plantation (300 ha) showed the similar symptoms. Initially, circular lesions appeared on young leaves as black spots (about 1 to 2 mm). Then, the spots gradually enlarged usually with an obvious yellowish margin (6 to 8 mm). Finally, the lesions coalesced and formed irregular, black, and large necrotic areas, resulting in the leaf abscission. For pathogen isolation, small pieces of tissue (5×5 mm) taken from 25 diseased leaves were sterilized with 75% ethanol for 30 s, subsequently, soaked in 0.1% HgCl2 for 2 min, rinsed three times in sterile distilled water, dried, and then placed aseptically onto the potato dextrose agar (PDA) plates, and incubated at 28 °C (12 h/12 h light/dark). Three days later, the isolates were placed on a new PDA plate for subsequent purification and sporulation. 20 pure fungal isolates were obtained from single spores. Of which, 15 isolates showed similar morphological characteristics.The colonies on PDA were round, dense, gray edge and dark gray in center area. Conidia in culture were appeared light brown, cylindrical in shape, with 0 to 8 septa, and 55 to 165 μm × 5.2 to 13.5 μm in size (mean = 106.2 μm × 8.6 μm, n = 30). These morphological characteristics resemble those of Corynespora sp. (Berk. & M.A. Curtis) C.T. Wei (Ellis et al. 1971). A single-spore isolate (ZD5) was selected from the 15 fungal isolates for a subsequent molecular identification. The genes of internal transcribed spacer (ITS) of ribosomal DNA, β-tublin, and actin were amplified with the primer pairs ITS-1/ITS-4 (White et al. 1990), β-tubulin 2-Bt2a/Bt2b (Glass and Donaldson 1995), ACT-512F/ACT-783R (Carbone and Kohn 1999), respectively. And the ITS, β-tublin, and actin sequences were deposited in the GenBank database with the accession numbers MW362446, MW367029, and MW533122. Blast analysis and neighbor-joining analysis based on ITS, β-tublin, and actin sequences using MEGA 6 revealed that the isolate was placed in the same clade as C. cassicola with 100% bootstrap support. Pathogenicity test was performed on the two-year-old potted S. glabra. Six-mm-diameter mycelial plugs were attached to the healthy leaves of S. glabra for co-culture, while the control group was attached with PDA. All plants were covered with plastic bags for 2 days in order to maintain high humidity and cultured in a greenhouse at 28 °C with a 12-h/12-h light/dark cycle. The symptoms appeared 2 days after co-culture were identical to those observed in the field. The same fungus was re-isolated from the lesions, and further morphological characterization and molecular assays, as described above.The control leaves remained symptomless during the pathogenicity tests. According to the previous literatures, C. cassicola is a plant pathogenic fungus with a broad host range, which can damage diverse tropical plants including Salvia miltiorrhiza (Lu et al. 2019), Solanum americanum (Wagner and Louise 2019), Vitex rotundifolia (Yeh and Kirschner 2017), Cucumis sativus, Lycopersicon esculentum (Hsu et al. 2002), Carica papaya (Tsai et al. 2015),and so on. To our knowledge, this is the first report of C. cassicola causing leaf spot on S. glabra in China.