Characterization of <i>Alternaria alternata</i> Associated with Black Spot Disease of <i>Isatis indigotica</i> in Yunan, China

Isatis indigotica Fortune, a biennial plant of the Cruciferae family, is widely cultivated in China for treating seasonal febrile illnesses, plague, mumps, rash disorders, skin redness, sore throats, and other inflammatory diseases (Ho et al. 2023). In 2024, a new leaf disease was discovered in an understory I. indigotica plantation in Kunming County, Yunnan Province, China, affecting nearly 40% of the cultivated area. Initially, irregular dark brown spots appear on the leaves, gradually spreading to form large necrotic areas, causing the affected plants to wither and eventually die. Ten disease samples were collected from different plants in the cultivation area to isolate the pathogens. The infected tissues were rinsed with sterile water, then disinfected with 75% ethanol for 90 seconds, followed by 3% hydrogen peroxide for 30 seconds, and finally rinsed three times with sterile water. The were transferred to water agar plates and incubated at 28°C for 3 days. Emerging hyphal tips were subcultured onto potato dextrose agar plates and incubated at 28°C in the darkness for 3 days in the dark. Eventually, 15 purified fungal isolates were obtained. Morphological assessment revealed that 11 isolates (73.3% isolation rate) exhibited characteristics consistent with the genus Alternaria. Three representative isolates (BLG-5, BLG-7, BLG-9) were selected for further study. The initial colonies were dark green with white fluffy hyphae on the surface, and the outermost edge showed a distinct white margin. Mature colonies turned blackish-brown and developed dense, cottony aerial hyphae. The conidia were light brown, elongated or ellipsoidal, with 0 to 5 transverse septa and 0 to 2 longitudinal septa, measuring 6.102 to 26.844 µm by 3.638 to 10.293 µm (n = 100). Based on the colony and spore morphology, the isolates were tentatively identified as belonging to the Alternaria genus. To further confirm the identity of the isolates, genomic DNA was amplified and sequenced for the internal transcribed spacer (ITS), translation elongation factor (TEF), and RNA polymerase II second largest subunit (RPB2) genes (Ramirez et al. 2023). The sequences were submitted to GenBank (BLG5: ITS, PV544773; RPB2, PV763591; TEF, PV763590; BLG7: ITS, PV717331; RPB2, PV890566; TEF, PV890565; BLG9: ITS, PV717332; RPB2, PV890568; TEF, PV890567). A combined phylogenetic tree was constructed based on the concatenated sequences of the three genes, and the results showed that the isolates were significantly clustered with Alternaria alternata. Pathogenicity tests were conducted on four healthy plants. For the experimental group, three young leaves were selected and inoculated with 6 × 6 mm PDA blocks moistened with spore solution, while sterile PDA blocks were used for the control group. The treated plants were placed in an incubator at 28°C and 80% humidity, with 12 hours of light per day for 7-10 days. Field-similar symptoms appeared on the leaf surfaces of the inoculated plants. Fungal strains re-isolated from these lesions exhibited colony characteristics identical to the original isolates. DNA sequencing confirmed them to be A. alternata, fulfilling Koch's postulates. A. alternata has been shown to cause black spot disease on Diospyros kaki L. (Xiang et al. 2023), Rosmarinus officinalis L. (Huang et al. 2023) and Ophiopogon japonicus (Xu et al. 2022). To our knowledge, this is the first report of A. alternata causing black spot disease in I. indigotica. These findings form the basis for the management of this leaf spot disease.