Leaf spot of Acorus calamus var. angustatus Caused by Alternaria alternata in Anhui Province, China

Acorus calamus var. angustatus Besser, a perennial herb of the Araceae family, was first reported in the Shen Nong's Herbal Classic and is widely distributed in southern China (Li 1979). It is important in traditional Chinese medicine for treating heart, stomach, and brain ailments (Lam et al. 2016). In March 2024, leaf spots were found on its leaves in a traditional Chinese medicine planting base in Yuexi County (30°91′56″ N, 116°19′24″ E), Anhui Province, with an incidence of about 35%. The symptoms began as small, light brown lesions that expanded, resulting in necrotic lesions ranging from 1 to 8 mm in diameter with brown halos. 3 × 3 mm sections, including both symptomatic and asymptomatic tissues, were cut from six infected plants. They were disinfected in 75% ethanol for 30 s, washed three times with sterile distilled water, transferred to Petri dishes containing potato dextrose agar (PDA) and incubated at 25 °C in the dark. Purified fungal isolates were obtained by the single-spore isolation method. The colonies on PDA initially appeared white, gradually became olive-green with 1 to 3 mm white margins and abundant aerial hyphae, while the reverse was greyish green to black. The conidia were light brown, ellipsoidal, obclavate, and 10.0 to 49.5 µm × 4.5 to 16.4 µm (mean 23.9 × 10.2 µm, n=50) in size, with 0 to 6 transverse septa and 0 to 6 longitudinal or oblique septa (n=50). Conidiophores were thick, dark brown, single-celled, with multiple conidial scars, measuring 12.8 to 146.8 × 2.9 to 6.1 (mean 50.6 × 4.2) µm (n=50). Based on above observations, the pathogen were identified as Alternaria spp. (Simmons 2007). Three representative isolates, SCP-1, SCP-2, and SCP-3 were selected for molecular identification. The Internal transcribed spacer (ITS), Alternaria major allergen (Alt a 1), and translation elongation factor 1-alpha (TEF1) genes were amplified with the primers ITS1/ITS4 (White et al. 1990), Alt-for/Alt-rev (Woudenberg et al. 2015) and EF1-728F/EF1-986R (Carbone and Kohn 1999), respectively. The sequences of the three isolates were consistent, and the sequences of isolate SCP-1 were submitted to NCBI GenBank (ITS, PP723104; Alt a1, PP708704; TEF1, PP708703). The ITS region of isolate SCP-1 was 100% similar to A. alternata TCS3002 (MN394880, Wang et al. 2023), the Alt a 1 gene was 100% similar to A. alternata CBS 620.83 (KP123868), and the TEF1 gene was 100% similar to A. alternata CBS 916.96 ex-type (KC584634). A phylogenetic tree based on sequences of ITS, Alt a 1 and TEF1 genes was constructed using the neighbor-joining method in MEGA 7 software, confirming the fungus as A. alternata. Three healthy plants of A. calamus var. angustatus were spayed with conidial suspension (1 × 10 7 conidia/ml) of isolate SCP-1. Three additional plants sprayed only with sterile distilled water were controls. All plants were covered with plastic bags to maintain a relative humidity of 90% for 48 h and incubated at 25 °C under a 12-h light photo-period. Twelve days post-inoculation, brown necrotic lesions developed on the inoculated leaves, enlarged, and the symptoms were similar to the original ones. The control plants remained healthy. The fungus was re-isolated from the infected plants and confirmed by morphological traits and molecular methods, fulfilling Koch's postulates. To our knowledge, there are no other reports of this fungus on A. calamus var. angustatus in Anhui, China. This report will help identify the disease based on field symptoms and provide a basis for disease management strategies of A. calamus var. angustatus.