First report of Alternaria alternata causing leaf spot on Saposhnikovia divaricata in China

Fangfeng (Saposhnikovia divaricata) is a perennial plant belonging to the Umbelliferae family, and is widely cultivated as a traditional Chinese medicine plant used to treat various diseases in northern China. In August 2022, a widespread leaf spot disease emerged on the Fangfeng leaves across a 2.5-acre farmland located in the Naiman District of Tongliao City, China ( 44°17' N; 121°29' E), where 5,000 acres of Fangfeng had been cultivated. The incidence of the disease approached 100% in the two-year-old Fangfeng crops within the specific fields. Small, pale yellow spots were observed on the affected leaves of the Fangfeng plant. Diseased tissue pieces (5 mm) from symptomatic leaves were sterilized in 75% ethanol for 5 s and 1% NaClO for 25 s, then rinsed three times with sterilized distilled water and placed on potato dextrose agar (PDA) at 26°C in the dark for 7 days. Thirty-three isolates were obtained from 11 diseased leaf samples, representing one fungal species. The cultures had a dark olive color on both sides, accompanied by the presence of loose, cottony mycelium on the surface (Simmons, 1992). The conidiophores were light brown with clear separation, solitary or in clusters and ranged from 5.5 to 44.4 μm. Conidia are obpyriform with a cylindrical beak ( 0 to 22 µm in length) in short chains on Potato Carrot Agar (PCA). Conidia averaged 15.3 × 47.4μm with one to five transverse septa and zero or two occasional longitudinal septa. It is similar to those described as Alternaria sp. based on morphological characteristics (Simmons, 2007). The ITS, TEF1 and RPB2 gene fragments of isolates FFYB2 and FFYB4 were amplified with the primer pairs ITS5/ITS4, EF1-728F/EF1-986R and RPB2-5F/RPB2-7cR (White et al, 1990; Carbone and Kohn, 1999; Berbee et al., 1999; Liu et al., 1999) and submitted to GenBank (ITS, PP976058 and PP976059; TEF, PQ096003 and PQ096004; RPB2, PQ120838 and PQ120839). The three gene sequences ITS+ TEF1+ RPB2 were aligned in Clustal X 1.83 with equal weight and a phylogenetic tree was constructed in Mega 7.0.26 software (He et al., 2021). Finally, it was identified as A. alternata based on morphological and molecular characteristics. 36 Fangfeng leaves from six 8-month-old plants were utilized to assess the pathogenicity of the FFYB2 isolate, encompassing the control. All of PDA plugs (0.5 cm in diameter) colonized with the isolate and without the isolate were placed on the pinholes by a sterilized needle of the Fangfeng leaf surface sterilized with 75% alcohol-tampon, then removed after 48 h. On the fifth day, brown lesions (5 mm) appeared on 15 out of 18 Fangfeng leaves inoculated. No lesions were observed on 18 control leaves inoculated with PDA plugs. The fungi were re-isolated from the new lesions after a 7-day post-inoculation period, utilizing the aforementioned method, and their morphology was found to be identical to that of Alternaria sp.. Although A. divaricata was previously identified as a pathogen in Badong County (30°28'N; 110°29'E) of Yichang City, Hebei Province, China (He et al., 2021), the research results were deficient in providing details on the severity, symptoms, pathogenicity tests of the disease, and other relevant aspects. To our knowledge, this is the first report of A. alternata causing Fangfeng leaf spot in China. Fangfeng Alternaria leaf spot has caused severe economic losses in the region. Effective management strategies for this disease were urgently needed.