First Report of Angelica keiskei Leaf Spots Caused by Alternaria alternata in China

Abstract

Ashitaba (Angelica keiskei) is a flowering plant native to Japan and planted in many Asian countries (Park 2013). It was introduced into China in the late 1990s, where its functional components, including chalcone, coumarin, and flavonoids, are found to promote human health. Consequently, ashitaba has been used in various herbal products, wine, tea, and other supplements (Wu et al. 2016). The current cultivated area is 150 to 200 ha in China. From May to August 2017, a severe disease was discovered in Wuhan, Hubei province, China. Symptoms appeared as dark brown, oval to irregular necrotic spots surrounded by a yellowish border on approximately 30 to 40% of plants. The disease is a potential risk for the development of the ashitaba industry. To identify the pathogen, 10 symptomatic leaves were collected from 10 individual ashitaba plants. Small pieces of tissue (5 × 5 mm) were cut from the lesion margin, surface disinfested in 75% ethanol for 30 s and 0.1% HgCl₂ for 30 s, rinsed twice in sterilized distilled water, plated on potato dextrose agar amended with streptomycin sulfate, and then incubated at 25°C in the dark. Ten fungal colonies (K1 to K10) displaying gray-brown and gray-white aerial mycelia were consistently isolated from leaf lesions. Single-spore cultures were obtained on potato carrot agar in the dark at 25°C for 7 days. Conidia (n = 100) were observed and ranged from 10.0 to 31.0 × 7.0 to 11.0 μm and had three to six transverse and zero to three longitudinal septa. Short conidiophores arose singly and were approximately 15 to 76 μm long by 3 to 6 μm wide. Morphological characteristics were similar to those described for Alternaria alternata (Fr.) Keissl (Simmons 2007). To confirm this identification, genomic DNA was extracted from single conidial cultures of a representative isolate K3, and internal transcribed spacer regions (ITS), translation elongation factor 1-alpha gene (TEF-1a), glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH), endopolygalacturonase gene (endoPGs), and histone gene (H3 genes) were amplified using primers ITS1 and ITS4, TEF-1a, GAPDH, endoPGs, and H3 genes (Woudenberg et al. 2015). The sequences (MG661807, MK210168, MK210169, MK210170, and MK210171) were compared on NCBI GenBank and showed 100% identity with A. alternata isolate CBS 916.96. The fungal specimens were kept in the Vegetable Disease Research Laboratory of the Institute of Economic Crops, Hubei Academy of Agricultural Sciences. Pathogenicity tests were performed by spraying apparently healthy leaves of 5-month-old A. keiskei plants with a spore suspension of 10⁶ conidia/ml. Plants sprayed only with water served as a control (Mehmood et al. 2018). Three pots (three plants per pot) were used for each treatment. Plants were covered with plastic bags for 4 days after inoculation and maintained in the same greenhouse at an average temperature of 28°C. Experiments were carried out twice. Lesions developed on leaves 7 to 10 days after the artificial inoculation, whereas control plants remained healthy. To fulfill Koch’s postulates, the fungus was reisolated from the diseased leaves and confirmed to be A. alternata based on morphological characteristics and sequence analysis. To our knowledge, this is the first report of A. alternata causing leaf spots on A. keiskei in China. According to the USDA-ARS Fungal Database, it might be also the first report on this host worldwide.