First Report of Leaf Spot of Atractylodes macrocephala Caused by Fusarium commune in Zhejiang province, China.

Abstract

Atractylodes macrocephala Koidz is a very common herbs in China, also famous for its high medicinal value (Lee et al., 2007). In summer of 2019, in Fuyang county of Zhejiang province, the main production area of China, 74 plants of A. macrocephala from a total of about 300 plants, showed black leaf spots . The incidence of the disease was 25% and increased under high temperature and humidity conditions. Initial leaf symptoms appeared as black or tan spots surrounded by brown margins and expanded irregularly. Finally, large blackish brown spots appeared on the leaves, elliptical or irregular, 1.0 to 1.5 cm in diameter, and then lesions turned necrotic. To isolate the pathogen, small pieces (5×5 mm) from the margin of symptomatic leaves were surface-sterilized with 75% ethanol for 30 s and 2% sodium hypochlorite for 2 min, rinsed five times with sterile water, and incubated on potato dextrose agar (PDA) at 28°C in darkness. Purified colonies were white to pink with densely floccose to fluffy aerial mycelium and peach-orange pigmentation. Macroconidia, usually three-septate, were 26.7 to 43.3×3.1 to 5.3μm (n=50), thin-walled, slightly curved, with apical and basal cells curved when cultured in continuous darkness. Microconidia were mostly aseptate, ovate-oblong, straight to slightly curved, and measuring 5.9 to 14.3×2.3 to 3.9μm in size (n=50). Spherical chlamydospores were produced singly or in pairs from mycelium and spores. These characteristics were consistent with the description of Fusarium spp. (Leslie and Summerell, 2006). To identify the species, the translation elongation factor-1 alpha regions (TEF-1α) and the mitochondrial small subunit (mtSSU) were amplified using primers EF-3/ EF-22 (Palmore et al.,2010 and O'Donnell et al., 1998) and MS3F/ MS3R (Stenglein et al., 2010), respectively. Sequences were deposited in GenBank (MT263720, OM203177, OM203178, OM203179, OM203180, OM203181 and MN853662, MZ028170.1, MZ028171.1, MZ028172.1, MZ028173.1, MZ028174.1). These six isolates clustered in the Fusarium commune clade with 100% and 98% similarity, respectively. To test pathogenicity of every isolate, five 8-week-old potted A. macrocephalae plants were wound-inoculated and mycelial discs of 5-mm diameter were used to inoculate. As a control, five plants were inoculated with 5-mm PDA plugs. All plants were individually covered with a plastic bag and kept in a greenhouse at 25 ± 2°C with a 12-h photoperiod at 70 to 80% relative humidity. Typical symptoms similar to those of the field appeared only in inoculated plants after five days. In addition, a conidial suspension (1×105 spores/ml) was sprayed onto young leaves of three potted healthy plants. Three plants sprayed with sterile distilled water served as controls. After 7 days, typical symptoms were observed on all inoculated leaves. Experiments were replicated three times. F. commune was successfully re-isolated from diseased plants. Based on morphological and molecular identification, the pathogen was identified as F. commune. In China, Alternaria alternata (Zhuang, 2005), A. longipes (Tan et al., 2012), Phyllosticta commonsii (Sang et al., 2006) and Phoma exigua (Zhang et al., 2018) were reported as causal agents of the leaf spot disease of A. macrocephalae. To our knowledge, this is the first report of leaf spot disease on A. macrocephalae caused by F. commune in China. Effective control strategies need to be established to reduce the losses.