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First Report of Colletotrichum spaethianum Causing Anthracnose in Atractylodes japonica in China

Guan, Y. M., Liu, Z. B., Li, M. J., Wang, Q. X., Zhang, Y. Y.
Plant disease 2018 v.102 no.1 pp. 239
Atractylodes, Colletotrichum, actin, agar, anthracnose, appressoria, chitin synthase, chlamydospores, conidia, culture media, cutting, droplets, fungi, glass, glyceraldehyde-3-phosphate dehydrogenase, greenhouses, histones, internal transcribed spacers, landraces, leaves, medicinal plants, mycelium, oils, pathogenicity, photoperiod, phylogeny, plastic bags, relative humidity, ribosomal DNA, sodium hypochlorite, solar radiation, China
Atractylodes japonica Koidz. ex Kitam. (Compositae) is a perennial medicinal herb that is widely cultivated in China. In August 2012, anthracnose-like symptoms were observed on the leaves of a 2-year-old A. japonica landrace in Qingyuan County in northeastern China. The leaves exhibited irregular gray, sunken, necrotic lesions that were irregular in shape. The lesions developed a white center gradually and multiple lesions coalesced eventually causing leaf blighting. The incidence was as high as 32% in 2014. Symptomatic leaves (n = 25) were collected from three commercial fields: Changbai City, Jilin City, and Qingyuan County, in 2012 and 2014. Fungal isolates were obtained by cutting symptomatic leaves into 5-mm² pieces, surface-disinfesting them in 1% NaOCl for 2 min, rinsing with sterile distilled water for 90 s three times, and plating them on half-strength acidified potato dextrose agar (PDA) at 25°C (12-h photoperiod). Each isolate was induced to produce conidia under black light and purified from a single spore. Three isolates were randomly selected for morphological observation. When cultured on synthetic nutrient-poor agar medium (SNA), the colonies produced grayish white, cottony aerial mycelia. Setae were dark brown with an acute tip, 2- to 3-septate, and 26.5 to 81.2 μm long. Conidia were rare, hyaline, smooth-walled, slightly curved, aseptate, with a truncate base and acute apex, 16.0 to 25.0 × 3.1to 4.9 μm, with a length/width ratio of 5.1, and slightly narrower than those reported by Damm et al. (2009). Each conidium contained one oil droplet. Conidia and mycelia produced dark brown, irregularly shaped appressoria, with an average length/width ratio of 1.1. Chlamydospores were not observed. Identification was confirmed by sequencing the internal transcribed spacer (ITS) region of ribosomal DNA, the actin (ACT), and chitin synthase 1 (CHS-1) (Carbone and Kohn 1999), partial glyceraldehyde-3-phosphate dehydrogenase (GAPDH), and the histone 3 (HIS3) of the three isolates. The consensus sequences of the three isolates (GenBank accession nos. KT122846–48 for ITS, KT122849–51 for CHS-1, KT122852–54 for ACT, KT122855–57 for GAPDH, and MF362663–65 for HIS3) showed 99 to 100% identity with related sequences of Colletotrichum spaethianum in GenBank (NR_111451 for ITS, GU228297 for CHS-1, GU227905 for ACT, GU228201 for GAPDH, and GU228005 for HIS3). Phylogenetic analysis showed that they were grouped into the clade with C. spaethianum (Fig. 2). The pathogenicity of the three isolates was tested with mycelial fragments suspensions (grounded with a glass rod). Mycelial suspensions were sprayed onto leaves of the plants, plants sprayed with distilled water served as a noninoculated control, and every isolate was inoculated on two 2-year-old healthy plants in the greenhouse (28°C, natural light, and 90% relative humidity). Clear plastic bags were used to maintain high humidity. After 4 days, the inoculated leaves exhibited similar symptoms as the original field plants, and the control remained asymptomatic. The same fungus was reisolated and confirmed by morphological and molecular methods previously described. To our knowledge, this is the first report of anthracnose of A. japonica caused by C. spaethianum in China. The disease poses a threat to production of A. japonica, and management strategies need to be developed.