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Identity of Epicoccum sorghinum Causing Leaf Spot Disease of Bletilla striata in China

Zhou, H., Liu, P. P., Qiu, S., Wei, S. J., Xia, K., Gao, Q.
Plant disease 2018 v.102 no.5 pp. 1039
sodium hypochlorite, phylogeny, leaf spot, chlamydospores, ultraviolet radiation, greenhouses, conidia, internal transcribed spacers, pycnidia, mycelium, leaves, antimicrobial properties, sequence analysis, plastic bags, pathogenicity, Epicoccum, leaf abscission, photoperiod, statistical analysis, culture media, Epicoccum sorghinum, Bletilla striata, fungi, China
Bletilla striata (Thunb.) Rchb. f. (Orchidaceae), known as a traditional Chinese herb, has been widely used for hemostatic and antimicrobial properties. In May 2017, a leaf spot disease was observed on 20 to 30% of B. striata in commercial fields located in Ziyuan County, Guangxi, China. Small, circular, brown spots were observed early on leaves, enlarging and developing into irregular dark brown and necrotic lesions. Necrotic lesions caused leaf abscission and occurred frequently during the plant’s life cycle. Three symptomatic leaves from three individual plants were surface sterilized in a solution containing 1% sodium hypochlorite for 2 min, rinsed three times in sterile water, plated on potato dextrose agar (PDA) medium, and then incubated at 26°C for 3 days. Seven isolates recovered from diseased plants and the colonies were villose and regular, pale gray, secreting scarlet pigment to the media. After 15 days of incubation, subspherical chlamydospores were observed, which were brown to black, with verrucose surface. Unicellular chlamydospores were 12.9 to 29.3 × 13.5 to 32.7 μm while multicellular chlamydospores were 6.2 to 13.6 × 29.7 to 89.4 μm. Fungal colonies grown under ultraviolet light for 12 h with a distance of 60 cm produced numerous pycnidia and conidia on PDA. Brown pycnidia ranged from 90 to 240 μm in diameter. Conidia were hyaline, ellipsoidal, unicellular, aseptate, 5.0 to 7.1 × 2.9 to 3.7 μm in size. Based on the above morphological characteristics, these isolates were identified as Epicoccum sp. (previously as Phoma, Yuan et al. 2016). The identity of isolate was confirmed by sequence analysis of rDNA-ITS, TUB, and ACT of strain BJ-F1 (GenBank MF948994, MF987525, and MF987526, respectively). The primers of ITS, TUB, and ACT were respectively ITS1/ITS4, T1/Bt2b, and ACT-512F/ACT-783R (Carbone and Kohn 1999; Glass et al. 1995; O’Donnell et al. 1997; White et al. 1990). BLAST results showed that the rDNA-ITS gene sequence was 99% identical to those of Epicoccum sorghinum (= Phoma sorghina) in GenBank (KJ767080 and KY454466), while the TUB and ACT gene sequences were 100% homologous to those of the above species (KU728638 and KU728639). Further molecular phylogenetic analysis by maximum likelihood method based on the combined ITS-TUB-ACT sequences revealed that strain BJ-F1 was closest to E. sorghinum (previously as E. sorghi, Aveskamp et al. 2010). Pathogenicity tests were conducted on six young plants (∼1.5 years old) by inoculating unwounded leaves with three 5 × 5 mm mycelial discs of two isolates on PDA. Three control plants were inoculated with sterile PDA discs. All inoculated and control plants were enclosed in transparent plastic bags and incubated in a greenhouse at 26°C for 13 days with a photoperiod of 12 h. The first lesions appeared 3 days after inoculation on leaves, and typical symptoms developed 10 days later. Uninoculated controls remained asymptomatic. The experiments were repeated three times with similar results. To fulfill Koch’s postulates, the fungus was reisolated from the inoculated leaves on PDA, and confirmed as E. sorghinum. As far as we know, this is the first report of E. sorghinum causing leaf spot on B. striata in China.