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First Report of Colletotrichum gloeosporioides sensu stricto Causing Anthracnose on Nandina domestica in Sichuan Province of China

Wang, T. T., Li, P. L., Li, H. W., Li, J., Gao, F., Liu, D., Yan, J. M., Gong, G. S.
Plant disease 2018 v.102 no.4 pp. 822
Euonymus japonicus, Glomerella cingulata, Nandina domestica, actin, anthracnose, appressoria, calmodulin, chitin synthase, conidia, culture media, genes, glyceraldehyde-3-phosphate dehydrogenase, greenhouses, hyphae, internal transcribed spacers, landscapes, leaf spot, leaves, mycelium, ornamental woody plants, pathogenicity, pathogens, phylogeny, plastic bags, sodium hypochlorite, spraying, trees, tubulin, China
Nandina domestica Thunb. is an evergreen ornamental shrub commonly used in both landscape and interior decoration. In September 2016, leaf spot symptoms were observed on N. domestica planted at Chengdu, Sichuan Province, China. Approximately 10% of the leaves exhibited symptoms of anthracnose. Symptoms began as small, light brown spots and then coalesced into larger, irregular lesions. The center of the lesions were pale gray and their borders black and surrounded by wide red areas. Most of the lesions dried and formed extensive necrotic areas that contained acervuli. Samples taken from the lesions were surface disinfected for 3 min in 5% sodium hypochlorite, rinsed in sterile water, and plated on potato dextrose agar (PDA). Single-spore cultures collected from several colonies were plated on PDA. After 5 days at 25 ± 2°C in the dark, the mycelium of a representative culture, NTZ, covered the entire plate surface (9 cm diameter). Hyphae were white at first and became dark with age. After 7 days, colonies showed abundant aerial, pale gray mycelium with a large number of pink conidia. The nonseptate conidia were hyaline, cylindrical, with two ends round or one end slightly acute, and ranged from 15.2 to 20.5 μm (mean 16.9 μm) × 4.7 to 5.9 μm (mean 5.3 μm) (n = 100). The conidial appressoria were ovoid to slightly irregular, brown, and ranged from 5.2 to 12.4 μm (mean 7.3 μm) × 3.6 to 6.5 μm (mean 5.0 μm) (n = 50). Based on these characteristics, isolates were tentatively identified as Colletotrichum gloeosporioides (Penz.) Penz. & Sacc (Cannon et al. 2008). Pathogenicity tests were conducted by spraying a conidial suspension of NTZ (1 × 10⁷ conidia/ml) to 10 wounded and 10 nonwounded leaves in total from 10 2-year-old N. domestica plants. Two cuticle areas on either side of the midrib were wounded by lightly scratching with a needle prior to inoculation (Langrell and Irvine 2001). As a control, distilled water was sprayed onto an equal number of wounded and nonwounded leaves. All inoculated and control plants sealed in plastic bags were incubated in a greenhouse (25 ± 2°C). Lesions similar to those observed in the field appeared on all wounded inoculated leaves within 7 days after inoculation, whereas all nonwounded inoculated leaves and controls remained symptomless. Reisolations of the putative pathogen were confirmed through morphological and cultural characteristics. The internal transcribed spacers, actin, glyceraldehyde-3-phosphate dehydrogenase, chitin synthase 1, beta-tubulin, and calmodulin genes were sequenced (Weir et al. 2012) and deposited in GenBank (accession nos. MF322895, MF322896, MF322897, MF322898, MF322899, and MF322900, respectively). BLAST analysis showed high identity with C. gloeosporioides sensu stricto (culture IMI 356878): accession nos. JX010152 (99%), JX009531 (99%), JX010056 (97%), JX009818 (100%), JX010445 (100%), and JX009731 (99%), respectively. All six gene sequences were analyzed together and a phylogenetic tree was generated via the neighbor-joining method using PAUP v4.0. The tree also placed the isolate in the group of C. gloeosporioides sensu stricto. A recent study reported that C. gloeosporioides caused anthracnose of many ornamentals, such as Euonymus japonicus (Huang et al. 2016). However, there are no reports of C. gloeosporioides causing anthracnose on N. domestica in China or worldwide (Farr and Rossman 2017). The identification of the causal agent of the disease make clear the pathogen causing anthracnose on N. domestica, and provide theoretical basis for the diagnosis and treatment of disease.