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Colletotrichum cymbidiicola Causes Leaf Spot of Cymbidium ensifolium in Jilin Province, China

Liu, Y. N., Mao, F. R., Zhang, H., Wang, L. B., Zheng, S. J., Wang, J. F., Lou, Q., Liu, J. L.
Plant disease 2018 v.102 no.7 pp. 1462
Colletotrichum, Cymbidium ensifolium, actin, agar, anthracnose, asci, ascospores, conidia, conidiophores, culture media, ethanol, fungi, gardening, glyceraldehyde-3-phosphate dehydrogenase, greenhouses, herbaceous plants, humidity, internal transcribed spacers, leaf spot, leaves, pathogenicity, perithecia, photoperiod, plastic bags, sodium hypochlorite, solar radiation, tubulin, China
Cymbidium ensifolium (L.) Sw. is a perennial herbaceous plant with high gardening and herbal value. In August 2014, a severe anthracnose disease was observed on C. ensifolium leaves in Changchun (125.24°E, 43.48°N), Jilin Province, China. More than 80% of the plants were symptomatic. In the early stage of disease development, small brown lesions (1 mm in diameter) were visible on infected leaves, which subsequently developed into oval spots and turned light brown in the center. Lesions ranged from 5 to 15 mm in diameter. As lesions enlarged and coalesced, some leaves dried up. When the humidity was high, the surfaces of the lesions were covered with dark brown acervuli. Small pieces of infected leaves were surface-sterilized in 75% ethanol for 60 s, sterilized for 1 min in 1.5% NaClO, rinsed twice with sterilized distilled water, and plated on potato dextrose agar (PDA) in Petri dishes at 25 ± 1°C for 7 days. Ten fungal isolates were transferred from PDA to synthetic nutrient-poor agar (SNA). White colonies were observed in 2 days with smooth margins, and they covered the Petri dishes after 7 days. Perithecia (n = 100) were brown to dark brown, globose to subglobose, and measured 136.24 to 154.5 × 145.42 to 236.68 µm. Asci and ascospores were absent. Conidiophores and setae were formed directly on the media. Conidiophores (n = 100) were cylindrical, septate, branched, and measured 5.35 to 18.28 × 2.43 to 6.56 µm. Conidia (n = 100) were hyaline, aseptate, smooth, cylindrical, contents granular, and were 12.21 to 18.42 µm in length, and 4.40 to 7.90 µm in width. Setae (n = 100) were brown, cylindrical to conical, measuring 40.62 to 160.58 µm in length, and were 1 to 4 septate. Based upon these morphological and cultural characteristics, the causal agent was identified as Colletotrichum cymbidiicola. Five sequences of the internal transcribed spacer (ITS, KX058529), glyceraldehyde-3-phosphate dehydrogenase (GAPDH, KX068532), actin (ACT, KX058531), and beta-tubulin (TUB, KX058530) regions were obtained and deposited in GenBank. BLAST search results revealed that all sequences showed 99% identity with C. cymbidiicola sequences. Pathogenicity of 10 isolates of C. cymbidiicola from C. ensifolium were confirmed by inoculating healthy leaves of C. ensifolium plants with a spore suspension (1 × 10⁶ spores/ml) produced on PDA. Each isolate was inoculated onto five plants and another five healthy plants were sprayed with sterilized water as controls. All plants were enclosed in plastic bags for 48 h in a greenhouse at 28°C and 12 h/day light cycle. After 7 days, inoculated plants showed similar symptoms to those on the original diseased plants, while control plants remained symptomless. C. cymbidiicola was successfully reisolated from symptomatic plants to fulfill Koch’s postulates. C. cymbidiicola causing disease was reported on cymbidium in Australia, New Zealand, Japan (Damm et al. 2012; Hou et al. 2016), and India (Cannon et al. 2012). To our knowledge, this is the first report of C. cymbidiicola on C. ensifolium in China.