Cymbidium sinense are among the most important commercial orchids cultivated in China for flower production. In April of 2010, a leaf spot was sporadically observed on C. sinense in fields located in Guangzhou, China. Symptoms first appeared as yellow to brown, irregular-shaped lesions on the leaf margin or tip. As the infection continued on the tissues, the spot expanded and became dark brown along the margins and developed gray brown centers. At later stages, numerous epidermal acervuli developed on the lesions and mucilaginous conidial masses appeared on the lesions under moist conditions. Ten samples from tissue along the margins of lesions were collected and surface sterilized by washing in 70% ethanol for 30 s, followed by washing in 1% sodium hypochlorite for 30 s, and rinsing in sterile distilled water. These samples were plated onto potato dextrose agar (PDA) and incubated at 25°C with a 12-h alternating light and dark cycle. After 5 days, fungal colonies that grew from the tissue were subcultured onto PDA and pure cultures were obtained using the single-spore method. The fungus was identified as Colletotrichum gloeosporioides based on typical cultural characteristics and conidial morphology (1). PDA cultures were white at first and subsequently became grayish white to gray and pink to reddish brown. Conidia were straight, one-celled, hyaline, oblong, or cylindrical, slightly curved with truncate base and rounded apex and measured 14.0 to 19.5 × 4.0 to 6.0 μm. Chlamydospores, sclerotia, and a teleomorph were not found. Genomic DNA was extracted from one isolate and the internal transcribed spacer (ITS) region of the ribosomal DNA (ITS1-5.8S-ITS2) was amplified using ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) and ITS4 (5′-CCTCCGCTTATTGATATGC-3′) primers. The ITS region was further cloned and sequenced and showed 100% homology with many GenBank sequences (e.g., HQ333546.1) of C. gloeosporioides as the closest match. Pathogenicity tests were done by transferring one 4-mm-diameter disk of PDA that was colonized by the test isolates to wounds (4 × 4 mm) made with a needle in the leaves of 1-year-old C. sinense plants. Control plants received a sterile agar plug in wound. Ten inoculated plants were covered with plastic bags to maintain a high relative humidity and maintained in a greenhouse at 25 ± 2°C for 72 h. Five days after inoculation, no symptoms developed on the control plants. Foliar lesions closely resembled those observed in the field. C. gloeosporioides was reisolated consistently from symptomatic tissue collected from greenhouse experiments. To our knowledge, this is the first report of C. gloeosporioides causing anthracnose on C. sinense in China.
Reference: (1) B. C. Sutton. Colletotrichum Biology, Pathology and Control. CAB International, Wallingford, UK, 1992.