In November 2009, cyclamen (Cyclamen persicum) plants with disease symptoms from a commercial greenhouse operation in the western part of North Carolina were sent to the Plant Diseases and Insect Clinic at North Carolina State University. Symptoms consisted of coalescing reddish and tan necrotic leaf spots with concentric circles. Other symptoms included darkened vascular tissue and decay of the corm, large roots, and petioles. Diseased leaves and stems were surface sterilized in 0.5% sodium hypochlorite for 3 min, air dried, and placed in petri dishes containing alkaline water agar. After 3 days of incubation at room temperature, fungal colonies were transferred to acidified potato dextrose agar. Isolation frequency after 5 days was 33% (three of nine pieces) and 16% (one of six pieces) from small leaf spots and petioles, respectively. Pure cultures of isolates were gray and black with abundant, aerial, gray whitish mycelia. Diseased plants were also incubated in a moist chamber at room temperature and sporulation was observed within 7 days. Conidia were tapered with rounded ends and produced in the acervulus and on the tips of setae, which is consistent with the morphology of described isolates of Colletotrichum fragariae. Similar setae were also observed directly on the fine roots of the original sample. The pathogenicity of single-spore cultures was tested by spraying four 2-month-old cyclamen plants with a conidial suspension (106 conidia/ml) and the plants were kept in a humid chamber for 24 h. Noninoculated controls (four plants) were sprayed with distilled water and subjected to the same conditions. The pathogenicity test was also repeated. Inoculated plants and controls were placed in a greenhouse with a temperature range from 22 to 25°C. After 7 to 10 days, symptomatic leaves and stems were observed on all the inoculated plants but not on the control plants. Fungi reisolated from 10 symptomatic leaf tissues had identical morphological features as the original isolates. Fungal DNA was extracted with DNeasy Plant Mini DNA Extraction Kits following the manufacturer's protocol (Qiagen Inc., Valencia, CA). Sequence analysis of the rRNA internal transcribed spacer (ITS) region of the cyclamen isolate (GenBank Accession No. HQ188923), based on the fragment amplified with ITS1 and ITS4 primers, showed 100% similarity to isolates of C. fragariae deposited in GenBank (Accession Nos. FJ172290 [ATCC MYA-4443 from cyclamen] and FJ810510 [ATCC MYA-4442 from silver date palm]) and Florida isolate C16 isolated from strawberry (1). In addition, the morphology and ITS sequences of the cyclamen isolate were identical to those of the C. fragariae voucher isolate from strawberry (GU174546). Results from disease symptoms, colony and spore morphology, pathogenicity tests, and ITS sequence analysis suggest that C. fragariae was the pathogen responsible for the disease symptoms on cyclamens. To our knowledge, this is the first report of a disease caused by C. fragariae on cyclamen in North Carolina and complements an earlier report from Florida (1).
Reference: (1) S. J. MacKenzie et al. Plant Dis. 92:1432, 2008.
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