First Report of Ramularia didyma Causing a Leaf Spot on Ranunculus (Ranunculus asiaticus) Hybrids in California

Ranunculus or Persian buttercups (Ranunculus asiaticus) are colorful, cool-season perennials or annuals grown as landscape bedding plants and for field-grown bulb and cut flower production in mild winter climates. In March of 2008, tan-to-brown lesions were observed on the foliage of containerized ranunculus growing in a greenhouse at a production and retail nursery in coastal San Mateo County, CA. Approximately 15% of the approximately 150 cv. Bloomingdale mixed shade ranunculus plants had leaf spot symptoms. Symptomatic plants were generally clustered together on two benches, with a double-flowered purple cultivar with picotee markings having the highest disease incidence and severity. Angular to round, necrotic lesions ranged between 0.25 and 1.0 cm in diameter and were often surrounded by chlorotic tissue. Whitish sporulation was observed on abaxial and adaxial sides of the larger lesions. Conidiophores were hyaline, aseptate, measured 45 to 75 × 3 to 5 μm (56 × 3.7 μm average), and were produced in fascicles on the leaf surface. One-celled fusiform to cylindrical conidia measured 22.5 to 30.0 × 9.0 to 15 μm (25.4 × 11.9 μm average) and two-celled conidia measured 26.0 to 47.5 × 10 to 14 μm (35.3 × 12.1 μm average). Most conidia were hyaline, although a few were pale brown. Morphologically, the fungus matched the description of Ramularia didyma Unger (1). Small (3-mm²) pieces were taken from the margin of the lesion, disinfested with 0.6% sodium hypochlorite for 2 min, and placed at room temperature on carrot piece agar. Colonies were white and grew slowly to approximately 3.5 cm in 25 days. No conidia were produced in culture. To conduct pathogenicity tests, the foliage of nine mixed-color ranunculus plants grown from tubers for 7 weeks at 16°C in a growth chamber were sprayed with an aqueous suspension of mycelia. Inoculum was produced by grinding five, 3.5-cm colonies on carrot piece agar in 120 ml of water with a mortar and pestle. The foliage of an equal number of plants was sprayed with water. Plants were incubated in a dew chamber at 20°C for 48 h in the dark and then moved into a growth chamber with a 12-h photoperiod where relative humidity was maintained at ~95% by placing plants over a tray of water and covering each plant group with a plastic tent. Small, angular spots developed on approximately half of the inoculated plants 20 days later when the fungus was reisolated from the lesions. No symptoms were observed on the noninoculated control plants. Sporulation was observed on diseased lesions following misting of plants and incubation in sealed plastic bags for an additional week. Pathogenicity tests were repeated with mycelia with similar results. Sequence of the internal transcribed spacer regions of the rDNA was deposited into GenBank (Accession No. HQ442297). The sequence matched R. coleosporii and R. carthami with 96% identity. R. didyma has been reported to cause a leaf spot on Ranunculus spp. in the United States (Delaware, Iowa, Indiana, Maryland, New York, and Vermont). To our knowledge, this is the first confirmed report of R. didyma on Ranunculus asiaticus in California. Introduction of this pathogen into commercial production fields could cause significant economic loss. The closest large-scale production fields are located approximately 430 miles south of San Mateo County near Carlsbad, CA where more than 50 acres of ranunculus are grown for cut flower and tuber production. Reference: (1) J. B. Ellis and B. M. Everhart. J. Mycol. 1:79, 1885.