First Report of Leaf Spot of Orange Coneflower (Rudbeckia fulgida) Caused by a Phoma sp. in Italy.

Abstract

Rudbeckia fulgida (orange coneflower) is an herbaceous species (Asteraceae) grown in full sun in flower beds and borders in gardens. In the summer of 2009, a previously unknown leaf spot was observed on R. fulgida plants in three private gardens located near Biella (northern Italy). Leaves of infected plants showed extensive and irregular, dark brown, necrotic lesions that were slightly sunken with a well-defined border. Lesions initially ranged from 0.5 to 3 mm in diameter and eventually coalesced to cover the entire leaf, which curled without falling. At a later stage, stems were also affected, causing death of the plant. The disease affected 90% of plants. Dark brown pycnidia, 68 to 195 × 60 to 165 (average 135 × 117) μm in diameter, containing hyaline (light gray in mass), and ellipsoid, nonseptate conidia measuring 4.0 to 7.0 × 2.4 to 3.5 (average 5.4 × 3.0) μm were observed on symptomatic tissue. On the basis of these morphological characteristics, the fungus was related to the genus Phoma. Diseased tissue was excised from the margin of lesions, immersed in a solution containing 1% sodium hypochlorite for 2 to 3 s, rinsed in sterile distilled water, and then cultured on potato dextrose agar (PDA) medium. Fungal colonies initially produced a white mycelium that became greenish gray when incubated at temperatures ranging between 22 and 25°C under alternating daylight and darkness (13 h of light and 11 h of dark). After 14 days of incubation, unicellular, cylindrical or truncated cone-shaped, light brown chlamydospores measuring 6 to 12 μm in diameter developed in long chains. The internal transcribed spacer (ITS) region of rDNA was amplified using the primers ITS4/ITS6 and sequenced. BLAST analysis (1) of the 498-bp segment showed 100% homology with a sequence of a Phoma sp. (EF585395). The nucleotide sequence of our isolate was assigned GenBank Accession No. GU573979. Pathogenicity tests were performed by placing 100 ml of a water homogenate of mycelium (1 × 105 mycelial fragments/ml) obtained from 15-day-old PDA cultures of the fungus on leaves of three healthy 4-month-old potted R. fulgida plants. Three plants inoculated with a homogenate of PDA served as controls. Plants were maintained in a greenhouse, in a high humidity chamber for 7 days after inoculation, at temperatures ranging from 18 to 22°C and under high relative humidity conditions (70 to 90%). The first foliar lesions developed on leaves 7 days after inoculation, and after 10 to 12 days, 80% of leaves were severely infected. Control plants remained healthy. The organism reisolated on PDA from leaf lesions was identical in morphology to the isolate used for inoculation. The pathogenicity test was carried out twice. To our knowledge, this is the first report of the presence of a Phoma sp. on R. fulgida in Italy. Mycosphaerella ligulicola was reported on Rudbeckia sp. (2), while M. rudbeckiae and Phoma exigua have been reported on R. hirta (3). Currently, the economic importance of this disease is limited, but may become a more significant problem if the cultivation of this species increases.