First report of curvularia blight on sunflower caused by Curvularia aeria in Mexico

Sunflower (Helianthus annuus) is an annual herbaceous flowering crop grown for its edible and ornamental uses. During the summer season in August 2016, brown spots on leaves and black lesions on stems were observed on sunflower (var. Sunbright) plants submitted by an ornamental business to the plant diagnostic clinic in Homestead, Florida. The samples were from Mérida, the state of Yucatan in Mexico. Approximately 70% of the plants were symptomatic and the average disease severity on a plant basis was 10%. Affected leaves initially exhibited small (2 to 10 mm in diameter) irregular brown spots on the upper surface. The spots enlarged and coalesced to form large areas of necrosis resulting in blight of leaves after 12 to 14 days. Initial symptoms on stems were eye-shaped light brown to black lesions that coalesced to form large areas of diseased tissues. Symptomatic tissues were cut into small pieces (5 mm), disinfected in 70% ethanol for 30 s, surface-sterilized in 10% Clorox (0.6% sodium hypochlorite) for 1 min, and rinsed in sterile distilled water for 1 min before the tissue was placed onto potato dextrose agar (PDA). A fungus was consistently isolated and the fungal colonies grew up to 7 cm (diameter) when the tissue was incubated on PDA at 25°C in the dark for 7 days. The colonies were black with velvety texture and smooth margins. Conidiophores were brown, unbranched, septate, and geniculate at the apical region. Conidia were straight to pyriform in shape, smooth-walled, transversely septate with mostly three septa. Conidia, 15.4 to 22 × 8.8 to 11 (average 18.8 × 9.9 µm, n = 50) in size, were produced apically in a sympodial mode and pale brown to brown in color with the middle two cells being darker than the end cells. DNA fragments of the internal transcribed spacer (ITS) region and the glyceraldehyde-3-phosphate dehydrogenase (GPD) gene were amplified using the universal ITS1/ITS4 (White et al. 1990) and GPD1/GPD2 primer pairs (Berbee et al. 1999), respectively. The resulting sequences were deposited in GenBank with accession numbers MF101868 for ITS and MF101867 for GDP. BLAST analysis revealed that the ITS partial fragment had 100% identity to Curvularia aeria isolates (KU856631.1, KP340066.1). The sequence of GPD gene showed 100% identity only with a C. aeria isolate (KU552162.1). Based on its morphological and molecular features, this fungus was identified as C. aeria (Nakada et al. 1994). To confirm pathogenicity, the foliage of 4-week-old sunflower (var. Sunbright) plants were inoculated by spraying a suspension of C. aeria conidia (1 × 10⁵ conidia/ml). The control plants were sprayed with sterile water. All plants were covered with clear plastic bags and maintained in a greenhouse overnight for 16 h at 28 ± 2°C. Then the plants were placed on benches in the same greenhouse after the plastic bags were removed. Symptoms similar to the original observations appeared on all inoculated plants 5 days later but no symptoms were observed on the control plants. The same fungus was reisolated from the lesions, and confirmed by morphology and molecular identification as stated above. Curvularia blight, caused by C. aeria, has been previously found on the medicinal and aromatic plant Etlingera linguiformis in India (Kithan and Daiho 2014). To our knowledge, this is the first report of Curvularia blight on sunflower caused by C. aeria in Mexico and in the world. The finding is important because this report will extend the host range of C. aeria to sunflower.