First report of Curvularia lunata causing Curvularia leaf spot of corn in Delaware.

Abstract

In mid-August 2020, small circular tan-colored lesions were detected in leaves of corn, Zea mays, in Sussex county Delaware. Symptoms were observed in many fields, affecting multiple hybrids that were at approximately the dough growth stage (R4). Lesions were present on most plants and individual ear leaf severity ratings ranged from 3 to 14 %. Symptomatic corn leaves were selected from the field and kept in a moist chamber for 24 hrs at 25 °C. In lesions, melanized spores were observed ranging from 21.94 µm to 30.51 µm in length and 7.83µm to 11.70µm in width (n=20). One cm2 leaf sections were extracted and sterilized in a 0.85% sodium hypochlorite solution for 30s followed by a sterile water rinse for 30s. Leaf pieces were then plated onto potato dextrose agar amended with 50µg/ml penicillin-G-sodium salt and streptomycin sulfate. Petri dishes were incubated at 25 °C with a 12-hr photoperiod for 14 days. Colonies were brown-black to lighter gray in color. Media was stained from orange, to dark brown, or not at all. Conidia were melanized and curved with three transverse septa matching the size above. Colony and spore morphology were consistent with the description of Curvularia lunata (Garcia-Aroca et al. 2018). Pure cultures were obtained, and a representative isolate was sequenced to confirm fungal identity by amplifying the internal transcribed spacer (ITS) region with primers ITS4 and ITS5. BLAST search results confirmed 100% similarity (483/531 bp) to the C. lunata reference sequence (MK623264). The sequence was deposited in GenBank as accession number MW794323. To complete Koch's postulates, corn (hybrid: Hubner H6187RCSS) was planted into 10 cm pots and maintained at 25°C and a 12 hr photoperiod. A conidial suspension was made by soaking a Petri dish of a 3-week-old fungal colony with 20 ml of sterile water and a drop of Tween-20 solution then, scraping mycelia to dislodge spores. The conidial concentration was calibrated to 5 x 105 spores/ml and 15 ml were applied to each whorl of four three-collar stage plants using a Preval sprayer (Nakoma Products, Bridgeview, IL). Four plants were inoculated with 15 ml sterile DI water as a control. This experiment was repeated twice. Each plant was moved to an incubator and covered with a plastic bag for 24 hr to maintain humidity. Conditions in the incubator were maintained at 25 °C with a 12-hr photoperiod. Inoculated plants displayed small, oval-shaped lesions within four days. No symptoms were observed on the control plants. Symptomatic leaves were harvested and placed into a moist chamber for 24 hrs to sporulate and lesions were plated onto PDA as described above. Culture morphology was consistent with the original isolate, with spores slightly larger ranging from 22.35 µm to 39.29 µm in length, and 8.36 µm to 11.69 µm in width (n=20). Isolates obtained from inoculated plants were sequenced and maintained 100% identity with the reference sequence described above. C. lunata was first reported in Louisiana in 2017 (Garcia-Aroca et al. 2018) and in Kentucky in 2018 (Anderson et al. 2019). This is the first report of Curvularia leaf spot on corn in Delaware. Symptoms developed late in the season, so it is unlikely that yield was affected in 2020. However, the economic impact of this disease in the United States is still unclear, it will be important to monitor potential impacts of this disease in Delaware corn production.