Occurrence of Frogeye Leaf Spot, Caused by Cercospora nicotianae, on Greenhouse Tobacco Transplants in Kentucky

Frogeye leaf spot (FLS), caused by Cercospora nicotianae, has traditionally been considered a minor disease occurring on older, low-growing leaves of tobacco (Nicotiana tabacum L.). Historically, FLS was even considered desirable by some burley tobacco grading experts as an indicator of crop maturity. In recent years, however, multiple cured crops grown in south-central Kentucky have been rejected by buyers due to excessive FLS. In May 2017, symptoms of FLS were identified for the first time in burley tobacco transplants grown in greenhouse floatbeds in Metcalfe, Allen, and Barren counties, the same region of Kentucky where FLS severity has vastly increased in recent years. Lesions were characterized by a tan-white center surrounded by a narrow brown ring with a chlorotic halo. Black sporulation was macroscopically visible in lesion centers under high humidity. Incidence of FLS was 0.5 to 5% of transplants, with a maximum of 10% severity on the total leaf area of an individual transplant. Diseased transplants were seeded between 1 and 23 March; transplant lots seeded outside these dates were asymptomatic. Varieties affected were KT 209, KT 212, and HB 4488. Symptomatic leaves were collected from three farms. Lesions were washed, surface disinfested with 0.06% sodium hypochlorite, and incubated for 48 h to induce sporulation. A single conidium was transferred to acidified 1/4-strength potato dextrose agar. Five to seven days later, isolates were transferred to V8 agar to induce sporulation. Average conidia size for isolates 17CN011, 17CN015, and 17CN021 was 118.2 × 3.8, 112.7 × 3.8, and 120.2 × 3.6 µm, respectively. Gray, multiseptate, filiform conidia were somewhat flexuous when dislodged from conidiophores, with septations increasing in frequency at the basal end. Conidiophores were dark and simple, arising in fascicles of three to 12. Average conidiophore size for isolates 17CN011, 17CN015, and 17CN021 was 168.8 × 3.7, 158.7 × 3.9, and 173.2 × 3.8 µm, respectively. Morphology was consistent with Cercospora spp. descriptions (Barnett and Hunter 1999), particularly C. nicotianae (Hsieh and Goh 1990). To confirm pathogenicity, three county-representative isolates described above were grown individually on V8 agar at 22 to 24°C in a 12-h photoperiod with equal parts fluorescent and black light. After 10 days, 40 mycelial plugs were harvested from each culture using a #5 cork borer. Plugs were combined with 30 ml sterile deionized water in a sterile 50-ml tube and agitated using a vortex for 30 s. Approximately 25, 6-week old, ‘KY14’ tobacco plants grown in a mini float tray were spray-inoculated with 30 ml of 2.5 × 10⁴ conidia/ml, which was repeated for each isolate. Another 25 plants in a mini float tray were sprayed with sterile water as a negative control. Inoculated plants in their respective trays were incubated in clear plastic bags on a greenhouse bench at 24 to 25°C with ambient light for 4 days, then bags were removed. Small chlorotic spots were visible 7 days postinoculation on plants sprayed with isolates 17CN011, 17CN015, or 17CN021; lesions enlarged over the next week. Disease incidence on inoculated plants was 32 to 60%; leaf severity was 5 to 20%. Negative controls were asymptomatic. Symptomatic leaves were incubated in Petri dishes at 22 to 24°C under equal parts fluorescent and black lights for 48 h to induce sporulation. Excised lesions were microscopically examined to measure conidia and conidiophores for successful confirmation of C. nicotianae, completing Koch’s postulates. Though FLS incidence was low (<5%) in all transplant lots sampled, FLS has never been documented on burley tobacco at this young growth stage. In addition, FLS occurring in transplants likely increases the risk of pathogen population resistance to azoxystrobin due to exposure to this fungicide in the greenhouse. Azoxystrobin is the only systemic fungicide labeled for fungal leaf spot management in greenhouse and field tobacco production in the U.S.