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Boscalid Insensitivity Documented in Didymella bryoniae Isolated from Watermelon in Florida and North Carolina
Katherine L. Stevenson, University of Georgia, Tifton, GA 31793; Anthony P. Keinath, Clemson University, Coastal Research and Education Center, Charleston, SC 29414; Anna Thomas and David B. Langston, Jr., University of Georgia, Tifton, GA 31793; Pamela D. Roberts, University of Florida IFAS, Southwest Florida Research and Education Center, Immokalee, FL 34142; Robert C. Hochmuth, University of Florida IFAS, North Florida Research and Education Center, Suwannee Valley, Live Oak, FL 32060; and Allan C. Thornton, North Carolina State University, Sampson County Extension Center, Clinton, NC 28328
Corresponding author: Katherine L. Stevenson. firstname.lastname@example.org
Stevenson, K. L., Keinath, A. P., Thomas, A., Langston, D. B., Jr., Roberts, P. D., Hochmuth, R. C., and Thornton, A. C. 2012. Boscalid insensitivity documented in Didymella bryoniae isolated from watermelon in Florida and North Carolina. Online. Plant Health Progress doi:10.1094/PHP-2012-0518-01-BR.
Boscalid is a pyridine-carboxamide fungicide that inhibits fungal respiration by binding to the enzyme succinate dehydrogenase. A commercial mixture of 2:1 pyraclostrobin:boscalid (Pristine) has been used widely on watermelon (Citrullus lantanus) and other cucurbits to control gummy stem blight, caused by the fungus Didymella bryoniae (Fig. 1). Since 2007, isolates of D. bryoniae insensitive to boscalid have been found in Georgia (3), Indiana (1), and South Carolina (2). To investigate the possible existence of boscalid insensitivity in D. bryoniae on watermelon grown in neighboring states, sensitivity to boscalid was determined for isolates of D. bryoniae obtained from discrete lesions on watermelon leaves collected from fields in Florida and North Carolina in 2009 and 2010.
Most isolates were tested for sensitivity to boscalid using a mycelial growth assay on boscalid-amended medium (3). Technical grade boscalid was dissolved in acetone and added to autoclaved potato dextrose agar (PDA) to yield a concentration of 3.0 mg/liter boscalid. Control media contained 1.0 ml/liter acetone. Mycelial plugs from 1-week-old cultures on PDA were transferred to boscalid-amended and control medium. After 4 days incubation in the dark at 25°C, the diameter of each colony was measured and corrected by subtracting the diameter of the mycelial plug. Relative growth was calculated as the corrected colony diameter on boscalid-amended medium divided by the corrected colony diameter on control medium. Isolates with relative growth greater than 0.20 were considered insensitive to boscalid. In 2009, four of six isolates from Desoto, Hendry and Collier Counties in south Florida were insensitive to boscalid; 10 of 11 isolates from Suwannee Co. in north Florida were insensitive to boscalid; and all 10 isolates from Sampson Co. in eastern North Carolina were insensitive to boscalid. In 2010, 33 of 40 isolates from Suwannee and Lafayette counties in Florida were insensitive to boscalid; and 16 of 17 isolates from Sampson Co. in North Carolina were insensitive to boscalid.
In 2010, diseased leaves were collected from a field in Collier Co., FL, where Pristine had been applied that season and in previous years, but did not provide the level of efficacy achieved previously (Fig. 2). Eleven isolates of the pathogen were obtained from these leaves and tested for insensitivity to boscalid using a spore germination assay on boscalid-amended medium. Technical grade boscalid dissolved in acetone was added to autoclaved water agar to yield concentrations of 0.01, 0.1, 1.0, and 10 mg/liter boscalid. Control media contained 1.0 ml/liter acetone. Spore suspensions prepared from 10- to 14-day-old cultures grown on one-quarter-strength PDA were spread on media. After 24-h incubation at 24°C with a 16-h photoperiod, 100 conidia and 50 ascospores on each plate were examined microscopically. A spore was considered germinated if it had a germ tube longer than the length of the conidium or ascospore. Relative germination, calculated by dividing the number of spores that germinated on boscalid-amended medium by the mean number of spores that germinated on control medium, was used to calculate EC50 values. All 11 isolates had EC50 values ≥9.68 mg/liter, indicating that they were insensitive to boscalid.
Gummy stem blight can cause major losses in watermelons, particularly in wet years. Thus, documenting insensitivity to boscalid in North Carolina, Florida, and other major watermelon-producing states, is important so that growers know where they need to choose other fungicides. Our results provide evidence of a high frequency of insensitivity to boscalid in sampled fields. In place of Pristine, several triazole fungicides that were registered on cucurbits in 2009 and 2010 have been used to effectively manage gummy stem blight.
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2. Keinath, A. P., DuBose, V., and Walters, E. 2009. First report from South Carolina of boscalid-insensitive isolates of Didymella bryoniae on field-grown watermelon treated with boscalid-pyraclostrobin. (Abstr.) Phytopathology 99:S62.
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