First Report of Occurrence of Benomyl Resistance in Botrytis cinerea Isolates on Raspberry in Serbia

Botrytis fruit rot, caused by Botrytis cinerea, is one of the major diseases limiting production of raspberries (Rubus idaeus) in Serbia. Yield losses in commercial fields can exceed 50%, especially during periods of rainy, wet weather before harvest. Development of resistance to fungicides with site-specific modes of action is a serious problem in the control of B. cinerea worldwide. To insure the longest possible useful life of a fungicide, an early detection of shifts of sensitivity in pathogen population is crucial (1). The goal of this study was to evaluate sensitivity of B. cinerea isolates from commercial raspberry fields in Serbia to several fungicides that are frequently used: vinclozolin, benomyl, pyrimethanil, and fenhexamid. Initial isolation was done from sporulating berries during harvest. Single-spore isolates were identified based on colony and conidial morphology and by PCR amplification of an expected 0.7-kbp DNA fragment using B. cinerea-specific primer pair C729+/729- (3). Sensitivity of 130 isolates from six localities (20 to 30 isolates per locality) was determined on potato dextrose agar (PDA) amended with fungicides at discriminatory concentrations (1 and 10 mg/liter). Fungicides were suspended in sterile distilled water and added to autoclaved media that had cooled to 50°C. Inverted mycelial plugs (10-mm diameter), which had been cut from the edge of 4-day-old colonies on PDA, were placed on fungicide amended media and incubated for 48 h at 20°C. Treatments were replicated four times and the experiment repeated once. Strain SAS 56, which is sensitive to benzimidazoles and dicarboximides, and strain SAS 405, which is resistant to these fungicide classes, originating from German Collection of Microorganisms and Cell Cultures, were used as standards in the experiment. Isolates that did not grow at 1 mg/liter were designated as sensitive, those that grew at 10 mg/liter were considered highly resistant, and those that grew at 1 mg/liter but not at 10 mg/liter were classified as weakly resistant to all fungicides tested. Values of EC50 for all highly resistant strains were determined in radial growth experiments on PDA supplemented with a range of concentrations (5,000, 2,500, 1,000, and 500 mg/liter) of benomyl or thiophanate-methyl, according to the method described by Leroux and Gredt (2). All tested isolates were sensitive to vinclozolin, pyrimethanil, and fenhexamid. Nine of 130 isolates were highly resistant to benomyl with EC50 values between 1,056 and 1,523 mg/liter. The reference strain SAS 56 had an EC50 value of 0.17 mg/liter, compared to an EC50 value for SAS 405 strain of 1,548 mg/liter. All benomyl resistant isolates were also resistant to thiophanate-methyl and EC50 values ranged from 2,328 to 7,699 mg/liter. To our knowledge, this is the first report of benomyl resistance in isolates of B. cinerea on raspberry in Serbia. References: (1) H. Ishii. Jarq 40:205, 2006. (2) P. Leroux and M. Gredt. Page 1 in: Laboratoire de Phytopharmacie, INRA, Versailles, 1972. (3) S. Rigotti et al. FEMS Microbiol. Lett. 209:169, 2002.