Resistance to Pyrimethanil and other Fungicides in Botrytis cinerea Populations Collected on Vegetable Crops in Spain

Forty-seven greenhouses of vegetable crops were surveyed in south-eastern Spain at the beginning of the epidemic (January 2000) to compare sensitivity of Botrytis cinerea populations to pyrimethanil (an anilinopyrimidine fungicide) after 4 years of treatment with an unexposed population from a 1992 collection. A standard method to test the sensitivity of B. cinerea to pyrimethanil in a defined minimal medium (1.75 g/ l of KH2PO4, 0.75 g/l of MgSO4, 4 g/l of glucose and 4 g/l of gelatine) was used to establish frequency distributions of pyrimethanil sensitivity in both populations. Two different distributions for sensitive and resistant isolates were obtained. ED50 of sensitive isolates in 1992 ranged from 0.05 to 0.5 mg a.i./l (mean ± SE, 0.23 ± 0.02), and 0.04–0.4 mg a.i./l in 2000 (0.11 ± 0.01). ED50 for resistant isolates ranged from 1 to 10 mg a.i./l in both surveys (5 ± 2.64 and 4.25 ± 2.14, in 1992 and 2000, respectively). No resistance build-up to pyrimethanil was developed in B. cinerea populations after exposition of 4 years to the fungicide. An in vitro monitoring procedure was developed based on testing on one single discriminatory dose of pyrimethanil (established at 0.7 mg a.i./l). Isolates resistant to pyrimethanil in the in vitro assay caused visible lesions on cucumber leaf discs treated with the fungicide. No significant differences in fitness (growth or sporulation) between resistant and sensitive isolates were obtained. The 307 isolates collected in January 2000 were tested in vitro using discriminatory doses to estimate the frequencies of resistance of B. cinerea to benzimidazoles (carbendazim), dicarboximides (procymidone), N-phenylcarbamates (diethofencarb), and anilinopyrimidines (pyrimethanil). Of the 307 isolates collected, 90% were resistant to benzimidazoles, 77% to dicarboximides, 23% to N-phenylcarbamates and 12% to anilinopyrimidines (in this case of 165 isolates). Dicarboximide and benzimidazole cross-resistant isolates were found in each of the surveyed greenhouses and accounted for 65.8%. Fourteen percentage of the population were resistant to dicarboximides, benzimidazoles and N-phenylcarbamates, and 3% were also resistant to anilinopyrimidines.