A Botrytis cinerea Population from a Single Strawberry Field in Germany has a Complex Fungicide Resistance Pattern

Gray mold, caused by the fungus Botrytis cinerea, is one of the most important diseases of strawberry in Germany. The application of site-specific fungicides remains the main strategy to reduce disease incidence and severity in the field. Isolates (n = 199) were collected from fungicide-treated strawberry fruit at a German research site with a long history of fungicide efficacy trials against gray mold. Sensitivities to the six site-specific botryticides registered in Germany were determined using microtiter assays. Values for the concentration of a fungicide at which fungal development is inhibited by 50% (EC50) ranged from 0.03 to ≥30 ppm for the succinate dehydrogenase inhibitor boscalid, 0.015 to ≥10 ppm for the hydroxyanilide fenhexamid, 0.009 to 0.739 ppm for the phenylpyrrole fludioxonil, 0.55 to 43.45 ppm for the dicarboximide iprodione, 0.021 to ≥3 ppm for the quinone outside inhibitor pyraclostrobin, and 0.106 to ≥30 ppm for the anilinopyrimidine pyrimethanil. Pyrosequencing revealed that amino acid substitutions in the target proteins Bos1 (I365S/N, V368F + Q369H), CytB (G143A), Erg27 (F412S), and SdhB (P225F, N230I, and H272R/Y) were associated with reduced sensitivity levels to the corresponding fungicide classes. In most cases, isolates with a decreased sensitivity to fludioxonil showed a reduced sensitivity to tolnaftate. This reduction is considered to be an indication of multidrug efflux pump activity. The amino acid change I365S, I365N, or V368F + Q369H in Bos1 and H272R in SdhB by itself showed EC50 values of 3.99 to 14.73 ppm, 3.87 to 5.37 ppm, 4.81 to 15.63 ppm, and 2.071 to ≥30 ppm, respectively. When isolates that contained one of these mutations were also multidrug resistant, the ranges of EC50 values shifted to 6.47 to 43.45 ppm for I365S, 7.28 to 29.84 ppm for I365N, 6.89 to 26.67 ppm for V368F + Q369H, and ≥30 ppm for H272R. The reported data suggest that the combination of multidrug resistance and an amino acid change in the target site may result in a lower sensitivity to the fungicides than one resistance mechanism by itself. Although 20% of the population analyzed was sensitive to all six different chemical classes, the majority showed reduced sensitivity to one (6%), two (13%), three (23%), four (17%), five (11%), and six (11%) different fungicides.