Phenotypic and molecular characterization of fenhexamid resistance in Botrytis cinerea isolates collected from pistachio orchards and grape vineyards in California

Abstract

Botrytis cinerea causes Botrytis blossom, shoot and fruit blight in pistachio and gray mold in grape. The hydroxyanilide (Hyd) fenhexamid, one of the most frequently used fungicides for Botrytis control, inhibits the 3-ketoreductase (Erg27) of the ergosterol biosynthesis pathway. Due to its site-specific mode of action, this fungicide is at-risk for resistance development. In this study, 74 and 58 B. cinerea isolates, collected from Californian pistachio orchards and grape vineyards, respectively, were evaluated for their in vitro sensitivity toward fenhexamid. Of the 58 grape isolates, 90% were sensitive to fenhexamid (HydS, EC50 < 1 μg/ml) whereas the remaining 10%displayed low resistance to this fungicide (HydLR, 1 < EC50 < 5 μg/ml). Among the 74 pistachio isolates, 69, 14, 1, 5, and 11% displayed sensitivity (HydS, EC50 < 1 μg/ml), low (HydLR, 1 < EC50 < 5 μg/ml), weak (HydWR, 5 < EC50 < 10 μg/ml), moderate (HydMR, 10 < EC50 < 50 μg/ml), and high resistance (HydHR, EC50 > 50 μg/ml) phenotypes toward fenhexamid, respectively. The 3-ketoreductase erg27 gene was sequenced from all the 29 B. cinerea fenhexamid-resistant phenotypes (HydLR, WR, MR, HR) detected in this study. The resulting sequences were then compared to corresponding sequences obtained from 21 fenhexamid-sensitive isolates (HydS). Of the 6 HydLR phenotype from grape, 3 had a substitution of proline to serine at position 238 (P238S). No mutations were found in the remaining 3 HydLR nor in the 10 HydLR pistachio isolates. The only HydWR phenotype from pistachio presented a change at position 309 resulting the replacement of valine to Methionine (V309M). Among the HydMR phenotype from pistachio, two had F412S alteration while the other two possessed the L400F alteration at position 400. Highly resistant isolates (HydHR) carried a substitution of the phenylalanine at position 412, either serine (F412S) or isoleucine (F412I). Inoculations on detached grape berries showed that field rates of Elevate® 50 WDG (fenhexamid) controlled HydS and HydLR, but not the HydMR and HydHR phenotypes. The presence of B. cinerea fenhexamid-resistant phenotypes in pistachio and grape fields in California must be monitored continually and resistance management practices implemented as needed for sustained Botrytis blossom, shoot and fruit blight and gray mold controls with Elevate® 50 WDG.