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Genotyping of Benzimidazole-Resistant and Dicarboximide-Resistant Mutations in Botrytis cinerea Using Real-Time Polymerase Chain Reaction Assays

First author: Plant Function Research Center, Toyo University, Itakura, Oura-gun, Gunma 374-0193, Japan; second, third, and seventh authors: Faculty of Life Sciences, Toyo University, Itakura, Oura-gun, Gunma 374-0193, Japan; fourth author: Agricultural, Food and Environmental Sciences Research Center of Osaka Prefecture, Shakudo, Habikino, Osaka, 583-0862, Japan; fifth author: Kanagawa Prefectural Agricultural Research Institute, Kamikisawa, Hiratsuka, Kanagawa 259-1204, Japan; and sixth author: Plant and Microbial Metabolic Engineering Research Unit, Discovery Research Institute (DRI), RIKEN, Hirosawa, Wako, Saitama 351-0198, Japan.

Botrytis cinerea, an economically important gray mold pathogen, frequently exhibits multiple fungicide resistance. A fluorescence resonance energy transfer-based real-time polymerase chain reaction assay has been developed to detect benzimidazole- and dicarboximide-resistant mutations. Three benzimidazole-resistant mutations—¹⁹⁸Glu to Ala (E198A), F200Y, and E198K—in β-tubulin BenA were detected using a single set of fluorescence-labeled sensor and anchor probes by melting curve analysis. Similarly, three dicarboximide-resistant mutations—I365S, V368F plus Q369H, and Q369P—in the histidine kinase BcOS1 were successfully distinguished. Unassigned melting profiles in BenA genotyping assay resulted in the identification of a new benzimidazole-resistant BenA E198V mutation. This mutation conferred resistance to carbendazim as do E198A and E198K mutations. The isolates with BenA E198V mutation showed a negative cross-resistance to diethofencarb, but to a lesser extent than the E198A mutants. A survey of 210 B. cinerea field isolates revealed that most of benzimidazole-resistant isolates possessed the E198V or E198A mutation in the BenA gene, and the I365S mutation in the BcOS1 gene was also frequently observed in Japanese isolates. However, benzimidazole-resistant isolates with BenA F200Y or E198K mutations, which confer the diethofencarb-insensitive phenotype, were rare. Our BenA and BcOS1 genotyping is a rapid and reliable method that is suitable for monitoring the fungicide-resistant field population.

Additional keywords:Botryotinia fuckeliana.