Quantification of allele E198A in beta‐tubulin conferring benzimidazole resistance in Monilinia fructicola using real‐time PCR

BACKGROUND: Thiophanate-methyl, a member of the benzimidazole class of fungicides, is used in California to control brown rot of stone fruit caused by Monilinia fructicola (G. Wint.) Honey. The goal of this study was to develop a real-time polymerase chain reaction (PCR) assay as an efficient method to quantify the E198A allele of beta-tubulin that confers benzimidazole resistance. RESULTS: Using the real-time PCR assay, the frequency of allele E198A (FEA) in a population was determined from the quantities of DNA amplified with the E198A allele-specific primer pair HRF/HRR and the M. fructicola-specific primer pair MfF6/MfR6. The average proportions of highly resistant isolates determined with the conventional fungicide sensitivity method were within the range of average FEA values determined with the real-time PCR assay. We also determined the FEAs of M. fructicola populations sampled from 21 stone fruit orchards in California. Only one orchard showed a high FEA over 0.20, seven orchards had values between 0.01 and 0.1, and 13 orchards had values less than 0.01. CONCLUSION: The real-time PCR assay developed in this study provides a potentially useful tool to efficiently quantify benzimidazole resistance for large M. fructicola populations.