Simultaneous quantification of alleles E198A and H6Y in the β -tubulin gene conferring benzimidazole resistance in Monilinia fructicola using a duplex real-time (TaqMan) PCR

Abstract

The benzimidazole fungicide thiophanate-methyl is commonly used for the control of brown rot of stone fruits. Low and high levels of resistance to this fungicide have been found in field isolates of the causal pathogen Monilinia fructicola. The minor groove binding (MGB) TaqMan probes specific for alleles E198A and H6Y conferring the high and low levels of resistance in the β-tubulin gene of M. fructicola were designed. A duplex real-time PCR assay based on these probes was developed for simultaneous quantification of both mutations in a pathogen population. The specificity tests for the primers and probes were conducted using different fungal species of stone and pome fruit pathogens. Similar results were obtained between the duplex real-time (TaqMan) PCR assay and the conventional method to quantify the frequencies of alleles E198A and H6Y of eight samples from different peach orchards. The MGB TaqMan probe based duplex real-time PCR provides a useful tool for simultaneous quantification of both alleles, E198A and H6Y, conferring high and low resistance, and has a potential in monitoring the benzimidazole-resistance in M. fructicola populations in stone and pome fruit orchards.