Occurrence and Detection of the DMI Resistance-Associated Genetic Element 'Mona' in Monilinia fructicola

Sterol demethylation inhibitor (DMI) fungicide resistance in isolates of Monilinia fructicola from Georgia has been linked to overexpression of the MfCYP51 gene and a corresponding 65-bp genetic element 'Mona' inserted in the upstream region of MfCYP51. In this study, a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was developed to detect the Mona element. Fourteen DMI fungicide-resistant (DMI-R) and six DMI fungicide-sensitive (DMI-S) isolates from Georgia, six DMI-R and 11 DMI-S isolates from South Carolina, seven DMI-R and nine DMI-S isolates from New York, and two DMI-R and three DMI-S isolates from Ohio were used in this study. The isolates from the southeastern United States and Ohio were collected from peach, whereas isolates from New York were collected from cherry. A 376-bp fragment containing the Mona element was consistently amplified with primer pair INS65-F and INS65-R from DMI-R isolates, and either a 311-bp or 1,815-bp fragment was amplified from DMI-S isolates. The primer pair did not amplify DNA fragments of similar sizes from isolates of five other common fruit rot pathogens of peach, including Alternaria alternata, Colletotrichum acutatum, Gilbertella persicaria, Penicillium expansum, and Rhizopus stolonifer. Gel electrophoresis of the PCR amplicon can distinguish between DMI-R and DMI-S isolates based on the 65-bp size difference of the amplicon; however, the restriction digestion assay can verify questionable results, especially in the absence of a positive control. Only the 376-bp fragment containing the Mona element was digestable with endonuclease BsrBI, resulting in two restriction fragments of 236 and 140 bp in size. In this study, a protocol for Mona detection from aerial fungal structures was developed that can yield results within a few hours of sampling. This study confirms that the Mona element is strongly linked to the DMI-resistance phenotype and reveals that overexpression of the MfCYP51 gene is a common DMI fungicide resistance mechanism in M. fructicola, not only in Georgia but throughout the eastern United States.