Survey and genetic analysis of demethylation inhibitor fungicide resistance in Monilinia fructicola from Michigan orchards.

Resistance to sterol demethylation inhibitor fungicides (DMIs) in Monilinia fructicola, causal agent of brown rot of stone fruit, has been reported in the southeastern and eastern United States and in Brazil. DMI resistance of some M. fructicola isolates, in particular those recovered from the southeastern U.S., is associated with a sequence element termed 'Mona' that causes overexpression of the cytochrome demethylase target gene MfCYP51. In this study, we conducted statewide surveys of Michigan stone fruit orchards from 2009-2011 and in 2019, and determined the sensitivity to propiconazole of a total of 813 isolates of M. fructicola. A total of 80.7% of Michigan isolates were characterized as resistant to propiconazole by relative growth assays but the 'Mona' insert was not uniformly detected, and was present in some isolates that were not characterized as DMI resistant. Gene expression assays indicated that elevated expression of MfCYP51 was only weakly correlated with DMI-resistance in M. fructicola isolates from Michigan, and there was no obvious correlation between the presence of the 'Mona' element and elevated expression of MfCYP51. However, sequence analysis of MfCYP51 from 25 DMI-resistant isolates did not reveal any point mutations that could be correlated with resistance. Amplification and sequencing upstream of MfCYP51 resulted in detection of DNA insertions in a wide range of isolates typed by DMI phenotype and the presence of 'Mona' or other unique sequences. The function of these unique sequences or their presence upstream of MfCYP51 cannot be correlated to a DMI-resistant genotype at this time. Our results indicate that DMI resistance was established in Michigan populations of M. fructicola by 2009 to 2011, and that relative resistance levels have continued to increase to the point that practical resistance is present in most orchards. In addition, the presence of the 'Mona' insert is not a marker for identifying DMI-resistant isolates of M. fructicola in Michigan.