Brown rot - Genetics of DMI Fungicide Resistance

Among the most effective fungicides for preharvest brown rot control are the sterol demethylation inhibitor (DMI) fungicides, which are widely used for controlling brown rot in the United States. DMI fungicides specifically bind to the cytochrome P450 lanosterol 14α-demethylase (CYP51) thereby inhibiting the biosynthesis of ergosterol, the primary fungal cell membrane sterol which is responsible for maintaining membrane fluidity and stability.  Molecular mechanisms leading to DMI resistance have been studied in several important plant fungal pathogens. Common mechanisms include mutations in the DMI fungicides target enzyme CYP51; overexpression of the CYP51 gene; and energy-dependent drug efflux mechanisms.

We cloned and sequenced potential genetic determinants for DMI fungicide resistance in M. fructicola, such as the 14alpha-demethylase gene (MfCYP51) of M. fructicola (Schnabel and Dai 2003;.pdf, 639 KB). When the MfCYP51 gene was introduced into PDR5::TN5 Saccharomyces cerevisiae, transformants revealed reduced sensitivity to the DMI fungicide myclobutanil. This result indicated that overexpression of the MfCYP51 gene is a potential mechanism of DMI fungicide resistance in M. fructicola.

MonaIn a later study, we confirmed overexpression of this gene in resistant filed isolates due to the presence of a 65-bp genetic element located upstream the nucleotide sequence of the MfCYP51 gene (see figure above). The element contained a putative promoter at position -117 bp from the translational start site in DMI-R isolates but not in DMI-S isolates (Luo et al. 2008;.pdf, 141 KB). This repetitive element was named Mona. The link between Mona and the DMI resistance phenotype became even more apparent after studying the genetic diversity between the isolates. In contrast to DMI-S isolates, DMI-R isolates contained an MfCYP51 gene of identical nucleotide sequence associated with Mona. Still, DMI-R isolates were not genetically identical as revealed by Microsatellite-PCR analysis. Also, real-time PCR analysis of genomic DNA indicated that the relative copy number of Mona among DMI-S and DMI-R isolates varied, suggesting its potential for mobility. Mona was found to be a major genetic determinant of DMI fungicide resistance not only in M. fructicola from the Southeast but also in Ohio and New York (Luo et al. 2008;pdf).  In the latter study, a simple, PCR-based technique for Mona detection is described that allows identification of DMI resistance within hours of sampling.

Reduced sensitivity to QoI fungicides and SDHI fungicides has been observed but could not be associated with point mutations in the target genes.