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.
In 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.
In an effort to investigate other molecular mechanisms of DMI and Qo fungicide resistance in M. fructicola, the ABC transporter gene MfABC1 and the alternative oxidase gene MfAOX1 were cloned to study their potential role in conferring fungicide resistance (Schnabel et al. 2003; pdf). The MfABC1 gene revealed high amino acid homologies with atrB from Aspergillus nidulans, an ABC transporter conferring resistance to many fungicides including DMI fungicides. MfABC1 gene expression was induced after myclobutanil and propiconazole treatment in isolates with low sensitivity to the same fungicides and in an isolate with high sensitivity to propiconazole. The results suggested that the MfABC1 gene may be a DMI fungicide resistance determinant in M fructicola. In our study published in 2008 (see above), constitutive expression of the MfABC1 gene in DMI-R isolates was slightly lower compared to DMI-S isolates, but expression of the MfABC1 gene in DMI-R isolates was induced in mycelium after propiconazole treatment. The results suggested that the MfABC1 gene may play a minor role in DMI fungicide resistance in M. fructicola. Our results strongly suggest that overexpression of the MfCYP51 gene is an important mechanism in conferring DMI fungicide resistance in M. fructicola field isolates from Georgia and that this overexpression is correlated with Mona located upstream of the MfCYP51 gene.
In the 2003 study (see above), the alternative oxidase gene MfAOX1 from M. fructicola was cloned and gene expression was analyzed. The amino acid sequence was 63.8, 63.8, 57.7% identical to AOX genes from Venturia inaequalis (ViAOX1), Aspergillus niger, and A nidulans. MfAOX1 expression in some but not all M. fructicola isolates was induced in mycelia treated with azoxystrobin. Azoxystrobin at 2 µg ml-1 significantly induced MfAOX1 expression in isolates with low MfAOX1 constitutive expression levels.