The Cytochrome P450 Lanosterol 14α-Demethylase Gene Is a Demethylation Inhibitor Fungicide Resistance Determinant in Monilinia fructicola Field Isolates from Georgia

Abstract

Resistance in Monilinia fructicola to demethylation inhibitor (DMI) fungicides is beginning to emerge in North America, but its molecular basis is unknown. Two potential genetic determinants of DMI fungicide resistance including the 14␣-demethylase gene (MfCYP51) and the ATP-binding cassette transporter gene MfABC1, were investigated in six resistant (DMI-R) and six sensitive (DMI-S) field isolates. No point mutations leading to an amino acid change were found in the MfCYP51 gene. The constitutive expression of the MfCYP51 gene in DMI-R isolates was significantly higher compared to DMI-S isolates. Gene expression was not induced in mycelium of DMI-R or DMI-S isolates treated with 0.3 g of propiconazole/ml. A slightly higher average MfCYP51 copy number value was detected in DMI-R isolates (1.35) compared to DMI-S isolates (1.13); however, this difference could not be verified in Southern hybridization experiments or explain the up to 11-fold-increased MfCYP51 mRNA levels in DMI-R isolates. Analysis of the upstream nucleotide sequence of the MfCYP51 gene revealed a unique 65-bp repetitive element at base pair position ؊117 from the translational start site in DMI-R isolates but not in DMI-S isolates. This repetitive element contained a putative promoter and 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. Interestingly, constitutive expression of the MfABC1 gene in DMI-R isolates was slightly lower than that of DMI-S isolates, but expression of the MfABC1 gene in DMI-R isolates was induced in mycelium after propiconazole treatment. Therefore, 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.

Overexpression of ShCYP51B and ShatrD in Sclerotinia homoeocarpa Isolates Exhibiting Practical Field Resistance to a Demethylation Inhibitor Fungicide

“…An additional phylogenetic analysis using the above-mentioned strategy was conducted for the ShatrD amino acid sequence and included amino acid sequences from the phylogeny of the G subfamily of ABC (ABC-G) transporters reported previously by Hayashi et al (12), with the addition of the amino acid sequence of the ABC-G gene ABC1 from M. fructicola (23).…”

“…Accordingly, several nonsynonymous point mutations in the gene sequence of CYP51 have been linked to decreased sensitivity to DMI fungicides in diverse groups of fungi (8,9,37). Moreover, the overexpression of the CYP51 gene and its paralogs has also been linked to reduced DMI sensitivity in many fungal species (11,21,23,24). Another frequently reported mechanism contributing to reduced DMI sensitivity is fungicide efflux through the action of energy-dependent ABC (ATP binding cassette) and MFS (major facilitator superfamily) transporters (2,12,26,29).…”

Reduced sensitivity to azoxystrobin is stable in Monilinia fructicola isolates

“…For the M. fructicola isolates, propiconazole resistance was not related to mutations in the target gene but rather with overexpression of the gene due to the 'Mona' element near this gene (Cox et al, 2007;Luo and Schnabel, 2008). Apparently, this overexpression produces an adaptive cost to the pathogen if the fungus is only resistant to propiconazole.…”

Isolation and Characterization of Antifungal Metabolites from Pterocarpus santalinus against Fusarium graminearum Causing Fusarium Head Blight on Wheat

“…DMI (Delye , 1998;Leroux , 2007;Luo Schnabel, 2008;Yin , 2009) . , terpenoids, alkaloids, flavonones, polyketides , , , (Copping Duke, 2007;Copping Menn, 2000;Le Dang , 2012).…”