Parallel evolution of multiple mechanisms for demethylase inhibitor fungicide resistance in the barley pathogen Pyrenophora teres f. sp. maculata

Wesley J Mair,Geoffrey J Thomas,Kejal Dodhia,Andrea L Hills,Kithsiri W Jayasena,Simon R Ellwood,Richard P Oliver,Francisco J Lopez-Ruiz

doi:10.1016/j.fgb.2020.103475

Abstract

The fungal pathogen Pyrenophora teres f. sp. maculata (Ptm), responsible for spot-form of net blotch (SFNB), is currently the most significant disease of barley in Australia and a major disease worldwide. Management of SFNB relies heavily on fungicides and in Australia the demethylase inhibitors (DMIs) predominate. There have been sporadic reports of resistance to DMIs in Ptm but the mechanisms remain obscure. Ptm isolates collected from 1996 to 2019 in Western Australia were tested for fungicide sensitivity levels. Decreased sensitivity to DMIs was observed in isolates collected after 2015. Resistance factors to tebuconazole fell into two classes; moderate resistance (MR; RF 6–11) and high resistance (HR; RFs 30–65). Mutations linked to resistance were detected in the promoter region and coding sequence of the DMI target gene Cyp51A. Solo-LTR insertion elements were found at 5 different locations in the promoter region. Three different non-synonymous mutations encoded an altered protein with a phenylalanine to leucine substitution at position 489, F489L (F495I in the archetype CYP51A of Aspergillus fumigatus). F489L mutations have also been found in DMI-resistant strains of P. teres f. sp. teres. Ptm isolates carrying either a LTR insertion element or a F489L allele displayed the MR1 or MR2 phenotypes, respectively. Isolates carrying both an insertion element and a F489L mutation displayed the HR phenotype. Multiple mechanisms acting both alone and in concert were found to contribute to DMI resistance in Ptm. Moreover, these mutations have emerged repeatedly in Western Australian Ptm populations by a process of parallel evolution.

Highlights

The demethylase inhibitor (DMI) or group 3 fungicides are the most important class of compounds for the control both of plant and human fungal pathogens (Cools, Hawkins, and Fraaije 2013)
Overall the results indicate constitutive overexpression of cytochrome P450 sterol 14α-demethylase (Cyp51A) in those isolates carrying insertion elements in the Cyp51A upstream region, which is further upregulated in the presence of DMI fungicides
Four sensitivity levels to DMIs were determined (S, MR1, MR2 and highly DMI-resistant (HR)) and these were correlated with eight different mutations across the coding sequence and upstream promoter region of the Cyp51A gene

Summary

Introduction

The demethylase inhibitor (DMI) or group 3 fungicides are the most important class of compounds for the control both of plant and human fungal pathogens (Cools, Hawkins, and Fraaije 2013). The DMI compound acts as a non-competitive inhibitor by binding with a nitrogen atom of the heterocyclic ring to the haem group iron within the active site of the enzyme, preventing the transfer of O2 to the substrate (Mullins et al 2011); while the side chains of the DMI compound interact with residues within the catalytic pocket, and this interaction determines the binding affinity (and the inhibitory effect) of a particular DMI compound to a given CYP51 (Warrilow et al 2013) The result of this is a depletion of ergosterol, an accumulation of 14α-methylated precursor sterols, and lethal membrane disruption (Ziogas and Malandrakis 2015, Becher and Wirsel 2012)

Methods

Results

Conclusion