Parallel evolution of multiple mechanisms for demethylase inhibitor fungicide resistance in the barley pathogen Pyrenophora teres f. sp. maculata
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. The necrotrophic 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 disease of increasing concern worldwide. The main basis for management of SFNB is by fungicide application, and in Australia the DMIs predominate. Although reduced sensitivity to DMI fungicides has recently been described in the closely related pathogen P. teres f. sp. teres (Ptt), the mechanisms of DMI resistance have not thus far been described for Ptm. In this study, several different levels of sensitivity to DMI fungicides were identified in Western Australian strains of Ptm from 2016 onwards, and reduced sensitivity phenotypes were correlated with a number of distinct mutations in both the promoter region and coding sequence of the DMI target gene encoding cytochrome P450 sterol 14-demethylase (Cyp51A). Five insertions elements of 134-base pairs in length were found at different positions within the upstream regulatory region of Cyp51A in both highly DMI-resistant (HR) and select moderately DMI-resistant (MR1) Ptm isolates. The five insertion elements had at least 95% sequence identity and were determined to be Solo-LTR (Long Terminal Repeat) elements, all deriving from Ty1/Copia-family LTR Retrotransposons. The 134-bp elements contained a predicted promoter sequence and several predicted transcription factor binding sites, and the presence of an insertion element was correlated with constitutive overexpression of Cyp51A. The substitution of phenylalanine by leucine at position 489 of the predicted amino acid sequence of CYP51A was found in both HR and select moderately DMI-resistant (MR2) Ptm isolates. The same F489L amino acid substitution has been previously reported in Western Australian strains of Ptt, where it has also been associated with reduced sensitivity to DMI fungicides. In Ptm, the F489L amino acid change was associated with either of three different single nucleotide polymorphisms in codon 489. This suggests that, in contrast to Ptt, in Ptm the F489L mutation has emerged as a result of three distinct mutational events. Moderately DMI-resistant isolates had one or the other of the F489L substitution or a promoter insertion mutation, whereas highly DMI-resistant isolates were found to have combinations of both mechanisms together. Therefore, multiple mechanisms acting both alone and in concert were found to contribute to the observed phenomena of DMI fungicide resistance in Ptm. Moreover, these mutations have apparently emerged repeatedly and independently in Western Australian Ptm populations, by a process of convergent or parallel evolution.