Ellwood, S. R. | bioRxiv Science

bioRxiv2021

Species hybridisation and clonal expansion as a new fungicide resistance evolutionary mechanism in Pyrenophora teres spp.

The barley net blotch diseases are caused by two fungal species of the Pyrenophora genus. Specifically, spot form net blotch is caused by P. teres f. sp. maculata (Ptm) whereas net form net blotch is caused by P. teres f. sp. teres (Ptt). Ptt and Ptm show high genetic diversity in the field due to intraspecific sexual recombination and hybridisation of the two species although the latter is considered rare. Here we present occurrence of a natural Ptt/Ptm hybrid with azole fungicides resistance and its implication to barley disease management in Australia. We collected and sequenced a hybrid, 3 Ptm and 10 Ptt isolates and performed recombination analyses in the intergenic and whole genome level. Eleven out of 12 chromosomes showed significant (P < 0.05) recombination events in the intergenic regions while variable recombination rate showed significant recombination across all the chromosomes. Locus specific analyses of Cyp51A1 gene showed at least four recombination breakpoints including a point mutation that alter target protein function. This point mutation did not found in Ptt and Ptm collected prior to 2013 and 2017, respectively. Further genotyping of fourteen Ptt, 48 HR Ptm, fifteen Ptm and two P. teres isolates from barley grass using Diversity Arrays Technology markers showed that all HR Ptm isolates were clonal and not clustered with Ptt or Ptm. The result confirms occurrence of natural recombination between Ptt and Ptm in Western Australia and the HR Ptm is likely acquired azole fungicide resistance through recombination and underwent recent rapid selective sweep likely within the last decade. The use of available fungicide resistance management tactics are essential to minimise and restrict further dissemination of these adaptive HR Ptm isolates.

evolutionary biology↗

bioRxiv2019

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.

plant biology↗