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.