Title Extensive structural variation in the promoter of erg11/CYP51 confers widespread resistance to DMI fungicides in the canola pathogen Leptosphaeria maculans Authors Jack L. Scanlan1 | Angela C. Mitchell1 | Stephen J. Marcroft2 | Leanne M. Forsyth3 | Alexander Idnurm1 | Angela P. Van de Wouw1 1School of BioSciences, The University of Melbourne, VIC 3010, Australia 2Marcroft Grains Pathology, Horsham, VIC 3400, Australia 3Syngenta Crop Protection, Macquarie Park, NSW, 2113 Australia Abstract Continued use of fungicides provides a strong selection pressure for the emergence of strains with mutations to render these chemicals less effective. Previous research has shown that resistance to the demethylation inhibitor (DMI) fungicides, which target ergosterol synthesis, in the canola pathogen Leptosphaeria maculans has emerged in Australia and Europe. The change in fungicide sensitivity of individual isolates was found to be due to DNA insertions into the promoter of the erg11/CYP51 DMI target gene. Whether or not these were the only types of mutations and how prevalent they were in Australian populations was explored in the current study. First, new isolates were obtained from screens on fungicide-treated plants, to reveal a wide diversity of different DNA elements that insert within the erg11 promoter. Second, the frequency of such insertions was measured from populations of ascospores fired from stubble, via a deep amplicon sequencing approach. This suggested that at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. We also found no evidence for coding mutations in erg11 conferring resistance in Australian populations. This research thereby provides a key method for assessing resistance frequency and a baseline from which additional surveillance can be conducted. Whether or not such frequencies are associated with loss of efficacy in the field remains to be established.