Deep amplicon sequencing reveals extensive allelic diversity in the erg11/CYP51 promoter and allows multi-population DMI fungicide resistance monitoring in the canola pathogen Leptosphaeria maculans

Continued use of fungicides provides a strong selection pressure towards 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. New isolates with reduced DMI sensitivity were obtained from screens on DMI-treated plants, revealing eight independent insertions in the erg11 promoter. A novel deep amplicon sequencing approach applied to populations of ascospores fired from stubble identified an additional undetected insertion allele and quantified the frequencies of all known insertions, suggesting that, at least in the samples processed, the combined frequency of resistant alleles is between 0.0376% and 32.6%. Combined insertion allele frequencies positively correlated with population-level measures of in planta resistance to four different DMI treatments. Additionally, there was no evidence for erg11 coding mutations playing a role in conferring resistance in Australian populations. This research provides a key method for assessing fungicide resistance frequency in stubble-borne populations of plant pathogens and a baseline from which additional surveillance can be conducted in L. maculans. Whether or not the observed resistance allele frequencies are associated with loss of effective disease control in the field remains to be established.