Sterol Demethylation Inhibitor Fungicide (DMI) Resistance And Molecular Mechanism In Leptosphaeria Maculans In Canola

Blackleg is a worldwide disease in canola（Brassica napus）,caused by a complex of fungal species in the genus Leptosphaeria,that impacts canola production and seed quality.Sterol demethylation inhibitor（DMI）fungicides are an integral part of disease control.Blackleg or phoma stem canker results in yield losses and limits canola trade worldwide,especially in the canola growing regions of Australia,Canada,Europe,and China.Fungicide is commonly employed as an integral part in blackleg controls combined with resistant cultivars breeding and other agricultural managements.Sterol demethylation inhibitor（DMI）is a widely used fungicide class in canola industry,targeting 14α-demethylase（EC1.14.13.70）coded by ERG11（also known as CYP51）which is an essential enzyme in biosynthesis of plasma membrane.DMI resistance has been largely reported in a range of plant pathogens including L.maculans.However,the mechanism of DMI resistance in L.maculans is not clear and needs to be addressed further.In present study,wild resistant isolates of L.maculans to DMI were identified and molecular mechanisms were reported as following:（1）Six DMI resistant isolates,causing significant lesions on canola seedlings treated with DMI fungicides,were identified by using“ascospore shower”assay in planta named as 18BL149 to 18BL154,respectively;Sensitivity tests in vitro showed isolates 18BL151 and 18BL152 were consistently resistant to the three DMI fungicides tested,both in vitro and in planta,whereas the resistance varied between fungicide active ingredients in the other four isolates.Moreover,these isolates have significant higher EC₅₀ value to at least one DMI fungicide than that in sensitive isolates.Despite the isolate 18BL153 exhibiting sporulation decline,the other isolates obtained DMI resistance with little limited fitness cost.（2）Sequencing analysis of ERG11 coding region in resistant isolates revealed there was no gene modifications detected.Analysis of the regulatory region of the DMI target gene ERG11 revealed a 275 bp insertion in two of the isolates（18BL151and 18BL152）and three insertions（5263 bp,5267 bp,and 5248 bp）occurred in the promoter region of three resistant isolates（18BL149,18BL150,and 18BL153）,respectively.There was no change found in both the coding and promoter region of ERG11 in isolate 18BL154.（3）The three insertions were identified as three long terminal repeat retrotransposons（class I）,zolly-2,submitted to GenBank as accessions MN331773-MN331776,which share the similar long terminal repeats（LTRs）.Furthermore,comparison of the three insertions found the presence of single nucleotide polymorphisms following a mainly C-to-T and G-to-A change pattern,which corresponds to the action of repeat-induced point mutation（RIP）acting on repetitive elements in the L.maculans genome.NCBI BLAST of the 275 bp insertion revealed there were multiple（>400）copies in the genome of L.maculans and it is related to the retrotransposon Pholy.（4）The induced ERG11 expression levels in isolate 18BL151 and 18BL153,containing 275 bp and 5263 bp insertions,respectively,were dramatically increased and exhibited positive correlations with EC₅₀ values to tebuconazole.Furthermore,genetic approaches confirmed that these elements are responsible for DMI resistance in L.maculans and crosses show segregation consistent with a single locus.（5）Transformations of a sensitive isolate with ERG11 promoter insertion alleles including the promoter,coding and terminator of ERG11,confer fungicide resistance.Moreover,the induced ERG11 expression levels in resistant transformants were significantly corelative with their EC₅₀ values.（6）To date,there is no mutation reported in the ERG11 of L.maculans,point mutations were introduced within ERG11 gene using PCR assay to assess the effect of gene mutations to DMI resistance in L.maculans.The combination of point mutations（I381V,Y459/G460 deletion,and W461G）dramatically increased the resistance to tebuconazole with little fitness penalties.Overall,either the ERG11 gene overexpression caused by insertions occurred in the ERG11 promoter region,or the ERG11 gene containing combination of gene mutations（I381V,Y459/G460 deletion,and W461G）results in DMI resistance in L.maculans.This is the first report of genetic modifications in L.maculans relating to DMI fungicide resistance.