• real-time PCR
• mutations
• Fungicide resistance
• CYP51
• azole fungicide
• Zymoseptoria tritici

Zymoseptoria tritici is the causal agent of STB (Septoria Tritici Blotch), a devastating wheat disease. Application of azole fungicides is one of the major practices to control STB. The repeated use of fungicides has led to the development of azole resistance in Z. tritici especially due to mutations in the CYP51 gene. Until today, more than 30 resistant haplotypes have been discovered. The aim of the present work was to investigate the occurrence of mutations in the CYP51 gene associated with azole resistance in Z. tritici populations from leaves sampled in wheat field experiments testing fungicide effects. Analysis of CYP51 gene sequences using HiPlex PCR and PacBio sequencing identified 8 cyp51 haplotypes (D134G, V136A, S188N, A379G, I381V, Y459del, G460del, y461H and N513K), which were shown to associated with the azole tolerance. In addition, we discovered the presence of an 867 bp insertion in the promoter region in the CYP51 gene of certain Z. tritici strains. In silico analysis of the insertion sequences showed the presence of TFBP (Transcription Factor Binding Proteins) motifs and several stress-related motifs, especially osmotic-stress. Then, gene expression of CYP51 gene from two pure culture isolates was investigated with qPCR. Strains with insertion in CYP51 gene promoter region showed a significant overexpression of the CYP51 gene compared with strains without insertion. Furthermore analysis of fungicide tolerance showed that the strain with insertion had significantly higher tolerance toward the azole fungicide compared with the strains with no insertion. So far, most of the studies have been focused on investigating new haplotypes; however, overexpression of CYP51 is one of the mechanisms that can lead to fungicide resistance. This study highlights how the control of STB represents a growing challenge.