Imazalil resistance linked to a unique insertion sequence in the PdCYP51 promoter region of Penicillium digitatum

Two mechanisms of resistance to the fungicide imazalil (IMZ) existed among California strains of Penicillium digitatum, cause of citrus green mold. Sensitive (S; n = 50) strains did not grow on IMZ above 0.1 μg mL−1, while those resistant (R; n = 59) grew ≥0.5 mg L−1. After amplification of the promoter region of the CYP51 gene, fragments 250, 450, and 750 bp in size were generated. All S strains had a 250 bp product, while among R strains, 47 had a 450 bp product and 12 had a 750 bp product. The 450 bp unit was common among R strains, while the 750 bp unit, reported previously by others, was not. The promoter region of all was identical; variations occurred in the region's transcriptional enhancer unit. S strains with a 250 bp product and R strains with a 750 bp product had one and five copies, respectively, of a 126 bp transcriptional enhancer unit. R strains with a 450 bp product had a unique 199 bp insert within the 126 bp transcriptional enhancer unit with no known sequence correlations (GenBank). Both types of R strains exhibited significantly elevated expression, approximately 10-fold, of the target site CYP51 gene, indicating its overexpression was the mechanism of resistance.