Mutations in CYP51A and not -B confer propiconazole resistance in Geotrichum citri-aurantii and are validated by transformation of a sensitive isolate

Moderate (MR) and high (HR) levels of propiconazole resistance (average EC₅₀ 1.88 and 27.44 µg/ml, respectively) were detected in the citrus postharvest pathogen Geotrichum citri-aurantii isolated from summer-stored decayed lemons in California packinghouses starting in 2020. We investigated the CYP51 target sites and found several alleles of GcaCYP51A and GcaCYP51B that correlated with the propiconazole resistance phenotypes. A forward genetics approach was used to determine if the point mutations detected result in propiconazole resistance. Each of the alleles was cloned into the pAG26 fungal expression vector with hygromycin B as the selection marker. The recombinant vectors were transformed into a sensitive reference isolate of G. citri-aurantii using a modified lithium acetate/polyethylene glycol protocol. Successful transformation was verified with allele-specific PCR and growth on hygromycin B-amended agar. We compared the growth of sensitive, MR and HR isolates to that of the transformants on agar amended with hygromycin B and/or discriminatory doses of 1 and 10 µg/ml propiconazole. Isolates transformed with the CYP51A-MR and CYP51A-HR alleles exhibited the MR and HR phenotypes, respectively, but those transformed with the CYP51A-S, CYP51B-S, and CYP51B-HR alleles were not changed in their propiconazole sensitivity. Therefore, we confirmed that only point mutations in CYP51A are responsible for propiconazole resistance in this fungus.