Moderate and High Propiconazole Resistance Characterized in the Citrus Postharvest Sour Rot Pathogen <i>Geotrichum citri-aurantii</i> Is Based on Point Mutations in <i>CYP51A</i> but Not <i>CYP51B</i>

Moderate (MR) and high resistance (HR) in <i>Geotrichum citri-aurantii</i> to propiconazole (PPZ) have been detected in California citrus packinghouses. Fitness cost and resistance mechanisms were characterized in this study. No fitness cost was identified in resistant isolates, and they were highly virulent and competitive in the presence of sensitive (S) isolates. F1 ascospore progeny from crosses between MR or HR isolates and an S isolate mostly segregated in a 1:1 ratio of S to R isolates, indicating involvement of a single gene in resistance. Resistance was associated with point mutations in <i>GcaCYP51A</i> resulting in Y143F and L389V amino acid substitutions in HR isolates and an A125V substitution in MR isolates. There were no sequence differences in the <i>GcaCYP51B</i> paralog between S and MR isolates, but missense and silent mutations were identified in HR isolates. Segregation of <i>GcaCYP51B</i> alleles was observed in crosses between S and HR isolates: some PPZ-S progeny carried the <i>GcaCYP51B-</i>HR allele, whereas some PPZ-HR isolates carried the <i>GcaCYP51B</i>-S allele. Therefore, mutations in <i>GcaCYP51B</i> have no major role in PPZ resistance. The direct involvement of mutations in <i>GcaCYP51A</i> in resistance was demonstrated by transformation of a PPZ-S isolate with cloned alleles from MR or HR isolates, where transformants exhibited the expected resistance phenotype. Transformants carrying the <i>GcaCYP51B</i> allele of HR isolates, however, remained sensitive. No differences between S and R phenotypes were detected in the promoter sequences of <i>GcaCYP51A</i> or <i>GcaCYP51B</i>. Our data indicate that PPZ resistance in <i>G. citri-aurantii</i> is solely determined by point mutations in <i>GcaCYP51A</i>.