Clustered mutations in CYP51 genes associated with prochloraz resistance in Penicillium italicum

Among 294 isolates of Penicillium italicum collected from citrus-growing regions of China in 2021 and 2022, 70 (23.8%) isolates exhibited resistance to prochloraz, with resistance factors ranging from 45.4 to 653.6. All resistant isolates harbored clustered mutations in two paralogous genes, PiCYP51A and PiCYP51B. For PiCYP51A (1754 bp; sensitive genotype AS), three genotypes with progressively increasing numbers of mutations were identified among the resistant isolates. Genotype AR1 carried 30 nucleotide mutations, genotype AR2 harbored three additional point mutations relative to AR1, and genotype AR3 carried two further point mutations relative to AR2. For PiCYP51B (1752 bp; sensitive genotype BS), genotype BR1 carried 17 nucleotide mutations, and genotype BR2 harbored two additional point mutations. AR1, AR2, and AR3 accounted for 31.0%, 14.1%, and 54.9% of the resistant isolates, respectively, suggesting that the clustered mutations did not arise through a simple stepwise accumulation process. The mean EC₅₀ values for the sensitive genotype (AS/BS), AR1, AR2, AR3, BR1, and BR2 were 0.0134, 2.28, 2.29, 2.26, 2.06, and 2.71 mg/L, respectively, indicating that the additional mutations in AR2 and AR3 did not confer a further increase in resistance. Nevertheless, two-way analysis of variance (ANOVA) revealed that the PiCYP51A genotype and PiCYP51B genotype, as well as their interaction, significantly affected resistance levels. Prochloraz treatment did not significantly affect the expression of the translesion DNA synthesis (TLS) polymerase-related genes pol Rev1 and pol Zeta. These findings provide insights into the clustered mutations involved in fungicide resistance and hence, into the emergence and evolution of prochloraz resistance in P. italicum.