Molecular Identification of Mutations Conferring Resistance to Azoxystrobin in Cercospora nicotianae

Cercospora nicotianae, the causal agent of frogeye leaf spot (FLS) of tobacco, has been exposed to quinone outside inhibitor (QoI) fungicides for more than a decade through azoxystrobin applications targeting other major foliar diseases. From 2016 to 2018, a total of 124 isolates were collected from tobacco fields throughout Kentucky. Sensitivity of these isolates to azoxystrobin was previously characterized by determining the effective concentration to inhibit 50% conidial germination (EC₅₀). Based on azoxystrobin EC₅₀, isolates were categorized into three discrete groups: high sensitivity (<0.08 µg/ml), moderate sensitivity (0.14 to 0.64 µg/ml), and low sensitivity (>1.18 µg/ml). Variability in sensitivity in a limited number of C. nicotianae isolates was previously shown to be a result of resistance mutations in the fungicide target gene. To improve understanding of C. nicotianae cytochrome b (cytb) structure, the gene was cloned from three isolates representing each EC₅₀ group, and sequences were compared. Our analysis showed that cytb gene in C. nicotianae consists of 1,161 nucleotides encoding 386 amino acids. The cytb sequence among the cloned isolates was identical with the exception of the F129L and G143A point mutations. To more rapidly determine the resistance status of C. nicotianae isolates to azoxystrobin, a polymerase chain reaction (PCR) assay was developed to screen for mutations. According to this assay, 80% (n = 99) of tested C. nicotianae isolates carried an F129L mutation and were moderately resistant to azoxystrobin, and 7% (n = 9) carried the G143A mutation and were highly resistant. A receiver operating characteristic curve analysis suggested the PCR assay was a robust diagnostic tool to identify C. nicotianae isolates with different sensitivity to azoxystrobin in Kentucky tobacco production. The prevalence of both the F129L and G143A mutations in C. nicotianae from Kentucky differs from that of other pathosystems where resistance to QoI fungicides has been identified, in which the majority of sampled isolates of the pathogen species have a broadly occurring cytb mutation.