<i>Cytochrome b</i> Mutations F129L and G143A Confer Resistance to Azoxystrobin in <i>Cercospora nicotianae</i>, the Frogeye Leaf Spot Pathogen of Tobacco

Abstract

Azoxystrobin is the only synthetic, systemic fungicide labeled in the United States for management of frogeye leaf spot (FLS) of tobacco (<i>Nicotiana tabacum</i> L.), caused by <i>Cercospora nicotianae</i>. Though traditionally considered a minor disease in the United States, FLS has recently become yield and quality limiting. In 2016 and 2017, 100 <i>C. nicotianae</i> isolates were collected from symptomatic tobacco from eight counties in Kentucky, United States. Prior to azoxystrobin sensitivity testing, some <i>C. nicotianae</i> isolates were found to utilize the alternative oxidase pathway and, after assay comparisons, conidial germination was utilized to evaluate sensitivity in <i>C. nicotianae</i> as opposed to mycelial growth. Azoxystrobin sensitivity was determined by establishing the effective concentration to inhibit 50% conidial germination (EC₅₀) for 47 (in 2016) and 53 (in 2017) <i>C. nicotianae</i> isolates. Distributions of <i>C. nicotianae</i> EC₅₀ values indicated three qualitative levels of sensitivity to azoxystrobin. Partial <i>cytochrome b</i> sequence, encompassing the F129L and G143A mutation sites, indicated single-nucleotide polymorphisms (SNPs) conferring the F129L mutation in <i>C. nicotianae</i> of moderate resistance (azoxystrobin at 0.177 ≤ EC₅₀ ≤ 0.535 µg/ml) and the G143A mutation in isolates with an azoxystrobin-resistant phenotype (azoxystrobin EC₅₀ > 1.15 µg/ml). Higher frequencies of resistant isolates were identified from greenhouse transplant (4 of 17) and conventionally produced (58 of 62) tobacco samples, as compared with field-grown tobacco (<4 weeks prior to harvest; 4 of 62) or organically produced samples (1 of 7), respectively. Together, these results suggest that resistance to azoxystrobin in <i>C. nicotianae</i> occurs broadly in Kentucky, and generate new hypotheses about selection pressure affecting resistance mutation frequencies.