Azoxystrobin is the only synthetic, systemic fungicide labeled in the United States for management of frogeye leaf spot (FLS) of tobacco (Nicotiana tabacum L.), caused by Cercospora nicotianae. Though traditionally considered a minor disease in the United States, FLS has recently become yield and quality limiting. In 2016 and 2017, 100 C. nicotianae isolates were collected from symptomatic tobacco from eight counties in Kentucky, United States. Prior to azoxystrobin sensitivity testing, some C. nicotianae isolates were found to utilize the alternative oxidase pathway and, after assay comparisons, conidial germination was utilized to evaluate sensitivity in C. nicotianae 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) C. nicotianae isolates. Distributions of C. nicotianae EC50 values indicated three qualitative levels of sensitivity to azoxystrobin. Partial cytochrome b sequence, encompassing the F129L and G143A mutation sites, indicated single-nucleotide polymorphisms (SNPs) conferring the F129L mutation in C. nicotianae 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 C. nicotianae occurs broadly in Kentucky, and generate new hypotheses about selection pressure affecting resistance mutation frequencies.
Cercospora nicotianae, the causal agent of frogeye leaf spot (FLS) of tobacco, has been exposed to quinone outside inhibitor (QoI) fungicides for over 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 azoxystrobin sensitivity (< 0.08 µg/ml), moderate azoxystrobin sensitivity (0.14 to 0.64 µg/ml) and low azoxystrobin 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 EC50group, and sequences were compared. Our analysis showed that cytb gene in C. nicotianae consists of 1161 nucleotides encoding 386 amino acids. 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 PCR assay was developed to screen for mutations. Using this assay, 80% (n=99) of testedC. 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 operator characteristic curve analysis suggested the PCR assay is 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 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.
Phytophthora-induced plant diseases are most effectively managed using an integrated strategy that includes host resistance, best cultural practices, and fungicide applications. Black shank, caused by Phytophthora nicotianae, is the most significant soilborne disease affecting tobacco, and extensive breeding for resistance has resulted in an array of varieties with a spectrum of black shank resistance. Estimates of plant survival are the most common measurement in tobacco black shank trials, however, survival may not be the most accurate indicator of yield for varieties with robust resistance. Through the presented analyses, at-harvest green yield estimates are shown to be more closely associated with cured tobacco yields than survival counts, irrespective of black shank resistance status. However, green yield may only be considered a standalone yield predictor for low resistance varieties, with other factors likely contributing to cured yield of moderate and high resistance varieties. This prevents development of a unified (variety-nonspecific) cured yield model, which would be useful not only for tobacco scientists, but also commercial growers in planning labor activities and the industry at-large in managing cured tobacco inventory. More broadly, this suggests that research in other Phytophthora pathosystems may benefit from yield estimates in addition to survival counts in resistant variety evaluations.
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