Author ORCID Identifier

Year of Publication


Degree Name

Master of Science (MS)

Document Type

Master's Thesis


Agriculture, Food and Environment


Plant Pathology

First Advisor

Dr. Emily Pfeufer

Second Advisor

Dr. Kiersten Wise


Burley and dark tobacco production are important to agriculture in Kentucky, worth $145.1 and $94.4 million in 2020, respectively. The price received for a tobacco crop is influenced by leaf quality, determined by multiple characteristics including leaf damage. Frogeye leaf spot (FLS), caused by Cercospora nicotianae, has historically been a minor disease of tobacco. However, when FLS infections reach the upper canopy of tobacco, lesions are seen as damage that negatively influence leaf quality. Fungicides are regularly used to manage foliar diseases of tobacco, and the only systemic fungicide active ingredient labeled for tobacco is azoxystrobin. Azoxystrobin belongs to the quinol outside inhibitor (QoI) class of fungicides, named for the outer quinol binding site of the cytochrome bc1 complex of the inner mitochondrial matrix. When azoxystrobin binds to the Qo site of cytochrome b (cytb), it stops the flow of electrons and halts the production of adenosine triphosphate (ATP). Azoxystrobin was first released for use in 1996, and in 1998 resistance was detected in Erysiphe graminis f.sp. tritici in Europe. Resistance to QoI fungicides has now been reported in over 50 phytopathogens. Resistance to QoI fungicides occurs as the result of single nucleotide polymorphisms (SNPs) in cytb that result in the substitution of phenylalanine for leucin at codon 129 (F129L), glycine for arginine at position 137 (G137R), and glycine for alanine at position 143 (G143A). The F129L and G137R mutations confer partial resistance, while G143A confers complete resistance to QoI fungicides. In 2017, QoI-resistant C. nicotianae was reported in Kentucky, with both F129L and G143A mutations detected in the population.

This research looks at the selectivity of azoxystrobin in C. nicotianae populations with mixed azoxystrobin sensitivity. By sampling FLS infected leaves we were able to identify cytochrome b mutations from recovered C. nicotianae isolates. In inoculation mixtures containing wild type, F129L and G143A cytochrome b, there was significant selection for the G143A mutation in one season. This strong selection could result in C. nicotianae populations that do not respond to azoxystrobin treatment. We also compared the biological fitness of C. nicotianae with wild type, F129L or G143A cytb mutations to determine if there is any fitness cost associated with QoI resistance. In vitro fitness was determined as the conidial viability or mycelial growth rate. We also looked at the survival of azoxystrobin-resistant C. nicotianae over winter. We found that there were no in vitro fitness penalties associated with azoxystrobin resistance and no difference in the survival of mutated individuals. Conventional FLS fungicide programs alternate azoxystrobin with mancozeb to manage QoI resistance development. We explored biological or organic fungicides, already labeled in tobacco, for efficacy against FLS. The biological fungicide programs were not as effective against FLS compared to the conventional regimen, but treatments using only biological products did not shift the populations toward QoI resistance. An integrated pest management approach is the best way to control FLS and manage resistance development. Crop rotation and sanitation will minimize the introduction of C. nicotianae in field production. Identification of QoI resistance mutations in C. nicotianae populations will allow producers to make informed fungicide applications and minimize resistance development. Further, there is a need to label new or existing chemicals for use in tobacco to rotate modes of action and slow QoI resistance development.

Digital Object Identifier (DOI)