Thesis (MScAgric)--Stellenbosch University, 2014. / ENGLISH ABSTRACT: Please refer to full text for abstract / AFRIKAANSE OPSOMMING: Sien volteks vir opsomming

O controle da podridão parda do pessegueiro (Monilinia fructicola) é realizado essencialmente por aplicações preventivas de fungicidas, em sua maioria sistêmicos com modo de ação específico. O uso contínuo desses fungicidas pode levar à seleção de indivíduos resistentes ao ingrediente ativo e, consequentemente, à perda da eficiência do controle nos pomares. A evolução da população de isolados resistentes ao longo do tempo, na ausência do fungicida, pode ser prevista com estudos de habilidade competitiva entre isolados resistentes e sensíveis. Portanto, a quantificação da adaptabilidade (fitness) e da estabilidade, necessárias para caracterizar os isolados encontrados em campo, auxiliarão nas estratégias antirresistência. Embora a importância da determinação da adaptabilidade de isolados resistentes seja reconhecida, há poucos métodos descritos para a análise da habilidade competitiva. Assim, esse trabalho verificou a adaptabilidade, a estabilidade e a habilidade competitiva de isolados de M. fructicola com diferentes níveis de sensibilidade à azoxistrobina em experimentos in vitro e em frutos de pêssegos (in natura e enlatado) utilizando metodologias de avaliação diversas. A caracterização dos isolados foi realizada in vitro, in vivo e molecularmente. Foram avaliados o diâmetro da colônia e a esporulação in vitro; o período de incubação e de latência, a incidência e a severidade da doença e a esporulação in vivo dos isolados separadamente. Os dados de esporulação in vitro foram submetidos ao teste de Kruskal-Wallis e os dados de crescimento micelial ao teste de Tukey com 5% de probabilidade. Para a análise dos dados in vivo, utilizou-se a análise multivariada. Dez transferências em meio de cultura sem fungicida foram realizadas para o estudo da estabilidade de dois isolados resistentes (SP08435 e SP09839) e um sensível (PR09638). A esporulação, a porcentagem de germinação de conídios e o diâmetro da colônia dos isolados em meio de cultura com fungicida foram avaliados em quatro transferências (inicial, terceira, sexta e última transferência). A habilidade competitiva foi verificada pela evolução da porcentagem de indivíduos resistentes no tempo entre um isolado resistente (SP09839) e um sensível à azoxistrobina (PR09638). Modelos lineares ou modelos lineares generalizados (GLM) foram utilizados para descrever o progresso da proporção do isolado resistente ao longo do tempo. Os isolados resistentes exibiram esporulação in vitro e taxa de crescimento micelial semelhantes aos sensíveis, além de apresentarem-se no mesmo grupo de similaridade que os isolados sensíveis. Nenhuma mutação no cyt b foi associada à resistência de M. fructicola à azoxistrobina (G143A, G137R, e F129L) e em todos os isolados foi observado o íntron antes do códon 143. Mesmo após 10 transferências em meio de cultura sem fungicida, os isolados resistentes à azoxistrobina não reduziram sua sensibilidade nem sua capacidade de esporulação e crescimento fora do pêssego. Independentemente da metodologia utilizada para avaliar a habilidade competitiva, a resistência à azoxistrobina não correspondeu a um custo adaptativo para M. fructicola, pois a proporção do isolado resistente manteve-se inalterada ao longo das transferências. O uso de pêssego enlatado é recomendado na substituição de frutos in natura para experimentos de habilidade competitiva. / Control of brown rot in peaches (caused by Monilinia fructicola) is mainly by preventive applications of fungicides, mostly single-target site. Repeated use of these fungicides can, however, select for resistant pathogen populations, which may result in failure of disease control. The evolution of resistant isolates in a population over time can be predicted with competitive ability assays between sensitive and resistant isolates. Therefore, studies quantifying pathogen fitness and stability are necessary to characterize field isolates and improve anti-resistant strategies. However, there are limited reports concerning the methodology used in competitive ability assays. This study determined the fitness of M. fructicola isolates with different levels of sensitivity to azoxystrobin in trials in vitro and in vivo (in natura and canned peaches) with different monocyclic components. Isolates characterization were carried out in vitro, in vivo and molecularly. Mycelial growth rate and sporulation were estimated in vitro, and disease incidence, severity, lesion sporulation, incubation and latent period were evaluated in peach fruit for sensitive and resistant isolates. Sporulation in vitro data were analyzed by Kruskal-Wallis test and mycelial growth rate with the Tukey test at 5% probability. For the in vivo experiments, multivariate analysis were carried out. Ten consecutive transfers on non-amended media were conducted to determine the stability of isolates (two resistant and one sensitive). Sporulation, percent spore germination and colony diameter on azoxystrobin amended media were also evaluated four times (the first, the third, the sixth and the last transfer). Competitive ability was investigated by testing the percentage increase of resistant individuals five generations after inoculation with a mix of azoxystrobin sensitive and resistant isolates. The competitive ability of sensitive and resistant isolates were compared with different variables as colony-forming unit and spore germination. Linear or generalized linear models (GLM) were adjusted to compare the progress of resistant isolates over time. All resistant isolates had in vitro sporulation and mycelial growth rate similar to sensitive isolates. All isolates sequenced presented an intron close to codon 143 and did not have any of the point mutations commonly associated with resistance to QoI fungicides (G143A, G137R, and F129L). After 10 transfers in PDA, colony diameter of resistant isolates was not reduced in azoxystrobin-amended media with 1 μg.ml-1 nor was their sporulation in vitro For all parameters used to measure competitive ability, no fitness cost was associated to M. fructicola isolates resistant to azoxystrobin. Canned peach is a viable option for studies aiming to determine fitness cost of resistant isolates.

Thesis (M.S.)--Montana State University--Bozeman, 2004. / Typescript. Chairperson, Graduate Committee: Barry J. Jacobsen. Includes bibliographical references.

Made available in DSpace on 2014-06-11T19:35:01Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-06Bitstream added on 2014-06-13T20:45:47Z : No. of bitstreams: 1 linhares_ai_dr_botfca.pdf: 473592 bytes, checksum: c28606702a04fcfa23732c203ae197de (MD5) / O presente trabalho teve como objetivos comparar métodos de monitoramento de resistência de Sphaerotheca fuliginea, causador de oídio em cucurbitáceas, a fungicidas; avaliar a sensibilidade de isolados procedentes de vários locais a fungicidas e verificar a influência de doses de fungicidas na sensibilidade do patógeno. Foram comparados três variações de métodos de avaliação de resistência com discos cotiledonares: flutuando e imersos em solução aquosa do fungicida e retirados de plantas de pepino pulverizadas com fungicida. As culturas foram incubadas por 12 dias em sala de crescimento (222ºC e 12 h luz) e então avaliadas pela área foliar colonizada pelo patógeno. A sensibilidade de isolados, coletados na BA, no DF, em MG, RS e SP, foi verificada pelo método do disco foliar flutuando em solução do fungicida e a inoculação, as condições de crescimento e a avaliação conforme descrito acima. Determinou-se a concentração mínima inibitória (CMI) dos isolados para os fungicidas fenarimol (FE) e tebuconazole (TE) (nas concentrações de 0; 0,001; 0,01; 0,1 e 1 g.mL-1); benomyl (BE) e tiofanato metílico (TM) (0; 6,3; 12,5; 25 e 50 g.mL-1) e azoxystrobin (AZ) (0; 0,001; 0,003; 0,005; 0,01; 0,03; 0,05; 0,1; 0,3; 0,5 e 1 g.mL-1). Para verificar as mudanças na sensibilidade e comparar a eficiência dos fungicidas, foram realizados dois ensaios, com dois cultivos. Plantas de pepino foram cultivadas em vasos em casa de vegetação e inoculadas naturalmente com oídio. No primeiro ensaio, as plantas foram tratadas com água, FE, TE e BE nas doses recomendadas. Foram feitas quatro aplicações com intervalo de 15 dias. Isolados foram coletados de cada tratamento antes das aplicações e o método do disco foliar flutuando em solução do fungicida foi utilizado para verificar a sensibilidade através da CMI... / The aim of this work was to compare methods for monitoring fungicide resistance of Sphaerotheca fuliginea, causal agent of powdery mildew of cucurbits; evaluate the fungicide sensitivity of isolates from many locals and to study the influence of fungicide doses in the sensitivity of the pathogen. Three variations of resistance methods were compared by floating and dipping cotyledon leaf disks in fungicide aqueous solution and obtained from cucumber plants treated with fungicide. The cultures were incubated at growth room (22±2ºC with a 12 h photoperiod) for 12 days before the evaluation of colonized foliar area by the pathogen. The fungicide sensitivity of isolates collected in BA, in DF, in MG, RS and SP, Brazil, was tested on cotyledon leaf disks floating on solution of the fungicide and the inoculation, the growth conditions and the evaluation were as described above. It was determined the minimum inhibit concentration (MIC) of the isolates for the fungicides fenarimol (FE) and tebuconazole (TE) (at the concentrations of 0; 0,001; 0,01; 0,1 and 1 g.mL-1); benomyl (BE) and thiophanate methyl (TM) (0; 6,3; 12,5; 25 and 50 g.mL-1) and azoxystrobin (AZ) (0; 0,001; 0,003; 0,005; 0,01; 0,03; 0,05; 0,1; 0,3; 0,5 and 1 g.mL-1). To verify the changes in the sensitivity and to compare the efficiency of the fungicides two tests was carried out, repeated twice. Cucumber plants were growth in pots in a greenhouse and inoculated with powdery mildew. In the first test, the plants were treated with water, FE, TE and BE in the recommended...(Complete abstract, click electronic access below)

Orientador: Raquel Ghini / Resumo: O presente trabalho teve como objetivos comparar métodos de monitoramento de resistência de Sphaerotheca fuliginea, causador de oídio em cucurbitáceas, a fungicidas; avaliar a sensibilidade de isolados procedentes de vários locais a fungicidas e verificar a influência de doses de fungicidas na sensibilidade do patógeno. Foram comparados três variações de métodos de avaliação de resistência com discos cotiledonares: flutuando e imersos em solução aquosa do fungicida e retirados de plantas de pepino pulverizadas com fungicida. As culturas foram incubadas por 12 dias em sala de crescimento (222ºC e 12 h luz) e então avaliadas pela área foliar colonizada pelo patógeno. A sensibilidade de isolados, coletados na BA, no DF, em MG, RS e SP, foi verificada pelo método do disco foliar flutuando em solução do fungicida e a inoculação, as condições de crescimento e a avaliação conforme descrito acima. Determinou-se a concentração mínima inibitória (CMI) dos isolados para os fungicidas fenarimol (FE) e tebuconazole (TE) (nas concentrações de 0; 0,001; 0,01; 0,1 e 1 g.mL-1); benomyl (BE) e tiofanato metílico (TM) (0; 6,3; 12,5; 25 e 50 g.mL-1) e azoxystrobin (AZ) (0; 0,001; 0,003; 0,005; 0,01; 0,03; 0,05; 0,1; 0,3; 0,5 e 1 g.mL-1). Para verificar as mudanças na sensibilidade e comparar a eficiência dos fungicidas, foram realizados dois ensaios, com dois cultivos. Plantas de pepino foram cultivadas em vasos em casa de vegetação e inoculadas naturalmente com oídio. No primeiro ensaio, as plantas foram tratadas com água, FE, TE e BE nas doses recomendadas. Foram feitas quatro aplicações com intervalo de 15 dias. Isolados foram coletados de cada tratamento antes das aplicações e o método do disco foliar flutuando em solução do fungicida foi utilizado para verificar a sensibilidade através da CMI...(Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this work was to compare methods for monitoring fungicide resistance of Sphaerotheca fuliginea, causal agent of powdery mildew of cucurbits; evaluate the fungicide sensitivity of isolates from many locals and to study the influence of fungicide doses in the sensitivity of the pathogen. Three variations of resistance methods were compared by floating and dipping cotyledon leaf disks in fungicide aqueous solution and obtained from cucumber plants treated with fungicide. The cultures were incubated at growth room (22±2ºC with a 12 h photoperiod) for 12 days before the evaluation of colonized foliar area by the pathogen. The fungicide sensitivity of isolates collected in BA, in DF, in MG, RS and SP, Brazil, was tested on cotyledon leaf disks floating on solution of the fungicide and the inoculation, the growth conditions and the evaluation were as described above. It was determined the minimum inhibit concentration (MIC) of the isolates for the fungicides fenarimol (FE) and tebuconazole (TE) (at the concentrations of 0; 0,001; 0,01; 0,1 and 1 g.mL-1); benomyl (BE) and thiophanate methyl (TM) (0; 6,3; 12,5; 25 and 50 g.mL-1) and azoxystrobin (AZ) (0; 0,001; 0,003; 0,005; 0,01; 0,03; 0,05; 0,1; 0,3; 0,5 and 1 g.mL-1). To verify the changes in the sensitivity and to compare the efficiency of the fungicides two tests was carried out, repeated twice. Cucumber plants were growth in pots in a greenhouse and inoculated with powdery mildew. In the first test, the plants were treated with water, FE, TE and BE in the recommended...(Complete abstract, click electronic access below) / Doutor

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-23T14:09:27Z No. of bitstreams: 1 Romulo Diniz Cavalcante.pdf: 683087 bytes, checksum: 8124aad4adf327b8c664300dfebcb472 (MD5) / Made available in DSpace on 2017-03-23T14:09:27Z (GMT). No. of bitstreams: 1 Romulo Diniz Cavalcante.pdf: 683087 bytes, checksum: 8124aad4adf327b8c664300dfebcb472 (MD5) Previous issue date: 2013-02-25 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Stem rot caused by Lasiodiplodia theobromae is an important postharvest disease of papaya in Brazil. The disease symptoms start on the stem region and then progressing around the fruit. The tissues remain with soaked aspect and with progression of symptoms, lesions become dark brown and depressed. The infected area becomes gradually devoid of parenchymal tissue and fruits lose their consistency and strength. It is recommended that the disease control starts in the field and continue until the commercialization phase since the symptoms are difficult to be perceived until an advanced stage of maturation. The use of Fungicides is one of the main disease management measures. However, there are no data available on the sensitivity of L. theobromae to thiophanate methyl, the most common fungicide used in papaya orchards in Northeastern Brazil. Thus, the EC50 of 109 isolates of the fungus, representing five populations of the pathogen was estimated in vitro for the fungicide. Of the 109 isolates, 20,2% were non-sensitive (NS) to the fungicide with EC50 values greater than 300 mg ml-1, for the remaining 79.8% sensitive (S) isolates the average EC50 was 1.87 μg ml−1. The EC50 values for the NS isolates were significantly (P≤0.05) higher than those for the sensitive isolates. Seven components of fitness were measured for the 10 isolates with lower and high values of EC50. When the fitness components were evaluated, the non-sensitive (NS) isolates showed sporulation capacity significantly lower than the sensitive isolates, indicating a fitness costs. / A podridão peduncular causada por Lasiodiplodia theobromae é uma doença em pós-colheita importante na cultura do mamoeiro no Brasil. Os sintomas da doença iniciam na região do pedúnculo e então progride para todo o fruto. Os tecidos permanecem com aspecto encharcado e com a progressão dos sintomas, as lesões se tornam marrom escura e deprimida. A área infectada torna-se gradualmente desprovida de tecido parenquimatoso e os frutos perdem sua consistência e vigor. É recomendado que o controle da doença seja iniciado no campo e continue até a fase de comercialização, uma vez que os sintomas são difíceis de serem percebidos até um estádio avançado de maturação. O uso de fungicidas é uma das principais medidas de manejo da doença. Todavia, dados sobre a sensibilidade de L. theobromae a tiofanato metílico, fungicida comumente empregado em pomares de mamoeiros no Nordeste brasileiro são inexistentes. Sendo assim, a CE50 de 109 isolados do fungo, representando cinco populações do patógeno foi estimada in vitro para o fungicida. Dos 109 isolados, 20,2% foram não sensíveis (NS) ao fungicida com valores de CE50 superiores a 300 μg ml-1, para 79,8% dos isolados remanescente denominados de sensíveis (S), a média de CE50 foi 1,87 μg ml-1. Os valores da CE50 dos isolados NS foram significativamente (P≤0.05) superiores quando comparados com os isolados S. Sete componentes de adaptabilidade foram mensurados para os 10 isolados com menores e maiores valores de CE50. Quando foram avaliados os componentes de adaptabilidade, os isolados não sensíveis apresentaram capacidade de esporulação significativamente inferior aos isolados sensíveis, indicando um custo de adaptabilidade.

O controle da podridão parda do pessegueiro (Monilinia fructicola) é realizado essencialmente por aplicações preventivas de fungicidas, em sua maioria sistêmicos com modo de ação específico. O uso contínuo desses fungicidas pode levar à seleção de indivíduos resistentes ao ingrediente ativo e, consequentemente, à perda da eficiência do controle nos pomares. A evolução da população de isolados resistentes ao longo do tempo, na ausência do fungicida, pode ser prevista com estudos de habilidade competitiva entre isolados resistentes e sensíveis. Portanto, a quantificação da adaptabilidade (fitness) e da estabilidade, necessárias para caracterizar os isolados encontrados em campo, auxiliarão nas estratégias antirresistência. Embora a importância da determinação da adaptabilidade de isolados resistentes seja reconhecida, há poucos métodos descritos para a análise da habilidade competitiva. Assim, esse trabalho verificou a adaptabilidade, a estabilidade e a habilidade competitiva de isolados de M. fructicola com diferentes níveis de sensibilidade à azoxistrobina em experimentos in vitro e em frutos de pêssegos (in natura e enlatado) utilizando metodologias de avaliação diversas. A caracterização dos isolados foi realizada in vitro, in vivo e molecularmente. Foram avaliados o diâmetro da colônia e a esporulação in vitro; o período de incubação e de latência, a incidência e a severidade da doença e a esporulação in vivo dos isolados separadamente. Os dados de esporulação in vitro foram submetidos ao teste de Kruskal-Wallis e os dados de crescimento micelial ao teste de Tukey com 5% de probabilidade. Para a análise dos dados in vivo, utilizou-se a análise multivariada. Dez transferências em meio de cultura sem fungicida foram realizadas para o estudo da estabilidade de dois isolados resistentes (SP08435 e SP09839) e um sensível (PR09638). A esporulação, a porcentagem de germinação de conídios e o diâmetro da colônia dos isolados em meio de cultura com fungicida foram avaliados em quatro transferências (inicial, terceira, sexta e última transferência). A habilidade competitiva foi verificada pela evolução da porcentagem de indivíduos resistentes no tempo entre um isolado resistente (SP09839) e um sensível à azoxistrobina (PR09638). Modelos lineares ou modelos lineares generalizados (GLM) foram utilizados para descrever o progresso da proporção do isolado resistente ao longo do tempo. Os isolados resistentes exibiram esporulação in vitro e taxa de crescimento micelial semelhantes aos sensíveis, além de apresentarem-se no mesmo grupo de similaridade que os isolados sensíveis. Nenhuma mutação no cyt b foi associada à resistência de M. fructicola à azoxistrobina (G143A, G137R, e F129L) e em todos os isolados foi observado o íntron antes do códon 143. Mesmo após 10 transferências em meio de cultura sem fungicida, os isolados resistentes à azoxistrobina não reduziram sua sensibilidade nem sua capacidade de esporulação e crescimento fora do pêssego. Independentemente da metodologia utilizada para avaliar a habilidade competitiva, a resistência à azoxistrobina não correspondeu a um custo adaptativo para M. fructicola, pois a proporção do isolado resistente manteve-se inalterada ao longo das transferências. O uso de pêssego enlatado é recomendado na substituição de frutos in natura para experimentos de habilidade competitiva. / Control of brown rot in peaches (caused by Monilinia fructicola) is mainly by preventive applications of fungicides, mostly single-target site. Repeated use of these fungicides can, however, select for resistant pathogen populations, which may result in failure of disease control. The evolution of resistant isolates in a population over time can be predicted with competitive ability assays between sensitive and resistant isolates. Therefore, studies quantifying pathogen fitness and stability are necessary to characterize field isolates and improve anti-resistant strategies. However, there are limited reports concerning the methodology used in competitive ability assays. This study determined the fitness of M. fructicola isolates with different levels of sensitivity to azoxystrobin in trials in vitro and in vivo (in natura and canned peaches) with different monocyclic components. Isolates characterization were carried out in vitro, in vivo and molecularly. Mycelial growth rate and sporulation were estimated in vitro, and disease incidence, severity, lesion sporulation, incubation and latent period were evaluated in peach fruit for sensitive and resistant isolates. Sporulation in vitro data were analyzed by Kruskal-Wallis test and mycelial growth rate with the Tukey test at 5% probability. For the in vivo experiments, multivariate analysis were carried out. Ten consecutive transfers on non-amended media were conducted to determine the stability of isolates (two resistant and one sensitive). Sporulation, percent spore germination and colony diameter on azoxystrobin amended media were also evaluated four times (the first, the third, the sixth and the last transfer). Competitive ability was investigated by testing the percentage increase of resistant individuals five generations after inoculation with a mix of azoxystrobin sensitive and resistant isolates. The competitive ability of sensitive and resistant isolates were compared with different variables as colony-forming unit and spore germination. Linear or generalized linear models (GLM) were adjusted to compare the progress of resistant isolates over time. All resistant isolates had in vitro sporulation and mycelial growth rate similar to sensitive isolates. All isolates sequenced presented an intron close to codon 143 and did not have any of the point mutations commonly associated with resistance to QoI fungicides (G143A, G137R, and F129L). After 10 transfers in PDA, colony diameter of resistant isolates was not reduced in azoxystrobin-amended media with 1 μg.ml-1 nor was their sporulation in vitro For all parameters used to measure competitive ability, no fitness cost was associated to M. fructicola isolates resistant to azoxystrobin. Canned peach is a viable option for studies aiming to determine fitness cost of resistant isolates.

Sclerotinia homoeocarpa F.T. Bennett, the causal agent associated with dollar spot, is a common and economically challenging turfgrass disease in North America. Acceptable turfgrass quality requires the optimization of cultural practices and the judicious use of fungicides. Sclerotinia homoeocarpa causes significant damage to turfgrass swards from May to October annually, therefore, requiring multiple fungicide applications to maintain satisfactory turfgrass quality throughout the growing season. Sterol demethylation inhibitor (DMI) fungicides are among the most widely used in the United States and frequent use has led to the development of fungicide resistance to the DMI fungicide class. The precise mechanism of DMI fungicide resistance in S. homoeocarpa is not completely understood, however over expression of the CPY51A gene or efflux transporter genes have been reported as the molecular mechanism for other fungal systems. Fungicide resistance to the DMI class exhibits a gradual population shift towards insensitivity that can be monitored using In vitro fungicide sensitivity assays. In vitro fungicide sensitivity assays have been used to detect fungicide sensitivity differences in different S. homoeocarpa isolates and currently serve as the most accurate methodology to detect DMI insensitivity in S. homoeocarpa. Documentation of DMI field efficacy on native S. homoeocarpa populations with differing in vitro sensitivities has not been examined thoroughly and would provide critical information regarding the association between in vitro fungicide sensitivity and DMI field efficacy. The objectives of this research are (i) to determine the association between in vitro propiconazole sensitivity and reduced field efficacy for five native S. homoeocarpa populations and (ii) to develop a qualitative in vitro sensitivity assay for detection of S. homoeocarpa isolates responsible for practical field resistance.

Soybean is an important source of protein in animal feed, and growing demand for meat consumption worldwide has led to increased soybean production. Over 120 million metric tons of soybean were harvested in the United States in 2018, approximately one-third of the world production. In the Mid-Atlantic region, soybean is one of the most valuable field crops. Major foliar diseases that reduce soybean yield in the Mid-Atlantic region are frogeye leaf spot (FLS) and Cercospora leaf blight. In addition to crop rotation and host resistance, foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage these soybean foliar diseases. Yield benefits of foliar fungicides have been inconsistent and this may be the result of low disease pressure, unfavorable environmental conditions for disease development, or the presence of fungal pathogen populations that have developed resistance to fungicides. The objectives of this research were 1) to develop a pyrosequencing-based assay to rapidly quantify QoI resistance frequencies in Cercospora sojina, the causal agent of FLS, 2) to examine the effects of fungicide application timings, disease pressure, and environmental factors on soybean yield, and 3) to develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. using a threshold decision rule. A pyrosequencing assay targeting the G143A mutation was designed, and a Virginia survey of C. sojina populations indicated that the G143A mutation conferring QoI resistance is widespread. In small plot fungicide application timing experiments, five weekly fungicide applications starting at beginning pod (R3) resulted in the greatest yield, but for single fungicide applications, R3 or 1 week after R3 resulted in the greatest yields. There was positive relationship between the cumulative number of disease favorable days (mean daily temperature 20-30°C and ≥ 10 hours of relative humidity >90%) from planting to R3 and disease severity at the full pod stage (r = 0.97, P = <0.01). Higher disease severity was associated with greater yield loss (r2 =0.53, P = 0.10) suggesting foliar fungicide applications are more likely to have yield benefits as the number of disease favorable days prior to R3 increase. A disease favorable-days threshold (FDT) using the environmental parameters indicated above was evaluated in on-farm experiments throughout Virginia, Maryland, and Delaware. Based on decision rules, FDT = 8 three weeks prior to R3 was the best predictor of a yield benefit with an R3 fungicide application. The decision aid was also able to correctly predict when a fungicide application would not be profitable ≥90% of the time. This weather-based decision aid along with monitoring of fungicide resistance development within the region will provide soybean growers in the Mid-Atlantic U.S. with tools to maximize yields and profitability. / Doctor of Philosophy / Soybean is the third most valuable field crop in the world, ranked only behind rice and wheat in value. Over 98% of the soybean crop is used for animal feed due to its high protein content. The United States is the largest soybean producer in the world, responsible for one-third of global production. Soybean is the top cash crop in the Mid-Atlantic region. Foliar fungal diseases can reduce the soybean yield by causing lesions on the leaves that reduce photosynthesis and cause premature defoliation. Frogeye leaf spot (FLS) caused by Cercospora sojina is a major yield reducing soybean foliar diseases in the Mid-Atlantic region. Foliar fungicides, often with quinone outside inhibitor (QoI) active ingredients, are used to manage the disease. However, fungicide efficacy has been inconsistent. Inconsistencies may be due to low disease pressure, improper application timing, or fungicide resistance. The purpose of this research was to investigate the fungicide efficacy inconsistencies and to develop management tools to improve yield and maximize profitability. Our objectives were to 1) develop a molecular assay to quantify frequencies of the mutation conferring fungicide resistance in Virginia populations of C. sojina, 2) examine the effects of fungicide application timings, disease severity, and weather on soybean yield, and 3) develop a weather-based soybean foliar fungicide application decision aid for the Mid-Atlantic U.S. The C. sojina fungicide resistance mutation was widespread in Virginia, but overall frequencies were relatively low compared to findings from Midwest and Southern states. In fungicide timing experiments, beginning pod (R3) applications resulted in the most consistent yield benefits, and disease severity and yield loss increased as the number of weather-based disease favorable days prior to R3 increased. We used data from on-farm experiments in Virginia, Maryland, and Delaware to develop a weather-based disease favorable-days threshold that increased the probability that a fungicide application at R3 would have a yield benefit in soybean. The results of our research have led improved fungal disease management recommendations for soybean in the Mid-Atlantic that will maximize yields and profitability.

Practical resistance of Erysiphe necator to quinone outside inhibitors (QoIs) is now widespread, and resistance to demethylation inhibitors (DMIs) has also developed.  The goal of this research was to characterize fungicide resistance by elucidating resistance mechanisms and determining its stability.  QoI resistance persisted for several years in a field population after QoI application ended.  Resistant isolates were highly competitive in mixed populations in competition assays under laboratory conditions, indicating a lack of fitness cost.  In one competition trial under field conditions, resistance frequency declined, possibly due to spore migration and influx of background inoculum, but in a second trial, it did not decline.  Double resistance to QoI and DMI was detected and DMI application may have been partially responsible for maintaining QoI resistance in the field.  One isolate with QoI resistance but an undetectable level of the major QoI mutation was shown to be heteroplasmic -- resistant strains could be selected from this isolate. DMI resistance mechanisms in E. necator included the Y136F mutation in CYP51 and cyp51 over-expression.  The first mechanism was present in almost all isolates with substantial levels of resistance, and cyp51 expression level was correlated with resistance level.  Three cyp51 genotypes were detected.  Wildtype isolates with the TAT genotype were sensitive to DMIs, while isolates with increased resistance had either a TTT or TWT genotype; TWT indicated the presence of both wildtype and mutant alleles.  Cyp51 was expressed 1.4 to 19 times more in mutants than in wildtype.  It is not known whether the significant differences in cyp51 expression level among isolates and among genotype groups are due to gene copy number variation.  DMI resistance was found to decline after years of subculturing, and the decline appeared to occur after a few culture transfers of field samples on fungicide-free host leaves.  The observed decline, together with the finding that isolates could be "trained" to increase resistance, and may be slightly induced in cyp51 expression when successively challenged to grow in increasing fungicide concentration, indicate instability of DMI resistance. / Ph. D.