Early blight caused by Alternaria solani and brown spot caused by the small-spored Alternaria spp., Alternaria alternata, Alternaria arborescens, and Alternaria tenuissima are observed annually in midwestern potato production areas. The use of foliar fungicides remains a primary management strategy. However, Alternaria spp. have developed reduced-sensitivity and/or resistance to many single-site fungicides such as quinone outside inhibitor (QoI), succinate dehydrogenase inhibitor (SDHI), and anilinopyrimidines (AP) fungicides in recent years. Boscalid, fluopyram, solatenol, and adepidyn are EPA-registered SDHI fungicides applied commercially to a variety of crops including potato. High intrinsic activity was observed in fluopyram, solatenol and adepidyn to A. solani isolates. Adepidyn and solatenol reduced disease severity caused by A. solani in field evaluations. Molecular characterization of 2018 A. solani field isolates determined that the frequency of the D123E and H134R SDH mutations increased. In contrast, the H278R/Y and H133R SDH mutations were found at low frequency. Adepidyn demonstrated the highest intrinsic activity against the small-spored Alternaria spp. but high intrinsic activity was also observed with boscalid, fluopyram, and solatenol. In vivo experiments demonstrated that adepidyn, solatenol, and fluopyram were more effective at managing A. arborescens and A. tenuissima than boscalid. Under greenhouse conditions, adepidyn and solatenol reduced brown spot severity caused by A. alternata to a greater extent than did fluopyram and boscalid. Results of these studies determined that accurate pathogen identification of small-spored Alternaria spp. may be important for brown spot management. Fludioxonil and cyprodinil exhibited a higher efficacy against of A. solani isolates when compared to pyrimethanil in greenhouse assays. Fludioxonil and cyprodinil were also highly efficacious against the Alternaria spp. evaluated and appear to be a good addition into fungicide rotation programs for early blight and brown spot management. Anilinopyrimidine (AP) (pyrimethanil and cyprodinil) and phenylpyrrole (PP) (fludioxonil) fungicides have demonstrated high intrinsic activity against other pathogens. Determining the efficacy of these fungicides on Alternaria spp. is important to the potato industry.

Frogeye leaf spot is a foliar disease of soybean caused by Cercospora sojina Hara, which until recently had been successfully managed by quinone outside inhibitor (QoI) fungicides. After widespread resistance to the QoI fungicides was reported throughout Mississippi, the next step in characterizing C. sojina was to study the fitness of selected isolates. Fitness measurements of resistance stability, colony growth, conidia production and germination, and virulence were assessed. A phylogenetic analysis was also conducted to assess the genetic similarity of the QoI-resistant and -sensitive C. sojina isolates. All isolates remained stable in terms of QoI resistance. Results of all fitness measurements indicated no significant differences between the QoI-resistant and -sensitive C. sojina isolates. The phylogenetic analysis supported these results revealing similarity between QoI-resistant and -sensitive C. sojina isolates. Based on these results no indication of a fitness cost is associated with QoI resistance in C. sojina isolates from Mississippi soybean.

Frogeye leaf spot (FLS), caused by Cercospora sojina Hara, is a foliar disease of soybean (Glycine max (L.) Merr.). FLS has re-emerged as an important disease due to the widespread QoI-resistant fungal population in Mississippi. Growers have transitioned from a stand-alone QoI fungicide to a multi-mode of action fungicide. One disadvantage of certain MOA’s is phytotoxicity. Phytotoxicity is a concern among soybean growers as to whether or not yield is lost. Disease, phytotoxicity, green stem, yield, plant morphology, and grain quality were analyzed to determine the impacts of fungicide phytotoxicity. Yield was significantly and negatively correlated as a result of phytotoxicity. Numerical differences in phytotoxicity severity were observed among all cultivars. Phytotoxicity severity were greatest in the Hornbeck 4950 LL with an average of 26.9%. Tank-mixing dodine with Manniplex B Moly, Megafol, and Veritas decreased phytotoxicity by 8.5, 2.7, 11.4% compared to dodine as a stand-alone treatment, respectively.

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.

Target spot, caused by Corynespora cassiicola, is a common lower canopy disease of soybean in the southern United States. Given the recent resurgence of target spot and increasing reports of resistance to the quinone outside inhibitor (QoI) fungicide class within C. cassiicola, a survey of C. cassiicola from the Mississippi soybean production system was initiated in 2019 to determine the nature of its resistance mechanisms. A total of 819 monoconidial isolates were collected from 228 geographic field locations in 75 Mississippi counties. The molecular mechanism of resistance was determined using a PCR-RFLP analysis by comparing nucleotide sequences in the cytochrome b gene. The percentage of isolates containing the G143A substitution increased from 71.3% in 2016 to 93.5% in 2021. In all, 85.8% of the C. cassiicola isolates carried the G143A substitution. The EC50 values of QoI-resistant and -sensitive isolates to azoxystrobin varied significantly with QoI-sensitive isolates exhibiting lower EC50 values than QoI-resistant isolates. Moreover, results of fitness evaluations indicated that QoI-resistant isolates are more competitive than QoI-sensitive isolates and there were no fitness costs associated with QoI resistance in C. cassiicola. Additionally, the sensitivity of six C. cassiicola isolates to eight fungicide active ingredients in four fungicide classes were evaluated. Results indicated that three succinate dehydrogenase inhibitors benzovindiflupyr, fluxapyroxad, and pydiflumetofen were the most effective in inhibiting mycelial growth regardless of isolate phenotype followed by the methyl benzimidazole carbamate thiophanate-methyl, two demethylation inhibitors (DMI) difenoconazole and flutriafol, the QoI pyraclostrobin, and the DMI prothioconazole. Furthermore, the efficacy of seven commercial fungicides on target spot was evaluated in the greenhouse and field. Pydiflumetofen + difenoconazole, fluxapyroxad + pyraclostrobin, and thiophanate-methyl delayed disease progress and protected soybean yield, which indicated their effectiveness in managing target spot. Pydiflumetofen + difenoconazole also significantly reduced defoliation. Notably, fungicides applied at R3 were more effective in reducing disease severity and defoliation than additional growth stage timings. The current study revealed a reduction in C. cassiicola sensitivity to QoI fungicides and a shift to QoI-resistant populations exhibiting fitness advantages. Our findings provide pertinent information for growers as to which fungicides should be recommended to manage target spot.

A citricultura brasileira se destaca no mercado global de citros, apresentando altos valores de produção e produtividade. Dentre as diversas doenças que afetam a cultura, a podridão floral dos citros (PFC) vem ganhando destaque com o deslocamento de áreas produtoras para regiões mais favoráveis à ocorrência desta doença. A PFC, causada por espécies dos complexos Colletotrichum acutatum e C. gloeosporioides, é especialmente problemática em anos de temperaturas amenas e alta umidade. Os citricultores realizam pulverizações preventivas para o controle da PFC todos os anos, embora condições climáticas favoráveis à doença ocorram apenas ocasionalmente. Além do impacto econômico, as frequentes pulverizações com fungicidas aumentam a pressão de seleção sobre isolados resistentes, o que pode interferir na eficiência do controle químico. A utilização de sistemas de previsão de epidemias pode evitar que pulverizações desnecessárias de fungicidas sejam realizadas caso não haja condições favoráveis à ocorrência da doença. Para o desenvolvimento destes sistemas, é imprescindível determinar um limiar de ação para a aplicação de fungicidas nos pomares. Dessa forma, o presente trabalho teve por objetivos: (i) estabelecer um limiar de ação para a aplicação de fungicidas com base na germinação de conídios de C. acutatum que permita o controle a doença igualmente ou de forma mais eficiente do que o sistema de pulverização adotado pelos citricultores do sudoeste paulista; (ii) caracterizar a sensibilidade de isolados dos complexos C. gloeosporioides e C. acutatum à trifloxistrobina e ao tebuconazol in vitro e molecularmente, para identificar possíveis mudanças de sensibilidade das espécies a estes fungicidas. Para a definição do limiar de ação, tratamentos baseados em índices de risco foram comparados ao tratamento testemunha e ao calendário fixo de aplicações, usualmente adotado pelos produtores. A aplicação de fungicidas quando limiar de 15% de germinação de conídios era atingido, foi eficiente em controlar a doença e reduzir o número de aplicações de fungicidas. Nos ensaios de sensibilidade a fungicidas dois métodos foram utilizados para a determinação da CE50: o da diluição em gradiente espiral para trifloxistrobina e tebuconazol e o da inibição da germinação de conídios para a trifloxistrobina. Foram utilizados isolados coletados entre 1999 e 2016. A CE50 média da coleção de isolados de acordo com o método da diluição em gradiente espiral variou de 0,158 a 0,297 μg/ml e 0,1 a 0,182 μg/ml para trifloxistrobina e tebuconazol, respectivamente. Para a trifloxistrobina, de acordo com o método da inibição da germinação, a CE50 média foi de 0,002 μg/ml. Não foram verificadas características moleculares nem valores de CE50 atrelados à mudança de sensibilidade dos isolados. / Brazilian citrus industry represents a significant share in the global citrus market. Amongst several diseases that affect the crop, postbloom fruit drop (PFD) has been gaining prominence in Sao Paulo with the displacement of citrus areas to regions in which weather conditions are more favorable to the occurrence of this disease. PFD, caused by species of the complexes Colletotrichum acutatum and C. gloeosporioides, is especially problematic in years of mild temperatures and high humidity. Citrus growers spray the orchards preventively for PFD control every year, although favorable climatic conditions do not occur regularly. In addition to the economic impact, this practice increases the selection pressure of fungicide resistant isolates, and may decrease the efficiency of chemical control in a long term. The use of disease forecasting systems is able to prevent unnecessary fungicide sprays. For the development of such systems, it is essential to determine an action threshold for the application of fungicides in the orchards. The objectives of this work were: (i) to establish an action threshold for fungicide sprays based on the germination of C. acutatum conidia; (ii) to characterize the sensitivity of C. gloeosporioides and C. acutatum isolates to trifloxystrobin and tebuconazole in vitro and molecularly. Regarding the definition of the action threshold, treatments based on risk indices were compared to the control treatments and calendar based sprays, usually adopted by growers. The 15% conidia germination threshold was efficient in controlling the disease and reducing the number of fungicide applications. In the fungicide sensitivity tests, two methods were used to determine the EC50, the spiral gradient dilution for trifloxystrobin and tebuconazole, and the method of conidial germination inhibition for trifloxystrobin. Isolates collected between 1999 and 2016 were used. The mean EC50 of the isolate collection determined by the spiral gradient dilution method ranged from 0.158 to 0.297 μg/ml and from 0.1 to 0.182 μg/ml for trifloxystrobin and tebuconazole, respectively. Mean EC50 of trifloxystrobin estimated by the conidial germination inhibition method was 0.002 μg/ml. No mutations or EC50 values indicated shifts of fungicide sensitivity on the isolates.

A citricultura brasileira se destaca no mercado global de citros, apresentando altos valores de produção e produtividade. Dentre as diversas doenças que afetam a cultura, a podridão floral dos citros (PFC) vem ganhando destaque com o deslocamento de áreas produtoras para regiões mais favoráveis à ocorrência desta doença. A PFC, causada por espécies dos complexos Colletotrichum acutatum e C. gloeosporioides, é especialmente problemática em anos de temperaturas amenas e alta umidade. Os citricultores realizam pulverizações preventivas para o controle da PFC todos os anos, embora condições climáticas favoráveis à doença ocorram apenas ocasionalmente. Além do impacto econômico, as frequentes pulverizações com fungicidas aumentam a pressão de seleção sobre isolados resistentes, o que pode interferir na eficiência do controle químico. A utilização de sistemas de previsão de epidemias pode evitar que pulverizações desnecessárias de fungicidas sejam realizadas caso não haja condições favoráveis à ocorrência da doença. Para o desenvolvimento destes sistemas, é imprescindível determinar um limiar de ação para a aplicação de fungicidas nos pomares. Dessa forma, o presente trabalho teve por objetivos: (i) estabelecer um limiar de ação para a aplicação de fungicidas com base na germinação de conídios de C. acutatum que permita o controle a doença igualmente ou de forma mais eficiente do que o sistema de pulverização adotado pelos citricultores do sudoeste paulista; (ii) caracterizar a sensibilidade de isolados dos complexos C. gloeosporioides e C. acutatum à trifloxistrobina e ao tebuconazol in vitro e molecularmente, para identificar possíveis mudanças de sensibilidade das espécies a estes fungicidas. Para a definição do limiar de ação, tratamentos baseados em índices de risco foram comparados ao tratamento testemunha e ao calendário fixo de aplicações, usualmente adotado pelos produtores. A aplicação de fungicidas quando limiar de 15% de germinação de conídios era atingido, foi eficiente em controlar a doença e reduzir o número de aplicações de fungicidas. Nos ensaios de sensibilidade a fungicidas dois métodos foram utilizados para a determinação da CE50: o da diluição em gradiente espiral para trifloxistrobina e tebuconazol e o da inibição da germinação de conídios para a trifloxistrobina. Foram utilizados isolados coletados entre 1999 e 2016. A CE50 média da coleção de isolados de acordo com o método da diluição em gradiente espiral variou de 0,158 a 0,297 μg/ml e 0,1 a 0,182 μg/ml para trifloxistrobina e tebuconazol, respectivamente. Para a trifloxistrobina, de acordo com o método da inibição da germinação, a CE50 média foi de 0,002 μg/ml. Não foram verificadas características moleculares nem valores de CE50 atrelados à mudança de sensibilidade dos isolados. / Brazilian citrus industry represents a significant share in the global citrus market. Amongst several diseases that affect the crop, postbloom fruit drop (PFD) has been gaining prominence in Sao Paulo with the displacement of citrus areas to regions in which weather conditions are more favorable to the occurrence of this disease. PFD, caused by species of the complexes Colletotrichum acutatum and C. gloeosporioides, is especially problematic in years of mild temperatures and high humidity. Citrus growers spray the orchards preventively for PFD control every year, although favorable climatic conditions do not occur regularly. In addition to the economic impact, this practice increases the selection pressure of fungicide resistant isolates, and may decrease the efficiency of chemical control in a long term. The use of disease forecasting systems is able to prevent unnecessary fungicide sprays. For the development of such systems, it is essential to determine an action threshold for the application of fungicides in the orchards. The objectives of this work were: (i) to establish an action threshold for fungicide sprays based on the germination of C. acutatum conidia; (ii) to characterize the sensitivity of C. gloeosporioides and C. acutatum isolates to trifloxystrobin and tebuconazole in vitro and molecularly. Regarding the definition of the action threshold, treatments based on risk indices were compared to the control treatments and calendar based sprays, usually adopted by growers. The 15% conidia germination threshold was efficient in controlling the disease and reducing the number of fungicide applications. In the fungicide sensitivity tests, two methods were used to determine the EC50, the spiral gradient dilution for trifloxystrobin and tebuconazole, and the method of conidial germination inhibition for trifloxystrobin. Isolates collected between 1999 and 2016 were used. The mean EC50 of the isolate collection determined by the spiral gradient dilution method ranged from 0.158 to 0.297 μg/ml and from 0.1 to 0.182 μg/ml for trifloxystrobin and tebuconazole, respectively. Mean EC50 of trifloxystrobin estimated by the conidial germination inhibition method was 0.002 μg/ml. No mutations or EC50 values indicated shifts of fungicide sensitivity on the isolates.

Stagonospora nodorum blotch (SNB), is a major foliar disease of wheat in the mid-Atlantic U.S., is caused by the necrotrophic fungus Parastagonospora nodorum. SNB is managed using cultural practices, resistant varieties, and foliar fungicides. There are increasing trends of severity and incidence of SNB in Virginia and the surrounding mid-Atlantic region, but it is not known if changes in the pathogen population are contributing to this trend. The overall goal of this research was to 1) determine the occurrence of quinone outside inhibitor (QoI) resistance in Virginia populations of P. nodorum infecting wheat, 2) quantify the distribution of G143A mutations conferring fungicide resistance in Virginia populations of P. nodorum, and 3) characterize genetic diversity of P. nodorum populations in Virginia and assess influences of cultivars and environments on population structure and SNB severity. For Objective 1, QoI resistant isolates of P. nodorum were identified from Virginia wheat fields, and this was the first report of QoI resistant P. nodorum in the United States. The G143A substitution in the cytochrome b gene of P. nodorum was associated with reduced QoI sensitivity, and in Objective 2, a state-wide, two-year survey of P. nodorum populations in Virginia determined that the G143A mutation was widespread in the state and among sampled fields the frequency ranged from 5-32% (mean = 19%). For Objective 3, P. nodorum was isolated from five different wheat cultivars across seven locations over two years in Virginia. SNB severity varied by cultivar but greater differences in disease severity were observed among locations and years suggesting environment plays an important role in SNB development. Among the necrotrophic effector (NE) genes examined, SnTox1 was predominant followed by SnTox3, and frequencies of NE genes did not vary by cultivar or location. P. nodorum populations in Virginia had high genetic diversity, but there was no genetic subdivision among locations or wheat cultivars from which individuals were isolated. Results also indicated that the P. nodorum population in Virginia undergoes a mixed mode of reproduction, but sexual reproduction made the greatest contribution to population structure. Overall, this work provides insights into the population biology of P. nodorum in Virginia and information on variability in fungicide sensitivity and cultivar susceptibility to SNB that has implications for the current and future efficacy of fungicides and host resistance for management of SNB. / Doctor of Philosophy / Wheat (Triticum aestivum L.) is one of the major cereal crops grown worldwide for food, feed, and other products. However, yields of this crop are often limited by fungal diseases including Stagonospora nodorum blotch (SNB) caused by Parastagonospora nodorum. Increasing trends of severity and incidence of SNB may be due to reduced sensitivity of P. nodorum to fungicides or increased virulence of P. nodorum populations on commonly grown cultivars. Fungicides such as quinone outside inhibitors (QoIs) are one of the major classes of fungicides used for disease control and G143A substitution is the most common point mutation associated with complete resistance to QoIs. Therefore, the overall goal of this research was to better understand genotypic and phenotypic variation in Virginia populations of P. nodorum in the context of fungicide sensitivity and susceptibility of wheat cultivars to SNB. The specific objectives were to 1) determine the occurrence of quinone outside inhibitor (QoI) fungicide resistance in Virginia populations of P. nodorum infecting wheat, 2) quantify the distribution of G143A mutations conferring QoI fungicide resistance in Virginia populations of P. nodorum, and 3) characterize genetic diversity of P. nodorum populations in Virginia and assess influences of cultivars and environments on population structure and SNB severity. Results from this research indicate that QoI fungicide resistance occurs in Virginia populations of P. nodorum due to a target site mutation (G143A substitution in the cytochrome b gene), and this mutation is widespread and relatively common in Virginia wheat fields. Based on a multi-year multilocation study, P. nodorum populations were genetically diverse, but there was no genetic subdivision among locations or wheat cultivars. SNB severity varied by location and cultivar, but disease severity was greatest at site-years with moderate springtime temperatures and high rainfall. Overall, this work contributes to a better understanding of P. nodorum populations including the current efficacy of fungicides and host resistance for management of SNB in the region.

<p>  </p> <p>Protecting crops from disease requires continuous research because plant pathogen incidence, geographical range, and pathogenicity, are constantly shifting variables as agronomic practices and climate continue to evolve. The objectives of this research are to i.) evaluate field-scale fungicide timing programs for corn (<em>Zea mays L.</em>) diseases at multiple locations in Indiana; ii.) evaluate field-scale fungicide timing programs for soybean (<em>Glycine Max</em> (L.) Merr.) diseases at multiple locations in Indiana; iii.) continue to identify, document, and confirm the distribution of populations of the soybean frogeye leaf spot pathogen (<em>Cercospora sojina)</em> that contain the G143A mutation conferring resistance to quinone outside inhibitor (QoI) fungicides in Indiana; and iv.) assess the incidence, severity, and prevalence of tar spot (<em>Phyllachora maydis</em>) in Indiana. For the first and second objectives, field scale trials were established at three locations in Indiana from 2019 to 2022. No application timings at any location provided significant yield protection for corn or soybeans. To achieve the third objective, 165 isolates of <em>C. Sojina </em>were tested. In total, 24 out of the 32 counties sampled in 2021 and 2022 were documented with QoI-resistance. The fourth objective was accomplished by surveying Indiana counties for incidence and severity of tar spot. As of 2022, 86 out of 92 Indiana counties have been confirmed for the presence of tar spot.</p>

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