Thesis (doctoral)--Christian-Albrechts-Universität zu Kiel, 1999.

Cercospora sojina, the causal agent of frogeye leaf spot, is an important pathogen of soybean that reduces soybean yield by an average of 9.1 million bushels each year in the United States. Management strategies include using resistant cultivars and fungicide applications. Hill plots were used to evaluate over 600 commercial and public varieties for resistance or susceptibility to C. sojina. Each hill plot was spaced 0.6 m apart, planted with 10 seeds from a single cultivar, and was replicated three times. At flowering, the plants were inoculated by spraying a spore solution across all plants in the field. Hill plots were rated for disease severity at 14, 28 and 35 days after inoculation. There were 259 varieties that were susceptible to FLS, with 181 varieties with intermediate resistance, and 161 resistant varieties. A greenhouse trial evaluated fungicides and their ability to hinder symptom production and the duration of this suppression. Spores of the pathogen were applied at 7, 14 and 21 days after application of fungicides. Disease ratings were collected at 5 weekly intervals following inoculation. The significantly different treatment with the lowest disease ratings was the fungicide Stratego YLD (a strobilurin and triazole mix). The fungicide Tilt was found to be significantly different from all other treatments in the number of lesions on a single tracked leaf, including the non-treated control. These results can be used to recommend to producers available varieties.

Cercospora leaf spot is an economically devastating disease of sugar beet caused by the fungus Cercospora beticola. It has been demonstrated recently that the C. beticola CTB cluster is larger than previously recognized and includes novel genes involved in cercosporin biosynthesis and a partial duplication of the CTB cluster. Several genes in the C. nicotianae CTB cluster are known to be regulated by ‘feedback’ transcriptional inhibition. Expression analysis was conducted in wild type (WT) and CTB mutant backgrounds to determine if feedback inhibition occurs in C. beticola. My research showed that the transcription factor CTB8 which regulates the CTB cluster expression in C. nicotianae also regulates gene expression in the C. beticola CTB cluster. Expression analysis has shown that feedback inhibition occurs within some of the expanded CTB cluster genes. The partial duplication of the CTB cluster was not found to be light activated or subject to feedback inhibition. / USDA Bolton Sugar Beet Pathology Lab

Cercospora leaf spot (CLS), caused by the fungal pathogen Cercospora beticola, is one of the most destructive foliar diseases on sugar beet. It severely affects productivity and profitability of the sugar beet industry. CLS is managed by using resistant cultivars, rotating with non-host crops, and applying effective fungicides in a timely manner. In North Dakota and Minnesota, site-specific fungicides, such as quinone outside inhibitors (QoIs) and demethylation inhibitors (DMIs), had been widely and extensively applied to control C. beticola which has developed resistances to these fungicides. The mycelial growth, spore production, spore germination, and aggressiveness of QoI and/or DMI resistant isolates were compared to sensitive isolates in a laboratory and greenhouse study. Results indicated that the QoI and/or DMI resistant isolates had a relatively slower disease development on sugar beet leaves due to their fitness penalty in sporulation and mycelial growth but still caused high disease severities as sensitive isolates. Fungicides were evaluated to manage the QoI and/or DMI resistant C. beticola isolates in a sensitivity study and a greenhouse and field study. Copper-based multisite activity fungicides were evaluated for controlling fungicide resistant C. beticola. The mean EC50 values for nine copper-based chemicals ranged from 1 to 10 ug/ml using a spore germination assay. In a greenhouse study, Fertileader (a copper-based fertilizer) caused leaf injury and was not evaluated, but the other tested chemicals provided significantly better control of CLS compared to the control check with Badge X2, Champion, Cuprofix, COCS and Ridomil having significantly small AUDPC. Newer site specific fungicides with different modes of action that have never been used for CLS management in North Dakota and Minnesota were also tested. The mean EC50 values were 4.9, 33.1, 99.4, and 481.6 ug/ml using mycelial growth assay and 5.7, 4.1, 9.2, and 4.2 ug/ml using spore germination assay for cyprodinil, fluazinam, pydiflumetofen, and Chlorothalonil, respectively. In a two-year field study, all the fungicides resulted in significantly better disease control, significantly higher beet tonnage, and recovered sucrose compared to the nontreated check. However, none of tested fungicide treatments performed better than the industry?s standard fungicide, triphenyltin hydroxide.

Maize (Zea mays) is globally considered as an important cereal crop, and a major staple food in developing countries such as Africa (WARD et al. 1999). In South Africa, maize is considered the most important grain crop as it is the main feed grain used for animals and a staple food for the population (FAO 2012). Maize can also be used for the production of maize-based ethanol, which can be used as a bio-fuel. In the USA, approximately 40% (11 million tonnes) of maize produced in 2012 was used for the production of bio-fuel (FAO 2012). Maize production in Africa was estimated to be less than two and on average 1.4 tons per hectare and remains below world average (FAO 2012). It was expected that South African crop production would decrease by approximately six percent during the 2012/2013 growing season as droughts during February and March 2013 in the North West and Free State provinces led to below-average maize yields in these production areas (FAO 2012; USDA 2013). Over the last few years maize production output has not been increasing together with the increasing population growth rate and thus puts pressure on commercial farmers to produce more maize for food security purposes and economical growth. The FAO states that agricultural production still needs to increase by up to 60% (80% in developing countries) within the next four years to be able to cope with an estimated global population growth of 39% by 2050. / Dissertation (MSc)--University of Pretoria, 2014. / National Research Foundation (NRF) / Plant Science / MSc / Unrestricted

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.

No description available.

La combinación de la siembra directa con el monocultivo, junto con los cambios climáticos y la difusión de genotipos susceptibles, han generado las condiciones óptimas para que los patógenos necrotróficos, que sobreviven en los rastrojos sean anualmente responsables por daños y pérdidas significativas en el cultivo de soja. Dentro de este grupo de enfermedades, deben destacarse por su prevalencia y severidad, a las enfermedades de fin de ciclo (EFC) y a la mancha ojo de rana (MOR), Entre las prácticas para su manejo, deben mencionarse a la rotación de cultivos, tratamiento químico de semillas, resistencia genética, y el uso de fungicidas en órganos aéreos. Esta última medida se ha incrementado considerablemente como respuesta al aumento del potencial de inóculo sobre variedades susceptibles y bajo monocultivo. Sin embargo, la determinación del momento oportuno de la aplicación de fungicidas para el manejo de estas enfermedades no resulta una tarea sencilla y práctica. Existe una gran complejidad en el abordaje de su estudio con variadas dificultades que deben enfrentarse principalmente cuando el objetivo es asistir a los productores y asesores con metodologías prácticas que aseguren la sustentabilidad económica y ambiental. A pesar de que muchos investigadores están de acuerdo en que los fungicidas deberían ser aplicados durante el período crítico de generación del rendimiento del cultivo de soja, no fue posible encontrar información científica publicada sobre el momento más apropiado de aplicación de fungicidas, dentro de esa amplia ventana fisiológica de aplicación, que relacione ambiente, epidemiología, daños y umbrales. Los objetivos de este trabajo fueron a) generar criterios de uso de fungicidas para el manejo químico sustentable de las EFC y la MOR en el cultivo de soja, b) cuantificar sus daños, c) elaborar y validar un sistema de puntuación que permita definir el momento más eficiente de aplicación para las EFC, y d) determinar los umbrales de control para la MOR. Para ello, se realizaron y analizaron numerosos ensayos de campo provenientes de zonas agro-ecológicas diferentes. Para el caso de las EFC, los resultados demostraron que la respuesta al uso de fungicidas (kg/ha) dependió de la ocurrencia de lluvias entre R3-R5 y no de las que ocurrieron entre R1-R3, por lo tanto dependiendo de las condiciones ambientales, la aplicación de fungicida podría realizarse en R3, R4 o R5. Una vez que se logró identificar el factor ambiental y el período fenológico que estadísticamente se asociaron con el impacto en los rendimientos por el uso de fungicidas, la siguiente etapa fue la de desarrollar un sistema de puntuación basado en la ponderación de diferentes factores agronómicos. Dicho sistema contó con 10 factores de valoración relativa y permitió proponer una herramienta fácil, práctica y dinámica para orientar la decisión de control químico. Los ensayos de validación del sistema de puntuación llevados a cabo en localidades y campañas agrícolas diferentes, permitieron analizar el impacto en el rendimiento agronómico y en la rentabilidad lograda, cuando se utilizó el sistema de puntuación en comparación con las aplicaciones fenológicas fijas de R3, R5 y con un testigo sin aplicación química. Cuando el sistema recomendó no aplicar, los rendimientos que fueron obtenidos en los tratamientos de R3 y R5 no se diferenciaron de los del testigo, indicando que no se justificaba aplicar. Por otro lado, se demostró que algunos ensayos mostraron el mayor impacto por el uso de fungicidas cuando se realizaron las aplicaciones en R3 ó R4 ó R5 y que su decisión final dependió de la puntuación arrojada por el sistema en cada ensayo, demostrando que la aplicación regida exclusivamente por fenología rígida y estricta, no resulta en una alternativa técnica fundamentada para orientar el momento de la pulverización. Para el estudio de la MOR, se lograron estimar los daños y generar recomendaciones para su manejo mediante umbrales de control. El número de lesiones por folíolo central constituyó una medida objetiva, rápida y sencilla para cuantificar la epidemia y desarrollar los diferentes UDE. De esta manera los resultados epidemiológicos obtenidos indican que la MOR no debe ser considerada una clásica EFC debido a su comportamiento policíclico. Para diferentes estadios reproductivos de la soja y para cada estrato foliar considerado, se propusieron umbrales de daño móviles que serán de utilidad para enmarcar el uso de fungicidas dentro de un programa de manejo Integrado.

Lipases são enzimas que catalisam a hidrólise de triacilgliceróis em ácidos graxos, mono e diacilgliceróis e glicerol. Em contraste com as esterases, lipases são ativadas apenas quando estão adsorvidas a uma interface óleo-água. Lipases têm sido amplamente utilizadas em muitos processos industriais, tais como química orgânica, formulações de detergentes e de produtos como cosméticos e farmacêuticos. A principal preocupação na produção de enzimas comerciais é a proteção da sua estabilidade em solução aquosa. A água facilita ou medeia uma variedade de vias de degradação física e química, durante as etapas de purificação, transporte e armazenamento. Por conseguinte, formulações sólidas são desenvolvidas para alcançar uma vida útil aceitável para essas substâncias. Spray drying é comumente usado como uma técnica de desidratação na indústria farmacêutica para fabricação de produtos em pó diretamente do estado líquido. No presente trabalho, a purificação e caracterização bioquímica de lipases produzidas pelo fungo endofítico Cercospora kikuchii, bem como os efeitos de adjuvantes no processo de secagem destas enzimas foram estudados. A lipase bruta foi purificada à homogeneidade através de cromatografia de interação hidrofóbica e gel filtração. A lipase foi purificada 5,54 vezes, com rendimento de 9% e a atividade específica de 223,6 U/mg. O peso molecular da enzima foi estimado em 65,1 kDa por SDS-PAGE e 73,5 kDa utilizando cromatografia de gel filtração, indicando que provavelmente trata-se de um monômero. A lipase mostrou um pH ótimo em 4,6 e uma temperatura ótima de 35°C. Cerca de 80,2% de sua atividade foi mantida após incubação a 40°C durante 2 horas. A Vmax e Km foram 10,28 mmol/min/mg de proteína e 0,03240 mM, respectivamente, utilizando pNPP como substrato. As lipases presentes no extrato bruto e as lipases ligadas ao micélio foram caracterizadas para avaliar o potencial de utilização em biocatálise. A lipases no extrato bruto apresentaram atividade máxima a 60ºC e pH 6,2, enquanto que as lipases ligadas ao micélio apresentaram atividade máxima a 50ºC e pH 5,4. Nos estudos de efeito da temperatura sobre a atividade enzimática, as lipases no extrato bruto mantiveram-se estáveis a 50°C, com 85,3% de atividade residual após 2 horas de incubação. As lipases ligadas ao micélio mantiveram pelo menos 75,1% de atividade residual após 2 horas de incubação a 80°C. Estes resultados mostram que as lipases de C. kikuchii têm propriedades cinéticas e termoestabilidade desejáveis para aplicações em biocatálise. As lipases presentes no extrato bruto foram secas em spray dryer com diferentes adjuvantes, e sua estabilidade foi avaliada. A recuperação da atividade enzimática após a secagem, com a adição de 10% de lactose, -ciclodextrina, maltodextrina, manitol, goma arábica, e trealose variou de 63 a 100%. A atividade da enzima foi totalmente perdida durante a secagem do extrato bruto na ausência de adjuvantes. A maioria dos adjuvantes utilizados manteve pelo menos 50% da atividade enzimática a 5°C e 40% a 25°C, após 8 meses de armazenagem. As lipases secas com 10% de - ciclodextrina mantiveram 72% da atividade a 5°C no mesmo período. A partir destes resultados preliminares foi realizada a otimização do processo de secagem utilizando -ciclodextrina, maltodextrina e lactose como adjuvantes. A análise estatística dos resultados experimentais permitiu a determinação das condições ótimas para a retenção da atividade enzimática (RAE), a saber: concentração de adjuvantes de secagem de 12,05%, temperatura de entrada do gás de secagem em 153,6oC e vazão do extrato enzimático alimentado de 9,36 g/min, para - ciclodextrina e maltodextrina como adjuvantes. Para lactose, o estudo mostrou que o aumento da quantidade de adjuvante de secagem e/ou diminuindo a temperatura do gás de entrada tem um efeito positivo sobre a retenção da atividade enzimática do produto seco. Após o processo de purificação foi realizada a secagem da enzima parcialmente purificada e da lipase pura, com estes três adjuvantes. A manutenção da atividade enzimática variou 90,6-100% quando foram utilizadas as condições ótimas para cada adjuvante de secagem. Concluindo, as lipases produzidas por C. kikuchii podem ser eficientemente secas por spray dryer, uma vez que a atividade enzimática foi mantida no extrato bruto, na lipase pura e na lipase semi-purificada submetidas à secagem. / Lipases are enzymes that catalyze the hydrolysis of triacylglycerols to fatty acids, mono and diacylglycerols, and glycerol. In contrast to esterases, lipases are activated only when they are adsorbed to an oilwater interface. They have been widely used in many industrial processes such as organic chemical, detergent and cleaning formulations and in products like cosmetics and pharmaceutical products. The main concern in the production of commercial enzymes is to protect their stability in aqueous solution. Water facilitates or mediates a variety of physical and chemical degradation pathways, active during protein purification, shipping and storage. Consequently, dry solid formulations are developed to achieve an acceptable protein shelf life. Spray drying is commonly used as a dehydration technique in the pharmaceutical industry for making powdery products directly from the liquid. In the present work, the purification and biochemical characterization of lipases produced by endophytic fungus Cercospora kikuchii as well as the effects of adjuvants on the spray drying process of theses enzymes were studied. The crude lipase was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. The lipase purified was 5.54-fold with 9% recovery and the specific activity was 223.6. The molecular mass of the lipase was estimated to be 65.1 kDa using SDS-PAGE and 73.5 using gel filtration chromatography, indicating that the lipase is a monomer. The lipase demonstrated an optimum pH at 4.6, an optimum temperature of 35°C. About 80.2% of its activity was retained after incubation at 40°C for 2 hours. The Vmax and Km were 10.28 mol/min/mg protein and 0.03240 mM, respectively, using pNPP as substrate. The lipases present in crude extract and the mycelium-bound lipases were characterized in order to evaluate the potential for use in biocatalysis. The crude extract showed maximum activity at 60ºC and pH 6.2 while the myceliumbound lipases showed maximum activity at 50ºC and pH 5.4. In tests of the temperature effect on the enzymatic activity, the lipases in the crude extract was stable at 50°C, with 85.3% residual activity after 2 hours of incubation. The mycelium-bound lipases maintained at least 75.1% of residual activity after 2 h incubation at 80°C. These results show that the lipases of C. kikuchii have kinetic properties and stability characteristics suitable to applications in biocatalysis. The lipases present in crude extract were spray dried with different adjuvants, and their stability was evaluated. The recovery of the enzyme after drying with 10% of lactose, -cyclodextrin, maltodextrin, mannitol, gum arabic, and trehalose ranged from 63% to 100%; but the enzyme activity was lost in the absence of adjuvants. Most of the adjuvants used kept up at least 50% of the enzymatic activity at 5°C and 40% at 25°C after 8 months. The lipase dried with 10% of -cyclodextrin retained 72% of activity at 5°C. From these preliminary results the optimization of drying process using -cyclodextrin, maltodextrin and lactose as adjuvants was carried out. Statistical optimization of the experimental results allowed the determination of the processing conditions that maximized the retention of the enzymatic activity (RAE), namely: concentration of drying adjuvants of 12.05 %, inlet temperature of the drying gas of 153.6oC, and flow rate of the enzymatic extract fed to the dryer of 9.36 g/min, for the b-cyclodextrin and maltodextrin as adjuvants. For lactose as adjuvant the study showed that increasing the amount of drying adjuvant and/or decreasing the inlet gas temperature has positive effect on the retention of enzymatic activity of the dried product. After the purification process was carried out the drying of the partially purified enzyme and pure lipase, using these three adjuvants. The retention of enzymatic activity ranged from 90.6 to 100% when was used the optimal conditions for each drying adjuvant. Concluding, the lipases produced by C. kikuchii may be efficiently spray dried since its activity enzimatic was retained in crude extract, pure lipase and in semi-purified lipase after drying.

A ocorrência das doenças de final de ciclo causadas pelos fungos Septoria glycines e Cercospora kikuchii é facilmente observada no campo. Entretanto, são necessárias informações precisas sobre a quantificação de danos e perdas na produtividade, bem como, a definição da melhor época para aplicação de fungicidas. A falta de um método padrão de quantificação visual pode levar a estimativas imprecisas da severidade das mesmas, induzindo a conclusões erradas. Com os objetivos de elaborar e validar uma escala diagramática para a quantificação das doenças de final de ciclo da soja, avaliar o efeito dessas doenças sobre a produtividade, identificar o melhor estádio fenológico da cultura para o controle dessas doenças, verificar a relação entre a severidade dessas doenças e a produtividade e avaliar os efeitos das mesmas sobre a duração e absorção da área foliar sadia das plantas de soja, foram instalados experimentos na Fazenda Areão (ESALQ/USP), localizada em Piracicaba - SP. Utilizou-se o cultivar de soja MG/BR - 46 (Conquista), considerado suscetível a essas doenças, adotando-se o delineamento experimental de blocos ao acaso, no esquema fatorial 2 x 3 (dois fungicidas: benomyl e tebuconazole e três momentos fenológicos de pulverização: R4, R5.3 e R6) com três tratamentos adicionais (testemunha, pulverização quinzenal de benomyl e pulverização quinzenal de tebuconazole), repetidos três vezes. Determinou-se a severidade dessas doenças; o número de vagens e de sementes por planta; a massa de 1000 sementes e a produtividade. Para quantificação da severidade dessas doenças foi elaborada uma escala diagramática a partir de folhas coletadas em campo, apresentando diferentes níveis de severidade. A área de cada folha e sua correspondente severidade foram determinadas e, seguindo-se a "Lei do Estímulo de Weber-Fechner", elaborou-se a escala com os níveis de severidade de doença: 2,4; 15,2; 25,9; 40,5 e 66,6%. A validação foi realizada por nove avaliadores, sem experiência na avaliação das doenças de final de ciclo, os quais estimaram a severidade de 30 folíolos de soja com sintomas destas doenças. A precisão das avaliações variou de acordo com o avaliador (0,84<R 2 <0,65), bem como a acurácia (0,00<a<3,40; 0,90<b<1,29), não ocorrendo erro sistemático na superestimativa ou subestimativa da doença entre os avaliadores. Com base nos resultados obtidos em três safras agrícolas consecutivas (1999/2000, 2000/2001 e 2001/2002), pôde-se concluir que: a) a escala diagramática desenvolvida é adequada para a quantificação da severidade das doenças de final de ciclo em soja; b) as doenças de final de ciclo, quando em severidade inferior a 10%, não afetam o número de vagens e sementes produzidos por planta mas, causam redução na produtividade da soja; c) os fungicidas benomyl e tebuconazole podem ser utilizados no controle dessas doenças; d) não foi possível determinar a época fenológica mais adequada para a pulverização de fungicidas, que proporcionasse aumentos na produtividade da soja; e) em severidade dessas doenças abaixo de 10%, não existe relação entre esta variável, a massa de 1000 sementes e a produtividade; f) a absorção da área foliar sadia é a variável que melhor expressa a relação entre a severidade dessas doenças e a produtividade da soja. / The occurrence of late season leaf diseases caused by the fungus Septoria glycines and Cercospora kikuchii is easily identified in field. However, there is a need of precise information to quantify the damage and yield losses as well as to define the best occasions for fungicide applications. The lack of a standardized visual method may lead to inaccurate estimates of their severity, inducing inexact conclusions. With the purposes: to elaborate and validate a diagrammatic scale to assess late season leaf soybean diseases; to evaluate the effect of theses diseases on yield; to identify the best soybean growth stages for chemical control; to verify the relation between severity of theses diseases and yield and to evaluate the effect of theses diseases on healthy leaf area duration and healthy leaf area absorption, were installed field experiments at Fazenda Areão (ESALQ/USP), in Piracicaba - SP. The soybean cultivar MG/BR - 46 (Conquista), susceptible to both diseases was sown in a complete randomized blocks design in factorial 2 x 3 (two fungicides: benomyl and tebuconazole and three application stages: R4, R5.3 e R6) and three extra treatments (control without application, fortnightly application of benomyl and fortnightly application of tebuconazole), at three replications. The severity, the pod number per plant, the seed number per pod, the mass of 1,000 seed and yield were determined. To quantify the severity of these diseases it was elaborated a diagrammatic scale, from leaves collected in the field presenting different levels of severity. The area of each leaf and its correspondent severity were determined, and following the "Stimulus Law by Weber-Fechner" a scale was elaborated with the severity levels: 2.4, 15.2, 25.9, 40.5 and 66.6%. The validation was carried out by nine appraisers, without previous practice in assessing late season leaf diseases, who estimate the severity on 30 leaflets of soybean with disease symptoms. The evaluation precision varied according to the appraiser (0.84<R 2 <0.65), as well as the accuracy (0.00<a<3.40; 0.90<b<1.29), do not occurring systematic mistakes in the super or underestimation of the disease among the appraisers. Based on the obtained results in three consecutive crop seasons (1999/2000, 2000/2001 e 2001/2002), it was possible to conclude that: a) the developed diagrammatic scale is appropriate to quantify the late season leaf diseases severity of soybean; b) the late season leaf diseases, when in severity below 10%, do not affect the number of pod and seeds per plant but cause reduction of yield; c) the fungicides benomyl and tebuconazole may be used to control these diseases; d) it was not possible to determine the most appropriate growth stage for fungicide application, which result in yield gain; e) under severity below 10% there are not relation between this variable and the mass of 1,000 seed and yield; f) the healthy leaf area absorption is the variable which most express the relation between the severity of this diseases and the soybean yield.