The following body of research provides a detailed overview of the interactive effects of biocontrol agents and environmental factors and how these influence both the host plant and pathogen populations within hydroponic systems. Pythium and other zoosporic fungi are pathogens well suited to the aquatic environment of hydroponics. Motile zoospores facilitate rapid dispersal through fertigation water, resulting in Pythium becoming a yield reducing factor in most hydroponic systems and on most crops. With increasing trends away from pesticide use, biocontrol is becoming an ever more popular option. Unfortunately, much of our knowledge of biocontrol agents and their formulation can not be directly transferred to the widely differing environments of hydroponic systems. Paulitz (1997) was of the opinion that if biocontrol was to be successful anywhere, it would be in hydroponics. This is primarily due to the increased ability, in hydroponics, to control the growing environment and to differentiate between the requirements of the pathogen versus those of the host plant and biocontrol agent. Key environmental factors were identified as soil moisture, root zone temperature, form of nitrogen and pH. A review of the literature collated background information on the effects of biocontrol agents and environmental manipulation on plant growth and disease severity in hydroponic systems. A commercial formulation of Trichoderma (Eco-T(R1)) was used as the biocontrol agent in all trials. Dose responses in Pythium control and plant growth stimulation in lettuce were first determined using a horizontal trough system (closed system). In such systems optimum application rates were found to be lower than in field application (1.25x10[to the power of 5] spores/ml). This is probably because Trichoderma conidia are not lost from the system, but re-circulate until being transported into the root zone of a host plant. No significant growth stimulation was observed, although at high doses (5x10[to the power of 5] and 2.5x10[to the power of 5] spores/ml) a significant reduction in yield was recorded. Possible reasons for this growth inhibition are suggested and a new theory is proposed and investigated later in the thesis. In an open system of cucumber production (drip irrigated bag culture) no statistically significant results were initially obtained, however, general trends still showed the occurrence of positive biocontrol activity. The initial lack of significant results was mostly due to a poor knowledge of the horticulture of the crop and a lack of understanding of the epidemiology behind Trichoderma biocontrol activity. These pitfalls are highlighted and, in a repeat trial, were overcome. As a result it could be concluded that application rates in such systems are similar to those used in field applications. Management of soil moisture within artificial growing media can aid in the control of Pythium induced reductions in yield. A vertical hydroponic system was used to determine the interactive effects of soil moisture and Trichoderma. This system was used because it allowed for separate irrigation regimes at all 36 stations, controlled by a programmable logic controller (PLC). With lettuce plants receiving optimum irrigation levels, no significant reduction in yield was observed when inoculated with Pythium. However, after Pythium inoculation, stresses related to over- or under-watering caused significant yield losses. In both cases, Trichoderma overcame these negative effects and achieved significant levels of disease control, especially under higher soil moisture levels. Growth stimulation responses were also seen to increase with increasing soil moisture. Similar results were obtained from strawberry trials. These results show that Pythium control is best achieved through the integration of Trichoderma at optimum soil moisture. However, where soil moisture is above or below optimum, Trichoderma serves to minimize the negative effects of Pythium, providing a buffering capacity against the effects of poor soil moisture management. Pythium, root zone temperature and form of nitrogen interact significantly. In greenhouse trials using horizontal mini troughs with facilities for heating or cooling recirculating water, nitrate fertilizer treatments resulted in statistically significant results. Lettuce growth was highest at 12°C, although no significant differences in yield were observed between 12-24°C. Pythium was effective in causing disease over the same temperature range. Pythium inoculation did not result in yield reduction at 6 and 30°C. Trichoderma showed a slight competitive advantage under cooler temperatures (i.e., 12 degrees C), although significant biocontrol occurred over the 12-24 degrees C range. Ammonium fertilizer trials did not generate statistically significant data. This is possibly due to complex interactions between root temperature, ammonium uptake, and competitive exclusion of nitrification bacteria by Trichoderma. These interactions are difficult to replicate over time and are probably influenced by air temperature and available light which are difficult to keep constant over time in the system used. However, the data did lead to the first clues regarding the effects of Trichoderma on nitrogen cycling as plants grown with a high level of ammonium at high temperatures were seen to suffer more from ammonium toxicity when high levels of Trichoderma were added. In further trials, conducted in the recirculating horizontal mini trough system, it was determined that Trichoderma applications resulted in an increase in the percentage ammonium nitrogen in both the re-circulating solution and the growing medium. This was a dose-related response, with the percentage ammonium nitrogen increasing with increasing levels of Trichoderma application. At the same time an increase in ammonium in the root tissue was observed, corresponding with a decrease in leaf nitrate levels and an increase in levels of Cu, Na, Fe and P in leaf tissue. In independent pot trials, populations of nitrifying bacteria in the rhizosphere were also seen to decrease with increasing Trichoderma application rates. This led to the conclusion that the increase in ammonium concentration was as a result of decreased nitrification activity due to the competitive exclusion of nitrifying bacteria by Trichoderma. The possibility that Trichoderma functions as a mycorrhizal fungus and so increases the availability of ammonium for plant uptake is not discarded and it is thought that both mechanisms probably contribute. Water pH provides the most powerful tool for enhancing biocontrol of Pythium by Trichoderma. Trichoderma shows a preference for more acidic pHs while Pythium prefers pHs between 6.0 and 7.0. In vitro tests showed that Trichoderma achieved greater control of Pythium at pH 5.0, while achieving no control at pH 8.0. In greenhouse trials with the recirculating horizontal mini trough system, yield losses resulting from Pythium inoculation were greatest at pH 6.0 and 7.0, with no significant reduction in yield at pH 4.0. Biocontrol activity showed an inverse response with greatest biocontrol at pH 5.0. / Thesis (Ph.D.)-University of KwaZulu-Natal, Pietermaritzburg, 2003.

Fungi and Oomycetes represent the two most important groups of eukaryotic plant pathogens. Besides chemical and physical control of these pathogens, biological control is an approach enjoying increasingly more focus. One of the biological agents increasingly employed in biological control of plant pathogenic fungi is ironically the fungus Trichoderma, more specifically Trichoderma harzianum. Besides control of the fungal plant pathogens, another interesting aspect observed when plants are treated with Trichoderma harzianum are effects such as complete and even stand of plants, faster seed germination, increases in plant height and overall enhanced plant growth. Though there have been various studies on this effect, almost no research has yet been conducted to elucidate the mechanism by which these effects occur. In particular, effects such as faster seed germination suggest that Trichoderma harzianum produces a metabolite that may mimic the plant growth hormone gibberellic acid. Through an evaluation of the various metabolites produced by Trichoderma harzianum; gliotoxin seemed structurally most similar to gibberellic acid. To verify that gliotoxin can indeed serve as an analogue for gibberellic acid and elicit similar physiological responses in plants, a two–pronged approach was followed. Firstly, molecular similarity evaluation through common pharmacophore evaluation was conducted, followed by docking simulations into the recently discovered receptor for gibberellic acid. Common pharmacophore evaluation between gibberellic acid and gliotoxin showed successful alignment of gliotoxin into the gibberellic acid based pharmacophore space. Furthermore, docking simulations further strengthened this by the similarity in docking scores calculated and the similar poses of the ligands (gliotoxin and gibberellic acid) in the receptor space. However, similarity in pharmacophore alignment and docking simulation results only suggest that gliotoxin should be able to occupy the receptor space, but it is not a guarantee that similar physiological responses will be elicited. In the second part of the project, the ability of gliotoxin to elicit similar physiological responses in plants to gibberellic acid was investigated. For this, a–amylase induction; plant emergence and height; and chlorophyll fluorescence were compared for both gliotoxin and gibberellic acid treatments. In terms of a–amylase induction, gliotoxin was able to induce production of the enzyme as visualised by starch–containing native gel electrophoresis (zymograms). Gliotoxin induced the strongest response at a 10–6 M dilution which is typically the range expected for hormones in biological systems in de–embryonated seeds of Phaseolus vulgaris. Gibberellic acid was able to induce the strongest response at a 10–7 M dilution. In essence, similar physiological responses were observed. In terms of plant emergence and plant height, treatment with gliotoxin or gibberellic acid resulted in plant emergence a day earlier than the untreated control. However, even though there were slight differences in plant height favouring the gliotoxin or gibberellic acid treated plants, the differences were not statistically significant. Thus, in this regard similar responses were again observed for both gliotoxin and gibberellic acid treatments. In the final evaluation the effect of gliotoxin and gibberellic acid treatments on the chlorophyll fluorescence of mature plants was investigated. Overall, both gliotoxin and gibberellic acid elicited beneficial effects on plant vitality, expressed through PI(Abs) with the gliotoxin treatment performing better than the equivalent gibberellic acid treatment. Overall, the physiological tests demonstrated that gliotoxin can indeed elicit similar positive physiological responses to gibberellic acid in Phaseolus vulgaris. Furthermore the test used in this project can serve as a standard evaluation bench for screening for gibberellic acid analogues on a laboratory scale before larger scale field trials are considered. / Thesis (Ph.D. (Microbiology))--North-West University, Potchefstroom Campus, 2012.

Fungi and Oomycetes represent the two most important groups of eukaryotic plant pathogens. Besides chemical and physical control of these pathogens, biological control is an approach enjoying increasingly more focus. One of the biological agents increasingly employed in biological control of plant pathogenic fungi is ironically the fungus Trichoderma, more specifically Trichoderma harzianum. Besides control of the fungal plant pathogens, another interesting aspect observed when plants are treated with Trichoderma harzianum are effects such as complete and even stand of plants, faster seed germination, increases in plant height and overall enhanced plant growth. Though there have been various studies on this effect, almost no research has yet been conducted to elucidate the mechanism by which these effects occur. In particular, effects such as faster seed germination suggest that Trichoderma harzianum produces a metabolite that may mimic the plant growth hormone gibberellic acid. Through an evaluation of the various metabolites produced by Trichoderma harzianum; gliotoxin seemed structurally most similar to gibberellic acid. To verify that gliotoxin can indeed serve as an analogue for gibberellic acid and elicit similar physiological responses in plants, a two–pronged approach was followed. Firstly, molecular similarity evaluation through common pharmacophore evaluation was conducted, followed by docking simulations into the recently discovered receptor for gibberellic acid. Common pharmacophore evaluation between gibberellic acid and gliotoxin showed successful alignment of gliotoxin into the gibberellic acid based pharmacophore space. Furthermore, docking simulations further strengthened this by the similarity in docking scores calculated and the similar poses of the ligands (gliotoxin and gibberellic acid) in the receptor space. However, similarity in pharmacophore alignment and docking simulation results only suggest that gliotoxin should be able to occupy the receptor space, but it is not a guarantee that similar physiological responses will be elicited. In the second part of the project, the ability of gliotoxin to elicit similar physiological responses in plants to gibberellic acid was investigated. For this, a–amylase induction; plant emergence and height; and chlorophyll fluorescence were compared for both gliotoxin and gibberellic acid treatments. In terms of a–amylase induction, gliotoxin was able to induce production of the enzyme as visualised by starch–containing native gel electrophoresis (zymograms). Gliotoxin induced the strongest response at a 10–6 M dilution which is typically the range expected for hormones in biological systems in de–embryonated seeds of Phaseolus vulgaris. Gibberellic acid was able to induce the strongest response at a 10–7 M dilution. In essence, similar physiological responses were observed. In terms of plant emergence and plant height, treatment with gliotoxin or gibberellic acid resulted in plant emergence a day earlier than the untreated control. However, even though there were slight differences in plant height favouring the gliotoxin or gibberellic acid treated plants, the differences were not statistically significant. Thus, in this regard similar responses were again observed for both gliotoxin and gibberellic acid treatments. In the final evaluation the effect of gliotoxin and gibberellic acid treatments on the chlorophyll fluorescence of mature plants was investigated. Overall, both gliotoxin and gibberellic acid elicited beneficial effects on plant vitality, expressed through PI(Abs) with the gliotoxin treatment performing better than the equivalent gibberellic acid treatment. Overall, the physiological tests demonstrated that gliotoxin can indeed elicit similar positive physiological responses to gibberellic acid in Phaseolus vulgaris. Furthermore the test used in this project can serve as a standard evaluation bench for screening for gibberellic acid analogues on a laboratory scale before larger scale field trials are considered. / Thesis (Ph.D. (Microbiology))--North-West University, Potchefstroom Campus, 2012.

Thèse (M.Sc.)--Université Laval, 2004. / Bibliogr.: f. 61-87. Publié aussi en version électronique.

Orientador: Luiz Antônio Graciolli / Banca: Luzia Doretto Paccola Meirelles / Banca: Marli de Fátima Stradioto Papa / Abstract: The occurrence of contaminants fungus and competitors are common during the shiitake cultivation in logs. In Brazil, few are the works that identify and they analyze the effects of these microorganisms in the shiitake production. However, it is known that the incidence of these fungus in great proportions can take to the unproductiveness the logs. Therefore, the present work had as objective to test the effect of the moisturized whitewash and of the fungicide benomyl in the control of contaminant fungus and his answer in the shiitake production in logs. For so much, it was tested in vitro previously the effect of the benomyl in different concentrations (0.5; 1.0; 2.0; 4.0; 8.0 and 16.0 mg/mL) in the mycelial growth of five shiitake lineages (JAB-L; JAB-K; LE-96/17; LE-95/01 and, LE-96/22), in way to select the most tolerant lineage to the concentrations of proposed benomyl, comparing with the growth of the same ones without the presence of this fungicide (control). A completely randomized design in a factorial scheme 5X7, tends in the total 35 treatments, each one with three repetitions, corresponding to a plate of Petri. It was observed that only shiitake lineage did not suffer any effect of the benomyl in the proposed concentrations was the LE-96/17, being the chosen to be used in the experiment in logs, when the largest benomyl concentration was submitted (16.0 mg/mL). The other study was conducted in logs in an interely randomized design, with three treatments: control; whitewash (8 kg of whitewash / 60 liters of water) applied in the production logs soon after the inoculation and after each induction shock; and benomyl (16.0 mg/mL) applied biweekly starting from the inoculation of the logs. Each treatment contained 60 repetitions, being the experimental unit corresponding to a log. The used logs were of Eucalyptus urophylla, which were... (Complete abstract, click electronic address below). / Resumo: A ocorrência de fungos contaminantes e competidores é comum durante o cultivo de shiitake em toros. No Brasil, poucos são os trabalhos que identificam e analisam os efeitos destes microrganismos na produção de shiitake. No entanto, sabe-se que a incidência destes fungos em grandes proporções pode levar à improdutividade dos toros. Portanto, o presente trabalho teve como objetivo testar o efeito da cal hidratada e do fungicida benomyl no controle de fungos contaminantes e sua resposta na produção de shiitake em toros. Para tanto, testou-se anteriormente in vitro o efeito do benomyl nas concentrações 0,5; 1,0; 2,0; 4,0; 8,0 e 16,0 mg/mL no crescimento micelial das linhagens de shiitake JAB-L; JAB-K; LE-96/17; LE-95/01 e LE-96/22, de modo a selecionar a linhagem mais tolerante as concentrações de benomyl propostas, comparando com o crescimento das mesmas sem a presença deste fungicida (testemunha). O delineamento deste experimento foi inteiramente casualizado em fatorial 5X7, tendo no total trinta e cinco tratamentos, cada qual com três repetições, sendo cada repetição correspondente a uma placa de Petri. Observou-se que a única linhagem de shiitake a não sofrer qualquer efeito do benomyl nas concentrações propostas foi a LE-96/17, sendo a escolhida para ser utilizada no experimento em toros, a qual foi submetida a maior concentração de benomyl (16,0 mg/mL). O delineamento do experimento em toros foi inteiramente casualizado, com três tratamentos: testemunha; cal (8 kg de cal/ 60 litros de água) aplicados nos toros de produção logo após a inoculação e após cada choque de indução; e benomyl (16,0 mg/mL) aplicados quinzenalmente a partir da inoculação dos toros. Cada tratamento conteve 60 repetições, sendo a unidade experimental correspondente a um toro. Os toros utilizados foram de Eucalyptus urophylla, os quais foram inoculados... (Resumo completo, clicar acesso eletrônico abaixo). / Mestre

Orientador: Fernando Carlos Pagnocca / Banca: José Eduardo Marcondes de Almeida / Banca: Odair Correa Bueno / Resumo: Os gêneros de formigas-cortadeiras, Atta e Acromyrmex, mantém uma associação mutualística com o basidiomiceto Leucoagaricus gongylophorus, o qual é responsável pela produção de enzimas que degradam o material vegetal transportado para o interior dos ninhos. As formigas-cortadeiras são consideradas pragas devido a enorme quantidade de material vegetal que é cortada, principalmente em ambientes alterados por atividades antrópicas, como os locais de agricultura e reflorestamento. Devido à necessidade de manejo das formigascortadeiras, o controle biológico representa um método promissor e ambientalmente amigável. Nesse contexto, devido a íntima relação entre as formigas e o fungo mutualista, é de grande importância selecionar linhagens de fungos filamentosos que possam interferir negativamente sobre o jardim de fungos. No presente trabalho isolamos fungos filamentosos contaminantes de colônias iniciais da formiga Atta sexdens rubropilosa. Após o isolamento e identificação foram realizados testes para avaliar o crescimento desses fungos utilizando o jardim de fungos como substrato, e também as linhagens consideradas melhores nesse aspecto foram testadas em colônias completas. A combinação de dois fungos (Trichoderma harzianum e Trichoderma virens) mostrou-se promissora para prosseguimento nos testes e futuro uso no controle biológico. Adicionalmente, foi avaliada e secreção de compostos orgânicos voláteis através de análises GC/MS de três linhagens de fungos filamentosos após sete dias de crescimento em culturas individuais e também frente ao fungo mutualista. Ainda, utilizando a microscopia eletrônica de varredura, avaliamos como ocorre a interação do fungo R. variabilis frente ao fungo mutualista, demonstrando que o fungo R. variabilis possui caráter micoparasita ao interagir com o fungo mutualista / Abstract: The leaf-cutting ants, Atta and Acromyrmex, maintain a mutualistic association with the basidiomycete Leucoagaricus gongylophorus, which is responsible for the production of enzymes that degrade the plant material transported to the interior of the nests. The leafcutting ants are considered pests due to an enormous amount of plant material they cut, mainly at areas altered by anthropic activities, such as agricultural and reforestation sites. Due to the need for management of the leaf cutting ants, biological control represents a promising and environmentally friendly method. In this context, due to the close relationship between ants and mutual fungi, it is of great importance to select strains of filamentous fungi that negatively impact the fungus garden. Thus, in the present study, we evaluated lineages of filamentous fungi isolated from the initial colonies of Atta sexdens rubropilosa. After isolation and identification of the contaminants filamentous fungi, assays were carried out in order to determine the fungal growth of the most suitable lineages on the fungus garden of the lab colonies The combination of two fungi (Trichoderma harzianum and Trichoderma virens) was promising for the development of methods aiming at the biological control of leaf-cutting ants. Additionally, the production of volatile organic compounds was evaluated by the GC / MS analysis of three of the filamentous fungal strains after 7 days of growth in individual cultures, and also in the presence of the mutualistic fungus. Images of scanning electron microscopy were taken during the co-culture assays / Mestre

Memoria para optar al Título Profesional de: Ingeniero Agrónomo / Se evaluó la capacidad bioantagónica in vitro de diferentes bacterias (Bacillus sp. cepa 1; Paenibacillus sp. cepas 1 y 2) y hongos (cepas 24-A y 24-B de Clonostachys rosea; cepas Th 650, Th 12A 10.1 y ThF 2-1 de Trichoderma harzianum) y los productos comerciales Actinovate® (Streptomyces lydicus WYEC 108) y Trichonativa® (Trichoderma virens, Trichoderma harzianum y Trichoderma parceramosum) en el control de 13 cepas de basidiomycotas recuperadas desde plantas de vid con síntomas de enrollamiento clorótico y degradación de la madera, provenientes de 3 regiones de Chile (IV Región de Coquimbo, V Región de Valparaíso y RM de Chile). Se caracterizaron las diferentes cepas de hongos aislados respecto a temperatura (19, 22, 25 y 28ºC) y pH (5, 6, 7 y 8). La capacidad bioantagónica se determinó a través de pruebas de antagonismo directo realizado mediante cultivos duales y antagonismo por metabolitos difusibles. En los resultados de caracterización de las cepas recuperadas, se observó que el mayor crecimiento miceliar se obtuvo a 28ºC a un pH 6 del medio, determinándose ambos valores como los óptimos para el desarrollo de los aislados investigados. Acorde con los resultados obtenidos en antagonismo directo, todos los bioantagonistas demostraron tener un cierto grado de capacidad antagónica sobre las cepas. Mediante la inhibición del crecimiento miceliar, la bacteria que presentó un mayor efecto fue la cepa 1 de Paenibacillus sp., con un máximo de 63,5% de inhibición. En el caso de los hongos fueron todas las cepas de T. harzianum utilizadas, oscilando el máximo porcentaje de control entre un 82,7% para la cepa Th 650, un 84,6% para Th 12A 10.1 y 87,7% para la cepa ThF 2-1. En relación a los productos comerciales, Trichonativa® logró un 61,5% de inhibición miceliar, mientras que Actinovate® logró un 100% de inhibición, siendo por lo tanto S. lydicus WYEC108 el antagonista que ejerció el mayor efecto. Los resultados obtenidos en el ensayo de antagonismo mediante metabolitos difusibles ayudaron a corroborar el tipo de mecanismo de biocontrol que ejercieron los antagonistas. Las bacterias utilizadas para este ensayo reflejaron que básicamente son los metabolitos difusibles que producen, los que ejercen la inhibición de crecimiento miceliar de las cepas de basidiomycotas, obteniéndose con ambas cepas 1, de Bacillus sp. y Paenibacillus sp., valores en promedio superiores a 70%; en relación al producto comercial Actinovate®, se obtuvo una completa inhibición miceliar. Con respecto a los hongos C. rosea 24-B y T. harzianum ThF 2-1, estos mostraron una baja inhibición mediante la producción de metabolitos difusibles, obteniéndose valores en promedio que no superaron el 14% de inhibición de crecimiento miceliar, lo que indica que poseen otro tipo de mecanismos como en el caso de las cepas de T. harzianum. Para el producto comercial Trichonativa®, se obtuvo en promedio un 16,7% de inhibición miceliar, siendo éste el mayor dentro de los hongos. Los resultados obtenidos señalan a S. lydicus WYEC108 (Actinovate®) como al bioantagonista de mayor efecto. / The in vitro bioantagonistic capacity of different bacteria (Bacillus sp. strain 1, Paenibacillus sp. strains 1 and 2) and fungi (24-A and 24-B strains of Clonostachys rosea, and Th 650, Th 12A 10.1 and ThF 2-1 strains of Thrichoderma harzianum) and of the biofungicides Actinovate ® (Streptomyces lydicus WYEC 108) and Trichonativa ® (Trichoderma virens, Trichoderma harzianum and Trichoderma parceramosum) was evaluated in the control of 13 basidiomycota strains recovered from grapevines grown in three regions of Chile (IV Region of Coquimbo, V Region of Valparaiso and Metropolitan Region). The vines showed wood degradation and chlorotic leafroll symptoms. Different isolated fungus strains were characterized with respect to temperature (19, 22, 25, and 28ºC) and pH (5, 6, 7, and 8). The bioantagonistic capacity was determined by tests of direct antagonism with dual cultures and by diffusible metabolite antagonism. The results of recovered strain characterization showed that mycelial growth was highest with the culture medium at 28ºC and pH 6, indicating both values as optimum for the development of the investigated isolates. According to the direct antagonism results, all bioantagonists demonstrated to have some degree of antagonistic capacity over recovered strains. Through mycelial growth inhibition, the bacterium that showed a better effect was Paenibacillus sp. strain 1, with a maximum of 63.5% of inhibition. In the case of fungi, all the used T. harzianum strains had excellent results with the highest control percentages being 82.7% for the Th 650 strain, 84.6% for the Th 12A 10.1 strain and 87.7% for ThF 2-1 strain. In relation to the biofungicides tested, Trichonativa ® achieved a maximum of 61.5% of mycelial inhibition, while Actinovate ® showed 100% of inhibition of the 13 basidomycota strains. The results obtained in the antagonism trial by diffusible metabolite helped to corroborate the type of biocontrol mechanism exerted by the antagonists. The bacteria used for this trial basically showed that the diffusible metabolites that they produce are the ones which exert mycelial growth inhibition of basidiomycota strains, with both Bacillus sp. and Paenibacillus sp. strains 1 showing average values over 70%. With respect to Actinovate ®, complete mycelial growth inhibition was obtained with this biofungicide. Regarding fungi C. rosea 24-B and T. harzianum ThF 2-1, these showed low inhibition by diffusible metabolite production with average values not exceeding 14% of mycelial growth inhibition, which indicates that they have other types of mechanisms as in the case of T. harzianum strains. With Trichonativa ®, an average of 16.7% of mycelial growth inhibition was obtained, this biofungicide being the higher within the fungi used. The obtained results showed S. lydicus WYEC108 (Actinovate®) as the bioantagonist with higher effect.

Made available in DSpace on 2016-06-02T20:21:35Z (GMT). No. of bitstreams: 1 6150.pdf: 1801179 bytes, checksum: d2366a6c320c54df184a270c81c7c653 (MD5) Previous issue date: 2014-02-27 / The present situation of energy and environmental setting in the world has favored the development of alternative energy sources, and has emerged as an option the use of biofuels, such as bioethanol. Besides ethanol produced from sugarcane, there is the alternative of to use plant biomass to get called second-generation ethanol. In Brazil, the major lignocellulosic material used to produce bioethanol is sugarcane bagasse, and this ethanol is obtained by fermentation of the hydrolyzate of the plant cell wall polysaccharides, this hydrolysis is performed normally by enzymes cellulolytic. Microorganisms, such as fungi of the genus Trichoderma, which are filamentous fungi present in the soil, have a variety of these enzymes that convert cellulose into glucose. In this context, the aim of this study was the recombinant production of Trichoderma harzianum IOC-3844 endoglucanase IV and its biochemical characterization. This Endo IV, which presents high similarity with Endo IV of other Thichoderma species, was expressed using the heterologous expression system of the yeast Pichia pastoris, and after be purified, assays were performed to test their activity. The recombinant enzyme hasn t show sign of degradation in any of the substrates used and can t be confirmed its hydrolytic action. However, the literature has been showed that these enzymes have capacity to increase the breaking of lignocellulose by cellulases (or a mix of them). Based on these data, a test was performed to evaluate the ability of endo IV supplemental activity in the cocktail Acellerase 1500 (Genencor) in the hydrolysis of biomass. In the experiment, copper was added as an electron donor and despite the increase in the hydrolysis of sugarcane bagasse the results showed that copper alone improves the cocktail performance. A circular dichroism analysis was performed and showed that the recombinant Endo IV is correctly coiled. The study showed that the enzyme produced at the laboratory has a high level of expression, but its activity couldn t be detected. The intention now is to perform tests with cocktails that don t contain expansins and isolated enzymes in order to verify that the Endo IV has effect upon them. / A atual situação do cenário energético e ambiental do mundo tem favorecido o desenvolvimento de fontes alternativas de energia, e como opção tem surgido a utilização de biocombustíveis, como o bioetanol. Além do etanol produzido a partir do caldo de cana, há a alternativa de utilizar a biomassa vegetal para obter o chamado etanol de segunda geração. No Brasil o principal material lignocelulósico utilizado para produção de bioetanol é o bagaço de cana-de-açúcar, e este etanol é obtido por meio da fermentação do hidrolisado dos polissacarídeos da parede celular dos vegetais, sendo esta hidrólise, realizada, normalmente, por enzimas celulolíticas. Microrganismos, como os fungos do gênero Trichoderma, que são fungos filamentosos presentes no solo, possuem uma variedade dessas enzimas capazes de converter a celulose em glicose. Nesse contexto, o objetivo deste trabalho foi a produção recombinante da enzima endoglucanase IV de Trichoderma harzianum IOC-3844 e sua caracterização bioquímica. Esta Endo IV, que apresenta alta similaridade com as Endo IV de outras espécies de Thichoderma, foi expressa utilizando-se o sistema de expressão heteróloga da levedura Pichia pastoris e, após ser purificada, foram realizados ensaios para testar sua atividade. A enzima recombinante não demonstrou sinal de degradação em nenhum dos substratos utilizados, não podendo ser comprovada sua ação hidrolítica. Porém, na literatura consta que essas enzimas têm a capacidade de aumentar a quebra de lignocelulose por celulases (ou uma mistura delas). Com base nesses dados, um teste foi realizado para avaliar a capacidade da endo IV de suplementar a atividade do coquetel Acellerase 1500 (Genencor) na hidrólise de biomassa. No experimento, foi adicionado cobre como doador de elétrons e apesar do aumento na hidrólise do bagaço-de-cana os resultados mostraram que o cobre sozinho melhora a performance do coquetel. Uma análise de dicroísmo circular foi realizada e revelou que a Endo IV recombinante se encontra corretamente enovelada. O estudo mostrou que a enzima produzida em laboratório possui alto nível de expressão, porém sua atividade não pôde ser constatada. Pretende-se agora realizar testes com coquetéis que não contêm expansinas e com enzimas isoladas, a fim de verificar se a Endo IV possui efeito sobre elas.

Made available in DSpace on 2014-06-11T19:33:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2013-04-16Bitstream added on 2014-06-13T19:23:49Z : No. of bitstreams: 1 000739516.pdf: 1650325 bytes, checksum: a1b57134d48fa771d7c3cfd4a0dce580 (MD5) / As espécies de Trichoderma spp. e Clonostachys rosea são bastante estudadas atualmente para o controle biológico de doenças de plantas. O objetivo deste trabalho foi avaliar o potencial dos antagonistas, Trichoderma asperellum (LQC96) e Clonostachys rosea (LQC62), no controle biológico das principais doenças fúngicas de begônia (mofo cinzento, mancha de Mirotécio e podridão do colo). A etiologia da podridão do colo em begônias foi estudada por meio das análises de suas características morfológicas e moleculares. Foram realizados ensaios in vitro a fim de verificar os mecanismos envolvidos no controle biológico dessas doenças, avaliados por meio da produção de compostos voláteis e compostos solúveis com ação inibitória aos patógenos, além da capacidade hiperparasítica dos antagonistas. A metodologia para avaliar os compostos voláteis consistiu em posicionar dois fundos de placas de Petri sobrepostas, contendo em uma delas cultura do antagonista e em outra, do patógeno. Para avaliar os compostos solúveis, foram utilizadas duas metodologias, o método em cultivo sobre o papel celofane para LQC96 e o método do filtrado da cultura líquida para LQC62. O hiperparasitismo foi avaliado por meio de culturas pareadas. Verificou-se a colonização e a supressão da esporulação em discos e em hastes de begônias. Após a análise do potencial in vitro, foram realizadas ensaios em casa de vegetação, a fim de verificar o controle dessas doenças em cultivo comercial de begônia. Nos ensaios in vitro, verificou-se que os antagonistas tiveram atividade antagônica contra esses patógenos, com a produção de compostos voláteis e solúveis, além de demonstrar a capacidade hiperparasítica de ambos antagonistas. Ambos agentes de controle biológico avaliados foram capazes de colonizar os discos e hastes de begônia e a aplicação preventiva na concentração de... / Currently the species of Trichoderma spp. and Clonostachys rosea are widely studied for biological control of plant diseases. The aim of this study was to evaluate the potential of these isolates these antagonists (T. asperellum LQC96 and C. rosea LQC62) in the biological control of major fungal diseases of begonia (gray mold, Myrothecium leaf spot and stem rot). The etiology of stem rot was studied using morphological and molecular analysis. In vitro assays were performed to verify the mechanisms involved in the biological control of these diseases, measured by the production of volatile and soluble compounds with an inhibitory effect on pathogens and the ability of antagonist hyperparasitism. The methodology for assessing the volatiles consisted of placing two Petri dishes superimposed on one culture containing the antagonist and another pathogen. To evaluate soluble compounds, two methods were used, the cultivation on cellophane for LQC96 and and the culture liquid filtrate method for LQC62. The hyperparasitism was assessed using paired cultures. There colonization and suppression of sporulation was evaluate on leaf disks and stem of begonias. After analysis of the potential in vitro assays, were performed in a greenhouse in order to verify the control of these diseases in commercial cultivation of begonia. In vitro assays was found that the antagonists were antagonistic activity against these pathogens, producing volatile and soluble compounds, and demonstrate the ability of hyperparasitism. Both biocontrol agents evaluated were able to colonize the leaf discs and stem of begonia and preventive application in concentration of 1x107 conidia mL-1 proved to be the most effective in suppressing pathogen sporulation on leaf discs and stem of begonia. The essay on commercial cultivation decreased the incidence of diseases with weekly application of antagonists. For control of gray mold and ...

Made available in DSpace on 2014-06-11T19:35:05Z (GMT). No. of bitstreams: 0 Previous issue date: 2005Bitstream added on 2014-06-13T18:46:07Z : No. of bitstreams: 1 adalberto_pr_dr_araiq.pdf: 786247 bytes, checksum: cf55691544529062c4d2288dd8709c7a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / A β-D-galactopiranosídeo hidrolase é uma enzima com ampla aplicação tecnológica, clínica e experimental. A enzima possui um íon de metal bivalente coordenado. Para se obter informações a respeito do centro metálico da enzima, algumas espécies de fungos filamentosos foram testadas para a produção de β−galactosidase, ente as quais, a linhagem de Trichoderma reesei FTKO foi selecionada. O extrato enzimático, ao passar pelo processo de purificação, revelou ao menos duas isoformas de β- galactosidase identificadas como BGT I e BGT II. As enzimas são termofílicas e termotolerantes. A dependência do pH para a atividade, os valores de medidas cinéticas e a atividade revelada em gel de poliacrilamida distinguem as isozimas. Para avaliar a necessidade de metais bivalentes foi padronizada uma metodologia de desativação da enzima, apoiado na competição entre o agente seqüestrante e a enzima pelos metais. A metodologia foi aplicada ao o extrato comercial Lactozym 3000 (Novozyme Latin America Ltd.) revelou a necessidade de metal para a sua atividade com a desativação seguida de reativação pelos íons Mg2+, Mn2+, Co 2+, Ni2+, mas não por Zn2+ e Eu3+. A enzima BGT I (pH ótimo de 4,5) mostrou-se, contudo, resistente à desativação, pois se mantém ativa mesmo em altas concentrações de EDTA. / The β-D-galactopiranosyde-hidrolase is an enzyme with large technological, clinic, and scientific application. The enzyme coordinates a bivalent metal ion. In order to investigate the metal center of the enzyme of moulds, some strains were tested for β−galactosidase production. Among then, Trichoderma reesei FTKO were selected. The partially purified enzymatic extract reveals the production of two isoforms of β-galactosidase, at least. This enzymes are identified as BGT I e BGT II. Both are termophylic and thermotolerant. The pH dependence, the measured kinetic values and the activity revealed in polyacrylamide electrophoresis gels distinguished the isozymes. The metal assistance of enzymatic activity was evaluated in a standard deactivation methodology of enzyme by competition for the metal by chelating agent and metal site of enzyme. When this methodology was applied in the deactivation of commercial extract Lactozym 3000 (Novozyme Latin America Ltd.), revealed the metal importance for activity. The deactivation was reverted by metals ions as Mg2+, Mn2+, Co2+, Ni2+, but not by Zn2+ or Eu3+. On the other hand BGT I enzyme (optimal pH of 4,5) maintained the activity, even in high concentration of EDTA.