Dissertationen zum Thema „Sorokiniana“

O fungo filamentoso Bipolaris sorokiniana é um fitopatógeno que causa moléstias em cereais de inverno, tais como a mancha marrom, a ponta preta dos grãos e a podridão comum da raiz. O controle deste fungo é dificultado pelo fato do mesmo apresentar uma ampla variabilidade morfológica, fisiológica e genética. Assim sendo, os objetivos deste trabalho foram estudar a variabilidade fisiológica, genética e virulência de isolados de B. sorokiniana. Foram utilizados 35 isolados de B. sorokiniana e um isolado de B. orizae, provenientes de diferentes regiões geográficas do Brasil e de outros países. Inicialmente foi realizado o agrupamento dos isolados, conforme as características morfológicas e a medida da taxa de crescimento dos mesmos, separando-os em grupos morfológicos. Os isolados foram avaliados quanto à atividade enzimática em meio sólido, a virulência em sementes e plântulas de trigo e perfil de proteínas totais em gel SDS-PAGE. Com os resultados obtidos, cinco grupos morfológicos foram formados, com diferenças na coloração micelial e crescimento. Foram encontradas variações entre os isolados quanto à atividade enzimática, sendo a esterase a enzima que apresentou os mais altos índices de atividade. Os resultados do ensaio de virulência mostraram diferenças na porcentagem de sementes e plântulas infectadas, entre isolados da mesma região geográfica e grupo morfológico. O perfil de proteínas totais apresentou uma variação no número e intensidade das bandas no gel, onde algumas destas podem ser características da espécie. Também foi avaliada a incompatibilidade vegetativa entre os isolados, a influência de diferentes meios de cultivo sobre a incompatibilidade e a análise eletroforética de proteínas totais dos isolados, quando crescidos isoladamente e em reações de incompatibilidade e compatibilidade. Dos 31 cruzamentos de incompatibilidade realizados, 18 mostraram-se totalmente incompatíveis e essas reações apresentaram alterações nos diferentes meios de cultivo utilizados, evidenciando a influência do substrato nesta reação. Alguns isolados quando crescidos em condição de pareamento mostraram bandas mais intensas na eletroforese, sugerindo que algumas proteínas poderiam ser expressas em níveis mais elevados durante o co-cultivo.
Bipolaris sorokiniana is a phytopathogenic fungus that causes diseases in cereal crops, such as leaf spot disease, black point of the grain and common root rot. Because of its high morphological, physiological and genetic variability, the fungus control is a difficult task. The aim of this work was to study the physiological and genetic variability and the virulence of B. sorokiniana isolates. For this, 35 B. sorokiniana and one B. orizae isolates were used, proceeding from different geographic regions in Brazil and other countries. Initially, the isolates were evaluated for their morphological variability, considering mycelia color, sector formation, and growth rate. With this result the isolates were grouped by their morphologic characteristics. Extra-cellular enzymatic activity was analyzed in solid medium for all isolates, pathogenicity in wheat seeds and seedlings and analysis of total proteins by SDS-PAGE was done. Five morphological groups were formed with the results obtained with the morphological and growth characteristics. Variations among the isolates were found for enzymatic activity, and esterase was the enzyme that presented highest activity indices. The results obtained from infection of seeds and seedlings showed that isolates from the same geographic region and morphologic group had different degrees of virulence. The total protein profile presented by the isolates showed a variation in the bands number and intensity, where some of them can be characteristic of the specie. The vegetative incompatibility between the isolates was evaluated and the influences that different media culture in this reaction. The total proteins profile of the isolates was analyzed when the isolates were cultivated separately and in compatibility and incompatibility reactions. Thirty one crossings were realized and 18 out of them showed vegetative incompatibility, and theses reactions had presented alterations with different media culture. This result strongly suggests the influence of the substratum in this reaction. The isolates when pareated shown more intense protein bands in SDS-PAGE, suggesting that some proteins could be expressed in higher levels during co culture of the fungus.

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)
In aquatic environments, phytoplankton consists mostly of photosynthetic microorganisms that serve as the basis of food chains. However, besides photoautotrophy, it is widely reported in the literature that many microalgae can take up dissolved organic matter present in the environment concomitantly with the photosynthesis, a metabolic pathway known as mixotrophy. Little is known about the ecophysiology of mixotrophy in microalgae, and almost all studies are focused on the use of this metabolic pathway to increase the production of algal biomass and stimulate the production of specific biomolecules. Another important issue, considering the current anthropic activity, is that most of the contaminants eliminated in aquatic environments, such as metals and nanoparticles, affect the phytoplankton. However, so far, no ecotoxicological study involving mixotrophic metabolism was found in the literature. To better understand mixotrophy in microalgae, this work chose the chlorophycean freshwater Chlorella sorokiniana as test organism. We divided the study into two parts: the first focused on the physiological/biotechnological interest through the study of growth, photosynthetic parameters, changes in cellular volume, and production of biomolecules (proteins, carbohydrates and lipids); the second part focused on the ecotoxicological effects of cadmium (Cd) and titanium dioxide nanoparticles (NPs-TiO2). To stimulate mixotrophy, glucose (1.0 g.L-1 or 5 x 10-3 mol.L-1) was used as the organic carbon source. The results showed that during mixotrophy, the microalga C. sorokiniana presented higher population growth and production of biomolecules, such as chlorophyll a and lipids, when compared to photoautotrophic cultures. It was also observed that the photosynthetic parameters were affected by mixotrophy, although they did not interfere in the growth of the microalga, and that the presence of bacteria in the cultures acted as a stimulant factor in the production of algal biomass. Regarding the ecotoxicological effects of contaminants, microalgae in mixotrophy were more resistant to both Cd and NPs-TiO2 than those in photoautotrophy, but with changes in the biochemical composition what can affected the energy transfer in the environment. In general, we can conclude that mixotrophy should be considered in studies with phytoplankton, since aquatic environments present a myriad of organic carbon that can be used by these microorganisms. As general conclusions, we can mention that organic carbon acted as an extra source of structural carbon and energy for microalgae, not necessarily relying solely on photosynthesis to survive, so stimulating the growth and production of biomolecules of biotechnological interest, and increased cellular viability in environments contaminated with metals and nanoparticles. This study is a contribution to the understanding of mixotrophy and photoautotrophy metabolisms in a freshwater Chlorophyta with biotechnological potential.
Nos ambientes aquáticos, o fitoplâncton é formado basicamente de microrganismos fotossintetizantes que servem como base das cadeias alimentares. Entretanto, além da fotoautotrofia, é vastamente citado na literatura que muitas microalgas alimentam-se de matéria orgânica dissolvida presente no ambiente concomitantemente à realização da fotossíntese, uma via metabólica conhecida como mixotrofia. Sabe-se pouco sobre a ecofisiologia em metabolismo mixotrófico nas microalgas, sendo os estudos, em sua quase totalidade, voltados ao uso dessa via metabólica para aumentar a produção de biomassa algal e estimular a produção de biomoléculas específicas. Outra questão importante, considerando a atividade antrópica atual, é que a maioria dos contaminantes eliminados nos ambientes aquáticos, como metais e nanopartículas, são estudados em fitoplâncton sob metabolismo fotoautotrófico, não sendo encontrados trabalhos ecotoxicológicos envolvendo o metabolismo mixotrófico na literatura. Para entender melhor o metabolismo algal em mixotrofia, este trabalho escolheu a microalga Chlorophyta de água doce Chlorella sorokiniana como organismo-teste. Para melhor organizá-lo, foi dividido em duas partes: a primeira focou no interesse fisiológico/biotecnológico através do estudo do crescimento, parâmetros fotossintéticos, volume celular, e produção de biomoléculas (proteínas, carboidratos e lipídeos); a segunda parte focou nos efeitos ecotoxicológicos de cádmio (Cd) e de nanopartículas de dióxido de titânio (NPs-TiO2). Para estimular a mixotrofia, glicose (1.0 g.L-1 ou 5 x 10-3 mol.L-1) foi utilizada como fonte de carbono orgânico. Os resultados mostraram que durante a mixotrofia, a microalga C. sorokiniana apresentou maiores crescimento populacional e produção de biomoléculas, como clorofila a e lipídeos, quando comparada com as culturas em fotoautotrofia. Também foi observado que os parâmetros fotossintéticos foram afetados em mixotrofia, porém não interferindo no crescimento da microalga, e que a presença de bactérias pode ter atuado como fator estimulante na produção de biomassa algal. Em relação aos efeitos ecotoxicológicos dos contaminantes, as microalgas em mixotrofia foram mais resistentes tanto ao Cd quanto às NPs-TiO2 do que em fotoautotrofia, porém com mudanças na composição bioquímica, podendo afetar a transferência de energia nos ecossistemas aquáticos. De modo geral, podemos concluir que a mixotrofia deve ser considerada em estudos com fitoplâncton, visto que os ambientes aquáticos apresentam uma miríade de fontes de carbono orgânico para esses microrganismos. Na mixotrofia, o carbono orgânico funciona como uma fonte extra de carbono estrutural e de energia para as microalgas, não dependendo obrigatoriamente somente da fotossíntese para sobreviver, estimulando o crescimento e produção de biomoléculas de interesse biotecnológico, além de aumentar a viabilidade celular em ambientes contaminados tanto com Cd quanto com NPs-TiO2. Este estudo é uma contribuição ao entendimento dos metabolismos mixotróficos e fotoautotróficos em uma Chlorophyta de água doce com potencial biotecnológico.
CNPq: 302175/2015-6
FAPESP: 2014/15894-0

Chapter 1: Introduction Wheat is the second most widely grown and consumed food crop of the world after rice, and is the staple food of around 35% of the world’s population. The present wheat production is about 749 million tons (FAO, 2016; http://www.fao.org/faostat) and to feed the world’s ever-growing population with annual growth rate of 2.6%, there will be a requirement to produce about 1040 million tons of wheat in 2020. To reach this target, it is crucial to keep the crop free from various biotic as well as abiotic stresses. In recent years, spot blotch caused by Bipolaris sorokiniana has emerged as a serious threat for wheat cultivation in warmer and humid regions of the world. It causes foliar spot blotch, root rot, black point on grains, head blight and seedling blight of wheat and barley. Estimates of yield losses due to spot blotch are reported to vary from 30-80% and can reach up to 100% under severe infection conditions. In spite of several efforts world over, no wheat variety highly resistant to spot blotch has been released for field cultivation. One of the main reasons for this is that the molecular mechanism behind resistance to spot blotch has not yet been fully understood. In order to develop measures to control plant diseases, it is very important to understand not only the characteristic features of the pathogen, but also the molecular mechanism behind the disease progression. With this purpose, the thesis encompasses the following objectives: Objectives of the study 1. To explore the mechanism of plant-pathogen interaction during spot blotch in susceptible and moderately resistant wheat varieties 2. To understand the mechanism of survival of Bipolaris sorokiniana on exposure to the fungicide propiconazole Chapter 2: Isolation and characterization of Bipolaris sorokiniana isolates from different geographical regions of India B. sorokiniana is a phytopathogenic fungus causing diseases in wheat, barley and other winter cereals. Previous studies involving large numbers of strains collected from around the globe suggest that B. sorokiniana exist as numerous forms of isolates varying in virulence and aggressiveness with specific and nonspecific interactions. B. sorokiniana has high morphological as well as pathological variations. We collected or isolated 12 strains of B. sorokiniana from three different wheat growing geographical regions of India. During microscopic examinations, some cultures were found to be polysporic and hence needed to be purified. Thus, monoconidial cultures were established for seven sporulating isolates of B. sorokiniana. These cultures were characterized at morphological level as well as by sequencing the ITS region of the isolates and confirmed to be B. sorokiniana. Like previous reports, our results also showed high morphological variability among the isolates. However, the morphological variation had no relationship with the geographical background. No correlation was observed between genetic similarity of the isolates and their geographical origin, concluding that the morphological characteristics expression is not conditioned solely by genes. Light microscopy and scanning electron microscopy of the spores showed several variations in conidial size, level of melanization and number of septa. For evaluation of disease reaction, the following reported methods of pathogen inoculation were attempted: leaf painting, sterile seed inoculation and inoculation at Zadok’s scale 12 stage. All these methods had some or other limitations for pathogenicity testing and hence another method, inoculation of germinated seeds, was developed. This method was found to be the best method for high throughput evaluation of pathogenicity as well as screening of germplasms. Our study showed that isolates from the same geographic region and morphological group could show differences in virulence levels. Chapter 3: Exploring the molecular interaction of wheat-Bipolaris sorokiniana during spot blotch disease Triticum dicoccum (emmer wheat) has superior organoleptic, therapeutic and nutritional qualities. However, dominance by high yielding hexaploid wheat varieties has restricted its cultivation to some niche areas in Europe and other regions including the peninsular India. T. dicoccum is resistant to various biotic stresses and rust diseases but highly susceptible to stripe rust and spot blotch. Spot blotch has become a major constraint in T. dicoccum cultivation in India. The hemibiotrophic disease cycle of this pathogen is observed only in the susceptible host. Interactive transcriptome sequencing is gaining importance in plant pathogen interaction studies and has enabled simultaneous analysis of expression of plant as well as pathogen genes. Similarly, next generation sequencing has enabled genome sequencing of organisms to a great extent. With the availability of the reference genome sequences from plants as well as pathogen, it has become much easier to align the reads from RNA-seq data and hence expression quantification. In order to explore the interaction, we performed global transcriptome analysis of spot blotch susceptible variety, DDK 1025 and a moderate resistant variety, Chirya 3 upon pathogen inoculation using Illumina HiSeq platform. To understand the infection process and mechanism of disease progression, we performed differential gene expression analysis of spot blotch susceptible variety, DDK 1025 upon pathogen inoculation. A time series comparative study was performed to understand the biotrophic (1 dpi, days post inoculation), early necrotrophic (4 dpi) and necrotrophic phase (6 dpi) responses. The numbers of differentially expressed genes (DEGs) from three stages were 1810, 1562 and 2908 individually. GO annotations were obtained using Blast2GO for 75.63%, 70% and 73.89% of these DEGs respectively. GO enrichment was performed using agriGo online tool (http://bioinfo.cau.edu.cn/agriGO/analysis.php) using Triticum aestivum transcript ID v2.2 as the reference. Biological processes associated with carbohydrate metabolic process, response to abiotic stress, photosynthesis, cell death, regulation of gene expression, secondary metabolic process and generation of precursor metabolites were enriched. Under molecular function category carbohydrate binding, catalytic activity, enzyme regulator activity, protein binding and hydrolase activity was enriched. Although cellular component distribution showed all cellular parts including endoplasmic reticulum, plastid etc., extracellular region was profoundly enriched. Since acceptable annotation of T. aestivum genome was not available, insights into functional annotation were achieved using blast against Oryza sativa japonica group using the STRING platform v10.5 (https://string-db.org/). Pfam enrichment was performed to gain comprehensive understanding about the gene families involved in the infection process. Pathways intricate to this interaction mechanism were explored by KEGG enrichment of DEGs using ClueGo (cytoscape plugin) using O. sativa blast hits. After several enrichments and annotations, major components involved in the interaction were recognized as glycolysis, phenylpropanoid biosynthesis, protein processing in endoplasmic reticulum, photosynthesis, glyoxylate and dicarboxylate metabolism, heat shock proteins, protein kinases and defense response genes like chitinases and hydrolases. Down-regulation of several defense responsive genes in biotrophic phase suggests the contribution of effector mediated susceptibility. Glutathione metabolism mediated regulation of glycolysis and pentose phosphate pathway was identified. Differential expressions of multiple components of ubiquitin mediated proteolysis emphasize their role in hormone signal transduction during spot blotch. Of these DEGs, 177 genes were differentially expressed across all the three time points irrespective of the phases. Co-expression analysis using k-means clustering showed six patterns. Annotations showed that these genes had activities like chitin catabolic process, defense response to fungus, Bowman-Birk proteinase inhibitor and phenylalanine ammonia-lyase. Further information about the significance of these genes during interaction with the pathogen needs to be revealed using over/under expression experiments. Likewise, to recognize the resistance phenomenon, we sequenced the transcriptome of spot blotch resistant (T. aestivum) hexaploid variety, Chirya 3 upon B. sorokiniana inoculation. Differential expression analysis was performed for three stages i.e. 1 dpi, 4 dpi and 6 dpi, which depicts the biotrophic, early necrotrophic and necrotrophic phases in the spot blotch susceptible variety. Our results showed that the number of upregulated genes was higher than downregulated genes. GO annotation was obtained for 64.38%, 66.6% and 64.25% genes from the DEGs. A higher number of genes were unannotated and were found to have significantly higher fold change expression. This suggests that these genes with unknown function could be novel defense responsive genes from wheat. Comparison of gene ontology enrichment showed that biological processes like photosynthesis and cell death were affected in susceptible variety but not in the resistant variety. Whereas, enhanced activity of extracellular proteinase inhibitors and peroxidases was observed in the resistant variety. Thus, early recognition and activation of defense pathways in resistant variety appears to hinder pathogen growth, survival and hence infection. The results from transcriptome sequencing analyses demand confirmation using other complementary techniques like the quantitative reverse transcriptase polymerase chain reaction (qRT PCR), which is an indispensable tool for gene expression analyses. The most adopted method for relative quantification of gene expression in qRT PCR is based on the DDCt method. However, accurate quantification by this method requires an appropriate internal reference gene with stable expression across all or most of the experimental tissues. Selecting an appropriate internal reference gene is very important to elucidate the target gene expression reliably. Several housekeeping genes including 18S rRNA, ACTIN, GAPDH and EF-1α have been proposed as standard reference genes for qRT PCR studies. However, in case of plant pathogen interaction analyses, selection of an appropriate reference gene is even more crucial due to the presence of RNA from both the plant as well as the pathogen in the infected tissues. As several of these genes are also present in the fungal pathogen genome, this could result in unintended cross amplification; which can cause improper quantification of the target genes. Hence, we aimed to identify a wheat gene with the most stable expression and unique primers, which would selectively amplify only the wheat gene and not the pathogen gene, providing accurate quantification of the target genes. Hence, we evaluated six previously reported genes with expression stability under different conditions using the wheat-Bipolaris sorokiniana system. We employed various statistical analysis methods, based on which, we identified two most stable genes, ubiquitin conjugation enzyme (ULE) and phytochelatin synthase (PCS) as the best reference genes for qRT-PCR based quantification in wheat pathosystem. We further confirmed the expression of several candidate defense genes in wheat using ULE as the reference gene. However, both the genes can be used either individually or together as internal reference genes. Chapter 4: Global gene expression analysis of Bipolaris sorokiniana after exposure to propiconazole Integrated disease management has been proposed to control the spot blotch disease. However, due to the unavailability of spot blotch resistant wheat varieties, the application of foliar fungicide is the most widely practiced measure. Propiconazole is a commonly used azole fungicide to manage the spot blotch disease in the field. However, due to its fungistatic mode of action, there is a possibility of emergence of fungicide resistant pathogen strains. Several mechanisms are reported for azole resistance in fungi. However, the strategies vary in different fungi. Resistance to the fungicide could be attributed to multiple molecular components in the fungus. Moreover, azoles have multiple modes of action out of which few are not explored yet. Global transcriptomics analysis of the pathogen after exposure to sub-lethal doses of the fungicide can reveal the mechanism of survival as well as the mode of action of the azoles in the fungi. Hence, a time series gene expression analysis was performed using RNA-seq. Transcriptome analysis using various tools showed overexpression of the target genes in the sterol biosynthesis pathway of the pathogen. In addition, this study also revealed altered expression of several metabolic pathways, transporters and stress regulators in the pathogen. The use of multiple analysis tools for transcriptomics analysis provided additional confidence on the observed results. The observed results were validated using qRT-PCR. We explored three strategies in B. sorokiniana against propiconazole stress: i) overexpression of target enzymes, ii) increased expression of transporter genes, and iii) expression modulation of stress responsive factors. This study revealed several novel putative targets such as ent-kaurene oxidase, ligninase lg6 precursor and spore germination protein. These genes help the fungi to overcome stresses and survive. Hence, the drugs targeting these genes can be developed, which are expected to impair the stress tolerance and hence survival of the pathogen. However, resistance is a polygenic phenomenon and to understand the functional contribution of each gene, knockout/knockdown studies are suggested. Chapter 5: Conclusions and Future Prospects Spot blotch is an emerging disease causing yield losses of economically important cereals. The worldwide distribution of the causal agent, B. sorokiniana makes it of global concern. In the present study, we aimed to explore the mechanism of interaction of the pathogen with spot blotch susceptible and resistant wheat varieties. Initially, we established monoconidial cultures of seven isolates of B. sorokiniana collected from three different wheat growing regions of India and characterized them at morphological and molecular level. Although pathogenicity cannot directly be correlated with morphology on culture media, melanization level might be considered an important aspect in determining the level of virulence. Effector mediated downregulation of innate immunity and delayed response by plants leads to successful establishment of the pathogen in susceptible variety. We found that differential expression of ubiquitin mediated proteolysis played a key role in development of the disease in susceptible variety. On the contrary, proteinase inhibitors and peroxidase secretion led to effective elimination of pathogen in the resistant variety. Genotypes with higher expression of these genes are likely to provide improved resistance against the spot blotch disease. As integrated disease management is a sustainable approach which also includes judicial use of fungicides to control the disease; we explored novel targets for developing efficient fungicides. However, essentiality of these genes for the pathogen survival needs to be confirmed through knock-out/down studies. Overall, this study helped in understanding the molecular paradigm of spot blotch disease in wheat. The outcome of this study will assist in advancement of controlling measures against spot blotch.
AcSIR

O trigo é o principal cereal componente dos produtos amiláceos atualmente consumido pela população. Além das limitações econômicas e políticas, a produção brasileira de trigo encontra obstáculos como a incidência de doenças, muitas delas causadas por fungos. O fitopatógeno Bipolaris sorokiniana é o agente etiológico da helmintosporiose, cujo controle é baseado principalmente em antifúngicos sintéticos. O fungo não é o principal causador de patologias em trigo, mas é importante do ponto de vista fitossanitário nas plantações, pois se mantém no solo por longo período de tempo podendo atacar as plantações em climas úmidos e quentes. O presente estudo teve objetivos avaliar a maior atividade antifúngica de três isolados de Bacillus sp. contra 34 isolados B. sorokiniana, selecionar uma linhagem de Bacillus sp., elucidar sua atividade inibitória in vivo e avaliar as melhores condições de cultivo, propriedades físicoquímicas do cultivo filtrado obtido e a atividade antifúngica in vitro do mesmo. A bactéria com a melhor ação antagonista foi analisada quanto à produção das enzimas caseinase e lipase, produção e atividade do filtrado de cultivo em diferentes meios e após tratamento térmico e variação de pH. Também foi realizado um teste in vivo do antagonista conta o isolado 98031 do fungo. Os três isolados de Bacillus foram capazes de inibir in vitro os isolados de B. sorokiniana, mas destacaram-se os isolados E164 e C98017. Quando testado in vivo, o isolado Bacillus E164 causou efeitos sobre a morfologia da planta, como redução significativa no comprimento das raízes. No entanto, não foi possível observar um nível elevado de proteção visto que mesmo as plantas infectadas não apresentaram os sintomas da doença. A produção do filtrado foi baseada em caldo triptona de soja, uma vez que a inibição foi similar ao meio com palha de milho e maior que o meio com extrato de malte. O filtrado antifúngico apresentou resistência à fervura por até 90 minutos, mas a atividade foi significativamente diminuída nesse tratamento quando comparada aos tratamentos de 50 a 80ºC e temperatura ambiente. A refrigeração e o congelamento não causaram diminuição na atividade do filtrado. A influência do grau de ionização foi percebida nos pHs 5, 6, 8 e 10. O isolado de Bacillus E164 apresentou atividade proteolítica e lipolítica em meios específicos. O controle exercido pelo isolado Bacillus E164 sobre B. sorokininana foi relevante nos testes in vitro. Porém, a influência e a importância da ação do(s) metabólito(s) produzido(s) por esse microrganismo devem ser mais estudados para uma possível aplicabilidade in vivo.
Wheat is the main cereal component of starch products consumed by the population. Brazilian wheat production has many limitations such as government policies, economic problems, and apart from there is a large loss in production due to diseases caused by fungi. Bipolaris sorokiniana is a phytopathogen that causes helminthosporiosis in cereal crops, whose control is mainly rely on synthetical antifungal agents. It has phytossanitary importance, since it can live for a long period in the soil, and attack crops on wet and warm weather conditions. The objectives of this work were to evaluate the antifungal activity of three Bacillus sp. Strains against 34 B. sorokiniana isolates, to select the best inhibitor of them, to elucidate its action in vivo and evaluate the best cultive conditions, physic and chemical properties of the filtrated obtained and in vitro antifungal capacity. The best bacterial strain was chosen and analyzed for the proteolytic and lipolytic activities, productions and activity of the cultive filtrated after growing on different culture media and after thermal treatment or pH variation. In vivo test was made on wheat infected by 98031 isolate. All bacterial isolates were active against B. sorokiniana but E164 and C98017T were better than OR13. On in vivo test the isolate E164 caused morphological effects on the plant as significant root length reduction. Increase of plants protection was not observed, as even infected ones did not presented disease symptoms. Filtrated production was based in tryptic casein soy broth as the inhibition degree was similar to the corn straw culture and greater than malt extract broth culture. The antifungal filtrated resisted until 90 minutes at 100ºC, but significant decrease of activity was observed in this treatment when compared to 50ºC to 80ºC and environment temperature. Refrigeration and freezing did not cause loss on filtrated activity. Ionization degree influence was observed in pH 5, 6, 8 and 10. Bacillus E164 showed proteolytic and lipolytic activities in specific media. Control exerted by Bacillus E164 over B. sorokiniana isolates was relevant in vitro, nevertheless the influence and importance of the metabolites produced must be elucidated for the application in vivo.

Growth media recycling during algae cultivation is necessary to increase the efficiency and reduce the cost of biofuel production from algae feedstocks. Without recycling media, the cost of algae based biofuel production would be prohibitively high and large scale algae based biofuel production would not be economically viable. The ratio of media recycled to media wasted assumed for algae farms is generally calculated to maintain salt concentrations below growth inhibitory levels, ignoring the influence of secondary metabolites which might decrease productivity. Secondary metabolites, which include allelopathic or auto-inhibitory biological contaminants, might lead to the accumulation of growth-inhibiting compounds in recycled media used in algae production. Chlorella sorokiniana (strain DOE1412) was a leading algae biofuel feedstock candidate and has not previously been evaluated for inhibitor production. To test the effects of water recycling on the growth of DOE1412, media was recycled through multiple rounds of algae cultivation. DOE1412 was grown in modified BG11 culture media until reaching the end of linear growth phase, at which point the biomass was removed, nutrients replenished to their initial concentrations, and the recycled culture media used for a subsequent round of growth. The culture media was recycled through five rounds of growth with cultures grown on recycled media compared to controls grown on freshly prepared growth media. Biomass density was monitored via optical density and the specific and productivity growth rates were used to quantify the extent of inhibition. Exploratory work was performed with the goal of identifying potential inhibitory substances produced by DOE1412 during cultivation. Samples of recycled media were analyzed for polyunsaturated fatty acids which have been demonstrated to be inhibitory. The carbohydrates content of used media was analyzed to assess the amount of organic materials shed by DOE1412 into recycled media during growth. The log phase growth rate (day-1) of DOE1412 was inhibited by 3±2%, 8±1%, 10±2%, and 18.6±0.9% when grown in media recycled 1-4 times, respectively, with a 99% level of confidence that inhibition was observed in each round of regrowth. The productivity growth rate (OD750/day) of DOE1412 was not inhibited in media recycled 1-3 times. The productivity growth rate of DOE1412 was inhibited by 13±3% when grown in media recycled 4 times with a 99% level of confidence that inhibition was observed. Zinc was found to accumulate in the recycled media to potentially toxic levels (>0.09 mg/L), therefore it is uncertain if the observed inhibition was due to an accumulation of inhibitory secondary metabolites or the accumulation of zinc. Two inhibitory polyunsaturated acids, linoleic and linolenic acid, were identified in media recycled 4 times. The carbohydrate content of recycled media fluctuated between 8-10% of total fixed carbon in media recycled 1-3 times and increased to 18% in media recycled 4 times. However, changes observed in media recycled 4 times may have been due to improper storage of used media.

Neste trabalho de mestrado, investigou-se o desempenho de membranas poliméricas de microfiltração para retenção de microalgas (Chlorella sorokiniana) em um fotobiorreator com capacidade volumétrica de 3,5 L, alimentado por um fluxo contínuo de nutrientes com pH 7,0 em condições controladas de temperatura (21°C no meio de cultura e 24°C no meio), borbulhamento de ar e luminosidade (2500 lux, em fotoperíodo de 12/12 horas claro/escuro). Membranas comerciais de tamanho médio de poros 0,8, 1,2, 3,0 e 5,0 μm foram testadas pelo tempo suficiente para esgotamento do limite da permeação da membrana. A concentração das microalgas no fotobiorreator foi analisada através de densidade óptica (espectofotometria) ao número de células (contagem de unidades de células em lâminas do tipo Fuchs Rosenthal - Microscopia Óptica) das amostras de concentrado e permeado. O fenômeno físico de polarização sobre a superfície da membrana está diretamente relacionado ao desempenho da mesma na retenção de microalgas, portanto, fotomicrografias (MEV) da membrana antes e após a microfiltração foram analisadas e comparadas. Para analisar a composição química das microalgas, bem como sua afinidade com as membranas, foram investigadas a composição e a caracterização do fenômeno químico sobre a superfície da membrana, por análise de Energia Dispersiva de Raio-X (EDX). Análises das diferentes fases de crescimento das algas e seus componentes foram feitas em amostras secas a 40°C, através de análise elementar e análises térmicas (TG- Análise Termogravimétrica e DTA- Análise Térmica Diferencial). Os resultados experimentais obtidos neste trabalho permitiram concluir que o uso de membranas de microfiltração em fotobiorreator proporciona a retenção das microalgas, o que contribui para o aumento da concentração da biomassa algal, permitindo a manipulação da sua composição de acordo com as condições pelas quais estes microrganismos são submetidos.
This dissertation investigates the performance of polymeric microfiltration membranes for microalgae retention (Chlorella sorokiniana) in photobioreactor with a volumetric capacity of 3.5 L. The reactor is fed by a continuous flow of nutrients under controlled conditions of pH (7,0) and the temperature (21°C in culture medium and 24°C in the environment), bubbling air and light (2500 lux, with a photoperiod of 12/12 hour light/dark). Commercial membranes of the average pore sizes of 0.8, 1.2, 3.0 and 5.0 μm were tested by depletion of time sufficient to limit the permeation of the membranes. The microalgae concentration in the photobioreactor was analyzed by optical density (spectrophotometry) and number of cells (cell units count on Fuchs Rosenthal type plates-Optical Microscopy). The physical phenomenon of polarization on the surface of the membrane is directly related to its performance in the retention of microalgae therefore micrographs (SEM) of the membrane before and after the microfiltration were compared to identify the chemical affinity between the membranescomposition and the microalgae. The chemical phenomenon on the membrane surface was characterized by Energy Dispersive Analysis of X-ray (EDX). The different phases of the growth of algae and their components were measured in samples dried at 40°C, by elemental analysis and thermal analysis (TG-DTA-Thermogravimetric analysis and differential thermal analysis). The experimental results indicate that the use of microfiltration membranes provides the retention of microalgae, contributing to the increase in the concentration of algal biomass and allowing the manipulation of the composition according to the conditions under which these microrganisms are subjected.

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Microalgas quando se desenvolvem intensamente recebem o nome de florações. Como conseqüência formam-se flocos ou cobertura laminar que podem obstruir os sistemas de tratamento de água para uso industrial ou doméstico. Tratamentos convencionais por insumos químicos no sentido de inibir o crescimento nem sempre são eficientes. Na busca de métodos alternativos para o controle da população de microalgas, procurou-se aplicar ondas ultra-sônicas para destruir as células destes microrganismos. As espécies Selenastrum capricornutum e Chlorella sorokiniana foram cultivadas em meio mineral sólido e líquido por cerca de cinco dias. Amostras de 50 mL foram irradiadas com ultra-som de freqüência 20 KHz com amplitude de 80 % e sonda de 9 mm de diâmetro nos tempos: 30 segundos; 01; 10; 12 e 15 minutos. As células foram quantificadas em Câmara de Neubauer procedendo-se cinco contagens para cada tempo de sonicação. Os resultados com a S. capricornutum demonstraram que o ultra-som promove a desagregação celular após irradiação de 30 segundos identificado pelo aumento do número de células, permitindo que se infira que em algumas situações os resultados das contagens podem não ser totalmente exatas. Entretanto, a sonicação após dez minutos produziu 88% e 82% de morte da Chlorella sorokiniana e S. capricornutum respectivamente.
Micro-algae when developing intensely under favorable conditions is said to be flourishing. As consequence, flakes or laminar layer formations can obstruct industrial or domestic water treatment plant installations. Conventional treatments utilizing chemicals are not always very efficient in inhibiting these formations. In the search for alternative methods to control the micro-algae population we tried the application of ultra-sound for the destruction of the cells of these microorganisms. Selenastrum capricornutum and Chlorella sorokiniana were cultivated in solid and liquid mineral media for about 5 days. 50 mL samples were irradiated with ultra-sound with a frequency of 20 KHz, 80% amplitude and a 9 mm probe for: 30 seconds; 01; 10; 12 and 15 minutes. The cells were quantified in a Neubauer Chamber with 5 counts being done for each individual exposure time. The results with the S. capricornutum showed that a 30 seconds application of ultra-sound promotes cellular desegregation, evidenced by the results from counts are not reliable. We were able to determine though that the application of ultra-sound for 10 minutes was able to kill 88% and 82% of the Chlorella sorokiniana and S. capricornutum respectively.

Philosophiae Doctor - PhD
Biofuel production from microalgae is currently not economically competitive with fossil fuels due to high operational costs. A sustainable system needs to be developed which considers cultivation, harvesting and conversion to fuels as a single loop. The harvesting step has been identified as a major bottleneck within the biofuel production process, contributing to a significant proportion of the operational cost (20-30%). Chemical flocculation is a more affordable alternative to centrifugation and filtration. Chemical flocculants however negatively impact the quality of biomass and conversion efficiency to biofuel by increasing biomass ash content. Bioflocculation with biopolymers or microbes have a minimal impact on the quality of biomass. In this study, the interaction between the filamentous fungus Isaria fumosorosea and the microalgae C. sorokiniana is investigated. Under strict autotrophic conditions at pH 7-8, co-culture of microalgae (2-20 μm) with fungal blastospores resulted in theidevelopment of large pellets (1-2 mm) which may be easily harvested by sedimentation or filtration at 95% harvesting efficiency. Fungal assisted bioflocculation was compared to other harvesting methods with respect to cost and impact on the hydrothermal conversion process. Low cost carbon sources, including waste hydrothermal nutrients, minimal sugar concentrations and algal exudate may reduce fungal cultivation costs. Waste products, such as organic carbon, N, P, CO₂ and trace metals can be recycled and used for algae and fungal cultivation, closing the loop to make the system sustainable.
National Research Foundation; Swiss Government

Microalgas quando se desenvolvem intensamente recebem o nome de florações. Como conseqüência formam-se flocos ou cobertura laminar que podem obstruir os sistemas de tratamento de água para uso industrial ou doméstico. Tratamentos convencionais por insumos químicos no sentido de inibir o crescimento nem sempre são eficientes. Na busca de métodos alternativos para o controle da população de microalgas, procurou-se aplicar ondas ultra-sônicas para destruir as células destes microrganismos. As espécies Selenastrum capricornutum e Chlorella sorokiniana foram cultivadas em meio mineral sólido e líquido por cerca de cinco dias. Amostras de 50 mL foram irradiadas com ultra-som de freqüência 20 KHz com amplitude de 80 % e sonda de 9 mm de diâmetro nos tempos: 30 segundos; 01; 10; 12 e 15 minutos. As células foram quantificadas em Câmara de Neubauer procedendo-se cinco contagens para cada tempo de sonicação. Os resultados com a S. capricornutum demonstraram que o ultra-som promove a desagregação celular após irradiação de 30 segundos identificado pelo aumento do número de células, permitindo que se infira que em algumas situações os resultados das contagens podem não ser totalmente exatas. Entretanto, a sonicação após dez minutos produziu 88% e 82% de morte da Chlorella sorokiniana e S. capricornutum respectivamente.
Micro-algae when developing intensely under favorable conditions is said to be flourishing. As consequence, flakes or laminar layer formations can obstruct industrial or domestic water treatment plant installations. Conventional treatments utilizing chemicals are not always very efficient in inhibiting these formations. In the search for alternative methods to control the micro-algae population we tried the application of ultra-sound for the destruction of the cells of these microorganisms. Selenastrum capricornutum and Chlorella sorokiniana were cultivated in solid and liquid mineral media for about 5 days. 50 mL samples were irradiated with ultra-sound with a frequency of 20 KHz, 80% amplitude and a 9 mm probe for: 30 seconds; 01; 10; 12 and 15 minutes. The cells were quantified in a Neubauer Chamber with 5 counts being done for each individual exposure time. The results with the S. capricornutum showed that a 30 seconds application of ultra-sound promotes cellular desegregation, evidenced by the results from counts are not reliable. We were able to determine though that the application of ultra-sound for 10 minutes was able to kill 88% and 82% of the Chlorella sorokiniana and S. capricornutum respectively.
Orientador: Antonio Carlos Simões Pião
Coorientador: Dejanira de Franceschi de Angelis
Coorientador: Roberto Naves Domingos
Banca: Antonio Carlos Simões Pião
Banca: Ana Paula de Arruda Geraldes Kataoka
Banca: Lucia Helena de Mendonça Vargas
Mestre

As actinobactérias endofíticas estão presentes nos tecidos das plantas e, por meio da produção de metabólitos ativos, às protegem e auxiliam em condições de estresse. Esses microrganismos tem sido amplamente estudados no controle de doenças fitopatogênicas, como a mancha marrom causada por Bipolaris sorokiniana. Este fungo é o agente causal da podridão comum da raiz, manchas foliares, morte de plântulas e ponto preto das sementes de trigo e cevada, causando redução significativa na produtividade. Neste contexto, os objetivos do presente estudo foram avaliar a virulência de isolados de B. sorokiniana e a atividade antifúngica de actinobactérias contra este fitopatógeno. Os antagonistas com elevada atividade contra o fitopatógeno foram caracterizados quanto à produção enzimática, fisiologia, condições de crescimento e produção de metabólitos, bem como sequenciamento do 16S rRNA para identificação dos antagonistas. A caracterização parcial dos metabólitos foi realizada por meio de sistemas de Cromatografia de Camada delgada (CCD) contendo diferentes solventes. Os resultados mostraram que os isolados de B. sorokiniana apresentaram elevada virulência às plântulas e sementes de trigo, sendo que a maior agressividade foi relatada à semente. Por outro lado, 69,6% das actinobactérias apresentaram elevada atividade antifúngica contra isolados de B. sorokiniana em meio sólido, e 17% a mantiveram em cultura submersa. A maior produção ocorreu a 30°C após 72h de incubação, para a maioria dos isolados. A detecção da produção de catalase, amilase, pectinase, lipase e esterase foi observada para a maioria das actinobactérias (100, 95,6, 91,30, 95,6, 100%, respectivamente). Enquanto que a degradação de caseína, carboximetilcelulose e gelatina foi realizada por 60,8, 34,78 e 47,82% dos isolados, respectivamente. Os isolados 6(2), 6(4), 16(3) e R18(6), selecionados devido à elevada atividade antifúngica e enzimática, apresentaram reação positiva para produção de compostos voláteis, quitinase e glucanase, sideróforos, fixação de nitrogêno, AIA e colonização de raizes. Somente isolado R18(6) não apresentou capacidade de solubilizar fosfatos. A caracterização molecular determinou que estes isolados pertencem ao gênero Streptomyces. Os metabólitos produzidos pelo isolado R18(6) foram mais estáveis a mudanças de temperatura e pH, bem como para a ação das proteases e EDTA, quando comparado aos demais. Os solventes acetato de etila e hexano foram mais eficientes na extração de metabólitos do extrato bruto, porém a melhor separação de metabólitos em CCD foi obtida com misturas de solventes.
Endophitic actinobacterias are present in plant tissues and by means of active metabolites they protect and help them in stress conditions. These microorganisms have been widely used in the control of phytopathogenic diseases, such as the spot blotch caused by Bipolaris sorokiniana. This fungus is a causal agent of common root rot, leaf spots, death of seedlings and black point of the seeds of wheat and barley, causing significant reduction in productivity. In this context, the aim of this study was to evaluate the virulence of the B. sorokiniana isolates and the antifungal activity of actinobacterias against this phytopathogen. The antagonists with the higher activity against the phytopatogen were characterize taking in consideration their physiology, enzyme production, growth conditions and metabolites production and 16S rRNA sequencing for identification of the antagonists. Partial characterization (nós não purificamos) of the metabolites was performed using thin layer chromatography (TLC) systems containing different solvents. The results showed that the isolates of B. sorokiniana have a high virulence on wheat seed and seedlings, however the greater aggressiveness was observed to seed. On the other hand, 69.6% of actinomycetes showed high antifungal activity against of B. sorokiniana isolates on solid medium, and 17% maintained this behavior in submerged culture. The highest yield happened, for most isolates, when grown at 30°C with agitation after 72h of incubation. The detection of catalase, starch, pectin, lipase and esterase production was observed for most of the actinomycetes (100, 95.6, 91.30, 95.6, 100%, respectively). While the hydrolysis of casein, carboxymethylcellulase and gelatin was performed by 60.8, 34.78 and 47.82% of the isolates, respectively. Isolates 6(2), 6(4), 16(3) e R18(6), selected due to the high antifungal and enzyme activity, showed a positive reaction for the production of volatile compounds, chitinase and glucanase, siderophores, nitrogen fixation, AIA and colonization of the roots. Only the isolated R18(6) showed no ability to solubilize phosphates. Molecular characterization of the isolates determined that they belong to the genus Streptomyces. The metabolites produced by isolate R18 (6) were more stable to temperature and pH changes, as well to the action of proteases and EDTA, when compared to the others. The solvents ethyl acetate and hexane were more efficient for the extraction of the metabolites from the crude extract, however a better separation of the metabolites in the TLC was obtained with mixture of solvents.

Bipolaris sorokiniana é um fungo fitopatogênico de gramíneas, sendo mais importante nas culturas de trigo e de cevada, ocasionando moléstias como a podridão comum da raiz, carvão do nó, ponta preta dos grãos e mancha marrom. No Brasil esse fitopatógeno encontra-se disseminado em todas as regiões tritícolas. O uso de sementes sadias ou tratadas adequadamente com fungicidas é de grande importância para o controle do fungo. O diagnóstico é dificultado pela grande variabilidade fisiológica e morfológica que o patógeno apresenta. O presente estudo teve por objetivos examinar a presença de polimorfismos intra-específicos das regiões do espaço transcrito interno (Spacer Transcribed Internal-ITS) do DNA ribossomal de isolados de B. sorokiniana e relacionar os padrões de polimorfismos com a região geográfica de origem dos isolados e com os diferentes tipos de hospedeiros. As regiões ITS1 e ITS2 do rDNA de 50 isolados de B. sorokiniana do Brasil e de outros países, um isolado de B. oryzae e seis de Drechslera teres foram amplificadas com oligonucleotídeos iniciadores universais ITS5-ITS2 e ITS3-ITS4. Os produtos da amplificação foram clivados com 18 endonucleases de restrição selecionadas e os polimorfismos foram analisados em gel não desnaturante de poliacrilamida 8%. A análise dos produtos de amplificação revelou a presença de dois fragmentos para ambas as regiões ITS em todos os isolados. Dois isolados de B. sorokiniana apresentaram variabilidade intra-específica de ITS com um terceiro fragmento nesta região. A partir da análise dos dendrogramas da clivagem das regiões ITS, verificou-se que os isolados do Brasil e de outros países formaram grupos intra-específicos e grupos interespecíficos. B.oryzae agrupou-se com até 75% de similaridade na análise da região ITS1 e 72,7% na região ITS2 com isolados de B. sorokiniana. Um isolados de D. Teres apresentou índice de similaridade de ITS1 de 75% com amostras de B. sorokiniana isoladas de trigo. Em todos os dendrogramas analisados não foi possível agrupar os isolados por região geográfica ou por tipo de hospedeiro.
Bipolaris sorokiniana is a worldwide pathogen of many cereal grasses. It is the most important causal agent of common root rot, leaf spot disease, seedling blight and black point of wheat and barley. In Brazil this fungus is disseminated in all wheat producing regions. Since this fungus is a seed borne pathogen, it is a very important to use in plantation seeds free of the fungus or seeds properly treated with fungicide. Diagnose of the phytopathogen is a difficult task because its high morphological and physiological variability. The purpose of this study was to examine the intra-specific polymorphism of internally transcribed spacer (ITS) region in the ribosomal DNA (DNAr) of Bipolaris sorokiniana isolates and to determine if there is some relationship among the isolates and their geographic origin or host. DNAr from 50 B. sorokiniana isolates, from Brazil and other countries, one B. oryzae and six Drechslera teres were used for the amplification of the ITS region using the universal primers ITS5-ITS2 and ITS3-ITS4. The amplification products were digested with 18 selected restriction endonucleases and the polymorphism was analyzed in 8% no denaturing polyacrylamide gel. The results of the amplification products showed two fragments for each ITS regions in all isolates. Two B. sorokiniana isolates presented an intra-specific variability with a third fragment for the ITS1 region. The dendrogram analyses of the polymorphism's, generated with the digestions of ITS region, from isolates of Brazil and from the other countries showed an intra and inter-specific groups. B. oryzae showed 75% of similarity with some B. sorokiniana isolates in the polymorphism analysis of ITS1 region and 72, 7% of in the ITS2 region. In the same way in the ITS1 region, one D. teres isolate presented 75% of similarity for B. sorokiniana isolated from wheat. In all the dendrograms analysis there was not possibility to group the isolates accordingly to their geographic origin or host type.

Mancha marrom ou Helmintosporiose é uma das principais doenças do trigo, causada pelo fitopatógeno Bipolaris sorokiniana, sendo responsável por grandes perdas econômicas no cultivo do trigo no mundo inteiro. Este fungo apresenta uma grande diversidade morfológica, fisiológica e genética. Com objetivo de caracterizar a diversidade molecular de amostras monospóricas de B. sorokiniana isolados de sementes do Brasil e outros países, foram utilizados 60 isolados monospóricos tendo o DNA genômico desses isolados sido submetidos à amplificação por PCR. Foram utilizados 12 oligonucleotídeos iniciadores universais construídos a partir de sequências repetidas do genoma do arroz (URP). Os perfis gerados foram analisados pelo método de médias aritméticas não ponderadas (UPGMA). O método PCR – URP permitiu obter informações importantes sobre o perfil genético das culturas monospóricas mostrando distinções entre os três conídios isolados oriundos da mesma cepa polispórica. Os oligonucleotídeos URP-30F, URP-6R, URP-17R e URP-38Famplificaram com um elevado índice de isolados. Em contra partida, os oligonucleotideos URP-13R, URP- 25F e URP-32F apresentaram um perfil de amplificação menor entre os isolados do fitopatógeno. O número total de fragmentos gerados com cada um dos oligonucleotídeos variou entre 41 e 77 fragmentos. Os oligonucleotídeos URP-17R, URP-30F, URP-32F e URP-6R apresentaram maior índice de fragmentos polimórficos. Os isolados apresentaram uma grande diversidade intra-especifíca entre os oligonucleotideos iniciadores URP e entre os conídios monospóricos. A análise forneceu informações relevantes sobre a variabilidade genética e a relação entre os isolados de B. sorokiniana.
Leaf spot disease or helmintosporiosis is one of the most important diseases of wheat cultures caused by the phytopathogem Bipolaris sorokiniana. It is responsible for great economic loss of wheat cultivation worldwide. This fungus presents a great morphologic, physiologic and genetic diversity. The aim of this study was to characterize the molecular diversity of B. sorokiniana monosporic isolates isolated from seeds from Brazil and other countries. For this purpose 60 monosporic isolates were used. The genomic DNA of this isolates were submitted to PCR amplification using 12 universal primers built from repeated sequences of rice genome (URP). The analysis of the amplification profile were calculated by Unweighted Pair Group Arithmetic Mean (UPMGA) Method. The PCR-URP method provided important information about the genetic profile to the monosporic culture, showing distinctions between the three spore isolates of the same polysporic strain. The primers URP-30F, URP-6R, URP-17R and URP-38F were able to amplify a higher percentage of the isolates been the more efficient in the study of this phytopathogen. However, the primers URP-13R and URP-32F have shown a lower amplification profile between the B. sorokiniana isolates. The number of fragments that resulted from the amplification of each primer had varied between 41 and 77 fragments. The primers URP-17R, URP-30F, URP-32F and URP-6R generated a higher number of polymorphic fragments with the isolates. The analysis provided relevant information about the variability and genetic relationship between B. sorokiniana isolates.

Growing interest in biofuel production from non-fossil fuel sources has resulted in several studies exploring different raw material sources as feedstock, including many algae species, for large-scale production of biofuel. Algae are promising feedstock due to advantages such as its short growth cycle, high biomass production, and lipid content. However, there are still challenges to overcome in order to use algae for commercial biofuel production. One of these challenges is the requirement for a large quantity of water and nutrients needed for growing large quantities of the algae. This work explores a potential solution to this challenge by studying the possibility of using industrial wastewater to grow algae for biofuel production. However, many industrial wastewaters, including effluents from semiconductor processing plants, are known to contain heavy metals that are toxic to humans and the environment. In this work, the effects of four of such metals ions, As(V), As(III), Ga(III), and In(III) on Chlorella sorokiniana and Scenedesmus obliquus strains were studied. In particular, the heavy metal toxicity on the strains, effects on its growth rate, biomass yield, lipid content and fatty acid methyl esters (FAME) were studied. Also, the effect of controlling pH on growth rate, biomass yield, lipid content, and FAME was studied for Chlorella sorokiniana in the presence of Ga(III). The results of the study confirmed the toxicity of these metals on both strains. However, Ga(III) and In(III) had the highest effect, while As(V) showed the least toxicity to the strains, with Chlorella sorokiniana withstanding concentrations of As(V) as high as 140mg/L. The heavy metals were slightly more toxic to Scenedesmus obliquus compared to Chlorella sorokiniana. In addition, the heavy metals reduced the growth rate of both strains. High percent changes in growth rate (more than 50%) were seen in cultures containing Ga(III) and In(III). Furthermore, concentration measurements with Inductively Coupled Plasma Optical Emission Spectrometer (ICP) before, during, and at the end of the growth period, showed that Scenedesmus obliquus adsorbed higher amounts of the heavy metals compared to Chlorella sorokiniana. Microalgae biosorption of heavy metals limits its end use, hence making Scenedesmus obliquus a less favorable option for this study, but may be a better choice for wastewater treatment applications. The effects of the four metals on the lipid content and FAME profile of Chlorella sorokiniana were studied. The result showed an increase in Chlorella sorokiniana lipid content in the presence of In(III), but a decrease in the presence of As(V) and As(III). The heavy metals had effects on the strain’s FAME compositions. The fatty acid composition included C16:0, C16:1, C16:2, C16:3, C18:0, C18:1, ω-6, C18:2, ω-6, and C18:3, ω-3 accounting for more than 97% of the total FAME composition. Furthermore, controlling the pH of the culture in the presence of Ga(III) at 6.5 led to higher adsorption of the heavy metal, increase in lipid content, but no significant change in FAME composition.

The fungal pathogen Bipolaris sorokiniana (teleomorph Cochliobolus sativus)causes the foliar disease spot blotch (SB) and the root disease common root rot (CRR). Spot blotch and CRR are serious disease constraints to barley production in warmer growing regions of the world, with estimated yield losses ranging from 30-70% from SB and 15-30% for CRR. Although chemical treatments may assist incontrolling spot blotch infections, the most effective and environmentally sound means of control for each disease is breeding for varieties with natural resistance. InAustralia, no commercially available varieties offer resistance to either SB or CRR. This study has sought to establish molecular markers that will be useful for selecting for resistance to each of these important fungal diseases.Barley cultivars derived from the breeding line NDB112 have provided durable SB resistance in the North Dakota region of the USA for over 40 years. The robustnessof this resistance had not been determined under Australian environmental conditions or with those B. sorokiniana pathotypes present within Australia. Toelucidate the genetics of resistance, two seedling and two field trials were conducted on an ND11231-12/VB9524 (ND/VB) doubled haploid (DH) population (180 lines).A molecular map of the ND/VB population was curated in order to provide a firm basis for mapping of resistance loci. Composite interval mapping revealed thatdifferent gene combinations are effective at different stages of plant development. Seedling resistance was found to be conditioned by a major locus on the short arm ofchromosome 7H and this region was validated in the related population ND11231-11/WI2875*17. A minor quantitative locus on chromosome 5HS was detected in one of the two seedling trials. However, this region requires further investigation to confirm its association to SB resistance in this population. Field resistance to SB in adult plants was found to be associated with two major quantitative trait loci (QTL)on chromosomes 7HS and 3HS; and a putative third minor QTL on chromosome 2HS. The 7H region is common between seedling and field resistance and is the most important locus for the expression of resistance at both stages of plant development. These findings largely concur with genetic studies of this trait in tworowed barley germplasm in North American environments.Common root rot is a difficult disease to phenotype for, and breeding programs will benefit from the identification of molecular markers linked to resistance. Data wasprovided from field trials of subsets of the population over four years. Using a novel approach combining the efficiency of bulked-segregant analysis with highthroughputDiversity Arrays Technology markers (BSA-DArT), CRR resistance was found to be conditioned by three putative QTL in an unmapped Delta/Lindwall population. QTL were identified on chromosomes 2HS, 4HS, and 7HS. To validatethe trait-linkage associations between the DArT markers and the CRR QTL,microsatellite (SSR) markers known to map to the regions identified by BSA-DArT were used. The 2H and 4H regions were validated using marker regression of the SSR markers in most seedling trials, whereas the 7H QTL, which is proximal to the location of the SB resistance QTL in the ND/VB population, was detected in only one seedling trial.The QTL identified in this study offer potential to combat the foliar and root diseases causes by this fungal pathogen. The chromosomal location of QTL for SB and CRR resistance have been found to differ in the ND/VB and D/L populations,which suggests that resistance to each disease is independently inherited. Further research is required to confirm the hypothesis that it is possible to combineresistance to both diseases into a single genotype. Such allelic combinations would provide elite germplasm that would benefit barley breeding programs world-wide.

201p.
Title page, contents and abstract only. The complete thesis in print form is available from the University Library.
Thesis (Ph.D.)--University of Adelaide, Depts. of Crop Protection and Plant Science 1992

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Brown spot (Bipolaris sorokiniana) and net blotch (Drechslera teres) are the main foliar diseases of barley in southern of Brazil. The objectives of this study were to evaluate the survival and viability of B. sorokiniana and D. teres on barley seeds during the off seasons and verify the influence of different barley seed treatments on: a. The fungi transmission to plants; b. The population of emerged plants; c. The brown spot and net-blotch intensity; d. The productivity; e. The seed health and f. the number of captured spores in the air. Barley seeds of six cultivars from four regions were used for viability analysis. Seed sanity tests were developed during ten months. Samples of 400 seeds were disinfected by sodium hypochlorite (4%) and whashed by sterile distilled water. The samples were plated in the Potato Dextrose Agar medium and incubated in growth chamber for seven to ten days. Field experiments were carried out with two barley cultivars sowed in two different dates of 2012 and 2013 growing season. The experiments were performed with treatments, control (without fungicide seed treatment), commercial treatment and additional laboratory treatments. The experiments were conducted in a randomized block design with four replications. The number of symptomatic plants with brown spot and net-blotch diseases was assessed from five to seven days intervals during 40 days. The incidence and severity foliar were quantified from 40 to 95 days after sowing in ten tillers randomly per plot. Collectors spores like windmill with microscope slide smeared with a mixture of phenol + hexane + Vaseline + paraffin were installed in the field. The microscope slides were changed weekly, and the collectors remained in the field up to the 38 plant ear emergence. Grain yield, grain classification and thousand kernels was assessed during the harvest and the seeds submitted to pathology test. The incidence and viability of B. sorokiniana and D. teres reduced with the storage. The viability average reduction of B. sorokiniana and D. teres was 27% and 30% in the off season, respectively. None of the simultaneus seed treatments eradicated the fungi. Seed treatments allowed fungi transmission to the plant leaves. Seeds commercial treatment was not effective in the fungi eradication, allowing up to 90% transmission to plants. Additional seed treatments reduced up tp 89% the fungi transmission. Commercial seed treatment showed the AUDPCS of 519.0 and 139.0 for net blotch and brown spot, respectively. The most efficient seed treatment was triadimenol + difenoconazol + carbendazim + thiamethoxam, reducing the B. sorokiniana and D. teres AUSPC in 11.4 and 120.5, respectively. The highest fungi conidia capturing occurred in field under commercial treatment seeds. There was a positive and significant correlation (r = 0.89 B. sorokiniana and r = 0.70 D. teres) between the number of spores in the air and severity. Seed treatment influenced the sanitary quality of barley seeds. There is a significant and positive correlation (r = 0.99) between the brown spot and net-blotch AUDPC and the incidence of B. sorokiniana and D. teres in the harvested seed. Commercial seed treatment did not reduce the B. sorokiniana and D. teres inocula in barley seeds. Additional, commercial seed treatment anticipated the begining of brown spot and net blotch epidemic, increasing yield costs. All other treatments did not eradicate the fungi. However, they delayed the bigining of diseases, enabling the farmer profit increase
A mancha-marrom (Bipolaris sorokiniana) e a mancha-em-rede (Drechslera teres) são as principais doenças fúngicas foliares da cevada no sul do Brasil. Os objetivos foram: quantificar a sobrevivência e a viabilidade de B. sorokiniana e D. teres em sementes de cevada durante a entressafra e verificar o efeito de diferentes tratamentos de sementes de cevada na transmissão dos fungos para a parte aérea das plantas, na população de plantas emersas, na intensidade da mancha marrom e da mancha-em-rede, na produtividade, na sanidade de sementes colhidas e no número de conídios capturados no ar. Foram utilizadas sementes de cevada de seis cultivares oriundas de quatro regiões para a análise de viabilidade. Os testes de sanidade foram desenvolvidos durante dez meses. Amostras de 400 sementes foram desinfestadas em hipoclorito de sódio (4%) e água destilada esterilizada, distribuídas em meio de cultura Batata-Dextrose-Ágar e encubadas em câmara de crescimento durante sete a dez dias. Os experimentos de campo foram conduzidos em 2012 e 2013 em duas épocas de semeadura e duas cultivares. Os tratamentos utilizados foram: testemunha (sem tratamento fungicida de sementes), tratamento comercial e tratamentos adicionais testados em laboratório. O modelo experimental foi em blocos casualisados e quatro repetições. O número de plantas sintomáticas com mancha marrom e mancha-em-rede foi quantificado em intervalos de cinco a sete dias até 40 dias após a semeadura. A incidência e a severidade foliar foram quantificadas desde os 40 até os 95 dias após a semeadura em dez perfilhos coletados ao acaso de cada parcela. Foram instalados coletores de esporos tipo cata-vento contendo lâmina de microscopia untada com mistura de fenol+hexano+vaselina+parafina. As lâminas foram trocadas semanalmente, e os coletores permaneceram no campo até o espigamento das plantas. Na colheita, foi quantificado rendimento de grãos, classificação e massa de mil grãos. As sementes colhidas foram submetidas ao teste de sanidade de sementes. Houve redução da incidência e da viabilidade de B. sorokiniana e D. teres com o armazenamento. A redução média viabilidade de B. sorokiniana e D. teres foi de 27% e 30% na entressafra, respectivamente. Nenhum dos tratamentos erradicou os fungos simultaneamente das sementes e possibilitaram transmissão para a parte aérea as plantas. O tratamento comercial de sementes não foi eficiente na erradicação dos patógenos com transmissão para a parte aérea de até 90%. Tratamentos de sementes adicionais reduziram a transmissão dos fungos em até 89%. O tratamento comercial de sementes antecipou as doenças foliares com AACPS de até 519,0 de mancha em rede e 139,0 de mancha marrom. O tratamento de semente mais eficiente (triadimenol + difenoconazol + carbendazim + tiametoxan) reduziu a AACPS das doenças em 11,4 e 120,5, respectivamente. A maior captura de conídios no ar dos fungos ocorreu nas plantas submetidas ao tratamento comercial de sementes havendo correlação positiva e significativa (r = 0,89 B. sorokiniana e r = 0,70 D. teres) entre número de conídios no ar e severidade das doenças. O tratamento de semente utilizado influenciou a qualidade sanitária das sementes de cevada produzidas, com correlação significativa e positiva (r=0,99) entre AACPS da mancha marrom e da mancha-em-rede e incidência de B. sorokiniana e D. teres nas sementes colhidas. O tratamento comercial de sementes não reduziu o inóculo de B. sorokiniana e D. teres das sementes de cevada, antecipa a epidemia da mancha marrom e mancha-em-rede e aumenta custo de produção. Os demais tratamentos apesar de não erradicar os fungos, retardam as doenças e aumentam o lucro do agricultor

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Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)
The cement industry, closely associated with the global warming question, accounts for significant emissions of CO2 and other air pollutants, such as SO2 and NO2 in the atmosphere. In search for ways to mitigate the atmospheric CO2, we performed semicontinuous cultures of Chlorella sorokiniana under phototrophic conditions to test the effect of a flue gas simulation (18% CO2, 9% O2, 300 ppm NO2 and 140 ppm SO2). This was provided once a day in six serial experiments, in which the exposure to the gas was increased through the increase of bubbling time. A constant flow rate allowed us to calculate the total volume of gas introduced into the system each day (0.1, 0.3, 0.8, 1.5, 6 and 48 L). Air-CO2 (18%) was used as control and its bubbling time was pHregulated. Culture medium acidification led to suboptimal growth conditions that affected cell density, photosynthetic activity, cell viability and the biochemical composition of C. sorokiniana. Compared to control, the specific growth rate decreased by 17 and 3,9% in cultures that received 6 and 48 L gas d-1, respectively. The pulseamplitude modulated (PAM) fluorometry was used for culture evaluation. It revealed low maximum quantum yield (ΦM 0.40) and operational quantum yield (Φ'M 0.47) values one day after 48 L gas bubbling. Light saturation curves confirmed the negative effects of long-time gas simulation stress. On the other hand, quenching analysis indicated an increase in photochemical light use and low values of non hotochemical quanching (qN and NPQ). Exposure of the cells to the flue gas simulation resulted in lower cell viability compared to control. Biochemical analysis showed that 6 and 48 L gas d-1 significantly increased protein content by 75% and 154%, respectively; total carbohydrates also increased in the presence of the gas, 148% and 195%, respectively. Despite the physiological changes, C. sorokiniana resisted suboptimal growth conditions imposed by the gas, supporting its vigorous nature and relevance in biotechnological aplications with flue gases.
Inserida na problemática do aquecimento global, a indústria de cimento é uma das que mais contribui para emissão de CO2 e de poluentes como SO2 e NO2 na atmosfera. Visando alternativas para mitigação desses gases, que são oriundos principalmente de processos de combustão, foram testados seis cultivos fototróficos semicontínuos de Chlorella sorokiniana, para avaliar o efeito de diferentes volumes (0,1; 0,3; 0,8; 1,5; 6 e 48 L d-1) de uma simulação gasosa composta por CO2 (18%), O2 (9%), NO2 (300 ppm) e SO2 (140 ppm). Os volumes variaram conforme o aumento do tempo de borbulhamento diário dos gases nos cultivos. O tratamento controle, composto por ar sintético e CO2 (18%), teve seu tempo de borbulhamento definido pela variacão de pH do meio. O fornecimento da simulação gasosa resultou na acidificação do meio de cultura e afetou a densidade celular, a atividade fotossintética, a viabilidade celular e a composição bioquímica de C. sorokiniana. Comparadas ao controle, as exposições diárias de 6 e 48 L gás d-1 reduziram a taxa específica de crescimento em 17 e 39%, respectivamente. Por meio da fluorescência de amplitude modulada (PAM), verificamos baixos valores de rendimento quântico máximo (ΦM 0,40) e operacional (Φ’M 0,47) um dia após o primeiro borbulhamento de 48 L gás. Curvas de saturação de luz confirmaram os efeitos negativos do estresse prolongado à mistura gasosa. A análise de decaimento da fluorescência da clorofila, por sua vez, indicou um aumento da energia luminosa direcionada à fotoquímica da fotossíntese (qP) e baixos valores de dissipação não fotoquímica da energia luminosa (qN e NPQ). A simulação gasosa resultou, ainda, em menor viabilidade celular, se comparada ao controle. Pelas análises bioquímicas, constatamos que 6 e 48 L gás d-1 levaram a um aumento significativo do conteúdo proteico de 75% e 154%, respectivamente; os mesmos tratamentos também aumentaram a quantidade de carboidratos totais em 148% e 195%. Apesar das alterações fisiológicas, C. sorokiniana resistiu às condições subótimas de crescimento, o que comprova sua robustez e relevância em aplicações biotecnológicas envolvendo gases de combustão.

A Mancha marrom é uma das principais doenças do trigo, causada pelo fitopatógeno Bipolaris sorokiniana, responsável por grandes perdas econômicas no cultivo do trigo em todo mundo. Este fungo apresenta uma grande diversidade morfológica, fisiológica e genética. O objetivo do trabalho foi utilizar marcadores moleculares e fenotípicos para avaliar a variabilidade genética em isolados monopóricos e polispóricos de Bipolaris sorokiniana de sementes de trigo oriundos do Brasil e outros países. A caracterização molecular envolveu as metodologias URP-PCR, PCR-RFLP, REP-PCR, BOX-PCR, ERIC-PCR assim como testes isoenzimáticos e de patogenicidade. A análise de patogenicidade revelou que isolados polispóricos são mais severos para as sementes que para as partes aéreas das plantas quando comparados com os monospóricos. A caracterização isoenzimática e molecular através das técnicas URP-PCR, PCR-RFLP, REP-PCR, BOX-PCR, ERIC-PCR exibiram uma grande diversidade intra-populacional. REP-PCR e ERIC-PCR revelaram maior diversidade entre os isolados, com uma similaridade inferior a 70%. No entanto, as amplificações realizadas com o BOX-PCR apresentaram um perfil com uma maior similaridade. Com os resultados obtidos a partir das amplificações com BOX-PCR um par de primers foi desenhado a partir de um fragmento comum a todos os isolados e que foi capaz de amplificar um produto único ao gênero Bipolaris sp. entre as amostras testadas. Com a realização de mais ensaios com outros microrganismos é possível que o mesmo possa ser utilizado como um marcador deste gênero. A técnica de PCR-RFLP utilizando as enzimas HaeIII, HinfI, HhaI, EcoRI e HindIII apresentaram perfis de restrição com variação no número de fragmentos e no peso molecular. Uma possível explicação para à variabilidade observada em todas as técnicas utilizadas pode ser atribuída à condição multinucleada das células de B. sorokiniana bem como a heterocariose, que pode levar a recombinação mitótica e ao polimorfismo.
The brown spot is a major disease of wheat caused by the pathogen Bipolaris sorokiniana, responsible for large economic losses in wheat cultivation worldwide. This fungus has a wide morphological, physiological and genetic diversity. The main objective of this work was to characterize monosporic and polisporic B. sorokiniana isolates of wheat seeds from Brazil and other countries. Pathogenicity tests were conducted to evaluate the feasibility of virulence genes as well as different molecular methodologies were tested as: URP-PCR, PCR-RFLP, REP-PCR, BOX-PCR, ERIC-PCR and isoenzyme patterns of the isolates. The pathogenicity assay reveals that the polysporic isolates caused a more severe disease to seeds than to aerial parts of the plants when compared to single spore isolates. Isoenzyme and molecular characterization through the URP-PCR, PCR-RFLP, REP-PCR, BOX-PCR, ERIC-PCR techniques showed a large intra-population diversity among the isolates. REP and ERIC-PCR revealed greater diversity among the isolates with a similarity below 70%. However, the amplification results using with BOX- PCR showed a highest similarity. The PCR-RFLP using HaeIII, HinfI , HhaI , EcoRI and HindIII restriction enzymes showed a profile variation between all isolates in relation to the number and molecular weight of the fragments. However the results obtained with BOX- PCR amplifications a higher similarity was obtained. With this result a primer was designed, based on a fragment common to all isolates, and the amplifications using these primers produced a unique fragment with the Bipolaris genus among others isolates tested. More phytopatogeneic isolates must be tested in order to confirme the specificity for Bipolaris sp. With the PCR-RFLP assay, using the enzymes HaeIII, HinfI, HhaI, EcoRI e HindIII, variability was observed among the restriction patterns realated to the number of fragments and molecular weight. One possible explanation for the observed variability in all techniques used may be attributed to the condition of multinucleated cells of B. sorokiniana and the heterokaryosis which can lead to mitotic recombination and polymorphis.

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The induced resistance efficiency in the control of pathogens is recognized, however there are costs related to its use that are still little researched, as the case of fitness and ecological costs. This research aims to verify the efficiency of use of the acibenzolar-S-methyl (ASM), mannanoligosaccharide (MOS) and B. cereus inductors in the control of spot blotch wheat disease, and its relation with the induction of enzymes related to the plant defense, as well as the interference on gas exchanges, on the non-target microrganisms A. brasilense, and on the crop production. The experiment was conduced in green house growing at the Núcleo de Estações Experimentais of the Universidade Estadual do Oeste do Paraná, Marechal Cândido Rondon Campus. The experimental design was made in randomized block design in a factorial schema 4 x 2 x 2, with four replications. The first factor "Resistance Inductors" was constituted by ASM, MOS, B. cereus inductors and water (control). The second factor "Pathogen" was constituted by the presence or absence of B. sorokiniana pathogen. The third factor "PGPR" was made up by the presence or absence of the PGPR A. brasilense. The seeds were inoculated with A. brasilense 24 h before sowing. The treatment with the resistance inductors was done 25 days after the plant emergency and 24 h after it, the inoculation with the pathogen was done. Evaluations for the quantification of peroxidase enzyme, phenylalanine ammonia-lyase and β-1.3-glucanase enzymes activities were done at 0 h, 24 h, 48 h, 72 h and 96 h after treatment. Gas exchanges were evaluated at 0 h, 24 h, 48 h, 72 h, 96 h and 120 h after treatment. The quantification of diazotrophic was made in the implementation of the experiment, at the moment of the use of the resistance inductors and in the flowering. At the flowering, biometric evaluations were performed, and at the end of the cycle, production analysis were held. The use of resistance inductors was efficient in the control of spot blotch wheat disease. The treatment with inductors did not show any interference on the endophytic diazotrophic microrganism. Higher activity of peroxidases for the MOS treatment was observed, that presented high efficiency on the control of the disease. No relation between the activities of phenylalanine ammonia lyase and β-1.3-glucanase and the control of spot blotch was observed. A higher tax of liquid assimilation of CO2 (A) in the absence of the pathogen and presence of the A. brasilense was observed, however, MOS reduced the “A”. The internal concentration of CO2 and the leaf transpiration showed as superior in the treatment with the absence of the pathogen. The stomatal conduce was affected by MOS treatment. The length of the flag leaf and leaf area were affected by ASM use. But, the total weight of grain did not suffer interference of the treatments, in spite of the weight of 100 grains to have been superior to ASM.
É reconhecida a eficiência da indução de resistência no controle de fitopatógenos, no entanto, existem custos relacionados à sua aplicação ainda pouco investigados, como é o caso dos custos adaptativos e ecológicos. No presente trabalho objetivou-se verificar a eficiência da aplicação dos indutores acibenzolar-S-metil (ASM), mananoligossacarídeo fosforilado (MOS) e Bacillus cereus no controle de mancha marrom em trigo, e sua relação com a indução de enzimas relacionadas a defesa, assim como a interferência sobre as trocas gasosas, sobre o microrganismo não alvo Azospirillum brasilense, e a produção da cultura. O experimento foi conduzido sob cultivo protegido no Núcleo de Estações Experimentais da Universidade Estadual do Oeste do Paraná, Campus de Marechal Cândido Rondon. O delineamento experimental foi em blocos ao acaso em esquema fatorial 4 x 2 x 2, com quatro repetições. O primeiro fator “Indutores de Resistência” foi constituído pelos indutores ASM, MOS, Bacillus cereus e a testemunha Água. O segundo fator “Patógeno” foi constituído pela presença ou ausência do patógeno Bipolaris sorokiniana. O terceiro fator “BPCV” constituiu-se pela presença ou ausência da bactéria promotora de crescimento vegetal A. brasilense. As sementes foram inoculadas com A. brasilense 24 h antes da semeadura. O tratamento com os indutores de resistência foi realizado 25 dias após a emergência e 24 horas após foi realizada a inoculação com o patógeno. Foram realizadas avaliações para quantificação das enzimas peroxidase, fenilalanina amônia-liase e β-1,3-glucanase às 0 h, 24 h, 48 h, 72 h e 96 horas após o tratamento. As trocas gasosas foram avaliadas às 0 h, 24 h, 48 h, 72 h, 96 h e 120 horas após o tratamento. A quantificação de diazotróficos foi realizada na implantação do experimento, no momento da aplicação dos indutores de resistência e no florescimento da cultura. No florescimento foram realizadas análises biométricas e no final do ciclo, análise dos componentes da produção. A aplicação dos indutores de resistência foi eficiente no controle da mancha marrom em trigo. Os tratamentos indutores não apresentaram interferência sobre os microrganismos diazotróficos endofíticos. Observou-se maior atividade de peroxidases para o tratamento MOS o qual apresentou grande eficiência no controle da doença. Não observou-se relação entre a atividade de fenilalanina amônia-liase e β-1,3-glucanase e o controle de mancha marrom. Observou-se maior taxa de assimilação líquida de CO2 (A) na ausência do patógeno e presença de A. brasilense, porém MOS reduziu a A. A concentração interna de CO2 e a transpiração foliar apresentaram-se superiores nos tratamentos com ausência do patógeno. A condutância estomática foi afetada pelo tratamento MOS. O comprimento da folha bandeira e a área foliar foram afetados negativamente pela aplicação de ASM. Porém, a massa total de grãos não sofreu interferência dos tratamentos, apesar de a massa de 100 grãos ter sido superior para ASM.

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The Chemical control of diseases is the most used practice in wheat. The increase in mineral nutrition with foliar fertilizers has been a promising alternative for the plant resistance against leaf diseases. However, foliar fertilizers have been applied in association with fungicides and can change the performance of the fungicide on diseases control. This study aimed to evaluate the applicability of foliar fertilizer in combination with azoxystrobin + cyproconazole fungicide in wheat, based on biochemical, physiological , nutritional and yield parameters and determine the interference caused by the fertilizer on the evolution of leaf diseases in wheat. Isolated application rates of fertilizer and application in combination with the fungicide were performed on field and in the greenhouse works. The application of fertilizer increased the plant growth, green leaves and enhanced pigments levels (Chl a, Chl b and carotenoids). When the fungicide was applied with fertilizer, it reduced the stresses effect generated by fungicide application; it increased parameters of chlorophyll fluorescence, Fv / Fm and ETR. The levels of N, P and K in the leaves increased after fertilizer application. The fertilizer mixed with fungicide did not reduce the fungicide uptake. The diseases control was better when fertilizer was mixed with fungicide. The fertilizer applied alone had no effect on the diseases. Yield parameters were increased due to application of fungicide and foliar fertilizer.
O controle químico de doenças com o uso de fungicidas é umas das práticas mais empregadas na cultura do trigo em função da eficácia de controle. Incrementos na adubação mineral com fertilizantes foliares tem sido uma alternativa promissora em busca de maior resistência as doenças. Entretanto, fertilizantes foliares são comumente aplicados associados a fungicidas e podem interferir no desempenho de controle do produto. Este trabalho teve por objetivo avaliar a aplicabilidade do fertilizante foliar em mistura com o fungicida azoxistrobina + ciproconazol na cultura do trigo, com base em parâmetros bioquímicos, fisiológicos, nutricionais e produtivos e determinar a interferência causada pelo fertilizante sobre a evolução de doenças foliares na cultura. A partir da aplicação isolada de doses do fertilizante e da aplicação em associação com o fungicida foram realizados trabalhos a campo e em casa de vegetação. A aplicação do fertilizante refletiu em maior crescimento das plantas, manutenção de folhas verdes e maiores teores de pigmentos (Chl a, Chl b e carotenóides). Quando aplicado junto ao fungicida, o fertilizante teve efeito mitigatório dos estresses gerados pela aplicação do fungicida, com reflexos positivos em parâmetros da fluorescência da clorofila a, Fv/Fm e ETR. Houve aumento dos teores de N, P e K nas folhas em função do fertilizante foliar. Não houve redução da absorção do ingrediente ativo azoxistrobina + ciproconazol em mistura com o fertilizante. Houve melhor resposta de controle das doenças em função da mistura do fertilizante com o fungicida. O fertilizante isolado não teve nenhum efeito sobre as doenças. Parâmetros produtivos foram incrementados em função da aplicação do fungicida e do fertilizante foliar.

Os isolados Streptomyces sp. R18(6) e 6(4) foram avaliados quanto à sua capacidade de controlar a mancha marrom e podridão comum de raiz, causados por Bipolaris sorokiniana em plantas de trigo. A atividade antifúngica desses isolados foi testada usando os ensaios de dupla camada e pareamento de cultura à 28°C. A atividade fisiológica e enzimática foi avaliada através de ensaios de sideróforo, ácido indol-3-acético, fixação de nitrogênio e solubilização de fosfato. O controle biológico da doença e a eficiência de crescimento das plantas de trigo foram avaliados utilizando ensaios in vivo em casa de vegetação. Nos ensaios de pareamento de cultura, ambos os isolados inibiram o crescimento micelial de B. sorokiniana, enquanto na dupla camada apenas o isolado R18(6) inibiu. Streptomyces sp. 6(4) produziu auxina, sideróforos, fixou nitrogênio e solubilizou fosfato, enquanto R18(6) não produziu sideróforos. Nos ensaios em casa de vegetação, o isolado R18(6) mostrou diferenças estatísticas na massa seca da parte aérea e na massa seca de raiz em comparação com a do isolado 6(4) na presença do fitopatógeno (P≤0,05). Estes resultados foram mais evidentes quando a temperatura foi maior. Na ausência do fitopatógeno, o isolado 6(4) aumentou a massa seca de raiz em comparação com a do controle durante o mesmo período. Portanto, esses isolados apresentaram potencial em controlar a podridão das raízes e mancha marrom e podem promover o crescimento das plantas de trigo.
Streptomyces sp. R18(6) and 6(4) strains were evaluated for their ability to control brown spot and common root rot caused by Bipolaris sorokiniana in wheat crops. The antifungal activity of these isolates was tested using a doublelayer assay and culture pairing at 28 °C. Physiological and enzymatic activity were evaluated through siderophore, indole-3-acetic acid, nitrogen fixation and phosphate solubilization assays. The biocontrol of the disease and growthpromoting efficiency of wheat seedlings were assessed using in vivo assays in greenhouse. In the culture pairing assays, both strains inhibited B. sorokiniana mycelial growth, while in the double-layer only R18(6). Streptomyces sp. 6(4) produced auxin, siderophores, fixed nitrogen and solubilized phosphate, whereas R18(6) did not produce siderophores. In the greenhouse assays, strain R18 (6) showed statistical differences in shoot dry mass and root dry mass compared with those of strain 6(4) in the presence of the phytopathogen (P ≤ 0.05). These results were more evident when the temperature was higher. In the absence of the phytopathogen, strain 6(4) increased the root dry mass compared with that of the control during the same period. Therefore, these isolates can potentially control root rot and brown spotting and may promote the growth of wheat plants.

Dans l'optique de mieux cerner le mécanisme moléculaire des désaturases, une étude consistant à incuber des cellules entières en présence d'analogues soufrés d'acides gras a été menée sur la microalgue verte Chlorella sorokiniana. L'analyse de la régiocryptochimie des D9 et D12-désaturases a permis de mettre en évidence l'implication d'un mécanisme séquentiel. L'utilisation d'analogues soufrés adéquats a également permis de démontrer la présence d'un transfert d'oxygène hautement stéréosélectif du site actif vers l'atome de soufre pour la D12-désaturase. La détermination des configurations absolues des sulfoxydes obtenus a été envisagée par l'intermédiaire de la résonance magnétique nucléaire à l'aide de réactifs de déplacement chimique. La validité de cette approche a été vérifiée sur des sulfoxydes synthétisés sous forme énantiomériquement pure. Enfin, l'aptitude de cellules entières à oxyder des thioéthers prochiraux a été déterminée en terme de rendement et d'énantiosélectivité.

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The objectives of the work were: a) to obtain the damage function equations for multiple pathosystem to calculate the EDT to time fungicides application; b) to quantify the control and damage of the yield components, and c) To relate the brown spot incidence and severity in different growth stages. The experiments were carry out at the NBN Seeds Company during the 2009 and 2010 crop seasons, located in Muitos Capões county, Rio Grande do Sul state, Brazil and in the Agro science Center at Santa Catarina State University only in 2010. The cultivar BRS Cauê was used as susceptible cultivar to brown spot and powdery mildew. The experiments design was in randomized block with four replications. The nine treatments consisting of different rates (half and recommended rates) and fungicide applications number (one, two, three and four) of mixture strobilurin and triazole fungicides, generating the disease gradients intensity. The total area of 5.0 x 2.5 m was the experimental unit in both experiments. The fungicide applications and incidence and severity assessment were done at EC 22, EC 31, EC 39, EC 45 and EC 56 plant development stages. Plants from central rows of each plot were manually harvested and grain yield (GY), one-thousand grain weight (TGW) and granulometry (G) were evaluated. In the first chapter, the damage function equations between disease intensity and GY for each plant development stages, obtained by regression analysis, in both 2009-10 crop growing seasons were significant and negative indicative that increasing disease severity lead into decreasing grain yield. The damage coefficients of these equations can be used to calculate the economic damage threshold. In the second chapter, the values of disease intensity were used to calculate the area under disease progress curve (AUDPC). The GY, TGW, G and AUDPC values were tested using the mean comparison between treatments and the percentage of damage and disease control were determinated. The largest 2009 crop damage was 45.87%, 15.47% and 25.84% for GY, G and TGW, respectively. The ultimate control was 68.11% when considered the severity and four foliar applications, independent of the used dose. In 2010, the greatest damage were 31.16%, 14.02% and 10.76% in Muitos Capões and 39.44%, 23.59% and 45.88%, in Lages, for GY, 10 TGW and G, respectively. The highest percentage of control, based on the leaf severity were 71.63% and 73.96% for Muitos Capões and Lages, respectively. The greater control, independent of used dose were obtained with three and four applications in Muitos Capões and four applications in Lages. In the third chapter, brown spot incidence and severity data were subjected to regression analysis and correlation and the obtained values were significant and positive. The brown spot and powdery mildew diseases incidence and severity recommended by the Technique Indication of crop (TIC) to initiate fungicide applications is 20% and 5% respectively. The severity average values for initiating chemical control are 0.77% and 0.34% respectively for 2009 and 2010 crop seasons when the incidence diseases were substituted in the equations. These values are lower than TIC recommended values
Os objetivos do trabalho foram: a) obter equações de função de dano para patossistema múltiplo para calcular o LDE servindo como critério indicador de aplicação de fungicidas; b) quantificar controle e dano nos componentes de rendimento; e c) relacionar incidência e severidade foliar da mancha marrom em diferentes estádios fenológicos. Os experimentos foram conduzidos nas safras agrícolas 2009 e 2010 na NBN Sementes no município de Muitos Capões, RS; e, na safra de 2010 no Centro de Ciências Agroveterinárias no município de Lages, SC. Em todos os experimentos foi utilizada a cultivar BRS Cauê suscetível à mancha marrom e oídio. O delineamento foi blocos casualisados, com quatro repetições e nove tratamentos constituídos de diferentes doses (meia dose e dose indicada) e número (uma, duas, três e quatro) de aplicações de fungicidas triazóis e estrobilurinas para gerar os gradientes de intensidade das doenças. A área de cada unidade experimental correspondeu a 5,0 x 2,5 m. As aplicações e as avaliações da incidência e severidade foliar ocorreram nos estádios de crescimento (EC) EC 22, EC 31, EC 39, EC 45 e EC 56. A colheita foi manual cortando as plantas das linhas centrais de cada parcela. Foram avaliados rendimento de grãos (RG), massa de mil grãos (MMG) e granulometria (G). No primeiro capítulo, as equações de função de dano entre intensidade de doença e RG para cada EC, obtidas por análise de regressão, em ambas as safras agrícolas, foram significativas e negativas, ou seja, à medida que aumentou a intensidade de doença, diminuiu o RG. Os coeficientes de dano obtidos podem ser utilizados no cálculo do limiar de dano econômico. No segundo capítulo, os valores de intensidade das doenças foram usados para o cálculo da área abaixo da curva de progresso da doença (AACPD). Os valores de RG, MMG, G e AACPD foram submetidos ao teste de comparação de médias. Foram determinados o percentual de dano e de controle das doenças. Na safra 2009, os maiores danos foram 45,87%, 15,47% e 25,84% para RG, MMG e G, respectivamente. O controle máximo foi 68,11% considerando a severidade foliar e quatro aplicações, independente da dose usada. Em 2010, os maiores danos foram 31,16%, 14,02% e 10,76% em Muitos Capões e 39,44%, 23,59% e 8 45,88% em Lages, respectivamente para R, MMG e G. Com base na severidade obteve-se controle de 71,63% e 73,96% em Muitos Capões e Lages, respectivamente. Em Muitos Capões três e quatro aplicações e em Lages quatro aplicações, independente da dose, apresentaram maior percentual de controle. No terceiro capítulo, os dados de incidência e severidade foliar de mancha marrom foram submetidos à análise de regressão e correlação. As equações obtidas foram significativas e positivas. A incidência e a severidade foliar recomendado pela Indicação Técnica da cultura (ITC) para iniciar as aplicações de fungicidas é de 20% e 5%, respectivamente. Substituindo esse valor de incidência nas equações obtidas têm-se valores médios de severidade para iniciar o controle químico de 0,77% e 0,34% respectivamente para 2009 e 2010, inferior ao recomendado pela ITC

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Leaf rust and leaf spots caused by Bipolaris sorokiniana and Drechslera tritici-repentis, are diseases that dramatically reduce the productivity of wheat. Depending on the difficulty in having cultivars resistant to all diseases, chemical control is an alternative, technically and economically viable. To maximize the residual of fungicides is necessary to optimize the factors related with spray minimizing the losses due to disease incidence. The goal of the work was to determine the influence of the number of drops in the penetration of fungicides and its control efficacy at various stages of application. The experimental design used was randomized block in Split plot design, totaling 20 treatments and four replications. The treatments were two wheat cultivars (Fundacep Cristallino and Fundacep Nova Era); three spray tips (XR 11002, TJ 60-11002 and TX 8002); 3 fungicides: Pyraclostrobin + Epoxiconazol; Epoxiconazol and Pyraclostrobin, and a control without any application. It was evaluated the density of drops per square centimeter, DMV, DMN, severity of disease, weight of hectoliter and productivity of the crop. The use of fungicides pyraclostrobin and epoxiconazol + piraclostrobia, respectively, provided the better control of diseases, higher productivity and greater weight of hectoliter in Nova Era cultivar. On the other hand, Cristalino cultivar with lower impact of the diseases did not show difference on efficacy of the fungicides used. The control of diseases was influenced by the DMV/DMN ratio due to the use of different spray tips. Tips with thin/medium drop spectrum performed better coverage at the lower part of the canopy, providing fewer establishments of the diseases. The correlation between the variables showed that the second application of fungicides was the one that crashed in the calculation of the area under the curve of progress of diseases (AACPD), hectoliter weight and yield. The greater disease pressure at flowering can explain this result.
A Ferrugem da folha e as manchas foliares causadas por Bipolaris sorokiniana e Drechslera tritici-repentis, são doenças capazes de reduzir drasticamente a produtividade da cultura do trigo. Em função da dificuldade em dispor de cultivares resistentes a todas as doenças, o controle químico é uma alternativa viável, técnica e economicamente. Para maximizar o residual dos fungicidas é necessário otimizar os fatores relacionados à tecnologia de aplicação minimizando os danos em função da incidência das doenças. O objetivo do trabalho foi determinar a influência do número de gotas na penetração dos fungicidas e na sua eficácia de controle em diferentes momentos de aplicação. O delineamento experimental utilizado foi de Blocos ao acaso em parcelas subsubdivididas, totalizando 20 tratamentos em quatro repetições. Os tratamentos foram constituídos por duas cultivares de trigo (Fundacep Cristalino e Fundacep Nova Era); três pontas de pulverização (XR 11002, TJ 60-11002 e TX 8002); 3 fungicidas: Piraclostrobina + Epoxiconazol; Epoxiconazol e Piraclostrobina, mais uma testemunha sem aplicação. Foi avaliada a densidade de gotas por centímetro quadrado, diâmetro mediano volumétrico (DMV), diâmetro mediano numérico (DMN), severidade das doenças, peso do hectolitro e produtividade da cultura. A utilização dos fungicidas epoxiconazol+ piraclostrobina e piraclostrobina, respectivamente, proporcionaram o melhor controle de doenças, melhor produtividade e maior peso do hectolitro na cultivar Nova Era. Por outro lado, na cultivar Cristalino submetida a um menor impacto das doenças, não houve diferença com relação aos fungicidas utilizados. O controle de doenças foi influenciado pelo espectro de gotas proporcionado devido a utilização das diferentes pontas de pulverização. Pontas com espectro fino/médio apresentaram melhor cobertura do terço inferior do dossel, proporcionando menor estabelecimento das doenças. A correlação entre as variáveis mostrou que a segunda aplicação dos fungicidas foi a que mais impactou no calculo da área abaixo da curva de progresso das doenças (AACPD), produtividade de grão e peso do hectolitro. Esse resultado pode ser explicado pela maior pressão de doença no estádio de florescimento.

Existe um grande potencial para a expansão da produção de microalgas, tanto pela variedade de usos quanto pelos produtos que delas podem ser derivados. Dentre esses usos, as características nutricionais e funcionais conferem às algas alto valor biológico para uso em nutrição humana e animal. No entanto, são poucos os estudos que avaliam seu potencial na nutrição de peixes, mais especificadamente de tilápia, um dos principais produtos da aquicultura mundial. O objetivo deste trabalho foi avaliar o potencial de uso de duas espécies de microalgas e o nível ótimo de inclusão da Schizochytrium em dietas para a tilápia. Para tanto, foram realizados ensaios para avaliação do coeficiente de digestibilidade aparente (CDA) de uma microalga rica em proteína, Chlorella sorokiniana, e outra rica em energia, a Schizochytrium sp., ao longo de ensaios de desempenho e digestibilidade de rações contendo níveis crescentes (0%; 1%; 2,5%; 5%; 7,5% e 10%) de Schizochytrium sp. a fim de determinar o nível ótimo de inclusão nas dietas. As microalgas apresentaram altos coeficientes de digestibilidade para a tilápia, com CDA para a proteína de 90,51 e 97,20 % e para energia de 84,22 e 82,55 %, para a Chlorella e Schizochytrium, respectivamente. Os níveis crescentes de inclusão da Schizochytrium alteraram a digestibilidade das rações de forma decrescente para a proteína, passando de um CDA de 88,07 para 86,01, e energia, de um CDA de 74,19 para 67,35. No ensaio de desempenho foi registrado aumento no consumo de ração e piora na conversão alimentar aparente, sem que outros parâmetros de desempenho zootécnico fossem alterados, à medida que aumentava os níveis de inclusão da Schizochytrium na dieta. Não foram registradas alterações nos conteúdos de macro nutrientes na carcaça dos peixes, no entanto, os níveis crescentes de inclusão alteraram o perfil de ácidos graxos aumentando a quantidade de ácidos graxos n-3 no filé, principalmente do ácido docosaexaenoico (DHA), e redução na relação n-6/n-3. Dessa forma, é possível a utilizar Schizochytrium sp. como aditivo nas dietas para alteração do perfil de ácidos graxos da tilápia, enriquecendo os filés com ácidos graxos n-3, com pouco prejuízo nos parâmetros de desempenho.
There is a great potential for expand the microalgae production as by the variety of it uses as for the products and processes which may derive from them. Amongst such uses, nutritional and functional characteristics provide them a high biological value for use in human and animal nutrition. Nevertheless, few studies have evaluated their potential for fish nutrition more specifically tilapia, which is one of the main aquaculture products in the world. The aim of this research was to evaluate the potential use of two microalgae and the optimal level of inclusion of Schizochytrium in diets for tilápia. Therefore, tests were conducted to evaluate the apparent digestibility coefficient (ADC) of microalgae with high protein content Chlorella sorokiniana and another with high energy, Schizochytrium sp. and also performance assay and feed digestibility containing inclusion levels (0%, 1%, 2.5%, 5%, 7.5% and 10%) of Schizochytrium in order to determine the optimum addition level. Both species were very digestible for tilapia with ADCs for protein of 90.51 and 97.20 and for energy 84.22 and 82.55 to Chlorella and Schizochytrium respectively. The increasing levels of inclusion of Schizochytrium changed the digestibility of feed for the protein, through a ADC from 88.07 to 86.01 and for energy from 74.19 to 67.35 ADC. In the growth test as the inclusion of Schizochytrium increased there was an increase in feed intake and feed conversion ratio but any other growth parameters were changed. Regarding the carcass composition, there were no changes in macronutrients composition however, the increasing levels of Schizochytrium altered the fatty acid profile by increasing the amount of omega 3 mainly docosahexaenoic acid (DHA) and reduced the n-6/n-3 ratio. Thus, it is possible to use the Schizochytrium to modify the fatty acid profile of tilápia by incorporating omega 3, with little loss in performance parameters.

Les travaux decrits dans ce memoire ont pour objectif de contribuer a une meilleure comprehension des mecanismes de desaturation des chaines aliphatiques saturees, par des systemes plurienzymatiques complexes, appeles desaturases. Nous nous sommes interesses plus particulierement a l'oleyl-desaturase (ou 12-desaturase), qui permet la biotransformation de l'acide oleique en acide linoleique, premier acide gras essentiel. Cette reaction est exclusivement limitee au regne vegetal. Notre strategie consiste a effectuer des tests competitifs d'inhibition substrat naturel marque-substrat modifie, realises in vivo, de maniere a obtenir des relations structure-activite. Le modele biologique choisi est une microalgue verte, chlorella sorokiniana. L'influence de chaque acide modifie sur l'activite de la 12-desaturase peut alors etre evalue via le taux de desaturation du substrat naturel. Notre choix s'est porte sur la synthese de deux series d'acides modifies en c18, plus specifiquement sur des acides thiaoleiques, possedant un atome de soufre sur la chaine entre les carbones c#1#2 et c#1#6, et sur des acides oleiques et linoleique dimethyles, comportant un groupe gem-dimethyle en de la double liaison, c'est a dire en position c#8 ou c#1#1. La premiere partie de ce memoire est une bibliographie relative a la biosynthese des acides gras vegetaux : nous y presentons un bilan des connaissances actuelles concernant les desaturases solubles et membranaires. La synthese des acides thiaoleiques est decrite dans le chapitre ii : elle est basee sur la voie des acetyleniques ; les resultats des tests biologiques y sont egalement commentes. Le chapitre iii est consacre a la deuxieme serie d'analogues : leur synthese, axee sur des reactions de wittig, ainsi que les premieres conclusions biologiques y sont presentees.

La 12-desaturase, enzyme localisee dans la membrane du reticulum endoplasmique des cellules vegetales, est responsable de la desaturation de l'acide oleique en acide linoleique. Afin d'etudier les interactions enzyme - substrat et de contribuer a l'elucidation du mecanisme moleculaire de la desaturation des acides gras, nous avons utilise une approche chimique qui consiste a incuber des cellules entieres d'une microalgue verte, c. Sorokiniana, en presence d'acides gras soufres, analogues structuraux du substrat vrai de l'enzyme. L'inhibition regiospecifique de la desaturation de l'acide oleique et l'oxydation regio- et stereoselective des 12- et 13-thiaoleates en sulfoxydes correspondants nous ont permis de mettre en evidence un transfert d'oxygene du site actif de l'enzyme sur l'atome de soufre lorsque l'abstraction des hydrogenes n'est pas possible. L'analyse stereochimique de tels sulfoxydes aliphatiques chiraux et quasi symetriques a ete realisee par rmn #1h et #1#3c, en utilisant des nouveaux reactifs de deplacements chimiques de la famille des acides arylmethoxyacetiques. A la suite de cette etude fondamentale et au vu de la capacite des cellules entieres de c. Sorokiniana a oxyder des composes soufres, les conditions d'incubation ont ete optimisees (temperature, etat de croissance de l'algue, concentrations des substrats) dans le but d'utiliser cette microalgue comme biocatalyseur d'oxydations asymetriques. La comparaison des resultats d'oxydation in vivo de divers thioethers (influence de la lipophilie, des effets stereoelectroniques) en terme de rendements de conversion, d'exces enantiomeriques et de stereochimie nous permettra d'envisager la modelisation du site actif de l'enzyme responsable de l'oxydation (monooxygenase a flavine ou a cytochrome p450).

O tratamento anaeróbio de esgoto doméstico em combinação com a produção de biomassa de algas é considerado uma excelente alternativa para a remoção de nutrientes. O processo de separação sólido-líquido de microalgas continua sendo um grande desafio técnico e econômico. A ozonização seguida de flotação por dissolvido é uma alternativa interessante visando a separação de microalgas e, consequentemente, a remoção de nutrientes. Os objetivos deste trabalho foram: avaliar a remoção de nitrogênio e fósforo de um efluente de reator anaeróbio através do crescimento da microalga Chlorella sorokiniana em fotobiorreator tipo flat panel e investigar o uso da ozonização associada à flotação por ar dissolvido para o processo de separação sólido-líquido da microalga Chlorella sorokiniana. O efluente anaeróbio foi obtido a partir de um reator anaeróbio com leito de fibras flexíveis que trata esgoto doméstico sintético. As microalgas foram cultivadas em fotobiorreator flat panel com tempo de cultivo de cinco dias, sob condições controladas: intensidade de luz (196 μmol m-2 s-1), fluxo de ar (0,2 vvm), temperatura (29 ± 1 °C) e fotoperíodo (12 horas com iluminação artificial e 12 horas no escuro). No final do período de cultivo, foram realizados ensaios para adequação da FAD e da ozonização, através da avaliação de parâmetros, tais como: dosagem de ozônio, dosagem de polímero, pH, tempos e gradientes de mistura rápida e floculação, velocidade de flotação e quantidade de ar necessária para flotação por ar dissolvido. O reator anaeróbio apresentou altas eficiências de remoção de demanda química de oxigênio e demanda bioquímica de oxigênio: 63 ± 6% e 53 ± 8%, respectivamente. Ao final do período de cultivo das microalgas no fotobiorreator, as remoções de nitrogênio total dissolvido e fósforo total dissolvido foram de 52,1% e 31,8%, respectivamente. Analisando os resultados referentes à etapa de separação sólido-líquido, verificou-se que o pH não influenciou significativamente na eficiência do tratamento; altas velocidades de flotação (24 cm.min-1 e 36 cm.min-1) podem ser empregadas; a etapa de floculação não contribuiu para melhora do tratamento; e a ozonização não contribuiu significativamente para a melhora da eficiência do processo de separação sólido-líquido.
Anaerobic treatment of wastewater in combination with the production of algae biomass is an excellent alternative for nutrient removal. The process of solid-liquid separation of microalgae remains a major technical and economic challenge. Ozonization followed by flotation by dissolved air is an interesting alternative for the separation of microalgae and removal of nutrients. The objectives of this work were: to evaluate the nitrogen and phosphorus removal of an anaerobic effluent through the growth of Chlorella sorokiniana microalgae in a flat panel photobioreactor and to investigate the use of ozonation associated with dissolved air flotation (DAF) for the solid-liquid separation of the microalga Chlorella sorokiniana. The anaerobic effluent was obtained from a fixed-bed anaerobic reactor with flexible fibers. The microalgae were grown in a flat panel photobioreactor for five days, under controlled conditions: light intensity (196 mol μm -2 s-1), Airflow (0.2 vvm), temperature (29 ± 1°C) and photoperiod (12 hours). At the end of the cultivation period, tests were carried out to adjust FAD and ozonization through the evaluation of parameters such as: ozone dosage, polymer dosage, pH, fast mixing and flocculation times and gradients, flotation velocity. The anaerobic reactor presented high efficiency of removal of COD and BOD, 63 ± 6% and 53 ± 8%, respectively. At the end of the microalgae culture period in the photobioreactor, the removal of total nitrogen, total phosphorus and total dissolved carbon of the effluent from the anaerobic reactor were 52.1%, 31.8% and 66.2%, respectively. Analyzing the results concerning the separation step, it was verified that the pH did not influence the removal efficiencies, high flotation rates can be employed in the separation process, the flocculation step in the treatment was not necessary and ozonation didnt contributed to an improvement the solid-liquid separation process.

A presente Tese de Doutorado objetivou: (1) definir um método eficiente de transformação genética, por bombardeamento de partículas, para a obtenção de plantas transgênicas de cultivares brasileiras de cevada e (2) identificar gene(s) codificante(s) de quitinase(s) potencialmente capaz(es) de conferir resistência ao fungo patogênico de cevada Bipolaris sorokiniana. Culturas de calos obtidos a partir de escutelos imaturos das cultivares Brasileiras de cevada MN-599 e MN-698 (Cia. de Bebidas das Américas, AMBEV) foram bombardeadas com partículas de tungstênio e avaliadas quanto à expressão do gene repórter gusA através de ensaios histoquímicos de GUS e quanto ao efeito dos bombardeamentos na indução estruturas embriogênicas e regeneração de plantas. As condições de biobalística analisadas incluíram a região promotora regulando a expressão de gusA, tipo e pressão de gás hélio de dois aparelhos de bombardeamento, distância de migração das partículas, número de tiros e a realização de pré e pós-tratamento osmótico dos tecidos-alvo. No presente trabalho foram obtidos um número bastante alto de pontos azuis por calo, a indução de calos embriogênicos e embriões somáticos em uma freqüência de até 58,3% e a regeneração de 60 plantas, sendo 43 de calos bombardeados. As melhores condições observadas foram o promotor e primeiro íntron do gene Adh de milho (plasmídeo pNGI), o aparelho de bombardeamento “ Particle Inflow Gun” (PIG) utilizando-se a distância de migração de partículas de 14,8 cm, dois tiros disparados por placa e a realização de tratamento osmótico dos explantes com 0,2 M de manitol e 0,2 M de sorbitol 4-5 horas antes e 17-19 horas depois dos bombardeamentos. Das 43 plantas obtidas de calos bombardeadas, 3 apresentaram atividade de GUS na base das suas folhas. A utilização de primers sintéticos definidos a partir de genes de quitinases descritos na literatura em PCRs resultou na amplificação de dois fragmentos de aproximadamente 700 e 500 pb a partir de DNA total das cvs. MN-599 e MN-698 de cevada e um fragmento, com aproximadamente 500 pb, a partir do DNA total do isolado A4c de Trichoderma sp. Estes fragmentos foram purificados dos géis de agarose e diretamente seqüenciados de forma manual e automática. Os fragmentos de 700 e 500 pb amplificados do genoma da cultivar MN-599 foram identificados como genes de quitinases de cevada e o fragmento de 500 pb do isolado A4c de Trichoderma sp. não apresentou homologia com seqüências conhecidas de quitinases depositadas no EMBL/GenBank. A utilização de novos pares de primers, representando seqüências conservadas de quitinases do fungo Metarhizium anisopliae, resultou na amplificação de 3 fragmentos a partir do DNA total do isolado A4b de Trichoderma sp., que estão sendo purificados para realização de seqüenciamento.
The present Tesis aimed: (1) to determine an efficient method of genetic transformation by particle bombardment to obtain transgenic plants of Brazilian barley cultivars and (2) to identify a gene coding for chitinase that could confer resistance to barley against the pathogen Bipolaris sorokiniana. Calli cultures derived from immature scutella of Brazilian barley cultivars MN-599 e MN-698 (American Beverage Company, AMBEV) were bombarded with tungsten particles and analyzed for gusA reporter gene expression by GUS histochemical assay, embryogenic structures induction and plant regeneration. Physical and biological biolistic conditions analyzed included two promoter regions regulating the gusA gene, two particle bombardment devices, different helium pressures, distances between target tissues and the initial position of particles, number of shots and use of osmotic pre- and post-treatment of tissues. In the present work there were observed high numbers of blue spots per callus, embryogenic calli and somatic embryos induction with a frequency until 58.3% and the regeneration of 60 plants, being 43 from bombarded calli. The best conditions were obtained with the employment of the Adh promoter and its first intron (pNGI plasmid), Particle Inflow Gun (PIG) device, 14.8 cm of distance, 2 shots per plate and osmotic treatment of tissues with 0.2 M mannitol and 0.2 M sorbitol during 4-5 hours prior to and 17-19 hours after bombardments. Leaves from 3 out of 43 regenerated plants showed positive GUS activity in histochemical assays. The use of primers defined from chitinase genes described in literature resulted in the amplification of two fragments with approximately 700 and 500 bp from genomic DNA of MN-599 e MN-698 barley cultivars and one fragment, with approximately 500 bp, from Trichoderma sp. strain A4c genomic DNA. These fragments were purified from agarose gel and manual and automatically sequenced. Fragments of 700 and 500 bp from cv. MN-599 were identified as chitinase genes of barley and fragment of 500 bp Trichoderma sp. A4c presented no homology with chitinase sequences deposited in the EMBL/GenBank. The use of new primers representing conserved chitinase sequences of Metarhizium anisopliae resulted in the amplification of three fragments from genomic DNA of Trichoderma sp. strain A4b. These fragments should be purified and sequenced

La production céréalière, plus particulièrement le blé et l'orge, subit des pertes importantes de rendement occasionnées par la fonte des semis et les pourritures racinaires. Deux champignons sont majoritairement responsables de ces maladies chez le blé et l'orge : Fusarium graminearum et Bipolaris sorokiniana. L'utilisation des ultrasons comme une alternative « verte » aux fongicides pourrait permettre une culture biologique et minimiser les pertes économiques. L'objectif principal de ce projet consistait à établir les paramètres optimaux (puissance des ultrasons, présence d'éthanol, débit d'oxygène et temps de traitement) afin de réduire la contamination par B. sorokiniana des grains de blé et d'orge sous un seuil de 30 % tout en conservant la germination supérieure au seuil de 85 %. L'ensemble du projet de recherche exploratoire a permis de vérifier l'efficacité de traitements novateurs pour le blé contaminé. Le traitement de ces lots de grains sous forme sèche a révélé un maintien de la germination pour tous les types de traitement (ultrasons 30 W, oxygène en bullage dans l'éthanol et les deux techniques combinées) ainsi qu'une réduction significative du taux de contamination par B. sorokiniana (en moyenne en dessous de 15 %). Pour l'orge, ces traitements semblables ont permis le maintien de la germination ainsi qu'une faible diminution de la contamination par B. sorokiniana. Cette nuance peut être expliquée par la morphologie différente des deux grains. Une réelle avancée dans le domaine du traitement des grains sous forme sèche peut être mise en évidence pour sa rapidité de traitement. Il restera à vérifier l'efficacité du traitement sur d'autres lots et sur des quantités plus importantes pour vérifier la mise à l'échelle du dispositif (supérieure à 100 g). Pour l'avenir, une étude se basant sur le microcalorimétrie sera préconisée pour étudier les prémisses de la germination des grains traités.

La fonte des semis et la pourriture des racines font partie des maladies céréalières des grandes cultures au Québec. Elles sont causées par deux agents pathogènes, le Fusarium graminearum et le Bipolaris sorokiniana contaminant la semence céréalière, en particulier le blé et l’orge, ce qui entraîne à la fois une baisse de la levée et un rendement moins élevé. Des agriculteurs biologiques tentent de trouver un traitement de semences sans fongicide afin de lutter contre ces champignons, qui sont néfastes pour l’agriculture céréalière. Notre projet a comme objectif d’utiliser l’action oxydative de l’oxygène ou l’ozone humidifié pour engendrer un stress oxydatif afin de diminuer l’impact des deux agents pathogènes responsables de cette maladie, le F. graminearum et le B. sorokiniana, tout en préservant le pouvoir germinatif de ces semences. La dose en agent oxydant, le débit des gaz oxydants (ozone et /ou oxygène) ainsi que le temps d’exposition constituent les paramètres clés à optimiser pour ce traitement oxydatif et la cinétique de germination des graines céréalières traitées. Un tel traitement semblait prometteur pour la semence de blé. Il l’était toutefois un peu moins pour la semence d’orge en raison de son enveloppe assez rigide qui a rendu difficile la pénétration de l’ozone et l’oxygène. Pour remédier à ce problème, nous avons fait des tests préliminaires sur l’orge en utilisant la sonication par ultrasons comme prétraitement de l’orge avant le traitement oxydatif. Les résultats obtenus suggèrent qu’un tel traitement est prometteuret non négligeable afin d’optimiser notre traitement oxydatif et de permettre par la suite de réduire les agents responsables de la maladie sans nuire à la qualité des semences de céréales. Ce point a été abordé dans nos travaux par un test préliminaire sur l’orge, et cette approche s’annonce très prometteuse pour nos recherches futures.
Seedling blight and root rot are part of cereal diseases of field crops in Quebec. They are caused by two pathogens, Fusarium graminearum and Bipolaris sorokiniana, which contaminate the seed grain, especially wheat and barley, which causes both decreased, lift and lower performance. Organic farmers are trying to find a seed without fungicide treatment to fight against these fungi, which are harmful to cereal farming. Our project has as goal to use the oxidative action of oxygen or ozone moistened to cause oxidative stress in order to reduce the impact of the two pathogens responsible for this disease, F. graminearum and B. sorokiniana, while preserving the germinability of the seeds. The dose in oxidizing agent, the flow of oxidizing gases (ozone and/or oxygen) and the time of exposure are key parameters to optimize for this oxidative treatment and germination kinetics treated cereal seed. Such treatment looked promising for the seed of wheat. It was however a little less for the seed of barley because it’s fairly rigid envelope which made difficult the penetration of ozone and oxygen. To remedy this problem, we have preliminary tests on barley using sonication ultrasonic as pre-treatment of barley oxidative pre-treatment. The results suggest that, such treatment is promising and significant in order to optimize our oxidative treatment and subsequently, reduce the causative agents of disease without harming the quality of cereal seed. This point has been addressed in our work by a preliminary test on barley, and this approach looks very promising for our future research.

碩士
國立臺灣海洋大學
食品科學系
102
Recently, Chlorella are attracting enormous attention, and the studies about biological activities have been discussed, including antioxidant, anti-diabetes, anti-hypertension, anti-cancer, anti-microbial, lipid metabolic regulation and hepatotective activities. Since phenolic acids have similar bioactivities, this study would analyze the phenolic acids contents of Chlorella sorokiniana, study the optimum conditions of extraction, and evaluate the effect of cell disruption methods. 　　Different factors that influence the phenolic acids extraction procedure such as solvent composition, organic solvent liquid-liquid extraction (LLE) process were optimized. In addition, three extraction systems: conventional orbital shake extraction (CSE), ultrasonic-assisted extraction at room (UAE-RT), and ultrasonic-assisted extraction at degrees Celsius (UAE-60℃) were studied. By comparing the extraction yields and phenlic acids contents, the most efficient method is UAE-60℃ with 70% aqueous methanol solution, ratio of sample to solvent is 1:20 (g/mL). Ultrasonic extract for 30 min, three times. After acidified the extracts, the aquesous solution of the extracts were partitioned with with hexane, ether/ethyl acetate (1/1, v/v) liquid-liquid extraction. After that, alkaline hydrolyzed aquesous layer for 4 hrs, acidified and LLE with hexane, ether/ethyl acetate again. 　　Analysis of Chlorella sorokiniana phenolic acids fractions has been carried out by high-performance liquid chromatography (HPLC). Diverse phenolic acids, namely chlorogenic acid, caffeic acid, p-comaric acid, and ferulic acid were detected in the analysed extract. The analysis conditions used Inertstil C4 column (5 um, 4.6 #westeur024# 250 mm), modified the ratio of mobile phase to methanol/water = 32/68 (v/v) with phosphoric acid 0.04% (v/v, pH 2.6), and injected 20 L, at room temperature, and detected with 280, 310, 320 nm. Retention times are 8.65 min (chlorogenic acid), 12.94 min (caffeic aci), 23.33 min (p-comaric acid) and 26.74 min (ferulic acid). 　　To investigate the relation between phenolic acids contents and cell disruption methods, Chlorella sorokiniana divided into three groups, unpulverized, physical pulverized and enzymatic hydrolyzed. The statistical results showed that different cell disruption treatments would affect phenolic acids contents. Enzymatic hydrolysis group contains highest phenolic acids contents: chlorogenic acid 1213.67 ± 8.38 (ug/g), caffeic acid 1118.95 ± 6.73 (ug/g), p-coumaric acid 86.79 ± 5.43 (ug/g) and ferulic acid 42.10 ± 0.90 (ug/g), that would be the optimum cell disruption method.

碩士
國立中興大學
基因體暨生物資訊學研究所
99
The heat-talent Chlorella sorokiniana T89 was cloned from Chiayi paddy field. The genomic DNA and cDNA annotated data was unpublished so far. The lipid content of C. sorokiniana T89 was up to 30% and rich in unsaturated fatty acids which were suitable as biodiesel and functional foods. Culturs of C. sorokiniana T89 at different growth stages were used for RNA extracted and converted into cDNA for future sequencing analysis. Then get the contigs sequence data by Illumina sequencing system. Contigs were predicted their function via sequence alignment by using BLAST tools from NCBI and KEGG to find gene functions. Among the annotated genes with differential expression, the superoxide dismutase (SOD) which plays an importante role at antioxidant pathways was selected for further cloning, expression and biochemical characterization. In the future, we can use the annotation database to study the genes involved in physiological pathway of C. sorokiniana T89 andto developict as a good resource of biodiesel.

碩士
國立中興大學
食品暨應用生物科技學系所
100
Glutaredoxins (Grxs) are ubiquitous oxidoreductases in aerobic organism. They catalyze glutathione-dependent reactions to reduce protein disulfide linkage caused by oxidative stress. A full length cDNA encoding a glutaredoxin gene with 321 nucleotides was isolated from Chlorella sorokiniana T89. Nucleotides sequence analysis showed that it deduced 106 amino acid residues, the molecular weight of protein is predicted approximately 11.6 kDa. The deduced amino acid sequence is conserved among the reported dithiol Grxs because the characteristic catalytic motif C25-P-Y-C28. Recombinant glutaredoxin was subcloned into an vector pET-20b(+) and expressed in Escherichia coli BL21(DE3) as a C-terminal histidine-tagged fusion protein. The expression of glutaredoxin was purified by TALON affinity chromatography and analyzed by 16 % tricine SDS-PAGE. glutaredoxin activity was assayed by β-hydroxyethyl disulfide (HED) assay. The optimal pH for the recombinant glutaredoxin was 8.5, and the optimal temperature was 50 C. The recombinant glutaredoxin exhibition a good thermal stability and pH stability (pH 3~11). The Michaelis constant (Km) values for HED was 0.17 mM. The enzyme activity was significantly inhibited by 1 mM Cu2+, Zn2+, and Fe3+. The enzyme was susceptible inactivation in iodoacetamide, SDS, and urea.

碩士
國立中興大學
基因體暨生物資訊學研究所
99
A cDNA encoding a cinnamyl alcohol dehydrogenase (CAD) gene with 1155 mucleotides was isolated from Chlorella sorokiniana T89. Recombinant CAD protein was expressed in Escherichia coli as a C-terminal histidin-tagged fusion protein and purified by DEAE-Sephacel ion exchange chromatography and TALON affinity chromatography. By these steps, the CAD was purified 1250 fold over the crude protein, and the total recovery was 3%. The optimal pH for the recombinant CAD protein was 6.5, and the optimal temperature was 33 °C. 1 mM Co2+, 1 mM Ni2+ and 1 mM Cu2+ significantly inhibited the activity of the enzyme. 1% 2-Mercaptoethanol, 1 mM dithiothreitol, 1 mM ethylenediaminetetraacetic acid, 0.1% sodium dodecyl sulfateand 2 M urea significantly inhibited the activity of th enzyme. The recombinant CAD protein sequence analysis and prediction three-dimensional structure. There were some features of recombinant CAD protein. First, it may be combination of two zinc ions which related to the structural zinc ion and catalytic zinc ion. Second, the [GX(X)GXXG] motif may be the NADP+-binding domain. Finally, two pairs of disulfide bonds may form between C131 and C134, C75 and C191, respectively.

碩士
國立中興大學
生命科學院碩士在職專班
103
Chlorella sorokiniana (CS) is a single-cell green algae, green algae rich in nutrients, such as protein, amino acids, antioxidant enzymes and minerals. In addition,Chlorella sorokiniana can also cause a variety of beneficial pharmacological effects.Therefore this study is focused on evaluating the ability Chlorella sorokiniana extract in improving chemotherapy supressed bone marrow function. One day after Taxol treatment, laboratory mice had been given hot water extracted Chlorella every day within following 7 days(Based on subject weight, group A given 17mg/kg, group B given 34mg/kg), laboratory mice had been given hot water extracted Chlorella sorokiniana every day within three weeks (Based on its weight,. 50mg/kg). Compare with mice which received only Taxol treatment, white blood cell counts in peripheral blood which sampled 7 (Group A) and 3 (Group B) days after additional Chlorella extract treatment don''t show significant difference. On the other hand, neutrophil level for both groups had been significantly increased. In vitro assays showed that Chlorella sorokiniana enhanced the colony-forming ability of both granulocyte macrophage colony forming unit (GM-CFU) and osteogenic cells from bone marrow preparations and promoted the differentiation of bone marrow mesenchymal stromal cells into adipocytes, alkaline phosphatase–positive osteoblasts, and bone tissue.his result could be attributed to enhanced expression of Cbfa1 (core binding factor a) and BMP-2 (bone morphogenetic protein) with concurrent suppression of ODF (osteoclast differentiation factor/RANK [receptor activator of NF-κB]) ligand. In summary, Based on results of this experinment, Chlorella extract not only affects bone marrow and osteoblast differentiation but also enhances recovery of mice from Taxol treatment induced leukopenia.

碩士
國立臺灣海洋大學
食品科學系
98
This study investigated the improvement of adding flavor modifying substances on the flavor perception of green algae (Chlorella sorokiniana), a kind of health food, because the algae possess a unique aroma that may be unacceptable for some consumers. In the experiments, flavor improvements were evaluated by sensory testes and volatiles were analyzed by electronic nose, GC and GC-MS. A difference-from-control test was used using seven trained panelists to score the differences in unique aroma intensity between control (4% green alga in water) and test samples, in which flavor additives were added. Finally, the lowest significant concentrations of each flavor additives were determined. Of which, several substances including -cyclodextrin, maltosyl trehalose, lactic acid, ethyl maltol, alanine, MSG and IMP/GMP having better effects, were chosen and tested continuously by mixing. Results showed that the lowest significant concentrations thus obtained became much lower than that added alone, with a reduction of 50%~99%. According to an acceptance test using 9-point hedonic scale, results showed that the score of adding ethyl maltol/maltosyl trehalose or alanine/MSG/IMP/GMP mix was significantly higher (P＜0.05) than that of control. On contrast, no significant difference was observed between the sample containing 3.52 mM of -cyclodextrin and control. From the analyses of electronic nose equipped with 12 kinds of sensors, it is found that the detection values obtained from 3 sensors were associated well to the difference in volatiles between 2~6% green algae samples. Based on principal component analysis, it is clear that except for maltosyl trehalose, the adding of flavor additives at its lowest significant concentrations resulted in a decrease in detection values as compared to control. Adding higher amounts of -cyclodextrin also revealed a continuous decrease in detection values. Solid phase micro-extraction was applied to adsorb volatiles of green algae test sample before GC and GC-MS analyses. The peak areas of most volatiles reduced markedly after adding -cyclodextrin. Among volatiles, that had been identified were as follows: heptanal, -cyclocitral, geranylacetone, -ionone, dihydroactinidiolide, heptadecene. But geosmin and 2-methylisoborneol were not detected.

碩士
國立中興大學
食品暨應用生物科技學系所
101
The gene of glutaredoxin (Grx) from Chlorella sorokiniana T89 (CsT89) has been cloned and overexpressed successfully in E. coli. The recombinant protein was biochemically characterized. In order to realize the structural and functional relation, in this study, based on the glutathione (GSH) binding sites and conserved residues of glutaredoxin, 12 single point mutations were designed from CsT89 Grx. Twelve mutations were designed as the following rCsT89 GrxK22R, rCsT89 GrxIQ5859TK, rCsT89 GrxQ59K, rCsT89 GrxQ59N, rCsT89 Grx68PV, rCsT89 GrxS70T, rCsT89 GrxG84A, rCsT89 GrxG84N, rCsT89 GrxG84T, rCsT89 GrxG84Y, rCsT89 GrxD85E and rCsT89 GrxD85T. The protein expressions of recombinant mutant glutaredoxins were induced by the addition of isopropyl β-D-1-thiogalactopyranoside (IPTG) in E. coli BL21(DE3). Target recombinant soluble proteins were purified by TALONR metal affinity resin and analyzed by 16% tricine SDS-PAGE. The activities of recombinant mutant glutaredoxins were determined by the HED (β-hydroxyethyl disulfide) assay. The optimal reaction pH and temperature of recombinant mutant glutaredoxins were pH 8.5-9 and 40-55°C, respectively. Compare with rCsT89 Grx activity, CsT89 GrxG84A and rCsT89 GrxD85E activities were slightly higher; rCsT89 GrxK22R, rCsT89 GrxQ59K, rCsT89 GrxG84Yand rCsT89 GrxD85T activities were less than 50% of CsT89 Grx activity; activities of rCsT89 GrxQ59N and rCsT89 GrxS70T were only remaining 20% of rCsT89 Grx. Analyze the kinetic studies of recombinant mutant glutaredoxins, both kcat and Km of rCsT89 GrxQ59K, rCsT89 GrxG84A and rCsT89 GrxD85E have elevated. However, kcat of rCsT89 GrxD85T has declined and Km has increased. Furthermore, rCsT89 Grx was mutated to rCsT89 GrxK22R and rCsT89 GrxG84Y caused the low stability of recombinant proteins and effected the reproducibility of experiments.

碩士
靜宜大學
食品營養學系
100
It is known that the biopolymers (mainly glycoproteins) in hot water extracts of Chlorella sp. show in vitro and in vivo immunostimulatory activities. The immunostimulatory activities may be influenced by coexisting starch in the extract and processing conditions during extraction or purification of the active glycoproteins involved. Therefore, the aim of this study was to investigate the effects of de-starching treatment, thermal treatment, and the variety and concentration of stabilizing agents on the surface hydrophobicity and in vitro immunostimulatory activity in murine macrophage RAW 264.7 model of Chlorella glycoproteins. The samples studied included the macromolecular fraction WS-F1 and its destarched fraction WS-ds-F1 (both molecular weights  10 kDa). The WS-F1 was prepared by extraction with water at 90oC for 1 hr on Chlorella sorokiniana powder, following by ultrafitration with a membrane of molecular-weight cut-off 10 kDa. The WS-ds-F1 was yielded as the above procedure but with an additional degradation with thermally resistant -amylase during extraction. The resultant WS-F1 yielded 8.5% (w/w) and contained a total protein content of 55% (w/w) and sugar composition of mainly glucose (58 mol% based on detectable carbohydrates), followed by ribose (18 mol%) and galactose (14 mol%). In contrast to the WS-F1, the destarched WS-ds-F1 showed a similar yield and total protein content, sugar composition of a higher molar percentage in glucose and galactose and less in ribose, and a greater in vitro immumostimulatory activity. The Ws-ds-F1 was accordingly used for the stabilizing formulation study. The stabilizers employed included two sugars (trehalose and mannitol; WS-ds-F1 protein: sugar ratio = 1: 0.5  1: 6 (w/w)) and eight salts (NH4Cl, NaCl, KCl, CaCl2, Na2SO4, KH2PO4, NaH2PO4 and Na2HPO4; 0.011 M). The ANS fluorescence probe was applied to detect the surface hydrophobicity change of proteins in stabilized WS-ds-F1 formulation solutions. The results illustrated that, among the stabilized formulations, the formulations with 0.5-fold weight of trehalose (i.e. WS-ds-F1 protein: trehalose = 1:0.5 (w/w)), 1-fold weight of mannitol (i.e. WS-ds-F1 protein: mannitol = 1:1 (w/w)), 10 mM NaCl, and 10 mM Na2SO4 exhibited a significantly lower ANS characteristic fluorescence (at an emission wavelength of 480 nm) than the control without stabilizers, showing the best stabilizing effect for WS-ds-F1 protein. The immunostimulatory activities of these four formulations in RAW 264.7 cell model indicated that most of samples with Chlorella glycoproteins at low concentrations displayed promoting effects on RAW 264.7 viability, especially the mannitol-WS-ds-F1 formulation at 5 g/mL that increased effectively the cell viability to 233%. All samples containing Chlorella glycoproteins stimulated a significant amount of NO secreted from RAW 264.7 cells. The effectiveness of stimulating NO secretion followed the order of: stabilized WS-ds-F1 formulations > WS-F1 and WS-ds-F1 alone > related sugars. The trehalose-WS-ds-F1 formulation showed the highest effect on increasing NO level (e.g. 3.44 M NO at 100 g/mL formuation). At 10100 g/mL, WS-F1, WS-ds-F1, and its stabilized formulations all promoted effectively the cytokines TNF- and IL-6 secreted from RAW 264.7 cells generally in a dose dependence. All WS-ds-F1 samples at 50100 g/mL caused a similar TNF- level but significantly higher IL-6 level than those did by 1 g/mL lipopolysaccharide. Conclusively, the in vitro immunostimulatory activity in RAW 264.7 of WS-ds-F1 was greater than that of WS-F1. Among the stabilizers examined for WS-ds-F1, trehalose, mannitol, NaCl, and Na2SO4 were the better stabilizers with negligible effects on the immunostimulatory activity of WS-ds-F1.

碩士
國立屏東科技大學
生物科技系所
105
Spirulina platensis and Chlorella sorokiniana belong to microalgae, both of them contain abundant nutrients, especially protein. Their protein contents are about four times higher than that of hen egg . The health effects, such as antioxidant, anti-inflammatory, anti-cancer and liver protection, caused by Spirulina platensis (SP) and Chlorella sorokiniana (CS) proteins have been widely reported. However, those studies only focused on proteins’ activities demonstrated using in vitro models. They did not explore the compatibility and absorption of these proteins with gastrointestinal digestion enzyme after oral administration. In the point of view of health food, small peptide can be readily absorbed which allows more industrial applications. In this study, proteins from SP and CS were extracted and hydrolyzed by various enzymes. The peptides with anti-inflammatory efficacy were screened by means of bioassay-guided fractionation. The results showed that the thermolysin hydrolysate of SP andCS proteins had better effect on the inhibition of nitric oxide (NO) production in LPS-stimulated RAW cells. Meanwhile, the expression of iNOS and COX-2 were simultaneously decreased dose-dependently in the presence of thermolysin hydrolysate of SP and CS (50 µg/µl to 400 µg/µl). This result indicated that the hydrolysate from the both microalgae has a preliminary anti-inflammatory, effect, and they are potential for the development of health food products or as anti-inflammatory drugs.

碩士
靜宜大學
食品營養學系
99
Chlorella sp. is known to have many kinds of healthy functionality, partly relating to their potential inhibition activity against glycosidase enzymes. This study was to investigate the major chemical compositions, antioxidant activities (DPPH and ABTS free radicals), and inhibitory activities against glycosidase (alpha-glucoside, beta-glucoside and beta-glucuronidase) of various solvent extracts and fractions from Chlorella sorokiniana, in order to develop isolation processes for bioactive Chlorella fractions of high glucosidase inhibitory activities. Five solvents (n-hexane, ethyl acetate, acetone, ethanol and water) were applied in single solvent or mixture. The chemical composition results showed that 50% ethanol (50ES) and water soluble (WS) extracts of Chlorella sorokiniana contain a higher content in protein, total sugar and uronic acid (2560%, 2030% and 26%, respectively), in contrast to the other extracts. The monosaccharide composition in both 50ES and WS is mainly glucose (>50 mol %), followed by galactose, or ribose. Differently, the n-hexane (HS), ethyl acetate (EaS) and acetone (AS) extracts contained a higher content in total phenolics (51-53 mg/g) and chlorophylls. In the case of antioxidant activities, AS and mHS showed the highest DPPH-scavenging effects among the samples examined (80% and 85%, respectively, at 2 mg/mL extract). While, all 50% ethanol extracts (50ES, 95EI-50ES, 95EI-mHWI-50ES, mHWI-50ES, mHEaI-95EI-50ES, 25WI-50ES and 90WI-50ES) displayed good ABTS-radical scavenging effects (~90% at 2 mg/mL). For glycosidase inhibitory activities, the mWS exhibited a better alpha-glucosidase-inhibition rate than the other extracts (66.38 ± 0.66% at 10mg/mL). The 95EI-mWS and mHEaI-95EI-50EI-90WS extracts revealed the highest beta-glucosidase inhibition rate (91-92% at 10mg/mL). However, the beta-glucuronidase inhibition rate was the highest for 95EI-mHS extracts (89% at 10mg/mL). Enzymatic kinetics showed that the glycosidase inhibition mechanism of the fractions(mWS, 95EI-mWS and mHEaI-95EI-50EI-90WS) involved partially noncompetitive inhibition, and the fractions(95EI-mHS) involved mixed-type noncompetitive inhibition. In conclusion, the extract of high alpha-glucosidase inhibition activity could be better prepared from the WS fraction of mixed n-hexane-water extracts. Those of high beta-glucosidase inhibitory activity could be prepared from the water subfraction in n-hexane-water extracts on the 95% ethanol insoluble materials of Chlorella. And, that of high beta-glucuronidase inhibition activity was the best could be prepared from the n-hexane subfraction in n-hexane-water extracts on the 95% ethanol insoluble materials of Chlorella.