Düsseldorf, Universiẗat, Diss., 2003.

Submitted in fulfilment of the requirements of the Degree of Master of Technology: Biotechnology, Durban University of Technology, 2011. / Eremothecium gossypii has the ability to utilize vegetable oils as a carbon source to produce riboflavin. This organism has been known to produce as much as 40 000 times more riboflavin than it requires after genetic modification on simple sugars. Adaptation of this organism to various oil substrates for riboflavin production has been poorly investigated. The aim of this research was thus to investigate the production of riboflavin by Eremothecium gossypii, on various oils and to improve production by mutating the organism and optimising media components using Design of Experiments (DOE). Nine overproducing mutants were obtained after mutating with various concentrations of ethylmethane sulphonate (EMS), n-methyl-n‟-nitro-n-nitrosoguanidine (MNNG) and Ultraviolet light. Riboflavin overproducing mutants were screened on an itaconate-containing medium; the colonies appeared yellow instead of white in the case of the wild-type. The itaconate screening medium isolated mutants with an isocitrate lyase that was insensitive to feedback inhibition. Mutations performed using EMS increased the ability of E. gossypii to produce riboflavin by 611% (7-fold) compared to the wild-type. This was achieved with soybean oil as a carbon source and was better than the other five oils used. Using DOE, fractional factorial experiments were carried out to optimise media components for riboflavin production on soybean oil. The total riboflavin produced by E. gossypii mutant EMS30/1 increased from 59.30 mg l-1 on a standard O&K medium using soybean oil as a carbon source to 100.03 mg l-1 on a DOE improved O&K medium, a 69% increase. The final optimised growth medium was determined from a central composite design using response surface plots together with a mathematical point-prediction tool and consisted of 5.0 g l-1 peptone, 5.0 g l-1 malt extract, 5.1 g l-1 yeast extract, 0.64 g l-1 K2HPO4, 0.6 g l-1 MgSO4 and 20 g l-1 soybean oil. Fractional factorial and central composite media optimization designs increased riboflavin production by several fold over their iterations. There was an overall increase of 1099% (12-fold) in riboflavin production by the mutant grown in an optimized medium compared to the initial riboflavin produced by the wild-type.

Zugl.: Bonn., Univ., Diss., 2007

Hohenheim, Univ., Diss., 2004.

University, Diss., 2002--Bonn.

Thesis (Ph. D.)--Duke University, 2007. / Includes bibliographical references.

Submitted by (lucia.rodrigues@ufrpe.br) on 2017-03-23T16:00:00Z No. of bitstreams: 1 Waleria Guerreiro Lima.pdf: 1037853 bytes, checksum: 76b8dc4974744a728c0fae8cae99709c (MD5) / Made available in DSpace on 2017-03-23T16:00:00Z (GMT). No. of bitstreams: 1 Waleria Guerreiro Lima.pdf: 1037853 bytes, checksum: 76b8dc4974744a728c0fae8cae99709c (MD5) Previous issue date: 2007-02-27 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / Colletotrichum gossypii South. var. cephalosporioides Costa is the causal agent of one of the most important cotton diseases, the ramulosis. The disease is characterized by necrotic lesions on leaves and petioles mainly on young leaves. As the disease progresses, shortening of the internódios, apical death and overbudding are also observed. The main way of fungal dissemination is through the seeds, but also able to be externally propagated, in the form of conidia, or internally, by dormant mycelia. The pathogen control has been achieved through the use of healthy seeds, use of resistant cultivars and chemical control. However, some of these control practices not only of limited efficiency, but also cause unwanted side in natural environments. The use of the biological activity of secondary compounds obtained from plant crude extracts and their essential oils can be an effective way to control plant diseases. Preliminary tests toevaluate the fungistatic and fungicidal effect of these products are essential for revealing new alternatives of control. The effect of different oil dosages (0, 500, 1000, 1500, 2000 and 2500 ppm) prepared from leaves of Hibiscus cannabinus, Cymbopogus nardus, Eucalptus citriodora, Pipper marginatum, Malpighia glabra, and Lippia gracillis were evaluated in the inhibition of C. gossypii var. cephalosporioides mycelia growth, conidia germination, and apressorium formation. The oils of Pipper marginatum, Lippia gracillis and Cymbopogus nardus presented the greatest mycelia growth inhibition effects. The oils of citronela, lípia, and eucalipto showed significant control when conidial germination and apressorium formation were analyzed. When the mycelia growth inhibition was evaluated, piper oil presented lower DL50, followed by the lípia oil. The best results in the inhibition of apressorium formation were presented bycitronela and eucalipto with lower DL50 in relation to other oils. The kenaf oil did notpresent any effect on the micelial growth, conidial germination and apressorium formation. In a second experiment it was evaluated the curative and preventive effect of citronela oil on the control of the cotton ramulosis based on epidemiological components. The Area Below the Curve of Disease Progress and Rate of Disease Progress, showed significant differences when compared with the control treatment. However, the parameter Index of Disease Final in the curative treatment did not differ significantly from control treatment. When the variables Area Below the Curve of Disease Progress, Index of Disease Final, and Rate of Disease Progress were analyzed, it was observed that the preventive treatments that used citronela oil and fungicide differed significantly from the control, showing that citronela oil had an excellent preventive effect on the cotton ramulosis under greenhouse conditions. / Colletotrichum gossypii South. var. cephalosporioides Costa é o agente causal de uma das mais importantes doenças do algodoeiro. Caracteriza-se pelo surgimento de manchas necróticas nas folhas e pecíolos, com predominância nas folhas mais jovens. Ao longo do tempo, também são observados encurtamento dos internódios, morte do broto apical e superbrotamento. A principal via de disseminação do fungo é a semente, podendo ser veiculado externamente, na forma de conídios, ou internamente, na forma de micélio dormente. O controle do patógeno tem sido feito através do uso de sementes sadias, cultivares resistentes e controle químico. Entretanto, em função da ineficácia desses métodos de controle e dos malefícios provocados pelo uso dos pesticidas na natureza, a exploração da atividade biológica de compostos secundários, presentes no extrato bruto ou óleos essenciais de plantas, pode constituir uma forma efetiva de controle de doenças em plantas cultivadas. Testes preliminares para avaliar o efeitofungistático e fungicida desses produtos são imprescindíveis para descobertas de novas alternativas de controle. Foram avaliados os efeitos de diferentes dosagens (0, 500, 1000, 1500, 2000 e 2500 ppm) dos óleos essenciais extraídos de folhas de kenaf (Hibiscus cannabinus), citronela (Cymbopogus nardus), eucalipto (Eucalptus citriodora), Pipper marginatum, acerola (Malpighia glabra), e lípia (Lippia gracillis) na inibição do crescimento micelial, germinação dos conídios e formação do apressório de C. gossypii var. cephalosporioides. Os óleos de piper, lípia e citronela foram os que apresentaram maior efeito na inibição do crescimento do fungo que o de acerola. Os óleos de eucalipto e acerola tiveram comportamento intermediário e semelhantes de inibição de crescimento, apesar do óleo de eucalipto ter apresentado um melhor controle do fungo. Em relação à germinação dos conídios e formação de apressórios, os óleos de citronela, lípia e eucalipto ocasionaram um significativo controle do fungo. Verificou-seque, para a inibição do crescimento micelial, o óleo de piper foi o que apresentou a menor DL50, seguido pelo óleo de lípia. Para a formação de apressórios, verificou-se que os valores das DL50 dos óleos de citronela, eucalipto foram os menores em relação aos outros óleos. O óleo de kenaf não apresentou nenhum efeito no crescimento micelial, germinação dos conídios e formação de apressórios. Em um segundo experimento, objetivou-se avaliar o efeito curativo e preventivo do óleo de citronela para controle da ramulose do algodoeiro com base em componentes epidemiológicos, contribuindo assim para redução dos níveis de poluição ambiental decorrentes do uso de agrotóxicos. No que diz respeito à Área Abaixo da Curva de Progresso das Doença (AACPD) e Taxa de Progresso da Doença (TPD), houve diferença significativa quando comparada ao tratamento testemunha. Entretanto, ao avaliar os parâmetros índice de Doença Final (IDF) sob o efeito do tratamento curativo, observa-se que o mesmo não diferiu significativamente da testemunha. Ao analisar as variáveis AACPD, IDF, PI e TPD, observou-se que os tratamentos preventivo do óleo de citronela e fungicida diferiram significativamente da testemunha, verificando assim, que o óleo de citronela apresentou um ótimo efeito preventivo da ramulose do algodoeiro sob condições de casa de vegetação.

Spore formation is an essential step in the fungal life cycle that contributes to the dispersal of the organism and also to survival under harsh environmental conditions. The morphology of spores shows an astonishing diversity in the fungal kingdom and varies from very simple round and small spores to very complex multi-armed or sigmoid structures. With exception of the regulation of ascospore formation in Saccharomyces cerevisiae and Schizosaccharomyces pombe, which are well-characterized model organisms for spore development in fungi, little is currently known about the regulation of more complex spore morphologies. In this study, the filamentous ascomycete Ashbya gossypii is used as a model system for the investigation of a complex and composite spore morphology. A. gossypii produces linear, needle-shaped spores possessing a length of 30 µm, which can be divided into three major segments: a rigid tip segment, a more fragile membrane compartment and a stable tail-cap. Furthermore, the different compartments were shown to correlate with distinct materials. While the tip segment and the tail-cap of the spores consist of stabilizing materials like chitin and chitosan, these materials are absent from the compartment in the middle. The actin cytoskeleton plays an essential role in several steps of spore formation in A. gossypii. Different regions of actin accumulation were identified that directly correlate with the developing spores. Especially the developing tip segment is characterized by heavy-bundled linear actin structures. Furthermore, proteins of the formin family, a class of actin organizing proteins, were identified to be directly involved in spore formation in A. gossypii. The formin AgBnr2 fulfills an actin-related key function during spore development by linking actin to the spindle pole body during sporulation. Downregulation of AgBNR2 leads to severe sporulation defects, indicating a central function in spore development. Moreover, AgBni1, another representative of the formin family, also has a regulatory function in size determination of the typical needle-shaped spores of A. gossypii. Using a modified yeast two-hybrid approach, four potential activators of the formin AgBni1 were identified: the Rho-type GTPases AgRho1a, AgRho1b, AgRho3 and AgRho4. The interaction of AgBni1 with the two Rho1 GTPases plays an important role during spore development. In this study, the Rho binding domain of AgBni1 was further examined to identify amino acids that are essential for the interaction with the Rho-type GTPases. Using random mutagenesis combined with a two-hybrid screen, the point mutation S250P in the Rho binding domain of AgBni1 was identified to reduce the interaction of the formin with the Rho1 GTPases. Integration of AgBni1 S250P causes an increase in spore length, suggesting a direct effect of this signaling pathway in spore length determination. An actin-regulating protein network that includes the formin AgBni1, the Rho-type GTPases AgRho1a and AgRho1b and the paxillin-like protein AgPxl1 was identified to be mainly involved in the regulation of the spore length. Thereby, this network seems to be involved in the arrangement of the different spore compartments via the actin cytoskeleton.

In schnell wachsenden Hyphen des filamentösen Pilzes Ashbya gossypii ist die Oberflächenvergrößerung bis zu 40-fach höher, als in den Knospen des nah verwandten Pilzes Saccharomyces cerevisiae. Um die Wachstumszonen auf die Hyphenspitzen zu begrenzen, müssen Polaritätsfaktoren wie Rezeptoren und Sensoren, sowie überschüssiges Membranmaterial in subapikalen Bereichen von der Zelloberfläche entfernt werden. Dies wird durch den Prozess der Endozytose erreicht. In S. cerevisiae ist der Hauptendozytoseweg die Clathrin- und Aktin-abhängige Endozytose und der Prozess ist bereits gut charakterisiert. A. gossypii besitzt Homologe zu fast allen Komponenten dieser endozytischen Maschinerie und ist daher besonders gut geeignet die Anpassung des endozytischen Prozesses an schnelles, polares Wachstum zu untersuchen. Um die Endozytose während des polaren Hyphenwachstums zu analysieren, wurden neun homologe Proteine des aus S. cerevisiae bekannten Endozytosemechanismus mittels „live cell imaging“ und TIRF-Mikroskopie sowohl in langsam, als auch in schnell wachsenden Hyphen untersucht. Hierbei zeigte sich, dass die Endozytoserate in den schnell wachsenden Hyphen in A. gossypii im Vergleich zu Hefe-Zellen deutlich erhöht ist. Dies wird sowohl durch die Beschleunigung des endozytischen Prozesses, als auch durch eine erhöhte Anzahl an endozytischen Ereignissen pro µm2 Zelloberfläche erreicht. Die fluoreszenzmikroskopischen Analysen zeigten zudem, dass sich die Endozytosezone bei hoher Wachstumsgeschwindigkeit um ca. 3 µm in den hinteren Hyphenbereich verlagert. Ein wesentlicher Unterschied des endozytischen Prozesses in A. gossypii im Vergleich zu S. cerevisiae ist die Funktion von Clathrin. Clathrin kolokalisierte mit keiner der getesteten endozytischen Komponenten und konnte ausschließlich an zellinternen Strukturen detektiert werden. Dies deutet darauf hin, dass Clathrin bei der Endozytose in A. gossypii keine Rolle spielt und seine Funktion auf interne Kompartimente wie die Endosomen oder das Golgi-Netzwerk beschränkt ist. Die Unterschiede in der Clathrin-Funktion zwischen S. cerevisiae und A. gossypii hängen vermutlich mit einer minimalen Abweichung im Genset endozytischer Komponenten in A. gossypii zusammen. So besitzt A. gossypii kein Homologes zu ScSla2, welches in Hefe sowohl mit Clc1, als auch mit dem Aktin-Zytoskelett interagiert. Der Sequenzvergleich der Clc1-Proteine aus S. cerevisiae und A. gossypii zeigt, dass in AgClc1 die Sla2-Bindedomäne fehlt. Mittels eines Komplementationstests konnte nachgewiesen werden, dass die Fusion dieser Bindedomäne an das AgCLC1-Gen ausreicht, um die endozytische Funktion von Clathrin in S. cerevisiae wieder herzustellen. In S. cerevisiae führt die Interaktion von Sla2 und Clc1 zu einer verminderten Aktin-Anlagerung an das entstehende Vesikel und dient als Regulationsmechanismus für die Membraneinstülpung. Das Fehlen dieses Mechanismus könnte in A. gossypii die Membraneinstülpung durch vermehrte Aktin-Anlagerung beschleunigen und auf diese Weise zur Anpassung an das schnelle Hyphenwachstum beitragen.

O cultivo de plantas oleaginosas é uma das principais atividades de subsistência para pequenos agricultores na Paraíba. Porém estes cultivos são afetados por pulgões. Alternativas agroecológicas têm sido consideradas como uma estratégia eficiente no controle de insetos sugadores. Este estudo tem o objetivo de relatar a influência do consórcio de erva-doce (Foeniculum vulgare) e algodão (Gossypium hirsutum) com fibra colorida nas populações de Hyadaphis foeniculi (Passerini, 1860) (Hemiptera: Aphididae) e Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) e do principal inimigo natural Cycloneda sanguinea (Linnaeus, 1763) (Coleoptera: Coccinelidae) ao longo do tempo. Os experimentos foram conduzidos nos anos agrícolas de 2009, 2010 e 2011 na Área Experimental da Embrapa - Algodão localizado no município de Lagoa Seca - PB, Brasil. Utilizou-se o delineamento experimental de blocos ao acaso com três tratamentos: cultivo de erva-doce solteiro, algodão solteiro e erva-doce consorciada com algodão, distribuído em quatro repetições. O levantamento populacional dos pulgões e seus respectivos inimigos naturais na área experimental ocorreu em intervalos semanais do início até o fim do ciclo produtivo da erva-doce e do algodão com fibra colorida em cada ano de estudo. A dinâmica e distribuição dos insetos tanto no algodão quanto na erva-doce em ambos os sistemas de cultivo foram analisados através da ANOVA e as médias foram comparadas pelo teste de Student-Newman-Keuls (P = 0,05). A análise de associação interespecífica ao longo dos três anos de estudo foi baseada na presença e ausência de pulgões e seus inimigos naturais nos dois tipos de cultivos. Foram utilizados modelos matemáticos de equações de diferença para explicar o comportamento dos pulgões e do seu predador. A taxa de crescimento da presa (K1), qualidade da planta de algodão (Qa) e qualidade da planta de erva-doce (Qe), taxa de chegada da praga na planta, resposta numérica do predador foram inclusos nos modelos e analisados em diagrama de bifurcação. Tanto no algodão quanto na erva-doce solteira foi encontrado mais pulgões ápteros e alados do que no sistema consorciado. Independentemente do sistema de cultivo, as regiões da planta de algodão com maior concentração de A. gossypii foram a apical e a mediana, enquanto que a região da planta de erva-doce com maior número de H. foeniculi áptero e alado foi a apical. A produção de picos populacionais de A. gossypii e H. foeniculi se deu em idade distinta das plantas solteiras ou consorciadas. A. gossypii e H. foeniculi na maioria dos casos não coocorreram no consórcio. A movimentação do predador C. sanguinea foi geralmente associada ao sistema de cultivo consorciado e isto se deve provavelmente em função de A. gossypii e H. foeniculi áptero ou alado que ocorreram em épocas distintas ao longo do ano. A qualidade da planta, tanto no sistema de cultivo de algodão ou erva-doce solteiro quanto em algodão ou erva-doce no sistema de cultivo consorciado, mudou o comportamento dos insetos com K1 superior a 1,4. Portanto, o consórcio de algodão com erva-doce pode ser ferramenta importante para o manejo integrado de pulgões. / The cultivation of oilseeds is considered as major activity of small farmers in Paraiba State. However, these crops are affected by aphids. Agroecology alternatives have been considered as an efficient strategy in sucking insects. This study has the objective of reporting the influence of colored fiber cotton (Gossypium hirsutum) intercropped with fennel (Foeniculum vulgare) on populations of Hyadaphis foeniculi (Passerini, 1860) (Hemiptera: Aphididae) and Aphis gossypii Glover, 1877 (Hemiptera: Aphididae) and their natural enemy Cycloneda sanguinea (Linnaeus, 1763) (Coleoptera: Coccinelidae) over the time. The study was conducted on 2009, 2010, and 2011 seasons in Experimental Station of the Embrapa - Cotton, placed in the municipality of Lagoa Seca, Paraiba, Brazil. A randomized block design was used with three treatments: sole fennel, sole cotton, and colored fiber cotton intercropped with fennel, distributed in four replications. The population study of aphids and their natural enemy was carried out weekly within of each season. The dynamic and distribution of insects in sole cotton, sole fennel, or in cotton-fennel intercropping systems were analyzed by the ANOVA and the means were compared by the Student-Newman-Keuls (P = 0.05) test. Interspecific association was used to analyze the dynamic herbivore-plant and predator-prey over the three years study, based in aphid and their natural enemy presence or absence in sole crop or intercropping systems. Difference equations have been used to explain the behavior of aphids and their predator. Growth rate of pest (K1), quality of cotton plant (Qa) and fennel plant (Qe), pest immigration rate and the numerical response of predator were included in the model and showed in bifurcation diagrams. Both sole cotton and sole fennel hosted more aphids than cotton-fennel intercropping system. Independently of the crop system, in the cotton plants, A. gossypii populations were highest in the apical and middle regions, whereas in fennel, H. foeniculi apterous and alate populations were highest in the apical regions. A. gossypii and H. foeniculi peaked in different ages of sole crop or intercropping system. In many cases there was no co-occurrence of A. gossypii and H. foeniculi in intercropping systems. C. sanguinea was associated with the intercropping systems in response to A. gossypii and H. foeniculi that were found in distinct times over the year. The plant quality in both cotton and fennel in sole crop or intercropping system, changes the insect behavior with K1 higher than 1.4. Therefore, cotton-fennel intercropping can be used in aphids integrated pest management.