Diss.--Universität von Münster, 1999. / Bibliogr. p. 86-100.

Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Abstracted in Dissertation abstracts, v. 19 (1959) no. 7, p. 1535-1536. Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references (leaves 101-103).

Thesis (Ph. D.)--University of Wisconsin--Madison, 1939. / Typescript. Includes abstract and vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

Doctora en Bioquímica / Las giberelinas (GAs) son fitohormonas diterpénicas sintetizadas como metabolitos secundarios por algunos hongos, entre ellos Fusarium fujikuroi, patógeno del arroz que produce grandes cantidades de ácido giberélico (GA3). Esta especie forma parte del complejo taxonómico Gibberella fujikuroi que incluye otras 10 especies relacionadas filogenéticamente denominadas poblaciones de apareamiento (A-K). Aunque todas las especies del complejo G. fujikuroi contienen genes de la biosíntesis de GAs la producción de estos diterpenos se ha descrito casi exclusivamente en F. fujikuroi, con excepción de dos cepas de Fusarium proliferatum y una cepa de Fusarium konzum que sintetiza muy bajas cantidades de GAs. Con el objeto de investigar si existen otras especies productoras de GAs en el complejo G. fujikuroi, se caracterizaron varias cepas de Fusarium sacchari (aislado de la caña de azúcar), de Fusarium konzum (aislado de pastos de praderas) y de Fusarium subglutinans (aislado del maíz). F. sacchari es una especie cercanamente relacionada con F. fujikuroi, agrupada en el clado Asiático del complejo en tanto que F. konzum y F. subglutinans son especies filogenéticamente alejadas de F. fujikuroi agrupadas en el clado Americano. Las cepas de F. sacchari investigadas presentaron diferencias con respecto a su capacidad de sintetizar GAs. Cinco aislados (B-12756; B-1732, B-7610, B- 1721 and B-1797) resultaron activos sintetizando principalmente GA3 (2,76-28,4 mg/mL) mientras que otros dos (B-3828 and B-1725) resultaron inactivos. Las etapas catalizadas por la geranilgeranil difosfato sintasa (GGS2) y/o la ent-kaureno sintasa (CPS/KS) son limitantes en las cepas productoras ya que los niveles de GA3 aumentaron 2,9 veces por complementación con los genes ggs2 y cps/ks de F. fujikuroi. Con respecto a F. konzum, de los seis aislados que se investigaron tres (I-10653; I-11616; I-11893) sintetizaron GAs, principalmente GA1, un producto final diferente al de las cepas activas de F. sacchari. Además, los niveles de GAs sintetizados por F. konzum fueron muy bajos (menos de 0,1 mg/mL). Tres cepas de F. konzum resultaron inactivas y no presentaron actividad de ninguna oxidasa de GAs lo que sugiere que los genes respectivos no se expresan. Las dos cepas de F. subglutinans ensayadas no sintetizan GAs ni presentan actividad de las oxidasas. Estos resultados indican que la capacidad de sintetizar GAs está presente en otras especies del complejo G. fujikuroi además de F. fujikuroi, pero puede diferir significativamente entre cepas. Finalmente se investigó la biosíntesis de GAs en un conjunto de 19 cepas híbridas (CxD) provenientes de un cruce entre F. fujikuroi y F. proliferatum, dos especies muy cercanas filogenéticamente agrupadas en el clado Asiático del complejo G. fujikuroi. Las dos cepas parentales (F. fujikuroi C-1995 y F. proliferatum D-4854) contienen los genes de la biosíntesis de GAs pero difieren en su capacidad biosintética: C-1995 sintetiza GAs, principalmente GA3, mientras que D-4854 no produce GAs. Las cepas híbridas no presentaron un patrón de segregación Mendeliano 1:1 como se esperaba sino que se encontró en la progenie un patrón de tres fenotipos: 8 híbridos producen GA3 en cantidades similares a la cepa parental C-1995 en tanto que 6 cepas resultaron inactivas para la biosíntesis de GAs. Además de los fenotipos parentales una parte de la progenie (5 cepas) produjo pequeñas cantidades de GAs, principalmente GA1 y presenta bajos niveles de expresión de los genes. Estos resultados evidencian que el cruce interespecies puede generar nuevos patrones de metabolitos secundarios, en este caso de GAs. El conjunto de resultados obtenidos sugiere que la capacidad de sintetizar GAs en niveles significativos estaría restringida a las especies de Fusarium agrupadas en el clado Asiático del complejo G. fujikuroi / Gibberellins (GAs) are diterpene phytohormones synthesized as secondary metabolites by some fungi like Fusarium fujikuroi, a plant pathogen that produces high levels of gibberellic acid (GA3). This Fusarium species belongs to Gibberella fujikuroi, a taxonomic species complex that includes 10 other phylogenetically related species denominated mating populations (A-K). Even when all the species within the G. fujikuroi complex contain GA biosynthesis genes, the production of these diterpenes has been almost exclusively found in F. fujikuroi, except for two Fusarium proliferatum strains and one Fusarium konzum isolate that synthesizes very low GA levels. In order to find out if other species within the G. fujikuroi complex synthesize GAs, several strains of three Fusarium species were characterized: Fusarium sacchari (isolated from sugar cane), F. konzum (isolated from prairie grasses) and Fusarium subglutinans (isolated from maize). F. sacchari is closely related to F. fujikuroi and grouped in the Asian clade of the complex while F. konzum and F. subglutinans are phylogenetically distant species grouped in the American clade. Analyzed F. sacchari strains differed in their ability to synthesize GAs. Five isolates (B-12756; B-1732, B- 7610, B-1721 and B-1797) were active and synthesized mainly GA3 (2,76-28,4 mg/mL) while two others (B-3828 and B-1725) were inactive. The steps catalyzed by geranylgeranyl diphosphate synthase (GGS2) and/or ent-kaurene synthase (CPS/KS) were limiting in F. sacchari active strains since it was found that GA3 levels increased by 2.9 fold upon complementation with ggs2 and cps/ks genes from F. fujikuroi. For F. konzum, six isolates were analyzed of which three (I-10653; I-11616; I- 11893) synthesized GAs, mainly GA1, a different final product than that synthesized by F. sacchari active strains. GA levels formed by F. konzum isolates were very low (less than 0,1 mg/mL). Three F. konzum strains were inactive in GA biosynthesis and contained no GA oxidase activities suggesting that the respective genes were not expressed. For F. subglutinans the two isolates assayed did not synthesize GAs and lacked activity of the GA oxidases. These results evidence that GA biosynthesis is present in other species within the G. fujikuroi complex besides F. fujikuroi but may differ significantly between isolates. Finally, GA biosynthesis was investigated in a group of 19 hybrid strains (CxD) from an interspecies cross between F. fujikuroi y F. proliferatum, two closely related species that belong to the American clade in the G. fujikuroi complex. Both parental strains (F. fujikuroi C-1995 and F. proliferatum D-4854) contain the GA biosynthetic genes but differed in their ability to produce GAs: C-1995 synthesizes GAs, mainly GA3, while D-4854 did not produce GAs. The hybrid strains did not show a Mendelian 1:1 segregation pattern as expected but we found in the progeny a three phenotype pattern: 8 CxD hybrids produced GA3 at similar levels than the parental strain C-1995 while 6 strains were inactive for GA biosynthesis. Besides the parental phenotypes 5 progeny strains produced low GA levels, mainly GA1 and showed low expression of the respective genes. These findings indicate that interspecies crosses may generate new profiles of secondary metabolites, like the GAs. Altogether obtained results suggest that GA biosynthesis at significant levels would be restricted to the Fusarium species of the Asian clade in the G. fujikuroi complex / FONDECYT; FONDAP; MECESUP

Zugl.: Hohenheim, Univ., Diss., 2008

Reproductive isolation is an essential stage in speciation. In Ascomycetes, the ubiquitous distribution of many species suggests that sympatric speciation through assertive mating should be an important factor. The MAT locus and the pheromone/receptor system could both potentially contribute to the development of such sexual isolation. Alterations at the MAT loci could lead to distinct reproductive habits or a change in mating system, both of which can reduce gene-flow between species. However, if deliberate pre-mating sexual preferences exist, they are more likely to be determined by the pheromone/receptor system. This study of Fusarium reproductive genes, and comparisons with other Ascomycetes, has yielded numerous interesting findings regarding the evolution of these mate-recognition mechanisms and the implications thereof. The G. fujikuroi and F. graminearum species complexes have offered an interesting comparison between heterothallic and homothallic MAT locus evolution. The value of comparative sequence analysis has been demonstrated in the discovery of a previously unknown gene, MAT1-2-3, which may be specific to members of the Order Hypocreales. While all MAT genes share similar regulatory elements, this is the first report of evidence that a transition to homothallism can be accompanied by the recruitment of distinct elements that could facilitate alternate expression of MAT genes. The MAT genes are also highly divergent between Fusarium spp., largely due to relaxed selective constraint, particularly in homothallic species. However, inter-specific gene-flow could curb MAT gene divergence among homothallic species. Despite strong reproductive barriers in the G. fujikuroi complex, the F. sacchari MAT1-1 sequence appears to have been acquired through lateral transfer from a distant relative. Analytical analysis of the MAT locus novelties reported here, including the new MAT gene, will be necessary to determine their biological significance. To investigate the extent of pheromone diversity in the Ascomycetes, and to gain clues as to its biological importance, pheromone peptides from seventy ascomycete species were compared. A number of reproductively incompatible species, such as those in the G. fujikuroi complex, share identical pheromones; which implies that another mechanism must be responsible for the observed reproductive barriers. However, on the whole, pheromones are highly divergent among species. Both adaptive and non-adaptive evolution could have contributed to this pattern. In fact the structure of the á-class pheromone precursor gene, which consists of multiple repeats of the pheromone module, could facilitate rapid diversification through “birth-and-death” evolution. Within species, selection maintains pheromone peptides, implying that much of the inter-specific variation is functionally relevant. This further suggests that pheromone evolution could contribute to the generation of reproductive isolation between species. The most general trend in the findings of this study is that ascomycete reproductive genes are highly divergent. This is in agreement with findings in other Kingdoms. A number of evolutionary forces are probably involved but weaker selective constraint, resulting from the fact that reproduction is not essential in these fungi, appears to be a common factor. This reproductive gene variability could be directly linked to speciation and, therefore, the great diversity in Ascomycetes. Additional information on the appendices is available on a CD, stored at the Merensky Library on Level 3 / Dissertation (MSc)--University of Pretoria, 2011. / Genetics / Unrestricted

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The gibberellic acid (GA3) is a natural hormone found in some plants, this hormone has been used in agricultural formulations, as growth regulator, highly relevance both economic and industrial. Currently, its industrial scale production is achieved by Submerged Fermentation (SF) using the fungus Gibberella fujikuroi. The main problem in the industrial process is related to the low yield of GA3, causing the purification process presents high costs. An alternative to this process is the Solid- State Fermentation (SSF) that allows to obtain higher concentrations of this product. The greatest advantage of the SSF front SF is related to increased production of GA3 using low cost substrates such as waste and by-products of agroindustry. Given the fact that Brazil is highlighted as one of the most prosperous countries in terms of agricultural production, and the Rio Grande do Sul accounts for about 30-40% of rice and barley production in the country, this research evaluated the use of Raw Rice Bran (RRB) and Wet Brewery Waste (WBW), rice processing and brewing industry residues, as substrates for GA3 production by the fungus Gibberella fujikuroi. Two experimental designs, a linear type 2n and CCRD, both for two variables were performed. The first design evaluated, on three levels, evaluated the effect of moisture content in the range of 50 to 70%, and the composition of the medium, with RRB content ranging from 30 to 70% of the total substrate mass (RRB and WBW mass). In turn, the second planning in 5 levels, evaluated the effect of the addition of glucose, the carbon source over the range 0 to 80 g/L, and ammonium nitrate (NH4NO3), the nitrogen source, in the range 0 - 5 g/L, by making use of the best conditions of the first planning. It was found that, for seven days of fermentation, the greater yield for the first research proposed, was at the test carried out with medium composition of 30% RRB and 70% WBW and moisture content equals 70%. At the second design It was observed that the highest concentration of NH4NO3 favored the formation of GA3 by the fungus, towards intermediate value to the glucose content. Finally, investigation of the kinetic behavior showed an increase in production of GA3, with the peak on the seventh day with maximal production of 10,10 g/kg of substrate, and subsequent tendency for stabilization. / O ácido giberélico (GA3) é um hormônio natural presente em algumas plantas, sendo empregado em formulações agrícolas como regulador de crescimento, apresentando grande importância econômica e industrial. Atualmente, sua produção em escala industrial é realizada por fermentação submersa (FSub) empregando o fungo Gibberella fujikuroi. O maior problema no processo submerso está relacionado aos baixos rendimentos de GA3, fazendo com que o processo de purificação apresente elevados custos. Uma alternativa a este processo é a fermentação em estado sólido (FES) que permite a obtenção deste produto em concentrações maiores. A maior vantagem da FES frente à FSub está relacionada à maior produção de GA3 empregando substratos de baixo custo, como por exemplo, resíduos e subprodutos da agroindústria. Dado o fato de que o Brasil é destacado como um dos mais prósperos países em termos da produção agrícola, e que o Rio Grande do Sul é responsável por cerca de 30 a 40% da produção de arroz e cevada, no país, avaliou-se a utilização de Farelo de Arroz Bruto (FAB) e Resíduo de Cervejaria Úmido (RCU), resíduos do processamento de arroz e da indústria cervejeira, como substratos para a produção de GA3 pelo fungo Gibberella fujikuroi. Foram realizados dois planejamentos experimentais, linear do tipo 2n e DCCR, ambos para duas variáveis. O primeiro planejamento, em três níveis, avaliou o efeito da umidade, na faixa de 50 a 70%, e a composição do meio, com o teor de FAB variando entre 30 e 70% do total de massa do substrato (massa de FAB e de RCU). Por sua vez, o segundo planejamento, em 5 níveis, avaliou o efeito da adição de glicose, fonte de carbono na faixa de 0 a 80 g/L, e do Nitrato de Amônio (NH4NO3), fonte de nitrogênio, na faixa entre 0 a 5 g/L, na produtividade de GA3, fazendo uso das melhores condições do primeiro planejamento. Foi verificado que, para sete dias de fermentação, o maior rendimento obtido, para a primeira investigação proposta, foi no ensaio realizado com composição do meio 30% FAB e 70% RCU e umidade do meio de 70%. No segundo planejamento constatou-se que a maior concentração de NH4NO3 favoreceu a formação de GA3 pelo fungo, para um valor intermediário do teor de glicose. Por fim, a investigação da cinética demonstrou um comportamento de crescimento na produção de GA3, com o pico no sétimo dia, com produção máxima de 10,10 g/Kg de substrato, e posterior tendência à estabilização.