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Made available in DSpace on 2016-08-29T15:38:12Z (GMT). No. of bitstreams: 1 tese_7215_31 - Francisco de Castro Rocha Neto- dissertação20150904-103939.pdf: 1515880 bytes, checksum: 492b726ff20f4173c65523fb754e2f2b (MD5) Previous issue date: 2013-11-27 / A pimenta-do-reino é o terceiro item na pauta das exportações agrícolas do Estado do Espírito Santo. A fusariose é uma das principais doenças da cultura por causar a morte da planta e inviabilizar a produção econômica. O estudo do comportamento espaço-temporal é uma importante ferramenta no diagnóstico e na orientação do manejo de doenças de plantas. Objetivou-se, com este trabalho, conhecer o comportamento espaço-temporal da fusariose em plantios de pimenta do-reino. O experimento foi conduzido em condições de campo, na região Norte do Estado do Espírito Santo, no período de julho de 2010 a janeiro de 2012. Foram selecionados quatro talhões de cada idade, 1, 2, 3, 4 e 5 anos, em diferentes lavouras de pimentado-reino, com 500 plantas cada. A incidência da fusariose foi avaliada bimensalmente. No estudo temporal, realizado por meio da curva de progresso da doença, o modelo monomolecular foi o que melhor se ajustou aos dados obtidos. O comportamento espacial mostrou uma distribuição inicial da doença de forma casualizada, seguindo a mudança do modelo para agregado, indicando o lento processo de disseminação da doença planta-a-planta, característico de patógenos habitantes do solo.

Made available in DSpace on 2014-12-02T11:16:47Z (GMT). No. of bitstreams: 0 Previous issue date: 2014-09-05Bitstream added on 2014-12-02T11:21:37Z : No. of bitstreams: 1 000800061.pdf: 1547565 bytes, checksum: 2df2d2d64d204de432cc47eb081a5653 (MD5) / A murcha de fusário, causada pelo fungo de solo Fusarium oxysporum f.sp. lactucae é uma das doenças mais severas que ataca m a cultura da alface em todo o mundo. Em 2011, foram encontrados em uma plantação de alface n os município s de Capão Bonito e Campin as n o estado de São Paulo, sintomas característicos do ataque do patógeno. Plantas sintomáticas foram coletadas para identificação do patógeno. O s exame s em microscóp io óptico revel aram a presença de macroconídios falcados de coloração hialina, microconídi os hialinos unicelulares e testes de patogenicidade revelaram a presença de Fusarium oxysporum f.sp. lactucae . Efetuou - se o re - isolamento do patógeno, completa ndo - se assim o Postulado de Koch . No total, for am encontrados 4 isolados patogê nicos atacando pla ntas de alface. A partir do s isolados identificados , fo ram caracterizada s as variações patogênicas através de diferenciadoras de raça. Para esta finalidade foram utilizadas Patriot ( suscetível às raças 1, 2 e 3 ), Costa Rica ( resistente à raça 1 ; suscetível às raças 2 e 3 ), Summer Green ( suscetível às raças 1 e 3 ; resistente à raça 2 ) e Banchu Red Fire ( suscetível às raças 1 e 3, resistente à raça 2 ) . Os resultados obtidos identificaram a ocorrência da raça 3 de Folac. Este é o primeiro relato de ocorrência desta raça no Brasil . A partir do isolado selecionado como sendo o mais agressivo , e caracterizado como pertencente a raça 3 , r ealizou - se a triagem de genótipos de alface, afim de identificar fontes de resistência para uso em programas de melhoramento . No total de 63 genótipos testados apenas Onondaga, Itha ca, Sudenvil e JP - 11 apresentara m - se como resistente a raça 3 de Fusarium oxysporum f.sp. lactucae / The fusarium wilt, caused b y the soil fungus Fusarium oxysporum f.sp. soil lactucae is one of the most severe diseases that attack lettuce crop worldwide. In 2011, were found in a pla nting lettuce in Capão Bonito and Campinas in São Paulo, symptoms characteristic of this pathogen. S ymptomatic plants were collected for pathogen identification. The optical microscope examinations revealed the presence of hyaline staining falcate macroconidia, microconidia unicellular hyaline and pathogenicity tests revealed the presence of Fusarium oxy sporum f.sp. lactucae. We performed the re - isolation of the pathogen, there by supplementing the Koch's postulate. For this purpose were used: Patriot(susceptible to races 1, 2 and 3), Costa Rica (susceptible to races 2 and 3, resistant to race 1); Summer Green (susceptible to races 1 and 3; resistant to race 2) and Banchu Red Fire (susceptible to races 1 and 3, resistant to race 2). The results show the occurrence of race 3 of Folac. This is the first report of the case in Brazil. From the isolated select ed as being the 3 most aggressive, and characterized as belonging to race 3 was held screening of lettuce genotypes in order to identify sources of resistance for use in breeding programs. From of 63 genotypes tested only Onondaga, Ithaca, Sudenvil and JP - 11 were identified as resistant to race 3 of Fusarium oxysporum f.sp. lactucae

Made available in DSpace on 2015-03-26T13:37:40Z (GMT). No. of bitstreams: 1 texto completo.pdf: 494315 bytes, checksum: ae779e1b9f8fc13a152740edf2e2b595 (MD5) Previous issue date: 2009-02-12 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / The fusarium wilt caused by Fusarium oxysporum f. sp. lycopersici (Fol), is one of the major problems in tomato cultivation especially in green house crop. The soil infestation with this pathogen can make the green house cultivation unviable, therefore periodic fumigation is recommended to maintain low inoculum level in soil. This study was done to evaluate the fumigant effect of the mustard essential oil (MEO), containing 90% allyl isothiocyanate, to control Fol. In vitro bioassays were done to determine its effect on mycelial growth, sporulation and germination of conidia and clamydospores, with use of a wild Fol and benomyl resistant mutant (Folm). The fungal cultures in Petri plates were fumigated with different concentration of the MEO for 24 or 48 h, and then incubated in MEO free atmosphere. For all fungal propagules, the estimated DE50 was lowest if the fumigation was done for 48 h. The mycelium and conidia of the Fol were more susceptible to MEO than chlamydospores. The MEO did not affect sporulation. Fumigation with MEO was also evaluated for eradication of the chlamydospores of Folm in soil. Initially, the interaction between dose (0, 50, 100 or 150μL/L) and exposure time was determined (2, 4, 6 or 8 days). The soil infested with 2000 ±200 chlamydospores/g was placed in flasks, and after adding the requited amount of MEO the flasks were hermetically sealed. After each exposure period, the inoculum density of the fungus was determined by plating the soil dilutions on benomyl enriched galactosenitrate agar. The regression equation revealed that at dose of 125μL/L an exposure period of 5.4 days was required to eradicate Folm. To determine the fumigant effect of MEO in the green house, 20L of soil infested with 4000 ±250 chlamydospores/g was placed in the plastic bags of 30L, and treated with 0, 50, 100 or 150μL/L of MEO. The bags were then sealed and stored. After 7-days exposure period, the soil was distributed into 4L-plastic pots, and one 20-day old tomato seedling was transplanted into each pot. At 15-day interval, soil from each pot was sampled at 15-day interval to follow the population dynamic of the fungus. The disease progress was accompanied by leaf chlorophyll analysis leaves, and the final severity was evaluated by use of a numerical at the end of 60 days. It was found that the soil fumigation with 150μL/L of MEO reduced the Folm inoculum density by 95% and the disease severity was less than 15%. / A murcha de fusário, causada por Fusarium oxysporum f. sp. lycopersici (Fol), é um problema comum em campos de produção de tomate, especialmente quando o cultivo é realizado em ambiente protegido. Solos infestados por este patógeno podem inviabilizar a produção em estufas, sendo recomendada a fumigação periódica, visando à manutenção de um baixo nível de inóculo no solo. Este trabalho teve como objetivo avaliar o efeito fumigante do óleo essencial de mostarda, que é composto por 90% de isotiocianato de alila (ITCA), na redução de inóculo e no controle da murcha vascular causada por Fol. Foram realizados bioensaios in vitro de crescimento micelial, formação de conídios e germinação de conídios e de clamidósporos. Para os testes, foram utilizados um isolado selvagem (Fols) e um mutante resistente ao benomil (Folm), os quais foram fumigados com ITCA, em diferentes doses, dentro de recipientes plásticos vedados, por períodos de 24 ou 48 horas. Após a fumigação, as placas contendo as culturas foram incubadas na ausência dos vapores do produto até a avaliação. Os menores valores de DE50 foram estimados para o período de 48 horas de exposição, tanto para o bioensaio de crescimento micelial como para os de germinação de conídios e de clamidósporos. Verificou-se que os conídios foram os propágulos de Fol mais sensíveis ao produto e os clamidósporos os mais resistentes. O ITCA não afetou significativamente a formação de conídios pelos isolados. Avaliou-se também a eficiência do produto na erradicação de clamidósporos de Folm no solo. Inicialmente, foi estudada a interação entre doses (0, 50, 100 e 150μL/L) e tempo de exposição (2, 4, 6 e 8 dias) ao ITCA. Solo infestado com 2000 ±200 clamidósporos/g foi transferido para erlenmeyers, que receberam a dose desejada, sendo, em seguida, hermeticamente vedados. Após exposição, a população do fungo foi determinada por meio de plaqueamento de diluições em série em meio seletivo para F. oxysporum acrescido de benomil. A partir da equação de regressão gerada, pôde-se estimar que seria necessária uma fumigação de solo com 125μL/L por períodos superiores a 5,4 dias para erradicação de Folm no solo. Para determinar o efeito de ITCA em casa de vegetação, 20L de solo infestado com 4000 ±250 clamidósporos/g foram colocados em sacos de polietileno de 30L, os quais receberam as doses de 0, 50, 100 ou 150μL/L sendo, posteriormente, vedados, permitindo a fumigação por 7 dias. Decorrido este período, o solo foi transferido para vasos de 4L, os quais receberam uma muda de tomate com 20 dias de idade. As plantas foram cultivadas por 60 dias, sendo retiradas amostras quinzenais de solo para acompanhamento da dinâmica populacional do fungo no solo. Através de análise do conteúdo de clorofila nas folhas, acompanhou-se o desenvolvimento da doença e a severidade final foi avaliada por meio de escala de notas. Foi verificado que a fumigação com 150μL/L de ITCA reduziu em mais de 95% a população de Folm no solo e que a severidade da doença aos 60 dias foi inferior a 15%.

Made available in DSpace on 2018-08-24T12:05:23Z (GMT). No. of bitstreams: 1 tese_11827_Dissertação_Wilker.pdf: 947833 bytes, checksum: 7493679906fbdfd0a73a61ba54d6007e (MD5) Previous issue date: 2018-02-23 / O tomate é a hortaliça de maior consumo no mundo, sendo uma cultura de importância econômica e social. Porém, em seu cultivo, é propício a incidência de diversas doenças, dentre elas pode-se destacar a murcha de Fusarium, causada pelo Fusarium oxysporum f.sp. lycopersici. Sendo assim, objetivou-se com este estudo avaliar a atividade in vitro dos óleos essenciais (OEs) de Thymus vulgaris e Cinnamomun zeylanicum e seus componentes majoritários. A caracterização química dos OEs foi realizada por CG/DIC e CG/EM, tendo identificado o eugenol (77,95%) como componente majoritário do OE canela, o carvacrol (23,93%) e o timol (20,23%) do OE de tomilho. No ensaio foram calculados os valores de CE50 e CE100 do crescimento micelial de cada tratamento, sendo: OE de canela (171,79 e 575,18 ppm); OE de tomilho (106,29 e 341,73 ppm); eugenol (187,46 e 374,93 ppm); timol (88,67 e 193,02 ppm) e carvacrol (52,06 e 170,92 ppm). E, para esporulação, foram calculados os valores de: OE de canela (262,20 e 342,72 ppm); OE de tomilho (67,47 e 255,42 ppm); eugenol (80,94 e 359,55 ppm); timol (97,0 e 194,02 ppm) e carvacrol (44,66 e 113,82 ppm). Com isso, os óleos essenciais e seus componentes majoritários podem ser uma alternativa para o controle do F. oxysporum f.sp. lycopersici.

Doctor of Philosophy / Genetics Interdepartmental Program / Christopher Toomajian / Secondary metabolites (SMs), low molecular weight molecules that are not essential for normal organism growth and development, may confer a selective advantage in some environments. Fungal SMs are structurally and functionally diverse and include mycotoxins, plant regulators and pigments, and the genes that work together in SM biosynthetic pathways are physically clustered in the genome. Fusarium, a genus of filamentous fungi, is noted for SM production, especially mycotoxins, which may contribute to plant pathogenesis. Fusarium species exhibit differences in their SM profiles, and comparative genomics studies have found corresponding differences in the SM gene clusters in some Fusarium species. The investigation of differences in the genomes and SM gene clusters between closely related species, such as F. proliferatum and F. fujikuroi, may help explain their phenotypic divergence, including differences in SM profiles. In addition, the study of intra-species SM variation may indicate how SM loci affect a pathogen’s fitness traits. My research includes three main projects that address different aspects of Fusarium SM variability. To carry out my projects, I established a feasible Genotyping-by-Sequencing (GBS) protocol for Fusarium. One project explored the genetic bases underlying phenotypic divergence related to SM profiles and pathogenicity between F. proliferatum and F. fujikuroi using a quantitative genetics approach. Specifically, I 1) constructed the first high density genetic map based on progeny from an interspecific cross between these two species; and 2) detected a novel regulatory locus for gibberellic acid production and identified a region affecting onion virulence that includes the fumonisin gene cluster. The second project characterized the F. proliferatum parent genome from the previous cross and its SM gene clusters using a comparative genomics approach. Specifically, I 1) assembled the F. proliferatum genome into 12 chromosomes with a combined length of ~43 Mb; 2) annotated this assembly and characterized its 50 SM gene clusters; and 3) detected over 100 F. proliferatum specific genes that might play roles in this species’ host specificity and plant pathogenicity. The third project used a population genomics approach to explore how different F. graminearum chemotypes, or isolates classified based on the accumulation of alternate trichothecene toxin types, may differ for fitness traits and whether trichothecene genes are directly responsible for these differences. Specifically, I 1) genotyped over 300 F. graminearum strains from New York and the upper Midwest in the U.S. and from South America using our GBS protocol; 2) detected two major subpopulations that were correlated, though imperfectly, to the predicted 3-acetyl deoxynivalenol (3ADON) and 15-acetyl deoxynivalenol (15ADON) chemotypes in the U.S.; 3) identified a rapid linkage disequilibrium decay over a few tens of kb followed by a slower decay to background levels over a distance of 200 kb to 400 kb in selected subpopulations in the U.S.; and 4) found that neither chemotype has a clear fitness advantage in a small set of isolates from New York, but that isolates belonging to one genetic subpopulation may on average have a fitness advantage over isolates from the other subpopulation.

Doctor of Philosophy / Genetics Interdepartmental Program / Christopher Toomajian / Secondary metabolites (SMs), low molecular weight molecules that are not essential for normal organism growth and development, may confer a selective advantage in some environments. Fungal SMs are structurally and functionally diverse and include mycotoxins, plant regulators and pigments, and the genes that work together in SM biosynthetic pathways are physically clustered in the genome. Fusarium, a genus of filamentous fungi, is noted for SM production, especially mycotoxins, which may contribute to plant pathogenesis. Fusarium species exhibit differences in their SM profiles, and comparative genomics studies have found corresponding differences in the SM gene clusters in some Fusarium species. The investigation of differences in the genomes and SM gene clusters between closely related species, such as F. proliferatum and F. fujikuroi, may help explain their phenotypic divergence, including differences in SM profiles. In addition, the study of intra-species SM variation may indicate how SM loci affect a pathogen’s fitness traits. My research includes three main projects that address different aspects of Fusarium SM variability. To carry out my projects, I established a feasible Genotyping-by-Sequencing (GBS) protocol for Fusarium. One project explored the genetic bases underlying phenotypic divergence related to SM profiles and pathogenicity between F. proliferatum and F. fujikuroi using a quantitative genetics approach. Specifically, I 1) constructed the first high density genetic map based on progeny from an interspecific cross between these two species; and 2) detected a novel regulatory locus for gibberellic acid production and identified a region affecting onion virulence that includes the fumonisin gene cluster. The second project characterized the F. proliferatum parent genome from the previous cross and its SM gene clusters using a comparative genomics approach. Specifically, I 1) assembled the F. proliferatum genome into 12 chromosomes with a combined length of ~43 Mb; 2) annotated this assembly and characterized its 50 SM gene clusters; and 3) detected over 100 F. proliferatum specific genes that might play roles in this species’ host specificity and plant pathogenicity. The third project used a population genomics approach to explore how different F. graminearum chemotypes, or isolates classified based on the accumulation of alternate trichothecene toxin types, may differ for fitness traits and whether trichothecene genes are directly responsible for these differences. Specifically, I 1) genotyped over 300 F. graminearum strains from New York and the upper Midwest in the U.S. and from South America using our GBS protocol; 2) detected two major subpopulations that were correlated, though imperfectly, to the predicted 3-acetyl deoxynivalenol (3ADON) and 15-acetyl deoxynivalenol (15ADON) chemotypes in the U.S.; 3) identified a rapid linkage disequilibrium decay over a few tens of kb followed by a slower decay to background levels over a distance of 200 kb to 400 kb in selected subpopulations in the U.S.; and 4) found that neither chemotype has a clear fitness advantage in a small set of isolates from New York, but that isolates belonging to one genetic subpopulation may on average have a fitness advantage over isolates from the other subpopulation.

Tesis presentada a la Universidad de Chile para optar al grado académico de Magister en Bioquímica área de especialización en Bioquímica Ambiental y Memoria para optar al título profesional de Bioquímico / Memoria de título de bioquímico / No autorizada por el autor para ser publicada a texto completo / El hongo filamentoso Fusarium fujikuroi produce diversos metabolitos secundarios entre los que se encuentran las giberelinas (GAs), diterpenoides tetracíclicos derivados del ácido mevalónico de interés agronómico debido al efecto regulador que presentan sobre el crecimiento y desarrollo de las plantas. El interés comercial de estas moléculas y la alta eficiencia del sistema fúngico han llevado a caracterizar su biosíntesis a nivel de las reacciones químicas, las enzimas y los genes implicados en el proceso. La secuencia biosintética consiste principalmente en reacciones de oxidación, las que son catalizadas por monooxigenasas (MO) P450 codificadas por genes agrupados en un cluster. En este hongo la biosíntesis de GAs se induce en condiciones de carencia de compuestos nitrogenados y es inhibida por amonio o glutamina debido a la represión de los genes. Este mecanismo de regulación está mediado principalmente por el regulador global AREA, un factor de transcripción cuya actividad depende de los niveles de nitrógeno, aunque también podrían participar otros elementos regulatorios específicos para esta vía metabólica. Con el objeto de contribuir a la comprensión de los mecanismos de regulación de la biosíntesis de las GAs fúngicas, en este trabajo de Tesis se investigó si los genes de GAs de F. fujikuroi se expresan como proteínas activas en tres especies de hongos filogenéticamente relacionadas a F. fujikuroi: Fusarium oxysporum, Fusarium graminearum y Aspergillus nidulans. Estos hongos no poseen los genes de la biosíntesis de GAs pero contienen el regulador global AREA. Interesó determinar si la complementación con el cluster de genes de F. fujikuroi es suficiente para generar la capacidad de biosintetizar GAs en estas especies y si las transformantes presentan el mecanismo de represión por amonio. Se caracterizó la producción de GAs en cultivos líquidos mediante cromatografía de gases acoplada a espectrometría de masas (GC-MS) y las actividades de las distintas oxidasas de GAs se ensayaron mediante sustratos marcados con [14C] e identificando los productos por cromatografía en capa fina y cromatografía líquida de alta resolución. Se encontró que las transformantes T6, T11 y T1 de F. oxysporum así como la transformante T2 de F. graminearum presentan la capacidad de biosintetizar GAs en un medio líquido carente de compuestos nitrogenados. Principalmente sintetizan GAs 3β-hidroxiladas y 19-γ10-lactónicas (19C) como GA4, GA7 y GA3, además algunas GAs no hidroxiladas (GA9, GA24, GA25) y entkaurenoides, que son productos laterales de la vía biosintética. Este patrón de productos corresponde al que presenta la cepa silvestre IMI28589 de F. fujikuroi, sistema fúngico que sintetiza GA3 como producto final a partir de intermediarios 3β-hidroxilados. Estos resultados indican que los genes de la biosíntesis de GAs de F. fujikuroi se expresan y generan enzimas activas tanto en F. oxysporum como en F. graminearum. En particular, la metabolización del ácido ent-[14C]kaurenoico hasta [14C]GA14 y posteriormente hasta [14C]GA3 demostró que la MO P450-1 (GA14 sintasa) presenta las actividades de 7-oxidasa y de 3β-hidroxilasa, como en F. fujikuroi. Por otra parte, la MO P450-2 (GA20 oxidasa) forma el producto lactónico [14C]GA9 y su derivado [14C]GA40, a partir del sustrato [14C]GA12. En estos ensayos también se detectaron las actividades de 13-hidroxilasa y de desaturasa aunque con diferentes proporciones en las distintas transformantes, en concordancia con el análisis de GAs endógenas. El efecto represor del nitrato de amonio se determinó a través de la actividad de la GA20 oxidasa en la transformante de F. graminearum T2, donde se encontró que la velocidad de utilización de [14C]GA12 fue 5 veces menor en un medio con nitrato de amonio 4,8 g/L que en un medio sin amonio, en forma similar a la cepa silvestre IMI28589 de F. fujikuroi (siete veces menor en presencia de amonio). Esto sugiere que estaría operativo en F. graminearum el mecanismo de represión por compuestos nitrogenados. Para la transformante T6 de F.oxysporum el resultado fue menos claro, ya que se obtuvieron productos en presencia de amonio que no corresponden a la lactona [14C]GA9 o a su derivado [14C]GA40 (los principales productos de la GA20 oxidasa). La identificación de los productos por GC-MS permitirá confirmar si corresponden a la actividad de oxidasas inespecíficas y si la represión por nitrato de amonio está presente en esta especie. A diferencia de las transformantes de F. oxysporum y de F. graminearum, en cultivos de la transformante de A. nidulans no se detectó actividad de ninguna de las oxidasas de GAs en los ensayos con los sustratos marcados con [14C]. Esto sugiere que los genes de F. fujikuroi no se expresarían en A. nidulans, una especie filogenéticamente más alejada de F. fujikuroi que F. oxysporum y F. graminearum, a pesar de que AREA está presente en esta especie. En conclusión, los resultados obtenidos sugieren que las dos especies de Fusarium investigadas, F. oxysporum y F. graminearum, contienen los elementos regulatorios (AREA y/u otros) requeridos para la expresión de los genes de la biosíntesis de GAs en condiciones de carencia de nitrógeno. Estos factores no serían específicos para esta vía, ya que se encontraron en dos especies fúngicas que no contienen genes de la biosíntesis de GAs y no sintetizan estos diterpenos. / The filamentous fungus Fusarium fujikuroi synthesizes several secondary metabolites including gibberellins (GAs), tetracyclic diterpenoids derived from mevalonic acid that have an agronomic interest since they are plant growth regulators. Because of the high efficiency of the fungal system and its commercial interest GA biosynthesis has been characterized at the level of chemical reactions, enzymes and genes. The fungal GA biosynthetic pathway is based on oxidative reactions catalyzed by P450 monooxygenases (MO) codified by a gene cluster. GA biosynthesis is induced in the absence of nitrogenated compounds and inhibited by ammonia or glutamine due to repression of gene expression. The global regulator AREA, a transcription factor dependent on nitrogen levels, mediates this effect, together with other possible specific regulatory elements. In order to contribute to the understanding of GA biosynthesis regulation in F. fujikuroi in this work we investigated if the F. fujikuroi GA biosynthesis genes are expressed as active enzymes in three fungal species phylogenetically related to F. fujikuroi: Fusarium oxysporum, Fusarium graminearum and Aspergillus nidulans. These species do not contain the GA biosynthesis genes but contain the global regulator AREA. It was investigated if complementation with the F. fujikuroi gene cluster is enough to restore GA biosynthesis in these fungal species and if the transformants present ammonium repression of the GA genes as in F. fujikuroi. GA biosynthesis was determined in liquid cultures by gas chromatography coupled to mass spectrometry (GC-MS) and the activities of GA oxidases were assayed with [14C]-labelled substrates by identifying the respective products by thin layer chromatography or high performance liquid chromatography. F. oxysporum transformants T6, T11 and T1 as well as F. graminearum T2 synthesized GAs in liquid cultures that do not contain nitrogenated compounds. These were mainly 19-γ10-lactonic, 3β-hydroxylated GAs (C19; GA4, GA7 and GA3) together with non-hydroxylated GAs (GA9, GA24,GA25) and ent-kaurenoids, lateral products of the GA biosynthetic pathway. This product pattern corresponds to that of the wild-type F. fujikuroi strain IMI28589 that synthesizes GA3 as final product from 3β-hydroxylated intermediates. These results demonstrate that the F. fujikuroi GA biosynthesis genes are expressed as active enzymes in F. oxysporum as well as in F. graminearum. Particularly the conversion of ent-[14C]kaurenoic acid into [14C]GA14 and further into [14C]GA3 demonstrated that in F. oxysporum and F. graminearum GA14 synthase presents 7- oxidase and 3β-hydroxylase activities as in F. fujikuroi. On the other hand, in these transformants GA20 oxidase gave the 19-γ10-lactonic product [14C]GA9 or its derivative [14C]GA40 from [14C]GA12. 13-hydroxylase and desaturase activities were also detected in these assays even when in different proportions in the distinct transformants in agreement with endogenous GA analysis. Ammonium nitrate repressor effect was investigated over GA20 oxidase that was assayed in F. graminearum T2 liquid cultures. The rate of [14C]GA12 conversion was found to be 5 times less in 4,8 g/L ammonium-containing media than in the absence of ammonium, similar to that found for F. fujikuroi IMI28589 cultures (7 times less [14C]GA12 conversion in media containing ammonia). This suggests that the mechanism of repression by nitrogenated compounds would be active in F. graminearum. For F. oxysporum T6 a less clear result was obtained since the products formed in the presence of 4,8 g/L ammonium nitrate do not include [14C]GA9 or [14C]GA40, the main products of GA20 oxidase. GC-MS identification of these products will allow to confirm if they correspond to unspecific oxidation products and if ammonium repression is also present in F. oxysporum. In contrast to F. oxysporum and F. graminearum, the cultures of A. nidulans complemented with the F. fujikuroi GA biosynthesis genes did not present activity of any of the GA oxidases in assays with ent-[14C]kaurenoic acid, [14C]GA12 or [14C]GA4. This suggests that the F. fujikuroi genes would not be expressed in A.nidulans, a species phylogenetically less related to F. fujikuroi, even when it contains AREA. Altogether, the results obtained suggest that the two Fusarium species investigated contain the regulatory elements required for the expression of the GA biosynthesis genes (AREA and/or others) in the absence of nitrogenated compounds. These factors would not be specific for the GA pathway since they are present in two fungal species that do not contain the GA biosynthesis genes and do not synthesize GAs. / Fondecyt