Submitted by Renata Lopes (renatasil82@gmail.com) on 2017-06-22T12:13:15Z No. of bitstreams: 1 daniellelucianaaurorasoaresdoamaral.pdf: 933775 bytes, checksum: ab2892397c2895f9047b1f683dec1351 (MD5) / Approved for entry into archive by Adriana Oliveira (adriana.oliveira@ufjf.edu.br) on 2017-08-07T19:20:42Z (GMT) No. of bitstreams: 1 daniellelucianaaurorasoaresdoamaral.pdf: 933775 bytes, checksum: ab2892397c2895f9047b1f683dec1351 (MD5) / Made available in DSpace on 2017-08-07T19:20:42Z (GMT). No. of bitstreams: 1 daniellelucianaaurorasoaresdoamaral.pdf: 933775 bytes, checksum: ab2892397c2895f9047b1f683dec1351 (MD5) Previous issue date: 2014-03-07 / CAPES - Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / FAPEMIG - Fundação de Amparo à Pesquisa do Estado de Minas Gerais / Desde a domesticação das plantas para a utilização humana, as doenças vêm causando grandes perdas na produção. Fungos fitopatogênicos tais como Sclerotium rolfsii, Botrytis cinerea, Fusarium oxysporum e Sclerotinia sclerotiorum são capazes de infectar diferentes espécies de plantas. A infecção por estes fungos leva a perdas consideráveis na época da colheita. A fase inicial da infecção envolve a produção e o acúmulo de grande quantidade de ácido oxálico (AO), que parece ser um dos maiores determinantes da patogenicidade. Fusarium oxysporum é a espécie mais comum do gênero e causa murcha vascular em diferentes espécies de plantas. Esse fungo causa perdas severas em muitas lavouras, como algodão, fumo, banana, café, morango e cana de açúcar. Genes que conferem resistência a fitopatógenos tornam-se de importância agronômica como recursos para melhoramento. Dentre esses, destacamos o da enzima oxalato descarboxilase (OXDC), capaz de catalizar a degradação do AO em ácido fórmico e dióxido de carbono, diminuindo a capacidade de infecção do fungo. Na geração T0 de fumo transformado com gene da OXDC, foram obtidas quatro linhagens resistentes ao fungo, estas foram analisadas na T1. O objetivo deste trabalho foi avaliar a segregação do transgene OXDC para a geração T1 de fumo e avaliar se a geração T1 de plantas transformadas é capaz de resistir ao fitopatógeno Fusarium oxysporum. Trinta plantas de cada linhagem T1 de fumo transformado com OXDC foram avaliadas, por PCR, quanto a presença do HPTII. Estas quatro linhagens foram analisadas apresentaram proporção de segregação de 3:1. Uma planta PCR positiva de cada linhagem foi submetida a bioensaios para verificar a resistência ao fungo e ao AO. No ensaio de resistência ao fungo Fusarium oxysporum, este não foi capaz de infectar as linhagens transgênicas, mostrando um aumento da resistência da T1 em relação a T0 quando os resultados foram comparados. Os níveis de expressão do transgene foram avaliados por RT-PCR. As linhagens T1, T4 e T6 mostraram níveis de expressão semelhantes, já a linhagem T2 apresentou menor nível de expressão que as demais linhagens, de maneira que este resultado pode ser correlacionado com menor resistência ao AO. Com base nestes resultados pode-se concluir que a enzima Oxalato Descarboxilase demonstrou ser eficiente no combate ao patógeno Fusarium oxysporum e potencialmente eficiente no combate a outros fungos que também utilizem AO no processo de infecção. / Since the domestication of plants for human use, the diseases are causing major production losses. Pathogenic fungi such as Sclerotium rolfsii, Botrytis cinerea, Sclerotinia sclerotiorum and Fusarium oxysporum are capable of infecting various plant species. Infection by these fungi leads to considerable losses at harvest. The initial phase of the infection involves the production and accumulation of large amounts of oxalic acid (OA), which seems to be one of the biggest determinants of pathogenicity. Fusarium oxysporum is the most common species of the genus and causes vascular wilt in different plant species. This fungus causes severe losses in many crops such as cotton, tobacco, bananas, coffee, strawberry and sugar cane. Genes that confer resistance to pathogens become of agronomic importance as resources for improvement. Among these we highlight the enzyme oxalate decarboxylase (OXDC) capable of catalyzing the degradation of OA in formic acid and carbon dioxide, decreasing the infectivity of the fungus. In the T0 generation of tobacco transformed with OXDC gene four resistant fungal strains were obtained, they were analyzed in T1. The objective of this study was to evaluate the segregation of the transgene OXDC for T1 generation of smoke and assess whether the T1 generation of transformed plants are able to resist the pathogen Fusarium oxysporum. Thirty T1 plants of each line of tobacco transformed with OXDC were evaluated by PCR for the presence of HPTII. These four strains were analyzed showed a segregation ratio of 3:1. A positive PCR plant of each strain was subjected to bioassays to verify resistance to the fungus and the OA. In the test of resistance to the fungus Fusarium oxysporum, this was not able to infect the transgenic lines , showing an increase of the resistance of T1 relative to T0 when the results were compared. The levels of transgene expression were assessed by RT-PCR. T1, T4 and T6 strains showed similar levels of expression, the T2 strain showed lower expression level than other strains , so that this result can be correlated with lower resistance to OA. Based on these results it can be concluded that the enzyme Oxalate Descarboxylase demonstrated to be effective in combating the pathogen Fusarium oxysporum and potentially efficient against other fungi which also use OA in the infection process.

Orientadores: Ljubica Tasic, Priscyla Daniely Marcato Gaspari / Tese (doutorado) - Universidade Estadual de Campinas, Instituto de Química / Made available in DSpace on 2018-08-25T19:45:44Z (GMT). No. of bitstreams: 1 Ballottin_DanielaPottMarinho_D.pdf: 4687735 bytes, checksum: 78a932e6ae81a2c4a51ae94b6bf0c65f (MD5) Previous issue date: 2014 / Resumo: Esta tese compreende a produção biogênica e extracelular de nanopartículas de prata (AgNP) utilizando o fungo Fusarium oxysporum, a caracterização físico-química e bioquímica de AgNP e de proteínas, respectivamente. As partículas obtidas foram caracterizadas por técnicas microscópicas, espectroscópicas e de espalhamento dinâmico de luz, as quais forneceram informações em relação a morfologia, tamanho, composição elementar, carga superficial e cristalinidade das partículas. A presença de proteínas estabilizadoras ao redor das partículas foi evidenciada por UV-Vis e TEM. Estas macromoléculas também foram estudas e caracterizadas por diferentes técnicas, tais como, fluorescência, dicroísmo circular, FTIR e Raman. Além disso, foram realizadas análises de eletroforese em gel sendo possível estimar a massa molar das proteínas que estabilizam as AgNP. Algumas destas proteínas foram identificadas por espectrometria de massas, a qual permitiu a obtenção de resultados promissores e inéditos, uma vez que não há na literatura nenhum relato sobre a identificação de proteínas envolvidas na síntese e/ou na estabilização das AgNP. Adicionalmente, foi estudada a atividade antimicrobiana das AgNP frente a diversos micro-organismos patogênicos, como duas espécies de Candida sp. (C. albicans e C. parapsilosis), as quais causam infecções hospitalares, e Xanthomonas axonopodis pv. citri (Xac), uma bactéria patogênica causadora do cancro cítrico, doença com sérias consequências para citricultura brasileira. Nestes estudos foi verificada a alta atividade antimicrobiana das partículas com a atividade inibitória mínima (MIC) da ordem de ?g mL-1. Cito- e genotoxicidade em diferentes organismos e células também foram investigadas, demonstrando que em concentrações utilizadas neste trabalho, as AgNP não apresentam efeito cito- ou genotóxico. São mostrados também resultados da impregnação das AgNP em tecido de algodão e a atividade antimicrobiana deste material frente a C. albicans, C. parapsilosis e Xac / Abstract: This thesis comprises the biogenic and extracellular production os silver nanoparticles (AgNP) using the Fusarium oxysporum fungus, the physic-chemical and biochemical characterization of AgNP and proteins, respectively. The obtained particles were characterized by microscopic, spectroscopic and dynamic light scattering techniques, which provided information regarding the morphology, size, elemental composition, particle surface charge and cristallinity. The presence of surrounding proteins, which stabilize particles was evidenced by UV-Vis and TEM. These macromolecules have also been studied and characterized by different techniques, such as, fluorescence, circular dichroism, Raman and FTIR. Moreover, del electrophoresis analysis were performed ans it was possible to estimate the molar weight of proteins. Some of these were identified by mass spectrometry, which allowed to obtain novel and promising results since there is no reports on the identification of proteins involved in synthesis and/or stabilization of AgNP. Additionally, it was studied the antimicrobial activity of AgNP against several pathogenic micro-organisms, such as, Candida sp. (C. albicans and C. parapsilosis), which cause nosocomial infections, and Xanthomonas axonopodis pv. citri (Xac), a pathogenic bacterium that causes citrus canker, a disease with serious consequences for the Brazilian citrus industry. In these studies it was investigated the high antimicrobial activity of particles through the minimum inhibitory concentration (MIC) values (order of ?g mL-1). Cyto- and genotoxicity in different organisms and cells have also been investigated, showing thet at determined concentrations, AgNP have no cyto- and genotoxic effects. Results of impregnation of AgNP in cotton fabrics and its antimicrobial activity are also shown against C. albicans, C. parapsilosis and Xac / Doutorado / Quimica Organica / Doutora em Ciências

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / The genus Fusarium is characterized by hyaline filamentous fungi that cause a wide spectrum of infections predominantly in immunocompromised patients. These fungal infections show high rates of morbidity and mortality and are difficult to diagnosis, to prevent and to treat. The remarkable primary resistance to antifungal agents of this genus requires the search for new therapeutic possibilities. An attempt is a combination of drugs with different mechanisms of action. This study aimed to evaluate the in vitro susceptibility of Fusarium to conventional antifungal agents (amphotericin B, itraconazole, and voriconazole) and their combinations with no antifungal drugs (azithromycin, ciprofloxacin, fluvastatin, ibuprofen, metronidazole, and rifampicin) against 27 isolates of Fusarium spp. including the species: F. chlamydosporum (3), F. nygamai (1), F. oxysporum (6), F. proliferatum (2), F. solani (11), F. sporotrichoides (1), and F. verticillioides (3). Based on the protocol M38-A2 (CLSI, 2008) the technique of checkerboard was used for the assessment of associations. Alone, the MICs for amphotericin B ranged from 0.125 - 4 μg/ml; for voriconazole they ranged from 1 - >16 μg/ml, and for itraconazole all isolates showed MICs > 16 μg/ml. All combinations with amphotericin B showed synergistic interactions, and the most effective were amphotericin B plus ibuprofen (44.4%), amphotericin B plus metronidazole (40.7%), and amphotericin B plus ciprofloxacin (37%). No antagonistic interactions were observed for associations with amphotericin B. Similarly, all associations with voriconazole showed synergism, and the most significant associations were voriconazole plus ciprofloxacin (33.3%) and voriconazole plus metronidazole (33.3%). The antagonism was observed in all associations with voriconazole against F. verticillioides. The associations with itraconazole showed indifferent interactions for 100% of the isolates tested. These results demonstrate that the in vitro antifungal activity of the drug combination showed better results than the isolated one, except for associations with itraconazole. The most significant associations with amphotericin B and voriconazole deserve in vivo evaluations in order to verify their potential in the treatment of fusariosis. / O gênero Fusarium é caracterizado por fungos filamentosos hialinos que causam um amplo espectro de infecções predominantemente em pacientes imunocomprometidos. Estas micoses evidenciam elevados índices de morbidade e mortalidade e são de difícil diagnóstico, prevenção e tratamento. A marcante resistência primária deste gênero aos antifúngicos impõe a busca por novas possibilidades terapêuticas. Uma tentativa é a combinação entre fármacos com diferentes mecanismos de ação. Este estudo objetivou avaliar a suscetibilidade in vitro do gênero Fusarium a antifúngicos convencionais (anfotericina B, itraconazol e voriconazol) e associações destes com fármacos não antifúngicos (azitromicina, ciprofloxacina, fluvastatina, ibuprofeno, metronidazol e rifampicina) frente a 27 isolados de Fusarium spp., incluindo as espécies: F. chlamydosporum (3), F. nygamai (1), F. oxysporum (6), F. proliferatum (2), F. solani (11), F. sporotrichoides (1) e F. verticillioides (3). Com base no protocolo M38-A2 (CLSI, 2008) empregou-se a técnica de checkerboard para a avaliação das associações. Isoladamente, as CIMs para a anfotericina B variaram de 0,125 - 4 μg/ml; para o voriconazol variaram de 1 - >16 μg/ml e, para o itraconazol todos os isolados evidenciaram CIMs > 16 μg/ml. Todas as combinações com anfotericina B demonstraram interações sinérgicas, sendo que as mais eficazes foram: anfotericina B + ibuprofeno (44,4%), anfotericina B + metronidazol (40,7%) e anfotericina B + ciprofloxacina (37%). Não foram observadas interações antagônicas para as associações com anfotericina B. Similarmente, todas as associações com voriconazol demonstraram sinergismos; sendo que as associações mais significativas foram: voriconazol + ciprofloxacina (33,3%) e voriconazol + metronidazol (33,3%). O antagonismo foi observado em todas as associações com voriconazol frente a F. verticillioides. As associações com itraconazol evidenciaram interações indiferentes para 100% dos isolados testados. Estes resultados demonstram que a atividade antifúngica in vitro de fármacos combinados evidenciaram melhores resultados do que isolados, exceto para as associações com itraconazol. As associações mais relevantes com anfotericina B e voriconazol merecem avaliação in vivo, a fim de verificar o potencial das mesmas no tratamento da fusariose.

La fusariose des épis est l’une des principales maladies des céréales, majoritairement causée par le champignon pathogène et toxinogène, Fusarium graminearum (Fg). Lors son développement in planta, le champignon produit des mycotoxines dommageables pour la santé humaine et animale, dont le déoxynivalénol (DON). De nombreux loci à effet quantitatif sur la résistance à Fg ont été identifiés chez le blé tendre. Certains d’entre eux ont été corrélés à la capacité à détoxifier le DON, en particulier par glucosylation sous l’action d’UDP-glucosyltransférases (UGT). Une UGT d’orge impliquée dans la conjugaison du DON a été identifiée en système hétérologue. Brachypodium distachyon (Bd) a récemment émergé comme modèle d’étude pour les céréales. Ce travail à l’aide d’approches transcriptomique et métabolomique a mis en évidence que lors de l’interaction avec Fg, Bd met en place des réponses macroscopiques, moléculaires et métaboliques similaires à celles connues chez le blé et l’orge. La recherche d’UGTs candidates capables de conjuguer le DON en DON-3-O-glucoside (D3G) chez Bd a permis l’identification d’un candidat. L’analyse fonctionnelle du gène correspondant a été conduite par des approches de mutagenèse et de surexpression. Ceci a montré une sensibilité accrue des lignées mutantes à la toxine et à l’agent pathogène. A l’inverse les lignées surexpresseurs ont montré une tolérance et résistance quantitative à la toxine et l’agent pathogène. Ces résultats ont été corrélés par la détection in planta de DON et D3G, dans des proportions variables selon les lignées. Ces résultats démontrent le rôle majeur que joue la glucosylation du DON dans l’établissement de la résistance observée chez Bd en réponse à Fg. / Fusarium head blight is a major cereal disease, mostly caused by the pathogenic and toxin-producing fungus, Fusarium graminearum (Fg). During its development in planta, the fungus produces mycotoxins harmful to human and animal health, including deoxynivalenol (DON). Many quantitative trait loci exhibiting an effect on resistance to Fg have been identified in wheat. Some of them were correlated with the ability to detoxify DON, particularly by glucosylation by UDP-glycosyltransferases (UGT). A barley UGT involved in the conjugation of DON was identified in a heterologous system. Brachypodium distachyon (Bd) has recently emerged as a model species for cereals. Using transcriptomic and metabolomic approaches, we show that when interacting with Fg, Bd implements macroscopic, molecular and metabolic responses similar to those known in wheat and barley. The search for UGT candidates able to conjugate DON into DON-3-O-glucoside (D3G) in Bd resulted in the identification of the Bradi5g03300 gene. Functional analyses of this gene showed increased sensitivity of the mutant lines to the toxin and to the pathogen. Conversely the overexpressor lines showed a tolerance to the toxin and quantitative resistance to Fg. These results were correlated with the detection of differential amounts of DON and D3G in the different lines. These results demonstrate the important role of DON glucosylation in the resistance establishment of Bd observed in response to Fg.

O Brasil tem uma posição de destaque mundial na produção de frutas e alguns grãos, os quais são comercializados in natura ou na forma de produtos processados. O processamento têm várias vantagens, porém gera grandes quantidades de resíduos, os quais têm sido aproveitados para a alimentação animal e geração de energia. Entretanto, muito tem sido relatado sobre o potencial bioativo desses materiais, dentre eles a ação fungitóxica de alguns de seus compostos sobre fungos fitopatogênicos. Assim, o objetivo desse trabalho foi avaliar o potencial de extratos de resíduos agroindustriais de abacate, uva, café, manga, abacaxi, maracujá e caju na inibição do crescimento micelial in vitro dos fungos Fusarium pallidoroseum, Colletotrichum dematium, Rhizoctonia solani e Phomopsis sp., bem como a composição química do resíduo mais promissor. Os resíduos foram liofilizados, moídos e submetidos a extração com etanol 80% (etanol:água; 8:2, v/v) em banho de ultrassom (extrato denominado bruto ou EB). O EB também foi tratado com a resina Amberlite XAD-2, visando à eliminação de açúcares e interferentes, dando assim origem ao extrato semi-purificado (EP). Os extratos que apresentaram alto rendimento foram avaliados quanto à atividade antifúngica in vitro e teor de compostos fenólicos totais. A amostra que apresentou os melhores índices de inibição foi selecionada para dar continuidade ao estudo, sendo a mesma fracionada em coluna de gel Sephadex LH-20. Os extratos e as frações ativas, detectadas pelo ensaio de bioautografia, foram analisadas quanto a composição química pelas técnicas de HPLC e GC/MS. Na análise de fenólicos totais os maiores teores encontrados foram para a casca de abacate, enquanto que para o ensaio de inibição de crescimento micelial in vitro o melhor resultado foi para a semente de abacate, tanto para o EB quanto para o EP. Desta maneira, a semente de abacate foi selecionada para as etapas posteriores. Para o fracionamento em gel Sephadex LH-20 foi eleito o EP para F. pallidoroseum enquanto que para os demais fungos foi eleito o EB. No fracionamento do EP obtiveram-se 13 frações, sendo que as frações 3 e 4 foram ativas, enquanto que no do EB obtiveram-se nove, sendo as frações 3, 4 e 5 ativas. Pela técnica de HPLC foram detectados em comum nas frações 3, 4 e 5 do EB dois compostos majoritários, e nas frações 3 e 4 do EP, sete compostos, os quais não puderam ser identificados pelos padrões comerciais disponíveis. Já pela técnica de GC/MS foi possível a identificação de quatro compostos em comum nas frações ativas do EB e de 11 nas frações do EP. Dentre os compostos presentes nas frações ativas, foram identificados ácidos graxos, os quais têm sido reconhecidos por apresentarem ação antifúngica. Assim, pode-se concluir que os resíduos agroindustriais estudados são fontes de compostos com atividade antifúngica, podendo assim ser uma alternativa para o controle de fungos fitopatogênicos à cultura da soja / Brazil holds an important position in the world production of fruit and some grains, which are commercialized in natura or in processed products. The processing has several advantages, but generates large amounts of by-products which have been used for animal feed and power generation. However, bioactive potential of these materials have been reported, including the fungitoxic effect. The goal of this study was to evaluate the potential of agro-industrial by-products extracts of avocado, grape, coffee, mango, pineapple, passion fruit and cashew to inhibit in vitro mycelial growth of fungi Fusarium pallidoroseum, Colletotrichum dematium, Rhizoctonia solani and Phomopsis sp. as well as the chemical composition of the most promising by-products. The by-products were freeze-dried, milled and the extraction was done with ethanol 80% (ethanol:water, 8:2, v/v) in an ultrasound bath (called crude extract or CE). The CE was also treated with Amberlite XAD-2 to eliminate sugars and interfering compounds so that the semi-purified extract (PE) could be obtained. Extracts with high yield were evaluated to in vitro antifungal activity and phenolic compounds content. Samples with the best inhibition rates were selected to continue this study, and it was fractioned on gel Sephadex LH-20. The extracts and the active fractions, detected by bioautography, were analyzed by HPLC and GC/MS. The highest phenolic content was found in the avocado peel, meanwhile for the mycelial growth inhibition in vitro, the best result was found in the avocado seeds for both extracts (CE and PE). Thus, avocado seed was selected for subsequent steps. For fractionation on Sephadex LH-20 gel, PE was chosen for F. pallidoroseum and CE for the other fungi. For PE, 13 fractions were obtained in which fractions 3 and 4 were active. For CE, 9 fractions were obtained and the actives were 3, 4 and 5. In CE, HPLC technique detected two major compounds in common in fractions 3, 4 and 5. In PE, seven major compounds were detected in fractions 3 and 4, which could not be identified by commercial available standards. By the GC/MS technique was possible to identify four compounds in common in the active CE fractions and 11 in PE. Among the compounds presented in the active fractions, fatty acids were identified. It has been reported that antifungal action has been found in these compounds. Therefore, the studied agroindustrial by-products are sources of compounds with antifungal activity and they can be used as an alternative to control phytopathogenic fungi on soybean

The diploma thesis concerns observing the health conditions of pea plants. The experiments were established in 2016–2017 in cooperation with AGRITEC, research, breeding and services, s.r.o. company in Šumperk. There were 9 varieties and 3 lines selected for the experimentations. The observation for each experiment was divided into two timing terms. The resistance to pea powdery mildew caused by pathogen Eryshipe pisi, was observed in greenhouse conditions. The highest level of resistance occurred line 1109/13 and variety FRANKLIN. The lowest level of resistance had variety ARVIKA. However this variety also occured to have the highest level of resistance in laboratory tests to resistance to Fusarium solani and Fusarium oxysporum. The pathogens causing the complex of root rot and black stem deseases were observed in conditions of infectious field. Variety ATLAS was infected the least.

Almsjukan är en vissnesjukdom orsakad av invasiva skadesvampar i släktet Ophiostoma. På grund av almsjukan är svenska almar kritiskt hotade och nya metoder att skydda almar behövs därför akut. Biologisk kontroll är en lovande strategi mot trädsjukdomar som almsjukan, men det behövs mer kunskap innan metoden kan tillämpas i praktiken. Endofytsvampar som lever inuti växter utan att orsaka symptom har visat sig kunna forma trädets motståndskraft. I tidigare in vitro studier har en endofytsvamp i släktet Fusarium uppvisat kemisk antagonism mot almsjukepatogener i odling på standardagar. I denna studie undersöktes om samma reaktion även uppkommer när svampar odlas på agar som berikats med almbarkextrakt som innehåller bl.a. fenoliska substanser. I in vitro tester med Fusarium-svampen och två Ophiostoma-isolater upptäcktes att den antagonistiska reaktionen fanns kvar trots almkemikalier, vilket antyder att reaktionen kan vara stabil även inuti almar. Resultaten tyder på att almens kemikalier kan förstärka Fusarium-svampens antagonism mot den aggressivare patogenen, O. novo-ulmi. Studien bekräftar därför Fusarium endofytens potential i biokontroll av almsjukan.

The fungal plant pathogen Fusarium graminearum Schwabe (teleomorph Gibberella zeae¬) produces a dangerous trichothecene mycotoxin called deoxynivalenol (DON) and causes a devastating disease of barley (Hordeum vulgare L.) called Fusarium head blight (FHB). Food and feed products derived from barley, such as dried distillers grains with solubles (DDGS), may be contaminated with DON and pose a threat to the health of humans and domestic animals. New methods to mitigate the threat of DON in barley need to be developed and implemented. TRI101 and TRI201 are trichothecene 3-O-acetyltransferases that modify DON and reduce its toxicity. The first objective of this research was to isolate unique TRI101 and TRI201 enzymes that modify DON efficiently. We hypothesized that TRI101/TRI201 enzymes from different species of Fusarium would have varying rates and abilities to modify DON. Using degenerate primers, an internal portion of TRI101 or TRI201 was identified in 54 strains of Fusarium. Full-length sequences of seven TRI101 or TRI201 genes were cloned and expressed in yeast. All seven genes acetylated DON, but at different rates. The second objective of this research was to utilize transformed yeast expressing TRI101 or TRI201 to reduce DON levels in barley mashes and ultimately in DDGS. We hypothesized that DON levels would be reduced in DDGS derived from mashes prepared with transformed yeast. Five different barley genotypes were used to prepare the fermentation mashes and DON levels were reduced in all DDGS samples derived from mashes prepared with transformed yeast. The third objective of this study was to characterize barley genotypes developed at Virginia Tech for resistance to FHB and DON. We hypothesized that significant differences in resistance would be observed among barley genotypes and FHB resistance would be associated with reduced DON accumulation. From 2006 to 2010, FHB resistance was assessed in hulled (22 to 37) and hulless (13 to 32) barley genotypes by measuring incidence and index, and DON resistance was determined by quantifying DON levels in ground grain using gas chromatography-mass spectrometry. Our study showed that FHB and DON resistance is significantly determined by genotype. The final objective of this study was to develop a robust tissue culture system necessary for future development of transformed barley plants with FHB resistance gene(s). We hypothesized that callus production would vary among barley genotypes. In our analysis of 47 Virginia barley genotypes, 76% (36/47) of the genotypes produced callus tissue and there were significant differences in callus size. Our work sets the stage for identifying and characterizing DON detoxification genes in the future. The development of commercial barley lines that do not accumulate DON and that are resistant to FHB will directly impact growers and producers of small grains in the eastern U.S. / Ph. D.

No description available.

Des études de l'interaction de la plante modèle des légumineuses Medicago truncatula avec des microorganismes pathogènes et symbiotiques ont été réalisées. Tout d'abord un pathosystème fongique a été caractérisé: M. truncatula en interaction avec Fusarium oxysporum spp., champignon responsable des fusarioses, soit du flétrissement vasculaire ou de la pourriture racinaire chez de nombreuses plantes cultivées. Deux lignées de M. truncatula: A17 et F83005.5 ont été identifiées comme sensible et tolérante respectivement à F. oxysporum f.sp. medicaginis, la forma specialis attribuée à la luzerne. De plus 9 souches de F. oxysporum isolées de différentes plantes hôtes et une souche non pathogène du sol ont été testées dans des expériences d'inoculation avec ces deux lignées. Elles ont toutes été capables d'induire les symptômes de la maladie chez M. truncatula. A l'aide d'une souche de F. oxysporum f.sp. medicaginis exprimant le gène rapporteur GFP, les étapes de colonisation de la racine par le champignon ont été caractérisées. Les observations en microscopie à fluorescence et microscopie confocale des racines d'A17 et F83005.5 ont indiqué un patron de colonisation inhabituel et ont montré que la tolérance de F83005.5 n'était pas corrélée à un mécanisme d'exclusion du cylindre central. Cependant, des différences dans l'expression de gènes de défense ont été détectées entre les deux lignées. Dans la deuxième partie, le rôle de l'acide salicylique a été étudié. Les résultats d'expériences avec l'acide salicylique exogène ont indiqué que le prétraitement de racines avec ce composé pouvait conférer une protection aux plantes vis-à-vis de F. oxysporum f.sp. medicaginis et la bactérie phytopathogène Ralstonia solanacearum. Avec l'objectif d'étudier le rôle de l'acide salicylique endogène dans les interactions avec les microorganismes, la transformation génétique de M. truncatula avec le gène NahG codant le salicylate hydroxylase a été entreprise. Cette enzyme dégrade l'acide salicylique en catechol. Seulement la lignée 2HA a pu être transformée et régénérée en plantes transgéniques. Ces plantes 2HA-NahG ont été inoculées avec des microorganismes pathogènes (R. solanacearum, Verticillium albo-atrum, F. oxysporum f. sp. medicaginis Colletotrichum trifolii et C. higginsianum) ainsi qu'avec le champignon endomycorhizien Glomus intraradices. Les limitations expérimentales n'ont pas permis de conclure définitivement, mais indiquent qu'il est possible que la signalisation par l'acide salicylique ne soit pas impliquée dans les défenses de M. truncatula face à ces microorganismes pathogènes et symbiotiques. / A study on the interactions of the plant model legume Medicago truncatula (M.t.) with pathogens and symbiotic microorganisms was undertaken. First, a fungal pathosystem was characterized: M. truncatula in interaction with Fusarium oxysporum spp., the causal agent of Fusariosis, of Fusarium wilt and of root rot in many crop plants. Two M. truncatula lines, A17 and F83005.5, were identified as susceptible and tolerant respectively to F. oxysporum f.sp. medicaginis, the forma specialis related to alfalfa. Besides, 9 strains of F. oxysporum isolated from different host plants and a non-pathogenic soil-borne strain were tested in inoculation experiments with both lines. All the strains were able to trigger disease symptoms in M. truncatula. Using the F. oxysporum f.sp. medicaginis strain transformed with the GFP reporter gene, the stages of the root colonization by the fungi were characterized. Fluorescence and confocal microscopy observations on A17 and F83005.5 roots showed an unusual pattern of colonization and showed that the F83005.5 tolerance was not related to an exclusion mechanism in the central cylinder. However, differences on defence gene expression were detected in both lines. In the second part, the role of salicylic acid was studied. Results of experiments with exogenous salicylic acid indicated that prior treatment of roots with this compound may confer a protection towards F. oxysporum f. sp. medicaginis and the phytopathogenic bacterium Ralstonia solanacearum. With the goal to study the role of endogenous salicylic acid, the genetic transformation of M. truncatula with the NahG gene was initiated. This gene codes for a salicylate hydroxylase which degrades salicylic acid to catechol. Only the highly embryogenic 2HA line could be transformed and regenerated into transgenic plants. These 2HA plants were inoculated with pathogenic microorganisms (Ralstonia solanacearum, Verticillium albo-atrum, Fusarium oxysporum f.sp. medicaginis Colletotrichum trifolii and C. higginsianum) as well as the mycorrhiza fungus Glomus intraradices. Experimental limitations did not allow us to conclude definitely, but it seems possible that the salicylic acid signaling way may not be implicated in the defence of M. truncatula against these pathogenic and symbiotic microorganisms