The focus of my research was on the secondary metabolites produced by crucifer plants under stress and their biological activity against fungi. Both cultivated and wild plants were investigated to isolate phytoalexins and phytoanticipins, and determine their metabolite profiles.<p>The first chapter of this thesis describes cruciferous plants and their most important pathogenic fungi. These plants are divided into three groups: oilseeds, vegetables and wild species. The metabolites isolated from these plants and their biosynthetic studies are reviewed. In addition economically important necrotrophic fungi such as <i>Leptosphaeria maculans</i>, <i>Alternaria brassicae</i>, <i>Sclerotinia sclerotiorum</i> and <i>Rhizoctonia solani</i> are also reviewed along with their phytotoxins. <p>The second chapter of this thesis describes the detection, isolation, structure determination, syntheses of stress metabolites and biological activity of these metabolites against <i>L. maculans</i>, <i>S. sclerotiorum</i> and <i>R. solani</i>. The investigation of cauliflower led to the isolation of seven phytoalexins: 1-methoxybrassitin (55), spirobrassinin (71), isalexin (64), brassicanal C (60), caulilexins A (106), B (107), and C (105). The phytoalexins caulilexins A (106), B (107) and C (105) were reported for the first time. Caulilexin A (106), having a disulfide bridge, showed the highest activity against S. sclerotiorum and R. solani among the known phytoalexins. Similarly four phytoalexins: 1-methoxybrassitin, brussalexins A (121), B (117) and C (118) along with four metabolites: ascorbigen (51), diindolylmethane (50), 1-methoxy-3,3-diindolylmethane (119) and di-(1-methoxy-3-indolyl)methane (120) were isolated from Brussels sprouts. The phytoalexins brussalexins A (121), B (117) and C (118) are new metabolites. Brussalexin A (121) is the only cruciferous phytoalexins having an allyl thiolcarbamate functional group. The metabolite 1-methoxy-3,3-diindolylmethane (119) is reported for the first time.<p>The investigation of brown mustard for polar metabolites led to the isolation of indole-3-acetonitrile (76) and spirobrassinin (71) along with isorhamnetin-3,7-diglucoside (134). Investigation of wild species such as Asian mustard, sand rocket, wallrocket, hedge mustard and Abyssinian mustard for production of stress metabolites led to the isolation of indole-3-acetonitrile (76), arvelexin (84), 1,4-dimethoxyindole-3-acetonitrile (137), rapalexins A (138) and B (142), methyl-1-methoxyindole-3-carboxylate (59) and metabolites bis(4-isothiocyanotobutyl)-disulfide (139), 5-(3-isothiocyanato-propylsulfanyl)-pentylisothiocyanate (136) and 3-(methylsulfinyl)-propylisothiocyanate (135). <p>Two metabolites were also isolated from Brussels sprouts and brown mustard; however, these structures are not yet determined. The metabolites 1,4-dimethoxyindole-3-acetonitrile (137) and 5-(3-isothiocyanato-propylsulfanyl)-pentylisothiocyanate (136) are reported for the first time.

Chemical investigation of the transformation of the crucifer phytoalexins brassinin, brassilexin and camalexin by the phytopathogenic fungus <i>Alternaria brassicicola</i> was carried out. The objectives of this study included:<p> 1) the isolation and characterization of the metabolites of biotransformation of brassinin, brassilexin and camalexin by <i>A. brassicicola</i>;<p> 2) determination of the antifungal activity of these phytoalexins and their metabolites against <i>A. brassicicola.</i><p> The phytoalexins were synthesized and characterized using HPLC retention time tR, 1H NMR, 13C NMR, LC-MS and HRMS-ESI data. The metabolites of the biotransformation were also synthesized and characterized similarly. The metabolism of each phytoalexin and their metabolites was studied by analyzing broth extracts by HPLC. The percent inhibition of growth of <i>A. brassicicola</i> was determined by radial growth mycelial assays.<p> The biotransformation of brassinin by <i>A. brassicicola</i> afforded Nb-acetyl-3-indolylmethylamine via indole-3-methylamine intermediate. Brassilexin was metabolized to 3-(amino)methyleneindoline-2-thione by the reduction of the isothiazole ring. Camalexin did not appear to be metabolized or the metabolism was very slow. The results of biotransformation and bioassay studies established that the metabolism of brassinin by <i>A. brassicicola</i> was a detoxification process. However, these studies using brassilexin did not provide a rigorous conclusion. Camalexin showed strong inhibition of growth against <i>A. brassicicola</i> suggesting its importance in defense against this pathogen.

Thesis (Ph. D.)--University of Texas at Austin, 2002. / Vita. Includes bibliographical references. Available also from UMI Company.

M.Sc. / Inducible defence responses in both a susceptible cotton cultivar ( Acala ) and a resistant cotton cultivar ( OR-19 ) in response to elicitors fromVerticillium dahliae were investigated. These oligosaccharin elicitors represent the heat solubilized, non-dialyzable fraction of the pathogen cell wall. This elicitorfraction consist of 6.57 % protein and 68 % carbohydrate and represent merely a discrete portion of the cell wall. Moreover, symptoms such as chlorosis and necrosis were induced by the elicitor in both cultivars, but the timing and magnitute of symptom development differed in that the symptoms occurred much faster and were more intense ( hypersensitive response) in the resistant cultivar. An effective elicitor concentration of 30 ug/ ml ( = 21 ug glucose equivalents) was determined and used throughout this study. Inducible defence responses i.e, the accumulation of PR-proteins and specifically activities of B-1,3-glucanase and chitinase were investigated in the intercellular environment as well as cellular extractions of both cultivars. An attempt was also made to analyse the expression of B-1 ,3-glucanase and chitinase genes at them RNA level ( level of transcription) and to correlate it to the determined levels of enzyme activities. Lignification as well as smaller plant metabolites i.e, sesquiterpenoid-phytoalexins relating to pathogenesis that are induced by the elicitor, were also investigated in both cultivars. In this study it is shown that differences are found between Acala and OR-19 and that some of these differences can be correlated to plant resistance. These differences found in the time studies ( intensity, time of response, qualitative and quantitative differences ) of the defence responses induced in both cultivars is discussed against the background of disease resistance.

A indução de resistência em plantas contra fitopatógenos é um método alternativo de controle de doenças, o qual envolve a ativação de mecanismos de resistência latentes da planta. Hoje no mercado existem poucos produtos que atuam segundo este princípio. Na busca de novas moléculas que possam ser utilizadas em campo, diversos compostos de origem microbiana, com capacidade de estimular uma ou mais respostas de defesa, já foram isolados e caracterizados. A Saccharomyces cerevisiae é uma levedura capaz de induzir resistência e elicitar respostas de defesa em algumas plantas. Com o objetivo de extrair da levedura um ou mais compostos capazes de induzir o acúmulo de fitoalexinas em cotilédones de soja e na proteção dessa leguminosa contra Microsphaera diffusa (agente causal do oídio da soja) e de pepino contra Colletotrichum lagenarium (agente causal da antracnose do pepino), células em suspensão foram autoclavadas. Os compostos assim extraídos foram inicialmente separados através de precipitação etanólica. Em seguida, foram realizadas cromatografias de troca iônica e de afinidade para separar as frações com maior poder elicitor das de baixo poder elicitor. A fração não adsorvida à resina DEAE-Celulose foi a que induziu maior acúmulo de fitoalexinas. No entanto, nenhum dos preparados testados foi capaz de conferir proteção a plantas de soja contra M. diffusa. Já no caso de pepino, plântulas tratadas com as frações resultantes da cromatografia de afinidade apresentaram reduções entre 50 e 70 % de área lesionada causada por C. lagenarium e aumento na atividade de peroxidases. Extratos incorporados à meio de cultivo não apresentaram efeito inibitório sobre o crescimento e esporulação de C. lagenarium. Com base nesses resultados, concluímos que existe na parede da levedura compostos capazes de induzir resistência local em pepino contra C. lagenarium, sendo que pelo menos um destes compostos é um carboidrato, contendo provavelmente manana e glucosamina. / The acquired resistance of plants to pathogens is an alternative method to control diseases which includes the activation of resistance mechanisms in the plants. A few products already commercially available have their action based upon this mechanism. In the search for novel molecules that can be used under field conditions, many compounds from microbes with the ability to stimulate one or more defense responses were already isolated and characterized. Saccharomyces cerevisiae is an yeast with the ability to induce defense responses and resistance in some plants. A suspension of cells from the yeast was autoclaved with the purpose of extracting one or more compounds with the ability to induce the accumulation of phytoalexins in soybean cotyledons and to protect soybean plants against Microsphaera diffusa (causal agent of powdery mildew) and plants of cucumber against Colletotrichum lagenarium (causal agent of anthracnose). The compounds extracted by this method were separated using ethanolic precipitation. After this step, the fractions of higher elicitation activity were separated from those of lower one by using ion exchange cromatography and affinity cromatography. The non-adsorbed fraction to DEAE-Cellulose was the one that induced the highest accumulation of phytoalexins. However, none of the fractions were able to protect soybean plants from M. diffusa. In the case of cucumber, seedlings treated with the fractions from affinity chromatography were able to reduce disease symptoms caused by C. lagenarium by 50 to 70 % and to increase the activity of peroxidases. Extracts that were incorporated into growing media did exhibit any inhibitory effect on in vitro growth and sporulation of C. lagenarium. According to these results, it is possible to conclude that there are compounds in the cell walls of the yeast that are able to induce local resistance to C. lagenarium in cucumber and that at least one of these compounds is a carbohydrate that likely contains mannan and glucosamine.

Not available / text

Orientador: Marcos Aparecido Gimenes / Resumo: O gênero Arachis possui 81, espécies distribuídas em nove seções taxonômicas, sendo o amendoim (A. hypogaea L.) a principal espécie, devido ao seu alto valor nutricional. O gênero possui também outras espécies de interesse econômico, que têm sido utilizadas em programas de melhoramento do amendoim (espécies da seção Arachis) e espécies forrageiras de outras seções do gênero. Com relação às demais espécies, o germoplasma da grande maioria está disponível para a caracterização e pode, sem dúvida, contribuir para seu uso futuro. O gênero Arachis é um dos poucos que produzem o resveratrol, uma fitoalexina que, além de ser uma resposta da planta contra estresses, também tem ação antioxidante, sendo indicado para prevenção de cânceres e cardiopatias. A avaliação do potencial de produção de resveratrol foi realizada somente em algumas espécies da seção Arachis, faltando avaliação em outras espécies do gênero e nos tetraploides sintéticos que estão sendo utilizados para facilitar a transferência de alelos de espécies silvestres para a cultivada. Assim, o objetivo desse trabalho foi ampliar o conhecimento sobre resveratrol no gênero Arachis. Para tanto foram analisados 21 acessos de espécies silvestres de cinco diferentes seções do gênero Arachis, oito poliploides sintéticos e três cultivares de amendoim. A produção de resveratrol em um conjunto menor de cinco espécies foi acompanhada também de avaliação da expressão do gene que codifica a enzima resveratrol sintase, RS, para entende... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The Arachis genus comprises 81 species divided into nine taxonomic sections, being peanut (A. hypogaea L.) the main species due to its high nutritional value. The genus also has other species of economic interest, which have been used in breeding programs for peanut (species of Arachis section) and to forage production. Regarding the other species of Arachis, germplasm of most of them is available and their characterization could undoubtedly contribute to their future use. Arachis genus is one of the few that produces resveratrol, a phytoalexin that is synthesized by the plant due to a stress and it is also a antioxidant that has been used to prevention of cancers and heart diseases. The resveratrol evaluation in Arachis was carried out mainly in Arachis hypogaea and in some species of the section Arachis, lacking the evaluation in other species of the genus and in the synthetic tetraploids that have been used to facilitate the transfer of alleles from wild species to the cultivated one. Therefore, the objective of this study was to increase the knowledge about resveratrol in the genus Arachis. Twenty one accessions of wild species from five different sections of the genus Arachis, eight synthetic polyploids and three peanut cultivars were analyzed. The production of resveratrol in a smaller set of five species was also accompanied of evaluation of resveratrol synthase to try to understand the variation observed in the resveratrol content among the species. Since each experim... (Complete abstract click electronic access below) / Doutor

Submitted by PAULA ANDRÉA SAMPAIO DE VASCONCELOS CARVALHO null (paulasvasconcelos@hotmail.com) on 2017-10-06T14:46:03Z No. of bitstreams: 1 tese pos defesa Paula Andréa Sampaio de Vasconcelos Carvalho.pdf1.pdf: 1988306 bytes, checksum: 46c0925bf8ca00155c63f2c83d48acac (MD5) / Rejected by Monique Sasaki (sayumi_sasaki@hotmail.com), reason: Solicitamos que realize uma nova submissão seguindo a orientação abaixo: O arquivo submetido está sem a ficha catalográfica. A versão submetida por você é considerada a versão final da dissertação/tese, portanto não poderá ocorrer qualquer alteração em seu conteúdo após a aprovação. Corrija esta informação e realize uma nova submissão contendo o arquivo correto. Agradecemos a compreensão. on 2017-10-09T17:38:04Z (GMT) / Submitted by PAULA ANDRÉA SAMPAIO DE VASCONCELOS CARVALHO null (paulasvasconcelos@hotmail.com) on 2017-10-18T21:09:07Z No. of bitstreams: 1 tese Paula Andréa Sampaio de Vasconcelos Carvalho.pdf com ficha.pdf: 2023210 bytes, checksum: 0f11f1ef3d125e88872b67188fcfff93 (MD5) / Approved for entry into archive by Luiz Galeffi (luizgaleffi@gmail.com) on 2017-10-23T13:23:01Z (GMT) No. of bitstreams: 1 carvalho_pasv_dr_bot.pdf: 2023210 bytes, checksum: 0f11f1ef3d125e88872b67188fcfff93 (MD5) / Made available in DSpace on 2017-10-23T13:23:02Z (GMT). No. of bitstreams: 1 carvalho_pasv_dr_bot.pdf: 2023210 bytes, checksum: 0f11f1ef3d125e88872b67188fcfff93 (MD5) Previous issue date: 2017-08-07 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O gênero Arachis possui 81, espécies distribuídas em nove seções taxonômicas, sendo o amendoim (A. hypogaea L.) a principal espécie, devido ao seu alto valor nutricional. O gênero possui também outras espécies de interesse econômico, que têm sido utilizadas em programas de melhoramento do amendoim (espécies da seção Arachis) e espécies forrageiras de outras seções do gênero. Com relação às demais espécies, o germoplasma da grande maioria está disponível para a caracterização e pode, sem dúvida, contribuir para seu uso futuro. O gênero Arachis é um dos poucos que produzem o resveratrol, uma fitoalexina que, além de ser uma resposta da planta contra estresses, também tem ação antioxidante, sendo indicado para prevenção de cânceres e cardiopatias. A avaliação do potencial de produção de resveratrol foi realizada somente em algumas espécies da seção Arachis, faltando avaliação em outras espécies do gênero e nos tetraploides sintéticos que estão sendo utilizados para facilitar a transferência de alelos de espécies silvestres para a cultivada. Assim, o objetivo desse trabalho foi ampliar o conhecimento sobre resveratrol no gênero Arachis. Para tanto foram analisados 21 acessos de espécies silvestres de cinco diferentes seções do gênero Arachis, oito poliploides sintéticos e três cultivares de amendoim. A produção de resveratrol em um conjunto menor de cinco espécies foi acompanhada também de avaliação da expressão do gene que codifica a enzima resveratrol sintase, RS, para entender a variação observada no teor de resveratrol entre as espécies. Tendo em vista que cada experimento foi realizado em uma época diferente do ano e de modo separado, não foi possível reunir todos os dados e fazer uma comparação entre eles. Em todos os experimentos foram avaliadas folhas submetidas a estresse abiótico por raios ultravioleta (UV), que foi o melhor indutor de síntese em dados de literatura. Os resultados mostraram que 21 acessos de 17 espécies das cinco seções são capazes de produzir resveratrol, sendo o maior teor detectado em A. lignosa, da seção Procumbentes. Em relação ao potencial de produção dos híbridos, foi verificado que a concentração de resveratrol na maioria dos anfidiploides sintéticos é significativamente maior em relação aos parentais silvestres diploides. Além disso, a concentração de resveratrol nos híbridos resultantes do cruzamento entre anfidiploides e A. hypogaea foi significativamente maior que nos anfidiploides, sugerindo que a combinação entre alelos de silvestres e do cultivado tem um efeito positivo no aumento do resveratrol. Em relação às análises moleculares, notou-se que a expressão do gene é imediata ao término da indução, seguindo de forma decrescente. Por outro lado, as concentrações de resveratrol são crescentes em relação ao passar do tempo de coleta após a indução por UV. Os resultados demonstram o potencial do gênero como fonte de resveratrol, seja via amendoim cultivado, por meio de introgressão de alelos silvestres utilizando-se anfidiploides, ou pelo uso per se dos acessos de espécies silvestres, incluindo de outras seções distantes do amendoim, como é a Procumbentes, da qual faz parte A. lignosa, que apresentou o maior teor de resveratrol no estudo. / The Arachis genus comprises 81 species divided into nine taxonomic sections, being peanut (A. hypogaea L.) the main species due to its high nutritional value. The genus also has other species of economic interest, which have been used in breeding programs for peanut (species of Arachis section) and to forage production. Regarding the other species of Arachis, germplasm of most of them is available and their characterization could undoubtedly contribute to their future use. Arachis genus is one of the few that produces resveratrol, a phytoalexin that is synthesized by the plant due to a stress and it is also a antioxidant that has been used to prevention of cancers and heart diseases. The resveratrol evaluation in Arachis was carried out mainly in Arachis hypogaea and in some species of the section Arachis, lacking the evaluation in other species of the genus and in the synthetic tetraploids that have been used to facilitate the transfer of alleles from wild species to the cultivated one. Therefore, the objective of this study was to increase the knowledge about resveratrol in the genus Arachis. Twenty one accessions of wild species from five different sections of the genus Arachis, eight synthetic polyploids and three peanut cultivars were analyzed. The production of resveratrol in a smaller set of five species was also accompanied of evaluation of resveratrol synthase to try to understand the variation observed in the resveratrol content among the species. Since each experiment was performed at a different time of year and separately, it was not possible to gather all the data and make a comparison between them. In all experiments, leaves treated with ultraviolet radiation (UV) were evaluated, which was the best inducer of synthesis in literature data. The results showed that 21 accessions of 17 species of the 5 sections are able to produce resveratrol, being the highest content detected in A. lignosa from Procumbentes section. It was verified that the concentration of resveratrol in the majority of the synthetic amphidiploids is significantly higher in relation to the diploid wild parents. In addition, the concentration of resveratrol in the hybrids resulting from the crossing between amphidiploids and A. hypogaea was significantly higher than in amphidiploids, suggesting that the combination of wild and cultivated alleles has a positive effect on the increase of resveratrol. It was noticed that the expression of the gene is immediate at the end of the induction, following in a decreasing way. On the other hand, the concentrations of resveratrol are increasing in relation to the time of collection after the induction of the material. The results demonstrate the potential of wild species of genus Arachis as a 10 source of resveratrol, either via cultivated peanuts, through introgression of wild alleles using amphidiploids, or through the use of wild species accesses, including from other sections distant from peanut, such as A. lignosa.

A indução de resistência em plantas contra fitopatógenos é um método alternativo de controle de doenças, o qual envolve a ativação de mecanismos de resistência latentes da planta. Hoje no mercado existem poucos produtos que atuam segundo este princípio. Na busca de novas moléculas que possam ser utilizadas em campo, diversos compostos de origem microbiana, com capacidade de estimular uma ou mais respostas de defesa, já foram isolados e caracterizados. A Saccharomyces cerevisiae é uma levedura capaz de induzir resistência e elicitar respostas de defesa em algumas plantas. Com o objetivo de extrair da levedura um ou mais compostos capazes de induzir o acúmulo de fitoalexinas em cotilédones de soja e na proteção dessa leguminosa contra Microsphaera diffusa (agente causal do oídio da soja) e de pepino contra Colletotrichum lagenarium (agente causal da antracnose do pepino), células em suspensão foram autoclavadas. Os compostos assim extraídos foram inicialmente separados através de precipitação etanólica. Em seguida, foram realizadas cromatografias de troca iônica e de afinidade para separar as frações com maior poder elicitor das de baixo poder elicitor. A fração não adsorvida à resina DEAE-Celulose foi a que induziu maior acúmulo de fitoalexinas. No entanto, nenhum dos preparados testados foi capaz de conferir proteção a plantas de soja contra M. diffusa. Já no caso de pepino, plântulas tratadas com as frações resultantes da cromatografia de afinidade apresentaram reduções entre 50 e 70 % de área lesionada causada por C. lagenarium e aumento na atividade de peroxidases. Extratos incorporados à meio de cultivo não apresentaram efeito inibitório sobre o crescimento e esporulação de C. lagenarium. Com base nesses resultados, concluímos que existe na parede da levedura compostos capazes de induzir resistência local em pepino contra C. lagenarium, sendo que pelo menos um destes compostos é um carboidrato, contendo provavelmente manana e glucosamina. / The acquired resistance of plants to pathogens is an alternative method to control diseases which includes the activation of resistance mechanisms in the plants. A few products already commercially available have their action based upon this mechanism. In the search for novel molecules that can be used under field conditions, many compounds from microbes with the ability to stimulate one or more defense responses were already isolated and characterized. Saccharomyces cerevisiae is an yeast with the ability to induce defense responses and resistance in some plants. A suspension of cells from the yeast was autoclaved with the purpose of extracting one or more compounds with the ability to induce the accumulation of phytoalexins in soybean cotyledons and to protect soybean plants against Microsphaera diffusa (causal agent of powdery mildew) and plants of cucumber against Colletotrichum lagenarium (causal agent of anthracnose). The compounds extracted by this method were separated using ethanolic precipitation. After this step, the fractions of higher elicitation activity were separated from those of lower one by using ion exchange cromatography and affinity cromatography. The non-adsorbed fraction to DEAE-Cellulose was the one that induced the highest accumulation of phytoalexins. However, none of the fractions were able to protect soybean plants from M. diffusa. In the case of cucumber, seedlings treated with the fractions from affinity chromatography were able to reduce disease symptoms caused by C. lagenarium by 50 to 70 % and to increase the activity of peroxidases. Extracts that were incorporated into growing media did exhibit any inhibitory effect on in vitro growth and sporulation of C. lagenarium. According to these results, it is possible to conclude that there are compounds in the cell walls of the yeast that are able to induce local resistance to C. lagenarium in cucumber and that at least one of these compounds is a carbohydrate that likely contains mannan and glucosamine.

Submitted in fulfillment of the requirements for the Degree of Master of Technology: Biological sciences at the ML Sultan Technikon, 2000. / Plant medicine is an important area of commercial activity in South Africa. This is a rapidly expanding market, thus we are evaluating natural and stressinduced compounds (phytoalexins) from plants as agents that may be able to control mycotoxins. Natural compounds from Bridelia micrantha, Warburgia salutaris, Lippia javanica and Scenecio serratuloides and stress-induced compounds (phytoalexins) from Citrus sinensis cv Valencia were screened for antitunqal and antimycotoxic activity by bioautography against a test organism (Cladosporium cladosporoides) and mycotoxin producing fungi (Fusarium moniliforme and Aspergillus flavus). / M