Universiẗat, Diss., 2005--Bonn.

Submitted by Reginaldo Soares de Freitas (reginaldo.freitas@ufv.br) on 2018-01-19T11:42:45Z No. of bitstreams: 1 texto completo.pdf: 804820 bytes, checksum: 1d6c01f9b6cbd5f4e3e61bbba11e4db6 (MD5) / Made available in DSpace on 2018-01-19T11:42:45Z (GMT). No. of bitstreams: 1 texto completo.pdf: 804820 bytes, checksum: 1d6c01f9b6cbd5f4e3e61bbba11e4db6 (MD5) Previous issue date: 2017-07-21 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A eficiência da hidrólise da biomassa lignocelulósica apresenta algumas limitações, dentre elas a possível adsorção inespecífica das enzimas à lignina. Os objetivos deste trabalho foram avaliar o perfil de adsorção de proteínas e enzimas do fungo fitopatogênico Chrysoporthe cubensis em bagaços de cana-de-açúcar pré-tratados e com diferentes conteúdos de lignina e avaliar a eficiência da recuperação destas enzimas e proteínas utilizando metodologias de reciclagem enzimática. De modo a obter o extrato enzimático, o fungo foi cultivado sob fermentação em estado semi-sólido com farelo de trigo. Os bagaços de cana foram pré-tratados com NaOH (BAL) e com H 2 SO 4 (BAC), de modo a conter 8,1% e 33,8% de lignina, respectivamente. A isoterma de Langmuir obtida pelo experimento em condições não hidrolíticas, revelou os parâmetros Pmax e Kp superiores para BAC, e K superior para BAL. Ao final da hidrólise, BAC foi capaz de adsorver maiores quantidades de proteínas, comparado ao substrato BAL. As enzimas celobiohidrolase e xilanase sofreram maiores adsorções em BAC, e de modo contrário, a endoglucanase adsorveu majoritariamente em BAL. A β-glicosidase foi a enzima que menos adsorveu em ambos os substratos. Esses resultados demonstraram o efeito da lignina presente em biomassas complexas ao promover uma maior adsorção de proteínas e de algumas enzimas do extrato enzimático de C. cubensis. Dentre os experimentos de reciclagem enzimática, todos demonstraram ser eficientes na recuperação de proteínas e enzimas adsorvidas na biomassa. A reciclagem com adição de substratos e enzimas frescas foi capaz de promover a maior eficiência de hidrólise da biomassa. Entretanto, para BAC, com elevado conteúdo de lignina, as reciclagens de enzimas e proteínas não demonstraram ser tão eficazes, bem como a hidrólise de açúcares. Desse modo, conclui-se que a lignina presente em altas concentrações nos substratos, além de promover maiores adsorções de proteínas e enzimas, também interfere nos métodos de reciclagem. Também foi comprovada a alta eficiência de reciclagem das enzimas de C. cubensis, mesmo no substrato contendo alta concentração de lignina, além da alta termoestabilidade dessas enzimas, mesmo após 216 h de hidrólise. Desse modo, é confirmado o grande potencial hidrolítico do extrato enzimático obtido desse fungo. / The efficiency of biomass hydrolysis by enzymes has some limitations, such as the possible non-specific adsorptions of enzymes in lignin. The aims of this work were to evaluate the adsorption profile of proteins and enzymes from the phytopathogenic fungus Chrysoporthe cubensis in pretreated sugarcane bagasses with different lignin contents and to evaluate the efficiency of recuperation of this enzymes and proteins using enzymatic recycling methodologies. In order to obtain the enzymatic extract, the fungus was cultivated under semi-solid state fermentation with wheat bran. The sugarcane bagasses were pretreated with NaOH (BAL) and H 2 SO 4 (BAC), in order to obtain 8.1% and 33.8% of lignin, respectively. The Langmuir isotherm obtained by the experiment under non-hydrolytic conditions showed the highest Pmax and Kp parameters for BAC, while K was higher for BAL. At the end of hydrolysis, BAC was able to adsorb greater amounts of proteins compared to BAL. The enzymes cellobiohydrolase and xylanase showed higher adsorption on BAC, and endoglucanase adsorbed mostly on BAL. β-glycosidase was the enzyme that suffered less adsorption on both substrates. These results demonstrate8d the negative effect of the lignin present in complex biomasses which promotes greater adsorption of proteins and some enzymes of the C. cubensis enzymatic extract. Among the enzymatic recycling experiments, all of them were efficient in the recovery of proteins and enzymes adsorbed in biomass. The recycling with the addition of fresh substrates and enzymes was able to promote higher biomass hydrolysis yields. However, for BAC, with high lignin content, the enzymes and proteins recycling did not show to be effective, as well as the sugars hydrolysis. Thus, it is concluded that high concentrations of lignin in the substrates, besides promoting greater adsorption of proteins and enzymes, also interferes in recycling methods. The results also demonstrated the high recycling efficiency of C. cubensis enzymes, even in the substrate containing high lignin concentration, besides the high thermostability of these enzymes, even after 216 h of hydrolysis. Thus, the great hydrolytic potential of enzymatic extract obtained from this fungus is confirmed.

Cryphonectria cubensis is an ascomycetous fungus that causes a serious canker disease on Eucalyptus trees in many parts of the world. The importance of the disease has led to numerous studies involving the taxonomy, genetic diversity and the control of Cryphonectria canker. However, there remain many questions pertaining to the disease that have not been considered. The objectives of the studies presented in this thesis were, therefore, to investigate the possibility of biological control of Cryphonectria canker, to evaluate the currently used disease screening strategy in South Africa and to establish a transfection system with dsRNA elements in Diaporthe, which is closely related to Cryphonectria. The introductory chapter of this thesis provides a review of the literature pertaining to Cryphonectria cubensis. In addition literature on hypovirulence in fungi is also extensively reviewed, with a special emphasis on the genus Cryphonectria. The aim of study in the second chapter of the thesis was to screen the South African C. cubensis population for the presence of dsRNA viruses. Two viruses were identified and the full sequence of these elements showed a strong homology to the mitochondrial viruses (mitoviruses) within the family Narnaviridae. We, therefore, named the viruses Cryphonectria cubensis mitovirus 1 (CcMV1) and Cryphonectria cubensis mitovirus 2 (CcMV2). The two viral genomes are 2601 nucleotides and 2639 nucleotides in size respectively and encode for a protein that probably functions as an RNA-dependant RNA polymerase (RdRp). Pathogenicity studies indicated that the viruses do not result in a significant reduction in pathogenicity of C. cubensis. In the third chapter, results of a study to consider whether different Eucalyptus clones responded similarly to various South African C. cubensis isolates, are presented. The aim was, therefore, to evaluate the current C. cubensis resistant screening method used on Eucalyptus spp. in South Africa. The statistical analysis of the inoculation data showed a significant isolate x clone interaction. This data also suggest the possibility of vertical resistance, which is different to previous assumptions. Transfection studies (Chapter 4) involving a positive stranded RNA virus, Diaporthe RNA virus (DaRV) from a South African D. perjuncta isolate are presented here. In this study, a virus free D. perjuncta isolate, a virulent C. cubensis isolate and a hypovirulent C. cubensis isolate containing the hypovirus CHV1-EP713 were chosen to be transfected with DaRV. By using electroporation, it was possible to infect a virus free D. perjuncta isolate with the Diaporthe RNA virus, thus extending the transfection range of this virus. The resulting transfection led to altered colony morphology but did not lead to a reduction in pathogenicity. We were also not successful in attempts to transfect isolates of C. cubensis with DaRV, indicating that the virus does not replicate in this host. In a previous study a virulent South African C. cubensis isolate was transfected with the Cryphonectria parasitica hypovirus CHV1-EP713. This resulted in the fungus becoming hypovirulent. Chapter five of this thesis presents the results of a study to evaluate the potential use of this virus in the biological control of Cryphonectria canker in South Africa. A field trial was established and existing cankers were treated with the transfected isolate. The treatment of the cankers did not lead to a significant reduction in canker size, but did alter the morphology of the cankers. The virus was also shown to be transmitted via hyphal anastomosis to the virulent canker causing isolates. In addition the co-inoculation on single trees with both the virulent and virus-containing isolate, resulted in a significant reduction in the size of the lesions. This study also showed that the transfected C. cubensis isolate are characterised by significantly smaller lesions than those associated with the virulent, virus-free isolate. Cryphonectria cubensis and the associated canker disease of Eucalyptus threaten the forestry industry in South Africa. The overall aims of the studies presented in this thesis were to gain a more complete understanding of this fungus and to evaluate potential control strategies. Each of these chapters should contribute towards a better understanding of the viruses associated with C. cubensis and other important aspects of Cryphonectria canker, which will hopefully lead to enhanced control strategies of the disease in South Africa. / Thesis (PhD)--University of Pretoria, 2008. / Microbiology and Plant Pathology / PhD / Unrestricted

Made available in DSpace on 2014-06-11T19:28:36Z (GMT). No. of bitstreams: 0 Previous issue date: 2008-12-15Bitstream added on 2014-06-13T18:58:00Z : No. of bitstreams: 1 souza_hg_me_botfca.pdf: 1426991 bytes, checksum: 7c8beacd93595a04b7e0ef427ee21af6 (MD5) / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O termo cancro é utilizado para designar lesões localizadas na casca do caule de plantas lenhosas que podem resultar no anelamento do caule e no secamento da parte superior da copa e a morte da planta. A melhor opção para o controle da doença é a resistência genética dos indivíduos quanto ao patógeno utilizado. Com o objetivo de estudar o comportamento de resistência e mecanismos moleculares de clones dos parentais e das progênies de eucalipto quanto ao cancro causado por Chrysoporthe cubensis e Botryosphaeria sp. foram executados ensaios em casa de vegetação e em campo. Os isolados utilizados foram Chrysoporthe cubensis e Botryosphaeria sp. sendo produzidos em meio BDA e inoculados nos indivíduos pelo método do disco. A avaliação foi realizada 60 dias após a inoculação, medindo a área da lesão de cada material. Para o patógeno C. cubensis inoculados nos parentais foi encontrado uma média de 3,20 e 6,14 cm2 de área de lesão do tronco causado pelos isolados provenientes de Piracicaba e Bofete, respectivamente, enquanto que para Botryosphaeria sp. a média variou de 7,29 cm ² a 9,72cm ² da área da lesão. Dos indivíduos testados, com C. cubensis, foram encontrados 46,66% de plantas classificadas como resistentes; 6,67% plantas moderadamente resistente, 13,33% planta moderadamente suscetíveis e 33,34% plantas suscetíveis. Já para o teste de clones com Botryosphaeria sp. verificou-se 91,67% resistentes, 5% moderadamente resistentes, 1,67% moderadamente suscetível e 1,67% suscetíveis. As variâncias fenotípicas encontradas foram de 41,32 a 0,23 inoculados com C. cubensis e 0,285 a 156,88, quando inoculados com Bothyosphaeria sp. Os coeficientes de variação foram de 125% a 15,05% para C. cubensis e 29,86% a 101,13% para Bothyosphaeria sp. Os resultados indicam que os materiais genéticos testados possuem boa fonte de resistência para os dois fitopatógenos... / The term canker is used to assign to injuries located in the bark of stems of woody plants that can result in the anelamento of stems and drying of the superior part of the crown and the death of the plant. The best option for the control of the illness is the resistance genetic of the individuals concerning to the used patógeno. The objective of this study the molecular behavior of resistance and mechanisms of clonals of parental and the lineages the eucalyptus concerning the canker caused for Chrysoporthe cubensis and Dothiorella sp. The essays executed in the greenhouse and field. The isolated ones used concerning Chrysoporthe cubensis and Dothiorella sp. being produced in inoculated medium BDA and in the individuals for the method of the disk. The evaluation was concluded 60 days after the inoculation, measuring the area of the injury of each material. For pathogeno C. cubensis inoculated in the parental ones a 6,14 was found average of 3,20 and cm² of area of isolated injury of the trunk caused for the proceeding ones from Piracicaba and Bofete, respectively. The average of the area of the injury for the studied lineages varied of 7,29 cm ² 9,72cm ² for Dothiorella sp. The clonals inoculated C. cubensis were grouped into the categories HS (highly susceptible – 33,34%), MS (moderately susceptible -13,3%), MR (moderately resistant- 6,67%), and HR (highly resistant -46,66%). The clonal tested, with Botryosphaeria sp., verified resistant 91.67%, 5% moderately resistant, 1.67% moderately susceptible and 5,26% susceptible. The phenotypic variation found was from 41,32 to 0,23, inoculated with C. cubensis while Botryosphaeria sp from 0,285 to156,88. The coefficient of variation from 125% to 15,95% for C. cubensis and 29,86% to 101,13% for Botryosphaeria sp. The results indicate that the tested genetic materials possess good source of resistance for the two fitopatógenos tested... (Complete abstract click electronic access below)

Orientador: Edson Luis Furtado / Banca: Marli Teixiera de Almeida Minhoni / Banca: Celso Garcia Auer / Resumo: O termo cancro é utilizado para designar lesões localizadas na casca do caule de plantas lenhosas que podem resultar no anelamento do caule e no secamento da parte superior da copa e a morte da planta. A melhor opção para o controle da doença é a resistência genética dos indivíduos quanto ao patógeno utilizado. Com o objetivo de estudar o comportamento de resistência e mecanismos moleculares de clones dos parentais e das progênies de eucalipto quanto ao cancro causado por Chrysoporthe cubensis e Botryosphaeria sp. foram executados ensaios em casa de vegetação e em campo. Os isolados utilizados foram Chrysoporthe cubensis e Botryosphaeria sp. sendo produzidos em meio BDA e inoculados nos indivíduos pelo método do disco. A avaliação foi realizada 60 dias após a inoculação, medindo a área da lesão de cada material. Para o patógeno C. cubensis inoculados nos parentais foi encontrado uma média de 3,20 e 6,14 cm2 de área de lesão do tronco causado pelos isolados provenientes de Piracicaba e Bofete, respectivamente, enquanto que para Botryosphaeria sp. a média variou de 7,29 cm ² a 9,72cm ² da área da lesão. Dos indivíduos testados, com C. cubensis, foram encontrados 46,66% de plantas classificadas como resistentes; 6,67% plantas moderadamente resistente, 13,33% planta moderadamente suscetíveis e 33,34% plantas suscetíveis. Já para o teste de clones com Botryosphaeria sp. verificou-se 91,67% resistentes, 5% moderadamente resistentes, 1,67% moderadamente suscetível e 1,67% suscetíveis. As variâncias fenotípicas encontradas foram de 41,32 a 0,23 inoculados com C. cubensis e 0,285 a 156,88, quando inoculados com Bothyosphaeria sp. Os coeficientes de variação foram de 125% a 15,05% para C. cubensis e 29,86% a 101,13% para Bothyosphaeria sp. Os resultados indicam que os materiais genéticos testados possuem boa fonte de resistência para os dois fitopatógenos... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The term canker is used to assign to injuries located in the bark of stems of woody plants that can result in the anelamento of stems and drying of the superior part of the crown and the death of the plant. The best option for the control of the illness is the resistance genetic of the individuals concerning to the used patógeno. The objective of this study the molecular behavior of resistance and mechanisms of clonals of parental and the lineages the eucalyptus concerning the canker caused for Chrysoporthe cubensis and Dothiorella sp. The essays executed in the greenhouse and field. The isolated ones used concerning Chrysoporthe cubensis and Dothiorella sp. being produced in inoculated medium BDA and in the individuals for the method of the disk. The evaluation was concluded 60 days after the inoculation, measuring the area of the injury of each material. For pathogeno C. cubensis inoculated in the parental ones a 6,14 was found average of 3,20 and cm² of area of isolated injury of the trunk caused for the proceeding ones from Piracicaba and Bofete, respectively. The average of the area of the injury for the studied lineages varied of 7,29 cm ² 9,72cm ² for Dothiorella sp. The clonals inoculated C. cubensis were grouped into the categories HS (highly susceptible - 33,34%), MS (moderately susceptible -13,3%), MR (moderately resistant- 6,67%), and HR (highly resistant -46,66%). The clonal tested, with Botryosphaeria sp., verified resistant 91.67%, 5% moderately resistant, 1.67% moderately susceptible and 5,26% susceptible. The phenotypic variation found was from 41,32 to 0,23, inoculated with C. cubensis while Botryosphaeria sp from 0,285 to156,88. The coefficient of variation from 125% to 15,95% for C. cubensis and 29,86% to 101,13% for Botryosphaeria sp. The results indicate that the tested genetic materials possess good source of resistance for the two fitopatógenos tested... (Complete abstract click electronic access below) / Mestre

Submitted by Carolina Rabal Biasetto null (carolinarabal@yahoo.com.br) on 2016-05-18T19:36:08Z No. of bitstreams: 1 Tese_Doutorado_Carolina_Rabal_Biasetto_2016.pdf: 15348096 bytes, checksum: 59245cf961f6aaf1a999156f53853b5d (MD5) / Approved for entry into archive by Ana Paula Grisoto (grisotoana@reitoria.unesp.br) on 2016-05-20T18:01:57Z (GMT) No. of bitstreams: 1 biasetto_cr_dr_araiq_par.pdf: 1896284 bytes, checksum: 8b4dcb25761fe239f657abc24e10c1e7 (MD5) / Made available in DSpace on 2016-05-20T18:01:57Z (GMT). No. of bitstreams: 1 biasetto_cr_dr_araiq_par.pdf: 1896284 bytes, checksum: 8b4dcb25761fe239f657abc24e10c1e7 (MD5) Previous issue date: 2016-04-27 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / A capacidade biossintética dos fungos endofíticos, aliado aos estudos químico e biológicos relatados para Eugenia brasiliensis, motivou a idealização do projeto, a prospecção química, biológica e enzimática em fungos endofíticos associados a folhas, caules e frutos saudáveis de Eugenia brasiliensis e a avaliação do co-cultivo e epigenética em Xylaria cubensis, Diaporthe sp. e Colletotrichum sp., na obtenção de novas substâncias. O isolamento dos fungos endofíticos, resultou em dezessete linhagens de fungos endofíticos, sendo estes cultivados em escala reduzida em meio líquido de batata e dextrose (PDB) e Czapek, a 25 oC, sob modo estático para obtenção dos respectivos extratos brutos em AcOEt. A avaliação metabólica destes extratos foi realizada por CCDC, HPLC-DAD e RMN de 1H, como também a potencialidade enzimática e biológica pela avaliação das atividades antifúngica, anticolinesterásica e citotóxica, sendo que estes extratos demonstraram ser promissores. A prospecção inicial conduziu a seleção de três fungos endofíticos identificados como Xylaria cubensis (Eb_caH_5), Diaporthe sp. (Eb_caS_4), e Colletotrichum sp. (Eb_frmH_1), os quais foram cultivados (escala ampliada) em PDB para isolamento e determinação/elucidação estrutural dos metabólitos secundários. O estudo de Xylaria cubensis, resultou no isolamento de 8 substâncias, sendo da classe dos nucleosídeos, dicetopirazinas, isocumarinas e citocalasinas. De Diaporthe sp. foi isolado e identificado 8 substâncias: duas dicetopiperazinas, ácido nitropropiônico, uracila, tirosol, zygosporina D, pirrolidona (inédita) e alternariol. Colletotrichum sp. resultou no isolamento de 6 substâncias, sendo três dicetopiperazinas, além das substâncias N-(2-feniletil)acetamida, N-acetiltriptamina e metanoato de 2-hidroxibutila 3-indol (inédita). Todas as classes de substâncias produzidas por estes fungos endofíticos apresentam diversas atividades biológicas relatadas na literatura. Destaca-se a atividade fitotóxica da citocalasina D frente a coleóptilos de trigo superior ao herbicida comercial GOAL®. Para verificar a influência na produção metabólica de Xylaria cubensis, Diaporthe sp. e Colletotrichum sp., utilizou-se estratégias como o co-cultivo em meio sólido (PDA) e líquido (PDB) e a epigenética (escala reduzida), nas quais os endófitos mostraram produção metabólica diferente em relação à produção das monoculturas e na ausência do modulador epigenético ácido hidroxâmico suberoilanilida (SAHA), respectivamente. As ferramentas estatísticas, como PCA (Análise de componentes principais) e PLS-DA (Análise discriminante com calibração multivariada por mínimos quadrados parciais) permitiram uma rápida identificação e localização dos perfis metabólicos dos co-cultivos comparados às monoculturas. Estes dados contribuem para o conhecimento dos perfis metabólicos, biológicos e enzimáticos dos fungos endofíticos isolados de Eugenia brasiliensis, bem como suas interações com o hospedeiro. / The biosynthetic capacity of endophytic fungi, allied chemical and biological studies reported to Eugenia brasiliensis motivated the idealization of the project, prospection chemical, biological and enzymatic in endophytic fungi associated with healthy leaves, stems and fruits from Eugenia brasiliensis and evaluation co-culture and epigenetic in Xylaria cubensis, Diaporthe sp. and Colletotrichum sp., in obtaining new substances. The isolation of endophytic fungi resulted in seventeen endophytics fungi, these being cultivated in small scale on potato dextrose broth (PDB) and Czapek at 25 °C under static mode to obtain the corresponding crude extracts in AcOEt. The metabolic evaluation of these extracts was performed by CCDC, HPLC-DAD and 1H NMR, also enzymatic and biological potential were performed by antifungal, cytotoxic, and anticholinesterase activities, and these extracts have shown promise. The preliminary prospection led to the selection of three endophytic fungi identified as Xylaria cubensis (Eb_caH_5), Diaporthe sp. (Eb_caS_4) and Colletotrichum sp. (Eb_frmad_1) which were cultivated (larger scale) in PDB for isolation and determination or structural elucidation of secondary metabolites. The study of Xylaria cubensis resulted in the isolation of eight substances, as follows: nucleoside, diketopiperazines, isocoumarins and cytochalasins class. From Diaporthe sp. was isolated and identified eight substances, as follows: two diketopiperazines, nitropropionic acid, uracil, tyrosol, zygosporin D, pyrrolidone (unpublished) and alternariol. Colletotrichum sp. resulted in the isolation of six substances, three diketopiperazines, besides the substances N-(2-phenylethyl) acetamide, Nβ-acetyltryptamine and 3-hydroxybutan-2-yl-1H-indol-3-ylacetate (unpublished). All classes of substances produced by these endophytic fungi present several biological activities reported in the literature. Noteworthy is the phytotoxic activity of cytochalasin D against wheat coleoptile higher than commercial herbicide GOAL®. To verify the influence on the metabolic production of Xylaria cubensis, Diaporthe sp. and Colletotrichum sp., used strategies such as the co-culture on solid (PDA) and liquid medium (PDB) and epigenetic (small scale), in which endophytes showed an interesting and different metabolic production when compared with the production of monocultures and the absence of epigenetic modulator SAHA, respectively. The statistical tools, such as PCA (Principal component analysis) and PLS-DA (discriminant analysis with multivariate calibration partial least squares) allowed quick identification and location of the metabolic profiles of the co-culture compared monocultures. These data will contribute to the knowledge of the metabolic profiles, biological and enzymatic of endophytic fungi isolated from Eugenia brasiliensis and their interactions with host. / CNPq: 140980/2012-1