Orientador: Paulo Mazzafera / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-23T10:23:33Z (GMT). No. of bitstreams: 1 Souza_UiaraRomero_M.pdf: 2641900 bytes, checksum: ad3d22ad5c736583fafb12fd75d1453c (MD5) Previous issue date: 2013 / Resumo: A rota de biossíntese de lignina é intensamente estudada. Em grande parte, o interesse neste polímero é vinculado à manipulação do teor de lignina em determinadas espécies de plantas, tais como nas gramíneas utilizadas como forrageiras e em espécies utilizadas para a produção de papel e, mais recentemente, em plantas utilizadas na produção de biocombustíveis. Muitos avanços foram realizados no sentido de alterar o teor de lignina em uma determinada espécie a partir da modificação da expressão de determinados genes da rota de biossíntese de lignina. No entanto, estudos mais recentes passaram a levar em consideração também os fatores de transcrição (FTs) envolvidos no controle da expressão destes genes e como estes fatores interagem entre si para modular a expressão destes genes alvo, de acordo com estímulos ambientais e/ou relacionados ao estádio de desenvolvimento. Neste estudo foram realizadas análises bioquímicas para a dosagem de lignina e de expressão gênica de fatores de transcrição relacionados com a via de biossíntese dos monolignóis em uma espécie de eucalipto e dois híbridos utilizados na produção de papel e celulose, e em duas regiões distintas do caule, ápice e base. Para induzir modificações no metabolismo e no nível de expressão os eucaliptos foram submetidos a estresses hídrico e por baixa temperatura. Sob baixa temperatura, Eucalyptus globulus teve aumento do teor de lignina e os FTs envolvidos na regulação positiva de genes da biossíntese de lignina, como VND7, MYB52 e LIM1, apresentaram aumento de expressão. Eucalyptus uroglobulus mostrou pouca variação dos teores de lignina quando submetido a estresse hídrico, assim como E. globulus. A via de regulação para a formação de parede secundária parece ter diferentes respostas nestes dois eucaliptos, visto que, em E. uroglobulus o aumento da expressão de VND7 não foi suficiente para induzir a expressão de seus fatores de transcrição alvo, MYB46 e EgMYB2. Já em E. urograndis P42, na base do caule de plantas estressadas por déficit hídrico, os alvos de VND7 tiveram expressão reduzida acompanhando os valores de lignina também menores. Deste modo, esta pesquisa pode evidenciar as diversas respostas ao estresse apresentadas em três genótipos diferentes de eucaliptos, de forma que foi possível encontrar relações pontuais coerentes com as análises bioquímicas e de expressão / Abstract: The lignin biosynthesis pathway is intensely studied largely because of the interest in modifying lignin content in some plant species used for the production of paper and more recently in plants used in producing biofuels. Many advances have been made to modify the lignin content in species by modification of the expression lignin biosynthesis pathway genes. However, recent studies consider transcription factors involved in control of gene expression and how these factors interact with each other to modulate the expression of target genes in accordance with developmental or environmental signals. In this study, biochemical analysis of lignin and transcription factors genes expression was performed in eucalyptus for two distinct regions of the stem (shoot tips and shoot bases). To induce changes in eucalyptus metabolism plants were exposed to water stress and low temperature. E. globulus, under low temperature stress, showed increased lignin content and increased expression to FTs VND7, MYB52 and LIM1, which plays a positive adjustment. E. uroglobulus and E. globulus showed no variation in lignin content when exposed to water stress condition. The hybrid E. uroglobulus showed increased expression of VND7, but the expression of their target transcription factors, MYB46 and EgMYB2 did not change, unlike E. urograndis P42 because at the dry group shoot base FT VND7 displayed reduced expression and lignin content. In summary, we demonstrate genes differentially expressed during stress in eucalyptus, so that it was possible to find consistent relations between biochemical analysis and expression / Mestrado / Biologia Vegetal / Mestra em Biologia Vegetal

Memoria para optar al Título Profesional de Ingeniero Forestal

The Koala bears housed and maintained by the Pretoria Zoo are relying on 20 different Eucalyptus species in the Eucalyptus arboretum in Pretoria for food. Species of the Botryosphaeriaceae are well known endophytes and pathogens of Eucalyptus in South Africa and elsewhere. This study was aimed at characterizing the Botryosphaeriaceae infecting these trees planted for the Koala bears. The experimental set-up also provided the opportunity to broaden our knowledge regarding the Eucalyptus species preferences of the Botryosphaeriaceae, as well as to reveal their potential movement to and from surrounding trees. In Chapter 1, the literature is reviewed regarding the biology of fungal endophytes, with a specific focus on Botryosphaeriaceae on Eucalyptus. Such a review clearly shows that, despite much previous work, we still only have a limited understanding of the biology and ecology of most fungal endophytes. This is particularly true for tree endophytes. Botryosphaeriaceae are clearly important and widespread canker and dieback pathogens of Eucalyptus. The taxonomic confusion that plagued the group, however, hindered a clear understanding of their diversity and biology. The use of molecular tools together with morphological characteristics has improved the ability to separate these fungi at species level. These tools are important for future work to better understand the true diversity. This would be a prerequisite if we are to gain a better understanding of the biology of these fungi and design with management strategies to control them. In Chapter 2, five species of Botryosphaeriaceae were identified from Eucalyptus species in the Pretoria arboretum, South Africa. Two species were described here as new species of Botryosphaeriaceae, within Neofusicoccum, namely N. ursorum prov. nom. and N. crypto-australe prov. nom. This is the first report of the latter species on Eucalyptus in South Africa. The other species included N. parvum, N. eucalypti comb. nov. (previously known as Dichomera eucalypti) and B. dothidea. Neofusicoccum parvum was the most common species isolated, followed by N. ursorum and N. eucalypti. The identifications were all based on the morphological characteristics, including cultural and conidial morphology, and DNA sequence data of the internal transcribed spacer region (ITS 1 and 4), and the translation elongation factor 1-α. The closely related species N. parvum and N. ribis were distinguished using a previously designed PCR-RFLP technique. As for the distinction between the cryptic species N. crypto-australe and N. australe, it was necessary to analyze a third gene region (β- tubulin) to confirm their separation using the phylogenetic species concept. All isolates obtained from this study produced lesions on stems of Eucalyptus camaldulensis (clone ZG-14) in the pathogenicity trial conducted under green house conditions. Of all species, N. eucalypti and N. crypto-australe were found to be the most pathogenic and B. dothidea the least pathogenic. There was, however, also significant variation in virulence between isolates of the same species. The results clearly show the potential threat of species of Botryosphaeriaceae to Eucalyptus. Field trials should be conducted in future studies to validate the findings in the greenhouse trials. Isolates representing different species identified in Chapter 2, were used for designing molecular tools for the in vitro and in vivo identification of Botryosphaeriaceae (Chapter 3). Sequences of the translation elongation factor 1-α were compared, and unique polymorphisms identified. Species specific primers were designed around these polymorphisms. All the primers designed were proven to be specific enough to distinguish the five different species from each other. The sensitivity of all primers were shown to detect fungal DNA concentration between 50 ng/μL and 0.01ng/μL. Preliminary tests of these primers on Eucalyptus leaves were done, and latent infections of N. parvum (the most common species) could be identified. More samples are likely to reveal the latent infections of other species using these tools. Future studies could now use these tools for the rapid identification of the fungi on Eucalyptus. It could be expanded to other hosts and more species of the Botryosphaeriaceae as well. The results presented in this study provided detailed information on species of the Botryosphaeriaceae from the 20 different species of Eucalyptus in a Pretoria arboretum. It yielded unique species, as well as well known pathogens of this host. All species isolated in this study were found to be pathogenic on Eucalyptus. The knowledge foundation, data and tools provided by this study can now be applied to characterize the infection biology, fine scale distribution and population diversity of these fungi. It is likely to bring new insights into the ecology of these organisms, their potential origin and movement between hosts. The presence of these fungi in other parts of South Africa should also be considered. / Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / Unrestricted

Please read the abstract in the section 00front of this document / Dissertation (MSc)--University of Pretoria, 2005. / Microbiology and Plant Pathology / Unrestricted

Thesis (PhD)--University of Pretoria, 2010. / Microbiology and Plant Pathology / Unrestricted

Studies presented in this thesis, highlights the importance of determining whether members of the Enterobacteriaceae can be associated with plants as epi- and endophytes. In particular, whether the causal agent of blight and die-back of Eucalyptus can survive both epi- and endophytically onlin its host as well as in weeds grown in close proximity to these hosts. This knowledge allows one a better understanding of the etiology and epidemiology of this disease. Appropriate management strategies can now be provided and the impact of the disease lessened in the nursery environment Chapter One presents an evaluation of the potential importance of Enterobacteriaceae as epi- and endophytes on/in plants. Some information is known about the epi- and endophytes associated with economically important agricultural crops. This information is largely lacking for tree species, especially those grown for commercial forestry purposes. Many Enterobacteriaceae occur both epi- and endophytically onlin plants including Pantoea ananatis. This pathogen is known to occur epiphytically on weeds as well as on its hosts where under ideal environmental conditions it is capable of causing disease symptoms. As an endophyte, P. ananatis occurs in dune grass where it fixes nitrogen and in sweet potato where it is believed to protect the plant against fungal pathogens. Chapter Two analyses healthy leaves, both young and mature, removed from various clones of the hydrid, E. grandis x E. nitens, for the presence of bacterial epi- and endophytes. Enterobacteriaceae were also isolated and these included Pantoea spp. and Enterobacter spp. P. ananatis was isolated both epi- and endophytically onlin healthy Eucalyptus tissue as well as from leaves removed from weeds growing in close proximity to the diseased plants. This thesis clearly indicates that P. ananatis can occur both epi- and endophytically in healthy Eucalyptus tissue. The movement of planting material into new environments where bacterial blight and die-back does not occur should be restricted. Irrigation practices in nurseries should be reviewed to prevent the accumulation of water on the plant surface which will allow for entry of the pathogen into the host through natural openings. Another management strategy that must also be recommended is that stringent weed control be implemented in the nursery environment. / Dissertation (MSc (Microbiology))--University of Pretoria, 2009. / Microbiology and Plant Pathology / unrestricted

Plantations of Eucalyptus spp. are expanding world-wide to serve growing global requirements for timber and pulp products. Together with this expansion, there has been a concomitant increase in diseases affecting these trees. Most of these are caused by fungi but there are a growing number of diseases caused by bacterial pathogens. Very little is known about them and the focus of this study was to consider species in the genus Pantoea and their association with diseases on Eucalyptus. Pantoea spp. are known pathogens of agricultural crops in South Africa and elsewhere in the world. They are also ubiquitous occurring in diverse ecological niches. Despite their prevalence, little is known about their association with plants, particularly where they occur as pathogens. The first chapter of this thesis presents an overview of the important aspects concerning the identification and classification of bacterial pathogens. Different techniques used for bacterial identification and classification were considered. These techniques are classified into different levels, based on their complexity and level of data resolution. As techniques have developed and been refined, our understanding of how organisms are related to each other has increased. An overview of the taxonomic history of the genus Pantoea was used to illustrate this point. An interaction between two Pantoea spp. and Colletogloeopsis zuluense, a serious fungal pathogen of Eucalyptus has been reported in the past. In the second chapter of this thesis, I considered the view that pathogenicity of C. zuluense is enhanced when infection occurs in conjunction with the two Pantoea spp. The identities of the two Pantoea spp. were confirmed as Pantoea ananatis and Pantoea stewartii subsp. indologenes. Both greenhouse and field inoculation trials with the two Pantoea spp. and C. zuluense failed to confirm that there is an increase in pathogenicity of C. zuluense when these bacteria are present. Studies in chapter three of this thesis, considered the identity of bacteria associated with diseased Eucalyptus leaf material from Uganda, Thailand and Uruguay. Symptoms observed in these countries were very similar and they were also similar to those of bacterial blight observed in South Africa. The majority of isolates obtained from Thailand were identified as Pantoea dispersa, based on both phenotypic and DNA-based data. This is the first report of Pantoea dispersa associated with disease on Eucalyptus. Uganda isolates were identified as Pantoea vagens prov.nom., a new species in the genus Pantoea. The majority of Uruguay isolates were identified as either Pantoea ananatis or Pantoea vagens prov.nom. The remaining isolates from Uruguay were found to belong to Pantoea eucalyptii prov.nom., a proposed new Pantoea sp., as well as Pantoea agglomerans. Pathogenicity results showed that the majority of isolates from all three countries were moderately pathogenic, eliciting moderate reactions in both tobacco and susceptible Eucalyptus grandis x nitens hybrid clones. Overall, results of studies presented in this thesis showed that Pantoea spp. can exist in complex interactions with both plants and fungi. These interactions are, however, not necessary for bacterial survival. We believe that the majority of Pantoea spp. are opportunistic pathogens based on their ability to selectively enter into interactions as well as occur epiphytically on plants. Variability in pathogenicity, both observed in this study and previously reported, further supports this view. Additional studies are needed to determine the conditions conducive to disease development in order to fully understand the threat these pathogens pose to global forestry. / Dissertation (MSc (Microbiology))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Orientador : Inacio Maria Dal Fabbro / Dissertação (mestrado) - Universidade Estadual de Campinas, Faculdade de Engenharia Agricola / Made available in DSpace on 2018-07-17T00:05:44Z (GMT). No. of bitstreams: 1 Seixas_Fernando_M.pdf: 3755433 bytes, checksum: f513adfb803fd9655231510a731b6c3a (MD5) Previous issue date: 1985 / Mestrado / Mestre em Engenharia Agrícola

As proteínas Dof (ligação ao DNA com um dedo, do inglês, DNA binding with One Finger) compreendem uma família de fatores de transcrição exclusiva de plantas, caracterizadas pela presença de um domínio de ligação ao DNA semelhante ao domínio ‘dedo-de-zinco’. Estas proteínas estão associadas a diferentes processos biológicos vegetais como germinação, florescimento e outros. O objetivo principal do presente trabalho foi a caracterização funcional de três fatores de transcrição Dof de Eucalyptus grandis a fim de avaliar a função dos mesmos na biogênese do sistema vascular desta planta. Baseado em resultados prévios do nosso grupo, foram escolhidos três genes (EgD01698, EgD00607 e EgK00405) que apresentaram um perfil de expressão significativo em tecidos vasculares de caules de E. grandis e, também, alta homologia a genes Dof previamente caracterizados em tecidos vasculares de outros vegetais. As regiões codificadoras desses genes foram amplificadas por PCR e clonadas no vetor de entrada pENTR/D-TOPO (Invitrogen). Por recombinação, os genes foram transferidos para o vetor pH7WG2D (VIB) para expressão em Arabidopsis thaliana. Os genes Dof também foram clonados no vetor pGEX-4T-1 (GE Healthcare) para a expressão heteróloga em Escherichia coli Plantas de A. thaliana foram transformadas com os vetores binários pelo método de imersão floral e as sementes foram selecionadas com o antibiótico higromicina. Apenas duas plantas para o gene D00607 e duas para o gene K00405 foram obtidas. As plantas transgênicas estão atualmente sob cultivo para serem geradas linhagens homozigotas e, assim, caracterizadas molecularmente. Além disso, a localização subcelular foi verificada pela expressão transiente de Dof::GFP em folhas de Nicotiana benthamiana. A expressão de GFP foi detectada difusa no citoplasma e no núcleo. Entretanto, esses ensaios deverão ser repetidos para confirmação dos resultados. Em paralelo, células de E. coli BL21 (DE3) pT-GroE foram transformadas com os vetores pGEX-4T-1-D01698 e pGEX-4T-1-D00607. A expressão gênica foi induzida durante 20 h a 20oC com 1 mM IPTG. As proteínas foram detectadas na fração solúvel por SDS-PAGE e western blot e, posteriormente, purificadas por cromatografia de afinidade. A expressão bem sucedida das proteínas Dof em E. coli permitirá a produção dessas proteínas para futuros estudos de caracterização funcional. / DNA binding with one finger (Dof) proteins comprehend a family of plant exclusive transcription factors characterized by the presence of the Dof DNA binding domain whose structure is similar to the zinc finger domain. They are associated with diverse biological plant processes such as germination, flowering and many others. The aim of this work was to functionally characterize three Dof genes from Eucalyptus grandis in order to evaluate their role in the biogenesis of vascular system. Based on previous results related to the E. grandis Dof gene family studies obtained by our group, we chose three genes (D01698, D00607 and K00405) with higher expression profile in vascular tissues of E. grandis stalk, as well as with higher homology to Dof genes previously characterized as critical to the genesis of vascular tissues. The CDS of these genes were amplified by PCR and cloned into pENTR/D-TOPO entry vector (Invitrogen). Via recombination, Dof genes were then transferred to the pH7WG2D binary vector (VIB) for the expression in Arabidopsis thaliana. They were also cloned into the pGEX-4T-1 vector (GE Healthcare) for Escherichia coli recombinant gene expression. A. thaliana plants were genetically transformed with recombinant binary plasmids by the floral dip method and seeds were selected by hygromycin resistance Only two plants for the D00607 gene and two plants for the K00405 gene were obtained. Plants are being cultivated in order to obtain homozygous individuals and they will be molecularly characterized. Additionally, subcellular localization was verified by transient expression of Dof::GFP in Nicotiana benthamiana and the expression of GFP was detected at the cytoplasm and nucleus. However, these assays need to be repeated in order to confirm the previous results. In parallel, E. coli BL21 (DE3) pT-GroE cells were transformed with pGEX-4T-1- D01698 and pGEX-4T-1-D00607. Gene expression was induced for 20 h at 20 oC with 1 mM IPTG for protein production. Proteins were detected in the soluble fraction by SDS-PAGE and western blot analysis and then were purified by affinity chromatography. The successful expression of Dof proteins in E. coli provides a way to produce these proteins for future functional characterization studies.

Memoria para optar al Título Profesional de Ingeniero Forestal / El agua es el principal factor limitante para el desarrollo de especies de cultivo agrícola y especies forestales. En Chile más de tres cuartos de la superficie continental corresponde a zonas áridas y semiáridas. Diversos estudios han demostrado que las especies del género Eucaliptus, poseen un amplio potencial en la tolerancia a condiciones de pluviometría limitada. Por esta razón, se evalúa el efecto de la disponibilidad de agua en algunas respuestas fisiológicas de tres especies de Eucaliptos: Eucalyptus globulus, Eucalyptus camaldulensis y Eucalyptus cladocalyx. El estudio comprende un total de 90 plantas de eucaliptos, 30 de cada especie, las cuales se disponen en un diseño factorial de cinco bloques al azar. Las plantas fueron sometidas a tres niveles de disponibilidad de agua, definidos como: sin estrés, estrés moderado y estrés severo. Las plantas se mantuvieron bajo condiciones semi-controladas en invernadero y las respuestas fueron evaluadas durante dos ciclos de desecamiento, el primero de cinco semanas y el segundo de nueve semanas. Al finalizar cada ciclo, las plantas fueron rehidratadas durante una semana.