Orientadores: Silvia Azucena Nebra de Perez, Marlene Rita de Queiroz / Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Engenharia Mecanica / Made available in DSpace on 2018-07-25T02:21:13Z (GMT). No. of bitstreams: 1 Lima_AntonioGilsonBarbosade_D.pdf: 14051982 bytes, checksum: 6e5d15e1657c56ad9d7c6fe992afe6b3 (MD5) Previous issue date: 1999 / Resumo: Neste trabalho foram desenvolvidos vários modelos matemáticos bidimensionais analíticos e numéricos para simular o fenômeno de difusão em sólidos esferoidais prolatos (elipsoidais axisimétricos). Na formulação numérica, o método de volumes finitos, usando uma malha regular, é explorado para discretizar a equação de difusão, considerando o fenômeno com ou sem encolhimento, com ou sem têmpera, com ou sem transporte simultâneos de umidade e calor. O conjunto de equações lineares é resolvido iterativamente pelo método de Gauss-Seidel, usando condição de fronteira de equilíbrio ou convectiva e coeficiente de difusão constante ou variável. Os modelos predizem a transferência interna de umidade e/ou calor no sólido, bem como o seu teor de umidade médio e/ou temperatura média ao longo do processo. Vários casos foram estudados, variando-se os números de Fourier e Biot para transferência de calor ou de massa e a razão de aspecto do corpo. Foi feita uma análise dos efeitos da geometria do corpo, do encolhimento e da secagem em multipasses (têmpera), no fenômeno de difusão de umidade. Como aplicação, os modelos foram usados para descrever a transferência de calor e massa durante a secagem de banana, variedade "nanicão". Os resultados obtidos foram comparados com dados experimentais da literatura. Equações para as difusividades térmica e de massa e o coeficiente de transferência de calor convectivo foram obtidas utilizando o método dos mínimos quadrados / Abstract: ln this work, various two dimensions mathematical models to simulate the diffusion phenomenon in prolate spheroidal solids (ellipsoids axisymmetric) were developed. ln the numerical formulation, the finite-volume method, a regular grid is employed to discretize the diffusion equation, considering the phenomenon with and without shrinkage, with or without tempering, and simultaneous moisture and heat transfer. The linear equations set was solved iteratively utilizing the Gauss-Seidel method, with convective or equilibrium boundary conditions and constant or variable diffusion coefficient. The models predict the temperature and/or moisture content distribution inside the solid and the mean moisture content and/or mean temperature along the time. Various cases were analized, changing the Fourier and Biot numbers applied to heat or mass transfer and the aspect ratios ofthe body. The effects in the diffusion phenomenon due to the body geometry, shrinkage and tempering were showed. As an application, the models were used to describe the heat and mass transfer during the drying of banana, variety "nanicão", and the results were compared with experimental data from the literature. Equations for mass and thermal diffusivity and convective heat transfer coefficient were obtained using the least square method / Doutorado / Termica e Fluidos / Doutor em Engenharia Mecânica

Dans le cadre d’une convention de thèse CIFRE en collaboration de recherche entre la société Pollenergie et le Centre de Coopération Internationale en Recherche Agronomique pour le Développement (CIRAD), le potentiel antifongique de la propolis a été étudié. A cet effet, six échantillons de propolis provenant de régions géographiques différentes (France et Brésil) et d’origines botaniques variées (genre Populus, Dalbergia ecastophyllum et Baccharis dracunculifolia) ont été analysés.Les Extraits Ethanoliques de Propolis (EEP) mis au point, riches en composéspolyphénoliques (de 12,8 ± 0,4 à 16,2 ± 0,3 g EAG.l-1) présentent une grande diversité de classes de molécules bioactives selon leur origine botanique. Les polyphénols identifiés dans les extraits de propolis de Populus (France) sont principalement des flavonoïdes et leurs dérivés (galangine, pinobanksine, chrysine) et des acides-phénols (acides p-coumarique et caféique) et dans les extraits de propolis verte de Baccharis dracunculifolia et rouge de Dalbergia ecastophyllum (Brésil) ce sont respectivement des acides-phénols et leur dérivés prénylés (artépilline C, acide p-coumarique) et des composés isoflavonoïdiques (vestitol, médicarpine).Tous les EEP ont démontré un potentiel antifongique in vitro, variable selon les espèces botaniques, à deux stades de développement d’une souche phytopathogène (Colletotrichum musae), isolée de la pourriture de couronne de la banane. Deux extraits de Populus (France) en particulier ont montré la plus forte efficacité contre C. musae.Un extrait aqueux de propolis rouge de Dalbergia ecastophyllum a démontré la plus grande efficacité in vitro. Cet extrait, testé ensuite in vivo sur trois maladies de conservation de la banane a prouvé tout son potentiel antifongique comme traitement alternatif et son efficacité en augmentant la durée de conservation des bananes à l’export. / In the framework of a CIFRE thesis agreement in collaboration between Pollenergie and the Center for International Cooperation in Agronomic Research for Development (CIRAD), the antifungal potential of propolis has been studied. For this purpose, six samples of propolis from different geographical regions (France and Brazil) and various botanical origins (genus Populus, Dalbergiaecastophyllum and Baccharis dracunculifolia) were analyzed.The Ethanolic Extracts of Propolis (EEP) finalized, rich in polyphenolic compounds (from 12.8 ± 0.4 to 16.2 ± 0.3 g EAG.L-1), exhibit a great diversity of classes of bioactive molecules according to their botanical origin. The polyphenols identified in propolis extracts of Populus (France) are mainlyflavonoids and their derivatives (galangin, pinobanksin, chrysin) and acids-phenols (p-coumaric and caffeic acids). In contrast, the extracts of green propolis of Baccharis dracunculifolia and red from Dalbergia ecastophyllum (Brazil) they are respectively phenol acids and their prenylated derivatives(artepillin C, p-coumaric acid) and isoflavonoid compounds (vestitol, medicarpin).All EEPs have demonstrated an in vitro antifungal potential, which varies according to botanical species, at two stages of development of a phytopathogenic strain (Colletotrichum musae), isolated from the crown rot of banana. Two extracts of Populus (France) in particular showed the greatest efficacy against C. musae.An aqueous extract of red propolis from Dalbergia ecastophyllum demonstrated the greatest efficiency in vitro. This extract, which was then tested in vivo on three banana conservation diseases, proved its antifungal potential as an alternative treatment and its effectiveness in increasing the shelf life of bananas for export.

Chez la banane dessert, les saveurs sucrée et acide, caractéristiques importantes pour les consommateurs, sont pilotées par les teneurs en acides citrique et malique. Ce travail a donc porté sur l’étude des processus physiologiques qui pilotent l’accumulation de ces acides dans la pulpe de banane (Musa sp. AA) en combinant analyse expérimentale et modélisation écophysiologique. Nous nous sommes notamment intéressés à l’effet du génotype et des conditions de croissance du fruit en adoptant une approche intégrative liant les phases pré et post récolte.Les effets de la charge en fruit, de la fertilisation potassique, et du stade de récolte sur l’accumulation du citrate et du malate dans la pulpe ont été étudiés expérimentalement. La variabilité génotypique a été prise en compte en choisissant trois génotypes présentant des acidités contrastées à maturité. Des différences d’évolution des teneurs en acides, dues à des modifications métaboliques, ont été observées entre les génotypes pendant les phases pré et post récolte. Le stade de récolte a eu un effet significatif sur les teneurs en acides des fruits pendant la maturation post récolte. La charge en fruit et la fertilisation potassique n’en ont eu aucun. Des modèles écophysiologiques ont été développés pour prédire différents critères d’acidité de la banane en pré et post récolte. Le pH et l’acidité titrable ont été prédits par un modèle d’équilibres acido-basiques, la teneur en malate par un modèle de stockage vacuolaire, et la teneur en citrate par un modèle du cycle de Krebs. Ces modèles ont permis d’identifier les processus physiologiques clés qui pilotent l’acidité de la banane. Des paramètres génotypiques ont été identifiés liés à l’activité de l’enzyme malique mitochondriale et à celle des transporteurs mitochondriaux du malate pour le modèle citrate, et à l’activité des pompes à protons vacuolaire ATPases pour le modèle malate. Ces modèles ont également permis de disséquer l’effet des conditions de croissance du fruit sur l’acidité de la banane. L’intégration des modèles développés dans un modèle d’élaboration de l’acidité et son utilisation potentielle pour l’amélioration variétale sont discutées. / Citric and malic acids determine the sourness and sweetness of banana pulp, which are the two main determinants of consumer preferences. The present work focused on the physiological processes controlling the accumulation of citric and malic acids in banana pulp (Musa sp. AA) using experimental analysis and ecophysiological modeling. We chose an integrative approach linking the pre and post-harvest stages, and focused on the effect of genotype and fruit growing conditions. Experiments were conducted to study the effect of fruit load, potassium fertilization and fruit age at harvest on the accumulation of citrate and malate in banana pulp. To account for genotypic variability, three genotypes with contrasting acidity at the eating stage were studied. Major differences in the pattern of citrate and malate accumulation were found in the three cultivars both during growth and post-harvest ripening and were shown to be the result of metabolic changes. The harvest stage had a significant effect on the concentrations of acids during post-harvest ripening. Fruit load and potassium fertilization had no effect.Ecophysiological models were developed to predict several banana acidity criteria during the pre and post harvest stages. pH and titratable acidity were predicted by a model of acid-base reactions; malate content by a model of vacuolar storage; and citrate content by a model of the TCA cycle. These models led to the identification of the key physiological processes that control banana acidity. Genotypic parameters were identified, which were related to the activity of the mitochondrial malic enzyme and of the malate mitochondrial carriers in the citrate model, as well as to the activity of the vacuolar proton pump, ATPase, in the malate model. The two models were also used to analyze the effects of fruit growth conditions on banana acidity.Combining the three models in a global model of banana acidity, and the possible use of this model for varietal improvement are discussed.

Early maturity is one of the most important aerial growth traits next to bunch size, in determining banana productivity. However, low seed fertility in banana and the lack of breeding lines limit the application of conventional breeding for this trait. Genetic transformation with a CyclinD2-type gene, responsible for the CyclinD2 protein sub unit, which modulates the cell cycle progression at the G1/S phase, enhanced growth of tobacco plants. On this basis, investigations were carried out on the possibility of increasing the growth rate of banana plants through transformation with and expression of a CyclinD2- type coding sequence. Arabidopsis thaliana;CyclinD2;1 (Arath;CycD2;1) gene and its banana ortholog were over-expressed in banana to test their growth enhancement potential. The banana CyclinD2;1 (Musac;CycD2;1) was isolated from an East African highland cooking banana (AAA) cultivar “Nakasabira” and a cDNA was created by PCR using degenerated primers which was followed by genome walking. Characterization of the banana cyclin protein revealed an IWKVHAHY motif that was found to be conserved across the Musaceae family. Phylogenic analysis revealed a higher protein sequence identity of this banana cyclin to CyclinD2;1 of monocot plants than that of Arabidopsis. This cyclin was also found to be expressed highly in meristematic tissue which linked it to the cell cycle. The coding sequence was submitted to the GeneBank under accession number HQ839770. Arabidopsis and banana CyclinD2;1 gene coding sequences under the control of a constitutive promoter were used to transform embryogenic cells of the banana cultivar “Sukalindiizi” (AAB) using the Agrobacterium transformation system. A higher relative expression of Arath;CyclinD2;1 was found in the shoot than in the root apices and expression reduced transcript amounts of the endogenous banana CyclinD2;1. Plants of transformed banana line D2-41 had the highest Arath;CyclinD2;1 transcript amount and exhibited a significantly faster leaf elongation rate, better root growth, faster first leaf opening and a bigger lamina composed of bigger epidermal cells than non-transformed control plants. Banana plants transformed with Musac;CyclinD2;1 had a higher transcript amount of the transgene in the root apices when compared to the shoot apices. The higher transcript amount in the roots of plants of transformed line NKS-30 was related to faster root growth and development of an extensive root system. Overall, this study has provided evidence that expression of cyclin coding sequences in transformed banana is related to growth promotion. Specifically, Arath;CyclinD2;1 promoted shoot growth while the Musa homolog promoted root growth. Shoot and root growth phenotypes obtained in this study might have the potential to improve banana productivity in terms of short plant growth cycle, increased bunch weight, improved plant anchorage and increased plant resistance to root nematode damage. Future work should assess the produced plants in the field to allow transformed plants to exhibit their full potential and to be able to fully evaluate the vegetative and flowering phases. / Thesis (PhD)--University of Pretoria, 2012. / Plant Science / unrestricted

Various research efforts have focused on Banana streak badnavirus (BSV), the causal agent of banana streak disease (BSD), since the discovery of endogenous sequences of the virus in the nuclear genome of several Musa (banana and plantain) species. In vitro propagation of Musa was identified as one of the main activation triggers of integrated BSV sequences to cause systemic (episomal) BSD. This was especially observed in B genome-containing tetraploid hybrids. Although, the South African banana industry is based on Cavendish varieties, some plantations with tetraploid hybrids have been established. In order to investigate the occurrence of episomally expressed BSV, a survey was done in the Kiepersol area of South Africa and episomal BSV was detected in six out of seven locations sampled. No episomal BSV was detected in the Cavendish cultivars sampled in close proximity to BSV infected cultivars. To determine the risk of vector-assisted spread of endogenous BSV, which has become episomally activated after tissue culture, transmission studies with local mealybug species (Planococcus citri (Risso), P. ficus (Signoret), Dysmicoccus brevipes (Cockerell) and Pseudococcus longispinus (Targioni-Tozzetti)) were conducted under controlled conditions. Virus-free FHIA-21 was multiplied in vitro and resulting progeny with, putatively episomally activated BSV, served as sources for mealybug-assisted transmissions to Cavendish. Activated, episomal BSV was transmitted by three mealybug species to Cavendish. Transmission with P. ficus was demonstrated for the first time. Limited antiserum stocks against BSV occur worldwide and detection of the virus remains crucial for the safe movement of Musa germplasm between continents. Antiserum is needed in order to detect the episomal form of the virus that causes BSD. Using conventional immunization methodology, antisera against a wide spectrum of BSV isolates were produced. Twenty diverse BSV isolates were characterized by IC-PCR and selected as sources for the production of the polyclonal antiserums in two animal species. An effective triple antibody sandwich (TAS) enzyme linked immunosorbent assay (ELISA) system; able to detect various serologically different species of BSV was developed. BSV was screened with a synthetically manufactured phage displayed antibody library; however, no satisfactory polyclonal or monoclonal antibodies were obtianed in using this approach. / Dissertation (MSc (Microbiology))--University of Pretoria, 2007. / Microbiology and Plant Pathology / unrestricted

Lectins, multivalent carbohydrate-binding proteins of non-immune origin, have the unique ability to decode the information contained in complex carbohydrate structures of glycoproteins and glycolipids by stereo-specifically recognizing and binding to carbohydrates and carbohydrate linkages. The ubiquitous distribution of lectins in all forms of life and viruses along with their involvement in various biological processes such as cell-cell communication, host-pathogen interaction, cancer metastasis, embryogenesis, tissue development and mitogenic stimulation further emphasizes the importance of lectins in biological systems. Although not much is known about the endogenous roles of plant lectins, they constitute the most thoroughly studied class of lectins. On the basis of their subunit folds plant lectins have been divided in six major classes. They include jelly roll fold lectins (or legume lectins), hevein domain lectins (or cereal lectins), β-trefoil fold lectins, β-prism II fold lectins (or bulb lectins), β-prism I fold lectins and the most recently discovered lectin homologous to cyanovirin-N (http://www.cermav.cnrs.fr/lectines). Interestingly, of these, lectin subunits harbor an approximate three-fold symmetry in three cases and each subunit is believed to have evolved through successive gene duplication, fusion and divergent evolution. One of the major research activities in this laboratory involves structural studies on plant lectins. Decades of extensive studies in the laboratory have shed light on various structural and functional aspects of lectins such as variability in quaternary association, lectin-carbohydrate interactions, strategies for generating ligand specificity and multivalency. Furthermore, the β-prism I fold was first identified as a lectin fold in this laboratory through the X-ray analysis of the methyl-α-galactose complex of jacalin, one of the two lectins from the seeds of Artocarpus integrifolia. Subsequently, many other lectins with the same fold have been structurally characterized here and else where (http://www.cermav.cnrs.fr/lectines). They include mannose specific tetrameric artocarpin and dimeric banana lectin studied in this laboratory. Also investigated here is the structure of first dimeric β-prism II fold lectin, namely, garlic lectin. The subsequent work, carried out by the author, on the structure and dynamics of three-fold symmetric lectins form the subject matter of this thesis. Different web-servers available at NCBI and EXPASY web sites were used for sequence annotation studies. MRBAYES and MEGA were used for phylogenetic analysis. Molecular dynamics (MD) simulations were carried out using the simulation package GROMACS v.3.3.1. OPLS-AA/L and GLYCAM-06 force fields were used for proteins and carbohydrates respectively. Simulations were performed in explicit water system with TIP4P water model under NPT conditions with unit dielectric constant. The hanging drop method was used for crystallizing banana lectin and its complexes. Intensity data were collected on a MAR 345 image plate mounted on a Rigaku RU200 rotating-anode X-ray generator. The Oxford cryosystem was used when collecting data at low temperature. The data were processed using DENZO and SCALEPACK of HKL suite of programs. The structure factors from the processed data were calculated using TRUNCATE of CCP4 suite of programs. The molecular replacement program MOLREP was used for structure solution. Structure refinements were carried out using the CNS software package and REFMAC of CCP4. Model building was done using the molecular graphics program COOT. INSIGHT II, ALIGN, CONTACT, MUSTANG and SC of CCP4 were used for analysis of structural features. PROCHECK and web-server MOLPROBITY were used for the validation of the refined structures. The β-prism II fold lectins of known structure, all from monocots, invariably have three carbohydrate-binding sites in each subunit / domain. Until recently, β-prism I fold lectins of known structure were all from dicots and they exhibited one carbohydrate-binding site per subunit / domain. However, the recently determined structure of the β-prism I fold lectin from banana, a monocot, has two very similar carbohydrate-binding sites. This prompted a detailed analysis of all the sequences appropriate for the two lectin folds and which carry one or more relevant carbohydrate-binding motifs. The recent observation of a β-prism I fold lectin, griffithsin, with three binding sites in each domain further confirmed the need for such an analysis. The detailed sequence and phylogenetic analysis of all the β-prism I fold lectin or lectin-like sequences, available then, with particular attention to their carbohydrate-binding sites in them, in conjunction with the analysis of available three-dimensional structures demonstrate substantial diversity in the number of binding sites, unrelated to the taxonomical position of the plant source. However, the number of binding sites and the symmetry within the sequence exhibit reasonable correlation. The distribution of the two families of β-prism fold lectins among plants and the number of binding sites in them, appear to suggest that both of them arose through successive gene duplication, fusion and divergent evolution of the same primitive carbohydrate-binding motif involving a Greek key. Analysis with sequences in individual Greek keys as independent units lends further support to this conclusion. It would seem that the prepondence of three carbohydrate-binding sites per domain in monocot lectins, particularly those with the β-prism II fold, is related to the role of plant lectins in defence. Jacalin is the most thoroughly studied β-prism I fold lectin. A wealth of structural and thermodynamic data, mostly from this laboratory, led to a thorough characterization of carbohydrate-recognition in the case of jacalin. One aspect of jacalin that has not been investigated so far was its dynamics. The issue was addressed through reasonably long MD simulations, in explicit solvent system using all atom force field, of all the jacalin-carbohydrate complexes of known structure, models of unliganded molecules derived from the complexes and also models of relevant complexes where X-ray structures are not available. Results of the simulations and the available crystal structures involving jacalin permit delineation of the relatively rigid and flexible regions of the molecule and the dynamical variability of the hydrogen bonds involved in stabilizing the structure. Local flexibility appears to be related to solvent accessibility. Hydrogen bonds involving side chains and water bridges involving buried water molecules appear to be important in the stabilization of loop structures. The lectin-carbohydrate interactions observed in crystal structures, the average parameters pertaining to them derived from simulations, energetic contribution of the stacking residue estimated from quantum mechanical calculations and the scatter of the locations of carbohydrate and carbohydrate-binding residues, are consistent with the known thermodynamic parameters of jacalin-carbohydrate interactions. The simulations, along with X-ray results, provide a fuller picture of carbohydrate binding by jacalin than provided by crystallographic analysis alone. The simulations confirm that in the unliganded structures water molecules tend to occupy the positions occupied by carbohydrate oxygens in the lectin-carbohydrate complexes. Population distributions in simulations of the free lectin, the ligands and the complexes indicate a combination of conformational selection and induced fit. Mannose-specific β-prism I fold lectins, like lectins belonging to other plant families, exhibit interesting variability in their quaternary association. Mannose specific artocarpin and MornigaM are tetrameric, heltuba is octameric in the crystal structure and banana lectin and calsepa are dimeric. The modes of the dimerization in the last two are however, entirely different. This variability was explored through modelling and molecular dynamics simulations based on the known three-dimensional structures. This study, which combines computational approaches and results of X-ray analyses, provides valuable insights into the origin of the variability in quaternary association. MD simulations on individual subunits and the oligomers provide insights into the changes in the structure brought about in the protomers on oligomerization, including swapping of the N-terminal stretch in one instance. The regions which undergo changes also tend to exhibit dynamic flexibility during MD simulations. The internal symmetries of individual oligomers are substantially retained during the calculations. Simulations were also carried out on models using all possible oligomers employing the four different protomers. The unique dimerization pattern observed in calsepa could be traced to unique substitutions in a peptide stretch involved in dimerization. The impossibility of a specific mode of oligomerization involving a particular protomer is often expressed in terms of unacceptable steric contacts or dissociation of the oligomer during simulations. The calculations also lead to a rationale for the observation of a heltuba tetramer in solution although the lectin exists as an octamer in the crystal, in addition to providing insights into relations among evolution, oligomerization and ligand binding. The known crystal structures of banana lectin in its native and ligand bound forms revealed interesting features including the presence of two functional carbohydrate-binding sites per subunit. However, some confusion remained on the role of glycosidic linkage in carbohydrate-binding. The three crystal structures reported in this thesis provide information on details of the interactions of mannose and mannosylα-1,3-mannose with banana lectin and evidence for the binding of glucosyl-α-1,2glucose to the lectin. The known structures involving the lectin include a complex with glucosyl-β-1,3-glucose. Modelling studies on the three disaccharide complexes with the reducing end and the non-reducing end at the primary binding site are also presented here. The results of the X-ray and modelling studies show that the disaccharides with an α-1,3 linkage prefers to have the non-reducing end at the primary binding site while the reducing end is preferred at the site when the linkage is β-1,3 in mannose/glucose specific β-prism I fold lectins. In the corresponding galactose-specific lectins, however, α-1,3 linked disaccharides cannot bind the lectin with the non-reducing end at the primary binding site on account of steric clashes with an aromatic residue which occurs only when the lectin is galactose-specific. MD simulations based on the known structures involving banana lectin enrich the information on lectin-carbohydrate interactions obtained from crystal structures. They demonstrate that conformational selection as well as induced fit operate when carbohydrates bind to banana lectin. Snake gourd seed lectin (SGSL) isolated from Trichosanthes anguina is a glycosylated, galactose-specific, non-toxic lectin similar to type II ribosome inactivating proteins (RIPs) with a molecular weight of ~53kDa. It was established through preliminary X-ray studies that chain A with molecular weight of ~23kDa adopts the same fold as that of type I RIPs and the toxic chain of type II RIPs. Chain B with molecular weight ~32kDa has two β-trefoil fold domains and is responsible for the lectin activity of the protein. The two chains are connected with a disulphide bond. The sequence of the protein could not be determined using conventional methods despite extensive effort. It was derived from X-ray data at 2.4 Å resolution, which was used for structure analysis. The non-toxicity of SGSL appears to result from a combination of changes in the catalytic site in chain A and sugar-binding site in chain B. Detailed analysis of the sequences of type II RIPs of known structure and their homologues with unknown structure, provide valuable insights into the evolution of this class of proteins. It also indicates some variability in carbohydrate-binding sites, which appears to contribute to different levels of toxicity exhibited by lectins from various sources. In addition to the work on plant lectins, the author was also involved in studies on the crystal structures of the adipic acid complexes of L- and DL-Lysine. This investigation is presented in an appendix. A part of the work presented in the thesis has been reported in the following publications. Sharma, A., Thamotharan, S., Roy, S., & Vijayan, M. (2006). X-ray studies of crystalline complexes involving amino acids and peptides. XLIII. Adipic acid complexes of L- and DL-lysine. Acta Cryst, C62, o148-o152. Sharma, A., Chandran, D., Singh, D.D., & Vijayan, M. (2007). Multiplicity of carbohydrate-binding sites in beta-prism fold lectins: occurrence and possible evolutionary implications. J Biosci, 32, 1089-1110. Sharma, A., Sekar, K., & Vijayan, M. (2009). Structure, dynamics, and interactions of jacalin. Insights from molecular dynamics simulations examined in conjunction with results of X-ray studies. Proteins, 77, 760-777.

Fusarium oxysporum f.sp. cubense Schlecht (Foc), causal agent of Fusarium wilt of banana (Panama disease), is considered to be one of the most serious threats to banana production in the world. There is no effective control measure for Fusarium wilt, except for the replacement of susceptible with resistant banana varieties. However, resistant varieties are not always acceptable to producers and local consumer markets. A greater awareness of the detrimental effect of chemicals on the environment has stimulated research on biological control of plant pathogens. The use of indigenous microorganims, such as non-pathogenic F. oxysporum and the bacterium Pseudomonas fluorescens, therefore, offers not only an environmentally safe but also an economical approach to combat Fusarium wilt of banana as part of an integrated disease management strategy. Non-pathogenic F. oxysporum and P. fluorescens isolates have previously been isolated from the root rhizosphere in disease suppressive soils. These isolates have the ability to reduce the incidence of Fusarium wilt in greenhouse pathogenicity trials. In this study we had hoped to expand on existing knowledge on the biological control of Fusarium wilt of banana with non-pathogenic endophytic F. oxysporum and P. fluorescens. Isolates that significantly suppress disease development in greenhouse trials were tested under field conditions. Physiological and histological studies were also performed to understand the modes of action of putative biological control agents. For the histological investigations, non-pathogenic F. oxysporum isolates were modified with green and red fluorescent proteins. Chapter 1 depicts a general overview of the biological control of Fusarium wilt diseases of agricultural crops. This chapter addresses the biology and pathogenesis of F. oxysporum, before strategies to control Fusarium wilt are discussed. The application of biological control organisms was analysed in terms of potentially useful organisms, where they can be isolated, and their possible modes of action. Finally, factors that influence biological control of Fusarium wilt diseases are discussed. A good source of prospective biocontrol agents is suppressive soils. In Chapter 2, non-pathogenic F. oxysporum isolates were collected from healthy banana roots in disease suppressive soil. Random Fragment Length Polymorphisms of the intergenic spacer region were then applied to group the non-pathogenic F. oxysporum isolates into genotypes, from which candidates were selected for biological control studies. The selected endophytes were then inoculated onto banana roots to determine their ability to act as biocontrol agents against Foc. The isolates that protected banana best against Fusarium wilt in the greenhouse, together with P. fluorescens WCS 417, were tested in the field to determine whether these isolates could effectively reduce disease incidence in an uncontrolled environment. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce systemic resistance in Cavendish banana plants against Foc was investigated in Chapter 3 with the use of a split-root technique. The putative biocontrol agents were inoculated, separately and in combination, on one half of the roots in a split-root experiment, while the other half was challenged by a pathogenic isolate of Foc. Five different phenolic acids were assayed which included total soluble phenolic acids, non-conjugated (free acids) phenolic acids, ester-bound phenolic acids, glycosidebound phenolic acids and cell wall-bound phenolic acids. The knowledge gained will contribute to the understanding of how the biocontrol agents may induce defense responses in banana roots against Foc. Non-pathogenic isolates of F. oxysporum were transformed with the green fluorescent protein (GFP) and DsRed-Express genes in Chapter 4. These isolates were used to visualise their interactions with a GFP-transformed Foc isolate on the banana root in a non-destructive manner by means of confocal laser scanning microscopy (CLSM) in Chapter 5. The ability of non-pathogenic F. oxysporum and P. fluorescens WCS 417 to induce structural changes was also investigated with a split-root system using the CLSM. Antibioses as a mode of action of the two potential biocontrol agents was tested in vitro. Understanding the modes of action of non-pathogenic F. oxysporum and P. fluorescens WCS 417 are important when considering strategies for the implementation of these isolates in an integrated disease management strategy against Fusarium wilt of banana. / Dissertation (MSc (Plant Pathology))--University of Pretoria, 2011. / Microbiology and Plant Pathology / unrestricted

Orientador: Ronei Jesus Poppi / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Quimica / Made available in DSpace on 2018-08-14T14:16:59Z (GMT). No. of bitstreams: 1 Gallo_LucianaViviani_M.pdf: 1018650 bytes, checksum: 36599aa45a93a7d16c638ac0c60d6716 (MD5) Previous issue date: 2008 / Resumo: Neste trabalho foi desenvolvida uma metodologia analítica alternativa para a determinação de parâmetros de qualidade em bananas: pH, acidez titulável e Brix utilizando as espectroscopias no infravermelho médio e próximo em conjunto com calibração multivariada. A estratégia adotada foi obter os espectros na região do infravermelho próximo e médio do fruto e estabelecer um modelo de calibração multivariada, baseada no método dos mínimos quadrados parciais, para prever os diversos parâmetros. Foram utilizadas 54 amostras de banana, em diferentes estágios de maturação. Os espectros foram obtidos a partir da análise da polpa do fruto macerada, da solução obtida após a centrifugação da polpa, e pela técnica ¿Dry Extract Spectroscopy by infrared Absorption¿ (DESIR). Um total de 9 modelos foram propostos para a determinação dos parâmetros de qualidade e o melhor resultado para a determinação de pH foi na região do infravermelho médio utilizando a técnica DESIR, com erro médio de 1,32%. Já para a acidez, o melhor modelo obtido foi na região do infravermelho próximo utilizando DESIR, com erro relativo médio de 6,20%. O parâmetro brix apresentou melhores resultados na região do infravermelho médio através da análise da solução, com erro médio de 3,78%. Dessa maneira, a espectroscopia vibracional possibilita viabilizar a obtenção de maior número de informações sobre a qualidade da fruta, através da relação dos espectros com várias propriedades de interesse. / Abstract: banana quality parameters as pH, acidity and Brix by using infrared spectroscopy in the mid and near regions in conjunction with multivariate calibration. The strategy adopted was to obtain the fruit spectra in mid and near regions and to establish a multivariate calibration model, based on the partial least squares, to predict the several parameters. It was used 54 banana samples, in different stages of maturation. The spectra were obtained from the pulp fruit, of the solution obtained after pulp centrifugation and by technique Dry Extract Spectroscopy by Infrared Absorption (DESIR). A total of 9 models were proposed for the determination of the quality parameters and the best result for pH determination was in the mid infrared region using the DESIR technique, with mean error of 1.32%. For the acidity parameter, the best model was in the near infrared region using the DESIR technique, with mean error of 6.20%. The Brix model furnished the best result in the mid infrared region by using the solution obtained after pulp centrifugation, with mean error of 3.78%. In this way, the vibrational spectroscopy make possible to achieve large quantity of information about the fruit quality through the relationship between the spectra and several properties of interest. / Mestrado / Quimica Analitica / Mestre em Química

O engaço de bananeira, suporte que sustenta o cacho de bananas, normalmente é descartado após a colheita da fruta, seja nas casas de embalagens (packing houses) ou em centros distribuidores, onde é considerado verdadeiro resíduo pelo grande volume gerado e por não ser aproveitado. Por essa razão e por constituir-se em material fibroso, o engaço foi avaliado para produção de polpa celulósica. O engaço in natura apresenta cerca de 93% de umidade e células de parênquima em abundância; em termos de composição química, o engaço apresenta 7,4% de lignina, 47,8% de extrativos totais, e 47,6% de holocelulose. Nesta pesquisa, a performance do engaço como matéria-prima para produção de polpa celulósica foi avaliada, usando o CaO como fonte álcali. Foram utilizadas lavagem e pré-extração aquosa (100 ºC, por 100 minutos) como pré-processos com o propósito de reduzir a grande quantidade de extrativos no bagaço do engaço de bananeira. O bagaço original e os materiais obtidos após os pré-processos foram submetidos à polpação com CaO com 8, 10, 12 e 14% de CaO, à temperatura de 120 ºC por 120 minutos em digestor rotativo. Para comparação foi utilizado o processo soda de polpação, sob as mesmas condições, usando-se 12% de NaOH como carga alcalina. Os resultados mostraram que a aplicação da lavagem e pré-extração aquosa ocasionaram a redução de cerca de 40% da massa inicial do bagaço. Considerando-se as condições de polpação e também a composição química do engaço de bananeira, os pré-processos avaliados não apresentaram um significativo efeito no processo de cozimento. Para a polpação do engaço, o processo de polpação cal pode ser considerado uma alternativa técnica para produção de polpa celulósica, apresentando níveis de deslignificação similar ao do processo de polpação soda. / The banana stem, grain stalk that supports the banana fruits, is normally discarded after the fruit harvesting, either in the 'packing house' or in the delivering centers, where it is considered a residue due to the great volume generated. For this reason and for being a fibrous material, stem was evaluated for pulp production. The stem in natura presents 93 % of humidity and parenchymatic cells in abundance; in terms of chemical composition the stem presents 7,4% of lignin, 47,0 % of total extractives and 45,6% of holocelullose. In this research, the performance of the stem as raw material for pulp production was evaluated, using the CaO as an alkali source. Washing and aqueous pre-extraction (100 ºC and 100 minutes) were considered as a pre-process in order to reduce the amount of extractives on the banana stem bagasse; the original bagasse and the materials obtained after the pre-processes were submitted to the CaO pulping with 8, 10, 12 and 14 % of CaO, at 120 ºC temperature during 120 minutes in rotating digester. For comparison, a soda cooking was carried out, under the same conditions using 12% of NaOH as alkaline charge. The results showed that the aplication of washing and aqueous pre-extraction caused a reduction of about 40% in the initial mass of bagasse. Considering the pulping conditions and also the chemical composition of the banana stem bagasse the pre-process evaluated did not show a significant effect on the cooking process itself. For the banana stem pulping, a CaO process can be considered a technical alternative for pulp production, with delignification rates similar to the NaOH process.

Fusarium wilt of banana has a long and devastating history in many of the world’s banana producing countries. The most pronounced damage caused by Fusarium oxysporum f.sp. cubense (Foc), the Fusarium wilt pathogen, occurred during the 20th century in Central America, where tens of thousands of virgin forests were lost to further banana production. No control strategy is effective against Fusarium wilt other than replacement of susceptible by resistant varieties. It is, therefore, important to develop or identify resistant replacements that would not only be able to resist the pathogen, but also be acceptable to consumers. Resistance in wild banana varieties has been identified, and hybrids have been developed by breeding programmes with good resistance to Fusarium wilt. These varieties, unfortunately, appear not to be acceptable replacements for Cavendish bananas, the sweet desert banana variety that serves as the primary export banana and constitutes almost 40% of all bananas planted in the world today. A field selection, GCTCV-218, now proved to be the Cavendish plant with the most resistance to Foc ‘tropical’ race 4 (VCG 0121) has saved the Cavendish-based banana industry in Taiwan from devastation. In this thesis, GCTCV-218 has been evaluated against Foc ‘subtropical’ race 4 (VCG 0120), the primary variant of the pathogen in subtropical banana-producing countries such as South Africa, Australia and the Canary Islands. Defence-associated genes that are differentially expressed and that were up-regulated early in the defence response against the pathogen were isolated and identified. Greenhouse and field trials conducted at the research facilities of the Forestry and Agricultural Biotechnology Institute, University of Pretoria and in Kiepersol, South Africa, respectively, showed that GCTCV-218 had a significantly higher level of disease tolerance against Foc ‘subtropical’ race 4 (VCG 0120) when compared to the commercially grown Williams cultivar. Phenolic assays revealed that total phenolics and cell-wall bound phenolics were expressed at higher levels in GCTCV-218 after pathogen attack and seemed to play an important role in the tolerance of GCTCV-218. It was, therefore, proposed that GCTCV-218 could be considered a replacement for other Cavendish banana varieties planted in South Africa. The genetic basis of defence mechanisms in banana to Foc is unknown. In this investigation, Suppression Subtractive Hybridisation (SSH) was used to construct a cDNA library, containing banana genes that were up-regulated early (3&6 hours after infection), in the GCTCV-218/Foc interaction. The efficiency of the procedure was confirmed by PCR amplification of a known defence gene (endochitinase) present in the subtracted tester material, as well as analysing the reduction of a known housekeeping gene, actin, in the subtracted material compared to unsubtracted material. Southern blot data further provided confidence in the subtraction process. A cDNA library containing 736 gene fragments was constructed and then subjected to a screening procedure to remove false positives that escaped the subtraction process. The screening of a banana cDNA library for defence-related genes involved the development of a high-throughput cDNA microarray technique. This novel technique removed all false positives, such as housekeeping genes that escaped the subtraction as well as clones representing rDNAs. Seventy-nine genes differentially expressed in GCTCV-218 and not in Williams were selected, sequenced and subjected to BLASTX, BLASTN and DBest searches. Of these, several gene fragments showed homology to defence-associated genes, and 20 unique genes fragments were identified. These include two different peroxidases, response regulator 6, catalase 2, metallothionein, pectin acetylesterase (PAE), two different unknown proteins, salt stress, trypsin inhibitor, unspecific monooxygenase cytochrome P450, Bowman Birk proteinase inhibitor, root control, xylanase inhibitor, inhibitor CII, hypothetical protein, putative senescence-associated protein, pathogenesis-related protein 1 (PR1) and ribosomal protein S3a. The significance of the defence reaction to Fusarium wilt diseases in agricultural crops depends on the tempo of plant response. When a host plant is able to respond early to pathogen invasion the pathogen is successfully contained, preventing further spread throughout the plant. The expression of genes with antimicrobial activity, such as endochitinase, suggests an induced biochemical defence response against Foc. The expression of PAE and PR1 results in the deposition of lignin and callose production for cell wall strengthening. Four defence associated genes (catalase 2, pectin acetyl esterase (PAE), PR-1 and endochitinase) were selected for expression profile analysis using Real-time reverse transcriptase PCR, with TaqMan® and Light Cycler technology. All four genes were shown to be differentially expressed in GCTCV-218 at 3 and 6 hrs after infection, confirming SSH results. PR-1 and PAE were induced very early (3 hrs after infection) in the GCTCV-218, while PR3 and catalase 2 followed with a significant induction at 6 hrs after infection. This study concludes that GCTCV-218 is able to respond rapidly in response to Foc infection by activating both a biochemical and structural defence mechanism. / Thesis (PhD (Plant Pathology))--University of Pretoria, 2006. / Microbiology and Plant Pathology / unrestricted