Phytopathogenic fungi parasitise plants for survival. Knowledge of the interactions between plants and these fungi is of importance in designing control strategies. In this thesis the interactions of Eucalyptus polygalacturonase-inhibiting proteins (PGIPs) and fungal polygalacturonases (PGs) were characterised. About thirty years of research on the interaction between PGIPs and PGs was reviewed in Chapter 1. The gene sequences of the mature PGIP peptides from selected Eucalyptus species were determined in Chapter 2. The PGIP genes have very high similarities among themselves, signifying conservation of the function of PGIPs in this genus. Molecular characterisation of the endoPG gene of C. cubensis was presented in Chapter 3. This endoPG gene occurs more than two times in the genome of C. cubensis. The gene sequences of the other endoPGs remain to be determined. EndoPG production by C. cubensis, a Eucalyptus canker pathogen, was presented in Chapter 4. It was found that polygalactw:onases probably have a minor role in determining the levels of pathogenicity in different C. cubensis isolates. However, the hypovirus CHVl-173 has a role in reducing polygalacturonase production in infected hypovirulent C. cubensis isolates. Chapter 5 dealt with the interaction between Eucalyptus PGIPs and endoPGs from different fungal pathogens. Coniothyrium zuluense and Botryosphaeria dothidea produced more PGs than C. cubensis and Phytophthora cinnamomi in liquid culture. Disease tolerant Eucalyptus TAG5 clones produced PGIPs that are more effective in inhibiting endoPGs from C. zuluense than the susceptible Eucalyptus ZG14 clones. In Chapter 6 the gene sequence of the endoPG of Fusarium circinatum was detennined. This :fungus causes the . pitch canker disease in pine trees. This endoPG gene occurs as a single copy and is related to those of other Fusarium spp. The results presented in this thesis add to the current scientific knowledge pertaining to the role of PGIPs and PGs in pathogenic interactions, especially, between Eucalyptus PGIPs and fungal endoPGs. Interaction of Eucalyptus PGIPs and fungal endoPGs was demonstrated; therefore it is possible to genetically engineer Eucalyptus for more disease tolerant Eucalyptus plants using PGIP genes, especially, against Coniothyrium zuluense. This means that more research is needed to identity genes that will give a more global protection against many fungal pathogens. / Thesis (PhD (Plant Biotechnology))--University of Pretoria, 2006. / Genetics / unrestricted

Extensive areas of Eucalyptus plantations are being established in the "cerrado" region of Brazil for both pulp and charcoal production. These plantations are being managed intensively using short rotations and high stand density. Because the soils of the area are very infertile, there is concern over the sustainability of yields in the absence of repeated fertilization. Therefore, the present study was undertaken to evaluate the effects of intensive management of these plantations on the P and N status of the "cerrado" soils and the implications thereof for future productivity. Biomass and P and N content of each tree component (including the root system) were determined over age sequences of E. grandis plantations growing on good and poor "cerrado" sites in Minas Gerais State, Brazil. The dynamics of P and N were studied by analysing litterfall, forest floor accumulation, decomposition of branches and leaves, and internal cycling due to heartwood formation and the shedding of branches and foliage. The data that were collected permitted both a traditional static inventory assessment and a dynamic assessment of the effects of management on the site nutrient capital. However, because these two approaches are inadequate for the development of long-term sustained yield strategies for the management of these plantations, the thesis work included the development of conceptual models of two important aspects of eucalypt management in the "cerrado": coppice regeneration and phosphorus dynamics in the mineral soil. Studies of coppice sprouting were conducted to provide guidance for the development of the coppice model. By using information on both accumulation and dynamics of biomass and nutrients over age sequences of E. grandis growing on two different "cerrado" sites, the following predictions were obtained. Based on the static approach, the maximum yield over an initial 18 year period was obtained for a 54 month rotation, on both sites. However the use of this method to assess the effects of intensive management on site nutrient status would give misleading results because there is no feedback between changes in nutrient availability and yield. Therefore, a dynamic approach to the assessment was also used. This showed that over an 18 year period harvesting only stems on the good site is likely to cause a reduction in productivity of 48% while whole tree harvesting is likely to result in a reduction of 61%. On the poor site, there is likely to be only a 12% decrease due to whole tree harvesting, while harvesting stems only is unlikely to result in any decrease in yield. If whole tree harvesting is adopted on the good site, fertilizer quantities required to maintain the same level of productivity in the second rotation would be approximately 24 kg/ha of P while the amount required using stem harvesting only would be approximately 15 kg/ha. The amount of P required to produce one unit of biomass on the poor site was less than that required on the good site. In addition, the tree root systems on the poor site contain a greater proportion of total tree P as compared to those on the good site. This reduces the proportion of the total tree P that is removed at harvesting and gives an advantage to sprout growth on the poor site in subsequent rotations. Thus, intensive biomass harvesting on the poor site is expected to have relatively less impact on future site productivity compared to that on the good site. However, the good site can be expected to yield a greater biomass than the poor site. The data on accumulation and dynamics of biomass and P and N proved to be helpful in understanding the effects of intensive forest management on site nutrient status. However, the evaluation of long-term effects is rather complex because of the interaction of several factors when different management conditions are considered. Therefore, computer simulation models are required to evaluate the long-term effects of tree harvesting on site nutrient status. Because the growth strategies of a tree growing from seed are different from that of a tree developing from a stump, and the dynamics of P in mineral soil (the most limiting nutrient for eucalypt growth in the "cerrado" region) differ from that of N, coppice and phosphorus models were developed that could be implemented in the future in ecosystems models used to evaluate long-term effects of intensive management. / Forestry, Faculty of / Graduate

Lignin is a highly abundant aromatic biopolymer deposited during the final stages of secondary cell wall formation in plants and it constitutes a substantial proportion of the dry weight of woody plant stems. Lignin contributes structural support to xylem cell walls and hydrophobisity to water-conducting vessels and forms a defence mechanism against pathogen invasion. Although being an essential part of normal plant cell development, lignin content and composition are targets for tree improvement, because residual lignin in paper pulp has negative effects on paper quality and lignin therefore has to be removed using treatments that are expensive and often detrimental to the environment. At present, little is known about the amount of allelic diversity in lignin biosynthetic genes and whether such diversity may be associated with variation in lignin content and composition. However, the identification of alleles associated with desirable lignin phenotypes is dependent on a detailed understanding of the molecular evolution and population genetics of these genes. This M.Sc. study was aimed at analysing nucleotide and allelic diversity in two lignin biosynthetic genes of Eucalyptus trees. Additionally, the study aimed to develop single nucleotide polymorphism (SNP) markers that could be used to assay allelic diversity for these genes in populations of two target species, E. grandis and E. smithii. Orthologues of the tobacco LIM-domain1 (NtLIM1) transcription factor gene involved in the regulation of lignin biosynthesis were isolated from E. grandis and E. smithii. Approximately 3 kb of genomic sequence including the promoter and full-length gene regions were isolated for the two orthologues, respectively labeled EgrLIM1 and EsLIM1. The predicted amino acid sequences of EgrLIM1 and EsLIM1 were 99.4% identical to each other and indicated that LIM1 is a small protein of only 188 residues in eucalypt trees and has a predicted molecular weight of 21.0 kDa. Quantitative, real-time RT-PCR analysis confirmed the expression of LIM1 in wood-forming tissues undergoing lignification. Ten putative cis-regulatory elements were observed in the promoter regions of EgrLIM1 and EsLIM1including a GA-dinucleotide microsatellite that appears to be specific to LIM1 promoters of Eucalyptus tree species. The full-length LIM1 gene sequences could subsequently be used in the assessment of nucleotide and allelic diversity, together with the full-length CAD2 sequences that were already available in the public domain. The level of nucleotide and allelic diversity and the distribution and decay of linkage disequilibrium (LD) were surveyed in 5’ and 3’ derived gene fragments of CAD2 and LIM1 obtained from 20 E. grandis and 20 E. smithii individuals. Each gene displayed a unique genetic diversity profile, but for the most part, nucleotide diversity (π) was estimated at approximately 0.0010 except for the E. grandis LIM1 gene where π lower than 0.0040 was observed. Generally, except for the high amounts of LD observed in the CAD2 gene of E. grandis (> 2.5 kb), LD decayed within 500 bp. A large number (13 to 45) of SNP sites (defined as single nucleotide changes with minor allele frequencies of at least 0.10 in each species) were observed in each gene of each species. The high SNP density (ranging from one per 45 to one per 155 bp) observed in the two genes facilitated the efficient development of SNP markers to be used in future aspects of LD mapping, association genetics and marker-assisted breeding. The allele sequences obtained for the CAD2 and LIM1 genes were used as templates for the development of SNP marker panels (a series of six or seven SNP markers analysed together) for the analysis (tagging) of SNP haplotype diversity in species-wide reference populations (100 E. grandis and 137E. smithii individuals) of the two species. Each tag SNP was assayed using a single base extension assay and capillary gel electrophoresis. High polymorphism information content (average PIC of 0.836) was observed for the SNP marker panels. Four SNPs in the CAD2 and two in the LIM1 genes were found to be polymorphic in E. grandis and E. smithii (i.e. trans-specific SNPs), suggesting a possible ancestral origin for these polymorphisms. Assessment of candidate gene variation in the genomes of forest trees is of importance to ultimately be able to predict the amount and structure of nucleotide diversity available for the future design of SNP assays at the whole-genome level. Such assays will be useful to study differentiation among tree species and populations, to associate nucleotide polymorphisms with desirable phenotypes and to increase the efficiency of tree improvement approaches. / Dissertation (MSc (Genetics))--University of Pretoria, 2009. / Genetics / unrestricted

Eucalyptus trees are an important source of wood and fibre. The wood (secondary xylem) of this genus is widely used for pulp and papermaking. However, our understanding of the mechanisms which control the wood formation process (xylogenesis) in Eucalyptus and other woody species is far from complete. One reason is that xylogenesis is a very complex developmental process. The major components of wood are lignin and cellulose. Many genes involved in lignin and cellulose biosynthesis have been characterized. For example, Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are two important lignin biosynthesis genes. Plant cellulose is synthesized by cellulose synthase enzymes with the aid of some other proteins, such as sucrose synthase (SuSy) and sucrose phosphate synthase (SPS). Another factor which makes it difficult to analyze the function of Eucalyptus wood formation genes in vivo, is the long generation times of Eucalyptus trees and the difficulty to obtain transgenic Eucalyptus plants. Therefore, in this study, we investigated the use of Arabidopsis thaliana as a model system for functional analysis of wood formation genes. We transformed a lignin and a cellulose biosynthesis gene isolated from Eucalyptus to wild-type and mutant genetic backgrounds of Arabidopsis in order to test our ability to modify the cell wall chemistry of Arabidopsis thaliana using tree genes. The Eucalyptus CCR (EUCCR) gene was transformed into wild-type Arabidopsis (Col-0) and irregular xylem 4 (irx4) mutant plants, in which the homolog of EUCCR is mutated. A Eucalyptus cellulose synthase gene (EgCesA1) was also transformed into irregular xylem 1 (irx1) mutant plants, in which the homolog of EgCesA1 is mutated. Transgenics were only obtained from wild-type Col-0 transformed with EUCCR and from irx1 transformed with EgCesA1. We studied the cell wall chemistry of wild-type Arabidopsis plants overexpressing the Eucalyptus CCR gene. Chemical analysis of inflorescence stems revealed the modification of lignin and cellulose content in transgenic plants. Total lignin content was increased in T2 (5%) and T3 (12%) lines as revealed by micro-Klason lignin and thioglycolic acid quantification methods, respectively. High Pressure Liquid Chromatography (HPLC) analysis revealed that cellulose content was significantly decreased (10%) in T2 transgenic plants. This suggested the reallocation of carbon from cellulose to lignin as a result of overexpression of EUCCR in transgenic plants. Interestingly, thioacidolysis analysis revealed that in T2 plants, monomethoxylated guaiacyl (G) monomer was increased (16%) and bimethoxylated syringyl (S) monomer was decreased (21%). Therefore, the S/G lignin monomer ratio was significant decreased (32%). This implied that EUCCR might be specific to G monomer biosynthesis. The results described above confirmed that Arabidopsis thaliana can be used to model the function of wood formation genes isolated from Eucalyptus. Two novel full-length Eucalyptus sucrose synthase (SuSy) genes, EgSuSy1 and EgSuSy3, and one putative pseudogene, EgSuSy2, were also isolated in this study. Degenerate PCR was used to amplify Eucalyptus SuSy fragments from cDNA and genomic DNA. 3’RACE was used to amplify the 3’ ends of two Eucalyptus SuSy genes. Genome walking was performed to obtain the 5’ ends of EgSuSy1 and EgSuSy2 whereas 5’RACE technology was used to isolate the 5’ end of EgSuSy3. However, 3’RACE analysis failed when we tried to identify the 3’ end of EgSuSy2. Sequencing results from the genome walking product of EgSuSy2 further revealed that the start codon of this gene was missing, and we hypothesize that this is a psuedogene in the Eucalyptus genome. The EgSuSy1 cDNA was 2498 bp in length with an open reading frame of 2418 bp encoding 805 amino acids with a predicted molecular mass of 92.3 kDa. The 2528 bp full-length EgSuSy3 cDNA contained the same length of open reading frame as EgSuSy1, but encoded a polypeptide with a predicted molecular mass of 92.8 kDa. The results of quantitative real-time RT-PCR, phylogenetic analysis and gene structure of the two genes revealed that both genes might be involved in cellulose biosynthesis in primary and secondary cell walls of Eucalyptus. These two genes, EgSuSy1 and EgSuSy3, could therefore be useful targets for genetic engineering of wood properties in Eucalyptus. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / unrestricted

Dissertation (MSc)--University of Pretoria, 2009. / Microbiology and Plant Pathology / Unrestricted

The fungal genus Botryosphaeria including its anamorphs has a cosmopolitan distribution and occurs on a wide range of monocotyledonous, dicotyledonous and gymnospermous hosts, including woody twigs and branches, herbaceous leaves, stems of grasses, and even lichen thalli. These fungi give rise to a wide variety of symptoms such as shoot blights, stem cankers, fruit rots, die-back and gummosis. They are also known as saprophytes on dead or dying stems, branches or leaves of plants. In Venezuela, the following species have been reported: Lasiodiplodia theobromae (the anamorph of Botryosphaeria rhodina), Diplodia pinea. D. mutila and Dothiorella spp. However, their characterization has been based only on morphological descriptions. The most common and well characterized species. L. theobromae, is associated with pines and other hosts. The focus of the studies presented in this thesis as to survey Eucalyptus and Acacia plantations in Venezuela for the presence and influence of Botryosphaeria spp., and to characterize these fungi using morphological characteristics and DNA sequence data. I also evaluated the pathogenicity and population biology of Botryosphaeria species present in the plantations. It was anticipated that the various studies would characterize a relatively large number of Botryosphaeria species, potentially recognize new species and provide some perspective of their relative importance to the Eucalyptus growing industry. The thesis has been divided into chapters that reflect discrete units prepared for future publication. The first chapter presents a review of the relevant literature. Two chapters treat the taxonomy of the Botryosphaeria spp. collected in this study. An additional two chapters consider the population biology of the most commonly encountered species and the remaining chapter deals with the pathogenicity of these fungi to Eucalyptus. In the literature review I treat the taxonomy of Botyosphaeria, which has been the subject of much uncertainty for many years. I also briefly review recent findings related to their molecular characterization. A focus is also placed on the pathogenicity of Botyosphaeria species on various host plants and their known importance in Venezuela. The focus concerns mainly Eucalyptus spp. Surveys that formed part of this study logically gave rise to a large collection of Botryosphaeria isolates. An important component of this thesis was to characterize these fungi. This was done based on morphological characteristics and also comparisons or DNA sequence data for various gene regions known to be informative for these fungi. Lasiodiplodia theobromae (anamorph of B.rhodina), B. ribis and B. parva were studied from a population biology perspective. These fungi were chosen for study because they were the most common species encountered on Eucalyptus in Venezuela. To study populations, I made use of simple sequence repeat markers (SS R). Three populations of each of L. theobromae (Venezuela, Mexico and South Africa) and B.ribis-B. parva complex (Venezuela, Colombia and Hawaii) were analysed and the data considered in terms of population differentiation, gene now, mode of reproduction, gene and genotype diversity. In the final chapter of this thesis, I evaluated the pathogenicity or the seven Botryosphaeria species identified from Venezuela. These seven species were thus inoculated on Eucalyptus urophylla x E. grandis hybrid clones. A second series of inoculations was then carried out with the two most pathogenic fungi to determine the relative tolerance or the most important clones to infection. This thesis was conducted over a period of four years. Work was undertaken both in Venezuela and South Africa and this necessitated long periods of time away from my home University and family. Surveys were conducted in many parts of Venezuela to collect the Botryospaeria spp. of interest for latter study in South Africa. The research chapters have been completed systematically and they evolved over time. Each represents a discrete unit implying that there is some overlap, at least in the references, between them. I would like to believe that the thesis will provide a firm foundation for further studies of Botryospaeria spp. and prove valuable to the small but important Eucalyptus growing industry in Venezuela. / Thesis (PhD)--University of Pretoria, 2008. / Microbiology and Plant Pathology / unrestricted

O eucalipto é a arbórea mais cultivada no mundo para a exploração comercial de madeira. No Brasil, a principal espécie do gênero Eucalyptus utilizada é E. grandis, cujo cultivo, principalmente de híbridos com outras espécies, é voltado para a produção de celulose e papel. A relevância econômica do eucalipto para o Brasil e para vários países do mundo tem estimulado avanços científicos e tecnológicos, desde a área de biologia molecular até a silvicultura e a indústria de processamento da madeira. Entre os tópicos de interesse em biologia molecular estão genes e proteínas responsáveis pelo crescimento, pela qualidade da madeira e pela resposta vegetal a estresses ambientais. A família de proteínas Dof (DNA binding with one finger) compreende fatores de transcrição exclusivos de plantas, caracterizados pela presença do domínio Dof de ligação ao DNA, que contém uma estrutura semelhante ao domínio “dedo-de-zinco”. Esses fatores estão relacionados à ativação e à inibição de promotores de genes envolvidos nos mais variados fenômenos metabólicos das plantas tais como desenvolvimento do endosperma, metabolismo de carboidratos, germinação de sementes, desenvolvimento vascular e resposta a fitormônios. Pelo presente trabalho, visou-se caracterizar os fatores de transcrição Dof de E. grandis realizando-se análises filogenéticas com ferramentas de bioinformática, além de ensaios de RT-qPCR para se determinar perfis de expressão relativa dos genes Dof de E. grandis em folhas, caules e raízes de plantas sob diferentes tratamentos bióticos e abióticos que incluíram ABA, NAA, KIN, NaCl e seca. As análises filogenéticas permitiram a identificação de dez pares de genes parálogos no genoma de E. grandis, além de 13 grandes clados e nove pequenos grupos de genes ortólogos entre E. grandis, Arabidopsis thaliana e Populus trichocarpa. Os resultados permitiram-nos sugerir funções aos fatores de transcrição Dof de E. grandis. Os perfis de expressão relativa exibidos levaram-nos a concluir que os genes Dof são expressos em diversos órgãos e apresentam diferentes graus de acúmulo de mRNAs sob diferentes tratamentos. / Eucalypt is the most widely cultivated tree in the world for commercial logging. In Brazil, the main species of the Eucalyptus genus used is E. grandis, whose cultivation, mainly done with hybrids, is geared primarily for the production of pulp and paper. The economic relevance of eucalypts in Brazil and many countries worldwide has stimulated scientific and technological advances, from molecular biology to forestry and wood processing industry. Among the topics of interest in molecular biology are genes and proteins responsible for growth, the quality of wood and the plant response to environmental stress. The Dof (DNA binding with one finger) family of proteins comprehends plant exclusive transcription factors characterized by the presence of the DNA binding Dof domain, which is similar to the zinc finger domain. Dof transcription factors are related to the activation and inhibition of the promoters of genes involved in various plant metabolisms such as endosperm development, carbohydrate metabolism, seed germination, vascular development and response to phytohormones. In this study, we aimed to characterize the Dof transcription factors of E. grandis through phylogenetic analyzes using bioinformatic tools, in addition to RT-qPCR assays to determine the relative expression profiles of E. grandis Dof genes in leaves, stalks and roots of plants under different biotic and abiotic treatments that included ABA, NAA, KIN, NaCl and drought. Phylogenetic analysis allowed the identification of 10 pairs of paralogous genes in the genome of E. grandis, in addition to 13 major clusters and 9 small groups of orthologous genes between E. grandis, Arabidopsis thaliana and Populus trichocarpa, which allowed us to suggest functions for the E. grandis Dof transcription factors. The relative expression profiles showed that the Dof genes are expressed in various organs and display different degrees of mRNA accumulation under different treatments.

Submitted by Marco Antônio de Ramos Chagas (mchagas@ufv.br) on 2017-07-11T18:06:56Z No. of bitstreams: 1 texto completo.pdf: 518257 bytes, checksum: a284ae16f3d43e9016579dfb01f9d2c4 (MD5) / Made available in DSpace on 2017-07-11T18:06:56Z (GMT). No. of bitstreams: 1 texto completo.pdf: 518257 bytes, checksum: a284ae16f3d43e9016579dfb01f9d2c4 (MD5) Previous issue date: 2001-04-13 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A base da silvicultura clonal para o gênero Eucalyptus está na utilização de clones de alta produtividade, normalmente identificados nas avaliações dos testes clonais. Em geral, quanto maior o número de clones avaliados por unidade de tempo, maior a possibilidade de sucesso com a seleção. Entretanto, as etapas de avaliação e seleção são as mais caras e demoradas do programa de melhoramento, dificultando a elaboração e a execução de extensos programas de seleção de clones. Assim, o presente trabalho teve como objetivos: a avaliação da influência da idade, do espaçamento e do local sobre o tamanho da parcela experimental em testes clonais, considerando-se os coeficientes de variação experimentais, coeficientes de variação fenotípicos e produtividade em volume; e a determinação do tamanho da parcela experimental, por meio do Método de Máxima Curvatura Modificado, do Coeficiente de Correlação Intraclasse e da Análise Visual. A partir de quatro testes clonais, estabelecidos em área da International Paper do Brasil Ltda., analisaram -se as características altura, dap e volume dos quatro testes clonais, dispostos em delineamento em blocos ao acaso, com quatro repetições e parcela experimental quadrada de 25 plantas (5 x 5). Após a simulação de diferentes tamanhos de parcela: 2, 3, 4, 5, 9, 10, 15, 20 e 25 plantas/parcela, procedeu-se às análises do efeito do tamanho da parcela na produtividade, na precisão experimental e na variação do coeficiente fenotípico. Determinou-se, também, o tamanho da parcela pelos Métodos de Máxima Curvatura Modificado, do Coeficiente de Correlação Intraclasse e da Análise Visual. De forma geral, o tamanho da parcela não alterou a estimativa de produção volumétrica dos clones nas diferentes idades analisadas. No entanto, o coeficiente de variação experimental (CV exp ) e o coeficiente de variação fenotípico (CV f ) apresentaram maiores valores nas idades mais avançadas e com tendência de queda com o aumento do número de plantas na parcela, independentemente da idade. O espaçamento de plantio pode alterar o tamanho da parcela, pois, quanto maior o espaçamento, menores o CV exp e o CV f . O comportamento da parcela foi pouco influenciado pelas avaliações realizadas nos diferentes locais. O tamanho mínimo da parcela experimental indicado pelos Métodos de Máxima Curvatura Modificado e pelo Coeficiente de Correlação Intraclasse variou de 1 a 8,6 plantas/parcela; já na análise visual, variou de 5 a 15 plantas por parcela, nos diferentes testes clonais e nas características analisadas. Com base no presente estudo, pôde-se concluir que, em programas iniciais para seleção de clones, parcela de 5 a 10 plantas indica boa precisão experimental, sendo recomendada, principalmente, em situação com limitações de mudas, teste de grande número de clones e avaliações de cunho preliminar e em idade precoce. No entanto, vale ressaltar que, para um melhor conhecimento do clone para uso comercial, parcelas quadradas maiores e, ou, plantios-piloto são os mais indicados. / The base of clonal forestry for the Eucalyptus gender is the use of high production clones, normally identified in clonal test evaluations. In general, the higher the number of evaluated clones per time unit, the greater the possibility of a successful selection. However, the evaluation and selection stages are the most expensive and time-consuming of the enhancement program, creating difficulties for working out and carrying out extensive clone selection programs. Therefore, the present study had the objectives: evaluation of age influence, spacing and site on the experimental parcel size in clonal tests, considering the coefficient of variation experimental, the coefficient of variation phenotype and the volume productivity, as well as the determination of the experimental parcel size, by means of the Maximum Modified Curvation, the Interclass Correlation coefficient and Visual Analysis methods. Based on four clonal tests, established in the area of International Paper of Brazil Ltda., the characteristics height, diameter at breast height and volume were tested in the four clonal tests, set up in design of random blocks in four repetitions, in experimental square parcels of 25 plants (5x5). After the simulation of different parcel sizes: 2, 3, 4, 5, 10, 15, 20 and 25 plants/parcel, the influence of the parcel size on the productivity, the experimental accuracy and the variation of the phenotype coefficient were tested. The parcel size was also determined by the Maximum Modified Curvation, the Interclass Correlation Coefficient and the Visual Analysis methods. In the main, the parcel size did not influence the volume production estimative of the clones at the different analyzed ages. However, the coefficient of variation experimental (CV exp ) and the coefficient of variation phenotype (CV ph ) presented higher values at more advanced ages, with a sinking tendency when the number of plants in the parcel was reduced, independent of the age. The plant spacing can change the parcel size, since the greater the spacing, the lower the CV exp and CV ph . The parcel behavior was little influenced by the evaluations performed in the different sites. The minimum size of the experimental parcel indicated by the Maximum Modified Curvation and the Interclass Correlation Coefficient methods ranged from 1 to 8.6 plants per parcel, while for the Visual Analysis it varied from 5 to 15 plants per parcel in the different clone tests and the analyzed characteristics. Based on the present study, the conclusion can be drawn that in initial clone selection programs, parcels of 5 to 10 plants provide good experimental accuracy, especially recommended when there is only a limited number of seedlings available, a high number of test clones and evaluations of preliminary type and at an early age. However, it must be emphasized that in order to obtain more knowledge about a clone for commercial use, larger square parcels and/or, pilot plantings are the most indicated. / Dissertação importada do Alexandria

Trees exhibit many unique aspects of plant biology, one of which is the formation of wood. Wood is one of the most important natural products with a multitude of applications. The formation of wood (xylogenesis) is a highly ordered developmental process involving the patterned division and differentiation of the vascular cambium into secondary xylem and phloem tissue types. The progression of xylogenesis developmental process requires differential gene expression across the different tissue types. The tight regulation of wood formation is mediated by genes that regulate cambial meristem differentiation and xylem cell fate. MicroRNAs (miRNAs) are a group of endogenous ~ 20 to 24 nt RNA molecules that down regulate gene expression at the post-transcriptional level. MicroRNAs have validated roles in developmental processes through the regulation of meristem cell differentiation and developmental patterning in plants. They have been shown to spatially regulate differential gene expression patterns at different developmental stages. Thus, the vascular cambium and its derivatives are excellent candidate tissues for miRNA discovery. The aim of this M.Sc. study was to isolate microRNAs from actively differentiating tissues of two tree species in order to determine possible gene regulatory networks involved in early meristem differentiation, tissue patterning and secondary vascular development. A small RNA library from two-month old in vitro Populus trichocarpa plantlets was constructed to identify putative miRNAs contributing to the early postembryonic development of trees. This library, in conjunction with computational prediction of poplar miRNA homologues and precursor secondary structures, was used to identify a total of 72 poplar miRNAs. Sixteen of these were putative novel miRNAs, belonging to nine new miRNA families. A genome-wide search identified 55 putative target genes for the newly identified miRNAs. The target genes had diverse biological roles in developmental events and maintenance of cellular homeostasis. A number of the predicted targets were involved in plant organ development such as leaf cell fate, floral organ development and meristem differentiation. Other targets were involved in response to hormones, such as growth regulating factors and signaling proteins. Additionally, several targets were related to cellular metabolic processes, such as protein modification and ubiquitination. By isolating miRNAs from developing poplar plantlets, we were able to suggest possible developmental programmes under the control of these molecules, possibly affecting early seedling development and growth. A similar approach was used to identify miRNAs from three differentiating vascular tissues of Eucalyptus grandis. Isolated small RNA sequences were used in a search against all available bacterial artificial chromosome (BAC) shotgun genomic sequences from an ongoing Eucalyptus camaldulensis genome sequencing initiative at the Kazusa DNA Research Institute in Japan. We were able to characterize the first Eucalyptus miRNAs, and identified 48 putative miRNAs grouping into thirteen gene families. Twenty of the miRNAs belong to five families previously identified in other plant species, whereas the remaining 28 miRNAs grouped into eight putative novel miRNA families. Searches of the Populus and Arabidopsis annotated genomes revealed 45 putative target genes for the new families. Targets of particular interest included transcription factors involved in cell fate determination, including a MADS-box transcription factor involved in xylem formation. Further targets included auxin signaling proteins and auxin response factors, which could play a significant role during auxin regulation of vascular development. Expression profiling of the putative miRNAs using quantitative RT-PCR revealed that a number of the miRNAs exhibited differential expression patterns across xylogenic and non-xylogenic tissues. One miRNA showed expression in a single vascular tissue, whereas others were expressed at varying levels across the vascular tissues. This observation indicates a possible role for these putative miRNAs during vascular development and differentiation in eucalypt trees. In this study we used a combination of small RNA library construction and computational prediction to identify microRNAs from two tree species. We identified a total of 120 putative miRNAs grouping into 31 families. Of these, 44 group into 17 putative novel tree-specific miRNAs. This study has allowed the identification of novel miRNAs from a unique set of tissues, and has contributed to the ever-growing number of plant-specific miRNAs. The results of this study further contribute to our expanding knowledge of the unique developmental process of vascular tissue differentiation of perennial woody plants such as Eucalyptus and Populus species. / Dissertation (MSc (Genetics))--University of Pretoria, 2006. / Genetics / unrestricted

Colombia covers an area of approximately 114 million hectares (ha) of which the potential forestly land has been estimated at 60.7 million ha, about 53% of the total area. Only 54 million ha are considered as natural forest, leaving approximately 29 million ha to be used for livestock and agriculture. In order to provide an alternative source of timber, Colombian groups have planted approximately 327 000 ha to different species of Pinus, Eucalyptus and native species. This clearly represents only a small proportion of the total area that might be used for forestry, which is set to grow in the future. In general, trees established in plantations have been grown as monocultures that allow for substantial productivity per management unit. But this is also a homogeneous genetic resource that is highly susceptible to damage caused by insect pests and diseases. It is therefore, surprising that relatively little work has been conducted on pests and pathogens negatively affecting plantation foresty in Colombia. In this regard, the studies presented in this thesis present the first comprehensive treatment of the topic for the country. It is consequently hoped that these will form a basis for the future management and sustainability of forestry in Colombia. In the first Chapter of this thesis, I provide a historical background and the current pest and disease situation for the forestry sector in Colombia. This includes an overview of the main pest and diseases affecting commercial nonnative trees, especially species of Pinus and Eucalyptus. A wide range of sites occurring in Colombia were considered and the document also includes the impact of different climatic conditions on the incidence and management of the various pests and diseases treated. Some of the most important defoliators in Colombia reside in the order Phasmatodea and one of these, Litosermyle ocanae, was treated in studies presented in chapter two. The overall aim was to contribute basic knowledge of L. ocanae including an understanding of the biology, egg population dynamics, and potential biological control assessments based on early detection of the insect. vi Chapter three includes the discovery of one of the members of the Geometridae that causes serious damage due to defoliation of Pinus and Eucalyptus plantations. This pest, known as Chrysomima semiluteraria, has been known in Colombia for many years and this study included a comprehensive evaluation of its biology and field monitoring contributing to an Integrated Pest Management strategy for it. The insect was studied under field and laboratory conditions and a special emphasis was placed on its biological control using the egg parasitoid Telenomus alsophilae. Pinus plantations in Colombia have been affected by numerous pests, including recently, the adelgid Pineus sp. To establish management strategies to assist commercial forestry operations, the life cycle of this insect and the susceptibility of different Pinus species were determined in Chapter four. In addition, the possible role of a Ceraeochrysa sp. as a biological control agent was investigated. In chapter five, Fusarium circinatum is recorded for the first time on Pinus spp. in Colombia. The discovery of this fungus, known to cause the disease Pitch Canker has had an important impact on forestry, especially with regard to management strategies aimed at reducing its impact. Studies in this chapter included those to evaluate the susceptibility of families and provenances of Pinus spp., which are currently used in forestry planting programs in Colombia. The first emergence of the foliage pathogen Dothistroma septosporum as an important constraint to pine forestry in Colombia is treated in chapter six. This fungus is a serious pathogen of many species of Pinus around the world. It was, however, not recognised as a serious threat to forestry in Colombia until it emerged as a serious source of damage to Pinus tecunumanii. In this chapter, the aim was firstly to confirm the identity of the pathogen based on DNA sequence data. Subsequently, the host range and distribution of the pathogen was established in different Colombian forestry areas. The impact of the disease and the susceptibility of different provenances of P. tecunumanii was also assessed. In chapter seven, the main objective was to described the susceptibility of E. grandis clones to a new species of Ceratocystis and to identify clones resistant to it. The fungus was described as Ceratocystis neglecta and management strategies are suggested for it. This thesis includes two Appendices treating new reports of insect pests and an important Eucalyptus pathogen. In Appendix 1, I report on the importance of three different pest insects. Appendix 2 includes a description of a new disease, as well as the potential risks that this disease poses for the Colombian forestry sector. / Thesis (PhD)--University of Pretoria, 2013. / gm2013 / Forestry and Agricultural Biotechnology Institute (FABI) / Unrestricted