At the time of commencing this study, there were only five papers published on Coniothyrium canker disease of Eucalyptus. These studies included the formal description of the fungus causing the disease and some aspects of its biology and physiology were characterized. The fungus was described, at that time, as Coniothyrium zuluense, which had a very simple morphology, lacked sexual reproductive structures, had small nondescript conidia and it was slow growing in culture. Nevertheless, the taxonomic status of the Coniothyrium canker pathogens changed in several occasions during this study including placement in genera such as Colletogloeopsis, Kirramyces and Teratosphaeria. After the first appearance of Coniothyrium canker in South Africa, the disease was found in many other parts of the world. DNA sequences from cultures of C.zuluense became easier to obtain and this made it possible to undertake phylogenetic comparisons of isolates from various areas. Such studies also showed that C.zuluense was closely related to Mycosphaerella species. The common appearance of Coniothyrium canker in new areas motivated further studies of this disease and it causal agent, particularly applying newly available rDNA-based techniques. This also provided the motivation for studies presented in this thesis. The thesis is introduced by means of a literature review that treats Coniothyrium canker on Eucalyptus. Briefly, the general characteristics of the host species, Eucalyptus, are described. Furthermore, trends relating to emerging diseases in plantations of Eucalyptus during the past two decades are treated with particular focus being placed on Mycosphaerella diseases. The phylogenetic relationships between Coniothyrium, Mycosphaerella and its anamorphs are considered together with the population biology of related pathogens. In chapter two of this thesis, DNA sequence comparisons were used to determine the phylogenetic position of C.zuluense related to other fungi. In particular, the question as to whether C.zuluense was correctly placed in the genus Coniothyrium and its relatedness to Mycosphaerella was considered. Comparisons with the type species of Coniothyrium, C.palmarum and a collection of sequences of Mycosphaerella species were also conducted. In addition, the identity of isolates obtained from China with similarities in colony morphology to C.zuluense was considered. The objective of the study presented in chapter three was to investigate whether all the available isolates in the FABI collection from different countries and associated with Coniothyrium canker represented a single phylogenetic species. An additional methodological objective of this chapter was to select the best DNA regions for phylogenetic studies on this fungus and its relatives. Four DNA regions were selected based on the informative content as well as ease and reproducibility for Polymerase Chain Reaction (PCR) amplification. The studies presented in Chapter 3 of this thesis showed that two species cause Coniothyrium canker and these are now known as Teratosphaeria zuluensis and Teratosphaeria gauchensis. Therefore, the objectives of the studies presented in chapters four and five were to develop highly variable markers to study the genetic variability and population parameters of populations of both species. This included the development of a robust protocol to isolate microsatellites on both fungi and that would also be informative for related genera. The protocol finally developed and used is presented in Appendix 2 of this thesis. In chapters six and seven, the microsatellite markers developed in the previous chapters were applied. The genetic structure of populations of T.zuluensis and T.gauchensis was thus studied. Analyses of the amplified alleles and their frequencies were used to determine the levels of genetic diversity, clonality and to draw preliminary conclusions regarding the origin and global movement of the pathogens. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / Unrestricted

Trees are large, biologically complex multi-cellular organisms that have adapted to terrestrial growth. This places specific demands on their physiology such as the ability to transport water over long distances and the ability to withstand extreme mechanical forces. Wood formation (xylogenesis) is the development of the secondary vascular system within trees, which mainly addresses these two physiological needs. Xylogenesis is a highly ordered developmental process, consisting of a number of overlapping yet distinct developmental phases. These phases are strictly regulated through a combination of biochemical signalling networks and gene expression regulation.<ul> <li>microRNAs (miRNAs) are endogenous non-coding, small (~22 nt) RNAs that function predominantly as negative regulators of gene expression at the post-transcriptional level. They have been implicated in the regulation of plant developmental processes, including determining cell fate in the apical meristem and timing of developmental events such as flowering and leaf morphogenesis. miRNAs have recently been found to have regulatory roles in plants placed under various conditions of abiotic stress such as drought, mechanical stress, cold and high levels of salinity.</li></ul> Trees placed under mechanical stress produce a specialised form of wood called reaction wood. Reaction wood is referred to as tension wood in angiosperms, as it forms on the outside of a bent trunk or branch in order to correct for the non-vertical growth. The formation of tension wood requires extensive reprogramming of wood development processes. This makes tension wood induction an ideal tool to study and refine our understanding of wood development. Recently, miRNA regulation has been implicated in the control of normal and tension wood formation in trees, but the full extent to which miRNAs are involved in tension wood induction is not known. To identify miRNAs potentially involved in the regulation of normal and tension wood development in fast-growing Eucalyptus plantation trees, real-time quantitative polymerase chain reaction (RT-qPCR) and Northern blot analyses were performed for a number of conserved and putatively novel Eucalyptus miRNAs. A total of 12 miRNAs representing 12 distinct miRNA families were profiled, including three novel miRNAs that are putatively specific to Eucalyptus trees. To more fully understand miRNA function in terms of tree development, the abundance profiles of the selected miRNAs were first determined at the whole-tree level. Of the conserved miRNAs profiled, five (miR160, miR166, miR167, miR172, miR408) were found to have abundance profiles consistent with their predicted roles in plant development. At the whole-tree level, miR90, putatively novel to Eucalyptus, was predominately expressed in the mature leaves and flowers. miR90 may target a MADS-box transcription factor which is not required for mature leaf and flower growth. miR408, a regulator of the expression of a plastocyanin gene involved in lignin polymerisation, was expressed at low levels in the immature and mature xylem, where cell lignification is most prominent. In the tension wood, miR166, a known regulator of wood development and miR408 displayed similar increasing abundance over time in tension wood xylem. These profiles support their potential role in wood development. miR160 and miR167, which target auxin response factors responded early to bending stress, with their abundance reaching maximum levels six hrs post-induction before decreasing again. This is consistent with the observed role of auxin response factors as a mechanism to rapidly respond to stimuli, such as bending. The miRNAs abundance profiles generated in this study suggest that some miRNAs do indeed play a role in normal and tension wood development, though not necessarily directly. These results provide further insights into the complex nature of miRNA regulation and their hypothesised roles in wood development. The miRNAs highlighted herein are strong candidates for further functional studies as their abundance profiles and predicted targets are consistent with roles in wood development. Copyright / Dissertation (MSc)--University of Pretoria, 2010. / Genetics / unrestricted

Wood is one of the most important products of world trade, due to its countless uses as a source of timber, fibre, and renewable energy. In addition to its economic importance, the formation of wood represents a global carbon sink which reduces the excess atmospheric CO2 that contributes to global warming. The formation of wood or xylogenesis is a complex example of cell differentiation, controlled by multiple interacting environmental factors and the coordinated expression of hundreds of genes. Genomic studies have proved a valuable tool in identifying the genes associated with xylogenesis. The expression of these genes has been shown to under strict spatial regulation in a developmental-stage specific fashion. Despite recent advances in the understanding of this process, there remains much to learn about the cellular, molecular and developmental processes involved. While the spatial regulation of wood formation has been well described, less attention has been devoted to the temporal regulation of this process. Most organisms are known to match their activities to the daily oscillation of night and day in what is known as a diurnal rhythm. A subset of these diurnal rhythms are termed circadian rhythms, and persist in the absence of environmental time cues, with a period of approximately 24 hours. Circadian rhythms are endogenous in nature, being generated by a small number of central oscillator genes, and illustrate an organism's ability to measure time. Circadian rhythms are found across a wide taxonomic spectrum, and are believed to confer an adaptive benefit, possibly due to the ability to anticipate regular changes in the external environment. As wood formation is a major sink for the products of light driven photosynthesis, it represents a likely target for circadian control in plants. A large proportion of photosynthesis genes themselves are known to be under circadian control, as are several cell wall formation genes. Most studies of temporal rhythms in plants, however, have used the herbaceous model species Arabidopsis, which does not have a woody stem. It is likely, therefore, that the circadian control of many wood formation genes remains to be discovered. We used a spotted cDNA mIcroarray carryIng 2608 elements to quantitatively measure daily changes in transcript abundance in the wood-forming tissues of a fast growing, Eucalyptus hybrid. Eucalyptus is a large genus of tree species, many of which are of great economic importance, and are widely grown in plantations for solid timber and pulp production. We found that almost ten percent of the genes on the microarray showed significant daily changes in expression (-loglOP>3.74). These genes included Eucalyptusorthologues of the Arabidopsis central clock genes CCA1 (CIRCADIAN CLOCK ASSOCIATED 1) and GIGANTEA (GI) which cycled with a period and phase matching that seen in Arabidopsis. The remaining genes were involved in pathways including carbohydrate metabolism, hormone signalling, transcription regulation and wood formation. The types of genes that were seen to be diurnally influenced, suggests a role for circadian control of various important plant metabolic pathways, including aspects of carbon allocation to wood formation. / Dissertation (MSc)--University of Pretoria, 2008. / Genetics / 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

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