Studies presented in this thesis, highlight the complexity and importance of Mycosphaerella leaf disease (MLD) on Eucalyptus spp., especially in South Africa. In Chapter I, a review of the literature dealing with Mycosphaerella and MLD of Eucalyptus spp. is presented. It is clear from this review that the disease is prevalent in most countries where Eucalyptus spp. are commercially grown, including Australia where they are native. The number of Mycosphaerella species known from Eucalyptus spp. is increasing and this suggests that their economic effect on commercial Eucalyptus forestry, will probably Increase. It will thus become important to effectively identifY species responsible for MLD. To do this, the existing complex taxonomy of this group of fungi, will undoubtedly prove to be an obstacle. However, DNA based identification methods are proving useful in identifying species and delimiting lineages within Mycosphaerella. Future commercial propagation of Eucalyptus spp. will need to seriously consider the use of hybrids resistant to infection by Mycosphaerella spp. Furthermore, there will be a serious need for effective quarantine measures to prevent the introduction of new, perhaps more pathogenic, Mycosphaerella spp. into areas where they do not already occur. Three species of Mycosphaerella, M. molleriana, M. M. nubilosa and M. juvenis have traditionally been regarded as the most important Mycosphaerella spp. in South Africa. At various times, each species has been considered to be the only pathogen causing MLD in the country. Results from Chapter 2 and Chapter 3 have shown that M. nubilosa is the main pathogen responsible for MLD, especially, on E. nitens in the KwaZulu-Natal province of South Africa. This is interesting as M. molleriana, which was originally thought to be the only species in South Africa, was not isolated. Moreover, the susceptibility of E. nitens to M. nubilosa appears to be high, resulting in severe defoliation. Considering that E. nitens is a popular species grown at higher altitudes of South Africa, the recognition of M. nubilosa is important. This fungus is well recognized in Australia as an important pathogen and comparisons of data from that country will be useful in the future. Several Mycosphaerella spp. have, in the past been found to occur within single stands of commercial Eucalyptus spp. As part of the research presented in Chapter 3, surveys conducted in South Africa showed that there are seven species of Mycosphaerella occurring in plantations. These include: M. ellipsoidea, M. irregulariramosa, M. juvenis, M. lateralis, M. marksii, M. nubilosa and one newly described species M. fori. All of these species, apart from M. fori, were previously known to occur in South Africa. It is interesting that M. juvenis, previously thought to be one of the main species causing MLD, was found only to occur in a low numbers. This suggests that species causing epidemics may change over time. The identification of M. fori from a previously well surveyed area was unexpected. This new species was dominant in Tzaneen and future surveys will be conducted to determine its distribution and host range within South Afiica. The identification of a new species also highlights the need for additional surveys in South Africa to identify new species and to recognize possible new introductions of exotic Mycosphaerella spp. The presence of M. ellipsoidea, M. irregulariramosa, M. lateralis and M. marksii in this survey was not unusual, as they were previously known in South Africa. However, they were found only to occur at low levels and, as such, do not seem to contribute significantly to outbreaks of MLD.Various taxonomic and DNA-based methods have been used for the identification of Mycosphaerella spp. However, some taxonomic characters are of little value at the species level. In Chapter 4, RFLP's were considered as an option to differentiate between species of Mycosphaerella on Eucalyptus. Results of this study showed that the restriction enzyme HaeIII may be used for RFLP identification of Mycosphaerella spp. From a total of twenty-one Mycosphaerella spp. tested, RFLP digestion with HaeIII could resolve six of these species to species level. However, other species formed groups that had similar restriction profiles. They could be further separated based on ascospore germination patterns. This study forms a foundation for future studies in which other enzymes may be used together with HaeIIi to elucidate groups of species. It is suggested though, that this technique be combined with existing methods such as ascospore germination patterns and anamorph associations to identify species of Mycosphaerella occurring on Eucalyptus spp. with confidence. This should negate the use of expensive sequencing techniques, which are currently necessary. In virtually every country where Eucalyptus is grown commercially, MLD is prevalent. However, the specific Mycosphaerella spp. in countries are generally not the same. In Chapter 5, I used DNA sequence data from the ITS region of the rDNA operon as well as morphological data to identify M. heimii from Brazil and Hawaii, U.S.A. This represents the first report of the species from these countries. M heimii was previously thought to occur only in Madagascar and Indonesia, where it is recognized as a primary pathogen of several Eucalyptus spp, including E. urophylla. This is one of the main Eucalyptusspp. in Brazil. It has thus been suggested that this species may have been introduced into these countries via infected seed lots. This highlights the need for effective quarantine measures within these and other South American countries to inhibit the further spread of this pathogen through South America. Copyright / Dissertation (MSc)--University of Pretoria, 2011. / Microbiology and Plant Pathology / Unrestricted

Eucalyptus species form an integral part of the South African forestry industry and their uses extend from paper and pulp production to the synthesis of essential oils which are used in various cosmetic products. Throughout their lifetime these hosts are naturally challenged with various pests and pathogens, most of which cause devastating diseases. An approach to curb the spread of pathogens is to enhance the defence response of the host. Most of the information pertaining to defence against pathogens stems from studies conducted in model organisms such as Arabidopsis, however such information is scarce in woody species such as Eucalyptus. It is understood, from model systems, that once the pathogen is perceived by the host, a cascade of defences are initiated such as the activation of salicylic acid (SA), jasmonic acid (JA) and ethylene (ET) signalling pathways. These pathways in turn activate the expression of genes involved in limiting the spread of the pathogen such as pathogenesis-related (PR) proteins. Certain PR genes have also been shown to be markers of the induction of a specific pathway e.g. PR2 is a marker for the SA pathway. This study aimed to elucidate marker genes specific to the SA (PR1, PR2 and PR5) and JA (PR3, PR4 and LOX) signalling pathways in Eucalyptus grandis using the genome sequence, bioinformatics tools and sequence information from other plant species. A co-phylogenetic approach using neighbour joining analysis and maximum likelihood was used to identify and add confidence in the selection of putative orthologs. Following the selection of orthologous markers, the expression profile of these candidate genes was assessed using Reverse transcriptase quantitative PCR (RT-qPCR). Transcript profiling was conducted under mock induction of the signalling pathways as well as under pathogen stress. For the mock induction of the pathways, the expression profiles of the putative marker genes were investigated under various concentrations of the inducer and at various time points. In the interaction with Chrysoporthe austroafricana it was observed that the SA signalling pathway could have a role in facilitating resistance due to the expression profile observed for EgrPR2. In the tolerant genotype (TAG5) this gene was induced at an earlier time point as opposed to the susceptible genotype (ZG14). These putative markers could provide a diagnostic tool for the screening of pathogen challenged eucalypts to determine which signalling pathway(s) are activated against various pathogens. In addition, this research adds to our knowledge of defence responses in E. grandis by elucidating genes that can be used as targets for improving resistance. Additionally this study provides a stepping stone for understanding mechanisms to curb future tree diseases. / Dissertation (MSc)--University of Pretoria, 2014. / Genetics / Unrestricted

Dissertation (MSc)--University of Pretoria, 2010. / Plant Science / Unrestricted