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

Much research has been published on Mycosphaerella spp. causing Mycosphaerella Leaf Disease (MLD) on Eucalyptus spp. The first chapter of this thesis presents a review of the literature on this topic and focuses on the taxonomy, phylogeny and population biology of Mycosphaerella spp. occurring on Eucalyptus. From the published literature, it is clear that the majority of research conducted on MLD has focussed on the epidemiology and taxonomy of Mycosphaerella spp and the susceptibility of Eucalyptus hosts to species of Mycosphaerella. Advances in DNA-based technologies have, however, lead to extensive DNA sequence datasets of Mycosphaerella spp occurring on Eucalyptus. These datasets have provided substantial insight into species concepts for Mycosphaerella and have led to the realisation that many morphological species are complexes of several cryptic phylogenetic taxa. Furthermore, a recent application to the study of Mycosphaerella spp. occurring on Eucalyptus is that concerning their population dynamics. Such studies will aid in our understanding of the genetic structure of Mycosphaerella populations and their movement between countries. These population-based studies will aid forestry companies in establishing Eucalyptus breeding programmes to produce tolerant Eucalyptus genotypes that may be deployed in commercial forestry operations. Mycosphaerella spp. are difficult to identify, due to their conserved teleomorph morphology and the lack of natural occurrences of anamorph structures. DNA sequence data have, therefore, become the definitive technique used to identify Mycosphaerella spp. The Internal Transcribed Spacer (ITS) region of the ribosomal RNA operon has traditionally been targeted for DNA sequence comparisons. However, this gene region does not offer sufficient resolution to discriminate cryptic taxa or resolve deeper nodes within Mycosphaerella. Results presented in chapter two of this thesis present a multi-gene phylogeny for the identification of Mycosphaerella spp. occurring on Eucalyptus. This is based on DNA sequence data from four nuclear gene regions. The generation of these sequence datasets has allowed for competent elucidation of cryptic taxa, species complexes and the greater resolution of deeper nodes within Mycosphaerella. Furthermore, these results have also led to recognising that Mycosphaerella ambipyhlla and M. vespa is a synonym of Mycosphaerella molleriana and Pseudocercospora epispermogonia is recognised as the asexual state of Mycosphaerella marksii. A serious foliar disease of Eucalyptus camaldulensis and hybrids of this species has been known from Thailand and Vietnam for many years. This disease has been known to be caused by a species of Pseudocercospora and was attributed to the cosmopolitan Pseudocercospora eucalyptorum. Results of a study presented in chapter three of this thesis have, however, clearly shown that P. eucalyptorum is not the causal agent of the disease observed on E. camaldulensis in Thailand. By employing classical morphological techniques and DNA sequence data from four nuclear gene regions, I have shown that an undescribed species of Pseudocercospora is responsible for epidemics of this leaf disease. This species is formally described as Pseudocercospora flavomarginata. P. flavomarginata is only known from Thailand and Vietnam. However, considering that E. camaldulensis is planted in other south-east Asian countries and that E. camaldulensis is the most commonly found Eucalyptus sp. in Australia, further surveys in these areas will most likely lead to the discovery of the pathogen from these countries. Techniques that have been used to identify Mycosphaerella spp. include classical morphological comparisons and analyses of DNA sequence data. These techniques have, however, allowed only for the study of the evolutionary history within Mycosphaerella and for species identification. Recent advances in the field of population biology have led to the study of many fungal pathogens at a population level. One of the main tools used to study population biology involves applying DNA-based microsatellite markers. Chapter four of this thesis focuses on the development of DNA-based microsatellite markers for the Eucalyptus leaf pathogen Mycosphaerella nubilosa. By employing specific enrichment protocols, I was able to develop ten polymorphic microsatellite markers for M. nubilosa. These microsatellite markers exhibit high specificity for M. nubilosa and did not cross amplify with other Mycosphaerella spp. that are closely related to M. nubilosa. Mycosphaerella nubilosa has been extensively studied with respect to its taxonomy and epidemiology. However, nothing is known regarding the population biology of this important Eucalyptus leaf pathogen. Therefore, DNA-based microsatellite markers developed in chapter four of this thesis were used to study the population biology of M. nubilosa from several different geographic locations. Results presented in chapter five of this thesis show that populations of M. nubilosa from eastern Australia are genetically more diverse than those populations from western Australia, Africa and Europe. This indicates that eastern Australia is the likely centre of origin for M. nubilosa. Furthermore, based on shared haplotypes between M. nubilosa populations used in this study, I have proposed a pathway of gene flow of M. nubilosa. This suggests that the pathogen moved from eastern Australia to both western Australia and South Africa and then from South Africa into other countries in Africa and finally into Europe. An interesting result emerging from the population biology study presented in chapter five, is the finding that M. nubilosa appears to employ a homothallic mating strategy. Thus, opportunities exist, in countries with limited genetic diversity of M. nubilosa, to breed for Eucalyptus resistance. From the high number of M. nubilosa haplotypes observed in Australia and South Africa, it is also important that this pathogen be added to quarantine action lists to prevent the movement of contaminated Eucalyptus germplasm. This is necessary to prevent novel M. nubilosa haplotypes from moving into new environments where susceptible Eucalyptus spp. are propagated. Mycosphaerella nubilosa is one of the most pathogenic Mycosphaerella spp. causing MLD on Eucalyptus. Surveys of diseased Eucalyptus plantations from several countries where this pathogen occurs, have resulted in an extensive collection of M. nubilosa isolates. Recently, DNA-based studies have led to the hypothesis that M. nubilosa may represent two distinct taxa. Results of studies presented in chapter six of this thesis indicate that two distinct ITS phylogenetic lineages are represented by M. nubilosa sensu lato. These are characterized by defined geographic distributions and Eucalyptus host associations. M. nubilosa ITS lineage 1 is found exclusively in New Zealand, Tasmania and Victoria, eastern Australia occurring on E. globulus. M. nubilosa ITS lineage 2 has a broader geographic distribution and can be found in Spain, Portugal, Tanzania, Kenya, Ethiopia, South Africa, western Australia, Victoria and New South Wales, eastern Australia, where it occurs on E. globulus and several other Eucalyptus spp. that are used in commercial forestry including E. nitens. It is envisaged that results presented in chapter six will lead to more extensive studies into M. nubilosa sensu lato that may result in the description of a new Mycosphaerella sp. represented by M. nubilosa ITS lineage 1. / Thesis (PhD)--University of Pretoria, 2011. / Microbiology and Plant Pathology / Unrestricted