The Koala bears housed and maintained by the Pretoria Zoo are relying on 20 different Eucalyptus species in the Eucalyptus arboretum in Pretoria for food. Species of the Botryosphaeriaceae are well known endophytes and pathogens of Eucalyptus in South Africa and elsewhere. This study was aimed at characterizing the Botryosphaeriaceae infecting these trees planted for the Koala bears. The experimental set-up also provided the opportunity to broaden our knowledge regarding the Eucalyptus species preferences of the Botryosphaeriaceae, as well as to reveal their potential movement to and from surrounding trees. In Chapter 1, the literature is reviewed regarding the biology of fungal endophytes, with a specific focus on Botryosphaeriaceae on Eucalyptus. Such a review clearly shows that, despite much previous work, we still only have a limited understanding of the biology and ecology of most fungal endophytes. This is particularly true for tree endophytes. Botryosphaeriaceae are clearly important and widespread canker and dieback pathogens of Eucalyptus. The taxonomic confusion that plagued the group, however, hindered a clear understanding of their diversity and biology. The use of molecular tools together with morphological characteristics has improved the ability to separate these fungi at species level. These tools are important for future work to better understand the true diversity. This would be a prerequisite if we are to gain a better understanding of the biology of these fungi and design with management strategies to control them. In Chapter 2, five species of Botryosphaeriaceae were identified from Eucalyptus species in the Pretoria arboretum, South Africa. Two species were described here as new species of Botryosphaeriaceae, within Neofusicoccum, namely N. ursorum prov. nom. and N. crypto-australe prov. nom. This is the first report of the latter species on Eucalyptus in South Africa. The other species included N. parvum, N. eucalypti comb. nov. (previously known as Dichomera eucalypti) and B. dothidea. Neofusicoccum parvum was the most common species isolated, followed by N. ursorum and N. eucalypti. The identifications were all based on the morphological characteristics, including cultural and conidial morphology, and DNA sequence data of the internal transcribed spacer region (ITS 1 and 4), and the translation elongation factor 1-α. The closely related species N. parvum and N. ribis were distinguished using a previously designed PCR-RFLP technique. As for the distinction between the cryptic species N. crypto-australe and N. australe, it was necessary to analyze a third gene region (β- tubulin) to confirm their separation using the phylogenetic species concept. All isolates obtained from this study produced lesions on stems of Eucalyptus camaldulensis (clone ZG-14) in the pathogenicity trial conducted under green house conditions. Of all species, N. eucalypti and N. crypto-australe were found to be the most pathogenic and B. dothidea the least pathogenic. There was, however, also significant variation in virulence between isolates of the same species. The results clearly show the potential threat of species of Botryosphaeriaceae to Eucalyptus. Field trials should be conducted in future studies to validate the findings in the greenhouse trials. Isolates representing different species identified in Chapter 2, were used for designing molecular tools for the in vitro and in vivo identification of Botryosphaeriaceae (Chapter 3). Sequences of the translation elongation factor 1-α were compared, and unique polymorphisms identified. Species specific primers were designed around these polymorphisms. All the primers designed were proven to be specific enough to distinguish the five different species from each other. The sensitivity of all primers were shown to detect fungal DNA concentration between 50 ng/μL and 0.01ng/μL. Preliminary tests of these primers on Eucalyptus leaves were done, and latent infections of N. parvum (the most common species) could be identified. More samples are likely to reveal the latent infections of other species using these tools. Future studies could now use these tools for the rapid identification of the fungi on Eucalyptus. It could be expanded to other hosts and more species of the Botryosphaeriaceae as well. The results presented in this study provided detailed information on species of the Botryosphaeriaceae from the 20 different species of Eucalyptus in a Pretoria arboretum. It yielded unique species, as well as well known pathogens of this host. All species isolated in this study were found to be pathogenic on Eucalyptus. The knowledge foundation, data and tools provided by this study can now be applied to characterize the infection biology, fine scale distribution and population diversity of these fungi. It is likely to bring new insights into the ecology of these organisms, their potential origin and movement between hosts. The presence of these fungi in other parts of South Africa should also be considered.