This study has significantly broadened and deepened the understanding of ecological aspects related to the spread and reproduction of Diplodia pinea as an endophyte, latent pathogen and causal agent of serious disease problems in plantations of Pinus spp. Analyses of genetic diversity in populations using microsatellite data has revealed very high levels of genetic diversity of populations of the pathogen at different spatial levels, ranging from within a single asymptomatic tree to within and between plantations over large geographic areas and in different countries where the fungus has been introduced. Analysis of the structure of the distribution of genotypes and the association of alleles within populations, suggest that sexual recombination is occurring in most environments in the Southern Hemisphere D. pinea populations. This indicates the presence of a cryptic sexual state in this fungus. The genetic diversity was structured and differentiated for regions separated by as little as 65 km to a country and continental scale. The diversity and likely sexual reproduction of D. pinea must complicate control strategies such as selection and breeding for resistance. It is thus essential to strengthen quarantine services aimed at minimizing the risk of introducing additional genotypes of D. pinea. In this regard, understanding the infection and spread between regions is essential. Results of this study demonstrate that this fungus infects seeds, but only at low levels, and is not transmitted vertically via seeds to seedlings. Diplodia pinea was also not isolated from seedlings in three commercial nurseries and open fields in South Africa. These results provide strong evidence that neither seeds nor seedlings are the primary sources of inoculum, but that the pathogen is mainly transmitted horizontally from mature trees and debris left in plantations. Finally extensive sampling conducted as part of this study led to the discovery of the sibling species, D. scrobiculata in South Africa and outside the Northern Hemisphere for the first time. / Thesis (PhD)--University of Pretoria, 2011. / Genetics / Unrestricted

The increasing frequency and severity of windthrow events affecting the forestry industry in New Zealand have raised important management issues surrounding the rate of colonisation of fallen trees by sapstain fungi and the time available for salvage harvesting before sapstain degradation limits potential economic returns. These fungi are known to be spread by a multitude of factors including wind, rain splash, harvesting processes and insect vectoring. Apart from the ecological interest in these interactions between fungi, plants and insects, sapstain fungi are also economically important because their hyphae discolour the sapwood and reduce the overall quality of the timber. The amount of time available to salvage harvest damaged trees is unknown, especially on seasonal and regional scales. Manipulative experiments were established in Pinus radiata forests to examine this seasonal and regional variation in sapstain attack following windthrow, and to investigate the importance of bark beetles as vectors of sapstain fungi. A range of methods were implemented to assess the role of bark beetles as vectors and to ascertain which sapstain fungi are associated with them. Experimental billet logs were caged to exclude beetles and subsequently analyse fungal attack in comparison with identical logs left exposed to beetles. In addition, individual beetles were sampled directly to determine whether they carried spores of particular fungal species and to assess the degree of association in vector-fungal dynamics. Finally, a novel application of DNA melt peak analysis was developed to investigate variation among the fungal communities associated with beetles potentially involved in vectoring sapstain spores. The moisture content of fallen trees was found to be the main factor regulating sapstain development, and when moisture content drops below 100% (on dry weight basis) sapstain fungi grew rapidly. The speed at which this level drops depends on the season, with much faster drying occurring in the warmer months of spring and summer. As a result, trees that fell in the previous winter or autumn did not develop significant sapstain levels until temperatures rose in the following summer, suggesting that storm-damaged trees that fall in winter can be left safely until just before the next summer before they are no longer suitable for salvage harvest. In New Zealand, the bark beetle species acting as vectors of sapstain fungi are not behaviourally adapted to colonisation of logs that are not in contact with the ground. Following windthrow events in pine forests, trees generally lie with their stems suspended above the level of the ground by their branches. As a result, under these circumstances, beetle colonisation of windthrown timber was low, and bark beetles were not a significant vector of stain. The caged and un-caged experimental log billets, however, were in contact with the ground, resulting in colonisation of the un-caged logs. In this case, bark beetles did play an important role in contributing to sapstain intensity, and the stain distribution within the logs mirrored that of the stain distribution. However, this effect may be due to the provision of access points for wind- or water-borne spores of the non-insect vectored stain fungus Diplodia pinea, or to the spread of hyphae through the tunnelling and feeding activities of beetles within the tree, rather than by bark beetles acting as vectors of spores. Bark beetles were confirmed as sapstain vectors with the isolation of seven different ophiostomatoid stain fungi from them, five of which were also found in wood. Finally, the development of a laboratory based, rapid species identification method was developed to identify fungal DNA. Melt peak analysis allowed the species-specific DNA melt temperatures to be compared with the melt temperatures of known species to be able to rapidly, and cheaply, identify an unknown species. Bark beetles are vectors of sapstain fungi in P. radiata forests, however the bark beetle species naturalised in New Zealand prefer to colonise wood when it is in contact with the ground. Following windthrow, trees are generally not attacked by beetles as they are held from the ground by their branches, leaving them to be stained predominantly be wind and rain dispersed stain fungus D. pinea. Stain did not occur until the moisture content of fallen trees dropped below 100%, which only happens in the warm months of summer and spring. In New Zealand, there are interactions between trees and bark beetles, and bark beetles with fungal pathogens from all around the globe resulting in a unique novel assemblage of species together for the first time. Understanding the dynamics of these species in their novel environment is crucial to effectively responding to potential pest threats.

Diplodia pinea causes Diplodia tip blight on more than 30 different pine species. During the past 10 years, Diplodia tip blight has emerged as a serious problem in landscape and Christmas tree farms in this region. Surveys of diseased and symptomless Austrian pines revealed that latent infections of symptomless shoots by D. pinea were common. Latent infections may account for the recently observed rapid decline of mildly diseased pines in our region. To investigate the colonization habits of D. pinea within its host, molecular cytology was attempted and traditional histology was performed on naturally infected, diseased and asymptomatic Austrian pine tissues. I devoted much effort to developing a transformation system for D. pinea. Ultimately I did not succeed in this goal, but I was able to develop a highly efficient protocol for Agrobacterium tumefaciens-mediated transformation of another pathogenic fungus, Colletotrichum graminicola, in the process. The work that I did should help in future efforts to transform D. pinea, something that will be essential if it is to become a tractable system for the study of fungal latency. Traditional histological methods were more successful, and provided important information about the nature of latent infections. Very sparse epiphytic and subcuticular fungal growth was observed in healthy shoots, however, no fungal tissues were present within the shoots. In diseased and latently infected shoots, crevices created between the needle bundles and the shoots were filled with fungal material, and hyphae were observed colonizing the needle sheaths. Hyphae were also observed breaching the shoot epidermal layer in these crevices and colonizing the underlying periderm. D. pinea colonization was extensive in all tissues of diseased shoots early in symptom development. In contrast, localized pockets of degradation were observed in the periderm and adjacent cortical cells located around areas of needle attachment in asymptomatic, latently infected shoots. The mechanism that operates to prevent expansion of these infected pockets in the latently infected shoots is still unclear. Obvious signs of pine defense mechanisms were only observed in 2 shoots. My observations were consistent with the idea that colonization progresses into the vascular tissues, and that this results in symptom development. Vascular colonization may occur more readily if the host is stressed. My research lays the groundwork for future efforts to understand the nature of the transformation from latent to pathogenic infection.