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

<p>Thesis submission for Caleb Kell</p>

<div>Anthropogenically driven global change is disrupting ecosystems and habitats of many plant species, straining the ability of native species to survive and reproduce. The overarching goal of this research was to holistically work towards restoration of a threatened tree species by connecting research from different disciplines. In order to do so, the threatened butternut tree (<i>Juglans cinerea</i>) and its hybrids were used as a case study. Hybridization can incorporate stress tolerance in plants and could be a potential restoration tool. Evidence in some wild butternut populations indicates that naturalized hybrids of butternut with Japanese walnut (<i>Juglans ailantifolia</i>) may be more tolerant to butternut canker disease (BCD) than butternut, but this has not been formally tested. Thus, chapter 2 examined potential BCD tolerance within and between unadmixed and hybrid butternut inoculated with two BCD fungal isolates. Differences in canker growth were observed by fungal isolate, which could help to explain some differences in BCD severity found among butternut populations. Smaller and fewer cankers and greater genetic gains were detected in hybrid families, demonstrating that hybrids warrant further evaluation as a possible breeding tool for developing BCD-resistant butternut trees.</div><div>However, even with increased disease tolerance, hybrids must possess similar ecophysiological tolerances to their native progenitor to be an effective replacement. Butternut is extremely cold hardy, but Japanese walnuts are native to a warmer ecosystem, indicating potential disparities in extreme temperature tolerances between the two species and their hybrids. Thus, samples from mature trees were subjected to cold and heat treatments to compare relative extreme temperature tolerances within butternut and between butternut, Japanese walnut, and their hybrids. Within butternut, trees from colder areas exhibited less cold damage than those from warmer areas. Differences in heat damage among provenances occurred but did not follow a clear trend. Butternut exhibited greatest cold tolerance, Japanese walnut exhibited greatest heat tolerance, and hybrids were intermediate. Thus, the utility of hybrids for restoration could be limited at the extremes of the species’ distributions.</div><div>A second, but different type of freeze test was conducted for chapter 4 using seedlings to gain a more nuanced understanding of cold tolerance within butternut and between butternut and its hybrids. No survival or damage differences were detected in butternut provenances, although seedlings from the coldest provenances experienced more delayed budbreak at the two warmest treatments than those from warmer provenances. Interspecific differences were not observed in dieback but were detected in survival and budbreak. The hybrids had greater survival than butternut from warmer provenances at the lowest temperature treatment (-38 °C), but given that temperatures that low are extremely unlikely to occur in those provenances, it is not anticipated to give the hybrids an advantage if planted in those areas. However, the hybrids’ earlier budbreak could limit the success of restoration with these hybrids in the coldest extents of butternut’s range. </div><div>If hybrids, as well as genetically modified (GM) trees, are successfully developed for effective disease tolerance and to serve as an ecologically suitable replacement, success of restoration using hybrids will ultimately depend on those directly responsible for replanting efforts. A survey was administered to land managers in 46 organizations in Indiana to gauge perceptions of hybrid and GM trees, as well as current use of hybrid trees. Land managers had stronger concern for ecological, rather than economic, issues. Agreement was highest for using hybrid and GM trees for “conservation and restoration of at-risk species”, “timber production”, and “non-timber products (fruit, syrup, etc.)”. However, perceptions varied by characteristics, such as concern type, age, and the type of land they managed. Ecological concern and the type of land being managed most strongly predicted current hybrid use. Overall, results indicate the majority of land managers in Indiana would likely be agreeable to recommendations towards using hybrids. However, most nonetheless had strong ecological concerns about their suitability as a native replacement. It is important to note, though, that consistent with the results of previous studies, great variation was seen within the performance and characteristics of the butternut hybrids in chapters 2-4. Thus, it may be possible with careful selection and breeding to harness this variation to develop disease tolerant and ecologically similar hybrids acceptable to land managers.</div>