Studies on Cryphonectria cubensis in South Africa with special reference to mycovirus infection

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.