Taxonomy and population genetics of Teratosphaeria causing stem cankers on Eucalyptus trees

Abstract

At the time of commencing this study, there were only five papers published on Coniothyrium canker disease of Eucalyptus. These studies included the formal description of the fungus causing the disease and some aspects of its biology and physiology were characterized. The fungus was described, at that time, as Coniothyrium zuluense, which had a very simple morphology, lacked sexual reproductive structures, had small nondescript conidia and it was slow growing in culture. Nevertheless, the taxonomic status of the Coniothyrium canker pathogens changed in several occasions during this study including placement in genera such as Colletogloeopsis, Kirramyces and Teratosphaeria. After the first appearance of Coniothyrium canker in South Africa, the disease was found in many other parts of the world. DNA sequences from cultures of C.zuluense became easier to obtain and this made it possible to undertake phylogenetic comparisons of isolates from various areas. Such studies also showed that C.zuluense was closely related to Mycosphaerella species. The common appearance of Coniothyrium canker in new areas motivated further studies of this disease and it causal agent, particularly applying newly available rDNA-based techniques. This also provided the motivation for studies presented in this thesis. The thesis is introduced by means of a literature review that treats Coniothyrium canker on Eucalyptus. Briefly, the general characteristics of the host species, Eucalyptus, are described. Furthermore, trends relating to emerging diseases in plantations of Eucalyptus during the past two decades are treated with particular focus being placed on Mycosphaerella diseases. The phylogenetic relationships between Coniothyrium, Mycosphaerella and its anamorphs are considered together with the population biology of related pathogens. In chapter two of this thesis, DNA sequence comparisons were used to determine the phylogenetic position of C.zuluense related to other fungi. In particular, the question as to whether C.zuluense was correctly placed in the genus Coniothyrium and its relatedness to Mycosphaerella was considered. Comparisons with the type species of Coniothyrium, C.palmarum and a collection of sequences of Mycosphaerella species were also conducted. In addition, the identity of isolates obtained from China with similarities in colony morphology to C.zuluense was considered. The objective of the study presented in chapter three was to investigate whether all the available isolates in the FABI collection from different countries and associated with Coniothyrium canker represented a single phylogenetic species. An additional methodological objective of this chapter was to select the best DNA regions for phylogenetic studies on this fungus and its relatives. Four DNA regions were selected based on the informative content as well as ease and reproducibility for Polymerase Chain Reaction (PCR) amplification. The studies presented in Chapter 3 of this thesis showed that two species cause Coniothyrium canker and these are now known as Teratosphaeria zuluensis and Teratosphaeria gauchensis. Therefore, the objectives of the studies presented in chapters four and five were to develop highly variable markers to study the genetic variability and population parameters of populations of both species. This included the development of a robust protocol to isolate microsatellites on both fungi and that would also be informative for related genera. The protocol finally developed and used is presented in Appendix 2 of this thesis. In chapters six and seven, the microsatellite markers developed in the previous chapters were applied. The genetic structure of populations of T.zuluensis and T.gauchensis was thus studied. Analyses of the amplified alleles and their frequencies were used to determine the levels of genetic diversity, clonality and to draw preliminary conclusions regarding the origin and global movement of the pathogens.