Molecular phylogeny and population biology studies on the Eucalyptus canker pathogen Cryphonectria cubensis

Abstract

Cryphonectria canker of Eucalyptus, caused by Cryphonectria cubensis, is considered to be one of the most important fungal diseases affecting Forestry in South Africa. This disease also occurs in other tropical and sub-tropical regions of the world, including South America, Australia and South East Asia. Due to the commercial importance of C. cubensis, several recent studies in South Africa have focused on the elucidation of population diversity and phylogenetic relationships of the fungus from diverse geographic origins. These studies have resulted in more effective tree breeding programmes, and the identification of the possible origin for C. cubensis in South Africa. In this thesis, various aspects of the biology of C. cubensis in South Africa and Colombia are addressed. These studies have focused on the elucidation of the diversity of populations of the fungus occurring in Colombia and South Africa, as well as determination of the phylogenetic relationships of C. cubensis from Colombia. The sexual reproductive system of the fungus from Colombia has also been investigated . . In an investigation into the phylogenetic relationships of C. cubensis isolates from Colombia (Chapter IT), it was found that these isolates are most closely related to other South American isolates. This finding suggests that properties displayed by populations in other South American countries can be extrapolated to the Colombian population, and vice versa. The Colombian isolates were distinct from C. cubensis found in South East Asia, implying a more distant relatedness between these isolates. A study of homothallism and the possibility of sexual outcrossing in Colombian C. cubensis isolates (Chapter III), revealed that the fungus is homothallic. This was shown by allowing single ascospore isolates to reproduce sexually on Eucalyptus twigs, followed by genetic analysis of progeny using DNA fingerprinting. The DNA fuigerprinting profiles of these progeny were identical, indicating that no outcrossing had occurred. The sexual event was, therefore, due to self-fertilisation. In contrast, when progeny from naturally occurring perithecia were analysed genetically using vegetative compatibility groups (VCGs) and DNA fingerprinting, results suggested that oucrossing had occurred, but only to a limited extent. C. cubensis in Colombia is therefore homothallic, but can outcross. Presumably, the same is true for other populations of the fungus in South America. The genotypic diversity of the Colombian population of C. cubensis was investigated using VCGs and RAPDs. Results of this study indicated that the genotypic diversity of this population was similar to diversities previously found for other South American populations of C. cubensis. However, the phenotypic (VCG) and genetic (RAPD) data for the Colombian population were significantly different. The estimation of genotypic diversity based on RAPDs was significantly higher than the same figure for VCG data The reason for the difference in obtained values is attributed to the low level of sensitivity ofVCGs to detect genetic differences between isolates. In Chapter V, a novel technique for obtaining polymorphic, micro satellite-like DNA markers from fungi is described. The technique is based on the identification and characterisation of polymorphic DNA fragments originating from a random amplification of micro satellite sequences using the polymerase chain reaction (PCR). Sequence data from these fragments were used to construct specific primers to amplify polymorphic loci from genomic DNA. The technique has a high success rate in comparison to traditional techniques for the isolation of polymorphic markers. It was also shown that markers obtained with the new technique can be used to differentiate C. cubensis isolates originating in South Africa from those originating in other countries. The last chapter (VI) of this thesis represents the first intensive molecular population diversity study of C. cubensis in South Africa Using polymorphic markers from an earlier study (Chapter V), it was possible to assess the molecular variation of the South African population, and to compare this with phenotypic data obtained previously. It was found that molecular and phenotypic data yield different estimations of population diversity. An estimation of the gametic disequilibrium of the South African C. cubensis population revealed that the tested alleles were randomly associated. Such a situation is expected for populations that preferentially reproduce sexually, and consequently outcross. These figures are, however, only an indirect indication that outcrossing occurs in the South African population. Future studies on the South African C. cubensis population would need to be based on a larger number of markers, and should also include a greater number of isolates of the fungus. Knowledge gained through the studies presented in this thesis will hopefully aid to develop more effective control strategies against Cryphonectria canker of eucalypts. However, new questions about the biology of C. cubensis have emerged that need urgent attention. Data from this thesis might, therefore, prove important to future studies of the fungus.