Doctoral Degrees (Microbial, Biochemical and Food Biotechnology)

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Browsing Doctoral Degrees (Microbial, Biochemical and Food Biotechnology) by Advisor "Harrington, T. C."

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    Molecular taxonomy and mating type genes in Ceratocystis sensu stricto
    (University of the Free State, 1999-01) Witthuhn (née Strydom), Regina Cornelia; Wingfield, B. D.; Wingfield, M. J.; Harrington, T. C.
    English: Most species of Ceratocystis sensu stricto are virulent pathogens of a wide variety of plants. In the research presented in this thesis, I have developed a rapid and reliable PCR-based RFLP identification method for species of Ceratocystis. A 1.6 kb fragment within the ribosomal DNA operon was directly amplified from living fungal tissue, without extracting DNA. The amplified fragment included part of the small and large sub-unit rRNA genes, the 5.8S rRNA gene and the internal transcribed spaeers 1 and 2. The PCR fragments were digested with eighteen restriction enzymes. Four of these (AluI, DraI, HaeIII and RsaI) produced RFLPs that separated the species of Ceratocystis. The 1.6 kb PCR products, from the better known species of Ceratocystis, were sequenced and phylogenetically analyzed. The delimitation of taxa was consistent with results of previous studies using isozymes and rDNA sequence analysis. Ceratocystis coerulescens is a well-known cause of blue stain in spruce and pine. It was shown that C. coerulescens encompasses at least five morphological types. I compared isolates of C. coerulescens sensu lato and morphologically similar species on the basis of lTS DNA sequences. A 600 bp fragment within the ribosomal DNA operon, including the 5.8S rRNA gene and ITS 1 and 2, was amplified, sequenced and analyzed using PAUP. The five morphological types previously known as C. coerulescens, and the two other taxa from conifers, formed a strongly supported monophyletic group that includes all the Ceratocystis species occurring primarily on conifers. The species from hardwood trees, C. eucalypti, Ch. australis and Ch. neocaledoniae, also formed a monophyletic group, sister to the conifer group. The fourth species from hardwoods, C. virescens, formed a group basal to the two sister groups. The phylogeny of species in the C. coerulescens complex based on ITS DNA sequences were compared to the phylogeny based on the MAT-2 HMG box DNA sequences. A 210 bp PCR fragment of part of the MA T-2 HMG box of species in the C. coerulescens complex was amplified. C.fimbriata was used as the outgroup taxon and was distinct from the other Ceratocystis species studied. The species from conifers formed a single clade, sister to the clade formed by the species from hardwoods. Phylogenetic analysis of the MAT-2 HMG box DNA sequences differed slightly from the phylogenetic analysis based on ITS DNA sequences. Based on ITS DNA sequences C. vireseens was basal to the other species in the C. coerulescens complex, while C. laricicola and C. polonica could not be separated from each other. Differences in the MAT-2 HMG box DNA sequences for the latter two species clearly showed them to be as distinct from each other. Recent mating studies on C. coerulescens have prompted a study of the expression of mating type genes in species of Ceratocystis sensu stricto. C. eucalypti is strictly heterothallic. Most other Ceratocystis species, including C. virescens, C. coerulescens and C. pinicola are homothallic. The MAT-2 strains are self-fertile, while MAT-1 strains are self-sterile and grow more slowly than MAT-2 strains. Part of the MAT-2 idiomorph in C. eucalypti, C. vireseens and C. pinicola was amplified using degenerate primers designed from the conserved MAT-2 HMG DNA binding motif. The expected ~300 bp PCR products were cloned and sequenced. Specific primers were designed that amplified a 210 bp fragment only in MAT-2 isolates of C. eucalypti, C. vireseens and C. pinicola. This fragment was absent from the self-sterile (MAT -1) progeny of C. vireseens and C. pinicola, confirming the deletion of MAT-2 during uni-directional mating type switching. The known DNA sequence data for the C. eucalypti MAT-2 mating type idiomorph was increased from 280 bp to 1 371 bp, using TAIL-peR and uneven PCR.