Molecular taxonomy and mating type genes in Ceratocystis sensu stricto - Witthuhn (née Strydom), Regina Cornelia (University of the Free State, 1999)
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.

Die meeste spesies in Ceratocystis sensu stricto is virulente patogene van 'n wye verskeidenheid plante. Die navorsing saamgevat in hierdie tesis sluit die ontwikkeling van 'n vinnige en betroubare PKR-gebaseerde, RFLP-identifikasie van die Ceratocystis spesies in. 'n 1.6 kb fragment binne die ribosomale DNA operon was direk vanaf lewendige fungus materiaal geamplifiseer, sonder dat die ekstraksie van DNA plaasgevind het. Die geamplifiseerde fragment het die klein en groot sub-eenhede van die rRNA gene, asook die ITS 1 en 2 gebiede ingesluit. Die PKR fragmente was met agtien beperkingsensieme gesny. Vier van hierdie ensieme (AluI, DraI, HaeIII en RsaI) het RFLPs geproduseer wat die species in Ceratocystis van mekaar onderskei het. Die basispaaropeenvolging van die 1.6 kb PKR produkte van die bekendste Ceratocystis spesies was bepaal en filogeneties ontleed. Die filogenie van die taksa was ooreenstemmend met resultate van vorige studies gebaseer op iso-ensieme en die analise van rDNA basispaaropeenvolgings. Ceratocystis coerulescens is 'n bekende oorsaak van blou vlek in sparden en dennebome. Daar is reeds uitgewys dat C. coerulescens uit ten minste vyf morfologies soortegelyke tipes bestaan. Die isolate van C. coerulescens sensu lato en morfologies soortgelyke spesies was op die basis van ITS DNA basispaaropeenvolgings met mekaar vergelyk. Die basispaaropeenvolging van 'n 600 bp fragment binne die ribosomale DNA operon, insluitende die ITS 1 en 2 gebiede, sowel as die 5.8 S rRNA gene, was bepaal en filogeneties ontleed. Die vyf morfologiese tipes, voorheen bekend as C. coerulescens, asook die ander twee taksa vanafkonifers, het 'n sterk ondersteunde monofiletiese groep gevorm, wat al die spesies van konifers ingesluit het. Die spesies van hardehoutbome, C. eucalypti, Ch. australis en Ch. neocaledoniae, het ook 'n monofiletiese groep gevorm, en was 'n sustersgroep van die spesies van konifers. Die vierde spesie van hardehout, C. virescens, was basaal tot die twee sustersgroepe. Die filogenie van spesies m die C. coerulescens kompleks, gebaseer op ITS DNA basispaaropeenvolging, is vergelyk met die filogenie gebaseer op die MAT-2 HMG boks DNA basispaaropeenvolging. 'n 210 bp PKR fragment van 'n gedeelte van die MAT-2 HMG boks van spesies in die C. coerulescens komplek was geamplifiseer. C.jimbriata was gebruik as die buite groep en het afsonderlik van die ander bestudeerde Ceratocystis spesies gegroepeer. Die spesies vanafkonifers het 'n enkele groep gevorm en was 'n sustersgroep van die hardehout spesiegroep. Filogenetiese analise van die MAT-2 HMG boks DNA basispaaropeenvolging het effens verskil van die filogenetiese studie wat op die lTS DNA basispaaropeenvolging gebaseer was. Volgens die ITS DNA basispaaropeenvolging was C. vireseens basaal tot die ander spesies in die C. coerulescens kompleks, terwyl C. laricicola en C. polonica nie van mekaar onderskei kon word nie. Verskille in die MAT-2 HMG boks DNA basispaaropeenvolging van die twee laasgenoemde spesies onderskei dié as twee afsonderlike spesies. Onlangse parings van C. coerulescens het 'n studie aangaande die uitdrukking van die paringstipe gene van spesies in Ceratocystis sensu stricto geïnisieer. C. eucalypti is streng heterotallies. Die meeste ander Ceratocystis spesies, insluitende C. virescens, C. coerulescens en C. pinicola, is homotallies. Die MAT-2 stamme is self-fertiel, terwyl die MAT-l stamme self-steriel is en stadiger groei as die MAT-2 stamme. 'n Gedeelte van die MAT-2 idiomorfin C. eucalypti, C. vireseens en C. pinicola was geamplifiseer deur gebruik te maak van gedegenereerde peilers. Die peilers was ontwerp vanaf die gekonserveerde MA T-2 HMG DNA bindingsmotief. Die verwagte ~300 bp PKR fragmente was gekloneer en die DNA basispaaropeenvolging was daarna bepaal. Spesifieke peilers was ontwerp vir die amplifikasie van 'n 210 bp fragment in slegs MAT-2 isolate van C. eucalypti, C. vireseens en C. pinicola. Hierdie fragment was nie teenwoordig in die selfsteriele (MAT -1) nageslag van C. vireseens en C. pinicola nie. Hierdie bevinding bevestig die verwydering van MAT-2 gedurende een-rigting paringstipe omskakeling. Die bekende DNA basispaaropeenvolging van die C. eucalypti MAT-2 paringstipe idiomorf was vanaf 280 bp tot 1 371 bp uitgebrei, deur van TAIL-PKR en ongelyke PKR gebruik te maak.
Thesis (Ph.D. (Microbiology and Biochemistry))--University of the Free State, 1999
Keywords: Biology -- Classification -- Molecular aspects; Cerato cystis -- Classification