B.J.H. Janse

Phylogenetic relationships of the genus Phanerochaete inferred from the internal transcribed spacer region.

Phanerochaete is a genus of resupinate homobasidiomycetes that are saprophytic on woody debris and logs. Morphological studies in the past indicated that Phanerochaete is a heterogeneous assemblage of species. In this study the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA was used to test the monophyly of the genus Phanerocthaete and to infer phylogenetic relationships of the 24 taxa studied. Maximum parsimony, maximum likelihood, and Bayesian analyses do not support the monophyly of the genus. However, a core group of species represented by Phanerochaete velutina, P. chrysosporium, P. sordida, P. sanguinea and others are closely related and group together in a clade. Other common Phanerochaete species including Phanerochaete rimosa, P. chrysorhiza, P. omnivora, P. avellanea, P. tiberculata, P. flava, and P. allantospora, however, do not cluster with the core Phanerochaete group.

Comparison of H2O2‐producing enzymes in selected white rot fungi

Using fungi grown on synthetic agar medium, we evaluated and compared the concentration of various H2O2-producing enzymes. Our results showed that oxidase production in solid medium was better than that found in liquid medium and as high as that detected in wood samples. High yields of oxidases made it possible to compare different oxidases in the same culture extracts and under different conditions. Our results also indicated that H2O2 production is ubiquitous in the white rot fungi tested and that enzyme levels are influenced by the substrate composition.

Regional sequence homologies in starch-degrading enzymes.

The enzymatic hydrolysis of starch, consisting of linear (amylose) and branched (amylopectin) glucose polymers, is catalyzed by α-, β- and glucoamylases (γ-amylases), cyclodextrinases, α-glucosidases, and debranching enzymes. Saccharomyces cerevisiae cannot utilize starch. Our laboratory has previously co-expressed the Bacillus amyloliquefaciens α-amylase (AMY) and the Saccharomyces diastaticus glucoamylase (STA2) genes in S. cerevisiae. A gene encoding a debranching enzyme (pullulanase) from Klebsiella pneumoniae ATCC15050 was cloned and its nucleotide sequence determined. This gene will be co-expressed with the α- and γ-amylase to produce an amylolytic S. cerevisiae strain. Extensive data base comparisons of the K. pneumoniae pullulanase amino-acid sequence with the the amino-acid sequences of other debranching enzymes and α-, β- and γ-amylases (from bacteria, yeasts, higher fungi and higher eukaryotes), indicated that these debranching enzymes have amino-acid regions similar to those found in α-amylases. The conserved regions in α-amylases comprise key residues that are implicated in substrate binding, catalysis, and calcium binding and are as follows. Region 1: DVVINH; region 2: GFRLDAAKH and region 4: FVDNHD. When comparing conserved regions, no similarity could be detected between debranching enzymes and β- and γ-amylases.

Characterization of Some Cylindrocladium Species with Three-Septate Conidia Using Morphology, Isozyme Banding Patterns and DNA Polymorphisms

Summary Morphological and growth characteristics, esterase isozyme banding patterns and total DNA profiles were obtained for isolates of Cylindrocladium citri Fawcett & Klotz, C. crotalariae (Loos) Bell & Sobers, C. ilicicola (Hawley) Boedijn & Reitsma, C. penicilloides (Tub.) Tubaki, C. spathulatum El-Gholl, Kimbrough, Barnard, Alfieri & Schoulties as well as seven previously unidentified isolates. The study of zymograms and total DNA banding patterns enhanced our ability to determine relationships among and within species. Each procedure allowed us to differentiate among all the species except between C. penicilloides and C. citri, which appeared distinct only on the basis of their esterase zymograms. C. penicilloides may therefore be regarded as an intraspecific variant of C. citri. Furthermore, a new species, C. variabile, and its teleomorph, Calonectria variabilis, are described. The new species has distinct vesicle morphology, esterase and total DNA banding patterns.

Genetic Variation in Cylindrocladium floridanum and other Morphologically Similar Cylindrocladium Species

Summary Species of Cylindrocladium (C.) are anamorphs of the genus Calonectria (C a .), and are important pathogens of numerous crops worldwide. C. floridanum is characterized by short, 1-septate, straight, cylindrical conidia and sphaeropedunculate vesicles. Isolates of Ca. kyotensis (anam. C. floridanum ), Ca. candelabra (anam. C. scoparium ), Ca. morganii (anam. C. candelabrum ), C. ovatum and C. naviculatum were compared based on morphology, sexual compatibility, radial growth on media with different osmotic potentials, RAPD markers and A + T-rich DNA (AT-DNA) polymorphisms. In CLUSTER analyses of the data using the average linkage method, all five species clustered separately. RAPD profiles of ex-type cultures of the two acknowledged synonyms of Ca. kyotensis ( Ca. floridana, Ca. uniseptata ) shared 78–97% similarity, supporting their conspecificity. Strains of the opposite mating type of the respective heterothallic species studied shared high similarity coefficients of 77% for Ca. candelabra , and 92% for Ca. morganii . Two opposite mating types of C. ovatum with 99% similarity mated to produce a new teleomorph, described here as Calonectria ovata . Based on their RAPD and AT-DNA profiles, two major groups could be distinguished within Ca. kyotensis . Group two (including most of the Canadian isolates) shared only 12–45% similarity with the ex-type strains (group one) and clustered with mean correlation coefficients of r = 0.56 (RAPD analysis) and r = 0.37 (AT-DNA analysis), respectively. An isolate similar to Ca. kyotensis but with curved conidia had distinct RAPD and AT-DNA profiles, and shared less than 35% similarity ( r = 0.00) with any of the species studied. These findings suggest that strains with curved conidia and sphaeropedunculate vesicles represent an undescribed taxon, and the name Cylindrocladium curvisporum is thus proposed for them.

DNA homology between Pyrenophora japonica and P. teres

Pyrenophora isolates associated with net- and spot-type net blotch of barley were compared with verified isolates of P. teres f. teres, P. teres f. maculata and P. japonica using total DNA banding patterns, morphology, symptomatology and mating studies. DNA of South African and overseas spot- and net-type isolates digested with Hae III and Msp I showed 74–100 % similarity. Furthermore, spot- and net-type isolates could be mated to produce viable progeny, indicating P. teres to have two-allele heterothallism. Single-ascospores inoculated on differential barley cultivars produced spot, net or mixed symptoms, suggesting that recombination has occurred. Based on the high degree of homology in DNA banding patterns between P. japonica and P. teres f. maculata , as well as similarities in symptom expression on differential cultivars and general morphology, P. japonica is proposed as synonym of P. teres .

Isolation and enzymic characterisation of South African white-rot fungi

Over 600 basidiomycetes were isolated from indigenous forests and commercial Eucalyptus and Pinus plantations in South Africa. One hundred and twenty of the cultures were identified and biochemical tests were done to screen the cultures for characteristics that are favourable for biopulping. Most of the white-rot fungi previously associated with biopulping elsewhere in the world were also isolated in South Africa, as well as an isolate with uniquely regulated ligninolytic systems. Phanerochaete chrysosporium was found to be a natural coloniser of wood chip piles in South Africa.

Expression of the Klebsiella pneumoniae pullulanase-encoding gene in Saccharomyces cerevisiae

A 3800-base pair (bp) DNA fragment encoding the mature pullulanase from Klebsiella pneumoniae was inserted between two different yeast expression-secretion cassettes and an yeast gene terminator. These cassettes were cloned into an yeast centromeric plasmid YCplacIII and transformed into laboratory strains of Saccharomyces cerevisiae. Transcription initiation signals were derived from the mating pheromone α-factor (MFα1p) and alcohol dehydrogenase (ADC1p) gene promoters. Secretion of pullulanase was directed by the leader sequence of the yeast mating pheromone α-factor (MFα1s). Transcription termination was effected by the yeast tryptophan synthase gene terminator (TRP5T). Southernblot analysis confirmed the presence of pulA in transformed yeasts and Northern-blot analysis revealed the presence of PUL1 mRNA. A pullulan agarose assay indicated the extracellular production of biologically active pullulanase by S. cerevisiae.

Plasmids in Leuconostoc oenos

A new procedure was used to isolate 11 plasmids from eight Leuconostoc oenos strains. Plasmid DNA was not detected in 34 other strains of this species. Plasmid sizes ranged from 2.47 to 4.61 kilobase pairs. This is the first report of extrachromosomal elements in L. oenos.

Biochemical and molecular characterization of South African strains of Phanerochaete chrysosporium

Fifty-five strains of Phanerochaete chrysosporium were isolated in South Africa, and screened for indicators of ligninolytic activity: lignin peroxidase (LiP), manganese peroxidase (MnP) and glyoxal oxidase (GLOX). MnP-production as a function of time was followed in all strains. Nine strains were selected for quantification of MnP, LiP and GLOX activities. Statistically significant variation in MnP and GLOX activities existed among the different strains. Under low nitrogen, LiP activity of selected strains showed no significant variation, whereas strain PP25 had significantly increased LiP levels under high nitrogen conditions. Probing genomic DNA with the genes encoding lignin peroxidase ( lipD and lipl1 ), manganese peroxidase ( mnp2 ), and glyoxal oxidase ( glox ) showed significant genetic diversity with lignin peroxidase and manganese peroxidase probes, but not with the glyoxal oxidase probe.