Leptographium is an anamorph genus within the Ophiostomatoid group of fungi and represents a unique case for molecular applications. The genus has a near complete sequence data available for three genes across all known species. This characteristic makes it a perfect test group for investigating applications of new diagnostic techniques within ascomycetes. Probes and primers, for microarrays, are designed from phylogenetically useful gene regions and are fabricated onto a solid substrate using printing technology. The sample is prepared using PCR and is hybridised to the probes under stringent conditions. The resulting fluorescent pattern is rigorously analysed to distinguish species from each other. Diagnostic PCR uses primers that are designed in similar way to the way probes are designed for microarrays and indicate the presence of a species through positive amplification. This research methodology will be applied to Leptographium to evaluate the efficacy of microarray technology for discriminating species within that genus. The data gained from this research study will be used in applications for other genera using microarray technology. / Dissertation (MSc)--University of Pretoria, 2009. / Genetics / Unrestricted

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Evolutionary relationships amongst strains of the fungal genus Leptographium and related taxa were inferred using the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat. To generate robust sequence alignments for phylogenetic analysis the relationship between DNA sequence variability and RNA structural conservation of ITS segments was examined. The results demonstrate that structural conservation of helical regions is facilitated by compensatory base changes, compensating insertions/deletions, and, possibly, RNA strand slippage. A high mol % G+C bias for ITS1 and ITS2 and structural constraints at the RNA level appear to limit the types of changes observed. Fifty strains of Leptographium were screened for the presence of introns within mitochondrial genes. Superimposing intron survey data onto the ITS-derived phylogenetic tree reveals that introns are absent from the small ribosomal RNA (rns) gene of all strains of L. procerum yet are found in all strains of L. lundbergii. Amongst members of L. wingfieldii, L. terebrantis, and L. truncatum intron distribution is stochastic and is not correlated to the evolutionary relationships amongst strains. A group II intron/LAGLIDADG homing endonuclease gene (HEG) composite element from the mt rns gene of L. truncatum strain CBS929.85 was characterized. Intron-catalyzed splicing was tested using ORF-less and ORF-containing precursor transcripts, and both versions of the intron readily self-splice under moderate temperature and ionic conditions (37 °C and 6 mM MgCl2). Cleavage activity of the intron-encoded protein (I-LtrII) was tested using an N-terminal His6-tagged and near native protein. The homing endonuclease cleaves double-stranded DNA 2 nucleotides upstream of the intron insertion site within the exon, generating 4 nucleotide 3’ OH overhangs. Intron splicing is not enhanced by the addition of I-LtrII and RNA-binding assays indicate that the His6-tagged protein does not bind to the intron. Phylogenetic relationships amongst the rns gene, intron, and amino acid sequences were inferred. An evolutionary model of the composite element is proposed in which the HEG invaded a group II intron and mobilized it. The mobile genetic element may be transmitted vertically amongst L. lundbergii strains and horizontally through lateral gene transfer amongst strains of L. wingfieldii, L. terebrantis, and L. truncatum.

Evolutionary relationships amongst strains of the fungal genus Leptographium and related taxa were inferred using the internal transcribed spacer (ITS) region of the nuclear ribosomal DNA repeat. To generate robust sequence alignments for phylogenetic analysis the relationship between DNA sequence variability and RNA structural conservation of ITS segments was examined. The results demonstrate that structural conservation of helical regions is facilitated by compensatory base changes, compensating insertions/deletions, and, possibly, RNA strand slippage. A high mol % G+C bias for ITS1 and ITS2 and structural constraints at the RNA level appear to limit the types of changes observed. Fifty strains of Leptographium were screened for the presence of introns within mitochondrial genes. Superimposing intron survey data onto the ITS-derived phylogenetic tree reveals that introns are absent from the small ribosomal RNA (rns) gene of all strains of L. procerum yet are found in all strains of L. lundbergii. Amongst members of L. wingfieldii, L. terebrantis, and L. truncatum intron distribution is stochastic and is not correlated to the evolutionary relationships amongst strains. A group II intron/LAGLIDADG homing endonuclease gene (HEG) composite element from the mt rns gene of L. truncatum strain CBS929.85 was characterized. Intron-catalyzed splicing was tested using ORF-less and ORF-containing precursor transcripts, and both versions of the intron readily self-splice under moderate temperature and ionic conditions (37 °C and 6 mM MgCl2). Cleavage activity of the intron-encoded protein (I-LtrII) was tested using an N-terminal His6-tagged and near native protein. The homing endonuclease cleaves double-stranded DNA 2 nucleotides upstream of the intron insertion site within the exon, generating 4 nucleotide 3’ OH overhangs. Intron splicing is not enhanced by the addition of I-LtrII and RNA-binding assays indicate that the His6-tagged protein does not bind to the intron. Phylogenetic relationships amongst the rns gene, intron, and amino acid sequences were inferred. An evolutionary model of the composite element is proposed in which the HEG invaded a group II intron and mobilized it. The mobile genetic element may be transmitted vertically amongst L. lundbergii strains and horizontally through lateral gene transfer amongst strains of L. wingfieldii, L. terebrantis, and L. truncatum.

Leptographium have been known since the early part of the 20th Century and include of many species causing blue stain of timber. Among these species are several species known or believed to be involved in causing diseases of trees. Leptographium spp. occur mainly on conifers and many species are recognized as anamorphs of Ophiostoma. Similar to Ophiostoma, Leptographium spp. are closely associated with insects. Their morphology thus reflects this association, and they thus have upright conidiophores with slimy masses that are produced in beetle galleries. Leptographium spp. are morphologically very similar to each other and this makes their accurate identification difficult. The first part of this thesis, presents dichotomous, as well as synoptic keys for the identification of these species. These keys are supported by comprehensive descriptions accompanied by both photographs and line drawings. The second part of this thesis. deals with several key taxonomic questions pertaining to Leptographium. Chapter one represents a phylogenetic study of the majority of species in Leptographium. Morphological characters were coded and analyzed. The results of the molecular and the morphological analyses are compared to determine whether any morphological characters might be used to infer phylogeny. The results indicate that morphology does not infer phylogenetic relatedness. Chapter two represents a comparison between Leptographium abietinum and L. engelmannii. These species are morphologically similar, and various authors have suggested that they are synonyms. Based on morphology, L. engelmanni was synonomised with L. abietinum. Furthermore, examination of various atypical isolates led to the description of the new species, L. hughesii. In chapter three, Ophiostoma europhioides, O. piceaperdum and Ceratocystis pseudoeurophioides are compared. These species have Leptographium anamorphs and are morphologically identical. Both O. europhioides and C. pseudoeurophioides are synonymised with O. piceaperdum, and a name is provided for the anamorph of O. piceaperdum. Chapter four represents a re-evaluation of Phialocepha/a phycomyces. The inconspicuous collarettes, characteristic of this fungus, are unlike the deep¬seated collarettes of the type species of Phialocepha/a (P. dimorphospora). Scanning and transmission electron microscopy revealed that conidiogenesis in P. phycomyces is phialidic, placing this species among other Phialocephala spp. However, P. phycomyces is able to tolerate high concentrations of cycloheximide, characteristic of Leptographium spp. DNA analysis indicates that this species does not belong in either Phialocepha/a or Leptographium. A new genus Kendrickiella is described to accommodate this species. In chapter five, a new species of Leptographium, L. eucalyptophilum, is described. This species is unique in that it occurs on Eucalyptus, which is an unusual host for this species. In addition, this species is one of several described from tropical regions and it is apparently adapted to this habitat. Chapter six represents a critical re-evaluation of isolates identified as L. procerum. Morphological comparison of these isolates revealed that L. procerum sensu lato, represents more than one taxon. From this study, three new species of Leptographium were described. These are L. alethinum, L. pityophilum and L. euphyes. These species can easily be distinguished from L. procerum s. str. and their incorrect identification is probably as a result of their shared habitat. In chapter seven, I describe an additional three species of Leptographium. Like most other Leptographium spp., these were isolated from conifers. The first of these, L. pineti, originates from Indonesia. The other two species is found in high elevation sites in Eastern North America. These are L. abicolens and L. peucophilum. These species are unique in that they are associated with the conifer swift moth, which is an unusual insect associate of Leptographium. Chapter eighth presents a description of a new species of Leptographium from Russia. This species, L. sibiricum, is associated with staining and mortality in siberian fir (Abies sibirica). The role of the fungus in the disease complex is still unknown, and awaits further study. This thesis represents a comprehensive review of all known, as well as newly described species. It should greatly facilitate plant pathologists and mycologists in the identification of Leptographium spp. This should lead to extensive pathogenicity tests, to determine the economic impact of species in this genus as blue-stain fungi and pathogens. It is my sincere wish that it will renew interest in this group of fungi, and will lead to the description of many more species in this genus. / Thesis (PhD)--University of Pretoria, 2006. / Microbiology and Plant Pathology / Unrestricted

L'inventaire de champignons ophiostomatoïdes isolés de quatre espèces de scolytes de l'écorce {Dryocoetes affaber, Ips borealis, I. perturbatus et Polygraphus rufipennis) récoltés dans des bûches d'épinette blanche, a été réalisé au Québec. Ces champignons ont été classés à l'aide de marqueurs moléculaires (PCR-RFLP-rDNA) et d'analyses phylogénétiques sur les gènes de PADNr et de la (3-tubuline. Un total de 23 taxa de champignons ophiostomatoïdes ont été répertoriés, dont 13 ne semblant pas avoir de description dans la documentation scientifique. Par ailleurs, il semble y avoir une différentiation des communautés fongiques selon l'espèce de scolyte d'où elles proviennent. Finalement, l'initiation de la caractérisation d'une nouvelle espèce de Leptographium, a été effectuée à l'aide d'analyses phylogénétiques et moléculaires sur les gènes de l'ADNr, de la P-tubuline, de l'actine et du facteur d'élongation — la. Grâce à ces analyses, nous avons pu obtenir des indices sur la phylogénie et l'évolution de cette nouvelle espèce. / A survey of ophiostomatoid fungi isolated from four bark beetle species (Dryocoetes affaber, Ips borealis, I. perturbatus and Polygraphus rufipennis) collected in white spruce logs, was carried out in the province of Québec. These fungi were classified by means of molecular markers (PCR-RFLP-rDNA) and phylogenetic analyses on the rDNA and ptubulin genes. A total of 23 ophiostomatoid fungi were listed, among which 13 do not seem to have been described in the scientific literature. Moreover, the fungal community seemed to be differentiated according to the bark beetle species from which fungi were collected. Finally, the characterization of a new Leptographium species was initiated with the aid of phylogenetic and molecular analyses of the rDNA, P-tubulin, actin and elongation factor — la genes. By using these analyses, we were able to acquire indications on the phylogeny and evolution of this new Leptographium species.

Procerum root disease of eastern white pine (Pinus strobus L.), caused by Leptographium procerum (Kendr.) Wingf., has been epidemic in Virginia Christmas tree plantations since 1990. Symptoms of chlorosis, wilt, and decreased apical growth resemble those of water stress. Resin infiltration of the xylem at the stem base may be responsible for vascular occlusion leading to severe water deficits and mortality. The pathogen has been isolated from the roots of ozone-sensitive eastern white pines in the field, although not from nearby tolerant trees, and it may be that ozone sensitivity predisposes the trees to infection. The objectives of my Studies were to investigate the physiology of diseased white pines, and to determine the effects of ozone fumigation on disease development. Impacts of vascular occlusion upon host water relations and gas exchange were investigated in 8-yr-old, plantation-grown, white pine Christmas trees. Disease severity was estimated as the proportion of resin-soaked cross-sectional area at the base of the stem. The linear response of a suite of six physiological variables to disease severity was highly significant. Individually, the variables pre-dawn water potential, daily change in pre-dawn to mid-day water potential, stomatal conductance, and photosynthetic and transpiration rates all decreased significantly with increasing disease severity. Fumigation studies were conducted on white and loblolly (P. taeda L.) pine seedlings to determine if ozone exposure increased the incidence of root disease or the amount of stem tissue colonized by L. procerum. Roots were inoculated by soil drenching with conidial suspension, and stems were wounded at the base and inoculated with mycelium. Beginning 24 h post-inoculation, and for 14 consecutive days, seedlings were fumigated in closed chambers with charcoal-filtered air or 200 ppb ozone for 5 h/day, then removed to a charcoal-filtered greenhouse. Six weeks post-inoculation, root and stem tissue were plated on a medium selective for L. procerum. Ozone treatment did not significantly affect the proportion of diseased roots per seedling or the vertical colonization of stem tissue in seedlings of either species. / Ph. D.

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