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Les interactions entre les plantes et les agents pathogènes fongiques conduisent habituellement à la mise en place par l’hôte de différents mécanismes de défense. Des exemples de ces mécanismes sont le renforcement de la paroi cellulaire par la subérine, la lignine et les composés phénoliques pariétaux, ainsi que l’accumulation de protéines PR et la production de phytoalexines qui peuvent être toxiques pour l’agent pathogène. Chez les plantes susceptibles, ces mécanismes ne réussissent pas à empêcher le développement de la maladie. La maladie hollandaise de l’orme est une maladie à caractère épidémique qui a causé la mort de millions d’ormes en Europe et en Amérique du Nord. Les bases moléculaires de cette maladie sont encore peu connues. Afin d’identifier des gènes impliqués dans l’interaction entre l’espèce susceptible Ulmus americana L. et son champignon pathogène Ophiostoma novo-ulmi Brasier, un système in vitro a été mis au point, celui-ci utilisant des cultures de cals auxquelles des cellules levuriformes du champignon ont été inoculées. Afin de valider son utilisation pour l’analyse génomique, ce système a d’abord fait l’objet d’une analyse histopathologique en microscopie photonique et électronique. Le développement du champignon dans les cals et les réactions de défenses de ces derniers face à la présence du champignon ont été observés à 4, 24 48, 72 et 96 heures post-inoculation (hpi). Le champignon a été détecté sous forme de réseau d’hyphes dans toutes les parties du cal à partir de 48 hpi. Les tests histochimiques ont montré l’importance de certaines réactions telles que l’accumulation de phénols, de lignine et de subérine dans les cellules des cals. Le pourcentage de cellules subérisées était 4,6 fois plus élevé à 96 hpi dans les cals infectés que dans les cals témoins. Une banque d’ADN complémentaire a été construite à partir de cals infectés (72 hpi) en utilisant la technique des hybridations suppressives et soustractives (SSH). Un total de 535 étiquettes de séquences exprimées (EST) a été obtenu et déposé dans la banque de données Genbank suite au séquençage partiel des clones. Ces étiquettes ont été regroupées en 314 uniséquences dont la majorité correspondait à des gènes d’orme identifiés durant l’interaction. Cinquante-trois uniséquences représentant des gènes impliqués dans différentes voies métaboliques associées à la défense ont été sélectionnées par un criblage différentiel et considérées comme étant induites durant l’interaction. Les profils d’expression à différents temps après inoculation ont été établis par PCR quantitative chez 18 uniséquences provenant de cals infectés ainsi que de cals traités à l’eau stérile. Ils confirment l’induction ou l’expression constitutive des gènes correspondants durant le processus de l’infection. Cette étude fournit pour la première fois une ressource génomique pour l’orme et révèle des mécanismes moléculaires impliqués dans l’interaction entre l’orme américain et l’agent pathogène responsable de la maladie hollandaise de l’orme. / Interactions between plants and fungal pathogens usually lead to the induction of different host defense mechanisms. Some of these mechanisms are the reinforcement of the plant cell wall by suberin, lignin and wall bound-phenolics, the accumulation of pathogenesis-related proteins, and the production of phytoalexins that could be toxic to the pathogen. Yet in susceptible plants, these mechanisms are not effective to produce resistance and disease develops. Dutch elm disease (DED) is a pandemic tree disease that killed millions of elm trees, especially in North America and Europe. The molecular bases of this disease are still poorly understood. With the objective of identifying genes involved in the interaction between the susceptible Ulmus americana L. and the pathogen Ophiostoma novo-ulmi Brasier, an in vitro system was developed using callus cultures inoculated with budding cells of the fungus. In order to validate the use of this system for genomic analyses of the interaction, a histopathological analysis was carried out using light and electron microscopy. Fungal colonization of the callus tissue and reactions of callus cells to the presence of the pathogen were observed at 4, 24, 48, 72 and 96 hours post-inoculation (hpi). The fungus was seen in its hyphal form by 48 hpi in all parts of the callus. Histochemical tests showed the importance of host reactions such as the accumulation of phenols, lignin, and suberin. The percentage of suberized cells was 4.6 times higher at 96 hpi in infected calli than in mock-inoculated control calli. A cDNA library using suppression subtractive hybridization (SSH) was constructed from infected elm callus tissue harvested at 72 hpi. A total of 535 expressed sequence tags were generated through partial sequencing and submitted to Genbank. These were grouped into 314 unisequences, the majority corresponding to elm genes identified during the interaction. Fifty-three unisequences representing genes involved in different pathways associated with plant defense were selected by differential screening and considered upregulated in the infected tissues. The expression profiles in mock and infected elm callus cultures of a subset of 18 elm genes were analyzed in more detail by quantitative reverse transcriptase polymerase chain reaction. These confirmed upregulation and constitutive expression of selected genes during the infection process. This study provides, for the first time, a genome-wide resource for the elm, and furthermore identifies molecular mechanisms likely involved during the interaction between U. americana and the DED pathogen.

Cartapip-treated pulps are evaluated for increased strength properties / Thesis (Ph.D.)--Institute of Paper Science and Technology, 1995.

The increasing frequency and severity of windthrow events affecting the forestry industry in New Zealand have raised important management issues surrounding the rate of colonisation of fallen trees by sapstain fungi and the time available for salvage harvesting before sapstain degradation limits potential economic returns. These fungi are known to be spread by a multitude of factors including wind, rain splash, harvesting processes and insect vectoring. Apart from the ecological interest in these interactions between fungi, plants and insects, sapstain fungi are also economically important because their hyphae discolour the sapwood and reduce the overall quality of the timber. The amount of time available to salvage harvest damaged trees is unknown, especially on seasonal and regional scales. Manipulative experiments were established in Pinus radiata forests to examine this seasonal and regional variation in sapstain attack following windthrow, and to investigate the importance of bark beetles as vectors of sapstain fungi. A range of methods were implemented to assess the role of bark beetles as vectors and to ascertain which sapstain fungi are associated with them. Experimental billet logs were caged to exclude beetles and subsequently analyse fungal attack in comparison with identical logs left exposed to beetles. In addition, individual beetles were sampled directly to determine whether they carried spores of particular fungal species and to assess the degree of association in vector-fungal dynamics. Finally, a novel application of DNA melt peak analysis was developed to investigate variation among the fungal communities associated with beetles potentially involved in vectoring sapstain spores. The moisture content of fallen trees was found to be the main factor regulating sapstain development, and when moisture content drops below 100% (on dry weight basis) sapstain fungi grew rapidly. The speed at which this level drops depends on the season, with much faster drying occurring in the warmer months of spring and summer. As a result, trees that fell in the previous winter or autumn did not develop significant sapstain levels until temperatures rose in the following summer, suggesting that storm-damaged trees that fall in winter can be left safely until just before the next summer before they are no longer suitable for salvage harvest. In New Zealand, the bark beetle species acting as vectors of sapstain fungi are not behaviourally adapted to colonisation of logs that are not in contact with the ground. Following windthrow events in pine forests, trees generally lie with their stems suspended above the level of the ground by their branches. As a result, under these circumstances, beetle colonisation of windthrown timber was low, and bark beetles were not a significant vector of stain. The caged and un-caged experimental log billets, however, were in contact with the ground, resulting in colonisation of the un-caged logs. In this case, bark beetles did play an important role in contributing to sapstain intensity, and the stain distribution within the logs mirrored that of the stain distribution. However, this effect may be due to the provision of access points for wind- or water-borne spores of the non-insect vectored stain fungus Diplodia pinea, or to the spread of hyphae through the tunnelling and feeding activities of beetles within the tree, rather than by bark beetles acting as vectors of spores. Bark beetles were confirmed as sapstain vectors with the isolation of seven different ophiostomatoid stain fungi from them, five of which were also found in wood. Finally, the development of a laboratory based, rapid species identification method was developed to identify fungal DNA. Melt peak analysis allowed the species-specific DNA melt temperatures to be compared with the melt temperatures of known species to be able to rapidly, and cheaply, identify an unknown species. Bark beetles are vectors of sapstain fungi in P. radiata forests, however the bark beetle species naturalised in New Zealand prefer to colonise wood when it is in contact with the ground. Following windthrow, trees are generally not attacked by beetles as they are held from the ground by their branches, leaving them to be stained predominantly be wind and rain dispersed stain fungus D. pinea. Stain did not occur until the moisture content of fallen trees dropped below 100%, which only happens in the warm months of summer and spring. In New Zealand, there are interactions between trees and bark beetles, and bark beetles with fungal pathogens from all around the globe resulting in a unique novel assemblage of species together for the first time. Understanding the dynamics of these species in their novel environment is crucial to effectively responding to potential pest threats.

A genetic catalogue was generated from expressed sequence tags (ESTs) from the pathogenic filamentous fungus Ophiostoma novo-ulmi. Rather than full sequencing of the entire genome, fragments of each gene being actively expressed at a specific point in time were catalogued and annotated for identity and function. This catalogue represents a resource of considerable depth for the purposes of gene discovery, genetic regulation, protein expression, pathogenicity, and growth state studies. An online database was generated to serve as a powerful tool for downstream applications, facilitating and enhancing future research in all of these important areas of fungal biology. The ascomycetous fungus Ophiostoma novo-ulmi represents an excellent model organism for genetic experimentation. A diversity of physiological functions, including dimorphism, pathogenicity, melanin biosynthesis, and glycoprotein secretion at high levels mean that principles elucidated from this fungus are likely of broad application. Ophiostoma novo-ulmi has been identified as the causative agent of Dutch elm disease, which has become an economic and horticultural pandemic in North America. As a result, the mechanisms of host-pathogen interaction of this fungus are of particular interest. Initial attempts at disruption of pathogenicity, most commonly by disruption of single genes identified as potential pathogenicity factors, have met with little success. As our understanding of the complexity and co-ordination of proteins involved with host-pathogen interaction deepens, the discovery of a single dominant pathogenicity gene is becoming increasingly unlikely. As such, a broader genomics approach was employed to work towards identification of groups, or networks of genes that operate in a concerted manner, regulating pathogenicity or parasitic fitness. A low redundancy library was constructed from Ophiostoma nova-ulmi complementary DNA, producing a total of 4386 readable expressed sequence tags (ESTs) from 5760 clones. Of these, 2093 sequences matched with sequences found in public databases while 2293 represented orphan sequences. Of the sequences in the former group, 1761 sequences matched with known proteins while 332 sequences matched with hypothetical/predicted proteins. Sequences matching known proteins included 880 singletons, corresponding to 49.97 % of the ESTs in this category. Extrapolating this proportion to the sequences matching hypothetical proteins estimated the number of singletons in this category to be 166. Similarly, 1835 orphan sequences were estimated to contain 917 unique sequences. Singletons matching entries in public databases (n=880) were manually annotated into functional categories as established by the Munich Information Centre for Protein Sequences (MIPS). Metabolism (21%), Protein Synthesis (10%), Subcellular Localization (10%), Biogenesis of Cell Components (8%), and Transcription (8%) categories were the most highly represented.

Thesis (Ph. D.)--Oregon State University, 2004. / Printout. Includes bibliographical references. Also available on the World Wide Web.

This thesis concerns the study of selected Ceratocystis species and Ophiostoma species infecting wounds on broad-leaved trees, particularly those occurring in Africa. However, two chapters also deal with these fungi from Australia, Norway, Sweden and Austria. The dissertation is comprised of a literature review, followed by four research chapters, addressing the occurrence of Ceratocystis spp. and Ophiostoma spp. in Africa, Australia and Norway. The first chapter of the dissertation is a review of Ceratocystis spp. and Ophiostoma spp. with particular reference to Africa, hardwood tree species and wound infections. The review highlights the importance of wood and trees, especially on the African continent, and discusses the threat of deforestation. This is despite efforts by many African countries to establish forests of non-native tree species to address the demand for wood and wood products on the continent. Reforestation is associated with increased risks of the introduction of pests and pathogens, including species of Ceratocystis and Ophiostoma. The taxonomic history of these two fungal genera is summarized, and the review further focuses on the economically important species in these genera, particularly those infecting hardwood tree species on the African continent. The lack of information regarding Ceratocystis spp. and Ophiostoma spp. on hardwood trees in Africa is thus highlighted. Furthermore, the review summarised the dispersal mechanisms of these pathogens, highlighting dispersal too and infection of wounds. Ceratocystis pirilliformis was described in 2003 and it is the only species in the Ceratocystis fimbriata species complex that has pear-shaped ascomatal bases. This fungus was first described from Australia where Eucalyptus spp. are endemic. It was later reported from South Africa on Eucalyptus grandis trees. Chapter two of this dissertation attempts to address questions regarding the geographic distribution, impact and origin of C. pirilliformis in South Africa. This was in line with the fact that it has been suggested that the fungus is likely native to Australia. To address this question, surveys were conducted in many Eucalyptus planting areas in South Africa and the genetic diversity of the fungus in the country was investigated using microsatellite markers previously developed for C. fimbriata. C. pirilliformis was found in three Eucalyptus-growing areas of South Africa, which has considerably increased the known geographic range of the fungus in South Africa. The gene diversity as well as the genotypic diversity for the fungus was found to be very low in the country and the population is apparently clonal. Results thus support the view that C. pirilliformis was accidentally introduced into South Africa. In chapter three of this dissertation, O. quercus is reported for the first time from wounds on non-native Acacia mearnsii in Uganda. In addition a new Pesotum sp., P. australi prov. nom. is described from wounds on native A. mearnsii in Australia. This fungus resembles other Pesotum anamorphs of Ophiostoma in many ways, especially species of the O. piceae complex. However, it can be distinguished from these species by many morphological traits and also based on phylogenetic inference. The closest phylogenetic neighbor of P. australi prov. nom. is O. quercus. The fact that it was isolated from A. mearnsii in Australia indicates that it is probably a native fungus in that area. In chapter four, two Ceratocystis spp. and one Ophiostoma sp. are described as new to science, from wounds on native broad-leaved tree in South Africa. Three other Ophiostomaspp. are also reported in this study. Until recently, very little research has been done with regard to Ceratocystis spp. and Ophiostoma spp. occurring on native tree species in Africa. However, results presented in this chapter strongly suggest that these fungi are common on native trees in Africa and many other species, including potential pathogens await discovery. Chapter five of the dissertation reports, for the first time, Ophiostoma catonianum, O. pluriannulatum and O. quercus from native broad-leaved trees in Norway. It also reports O. catonianum for the first time from Austria and O. quercus for the first time from Sweden. In the past, very little research has been undertaken to explore the diversity of these fungi on hardwood trees in the Nordic countries or other parts of Europe, where most research has been focused on Ceratocystis spp. and Ophiostoma spp. associated with conifer-infesting bark beetles. This chapter represents a preliminary study with important discoveries. It indicates that these fungi are common on wounds on hardwood trees in Europe and emphasizes the importance of expanding these studies in the Nordic countries, to include more hosts and geographic areas. Such studies will almost certainly reveal more species and possibly new species of Ceratocystis and Ophiostoma. / Dissertation (MSc)--University of Pretoria, 2010. / Microbiology and Plant Pathology / Unrestricted

One great issue of reforestation is the infestation of pest insects. The pine weevil (Hylobius abietis L.) is one of the economically most important pest insects in Europe and Asia. When insects feed on the trees the plants emit compounds that repel the insects. Methyl salicylate (MeSA) is one substance known to be emitted by conifer seedlings after herbivore feeding. Similar compounds are emitted by fungus isolated from pine weevil frass. Female pine weevils add frass to the egg laying site to protect the eggs from other pine weevils. The goal of the project was to cultivate the fungi Ophiostoma pluriannulatum and analyze the volatile substances. The fungus was cultivated using natural pine weevil frass (NF) as main substrate. Later new batches were prepared using lignin and potato dextrose broth (PDB), as feeding media to compare the results. Two sampling methods were used, SPME and adsorption with Porapak™ polymer column packing material. These samples were analyzed using GC-MS. Methyl salicylate was found in NF fungi cultures, peaking after ten days. Another, unexpected, product of the fungi was a sesquiterpene hydrocarbon. The lignin and the PDB samples did not produce MeSA. However, the sesquiterpene could be found in the lignin samples.

Almsjukan är en vissnesjukdom orsakad av invasiva skadesvampar i släktet Ophiostoma. På grund av almsjukan är svenska almar kritiskt hotade och nya metoder att skydda almar behövs därför akut. Biologisk kontroll är en lovande strategi mot trädsjukdomar som almsjukan, men det behövs mer kunskap innan metoden kan tillämpas i praktiken. Endofytsvampar som lever inuti växter utan att orsaka symptom har visat sig kunna forma trädets motståndskraft. I tidigare in vitro studier har en endofytsvamp i släktet Fusarium uppvisat kemisk antagonism mot almsjukepatogener i odling på standardagar. I denna studie undersöktes om samma reaktion även uppkommer när svampar odlas på agar som berikats med almbarkextrakt som innehåller bl.a. fenoliska substanser. I in vitro tester med Fusarium-svampen och två Ophiostoma-isolater upptäcktes att den antagonistiska reaktionen fanns kvar trots almkemikalier, vilket antyder att reaktionen kan vara stabil även inuti almar. Resultaten tyder på att almens kemikalier kan förstärka Fusarium-svampens antagonism mot den aggressivare patogenen, O. novo-ulmi. Studien bekräftar därför Fusarium endofytens potential i biokontroll av almsjukan.

Des éléments mobiles de type 2 ont été mis en évidence chez Ophiostoma ulmi et O. novoulmi, agents pathogènes de la maladie hollandaise de l'orme. Dans un premier temps, ces transposons à ADN, nommés OPHIOl, OPHI02 et OPHI03, ont été subséquemment caractérisés, tant au niveau structural qu'au niveau de leur répartition dans les espèces concernées. L'étude approfondie de leur séquence a permis de faire ressortir des mutations particulières de type RIP (Repeat induced point mutations) uniquement présentes chez OPHI03. Ces dernières, ont par ailleurs, permis de mettre au point une nouvelle technique de visualisation de ce type de mutations (CTS visualizatiori), applicable à l'ensemble de ces éléments mobiles. Dans un second temps, la mobilité & OPHIOl et OPHI02 a fait l'objet d'une étude détaillée. Grâce à divers stress abiotiques, nous avons démontré que ces éléments sont mobiles au sein des génomes des champignons responsables de la maladie hollandaise de l'orme. En dernier lieu, des analyses bioinformatiques ont permis de mettre en évidence des zones de sélection positive au sein de domaines précis des transposases, l'enzyme requise pour la mobilité d'OPHIOl et d'OPHI02. L'ensemble de ces résultats a permis d'accroître la connaissance des TE ainsi que d'essayer de comprendre la dynamique complexe existant entre les transposons et leurs génomes hôtes. / Type 2 mobile elements were detected in Ophiostoma ulmi and O. novo-ulmi sp., the causal agents of the Dutch elm disease. Firstly, the structure and the distribution in Ophiostoma species of these DNA transposons, named OPHIOl, OPHI02 and OPHI03, were characterized. A precise analysis of their sequence demonstrated some particular RIP mutations (Repeat induced point mutations) in the case of OPHI03. These mutations allowed us to develop a new visualization (CTS visualization) for these types of mutations, applicable to ail mobile elements. Secondly, the mobility of OPHIOl and OPHIOl was the main investigation of a detailed study. We demonstrated that abiotic stresses have a direct impact on the induction of mobility of the transposons within Ophiostoma sp. To finish our investigation, bioinformatics analyses were performed on OPHIOl and OPHIOl (considered to be active transposons) and allowed the presence of regions under positive selection inside the transposase (enzyme required for their mobility). Taken together, these results lead to a better understanding of a part of the complex dynamics that links mobile elements to their host genomes.