There are generally two contrasting alternatives to what limits recruitment in plants, namely the availability of seeds (seed limitation) or the quality or quantity of suitable sites (microsite limitation). Dust seeds, the smallest existing seeds, lack or have minimal nutrient reserves. During germination and initial development they consequently parasitize on mycorrhizal fungi. This is called mycoheterotrophy, and can vary in degree of fungal dependency in adult plants from full, partial or initial mycoheterotrophy. The aim of this thesis was to investigate the recruitment ecology of mycoheterotrophic Ericaceae (tribe Pyroleae) species with dust seeds, and to determine what limits their recruitment. The investigated species were: Chimaphila umbellata, Moneses uniflora, Orthilia secunda, Pyrola chlorantha, P. minor and P. rotundifolia. This aim was achieved by combining field experiments (seed sowing) with isotope analysis and fungal host pyrosequencing. Results provide evidence that the species in Pyroleae are heterogeneous, not only with regard to their degree of mycoheterotrophy, but also concerning germination and early seedling development. A combination of microsite and seed limitation is thus likely to be of importance for all studied species, but the relative importance of these limitations varies among species. Despite having adaptations for wind dispersal the majority of the seeds were deposited in close vicinity of the seed source. But with high seed production at least some seeds should be able to disperse long-distance. Seedlings of all studied species were found to associate with a wide range of ectomycorrhizal fungi, at least during their initial developmental stages. There seems to be a tendency for host narrowing in some Pyroleae species, but not as strict as the host specialization seen in fully mycoheterotrophic Monotropa hypopitys, supporting the hypothesis of geographical and developmental host shifts. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 3: In press. Paper 4: Manuscript.</p>

Abstract Disturbance may shift microbial communities from one state to another. However, species differ in their ecological characteristics and their abilities to withstand disturbance. No single species or individuals of a species exist alone, but they are parts of complex interaction networks including species above- and belowground. In boreal and subarctic forests, almost all plants and a high number of fungi form mycorrhizas at the plant roots. In mycorrhiza, the fungal partner harvests nutrients for the host plant and, in return, gains carbon from the plant. In general, these common associations benefit both partners, but as heterotrophs, fungi are dependent on carbon photosynthesized by plants, whereas plants can survive alone as autotrophs. In addition to mycorrhizal fungi, also other fungi, such as endophytes, saprotrophs and pathogens, live in and on plant roots. This thesis concerns the impacts of disturbance on fungi living in plant roots and in soil near the roots. I hypothesized that i) root-associated fungal (RAF) and soil fungal communities and colonization types change after disturbance, that ii) the observed shifts relate to disturbance intensity and that iii) they co-occur with changes in soil conditions and vegetation. Changes in RAF were studied as changes in root fungal colonization, or in fungal community composition. The latter were detected with next-generation sequencing methods. The responses of RAF to disturbance seemed to be context dependent and related to sources of fungal communities (e.g. soil, RAF networks), environmental conditions (e.g. soil pH and nutrients) and host performance. It seems that abundances of those RAF species, which are present in the roots first (priority effect), may be increased by disturbance. Research produced new information related to ecological roles of the genera Phialocephala and Meliniomyces. Altogether, the results indicate connections between both abiotic and biotic environments and RAF, and host species viability and RAF. / Tiivistelmä Häiriöt voivat siirtää eliöyhteisön tilasta toiseen. Lajien ominaisuudet ja häiriönsietokyvyt eroavat toisistaan. Mikään laji tai yksilö ei elä yksin, vaan lajit ovat osa maan ylä- ja alapuolelle ulottuvia monimutkaisia vuorovaikutusverkostoja. Boreaalisissa ja subarktisissa metsissä lähes kaikki kasvit ja useat sienet muodostavat sienijuuren eli mykorritsan. Mykorritsassa sieniosakas hankkii isäntäkasville ravinteita ja saa vastavuoroisesti kasvilta hiiltä. Tavallisesti nämä vuorovaikutussuhteet hyödyttävät molempia sienijuuren osakkaita, mutta toisenvaraisina (heterotrofeina) sienet ovat riippuvaisia kasvien yhteyttämästä hiilestä, kun taas tuottajina (autotrofeina) kasvit voivat elää itsenäisesti. Mykorritsasienten lisäksi kasvien juurissa elää yleisesti myös muita sieniä kuten endofyyttejä, saprotrofeja ja patogeeneja. Tämä väitöskirja käsittelee häiriön vaikutuksia sieniin, jotka elävät kasvien juurissa ja juuria ympäröivässä maassa. Hypoteesieni mukaan i) juurissa ja maassa elävien sienten yhteisöt ja kolonisaatiotyypit muuttuvat häiriön jälkeen, ii) muutokset liittyvät häiriön voimakkuuteen ja iii) muutokset tapahtuvat samanaikaisesti maan olosuhteiden ja kasvillisuuden muutoksien kanssa. Juurissa elävien sienten esiintymisen muutokset tutkittiin sienten kolonisaation tai yhteisörakenteen muutoksina. Sieniyhteisöt selvitettiin NGS-menetelmien avulla. Juurissa elävien sienten vasteet häiriöön vaikuttavat olevan tilannesidonnaisia ja liittyvän sienilajien lähteisiin (esim. maa, juurisieniverkostot), ympäristömuuttujiin (esim. maan pH, ravinteet) ja isäntäkasvin menestymiseen. Häiriö voi vahvistaa juurissa ensimmäisenä läsnä olevien sienilajien menestymistä (prioriteettivaikutus). Uutta tietoa tuotettiin Phialocephala ja Meliniomyces –sienisukujen ekologiasta, jota tunnetaan huonosti. Kaiken kaikkiaan, tulokset osoittavat yhteydet sekä elottoman ja elollisen ympäristön ja juurten sieniyhteisön että isäntäkasvin elinkyvyn ja juurten sieniyhteisön välillä.

La phyllosphère est l’habitat fourni par la partie foliaire des plantes. De nombreuses espèces microbiennes - pathogènes, saprophytes ou mutualistes des plantes - peuplent cet environnement. Ce compartiment microbien influence donc la dynamique et la structure des communautés végétales. L’objectif principal de cette thèse était d’étudier les effets potentiels du changement climatique sur la structure des assemblages fongiques de la phyllosphère des arbres forestiers, et sur la niche écologique des espèces fongiques pathogènes des arbres forestiers. Nous avons pour cela utilisé deux approches, i) l’étude de gradients altitudinaux et ii) la construction de modèles de niche bioclimatique.Les assemblages fongiques de la phyllosphère des arbres forestiers étant encore peu connus, nous avons dans un premier temps décrit leur diversité et quantifié leur variabilité spatiale à l’échelle d’une parcelle forestière.Nos résultats montrent que la phyllosphère d’un arbre forestier abrite quelques centaines d’espèces fongiques, avec quelques espèces dominantes et beaucoup d’espèces rares. Les facteurs structurant ces assemblages incluent à la fois des facteurs abiotiques et biotiques : la température apparaît comme la variable climatique la plus explicative le long d’un gradient altitudinal ; à l’échelle d’une parcelle, la proximité génétique entre arbres est plus déterminante que leur distance géographique.L’analyse des modèles de niche des champignons pathogènes forestiers à l’échelle de la France met en évidence des limitations climatiques, les pluies estivales étant une variable explicative importante.Toutefois, plusieurs espèces introduites occupent déjà la plus grande part de la distribution de leur hôte,sans limitation apparente par le climat. Les effets du changement climatique sur la plupart des pathogènes s’exerceront d’abord indirectement par des effets dépressifs très importants sur l’abondance de leurs arbres-hôtes. Seuls les pathogènes adaptés au biotope méditerranéen verraient leur impact s’accroitre. / Phyllosphere is the habitat provided by the leaves of living plants. Many microbial species -pathogens, saprophytes or mutualists of plants - inhabit this environment. These microbes therefore influence the dynamics and structure of plant communities. The main objective was to study the potential effects of climate change on the structure of phyllosphere fungal assemblages, and on the ecological niche of pathogenic fungal species of forest trees. We used two approaches, i) the study of altitudinal gradients and ii) the construction of bioclimatic niche models. Since phyllosphere fungal assemblages of forest trees are still poorly known, we first described their diversity and quantified their spatial variability at the scale of a forest stand.Our results show that the phyllosphere of a forest tree houses hundreds of fungal species, with few dominant species and many rare species. Factors structuring these assemblages include both abiotic and biotic factors: the temperature appears as the most explanatory variable along an elevation algradient. At the scale of a forest stand, the genetic proximity between trees is more important than the geographic distance. Analysis of the bioclimatic niche models of pathogenic fungi forest at the French scale highlights some climatic limitations, and the summer rainfall is an important explanatory variable. However, many introduced species already occupy the distribution of their host, without apparent climatic limitation. The effects of climate change on most pathogens will be exercised indirectly by very important depressive effects on the abundance of their host trees. Only pathogens adapted to the Mediterranean biotope would increase their impact.

Biocrusts and the ecosystem services they provide are becoming more susceptible to fire as exotic annual grass invasions facilitate the spread of desert wildfires. Further, precipitation patterns across the western United States are predicted to change over the next century, and have the potential to dramatically influence fire regimes and the recovery of burned biocrusts. Despite these changes to desert fire and precipitation cycles, our understanding of post-fire biocrust recovery is limited, especially regarding the first two years after fire. To investigate biocrust recovery, we created burn manipulations (i.e., unburned and burned) and tracked crust form and function over two years in one cold and one hot desert ecosystem (UT, USA). We evaluated the entire bacterial community, but focused on Cyanobacteria species that confer soil stability and N fixation capabilities to biocrusts. Specifically, we quantified shifts in biocrust bacterial community composition using target metagenomics of 16S rDNA; monitored biocrust moss and lichen cover; measured N fixation potential; and assessed soil infiltration rates and soil stability. We found little evidence that biocrust form or function recovered from fire within two years. Based on pyrosequencing results, fire altered biocrust community composition in interspace and shrub biocrusts. Cyanobacteria species were almost completely eliminated by fire, constituting 9-21% of unburned plots and less than 0.01% of burned interspace and shrub biocrust communities. Based on cover estimates, no lichen or moss species survived the fire or recovered within two years. N fixation potentials decreased by at least six-fold in burned interspace biocrusts, representing a reduction in soil N inputs into already N-limited desert soils. Soil infiltration rates also drastically declined in burned biocrusts and remained depressed, but only remained depressed for one year. To investigate the interactions between biocrust recovery, fire, and precipitation, we nested precipitation treatments manipulating the amount of monthly rainfall (i.e., ambient, plus 30% and minus 30%) within burn treatments during the second year. Soil NH4+ was the only parameter to be affected by precipitation, and exhibited a positive relationship with precipitation magnitude at the end of one year. Our results demonstrate that fire is a strong destabilizer of the bacterial components of biocrust communities and that the ecosystem services provided by crusts recover at different rates, with N dynamics recovering more slowly than soil ecohydrology.

Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012

L’aulacode (Thryonomys swinderianus) est un rongeur herbivore récemment domestiqué en Afrique pour la production de viande. Quelques études antérieures ont portés sur l’alimentation de cet animal, dans le but d’améliorer la productivité des élevages. A ce jour, nos connaissances sur la digestion et les besoins nutritionnels de cet animal sont encore très parcellaires. Le premier objectif de notre étude était d’améliorer nos connaissances sur la physiologie digestive de l’aulacode en croissance, en particulier en relation avec les apports de fibres alimentaires, avec pour finalité de proposer des recommandations nutritionnelles en fibres pour optimiser la croissance et la santé digestive de cet animal. Notre second objectif était d’analyser les effets d’une diminution du ratio protéines digestibles / énergie digestible parallèlement à une hausse des apports d’amidon, sur la digestion et les performances. La finalité était d’analyser les possibilités de formuler un aliment complet moins onéreux pour les éleveurs et qui respecte les besoins de l’aulacode en croissance. Notre étude a permis de savoir que le caecum est le compartiment digestif le plus important du jeune aulacode entre 1 et 3 mois d’âge, avec plus de 40% du contenu digestif total. L’activité microbienne caecale (100 mM d’acides gras volatils totaux (AGVt) par gramme de contenu frais) est élevée, et similaire à celle des ruminants ou d’autres herbivores monogastriques. Le profil fermentaire est caractérisé par une prédominance de l’acétate (75 % des AGVt) et un ratio propionate / butyrate supérieur à 1. Le pyroséquençage 454 de l’ADN16S bactérien a permis de caractériser le microbiote caecal. Au sevrage, nous observons une prédominance du phylum des Bacteroidetes, avec 51 % d’abondance relative, alors que le phylum des Firmicutes devient majoritaire (50%) à 3 mois d’âge. Le microbiote caecal est caractérisé par la présence de genres souvent identifiés dans d’autres écosystèmes digestifs d’herbivores, tels que : RC9 (2 à 8%), Parabacteroides (1 à 8%), Prevotella (3 à 6%) et Xylanibacter (1%), Erysipelotrichaceae Turicibacter (1 à 7%), Lachnospiraceae Incertae_Sedis (4 à 5%), Ruminococcaceae Incertae_Sedis (1 à 2%) et Ruminococcus (1 à 3%). D’autres genres, absents chez des espèces voisines comme le lapin et le cobaye, semblent plus spécifiques de l’aulacode, tels que Termite_Treponema_cluster (1.7 à 2.2%) et Treponema (7 à 13%), du phylum des Spirochaetes. L’analyse des performances zootechniques indique qu’un taux de fibres compris entre 17 et 21 % d’ADF représenterait un bon compromis entre santé digestive et croissance de l’aulacode après son sevrage. Descendre au dessous de 6 g de protéines digestibles par MJ d’énergie digestible, via une hausse importante des apports d’amidon et une baisse importante du taux de protéines brutes (en dessous de 11 %) et de fibres, est préjudiciable à la croissance des animaux. / The cane rat or grasscutter (Thryonomys swinderianus) is a rodent herbivore recently domesticated in Africa for meat production. Some previous studies focused on the feeding of this animal, in order to improve the productivity of farms. To date, our knowledge of digestion and nutritional requirements of this animal are still very scarce. Our first objective was to improve our knowledge of digestive physiology of the young grasscutter, particularly in relation to dietary fibre supply, in order to improve the recommendations for dietary fibre content of diets to optimize growth and digestive health. Our second objective was to analyze the effects of a decreased digestible protein / digestible energy ratio, along with an increased intake of starch, on digestion and performances. The final aim was to analyze the possibilities to formulate a complete feed, cheaper for farmers and that meets the requirements of the young grasscutter. Our study found that the caecum is the most important digestive compartment of the young grasscutter between 1 and 3 months of age, with more than 40% of the total gut contents. The caecal microbial activity (100 mM of total volatile fatty acids (VFA) per gram of fresh content) is high and similar to that of ruminants or other herbivorous monogastric animals. The fermentation profile is characterized by a predominance of acetate (75% of total VFA) and a propionate / butyrate ratio greater than 1. A pyrosequencing of bacterial 16S-DNA was used to characterize the caecal microbiota. At weaning (one month), we observe a predominance of the Bacteroidetes phylum, with 51% of relative abundance, whereas the Firmicutes phylum becomes predominant (50%) at 3 months of age. Caecal microbiota is characterized by the presence of genera often identified in other digestive ecosystems of herbivores, such as: RC9 (2-8%), Parabacteroides (1-8%), Prevotella (3.6%) and Xylanibacter (1%), Erysipelotrichaceae Turicibacter (1-7%), Lachnospiraceae Incertae_Sedis (4-5%), Ruminococcaceae Incertae_Sedis (1-2%) and Ruminococcus (1-3%). Other genera, absent in related species such as rabbits and guinea pigs, seemed more specific of the grasscutter, such as Termite_Treponema_cluster (1.7-2.2%) and Treponema (7-13%) of the Spirochaetes phylum. The analysis of growth performances indicated that a dietary fibre content between 17% and 21% of ADF represents a good compromise between digestive health and growth of the grasscutter after weaning. Decreasing below 6g of digestible protein / MJ of digestible energy, via a high increase in starch intake and a significant decline in crude protein content (below 11%) and fibre, is detrimental to the growth of animals.

The remediation of persistent chlorinated aromatic compounds has become a priority of great relevance due to the teratogenic, carcinogenic and endocrine-disrupting properties of these xenobiotics. The use of biological methodologies for the clean-up of contaminated sites, collectively referred to as "bioremediation", has been gaining an increasing interest in recent years because it represents an effective, cost-competitive and environmentally friendly alternative to the physico-chemical and thermal treatments. In this respect, "white rot" fungi, an ecological subgroup of filamentous fungi, display features that make them excellent candidates to design an effective remediation technology ("mycoremediation"). In spite of this, fungi have not been widely exploited for their metabolic capabilities and the mechanism by which they are able to degrade the aforementioned pollutants has not been fully elucidated yet. Within this frame, the present Ph.D thesis was aimed at: i) assessing the efficiency of different mycoremediation strategies for the clean-up of a polychlorinated biphenyl (PCBs)-contaminated soil; ii) understanding the fungal degradation pathways of polychlorinated biphenyls and their major metabolites, namely chlorobenzoic acids (CBAs) and hydroxylated polychlorinated biphenyls (OH-PCBs). i)...

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Despite the global licensure of two live-attenuated rotavirus vaccines, Rotarix® and RotaTeq®, rotavirus remains the major cause of severe dehydrating diarrhoea in young mammals and the need for further development of additional rotavirus vaccines, especially vaccines effective against regional strains in developing country settings, is increasing. The design and formulation of new effective multivalent rotavirus vaccines is complicated by the wide rotavirus strain diversity. Novel rotavirus strains emerge periodically due to the propensity of rotaviruses to evolve using mechanisms such as point mutation, genome segment reassortment, genome segment recombination and interspecies transmission. Mutations occurring within the primer binding regions targeted by the current commonly employed sequence-dependent genotyping techniques lead to difficulties in genotyping novel mutant rotavirus strains. Therefore, use of sequence-independent techniques coupled with online rotavirus genotyping tools will help to understand the complete epidemiology of the circulating strains which, in turn, is vital for developing intervention measures such as vaccine and anti-viral therapies. In this study, sequence-independent cDNA synthesis that uses a single set of oligonucleotides that do not require prior sequence knowledge of the rotavirus strains, 454® pyrosequencing, and an online rotavirus genotyping tool, RotaC, were used to swiftly characterise the whole genome of rotaviruses. The robustness of this approach was demonstrated in characterising the complete genetic constellations and evolutionary origin of selected human rotavirus strains that emerged in the past two decades worldwide, human rotavirus strains frequently detected in Africa, and the whole genomes of some common strains frequently detected in bovine species. Most of the characterised strains emerged either through intra- or interspecies genome segment reassortment processes. The methods used in this study also allowed determination of the whole consensus genome sequence of multiple rotavirus variants present in a single stool sample and the elucidation of the evolutionary mechanisms that explained their origin. The 454® pyrosequence-generated data revealed evidence of intergenotype rotavirus genome segment recombination between the genome segments 6 (VP6), 8 (NSP2) and 10 (NSP4) of Wa-like and DS-1-like origin. The use of next generation sequencing technology combined with sequence-independent amplification of the rotavirus genomes allowed the determination of the consensus nucleotide sequence for each of the genome segments of the selected study strains directly from stool sample. The consensus nucleotide sequences of the genome segments encoding VP2, VP4, VP6 and VP7 of some of the study strains were codon optimised for insect cell expression and used to generate recombinant baculoviruses. The Bac-to-Bac baculovirus expression system was used to generate chimaeric rotavirus virus-like particles (RV-VLPs). These chimaeric RV-VLPs contained inner capsids (VP2 and VP6) derived from a South African RVA/Humanwt/ ZAF/GR10924/1999/G9P[6] strain, on to which outer capsid layer proteins composed of various combinations of VP4 and VP7 were assembled. The outer capsid proteins were derived from the dsRNA of G2, G8, G9 or G12 strains associated with either P[4], P[6] or P[8] genotypes that were directly extracted from human stool faecal specimens. The structures of these chimaeric RV-VLPs were morphologically evaluated using transmission electron microscopy (TEM). Based on the size and morphology of the particles, doublelayered (dRV-VLPs) and triple-layered RV-VLPs (tRV-VLPs) were produced. Recombinant rotavirus proteins readily assembled into dRV-VLPs, whereas approximately 10 – 30% of the assembled RV-VLPs from insect expressed recombinant VP2/6/7/4 were chimaeric tRVVLPs. These RV-VLPs will be evaluated in future animal studies as potential non-live rotavirus vaccine candidates. The novel approach of producing RV-VLPs introduced in this study, namely by using the consensus nucleotide sequence derived from dsRNA extracted directly from clinical specimens, should speed up vaccine research and development by bypassing the need to adapt the viruses to tissue culture and circumventing some other problems associated with cell culture adaptation as well. Thus, it is now possible to generate RV-VLPs for evaluation as non-live vaccine candidates for any human or animal field rotavirus strain. / Thesis (PhD (Biochemistry))--North-West University, Potchefstroom Campus, 2012