En utilisant le binôme Cupriavidus metallidurans CH34-Solanacées comme un modèle et comme un point de départ, une étude sur l'interaction entre les plantes et les bactéries sur un substrat pollué par le cuivre a été menée dans deux directions: 1 °) une étude en conditions de laboratoire sur les capacités de C. metallidurans CH34 à interagir avec Nicotiana plumbaginifolia (les solanacées) 2 °) une étude sur le terrain visant à examiner les interactions entre cuprophytes et bactéries résistantes aux métaux des régions minières du Katanga. La première partie inclut une étude in silico visant à établir un catalogue des gènes C. metallidurans CH34 potentiellement impliqués par les interactions plantes-bactéries. Ce catalogue, tout en se reposant sur le génome proche de Ralstonia solanacearum, bactérie phytopathogène de plusieurs espèces végétales appartenant principalement à la famille des Solanaceae, il n'a pas pris en compte les orthologues des gènes clés de la virulence de cette phytopathogène. Les gènes correspondants de C. metallidurans étaient situées sur les deux chromosomes et ont des orthologues dans tous les génomes séquencés des Cupriavidus / Ralstonia et dans Enterobacter sp. 638, endophyte de peuplier. L'étude transcriptomique, à l'aide de «microarray» a montré que certains de ces gènes étaient induits, notamment des gènes impliqués dans la mobilité flagellaire (comme motA) et dans la synthèse des polysaccharides extracellulaires étaient surexprimés pendant le contact entre les plantes et les bactéries, tandis que phcA (impliqué dans la détection de la densité de population et dans la conversion phénotypique) et des gènes impliqués dans la biosynthèse de pili étaient sousexprimés dans les conditions expérimentales testées. En outre, le contact avec les plantes semble avoir induit la surexpression des gènes impliqués dans la réponse de cuivre et d'autres métaux. La capacité de C. metallidurans CH34 à coloniser l'endosphere de N. plumbaginifolia a été confirmée in vitro ainsi qu'un effet de promotion de la croissance des plantes dans certaines conditions. Mais la densité de la colonisation (104-106 c.f.u/g. poids frais) est considérablement réduite dans des conditions non stériles et en l'absence de pression de sélection métallique. La deuxième partie de l'étude s'est concentrée sur la microbiologie de cuprophytes (Haumaniastrum katagense et Crepidorhopalon tenuis) dans l'arc cuprifère du Katanga: des isolats Cuprorésistants appartenant aux genres Stenotrophomonas et Sphingomonas prédominent dans la rhizosphère alors que des isolats appartenant aux genres Methylobacterium, Xanthomonas et Variovorax prédominent dans l'endosphere. Certaines de ces bactéries sont plus résistantes au Cu(II), à des concentrations minimales inhibitrices (MIC) allant jusqu'à 5 mM, que C. metallidurans CH34 (MIC: 1,5 mM) et la plupart d'entre elles résistent également aux Zn(II), Co(II) et Cd(II). Des isolats appartenant au genre Cupriavidus/Ralstonia ont été détectés dans la rhizosphère des cuprophytes ainsi que les séquences 16S rDNA de C. metallidurans ont été également détectées dans l'ADN total extrait des cuprophytes. La détection via la réaction de la polymérase en chaîne (PCR) de gènes de résistance au cuivre correspondant à des protéines periplasmiques a confirmé la présence dans les bactéries cuprorésistantes, principalement de copA et dans une moindre mesure celle de copK. Mais les gènes homologues de copA et de copK n'ont pas été détectés dans tous les bactéries du genre Methylobacterium dont les membres ont été pourtant les plus résistants aux métaux. Certaines bactéries isolées sont capables d'interagir avec le système hormonal végétal et quelques unes semblent également manifester un effet de promotion de la croissance des plantes. Les premières tentatives d'élaboration de protocoles de reinoculation des bactéries endophytic cuprorésistantes dans Haumaniastrum katagense ont été effectués. La biologie moléculaire et l'écologie des interactions plantes-bactéries et des mécanismes de résistance métallique décrits dans ce travail peuvent préparer la voie à de nouvelles applications en bioremédiation (phytostabilization / phytoextraction de métaux toxiques).

My work focuses on the community ecology of symbioses, specifically of fungal endophytes and their hosts. This thesis describes how plant defense compounds and a seed endophyte influence community structure of maize fungal endophytes. Maize produces benzoxazinoids (BXs), compounds toxic to microbes and insects. I assessed the influence of three factors on endophyte communities: host BX production, host neighbor identity and presence of a BX-detoxifying endophyte, Fusarium verticillioides (FV). To determine the influence of BXs on communities, two BX-producing (BX+) and one BX-nonproducing (BX–) genotype were planted in Ridgetown and Harrow, Ontario (triculture). Fungi were isolated and tested for tolerance to 2-benzoxazolinone (BOA), a toxic BX byproduct. Species and functional diversity (community distribution of BOA tolerance levels) was calculated. In seedling roots and mature leaves, the community proportion with low BOA tolerance was greater in BX– than BX+ plants. Fusarium abundance was up to 35 times higher in mature leaves of BX+ than BX– plants. Next, to assess the effect of host neighbor identity on communities, BX– monocultures were planted, and communities from BX– plants in monoculture and triculture compared. Monoculture root communities had higher species diversity than those in triculture. In vitro experiments were conducted to evaluate the influence of BOA on endophyte species interactions. FV facilitated species with lower BOA tolerance in the presence of BOA. Finally, fields were planted with a BX+ and BX– genotype in Ontario, Canada and Georgia, USA. Seed was inoculated with FV (FV+) or sterilized (FV–). FV abundance was highest in BX+FV+ plants, and Fusarium abundance was greater in BX+ than BX– plants in mature leaves. In Georgia, BX+FV+ communities in below ground tissue had lower abundance of BOA sensitive species than BX+FV–. Overall, results suggest that BXs are a habitat filter that increased colonization by horizontally transmitted and seed-born Fusarium species. This invokes the hypothesis that selective breeding for enhanced BX concentrations increased abundance of Fusarium species in maize. The in vitro study indicated that FV could facilitate other species. In contrast, field results suggest that FV interacts competitively with community members, a trait enhanced in the presence of BXs.

My work focuses on the community ecology of symbioses, specifically of fungal endophytes and their hosts. This thesis describes how plant defense compounds and a seed endophyte influence community structure of maize fungal endophytes. Maize produces benzoxazinoids (BXs), compounds toxic to microbes and insects. I assessed the influence of three factors on endophyte communities: host BX production, host neighbor identity and presence of a BX-detoxifying endophyte, Fusarium verticillioides (FV). To determine the influence of BXs on communities, two BX-producing (BX+) and one BX-nonproducing (BX–) genotype were planted in Ridgetown and Harrow, Ontario (triculture). Fungi were isolated and tested for tolerance to 2-benzoxazolinone (BOA), a toxic BX byproduct. Species and functional diversity (community distribution of BOA tolerance levels) was calculated. In seedling roots and mature leaves, the community proportion with low BOA tolerance was greater in BX– than BX+ plants. Fusarium abundance was up to 35 times higher in mature leaves of BX+ than BX– plants. Next, to assess the effect of host neighbor identity on communities, BX– monocultures were planted, and communities from BX– plants in monoculture and triculture compared. Monoculture root communities had higher species diversity than those in triculture. In vitro experiments were conducted to evaluate the influence of BOA on endophyte species interactions. FV facilitated species with lower BOA tolerance in the presence of BOA. Finally, fields were planted with a BX+ and BX– genotype in Ontario, Canada and Georgia, USA. Seed was inoculated with FV (FV+) or sterilized (FV–). FV abundance was highest in BX+FV+ plants, and Fusarium abundance was greater in BX+ than BX– plants in mature leaves. In Georgia, BX+FV+ communities in below ground tissue had lower abundance of BOA sensitive species than BX+FV–. Overall, results suggest that BXs are a habitat filter that increased colonization by horizontally transmitted and seed-born Fusarium species. This invokes the hypothesis that selective breeding for enhanced BX concentrations increased abundance of Fusarium species in maize. The in vitro study indicated that FV could facilitate other species. In contrast, field results suggest that FV interacts competitively with community members, a trait enhanced in the presence of BXs.

This work describes progress toward developing a model system to investigate <i>in plantae</i> suppression of human pathogens by <i>Stemphylium</i>-like fungal endophyte strains.

Algae are increasingly being used in human affairs. Alongside with higher volumes, threats for seaweed natural reservoirs and seaweed farming are starting to appear. Epidemics seems a key complication, similar to agriculture and animal aquaculture. However, phycopathologies are much more under documented, and future diseases are currently difficult to foresee. Thus, efforts should be stressed on biodiversity of algal pathogens and algal defense strategies against diseases. This thesis is divided in two parts: the first section comprises Chapters Two and Three, which describe novel algal pathosystems from cell biology and molecular phylogeny perspectives. Chapter Two characterizes the obligate parasite Maullinia braseltonii sp. nov., a phytomyxid infecting Durvillaea (bull kelp) in the Southern hemisphere. Chapter Three reports new records, cell plasticity and epidemiology of the endophyte Laminariocolax aecidioides infecting Lessonia berteroana (huiro negro) in northern Chile. The two hosts aforementioned have commercial importance, and remarkable roles in coastal ecology in temperate marine habitats in the Southern hemisphere. Chapters Four, Five and Six correspond to a second section and describe cellular processes in novel brown algal pathosystems, related with infection and immunity responses. In Chapter Four, using the Macrocystis/Anisolpidium as a model, microscopy evidence indicate that autophagy is a key cellular process used either to help starving pathogen syncytia to survive (controlled) or to kill themselves (abortive), probably induced by the host. Likewise, host autophagy was also widely observed and may be involved in the neutralization of early-stage oomycetes or nutrient recycling to mount defences. Chapter Five shows the results on a screening of ca. 40 strains from different brown algal orders, to phenotype cellular defense markers against two phylogenetic distant pathogens (Anisolpidium ectocarpii and Maullinia ectocarpii). Overall, responses such as cell wall re-arrangements, programmed cell death, autophagy and ROS accumulation were confirmed to be at least partially conserved across different Phaeophycean lineages. Chapter 6 reports a boost on the phlorotannin metabolism in the model brown alga Ectocarpus (e.g. physode formation and polyketide synthase gene expression) after pathogen challenging, suggesting a similar protective role of this molecule as against grazers and deleterious light regimes. Phlorotannins role however needs to be elucidated.

Prairies of the Pacific Northwest are threatened systems, with only ~2% of historic land remaining. The combined risk of global climate change and land use change make these systems a high conservation priority. However, little attention has been paid to the microbiota. Fungal endophytes are ubiquitous in plants and are important in ecosystem functioning and host dynamics. To understand fungal community assembly, we used high-throughput sequencing to investigate the composition of fungal foliar endophyte communities in two native, cool-season (C3) bunchgrasses along a natural latitudinal gradient. We quantified the importance of host, host traits, climate, edaphic factors, and spatial distance in microbial community composition. We found that spatial distance was the strongest predictors of endophyte community, while host traits (e.g., plant size, density) and abiotic environment were less important for community structure. These findings underline the importance of dispersal in shaping microbial communities. This thesis includes previously unpublished, co-authored material.

The Neotropical cloud forest inhabiting orchid Dracula felix has long been postulated to be a fungal mimic due to the form of its lower labellum and attraction to it by drosophilid flies that are often found feeding on fungal fruiting bodies in the surrounding area. The low number of co-occurring flowers in the area combined with the high number of fruiting fungi appears to have driven the evolution of the orchid genus Dracula to mimic these co-occurring fungi so that pollinators may be recruited. Over several years of working with these orchids we have noticed a particular lapping behavior by the pollinating flies on the labella and sepals of the Dracula flowers. In this study we have first surveyed floral yeasts and molds associated with Dracula flowers and then investigated the role of these fungi in attracting pollinators and offering a food reward to retain them for pollination purposes. In addition to the floral yeasts, leaf endophytes and root associated fungi were cultured and identified, and their frequencies were determined.

published_or_final_version / Ecology and Biodiversity / Doctoral / Doctor of Philosophy

In this dissertation, I explore the diversity of endophytic fungi associated with above-ground tissues of neotropical ferns, assess factors shaping their community structure in three forests, and explore the contributions of endophytes and related strains to our understanding of the diversity and ecology of a representative genus of Ascomycota. In Appendix A, I report a survey of endophytic fungi from seven species of ferns at La Selva, Costa Rica. Using both species-level and phylogenetic analyses, I compare culturable endophytes as a function of fern taxonomy, frond tissues (blades vs. stalks), habits (terrestrial vs. epiphytic), and vegetation types (arboretum vs. primary and secondary forest) during two consecutive years. Analysis of>500 strains provides a first taxonomic overview of fern endophytes at a community scale and reveals high diversity, host preference, and interannual variation in fungal assemblages. However, when variation due to host species is taken into account, community structure is similar among fern tissue types, habits, and sampling sites over a small spatial scale. In Appendix B, I expand my work to evaluate the abundance, species-level and phylogenetic diversity, and distributions of fern-affiliated endophytes in three neotropical forests (La Selva, Costa Rica; Barro Colorado Island, Panama; Los Tuxtlas, Mexico). My analysis of > 2000 isolates reveals that communities differ significantly among fern species within sites, and among ferns in different sites. Intersite differences in communities are correlated with geographic distance and environmental dissimilarity. My work suggests that extrapolative estimates of fungal diversity should take into account not just host species, but locality and environmental variation as well. In Appendix C I focus on a representative genus of Ascomycota (Lecythophora) to evaluate how multi-locus phylogenetic analyses of endophytes can detect new species. Lecythophora is a geographically widespread genus that includes opportunistic human pathogens, produces novel secondary metabolites, and consists presently of six described species. Multi-locus analysis of 33 newly collected strains suggested seven phylogenetic species that are new to science and highlighted their capacity to inhibit growth of two plant pathogenic fungi. This work shows how recognition of cryptic species even in well-characterized genera has major implications for estimating fungal biodiversity.

Fungal endophytes can improve plant tolerance to abiotic stresses such as heat and drought. I hypothesized that the six endophytic fungi SMCD 2204, 2206, 2208, 2210, 2214 and 2215 would promote heat and drought tolerance in wheat during both seed germination and at later developmental stages. The Vujanovic and Germida laboratories originally discovered these fungi from the roots of Saskatchewan grown wheat (Triticum turgidum L.). I assessed mycomediated enhancement of seed germination (mycovitality) including seedling performance, in vitro in terms of percent germination, seedling fresh weight, energy of germination (EG) and hydrothermal time (HTT) of germination. Endophytes SMCD 2206, 2210 and 2215 improved seedling heat or drought resistance, while SMCD 2204, 2208 and 2214 did not. In the greenhouse and phytotron, I evaluated the ability of the same six endophytes to enhance wheat tolerance for heat or drought stress by measuring photosynthetic stress (PS), carbon isotopic discrimination (∆), average seed weight (ASW), total seed weight (TSW) and the EG and percent germination of the F1 seeds produced. SMCD 2206, 2201 and 2215 increased performance of pot-grown wheat under heat and drought. Epigenetic modifications frequently involve changes in DNA methylation. Methyl-sensitive amplified polymorphism (MSAP) revealed that drought stressed wheat seedlings colonized with SMCD 2206 had DNA methylation patterns more similar to those of unstressed plants (with or without the endophyte) than to uncolonized drought stressed plants. Plant DNA sequences – similar to a cytochrome p450 EST and three transposable elements (TEs) – were differentially methylated between endophyte-free and endophyte colonized drought stressed plants. I tested the hypothesis that the endophyte-free progeny of SMCD 2206 colonized wheat grown in the phytotron or greenhouse under heat or drought stress would have heightened resistance for the same abiotic stressors to which their parents were exposed, compared to uninoculated first generation plants. Data on PS, ASW, TSW and ∆ showed that F2 plants incompletely inherited stress tolerance. This research demonstrated that fungal endophytes SMCD 2206, 2210 and 2215 improve wheat tolerance for heat and drought both in vitro and in pot studies. If field trials produce similar results, these isolates could be agriculturally important.