Orchids are characterized by producing minute endosperm-lacking seeds, which depend on mycorrhizal fungi for germination and embryo development. Some aclorophyllous orchids remain dependent on the mycorrhizal association for carbon acquisition during their whole life history, whereasother orchids develop photosynthesis. Despite the biological significance of orchid mycorrhiza, gene expression studies are lacking. We have used different highthroughput approaches in order to understanding the mechanisms regulating orchid mycorrhiza development and functioning. Firstly, we have used a 2D-LC-MS/MS approach coupled to isobaric tagging for relative and absolute quantification (iTRAQ) to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense related responses, phytoalexins and carotenoid biosynthesis, suggesting that orchid protocorms undergo profound metabolic changes during the switch from the fully mycoheterotrophic to the photosynthethic stage. Secondly, three different proteomic techniques were carried out in independent experiments aiming to identify changes in protein accumulation in mycorrhizal roots of the terrestrial orchid Oeceoclades maculata.Finally, O. maculatamycorrhizal roots were used for transcriptome analyses. The data revealed a strong increase in general stress responses, accompanied by changes in signaling pathways possibly related to fungal recognition and establishment of a compatible interaction. Some of the upregulated genes may be involved in the reorganization of cell structure, likely related to accommodation of the fungal symbiont in the plant roots. We have also observed in mycorrhizal roots up-regulation of genes involved in carbon metabolism, including glycolysis/gluconeogenesis and amino sugars metabolism, as well as genes involved innitrogen assimilation. The down-regulation of genes involved in the jasmonate and ABA transduction pathways, and key genes encoding anti-fungal proteins, such as chitinase and a mannose-specific binding lectin, strongly suggests an alleviation of plant defense responses in O. maculata mycorrhizal roots. In general, our data suggest that the physiology of an orchid mycorrhiza is more similar to a compatible interaction than to an arm-race between plant and fungi. Overall orchid mycorrhiza have proved to be a promising model for investigating plantfungal interactions and further studies should now address the specific roles of the genes showing differential regulation in this study. / As orquídeas são caracterizadas por produzirem sementes diminutas, que não possuem endosperma. Necessitam, portanto, da interação com fungos micorrízicos para germinação e desenvolvimento do embrião. Algumas orquídeas aclorofiladas se mantêm dependentes dos fungos micorrízicos para a aquisição de carbono, enquanto outras desenvolvem a maquinaria fotossintética. Apesar do significado biológico das micorrizas de orquídeas, alterações na expressão gênica e no acúmulo de proteínas foram altamente negligenciads nos últimos anos. Neste trabalho, foram utilizadas diferentes técnicas sequenciamento e identificação de genes e proteínas em larga-escala para acessar as alterações moleculares responsáveis pela regulação das micorrizas de orquídeas. Uma abordagem baseada em 2D-LC MS/MS acoplada a técnica de quantificação absoluta e relativa iTRAQ, foiutilizada para identificar proteínas com acúmulo diferencial em Oncidium sphacelatum em diferentes estágios do desenvolvimento do protocormo (protocormos aclorofilados versus protocormos fotossintetizantes), após inoculação com um fungo do gênero Ceratobasidium. As análises mostraram que, as alterações esperadas no metabolismo do carbono foram acompanhadas de um acúmulo aumentado de proteínas envolvidas na modulação de espécies reativas de oxigênio, respostas de defesa, biossíntese de fitoalexinas e carotenóides, sugerindo que os protocormos de orquídeas passam por profundas alterações metabolicas durante a transição do metabolismo micoheterotrófico para o fotossintético. Posteriormente foram utilizadas três diferentes técnicas de proteômica quantitativa para explorar alterações fisiológicas em raízes micorrizadas e não-micorrizadas de Oeceoclades maculata.Este estudo foi ampliado, pela utilização de uma abordagem transcritômica ao mesmo modelo biológico. Em conjunto, os dados revelaram um forte aumento em respostas relacionadas ao estresse, acompanhadas de alterações em vias de transdução de sinal possivelmente relacionadas ao reconhecimento do simbionte fúngico e estabelecimento de uma interação compatível. Alguns genes com expressão aumentada devem estar envolvidos na reorganização celular, provavelmente ligada a acomodação do simbionte fúngico nas raízes das plantas. Também foi observado o aumento de genes envolvidos no metabolismo do carbono e de açúcares aminados, juntamente a genes relacionados a assimilação de nitrogênio em raízes micorrizadas. A expressão diminuída de genes envolvidas nas vias do jasmonato e ácido abscícico, juntamente a genes-chave que codificam para proteínas anti-fúngicas sugerem fortemente uma atenuação das respostas de defesa da planta em raízes micorrizadas de Oeceoclades maculata. No geral, parece que as micorrizas de orquídeas são fisiológicamente mais próximas de uma simbiose compatível do que de uma interação unilateral em favor da planta. Sobretudo, este sistema biológico provou ser promissor para investigação de interações planta-fungo e, próximas pesquisas devem agora ser focadas em funções específicas dos genes que mostraram regulação diferencial neste estudo.

Orchids are characterized by producing minute endosperm-lacking seeds, which depend on mycorrhizal fungi for germination and embryo development. Some aclorophyllous orchids remain dependent on the mycorrhizal association for carbon acquisition during their whole life history, whereasother orchids develop photosynthesis. Despite the biological significance of orchid mycorrhiza, gene expression studies are lacking. We have used different highthroughput approaches in order to understanding the mechanisms regulating orchid mycorrhiza development and functioning. Firstly, we have used a 2D-LC-MS/MS approach coupled to isobaric tagging for relative and absolute quantification (iTRAQ) to identify proteins with differential accumulation in Oncidium sphacelatum at different stages of mycorrhizal protocorm development (achlorophyllous and green protocorms) after seed inoculation with a Ceratobasidium sp. isolate. Quantitative analysis showed that the expected changes in carbon metabolism in green protocorms were accompanied by enhanced accumulation of proteins involved in the modulation of reactive oxygen species homeostasis, defense related responses, phytoalexins and carotenoid biosynthesis, suggesting that orchid protocorms undergo profound metabolic changes during the switch from the fully mycoheterotrophic to the photosynthethic stage. Secondly, three different proteomic techniques were carried out in independent experiments aiming to identify changes in protein accumulation in mycorrhizal roots of the terrestrial orchid Oeceoclades maculata.Finally, O. maculatamycorrhizal roots were used for transcriptome analyses. The data revealed a strong increase in general stress responses, accompanied by changes in signaling pathways possibly related to fungal recognition and establishment of a compatible interaction. Some of the upregulated genes may be involved in the reorganization of cell structure, likely related to accommodation of the fungal symbiont in the plant roots. We have also observed in mycorrhizal roots up-regulation of genes involved in carbon metabolism, including glycolysis/gluconeogenesis and amino sugars metabolism, as well as genes involved innitrogen assimilation. The down-regulation of genes involved in the jasmonate and ABA transduction pathways, and key genes encoding anti-fungal proteins, such as chitinase and a mannose-specific binding lectin, strongly suggests an alleviation of plant defense responses in O. maculata mycorrhizal roots. In general, our data suggest that the physiology of an orchid mycorrhiza is more similar to a compatible interaction than to an arm-race between plant and fungi. Overall orchid mycorrhiza have proved to be a promising model for investigating plantfungal interactions and further studies should now address the specific roles of the genes showing differential regulation in this study. / As orquídeas são caracterizadas por produzirem sementes diminutas, que não possuem endosperma. Necessitam, portanto, da interação com fungos micorrízicos para germinação e desenvolvimento do embrião. Algumas orquídeas aclorofiladas se mantêm dependentes dos fungos micorrízicos para a aquisição de carbono, enquanto outras desenvolvem a maquinaria fotossintética. Apesar do significado biológico das micorrizas de orquídeas, alterações na expressão gênica e no acúmulo de proteínas foram altamente negligenciads nos últimos anos. Neste trabalho, foram utilizadas diferentes técnicas sequenciamento e identificação de genes e proteínas em larga-escala para acessar as alterações moleculares responsáveis pela regulação das micorrizas de orquídeas. Uma abordagem baseada em 2D-LC MS/MS acoplada a técnica de quantificação absoluta e relativa iTRAQ, foiutilizada para identificar proteínas com acúmulo diferencial em Oncidium sphacelatum em diferentes estágios do desenvolvimento do protocormo (protocormos aclorofilados versus protocormos fotossintetizantes), após inoculação com um fungo do gênero Ceratobasidium. As análises mostraram que, as alterações esperadas no metabolismo do carbono foram acompanhadas de um acúmulo aumentado de proteínas envolvidas na modulação de espécies reativas de oxigênio, respostas de defesa, biossíntese de fitoalexinas e carotenóides, sugerindo que os protocormos de orquídeas passam por profundas alterações metabolicas durante a transição do metabolismo micoheterotrófico para o fotossintético. Posteriormente foram utilizadas três diferentes técnicas de proteômica quantitativa para explorar alterações fisiológicas em raízes micorrizadas e não-micorrizadas de Oeceoclades maculata.Este estudo foi ampliado, pela utilização de uma abordagem transcritômica ao mesmo modelo biológico. Em conjunto, os dados revelaram um forte aumento em respostas relacionadas ao estresse, acompanhadas de alterações em vias de transdução de sinal possivelmente relacionadas ao reconhecimento do simbionte fúngico e estabelecimento de uma interação compatível. Alguns genes com expressão aumentada devem estar envolvidos na reorganização celular, provavelmente ligada a acomodação do simbionte fúngico nas raízes das plantas. Também foi observado o aumento de genes envolvidos no metabolismo do carbono e de açúcares aminados, juntamente a genes relacionados a assimilação de nitrogênio em raízes micorrizadas. A expressão diminuída de genes envolvidas nas vias do jasmonato e ácido abscícico, juntamente a genes-chave que codificam para proteínas anti-fúngicas sugerem fortemente uma atenuação das respostas de defesa da planta em raízes micorrizadas de Oeceoclades maculata. No geral, parece que as micorrizas de orquídeas são fisiológicamente mais próximas de uma simbiose compatível do que de uma interação unilateral em favor da planta. Sobretudo, este sistema biológico provou ser promissor para investigação de interações planta-fungo e, próximas pesquisas devem agora ser focadas em funções específicas dos genes que mostraram regulação diferencial neste estudo.

As sessile organisms, plants evolved a plethora of defenses against their attackers. Given the role of plants as a primary food source for many organisms, plant defense has important implications for community ecology. Surprisingly, despite the potential to alter entire food webs and communities, the factors determining plant investment in defense are not well-understood, and are even less understood considering the numerous symbiotic interactions in the same plant. Legume-rhizobia symbioses engineer ecosystems by fixing nitrogen from the atmosphere in trade for plant photosynthates, yet connecting symbiotic resource exchange to food web interactions has yet to be established. Here I test how rhizobia influence plant defense and tritrophic interactions in lima bean (Fabaceae - Phaseolus lunatus L.): a model plant in chemical ecology research characterized by a broad range of different defenses. Examining suites of traits among lima bean genotypes, highly cyanogenic cultivars and wild type plants (high cyanotypes) produce more hook-shaped trichomes, as a putative combined approach of chemical and mechanical defenses, forming defense syndromes to protect against multiple feeding guilds (Chapter 2). Testing costs that may have contributed to forming tradeoffs among strategies, high cyanotypes show reduced fitness under plant-plant competition relative to low cyanotypes, but when challenged with herbivory, high cyanotypes fitness reductions are no longer evident (Chapter 3). Young leaves, not reproductive organs, are the most cyanogenic lima bean organ, and removal quantitatively decreases fitness, supporting assumptions that the most valuable tissues will be most highly defended (Chapter 4). Testing the degree to which nitrogen-fixing rhizobia contribute to cyanogenesis, high cyanotypes form more nodules than low cyanotypes. Quantitative relationships between nodule number and plant traits highlight the role symbiotic investment plays a role in plant defense and nutritive phenotype, while simultaneously, genotypically-determined levels of defense shape plant investment in symbiosis (Chapter 5). Interestingly, traits that trade off by cyanotype (i.e. high cyanogenesis but low indirect defense) reflect the patterns in plants with nitrogen-fixing rhizobia. Rhizobia-inoculated lima beans show reduced indirect defenses, recruiting fewer parasitoid wasps (Chapter 6) and predatory ants (Chapter 7). Examining plant-ant attraction in greater detail, ants prefer headspace regions above EFN droplets, corresponding with species-specific differences in suites of volatiles, indicating EFN, like floral nectar, can be scented to manipulate insect behavior (Chapter 8). Overall, understanding when investing in traits to recruit predators is more effective than investing in defensive chemistry, and how particular ecological contexts, such as symbioses can influence the outcome of defense allocation strategies remains a fascinating area of research. Determining the mechanisms underlying why rhizobia and other belowground microbial symbionts influence their host plants' above ground interactions, whether plants traits affected by symbiotic microbes are simply a function of the costs and benefits from resource exchange, or whether symbionts can influence the success of primarily direct versus indirectly defended plants is an important question for understanding complex trophic systems and connecting to agricultural implications for more effective biological pest control.

Some plant-beneficial bacteria with biopesticide potential produce antimicrobial compounds that are tightly involved in multitrophic interactions occurring in the phytosphere among which antagonism toward a diverse range of phytopathogens. However, there is a crucial need for an accurate assessment of antibiotic production rate in this environment colonized by these bacteria. In this context, we conducted the present thesis work with the scope to better understand how two different Bacillus genus strains produce lipopeptides in the rhizosphere of plants. In the first part, one of the strains (B. amyloliquefaciens S499) was selected out of other isolates for its technological traits and in vitro inhibition growth activity of plant pathogenic fungi. We further demonstrated its biocontrol potential in tomato open-field experiments where plantings have been devastated by a local fungus preliminary identified as Fusarium semitectum. In a second part, we combined two mass spectrometry-based approaches (electrospray ionization and imaging) to analyze the pattern of surfactin, iturin and fengycin lipopeptide families produced in planta by strain S499. Our results show that rhizosphere conditions are conducive for surfactin synthesis but not for other types of lipopeptides and that the lipopeptide pattern can be markedly influenced by nutritional factors, biofilm formation and oxygen availability. In a last part, surfactin gene expression (srfA) level was evaluated in situ on tomato root using the reporter gene (LacZ) inserted in B. subtilis strain BGS3. Results showed effective expression of srfA and production of surfactin in biologically important level quantities upon establishment of bacterial population on roots. Our results also demonstrate that BGS3 developing in colonies, efficiently utilizes the main substrates from plant exudates to produce surfactins. The production may also be favored in bacteria growing slowly in the rhizosphere. Globally, this work contributes to better appreciate the impact of some environmental factors on the in situ biosynthesis of lipopeptides by strains of Bacillus which is probably an essential step for improving the level and reliability of their efficacy as biological agents for the control of plant diseases.

Thesis (Ph.D.) -- University of Western Sydney, Nepean, 1999. / Bibliography : p. 213-233.

Vegetation is known to influence the composition of microbial communities. Bacteria can act as roots symbionts or be involved in the decomposition of plant biomass. They can be influenced by soil chemistry but also by plant exudates. Some plants produce targeted exudates to attract specific bacteria to their roots. Bacteria associate with plants frequently but the effect of plant diversity on bacterial communities on their roots and in the surrounding soil remains unclear. The aim of this work was to describe the relationship between the diversity and community composition of bacteria and the diversity of vegetation in forest and grassland ecosystems. The study areas were selected to represent a gradient of vegetation in Bohemian Forest NP and in White Carpathian flowery grasslands. I hypothesized that the diversity and evenness of bacterial community increase with increasing plant diversity. The composition of bacterial community was characterized by 16S rRNA sequencing. The composition of vegetation was determined by phytocenological relevées and by molecular markers trnL. In grassland ecosystem, there was a positive relationship between plant and bacterial diversity only in shoots. The space and vegetation were identified as an important drivers of bacterial community composition in shoots. The...

Os microrganismos endofíticos são aqueles, cultiváveis ou não, que habitam o interior da planta hospedeira sem causar danos aparentes ou estruturas externas visíveis. Essa interação microrganismos-planta é intrínseca a determinadas espécies de plantas e/ou bactérias. Nas últimas décadas os estudos de microrganismos endofíticos têm sido realizados em diversas plantas hospedeiras, sendo esses estudos direcionados principalmente para a diversidade e características benéficas induzidas, inclusive o controle biológico de doenças. A doença causada pelo fungo Ceratocystis fimbriata é considerada emergente no setor florestal. O Brasil está entre os maiores produtores mundiais de eucalipto e a expansão do setor juntamente com o cultivo clonal tem acarretado o aumento da incidência de patógenos. O surgimento de novas doenças exige estudos relacionados tanto a interação do agente patogênico com hospedeiro quanto de todos os componentes do patossistema. Neste contexto, os microrganismos endofíticos têm sido descritos como potenciais controladores biológicos de doenças. Dessa forma, o presente trabalho teve por objetivos avaliar a interação de C. fimbriata com a comunidade bacteriana associada à Eucalyptus sp. Adicionalmente, foi estudada a possível transferência desses endófitos via sementes e o padrão de colonização de Pantoea agglomerans em plântulas. Foi observado que plantas não infestadas por C. fimbriata apresentaram maior densidade bacteriana no rizoplano (20,66 x 104 UFC.cm2 -1 de raiz), enquanto que para a comunidade endofítica, a maior densidade foi observada em plantas infectadas pelo fungo (25,13 x 104 UFC.g-1 de raiz). As análises por ARDRA possibilitaram a obtenção de 8 e 13 ribotipos nas comunidades endofítica de raiz e do rizoplano, respectivamente. Os ribotipos mais freqüentes foram identificados como Bacillus cereus. As análises de diversidade por meio de DGGE das comunidades do rizoplano e endofítica de raiz mostraram que a infestação pelo fungo interfere na colonização de Eucalyptus. Foi observado também que bactérias endofíticas estão presentes no interior de sementes de Eucalyptus spp. em uma densidade de 0,33 a 1,83 X 102 UFC.g-1, para as espécies E. camandulensis e E. urophylla, respectivamente. A densidade bacteriana endofítica de plântulas obtidas de sementes desinfectadas superficialmente variaram entre 0,27 X 102 a 0,87 X 102 UFC.g-1, para E. citriodora e o híbrido E. robusta x E. grandis, respectivamente. Em algumas espécies de Eucalyptus não foram isoladas bactérias endofíticas das sementes e plântulas. Os resultados mostraram que algumas espécies de bactérias endofíticas podem ser transmitidas verticalmente por sementes. P. agglomerans inoculada nas sementes foi capaz de colonizar as plântulas após a germinação da semente, indicando que esta pode ser uma das formas utilizadas pelos microrganismos para colonizar e se estabelecer na planta hospedeira. Assim, os resultados obtidos neste trabalho mostram ainda que possa existir interação entre a presença de C. fimbriata e a comunidade bacteriana endofítica e do rizoplano de Eucalyptus. Foi possível observar também que estas bactérias endofíticas que são transmitidas por meio de sementes, permitindo que plântulas previamente inoculadas com bactérias benéficas possam ser produzidas antes de serem levadas a campo. / The endophytic microorganisms are those, cultivated or not, that inhabit the interior of the plant host without causing apparent damages or visible external structures. This interaction microorganisms-plant is specific to certain species of plants and/or bacteria. In the last few years studies of endophytic microorganisms have been carried out in several plant hosts, being these studies focused mainly to diversity and biotechnological potential, such as biological control of disease. The disease caused by the phytopathogenic fungi Ceratocystis fimbriata is considered emerging by the reforestation companies. Brazil is one of the largest world eucalyptus producers and the increasing of the eucalyptus production associated to clonal reproduction has allowed the increase in pathogen incidence. Studies that evaluate the interaction between pathogens and the microbial community associated to the host plant may allow understanding how disease symptoms come up. Endophytic microorganisms have been described as potential biological control of diseases and therefore, the aims of the present work were to i) study the interaction between C. fimbriata and the bacterial community associated to the Eucalyptus sp.; ii) evaluate the bacterial dissemination by seeds; iii) evaluate the colonization profile of Pantoea agglomerans in seedlings after seed inoculation. It was observed that the highest bacterial density on the rhizoplane (20.66 x 104 CFU.cm2 -1 of root) was observed in C. fimbriata uninfectedplants, while for endophytic community the highest density was observed in C. fimbriata infected plants (25.13 x 104 CFU.g-1 of root). The ARDRA analyses showed that the bacterial community of eucalyptus is composed by 8 and 13 ribotypes on rhizoplane and inside the roots (endophytic), respectively. The most frequent ribotypes were identified as Bacillus cereus. The DGGE analyses of diversity of endophytic and rhizoplane community showed that fungi infection shift the colonization of Eucalyptus associated bacteria. The bacterial community inside Eucalyptus spp. seeds ranged from 0.33 to 1.83 X 102 CFU.g-1, for E. camandulensis and E. urophylla, respectively. After seed germination the endophytic bacterial density in seedlings ranged from 0,27 X 102 to 0,87 X 102 CFU.g-1, for E. citriodora and the hybrid E. robusta x E. grandis, respectively. Although, endophytic bacteria have been isolated from seeds, for some plant species, bacteria were not isolated from seedlings. Also, some bacteria may be vertically transmitted from seed to seedlings, but some is specific for seeds. Seed inoculation of P. agglomerans resulted in seedlings colonized by these bacteria, suggesting that these bacteria could be seed transmitted. The results obtained in the present study show that the fungi C. fimbriata inside the Eucalyptus host can shift the endophytic and rhizoplane bacterial diversity. Also, these endophytic bacteria could be transmitted vertically by seeds, allowing that seeds previously inoculated with beneficial bacteria may result in protected plants before planting in the field.

A gradual depletion of high-grade ores, coupled with the growing demand for mineral commodities across the world has culminated in the increased exploitation of lean-grade ores with complex mineralogy. The mineral processing industry commonly uses an extensive range of inorganic, naturally derived or synthetic organic reagents in the separation of valuable minerals from the ore. Froth flotation is a commonly used separation technique to float or depress different sulfide minerals from the ore, based on their surface properties. In recent times, biological processes have been attracting attention in mineral processing and metal recovery operations due to a number of factors, especially lower operating costs, lesser energy consumption and their environment friendly nature. The use of microorganisms and their direct derivatives in mineral processing, hydrometallurgy and in the bioremediation of mineral industry discharges has led to the emerging area of “Mineral Bioprocessing”. In this study, a family of four microorganisms belonging to the Bacillus species, viz., Paenibacillus polymyxa, Bacillus circulans, Bacillus megaterium and Bacillus subtilis was used to ascertain the selective floatability of sphalerite from a sphalerite-galena mineral mixture. These bacteria are Gram positive, mesophilic, neutrophilic, aerobic and spore forming. The major objectives of the investigation include: a) Identification and characterization of bioreagents derived from Bacillus species for the flotation of sphalerite from a sphalerite-galena mixture b) Optimization of the flotation process for the enhanced recovery of sphalerite using specific bioreagent combinations c) Modes and mechanisms of bacterial adaptation to minerals and their consequent effects on the flotation of sphalerite and galena d) Elucidation of the mechanisms of microbe-mineral interactions and the role of extracellular secretions in sphalerite flotation column and their N-terminal residues were identified using Edmann N-terminal sequencing. Additionally, sequences of several internal peptides from both the proteins were determined using Tandem Mass Spectrometric techniques. A database search revealed that the sequences of these peptides are unique and have not been reported earlier. It was established that the bacterial cells give high flotation recovery of sphalerite under buffered conditions and that it took place only in the presence of anionic buffers. Additionally, the viability of the bacterial cells was not required for the flotation of minerals. A major finding of this study was that other than extracellular DNA (eDNA), none of the other bacterial surface components like teichoic acids, surface proteins, polysaccharides played a positive role in the flotation process. Nucleic acids, more particularly single stranded DNA (ssDNA), facilitated sphalerite flotation relative to double stranded DNA (dsDNA). A probable mechanism of ssDNA -mediated selective flotation of sphalerite has been presented. A negative role for non-DNA surface components was also observed. This led to the realization of the need for an optimum ratio of DNA to non-DNA components in the selective flotation of sphalerite from a sphalerite-galena mixture. It was found that the surface physiochemical properties of the mineral adapted bacteria differed significantly from that of the unadapted bacteria. Adaptation enhanced the flotation recoveries of the corresponding mineral vis-à-vis the unadapted bacteria. Sphalerite adapted bacteria secreted more extracellular proteins while the galena adapted bacteria secreted more polysaccharides compared to the unadapted bacteria. Sphalerite adapted bacteria selectively floats more sphalerite from the mineral mixture than the galena adapted as well as the unadapted bacteria. It was evident from the electrokinetic studies that the surface charge of the chosen sulfide mineral adapted bacteria was less negative relative to the unadapted bacteria. This phenomenon was observed with all the four bacterial species used in this study. A noteworthy finding was that the bacteria especially B.circulans induce a change in morphology from rod to sphere as a strategy during adaptation to a toxic mineral such as galena. This phenomenon has been shown to involve changes in crucial cell wall components as well as changes in the levels of expression of bacterial cytoskeleton elements involved in the maintenance of the rod shape. This aspect of the study involved the partial sequencing of the B.circulans homolog of the key cytoskeleton gene, mreB (B gene in murien cluster e), using the Polymerase Chain Reaction (PCR) followed by DNA sequencing. A Genbank search indicated that this is the first report of the sequence of B.circulans mreB gene. This was followed by measuring the hypothesized downward changes in the levels of expression of the mreB gene by Reverse Transcriptase Polymerase Chain Reaction (RT-PCR). The possible mechanisms of the adaptive morphological changes and of the interaction of the chosen sulfide minerals with the family of microorganisms studied have been discussed with respect to their bioflotation efficiency.

Pendant l’infection, les agents phytopathogènes sécrètent un arsenal de molécules, appelées effecteurs, éléments clés de la pathogénie qui modulent l’immunité innée de la plante et facilitent l’infection. Leptosphaeria maculans est le champignon responsable de la nécrose du collet du colza. Plus de 650 gènes codant des effecteurs potentiels ont été identifiés dans son génome, dont 7 ont un rôle reconnu dans l’avirulence du champignon. Les effecteurs fongiques correspondent principalement à de petites protéines potentiellement sécrétées (PPS), n’ayant pas d’homologues dans les bases de données et pas de motifs connus. Par conséquent, leur fonction biologique est difficile à prédire, et très peu de choses sont connues sur le mode d’action des effecteurs de L. maculans au cours de l’infection.L’objectif de ma thèse était de caractériser fonctionnellement des effecteurs de L. maculans afin de mieux comprendre leur rôle au cours du processus infectieux. Cette caractérisation fonctionnelle a consisté en : i) la détermination de la localisation subcellulaire de ces effecteurs dans Nicotiana benthamiana et Arabidopsis thaliana ; ii) la recherche de cibles végétales ciblées par ces effecteurs ; et iii) la détermination des processus cellulaires impactés par ces effecteurs par expression stable dans A. thaliana et tests de suppression de mort cellulaire dans N. benthamiana. Quatre effecteurs ont été choisis pour cette étude : AvrLm10-1, AvrLm10-2, AvrLm4-7 et AvrLm3.AvrLm10-1 et AvrLm10-2 sont tous les deux nécessaires pour induire une reconnaissance par le gène de résistance Rlm10. Des orthologues d’AvrLm10-1 et AvrLm10-2 ont été identifiés chez des Dothidéomycètes et des Sordariomycètes phytopathogènes ainsi que plusieurs paralogues exprimés spécifiquement pendant l’infection chez L. maculans. AvrLm10-1 et AvrLm10-2 présentent toutes les deux une localisation nucléo-cytoplasmique. Une interaction physique entre AvrLm10-1 et AvrLm10-2 a été mise en évidence, ainsi qu’une interaction potentielle de ces deux protéines avec une protéine PR1 (Pathogenesis-related 1) et une cystéine-protéase végétale.AvrLm4-7 est reconnu par deux gènes de résistance, Rlm4 et Rlm7, et sa présence empêche la reconnaissance d’AvrLm3 par Rlm3. AvrLm4-7 est capable de supprimer la mort cellulaire provoquée aussi bien par des inducteurs généraux de la mort cellulaire que par des inducteurs de la PAMP-Triggered Immunity (PTI) et de l’Effector-Triggered Immunity (ETI). AvrLm4-7 présente une localisation nucléo-cytoplasmique, qu’il soit exprimé avec ou sans son peptide signal, ce qui suggère un mode d’action intracellulaire. AvrLm4-7 interagit potentiellement avec une protéine ribosomale végétale, de la même manière qu’un effecteur de Blumeria graminis avec lequel il partage des analogies structurales. Cependant, des lignées d’A. thaliana exprimant AvrLm4-7 de façon constitutive ne présentent aucune différence morphologique ou de sensibilité aux maladies comparativement à l’écotype sauvage Col0.AvrLm3 est un gène d’avirulence très conservé dans les populations de L. maculans dont la reconnaissance par le gène de résistance Rlm3 est supprimée en présence d’AvrLm4-7. AvrLm3 est capable de supprimer la mort cellulaire associée à la PTI et à l’ETI. Cet effecteur est localisé dans l’apoplasme des cellules foliaires lorsqu’il est exprimé avec son peptide-signal, suggérant un mode d’action extracellulaire. AvrLm3 interagit potentiellement avec une myrosinase-associated proteine sécrétée impliquée dans le système myrosinase-glucosinolate, suggérant qu’AvrLm3 perturberait la synthèse des glucosinolates, ce qui est un mode d’action inédit pour un effecteur d’agent phytopathogène.Cette thèse a permis de mieux comprendre le mode d’action des effecteurs de L. maculans et de proposer de nouvelles stratégies de contrôle des maladies fongiques. / During infection, plant pathogens secrete an arsenal of molecules collectively known as effectors that circumvent plant innate immunity and trigger infection. The phytopathogenic fungus Leptosphaeria maculans is the causal agent of stem canker of oilseed rape. More than 650 putative effector-encoding genes have been identified in its genome, 7 of them corresponding to avirulence genes. Fungal effectors mainly correspond to small secreted proteins (SSP) with no known homologs and no predicted functions. Their biological function is therefore difficult to predict, and very little is known about the mode of action of L. maculans effectors during infection.The objective of my thesis was to elucidate the role of L. maculans effectors during infection through their functional characterization which included: i) the determination of their subcellular localization in Nicotiana benthamiana et Arabidopsis thaliana; ii) a search for their plant targets; and iii) the determination of the cellular processes targeted by those effectors through their stable expression in A. thaliana and by testing their ability to suppress cell-death in N. benthamiana. We investigated four effectors in that study: AvrLm10-1, AvrLm10-2, AvrLm4-7 and AvrLm3.AvrLm10-1 and AvrLm10-2 are both necessary to trigger recognition by the Rlm10 resistance gene. We have identified orthologs for AvrLm10-1 and AvrLm10-2 in Dothideomycetes and Sordariomycetes phytopathogens, and several paralogs in L. maculans which are expressed specifically during oilseed rape infection. AvrLm10-1 and AvrLm10-2 both have a nucleo-cytoplasmic localization. AvrLm10-1 and AvrLm10-2 physically interact, and may also interact with a PR1 (Pathogenesis-related 1) protein and a secreted cysteine-protease. AvrLm4-7 is recognized by two resistance genes, Rlm4 and Rlm7, and suppresses recognition of AvrLm3 by Rlm3. AvrLm4-7 suppresses cell-death triggered by general inducers, PAMP-Triggered Immunity (PTI) and Effector-Triggered Immunity (ETI). AvrLm4-7 has a nucleo-cytoplasmic localization, whether expressed with or without its signal peptide, suggesting an intracellular mode of action. One of the potential plant targets for AvrLm4-7 is a ribosomal protein, just like a Blumeria graminis effector with structural analogy to AvrLm4-7. Transgenic lines of A. thaliana constitutively expressing AvrLm4-7 do not show any morphological phenotypes or any difference in their susceptibility to diverse fungal pathogens. AvrLm3 is an avirulence gene strongly conserved in L. maculans populations. Recognition of AvrLm3 by Rlm3 is suppressed by the presence of AvrLm4-7. AvrLm3 suppresses cell-death triggered by several inducers of PTI and ETI. AvrLm3 is localized in plant apoplasm when expressed with its signal peptide, suggesting an extracellular localization. AvrLm3 potentially interacts with a secreted myrosinase-associated protein implicated in the myrosinase-glucosinolate system, suggesting that AvrLm3 could disturb glucosinolate production, which is a novel mode of action never described for a plant pathogen effector.My thesis allowed us to improve our knowledge on fungal effector function during infection and to propose new strategies for plant diseases management

As of January 2017, the U.S. poultry industry banned the use of antibiotics and now relies on alternatives such as probiotics to help protect animal health. Although probiotic use is not a new concept in the poultry industry, identifying the best combination of bacterial strains to generate an effective probiotic formula requires further investigation. This study aimed to detect a probiotic product of four bacterial strains (Pedioccoccus acidilactici, Pediococcus pentosaceus, Lactobacillus plantarum, and Bacillus subtilis) in a feeding trial with broiler chickens. Birds given the probiotic were predicted to show an improved growth performance with the probiotics colonizing the gut. Ninety-six broiler chickens were equally divided into 3 treatment and 3 control pens. During the 25-day experiment, birds were fed a starter diet (days 0-11) and a grower diet (days 12-25). Experimental birds were administered the probiotic product via the drinking water at a concentration of 3.1×104 CFU/ml. Control birds had an equivalent amount of dextrose filler added to their water supply. Feces were collected hourly on day one and daily thereafter. On days 1, 22, and 25 of the experiment, 2 birds from each pen were euthanized for gut sampling. Lumen and mucosa samples were collected from the duodenum, jejunum, ileum, and ceca. Species-specific and strain specific PCR primers were employed for probiotic detection. Wild strains of P. acidilactici, P. pentosaceus, and L. plantarum were detected in the feeds, inhibiting detection of the probiotic strains when using species-specific PCR primers. Strain-specific primers were used to detect the probiotic Pedioccoccus acidilactici and Lactobacillus plantarum strains. B. subtilis was detected in feces within one hour of probiotic administration and was predominantly detected in experimental birds only. Both P. acidilactici and L. plantarum probiotic strains were initially detected in the feces of treated birds within two hours of probiotic administration and again ten days later. Both L. plantarum and B. subtilis were seen only in treated bird gut samples. L. plantarum was predominantly detected in the ceca near the end of the small intestine. P. pentosaceus was observed more often in treated gut samples and P. acidilactici was the least commonly detected probiotic strain. All administered bacteria were rarely seen in mucosa samples. Feed-endogenous P. acidilactici and L. plantarum strains became progressively more detectable in the mucosa along the gastrointestinal tract suggesting gut colonization, however, probiotic strains did not appear to colonize the mucosa of treated birds. Although probiotic strains were no longer detected after product removal, all probiotic strains were detected in feces and gut samples during probiotic administration, suggesting the bacteria can colonize the gut. Probiotic supplementation did not result in significant differences in body weight gain, feed intake, or feed conversion ratio. However, birds growing in a more stressful environment than the carefully controlled experimental set up used here may show probiotic-related effects. This study identified that the probiotic bacteria appeared to survive the gastrointestinal tract, exhibited a transit time of 1-2 hours, could possibly colonize chickens, and localized near the end of the chicken gut.