La connaissance de la diversité microbienne des milieux ultramafiques est essentielle pour établir le fonctionnement écologique de ces milieux, qui présentent de fortes teneurs en Ni et sont caractérisés par une flore particulière, e.g. plantes hyperaccumulatrices de Ni. La rhizosphère des hyperaccumulateurs comporte une forte proportion de bactéries résistantes au Ni, qui peuvent aussi agir sur la nutrition des plantes et sur les propriétés physico-chimiques du sol. Le premier défi de cette thèse a été de cerner le déterminisme de la diversité bactérienne de la rhizosphère d’hyperaccumulateurs de Ni. Le second a été de tester l'intérêt de souches PGPR (Plant Growth Promoting Rhizobacteria) pour optimiser l'agromine à partir d'interactions entre les rhizobactéries et les hyperaccumulateurs de Ni. La démarche s'est appuyée sur un ensemble de prospections dans deux régions climatiques et sur des analyses de séquençage haut débit. Des tests de cultures de plantes hyperaccumulatrices inoculées ont également été conduits. Les résultats montrent que le déterminisme de la diversité bactérienne est variable selon l'échelle spatiale. A l'échelle mondiale, le type de végétation est le facteur majeur structurant les communautés bactériennes, elle-même contrôlée indirectement par le climat. L’influence directe du climat (température et humidité) sur la diversité est significative mais moindre. A l'échelle d'une région climatique, la physico-chimie des sols ultramafiques structure et détermine la diversité des communautés bactériennes rhizosphériques. Enfin, l'inoculation de souches PGPR fortement bioaccumulatrices de Ni modifie la dynamique du Ni dans le sol, ce qui démontre qu'il existe une compétition pour le Ni entre la plante et la bactérie inoculée. En conclusion, le déterminisme de la diversité des communautés bactériennes rhizosphériques est dépendant de l'échelle spatiale considérée. En outre, le choix de la souche PGPR à inoculer, dans un contexte d'amélioration de l'agromine du Ni, est primordial. / Knowledge of the microbial diversity in ultramafic areas is essential to establish the ecological functioning of these environments, which display high level of Ni and are characterized by the presence of particular plants, e.g. Ni hyperaccumulators. The rhizosphere of these plants promotes a high proportion of Ni resistant bacteria that can act on plant nutrition and soil physicochemical properties. The first challenge of this thesis was to understand the bacterial rhizosphere diversity of Ni hyperaccumulators. The second was to test the interest of PGPR (Plant Growth Promoting Rhizobacteria) strains in order to improve agromining based on rhizobacteria and Ni hyperaccumulators interactions. The approach was based on two-contrasted climatic areas prospection and on high-throughput sequencing analyzes. Tests on culture of hyperaccumulator plants inoculated were also conducted. The results show that the determinism of this bacterial diversity is variable according to the spatial scale. On a global scale, the vegetation type, indirectly influenced by the climate, is the major factor structuring bacterial communities. The direct influence of the climate (temperature and humidity) on bacterial diversity is significant but lower. At the scale of a climatic region, the physic-chemistry of ultramafic soils structures and determines the rhizosphere bacterial community diversity. Finally, the inoculation of highly Ni bioaccumulative PGPR strains modifies the Ni dynamic in the soil, demonstrating that there is a competition for this metal between the inoculated bacteria and the hyperaccumulator plant. In conclusion, the rhizosphere bacterial community diversity is dependent on the considered spatial scale. Furthermore, these results emphasize how the choice of the PGPR strain to inoculate is important in order to improve Ni agromining.

As rizobactérias promotoras de crescimento (PGPR) são microrganismos que ocorrem naturalmente no solo, são conhecidas por proporcionar melhorias no desenvolvimento das plantas atuando de diversas formas. Bacillus subtilis (cepa GB03) é uma PGPR disponível para comercialização como fungicida biológico concentrado, utilizada no tratamento de sementes de diversas culturas como algodão, soja, amendoim, trigo e cevada. Destaca-se pela capacidade de promover o crescimento de plantas por meio da emissão de voláteis. Vários estudos com Arabidopsis thaliana já comprovaram que B. subtilis (GB03) é capaz de auxiliar no desenvolvimento da planta por meio da promoção de crescimento e pela supressão de patógenos habitantes de solo. No entanto, o seu papel na proteção de plantas contra a herbivoria de insetos ainda não é bem caracterizado. Deste modo, buscou-se avaliar os efeitos da interação de B. subtilis (GB03) com plantas de rúcula (Eruca sativa) e Plutella xylostella (traça-das-crucíferas). Este inseto pertence à ordem Lepidoptera, considerado praga de maior importância no cultivo de Brassicaceae ao redor do mundo. Devido à sua alta prolificidade e capacidade de adaptação e ao seu curto ciclo de vida, tornou-se uma das pragas mais resistentes e de difícil controle da agricultura. Atualmente, os custos em escala mundial com o controle da praga anualmente chegam em torno de US $ 4 a 5 bilhões. Foram utilizados dois tratamentos, plantas inoculadas com B. subtilis (GB03) e controle (plantas não inoculadas). Avaliou-se o crescimento de plantas de rúcula e o peso seco de parte aérea. Para avaliar o desempenho e dano de P. xylostella em ambos tratamentos, previamente foram pesados grupos de quinze lagartas e submetidas a alimentação de plantas de rúcula durante 24 horas. Posteriormente, as lagartas foram retiradas e pesadas novamente e a área foliar consumida foi calculada por meio do software editor de imagens ImageJ®. A preferência de oviposição do inseto foi testada por meio de olfatometria, composta apenas de pistas olfativas e em arenas contendo tanto pistas olfativas quanto visuais. A emissão de voláteis foi caracterizada quantitativamente e qualitativamente por cromatografia gasosa e analisada por espectrometria de massas. A inoculação com GB03 em plantas de rúcula promoveu melhor crescimento das plantas em relação ao tratamento controle, ao mesmo tempo em que diminuiu os danos pelo consumo alimentar do inseto na planta. P. xylostella não apresentou distinção entre os odores das plantas nos testes de olfatometria. Entretanto, observou-se menor número de ovos em plantas com GB03 nos bioensaios de arena. Não foram constatadas diferenças significativas na emissão total de voláteis entre os tratamentos com e sem GB03, no entanto, foram encontradas concentrações diferentes dos compostos (Z)-3-hexenol e 2-ethyl-1-decanol. Outros testes devem ser realizados com a finalidade de estabelecer o papel desempenhado por GB03 na indução de defesas de plantas contra insetos. / The plant growth promoting rhizobateria (PGPR) are microorganisms that naturaly live in the soil, known by improving the plants\' development in many ways. Bacillus subtilis (strain GB03) is a comercial available PGPR, sold as a concentrated biological fungicide, applied in seed treatment of different cultures as cotton, soybean, peanut, wheat and barley. Moreover, it stands out by its capacity of plant growth promoting via volatiles emission. Several studies with Arabidopsis thaliana showed that B. subtilis (GB03) can help the plant development via growth promotion and by soil pathogens supression. However, its role in plant protection against insect herbivory has not been characterized yet. Thus, it aimed to evaluate the interaction effects among B. subtilis (GB03), arugula plants (Eruca sativa) and Plutella xylostella (Diamondback moth). This insect belongs to the order Lepidoptera and have been considered the main pest in Brassicaceae fields around the world. Due to its high prolificacy and plasticity in field survival, and its short life cycle, it has become one of the most resistant and hard control pest in agriculture. Currently, the annualy world costs with this pest control is about US $ 4-5 bilions. Here, it was used two treatments, innoculated plants with B. subtilis (GB03) and control (non-innoculated plants). The arugula plants growth and dry mass of shoots were evaluated. To analyze the performance and damage by P. xylostella in both treatments, it was previously weighed groups with fifteen caterpillars and submitted to feeding on arugula plants during 24 hours. After that, the caterpillars were removed and weighed again and the consumed leaf area was calculated by the image editor software ImageJ®. The insect oviposition preference was tested by olfactometry, with only olfactory cues and in arenas containing both, olfactory and visual cues. Volatiles emission was quantitatively and qualitatively characterized by gas chromatography and analyzed by mass spectrometry. GB03 innoculation in arugula plants promoted a better growth when compared to control, and, at the same time, there was an increasing in the plant damage by insect food consumption. P. xylostella did not show distinction between odors of the plants in olfactometry tests. Although, it was observed less number of eggs in plants with GB03 in arena bioassays. It was not found significant differences in total volatile emission between the treatments with and without GB03, even though different concentrations of (Z)-3-hexenol and 2-ethyl-1-decanol were observed. Other tests must be performed in order to estabilish the role played by GB03 in plant induction defense against insects.

Les plantes dans leur environnement établissent des interactions avec des micro-organismes du sol. Parmi ces interactions nous pouvons distinguer les symbioses associatives mettant en jeu des bactéries PGPR (Plant Growth Promoting Rhizobacteria). L’impact de ces microorganismes phytobénéfiques (Azospirillum, Pseudomonas…) sur le métabolisme de la plante hôte est encore mal connu. Le modèle d’étude que nous avons choisi dans le cadre de ce travail est Zea mays L. qui peut établir de nombreuses symbioses associatives avec des PGPR. Pour étudier les effets de ces micro-organismes sur le maïs, deux approches ont été développées faisant notamment appel à des outils de profilage métabolique pour i) déterminer l’impact de la simple inoculation micro-organismes sur le métabolisme secondaire racinaire et des parties aériennes de la plante hôte, et ii) évaluer les effets physiologiques de consortia microbiens comprenant Azsopirillum, Pseudomonas et Glomus. Les résultats de ce travail démontrent la place prépondérante des composés de type benzoxazinoide (benzoxazolinone et benzoxazinone) dans les interactions et la modulation de leur synthèse induite par les inocula. Par ailleurs nos travaux mettent également en évidence que la réponse métabolique de la plante à l’interaction avec les micro-organismes est dépendante de l’espèce et de la souche bactérienne considérée suggérant ainsi un phénomène de reconnaissance entre les deux organismes / In environment, plant performed some interactions with soil microorganisms. From these interactions, associative symbiosis involving PGPR bacteria (Plant Growth Promoting Rhizobacteria) can be considerate. Impact of phytobenefic microorganisms (Azospirillum, Pseudomonas…) leading to associatives interactions, on host plant metabolisms, still poorly understood. Zea mays L. was choose as study model because it can enter in various associatives symbiosis with Plant growth Promoting Rhizobacteria. To study effects of these microorganisms on maize, two approaches were developed thanks to metabolite profiling tools to (i) determine the impact of a single microorganism inoculation on host plant roots and shoots secondary metabolisms and (ii) evaluate physiological effect of microbial consortia including Azospirillum, Pseudomonas and Glomus species. Results of this work showed the major place of benzoxazinoids compounds (benzoxazolinone and benzoxazinone) in plant/microbe interaction and their synthesis modulation induced by inocula. Besides, our works brings to light that the metabolic answer of the plant to the interaction with microorganisms is dependent on species and bacterial strain suggesting a recognition phenomenon between both organisms

Ce travail a comme objectif d’explorer le potentiel de bioremédiation de plantes et de rhizobactéries pour la réhabilitation des sols de sites miniers dans la région de Marrakech. Nous avons isolé des souches rhizosphériques et nodulaires des racines de fève et de plantes indigènes des sites miniers Draa Sfar et Kettara. Plusieurs isolats ont montré une bonne tolérance aux ETM allant jusqu’à 25 mg/l de Cu, 200 mg/l de Pb et 150 mg/l de Zn. Les souches tolérantes aux métaux ont fait l’objet de criblage pour déterminer leurs caractères PGPR (production d’exopolysaccharides, sidérophores, AIA et activité antifongique). En présence du sol minier amendé avec le sol agricole, la fève a montré une capacité à accumuler les métaux différemment d’un métal à l’autre. Le Pb a été peu mobile dans les parties aériennes des plantes, et n’a pas atteint les graines. Le Zn a été le métal le plus transloqué vers les parties aériennes bien que sa quantité soit importante au niveau des racines. Le Cu a été accumulé plutôt dans les racines des plantes. Le Zn et le Cu ont été transloqués aux graines mais à des teneurs significativement faibles. L’inoculation par les bactéries PGPR a permis de mieux accumuler le Cu dans les plantes notamment dans la partie racinaire. L’inoculation mixte PGPR-rhizobia a amélioré l’absorption du Zn dans les plantes notamment dans la partie aérienne. Les analyses in natura sur ces sites miniers fortement contaminés par des métaux lourds indiquent que le microbiote a évolué, devenant tolérant à la pollution à long terme par les métaux lourds et adapté aux plantes indigènes elles-mêmes capables de tolérer et d'accumuler les métaux lourds. / This work aims to explore the potential of plant and rhizobacteria for bioremediation in mining soil in Marrakech region. Rhizospheric and nodules strains were isolated from faba bean roots and native plants growing in Draa Sfar and Kettara mining sites. Several isolates showed good tolerance to metals up to 25 mg/l of Cu, 200 mg/l of Pb and 150 mg/l of Zn. The tolerant strains to metals were screened for their PGPR characteristics (production of exopolysaccharides, siderophores, IAA and antifungal activity). In mining soil amended with agricultural soil, faba bean showed an ability to accumulate metals differently. Pb was not very mobile in shoots, and did not reach the seeds. Zn was the most translocated metal to the aerial parts although its amount was important in roots. Cu was accumulated rather in the roots of plants than shoots. Zn and Cu were translocated to seeds but at significantly lower levels. The inoculation with PGPR bacteria allowed Cu to accumulate better in plants, particularly in roots. Whereas the PGPR-rhizobia mixed inoculation improved the absorption of Zn in plants, especially in shoots. In natura investigation of mining sites heavily contaminated with heavy metals (HMs) indicates that microbiota has evolved, becoming tolerant to long-term HMs pollution and adapted to indigenous plants themselves able of tolerating and accumulating HMs.

Paraburkholderia phytofirmans souche PsJN est une bactérie endophyte capable d’induire une promotion de croissance de la vigne et de lui conférer une tolérance vis-à-vis des basses températures. Puisque la bactérie PsJN induit également une tolérance de la vigne contre la pourriture grise causée par le champignon Botrytis cinerea, l’objectif de ce travail était de caractériser les mécanismes de la résistance induite par P. phytofirmans suite à une infection par B. cinerea chez la vigne.Nos résultats indiquent que l’inoculation des racines de vitroplants par la bactérie PsJN entraîne des modifications du métabolisme phénolique au niveau des plantules bactérisées. En effet, des tanins d’origine gallique et ellagique sont accumulés au niveau des racines en réponse à PsJN alors qu’une accumulation de lignine a été observée dans la tige et d’anthocyanes dans les feuilles. De même, PsJN induit une résistance contre B. cinerea en potentialisant l'expression de gènes de défense dépendants de l’acide salicylique et de l’acide jasmonique dans les feuilles de vigne. Parallèlement, une accumulation de callose et d’H2O2 a été observée ainsi qu’une meilleure mobilisation des glucides dans les plantules bactérisées, corrélée à une réduction des dommages de l’activité photosynthétique.Suite aux résultats obtenus avec le modèle vitroplant, nous avons optimisé la bactérisation des boutures fructifère et évaluer l’impact de cette bactérie sur la résistance des inflorescences contre B. cinerea. Les résultats présentés dans ce travail permettent d’envisager l’utilisation de la bactérie PsJN comme agent de lutte biologique. / Paraburkholderia phytofirmans strain PsJN is an endophytic beneficial bacterium able to promote the growth of grapevine and to induce the plant resistance to biotic and abiotic stresses. Indeed, the interaction between PsJN and grapevine confers to the grapevine a protection towards cold stress (4 °C), by reducing damages on the photosynthetic system and modulating the carbon metabolism. The PsJN strain also induces a protection against gray mold caused by Botrytis cinerea. In this context, the aim of this work was to better characterize this P. phytofirmans strain PsJN-induced resistance in grapevine against B. cinerea.Our results indicate that the inoculation of in vitro-plantlets roots with PsJN altered the phenolic metabolism in bacterized-plantlets. Gallic and ellagic derived tannins are clearly accumulated in roots of bacterized-plantlets whereas an accumulation of lignin in stems and anthocyanins in leaves was observed. We also showed that PsJN induces a protection against B. cinerea by priming the expression of genes involved in salicylic acid and jasmonic acid pathways. In parallel, an accumulation of callose and H2O2, as well as a better mobilization of carbohydrates in bacterized plantlets were observed. In addition to the defense mechanisms setting up by plants, we demonstrated that PsJN reduces in vitro and in vivo development of B. cinerea, in a dose-dependent manner.Following results obtained with the in vitro-plantlets model, we optimized the protocol of bacterization of grapevine fruiting cuttings by the PsJN strain, and evaluate the impact of the bacteria on to induce the resistance of inflorescences to B. cinerea. Our results provide new insights for the use of PsJN and suggest its commercial use as a biological control agent.

Les systèmes à deux composants jouent un rôle prépondérant dans l'adaptation des bactéries à leur environnement. L'objectif de ce travail de thèse était d'identifier et de caractériser des systèmes à deux composants chez la bactérie phytostimulatrice Azospirillum nécessaires à l'adaptation à la rhizosphère de sa plante-hôte. L'analyse de la distribution génomique des gènes appartenant à la famille des systèmes à deux composants dans les génomes d'Azospirillum disponibles a révélé l'existence d'un grand nombre de gènes codant des hisitidine kinases hybrides, et une analyse plus approfondie a montré une organisation multidomaines complexe de cette famille de protéines. Afin de comprendre leur rôle chez Azospirillum, nous avons, dans un premier temps, sélectionné et inactivé quatre gènes codant des histidine kinases hybrides présentant une architecture multidomaines complexe. A l'aide d'une approche multidisciplinaire combinant génétique, biochimie et phylogénie, nous avons mis en évidence pour la première fois chez Azospirillum, un système atypique à trois-composants nommé PreSKR contrôlant un grand nombre de processus impliqués dans la survie et la colonisation de la rhizosphère, qui agirait en modulant le taux intracellulaire de c-di-GMP. Dans un second temps, nous nous sommes focalisés sur une histidine kinase hybride exprimée au contact de la plante hôte ; cette protéine, appelée RsiK, s'avère être impliquée dans la perception de surfaces et la régulation de la formation de biofilms. L'analyse du régulon par RNA-seq a révèlé que 78 gènes étaient contrôlés par ce système. La prévalence de la famille des histidine kinases hybrides chez Azospirillum couplée à l'approche fonctionnelle réalisée sur deux d'entre elle souligne l'importance des phosphorelais encore largement méconnus chez les bactéries rhizosphériques / Bacterial two-component systems play an important role in the ability of bacteria to adapt to various environments. The aim of this thesis was to identify and characterize two-component systems involved in the adaptation of the phytostimulatory bacteria Azospirillum to its host plant. Analysis of the genomic distribution of genes encoding two-component systems across Azospirillum available genomes revealed the existence of a high number of genes encoding hybrid histidine kinases, and further analyses highlighted a complex multi-domain organization of this family of proteins. In order to understand their role in Azospirillum, as a first step we selected and inactivated four genes encoding complex hybrid histidines kinases. Using a multidisciplinary approach which combines genetics, biochemistry and phylogeny, we brought to light for the first time in Azospirillum, an atypical three-component system named PreSKR which controls a wide variety of processes involved in survival and rhizosphere colonization likely by modulating c-di-GMP levels. As a second step, we focused on a gene encoding a hybrid histidine kinase named RsiK which is induced in contact with its host plant. RsiK is involved in surface sensing and biofilm formation regulation. Transcriptomic analysis of rsiK regulon by RNA-seq showed that 78 genes were under the control of this system. The prevalence of genes encoding hybrid histidine kinase family in Azospirillum, coupled with the functional characterization of two of them, highlight the importance of phosphorelays, still largely unrecognized in rhizospheric bacteria

L'agricoltura moderna sta affrontando sfide come la perdita di fertilità del suolo, la variabilità climatica e gli attacchi di agenti patogeni in continuo aumento. Le pratiche agricole si stanno evolvendo verso sistemi sostenibili e rispettosi dell'ambiente. L'uso di biostimolanti (PBS, plant biostimulant) è una soluzione innovativa per affrontare le sfide di un’agricoltura sostenibile che garantisce un assorbimento ottimale dei nutrienti, una resa delle colture e tolleranza agli stress abiotici. In particolare, tra i diversi tipi di biostimolanti presenti sul mercato, i rizobatteri, classificati come Plant Growth Promoting Rhizobacteria (PGPR), offrono un nuovo approccio per promuovere la crescita delle piante, la mitigazione degli stress e l’aumento della resa colturale. Pertanto i PGPR sono considerati come una sorta di "probiotici" vegetali, poiché contribuiscono in modo efficiente alla nutrizione e all'immunità delle piante. L'obiettivo principale di questa tesi è isolare e identificare batteri presenti nella rizosfera di pomodoro (Solanum lycopersicum L.) che mostrano proprietà PBS, nonché valutare i meccanismi coinvolti nell'azione di promozione della crescita delle piante (Capitolo 2) e la genetica alla base di questi meccanismi (Capitolo 3 e 4). Infatti, una profonda comprensione dei meccanismi d’azione dei PGPR potrebbe colmare la mancanza di coerenza del dato di efficacia tra gli studi di laboratorio e gli studi in campo e stimolare la ricerca per la produzione e la commercializzazione di nuovi prodotti biostimolanti microbici. / Modern agriculture faces challenges such as loss of soil fertility, fluctuating climatic factors and increasing pathogen and pest attacks. Agricultural practices have been evolving towards organic, sustainable and environmentally friendly systems. The use of natural plant biostimulants (PBS) is an innovative solution to address the challenges in sustainable agriculture, to ensure optimal nutrient uptake, crop yield, quality and tolerance to abiotic stress. In particular, among different types of biostimulants present on the market, plant growth promoting rhizobacteria (PGPR) offer a novel approach for promoting plant growth, mitigate stress and increase crop yield. Hence, PGPR inoculants are now considered as a kind of plant ‘probiotics’, since they efficiently contribute to plant nutrition and immunity. The main goal of this thesis was to isolate and identify bacteria symbionts of tomato (Solanum lycopersicum L.) rhizosphere, which showed PBS properties and evaluate mechanism involved in the action of PGPR (Chapter 2), underlying genetics and physiological pathways (Chapter 3 and 4). Indeed, a deeply understanding of the mechanisms of plant growth promotion, could fulfill the lack of consistency between lab, greenhouse and field studies, and support commercialization of novel plant biostimulant products.

O uso de fertilizantes minerais nas culturas, inclusive no milho, é uma prática agrícola que provoca danos ambientais e prejuízos econômicos. Uma alternativa promissora, visando melhorar a produtividade e reduzir o uso de fertilizantes, é a utilização de microrganismos benéficos associados às plantas, particularmente as rizobactérias promotoras de crescimento. Essas bactérias vivem na rizosfera e são capazes de colonizar diversos tecidos vegetais, beneficiando o desenvolvimento das plantas através de mecanismos de promoção de crescimento. Na busca por alternativas sustentáveis e mais rentáveis, o presente trabalho teve como objetivo isolar, caracterizar, selecionar e monitorar rizobactérias associadas ao guaranazeiro da Amazônia que possuíssem características promotoras de crescimento vegetal para serem usadas como inoculantes em sementes de milho. Amostras de solo rizosférico de cinco plantas de guaranazeiros foram coletadas e foi realizado o isolamento das rizobactérias. A caracterização molecular foi realizada através do sequenciamento do gene 16S rDNA para análise da diversidade microbiana e identificação das linhagens. Avaliou-se a capacidade das linhagens de produzir ácido indol acético (AIA), fixar nitrogênio atmosférico, solubilizar fosfato inorgânico e de produzir sideróforos. A análise da diversidade microbiana indicou semelhança entre a comunidade bacteriana isolada da rizosfera do guaranazeiro e a do milho encontrada na literatura. Foi observada predominância do filo Proteobacteria, sendo em sua maioria representado pelo gênero Burkholderia. Do total das 101 linhagens obtidas, 89% foram capazes de produzir AIA, 23% fixaram nitrogênio atmosférico, 43% solubilizaram fosfato inorgânico e 24% produziram sideróforos. Cinco linhagens foram selecionadas para o ensaio de promoção de crescimento de milho em casa de vegetação, essas foram identificadas pelo sequenciamento completo do gene 16S rDNA e compuseram os tratamentos como segue: RZ2MS9 - Bacillus sp. (T1), RZ2MS16 - Burkholderia ambifaria (T2) e consórcio (T3) de 5 linhagens (RZ1MS6 - Burkholderia vietnamiensis, RZ1MS11 - Burkholderia sp., RZ2MS9 - Bacillus sp., RZ2MS16 - Burkholderia ambifaria e RZ4MS18 - Delftia acidovorans). As análises estatísticas comprovaram que as linhagens RZ2MS9 (Bacillus sp.) e RZ2MS16 (Burkholderia ambifaria) foram eficientes como promotoras de crescimento em milho, aumentando a altura cerca de 39 e 33%, respectivamente, em relação ao controle, o peso seco da parte aérea cerca de 236 e 114% e do sistema radicular cerca de 248 e 136%, respectivamente, comparado ao controle não inoculado. A linhagem RZ2MS9 (Bacillus sp.) aumentou o conteúdo de Ca nas plantas inoculadas. Para o monitoramento da colonização da bactéria na planta, a linhagem RZ2MS16 (Burkholderia ambifaria) foi transformada com o plasmídio pCM88 e passou a expressar a proteína GFP, sendo possível observar, por microscopia óptica de fluorescência, que, 12 dias após a inoculação na planta, a bactéria encontra-se concentrada no cilindro central da raiz da mesma de onde pode se inserir em algum vaso condutor e colonizar a planta sistematicamente, o que demonstra que a mesma se comporta como endofítica da planta de milho. Assim, fica evidente a importância da exploração de plantas de clima tropical, como o guaranazeiro, como reservatórios de bactérias com enorme potencial biotecnológico. As bactérias estudadas nesse trabalho tem grande potencial para serem utilizadas futuramente como inoculantes. / The use of mineral fertilizers on agricultural crops, including maize, is a practice that causes environmental damage and economical losses. A promising alternative, to improve productivity and reduce fertilizer use is the use of benefical microrganisms associated with plants, particulary the growth-promoting rhizobacteria. These bacteria live in the rhizosphere and are capable of colonizing different plant tissues, benefiting plant growth through mechanisms of growth promotion. In the search for sustainable and more profitable alternative, this study aimed to isolate, characterize, monitor and select rhizobacteria associated with Amazonian guarana that possessed characteristics of plant growth promoters for use as inoculants in maize seeds. Rhizosphere soil samples from five guarana plants were collected and the isolation of rhizobacteria was performed. Molecular characterization was performed by sequencing the 16S rDNA for analysis of microbial diversity and identification of strains. It was evaluated the ability of strains to produce indole acetic acid (IAA), fix atmospheric nitrogen, solubilize inorganic phosphate and produce siderophores. The analysis of microbial diversity indicated similarity between the bacterial community isolated from the rhizosphere of guarana and that found in the literature to maize. It was observed predominance of Proteobacteria phylum, being mostly represented by the genus Burkholderia. Of the total 101 strains obtained, 89% were able to produce IAA, 23% fixed atmospheric nitrogen, 43% solubilized inorganic phosphate and 24% produced siderophores. Five strains were selected for testing growth promotion of maize under greenhouse conditions; these were identified by complete sequencing of the 16S rDNA and compose the treatments as follows: RZ2MS9 - Bacillus sp. (T1), RZ2MS16 - Burkholderia ambifaria (T2) and consortium (T3) of 5 strains (RZ1MS6 - Burkholderia vietnamiensis, RZ1MS11 - Burkholderia sp., RZ2MS9 - Bacillus sp., RZ2MS16 - Burkholderia ambifaria and RZ4MS18 - Delftia acidovorans). Statistical analyzes showed that the strains RZ2MS9 (Bacillus sp.) and RZ2MS16 (Burkholderia ambifaria) were effective as growth promoters in maize, increasing the height about 39 and 33%, respectively, compared to control, shoot dry weight about 236 and 114% and root system about 248 and 136%, respectively, compared to uninoculated control. The strain RZ2MS9 (Bacillus sp.) increased Ca content in inoculated plants. For monitoring of colonization of the bacteria in the plant, the strain RZ2MS16 (Burkholderia ambifaria) was transformed with the plasmid pCM88 and passed to express GFP, being possible to observe by fluorescence microscopy that, 12 days after inoculation on the plant, the bacteria is concentrated in the root central cylinder where the same can be inserted into a vessel conductor and consistently colonize the plant, proving the endophytic life style of this strain during maize interaction. Thus, it is clear the importance of tropical plants, like guarana, as reservoirs of bacteria with great biotechnological potential. The evaluated bacteria accessed in this work have great potential to be used in future as inoculants.

Para alimentar a população mundial crescente é necessário um aumento sustentável na produtividade agrícola. Nesse sentido, Rizobactérias Promotoras de Crescimento de Plantas (RPCPs) têm sido continuamente buscadas para formulações inoculantes por sua capacidade de incremento na produção vegetal aliado ao seu potencial de redução e/ou substituição do uso de fertilizantes minerais, insumos que causam grandes impactos ambientais, na saúde humana e econômicos. A RPCP Bacillus sp. RZ2MS9, um representante da biodiversidade amazônica brasileira, é uma forte candidata a bionoculante por seu efeito benéfico, previamente descrito, em uma ampla gama de culturas, incluindo milho e soja. Essas duas culturas representam mais de 80% da área cultivada com grãos no Brasil, de forma que incrementos relativamente modestos de crescimento e produtividade poderiam gerar ganhos significativos. Membros do gênero Bacillus apresentam vantagem em formulações inoculantes, principalmente devido a sua capacidade de formação de esporos resistentes ao calor e dissecação. Seus modos de ação são diversos, tornando o entendimento da sua interação com plantas bastante desafiador. Bacillus sp. RZ2MS9 apresentou, dentre os mecanismos envolvidos na promoção de crescimento vegetal, a produção de Ácido Indol Acético (AIA) e sideróforos, solubilização de fosfato e fixação biológica de nitrogênio, in vitro. No presente trabalho, foi buscado um entendimento detalhado dos mecanismos de ação dessa rizobactéria, explorando desde seu genoma até seu desempenho em condições de campo. O draft genômico (genoma parcial) bacteriano foi obtido utilizando a tecnologia de sequenciamento Illumina, o qual possibilitou a detecção de genes envolvidos nos mecanismos potencialmente relacionados ao efeito benéfico dessa bactéria, que vão desde sua formação de esporos, atração por exsudatos radiculares, motilidade e competição na rizosfera até mecanismos de solubilização de fosfato, produção de sideróforos, entre outros. As informações obtidas permitem uma exploração genética desses mecanismos, fornecendo uma oportunidade de maximizar essa interação e, futuramente, favorecer os benefícios em campo. Adicionalmente, foi demonstrado o potencial de quimiotaxia (atração) de RZ2MS9 em direção a raízes de milho. Um estudo filogenético dessa RPCP, utilizando um método de tipagem com o gene pycA (piruvato carboxilase), mostrou que o Bacillus sp. RZ2MS9 apresentou-se distante do clado altamente monomórfico de B. anthracis, patógeno humano, e se afiliou a um grupo composto por linhagens de B. thuringiensis (Bt) comercializadas como produtos biopesticidas há mais de 60 anos, o que sugere a potencial possibilidade de seu uso seguro no campo. Sabe-se que a maioria, se não todas, atividades fisiológicas das plantas é regulada por fitormônios como a auxina AIA, os quais podem ser sintetizados também por RPCPs. Com mais detalhamento, os genes envolvidos nas vias biossintéticas desse fitormônio foram detectados no draft genômico de RZ2MS9, indicando que sua produção ocorre através da via IPA (Indol-3-Piruvato). Além disso, plantas de tomate anão Micro-Tom (MT) e seu mutante Δdgt, defectivo na sensibilidade a auxinas, foram utilizadas para caracterizar especificamente o efeito do AIA produzido por Bacillus sp. RZ2MS9 na promoção de crescimento vegetal. A aplicação de RZ2MS9 causou inibição no crescimento de raízes primárias, aumento no comprimento de raízes laterais e na área superficial total de raízes de plantas MT, efeitos característicos daqueles proporcionados por auxinas. Esse incremento radicular refletiu, ainda, em aumento da biomassa da parte aérea de plantas MT. Os mesmos efeitos não foram observados em plantas Δdgt, insensíveis a auxinas, indicando que a elicitação de promoção de crescimento em MT por RZ2MS9 ocorre por meio desses fitormônios. Finalmente, foi demonstrado o efeito sobre o desenvolvimento e produtividade de milho e soja da aplicação de Bacillus sp. RZ2MS9 em condições de campo, sendo comparado com o desempenho de bioinoculantes comerciais. No milho, o efeito da inoculação bacteriana foi, ainda, associado à adubação nitrogenada para verificar a possibilidade de redução desses insumos. Bacillus sp. RZ2MS9 apresentou efeitos significativos sobre o desenvolvimento tanto da soja (comparáveis aos efeitos de rizóbios) quanto do milho, os quais, porém, não refletiram em aumento significativo de produtividade em ambas as culturas. No entanto, o potencial dessa rizobactéria é bastante claro pois, com um custo de produção inferior a R$1,00 por hectare, sua inoculação causou incremento de 16 sacas de milho por hectare com redução de 30% na adubação nitrogenada, assim como um incremento de 11 sacas de soja por hectare, ambos comparados ao controle não inoculado. Os resultados apresentados no presente trabalho vão, portanto, de encontro à grande expectativa na obtenção de linhagens microbianas promissoras visando sistemas agrícolas mais sustentáveis. / To feed the growing global population, a sustainable increase of agricultural production and crop yield is required. In this sense, Plant Growth Promoting Rhizobacteria (PGPR) have been continuously sought to inoculant formulation due to their capacity to increase plant yield along with their potential to reduce and/or replace the use of mineral fertilizers, inputs that cause serious impacts on environment, human health and economy. The PGPR Bacillus sp. RZ2MS9, a representative of the Brazilian Amazonian biodiversity, is a great candidate to bioinoculant because of its beneficial effect on a broad range of crops, including maize and soybean. These two crops represent more than 80% of the area planted with grains in Brazil, so relatively modest growth and yield increases could generate significant gains. Bacillus spp. have advantage in inoculant formulations, mainly due to their ability to form heat- and dissecation-resistant spores. Their modes of action are diverse, making the understanding of its interaction with plants quite challenging. Bacillus sp. RZ2MS9 displays, between the mechanisms involved in plant growth, Indole Acetic Acid (IAA) and siderophore production, phosphate solubilization and biological nitrogen fixation, in vitro. In the present work, we seek a detailed understanding of this rhizobacterium mechanisms of action, exploring from its genome to its performance in field conditions. The bacterial draft genome was obtained using Illumina sequencing technology, making possible the detection of genes involved in mechanisms potentially related to the beneficial effect of this bacterium, and range from its spore formation, attraction by root exudates, motility and competition in the rhizosphere to mechanisms of phosphate solubilization, siderophore production, among others. The information obtained allow a genetic exploration of these mechanisms, providing an opportunity to maximize this interaction and, in the future, favor benefits in field. Additionally, it was demonstrated the chemotaxis (attraction) potential of RZ2MS9 towards maize roots. A phylogenetic study of this PGPR, using a typing method with the pycA (pyruvate carboxylase) gene, showed that Bacillus sp. RZ2MS9 was distant from the highly monomorphic clade of B. anthracis, a human pathogen, and affiliated with B. thuringiensis (Bt) strains marketed as biopesticides for more than 60 years, suggesting the potential possibility of its safe use in the field. It is known that most, if not all, physiological activities of plants are regulated by phytormones such as the auxin IAA, which can also be synthesized by PGPRs. With more detail, genes involved in biosynthetic pathways of this phytormone were detected in the RZ2MS9 draft genome, indicating that its production occurs via the IPA (indole-3-pyruvate) pathway. In addition, plants of the dwarf tomato Micro-Tom (MT) and its mutant Δdgt, impaired in auxin sensibility, were used to specifically characterize the effects of IAA produced by Bacillus sp. RZ2MS9 in the plant growth promotion. The inoculation of RZ2MS9 caused inhibition in the primary roots growth, increase in lateral roots length and in roots total surface area of MT plants, characteristic effects of those provided by auxins. This root growth also reflected in an increase of MT plants shoot biomass. The same effects were not observed in Δdgt plants, insensitive to auxins, suggesting that the elicitation of growth promotion in MT by RZ2MS9 occurs through these phytormones. Finally, we demonstrated the effect of inoculation with Bacillus sp. RZ2MS9 on maize and soybean development and productivity under field conditions, being compared with the performance of commercial bioinoculants. In maize, the effect of bacterial inoculation was also associated with nitrogen fertilization to verify the possibility of reducing these inputs. Bacillus sp. RZ2MS9 showed significant effects on the development of both soybean (comparable to the effects of rhizobia) and maize, which, however, did not reflect a significant increase in productivity in both crops. However, the potential of this rhizobacterium is very clear because, with a cost of production of less than R$1.00 per hectare, its inoculation caused an increase of 16 sacks of maize per hectare with a 30% reduction in nitrogen fertilization, as well as an increase of 11 sacks of soybean per hectare, both compared to uninoculated control. The results presented in this study meet the great expectation of obtaining promising microbial strains aiming at more sustainable agricultural systems.

Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-01T14:56:39Z No. of bitstreams: 1 DIS_MAILA_PACHECO_DIAS_COMPLETO.pdf: 1237790 bytes, checksum: 5f0ce43f31e6bb7c9d9d5d922daa8668 (MD5) / Made available in DSpace on 2017-06-01T14:56:39Z (GMT). No. of bitstreams: 1 DIS_MAILA_PACHECO_DIAS_COMPLETO.pdf: 1237790 bytes, checksum: 5f0ce43f31e6bb7c9d9d5d922daa8668 (MD5) Previous issue date: 2016-03-29 / Solanum lycopersicum L., tomato, is an herbaceous plant belonging to the Solanaceae family. Its fruits are consumed worldwide, reaching the world production of 160 million tons per year. In Brazil, it is the second vegetable in economic importance. However, the tomato is attacked by numerous diseases that lead to loss of production and /or poor quality of the fruit, such as the hollow stem, the disease caused by Pectobacterium spp. Due to the large number of diseases caused by plant pathogens, the tomato is a culture in which a significant amount of agrochemicals is used. Therefore, this species is among the vegetables with the greatest amount of residual pesticides. Under these circumstances, it is essential to develop sustainable plant defense techniques in order to reduce the use of agrochemicals. Then, changes in plant metabolism related to defense must be understood so that new strategies and new products can be developed. Disease control using soil microorganisms has been considered as an alternative, since the rhizobacteria, in addition to promoting plant growth, may induce resistance as the result of activation of the natural plant defenses. These, calls plant growth promoting rhizobacteria (PGPR), has been explored for their biofertilizers, biopesticides and phyto-stimulating abilities. The aims of this study were to characterize biochemically the Streptomyces spp. isolates, to determine the antagonism against Pectobacterium carotovorum subsp. brasiliensis (Pcb), to determine the ability of Streptomyces spp. on promoting growth of tomato plants and to evaluate the modulation of the defense-related metabolism of tomato plants when treated with Streptomyces spp. The possible influence of Streptomyces spp. on reducing soft rot disease in tomato plants was also evaluated. Biochemical characterization was evaluated through the ability of Streptomyces spp. on producing siderophores, solubilizing phosphate, and activity of amylase and lipase, as well as volatile organic compounds (VOC) production. Antagonism of Streptomyces spp. against Pcb was determined by dual-culture method and I-plate for VOC effect analysis. Plant growth promotion was evaluated through VOC emission and by direct interaction with Streptomyces spp. isolates (PM1, PM3, PM4, PM5, PM6 e PM9). Enzymes related to plant defense were colorimetric analyzed in plants treated with isolates of Streptomyces spp. Evaluation of soft rot disease was performed on plants treated with Streptomyces spp. and challenged with Pcb through the area under the disease progression curve (AUDPC) and plant mortality. Isolates of Streptomyces spp. displayed characteristics of PGPR and 32 volatile compounds were identified from the different isolates. PM3 was the isolate showing efficient antagonism against Pcb. Most of the isolates promoted increase of root and shoot length of tomato plants by VOC although PM5 was efficient on promoting growth by direct interaction with Streptomyces spp. Treatment with Streptomyces spp. modulated the activity of defense-related enzymes and decrease incidence of soft rot disease. / O tomateiro (Solanum lycopersicum L.) ? uma planta herb?cea pertencente ? fam?lia Solanaceae. Seus frutos s?o consumidos mundialmente, chegando ? produ??o mundial de 160 milh?es de toneladas por ano. No Brasil, ? a segunda hortali?a em import?ncia econ?mica. Contudo, o tomateiro ? alvo de in?meras doen?as que levam ? perda de produ??o e/ou m? qualidade dos frutos, como por exemplo, a doen?a Talo oco causada por Pectobacterium spp. Devido ao elevado n?mero de doen?as causadas por fitopat?genos, o tomateiro ? uma cultura onde se utiliza uma quantidade expressiva de agroqu?micos, estando entre as hortali?as que apresentam maior quantidade de agrot?xicos residuais. Por isso, torna-se imprescind?vel o desenvolvimento de t?cnicas sustent?veis de defesa para o vegetal, a fim de reduzir o uso destes compostos. Para este fim, ? fundamental compreender as altera??es no metabolismo vegetal relacionado ? defesa, para que novas estrat?gias e novos produtos agr?colas possam ser desenvolvidos. O controle de doen?as utilizando microrganismos de solo tem sido considerado uma alternativa, uma vez que as rizobact?rias, al?m de promoverem o crescimento vegetal, podem induzir ? resist?ncia como consequ?ncia da ativa??o da defesa vegetal. Estas, chamadas rizobact?rias promotoras de crescimento vegetal (PGPR), v?m sendo exploradas quanto ? capacidade biofertilizante, fito-estimuladora e biopesticida. Os objetivos deste estudo foram caracterizar seis isolados de Streptomyces spp. como PGPR, determinar o antagonismo contra Pectobacterium carotovorum subsp. brasiliensis (Pcb), determinar a capacidade de isolados de Streptomyces spp. na promo??o do crescimento de plantas de tomate e avaliar a modula??o do metabolismo relacionado ? defesa das plantas de tomate quando tratadas com Streptomyces spp. A poss?vel influ?ncia de Streptomyces spp. na redu??o da doen?a Talo oco em plantas de tomate tamb?m foi avaliada. A caracteriza??o bioqu?mica de isolados de Streptomyces spp. foi realizada por meio da capacidade de produzir sider?foros, solubilizar fosfato, e da atividade de amilase e lipase, bem como a produ??o de compostos org?nicos vol?teis. O antagonismo de Streptomyces spp. contra Pcb foi determinado pelo m?todo de dupla cultura e placa com barreira para an?lise do efeito de compostos org?nicos vol?teis (VOC). A promo??o do crescimento das plantas foi avaliada por meio de emiss?o de VOC e pela intera??o direta com os isolados de Streptomyces spp. (PM1, PM3, PM4, PM5, PM6 e PM9). Enzimas relacionadas ? resposta de defesa foram analisadas colorimetricamente em plantas tratadas com isolados de Streptomyces spp. A avalia??o da doen?a Talo oco foi realizada em plantas tratadas com Streptomyces spp. e desafiadas com Pcb atrav?s da ?rea sob a curva de progresso da doen?a e da mortalidade das plantas em 24 dias. Os isolados de Streptomyces spp. mostraram caracter?sticas de PGPR e 32 compostos vol?teis foram identificados como produtos dos diferentes isolados. PM3 foi o isolado mais eficiente quanto ao antagonismo contra Pcb. A maioria dos isolados promoveu o aumento do comprimento de raiz e da parte a?rea do tomateiro por VOC, embora PM5 tenha sido tamb?m eficiente na promo??o do crescimento atrav?s da intera??o direta com Streptomyces spp. O tratamento com Streptomyces spp. modulou a atividade de enzimas relacionadas ? defesa e diminuiu a incid?ncia da doen?a Talo oco.