Submitted by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T17:30:00Z No. of bitstreams: 1 DIS_JULIANA_LOPES_HORSTMANN_COMPLETO.pdf: 1711527 bytes, checksum: 7681ac709a014d574046b6207b8a9728 (MD5) / Approved for entry into archive by Caroline Xavier (caroline.xavier@pucrs.br) on 2017-06-30T17:30:08Z (GMT) No. of bitstreams: 1 DIS_JULIANA_LOPES_HORSTMANN_COMPLETO.pdf: 1711527 bytes, checksum: 7681ac709a014d574046b6207b8a9728 (MD5) / Made available in DSpace on 2017-06-30T17:30:17Z (GMT). No. of bitstreams: 1 DIS_JULIANA_LOPES_HORSTMANN_COMPLETO.pdf: 1711527 bytes, checksum: 7681ac709a014d574046b6207b8a9728 (MD5) Previous issue date: 2017-03-31 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The plant growth promoting rhizobacteria (PGPR) can increase agricultural productivity by promoting growth through production of plant hormones, facilitating the uptake of nutrients and chemicals on the soil, as well as inhibiting plant stress factors. Streptomyces spp. (Stm) are bacteria with great biotechnological potential, because in addition to its growth promotion and plant defense induction, they are also known as great producers of secondary metabolites, including antibiotics and phenazines. Soybean [Glycine max (L) Merrill] is one of the main legume crop grown around the world and Brazil is the second largest producer. Its production is affected by many diseases, among which bacterial pustule caused by the pathogen Xanthomonas axonopodis pv. glycines (Xag). The objective of this project was to evaluate isolates of Streptomyces spp. obtained from the rhizosphere of Fabaceae plants regarding characteristics of PGPR, as well as the modulation capacity of soybean defenses in response to the phytopathogen Xag. Eleven isolates of Streptomyces spp. were screened for PGPR traits by siderophores production, 1-aminocyclopropane-1-carboxylate (ACC) deaminase, indole-3-acetic acid (IAA) and phenazines. For a taxonomic identification and growth evaluation of soybean plants, three isolates were selected for their biochemical characteristics. The growth promoting assay was performed in greenhouse using bacterized seeds with the selected isolate and sterile distilled water was use for the control. Length, fresh and dry weight from shoot and root at 15, 30 and 45 days of cultivation were the evaluated parameters. For evaluation of the induction capacity of defense mechanisms of soybean plants, the isolate that obtained the best performance in the growth promotion test was selected. Seeds of soybeans, from sensitive cultivars and resistant to Xag, were bacterized with the selected Stm isolate and grown under greenhouse conditions. The plants were challenged with Xag 15 days after emergence. The treatments consisted of (a) plants treated with sterile distilled water (absolute control); (b) plants bacterized with Stm CLV45 (Stm45); (c) water-treated and Xag challenged plants; and (d) plants bacterized with StmCLV45 and challenged with Xag (Stm45+Xag). The enzymatic responses related to the defense pathways were evaluated biochemically, by analyzing the activity of phenylalanine ammonia lyase (PAL) and by the production of phenolic compounds at times 0, 24, 48, 72 and 144 hours post infection (hpi) of Xag. The expression of the genes related to the defense in Xag challenged soybean plants was determined by the relative expression of the genes PAL, JAZ, ERF5 and PR1 by qPCR at times 0, 12, 24 and 48 hpi. The results of the biochemical analysis indicated the isolates CLV42, CLV44 and CLV46 as the major producers of siderophores and CLV41, CLV45 and CLV46 isolates with higher ACC deaminase activity. All isolates were able to produce IAA, highlighting the isolate CLV45, which produced 398.53 ?g AIA g-1 cell. Phenazine pyocyanin (PYO) was also detected in all isolates, but the same did not occur for the 1-carboxylic acid phenazine (PCA), only produced by CLV41, CLV43 and CLV45. The isolates CLV42, CLV44 and CLV45 were selected for their PGPR characteristics for the growth promotion trial of greenhouse soybean plants and taxonomically characterized as species of the genus Streptomyces. None of the isolates evaluated in the trial caused a growth deficit in soybean plants. The CLV45 isolate significantly promoted the growth of soybean shoots in 36.63%, corroborated by the highest dry mass, 17.97%, in relation to the control group, being selected for soybean defense pathways induction. Expression of PAL gene was moderately enhanced in susceptible Stm45+Xag plants at 12 hpi, followed by increase of PAL enzyme activity from 48 to 144 hpi, although corresponding accumulation of phenolic compounds was not recorded. In the resistant cultivar, the highlighted expression of PAL in Stm45+Xag plants resulted in high activity of this enzyme. Enhanced expression of ERF5 and decrease on JAZ gene at 12 hpi in Stm45+Xag plants from both cultivars suggested that ET and JA play a concert role on induced systemic defense by Streptomyces sp. CLV45 against Xag in soybean. / As rizobact?rias promotoras de crescimento de plantas (PGPR) podem aumentar a produtividade agr?cola, atuando atrav?s da promo??o de crescimento vegetal por meio de fitorm?nios reguladores de crescimento, facilitando a capta??o de nutrientes e de compostos qu?micos no solo, bem como inibindo fatores de estresse vegetal. Bact?rias do g?nero Streptomyces spp. (Stm) apresentam grande potencial biotecnol?gico, pois al?m de promoverem o crescimento e a indu??o de defesa vegetal, tamb?m s?o conhecidas pela grande produ??o de metab?litos secund?rios, incluindo antibi?ticos e fenazinas. A soja [Glycine max (L.) Merrill] ? uma das principais leguminosas cultivadas no mundo, sendo o Brasil o segundo maior produtor. Sua produ??o ? afetada por in?meras doen?as, como a P?stula bacteriana causada pelo fitopat?geno Xanthomonas axonopodis pv. glycines (Xag). O objetivo deste trabalho foi avaliar 11 isolados de Streptomyces spp. oriundos da rizosfera de plantas de Fabaceae quanto ?s caracter?sticas de PGPR, bem como, ? capacidade de modula??o das vias de defesa de plantas de soja em resposta ? fitobact?ria patog?nica Xag. Os isolados foram avaliados quanto ?s caracter?sticas de PGPR pela produ??o de sider?foros, de ?cido indolac?tico (AIA), da enzima 1-aminociclopropano-1-?cido carbox?lico (ACC) desaminase e de fenazinas. Para a identifica??o taxon?mica e a avalia??o da promo??o de crescimento de plantas de soja foram selecionados tr?s isolados com caracter?sticas de PGPR. O ensaio de promo??o de crescimento ocorreu em casa de vegeta??o por meio da microbioliza??o das sementes pelos isolados selecionados e o controle com ?gua destilada est?ril. Os par?metros avaliados foram: comprimento, massa fresca e seca, de parte a?rea e raiz, aos 15, 30 e 45 dias de cultivo. Para a avalia??o da capacidade de indu??o dos mecanismos de defesa de plantas de soja, foi selecionado o isolado que obteve o melhor desempenho no ensaio de promo??o de crescimento. Sementes de soja, de cultivar sens?vel e resistente ? Xag, foram microbiolizadas com o isolado de Stm selecionado e cultivadas em casa de vegeta??o. As plantas obtidas foram desafiadas com Xag, 15 dias ap?s a sua emerg?ncia. Os tratamentos consistiram de (a) sementes tratadas com ?gua destilada est?ril (controle absoluto); (b) sementes microbiolizadas com StmCLV45 (Stm45); (c) sementes tratadas com ?gua destilada est?ril e plantas desafiadas com Xag (Xag); e (d) sementes microbiolizadas StmCLV45 e plantas desafiadas com Xag (Stm45+Xag). As respostas enzim?ticas relacionadas ?s vias de defesa foram avaliadas bioquimicamente, pela an?lise da atividade da fenilalanina am?nia liase (PAL) e pela produ??o dos compostos fen?licos, nos tempos 0, 24, 48, 72 e 144 horas p?s inocula??o (hpi) da Xag. A express?o dos genes relacionados ? defesa das plantas de soja desafiadas com Xag foi determinada pela express?o relativa de JAZ, ERF5, PAL e PR1 por qPCR, nos tempos 0, 12, 24 e 48 hpi. Os resultados da an?lise bioqu?mica indicaram os isolados CLV42, CLV44 e CLV46 como maiores produtores de sider?foros e os isolados CLV41, CLV45 e CLV46 com maior atividade de ACC desaminase. Todos os isolados foram capazes de produzir AIA, com destaque para o isolado CLV45, que produziu 398,53 ?g AIA g-1 de c?lulas. A fenazina piocianina (PYO) tamb?m foi detectada em todos os isolados, entretanto o mesmo n?o ocorreu para a fenazina 1-?cido carbox?lico (PCA), somente produzida por CLV41, CLV43 e CLV45. Os isolados CLV42, CLV44 e CLV45 foram selecionados por suas caracter?sticas de PGPR para o ensaio de promo??o do crescimento de plantas de soja em casa de vegeta??o e caracterizados taxonomicamente como esp?cies do g?nero Streptomyces. Nenhum dos isolados avaliados no ensaio causou d?ficit de crescimento em plantas de soja. O isolado CLV45 promoveu significativamente o crescimento de parte a?rea de plantas soja, em 36,63%, corroborado pela maior massa seca, 17,97%, em rela??o ao grupo controle, sendo selecionado para avalia??o nas vias de defesa da soja. A express?o do gene PAL foi moderadamente aumentada em plantas suscet?veis Stm45+Xag em 12 hpi, seguido por aumento da atividade da enzima PAL de 48 a 144 hpi, embora o ac?mulo correspondente de compostos fen?licos n?o tenha sido registrado. Na cultivar resistente, a express?o de PAL em plantas Stm45+Xag resultou em alta atividade desta enzima. A express?o aumentada de ERF5 e a diminui??o de express?o do gene JAZ em 12 hpi em plantas Stm45+Xag de ambas as cultivares sugeriram que etileno e ?cido jasm?nico desempenharam fun??o na defesa sist?mica induzida por Streptomyces sp. CLV45 contra Xag em plantas de soja.

Résumé Certaines bactéries de la rhizosphère (PGPR, rhizobactéries promotrices de la croissance des plantes) exercent un effet bénéfique sur la croissance des plantes en stimulant des mécanismes de défense inductibles chez lhôte, rendant celle-ci moins susceptible vis-à-vis dune infection ultérieure par un agent pathogène. Ce phénomène appelé résistance systémique induite (ISR) a été mis en évidence chez plusieurs plantes pour lutter contre une gamme relativement large de pathogènes fongiques, bactériens ou viraux. Cependant, les bases moléculaires des mécanismes de défense proprement dits stimulés lors de lISR restent assez méconnues malgré les nombreux travaux réalisés cette dernière décennie. Dans ce contexte, des souches de PGPR (Bacillus subtilis et Pseudomonas putida) capables de protéger certaines plantes via linduction de lISR sont étudiées depuis plusieurs années au laboratoire. Dans le cadre de cette thèse de doctorat, nous avons étudié l´effet protecteur de ces bactéries dans deux pathosystèmes différents, tomate/Botrytis cinerea et concombre/Colletotrichum lagenarium. Nos résultats ont montré la capacité de P. putida BTP1 à induire l´ISR chez la tomate et le concombre sur base de la réduction des symptômes de maladie observée et sur base de la séparation spatiale des deux agents, bénéfique au niveau racinaire et pathogène au niveau foliaire. Cette résistance a été clairement associée avec la stimulation de la voie des oxylipines chez la tomate. Linduction de cette voie métabolique a dabord été mise en évidence biochimiquement par une augmentation des activités lipoxygénase et lipide hydroperoxydase dans les feuilles des plantes traitées avec Pseudomonas. De plus, au niveau moléculaire, les analyses par northern blot nous ont permis d´identifier un nouvel isoforme de gène Lox chez la tomate qui est exprimé différentiellement chez les plants traités par la bactérie. Ce gène dénommé LoxF montre une homologie de 82% avec un des cinq isoformes connus chez cette plante, LoxC. LoxF est essentiellement surexprimé dans les feuilles de tomates pré-inoculées avec BTP1 suite à l´infection par le pathogène. Il en est de même pour lactivité enzymatique correspondante de la lipoxygénase qui naugmente significativement chez les plants traités par rapport aux témoins quaprès infection par Botrytis. Ces résultats sous-entendent un phénomène de « priming » ou « mise en alerte » étroitement associé avec linduction de résistance systémique chez les plantes. La reconnaissance de la bactérie au niveau racinaire met en alerte la plante sans que des bouleversements majeurs au niveau métabolique ou génétique ne soient observés. Lhôte réagit alors plus fortement et plus rapidement pour mettre en uvre ses mécanismes de défense une fois le pathogène perçu. Par biotest sur TLC et analyses HPLC, CPG et LC-MS, nous avons mis en évidence laccumulation dune molécule dans les feuilles de tomate prétraitées avec BTP1. Cette molécule semble être de nature apolaire mais non phénolique et ne correspond pas aux phytoalexines connues de la tomate. Des études complémentaires doivent être réalisées pour lidentifier chimiquement mais sa cinétique daccumulation dans les tissus de la plante est étroitement associée à celle de la stimulation de la lipoxygénase. Elle pourrait donc dériver de cette voie métabolique. Des réactions de défense similaires ont été observées suite au traitement avec Bacillus subtilis chez la tomate et ces essais ont permis de mettre en évidence le rôle des lipopeptides produits en tant quéliciteurs impliqués dans linduction du phénomène de lISR par cette souche. Par contre, bien que les deux souches soient capables dinduire la résistance chez le concombre, aucune accumulation claire de phytoalexines ni de stimulation significative de la voie de la lipoxygénase nont pu y être associées chez cette plante. Globalement, nos résultats suggèrent que les voies métaboliques activées dans le cadre de lISR varient en fonction de lespèce végétale même si le microorganisme inducteur est identique. Abstract Some plant growth promoting rhizobacteria (PGPR) are able to stimulate inducible defence mechanisms that render the host plant less susceptible to a subsequent pathogen attack. This phenomenon called induced systemic resistance (ISR) can occur in several plant species against a wide range of bacterial, viral and fungal pathogens. Despite extensive work carried out this last decade, many aspects of the molecular basis underlying this rhizobacteria-mediated ISR remain unclear. In this context, Bacillus subtilis and Pseudomonas putida strains able to protect plants via induction of ISR have been studied for several years in our laboratory. In this thesis, we first aimed at evaluating the protective effect of these strains in different pathosystems and second at identifying the associated defence mechanisms. Our results showed the capacity of P. putida BTP1 to induce ISR in the tomato/Botrytis cinerea and cucumber/Colletotrichum lagenarium pathosystems on the basis of reduction of disease symptoms and on the basis of the spatial separation of the two agents, beneficial at the root level and pathogenic at the leaf level. This resistance was clearly associated the stimulation of the oxylipin pathway in tomato. The induction of this metabolic pathway was evidenced biochemically by an increase in the lipoxygenase and lipid hydroperoxydase activities in the leaves of plants treated with Pseudomonas compared to controls. Moreover, at the molecular level, northern blot analyses enabled us to identify a new isoforme of Lox gene in tomato which is expressed differentially in plants treated with the bacterium. This gene called LoxF shows a homology of 82% with one of the five isoformes known in this plant, LoxC. LoxF is mainly expressed in tomato leaves pre-inoculated with BTP1 after infection by pathogen. The same applies for the lipoxygenase enzyme activity which increases significantly in treated plants only after infection by Botrytis. These results imply a phenomenon of "priming" closely associated with the systemic resistance induction in plants. The recognition of the bacterium at the root level primes the plant but in most cases major changes at the metabolic or genetic level are not observed. The host then reacts more strongly and more quickly to express defence mechanisms once the pathogen is perceived. In addition, by combining the use of TLC-based biotests and analytical methods such as HPLC, CPG and LC-MS analyses, we highlighted the accumulation of a molecule in BTP1-pretreated tomato leaves. This molecule seems to be of a non-polar nature but not phenolic and does not correspond to any phytoalexin known in tomato. Complementary studies must be carried out to identify its structure but its kinetic of accumulation in the plant tissues is closely associated with the stimulation of the lipoxygenase enzyme. It could thus derive from this metabolic pathway. Similar defence reactions were observed in tomato following treatment with Bacillus subtilis and through these tests, we also highlighted the role of some lipopeptides produced by this strain as elicitors responsible for the induction of the ISR phenomenon. On the other hand, although the two strains are able to induce resistance in cucumber, no clear accumulation of phytoalexins nor of significant stimulation of the lipoxygenase pathway could be associated with disease reduction in this plant. All together, our results suggest that the metabolic pathways activated during ISR vary in function of the plant and pathogen species even if the inducing micro-organism is identical.

Soil salinity is a widespread problem that limits crop yield throughout the world. The accumulation of soluble salts in the soil can inhibit plant growth by increasing the osmotic potential of interstitial water, inducing ion toxicity and nutrient imbalances in plants. Over the last decade, considerable effort has been put into developing economical and effective methods to reclaim these damaged soils. Phytoremediation is a technique that uses plants to extract, contain, immobilize and degrade contaminants in soil. The most common process for salt bioremediation is phytoextraction which uses plants to accumulate salt in the shoots, which is then removed by harvesting the foliage. As developing significant plant biomass in saline soils is an issue, a group of free-living rhizobacteria, called plant growth promoting rhizobacteria (PGPR), can be applied to plant seeds to aid plant growth by alleviating salt stress. The principle objective of this research was to test the efficacy of PGPR in improving the growth of plants on salt-impacted soils through greenhouse and field studies. In this research, previously isolated PGPR strains of Pseudomonas putida. UW3, Pseudomonas putida UW4, and Pseudomonas corrugata CMH3 were applied to barley (Hordeum valgare C.V. AC ranger), oats (Avena sativa C.V. CDC baler), tall wheatgrass (Agropyron elongatum), and tall fescue (festuca arundinacea C.V. Inferno). PGPR effects on plant growth, membrane stability, and photosynthetic activity under salt stress were examined. Greenhouse studies showed that plants treated with PGPR resulted in an increase in plant biomass by up to 500% in salt-impacted soils. Electrolyte leakage assay showed that plants treated with PGPR resulted in 50% less electrolyte leakage from membranes. Several chlorophyll a fluorescence parameters, Fv/Fm, effective quantum yield, Fs, qP, and qN obtained from pulse amplitude modulation (PAM) fluorometry showed that PGPR-treated plants resulted in improvement in photosynthesis under salt stress. Field studies showed that PGPR promoted shoot dry biomass production by 27% to 230%. The NaCl accumulation in plant shoots increased by 7% to 98% with PGPR treatment. The averaged soil salinity level at the CMS and CMN site decreased by 20% and 60%, respectively, during the 2008 field season. However, there was no evidence of a decrease in soil salinity at the AL site. Based on the improvements of plant biomass production and NaCl uptake by PGPR observed in the 2008 field studies, the phytoremediation efficiency on salt-impacted sites is expected to increase by 30-60% with PGPR treatments. Based on the average data of 2007 and 2008 field season, the time required to remove 25% of NaCl of the top 50 cm soil at the CMS, CMN and AL site is estimated to be six, twelve, and sixteen years, respectively, with PGPR treatments. The remediation efficiency is expected to accelerate during the remediation process as the soil properties and soil salinity levels improve over time.

Soil salinity is a widespread problem that limits crop yield throughout the world. The accumulation of soluble salts in the soil can inhibit plant growth by increasing the osmotic potential of interstitial water, inducing ion toxicity and nutrient imbalances in plants. Over the last decade, considerable effort has been put into developing economical and effective methods to reclaim these damaged soils. Phytoremediation is a technique that uses plants to extract, contain, immobilize and degrade contaminants in soil. The most common process for salt bioremediation is phytoextraction which uses plants to accumulate salt in the shoots, which is then removed by harvesting the foliage. As developing significant plant biomass in saline soils is an issue, a group of free-living rhizobacteria, called plant growth promoting rhizobacteria (PGPR), can be applied to plant seeds to aid plant growth by alleviating salt stress. The principle objective of this research was to test the efficacy of PGPR in improving the growth of plants on salt-impacted soils through greenhouse and field studies. In this research, previously isolated PGPR strains of Pseudomonas putida. UW3, Pseudomonas putida UW4, and Pseudomonas corrugata CMH3 were applied to barley (Hordeum valgare C.V. AC ranger), oats (Avena sativa C.V. CDC baler), tall wheatgrass (Agropyron elongatum), and tall fescue (festuca arundinacea C.V. Inferno). PGPR effects on plant growth, membrane stability, and photosynthetic activity under salt stress were examined. Greenhouse studies showed that plants treated with PGPR resulted in an increase in plant biomass by up to 500% in salt-impacted soils. Electrolyte leakage assay showed that plants treated with PGPR resulted in 50% less electrolyte leakage from membranes. Several chlorophyll a fluorescence parameters, Fv/Fm, effective quantum yield, Fs, qP, and qN obtained from pulse amplitude modulation (PAM) fluorometry showed that PGPR-treated plants resulted in improvement in photosynthesis under salt stress. Field studies showed that PGPR promoted shoot dry biomass production by 27% to 230%. The NaCl accumulation in plant shoots increased by 7% to 98% with PGPR treatment. The averaged soil salinity level at the CMS and CMN site decreased by 20% and 60%, respectively, during the 2008 field season. However, there was no evidence of a decrease in soil salinity at the AL site. Based on the improvements of plant biomass production and NaCl uptake by PGPR observed in the 2008 field studies, the phytoremediation efficiency on salt-impacted sites is expected to increase by 30-60% with PGPR treatments. Based on the average data of 2007 and 2008 field season, the time required to remove 25% of NaCl of the top 50 cm soil at the CMS, CMN and AL site is estimated to be six, twelve, and sixteen years, respectively, with PGPR treatments. The remediation efficiency is expected to accelerate during the remediation process as the soil properties and soil salinity levels improve over time.

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

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.

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.

Salt stress is a severe environmental challenge in agriculture, limiting the quality and productivity of the crops around the globe. Plant growth promoting rhizobacteria (PGPR) is proposed as a friendly solution to overcome those challenges. The desert plant endophytic bacterium, Enterobacter sp. SA187 has shown plant growth promotion and salt stress tolerance beneficial effect on the model plant Arabidopsis thaliana in vitro as well as under the field conditions on different crops. SA187 has a distinguished morphology of yellow colonies (SA187Y) that could be due to carotenoid biosynthesis. However, the bacteria tend to lose the yellow color upon incubation with the plants and the colonies turn to white (SA187W). In comparison to SA187Y, SA187W shows 50% reduction on the beneficial impact on A. thaliana fresh and dry weight of root and shoot system. By counting the CFU/plant, we showed that SA187Y and SA187W both have similar colonization rate in both shoots and roots. Under non-salt conditions, optimal bacterial colonization was observed on day 8 after inocubation, however, under the salt stress condition, the optimal colonization was observed at day 4. Moreover, during the time period of the incubation of the SA187Y with the plants, there was a consistent noticeable loss of the yellow color of the colonies. This change in color is only observed eight days after transfer and the number of white colonies increases with the increase of the incubation time. In addition, SA187W was GFP-tagged by Tn7 transposon system and visualized by confocal laser scanning microscopy. The SA187W-GFP colonies have shown a similar colonization pattern as SA187Y-GFP, bacteria were colonizing the differentiation zone and cell elongation zone in the roots. Finally, the gene expression of the carotenoid biosynthesis pathways genes in SA187Y showed an overall higher gene expression compared to SA187W. In conclusion, the color loss seems to affect the beneficial impact of the bacteria on plants. However, the reduced beneficial impact is not due to the colonization efficiency of bacteria on the plant roots but could be due to a regulation of gene expression of carotenoid biosynthesis.

Wheat (Triticum aestivum) is a crop that dominates the diets of about 35% of the world's human population. But wheat yields can be severely affected by drought. Therefore, in this experiment, root exudation of winter wheat was compared to find out how exudation changes during drought stress. This was done by using control plants that were compared to plants that experienced 8 days of drought and subsequently, 3 days of rewetting. Moreover, it was explored which one of two wheat genotypes, Capo or Aristaro, is better adapted to drought by measuring plant physiology and if beneficial rhizosphere microorganisms could help alleviate drought in wheat. Exudations were analysed using a photometer. It was found that drought and rewetting treatment influenced shoot dry weight, shoot water content, relative chlorophyll, as well as exuded phenols, sugars and amino acids. Aristaro was found to be more drought tolerant, because Aristaro plants had higher and more stable shoot water content, lower non-photochemical quenching (NPQ(T)), and exuded more phenols and amino acids, which could help recruit plant-growth promoting rhizobacteria. However, Capo might be able to avoid drought through early maturity and can therefore be used in regions where drought occurs later in the year. The implications of this experiment are therefore useful for improving wheats resilience to drought and food security with use of microorganisms.

Au cours de cette thèse, un isolat de sol de désert, Saccharothrix algeriensis NRRL B-24137, a été évalué pour ses propriétés bioactives contre le champignon phytopathogène Botrytis cinerea, pour sa colonization sur Vitis vinifera L., et Arabidopsis thaliana ainsi qu’en vue d’étudier les méchanismes de résistance systémique induite (ISR) contre B. cinerea. Les résultats obtenus nous ont permis premièrement de montrer que Sa. algeriensis NRRL B-24137 peut présenter des activités antifongiques contre B. cinerea et que des métabolites peuvent être responsables de cette activité antifongique. Bien que ces métabolites soient encore en cours d’étude et que cette étude mérite d’être approfondie, nous avons démontré ensuite les propriétés de colonisation de l’isolat du sol du désert chez la vigne. Les résultats ont permis de montrer que la souche peut former des populations rhizosphèriques ainsi que des sous-populations endophytiques chez des plants de vigne (Cabernet Sauvignon sur porte-greffe 44-53 M) à des étapes précoces de colonisation. Puis nous avons démontré que la souche bénéfique peut induire une résistance systémique contre B. cinerea. Bien que les mécanismes impliqués ne soient pas encore compris, des parties préliminaires de ces travaux démontrent que les expressions de gènes responsables de la production de glucanase, chitinase ainsi qu’un inhibiteur de polygalacturonase ne semblent pas potentialisés pendant le phénomène de résistance systémique. Enfin nous avons démontré l’interaction entre Sa. algeriensis NRRL B-24137 et Arabidopsis thaliana qui résulte dans une association intime dûe également à colonisation rhizosphèrique et endophytique de la plante modèle. La souche bénéfique peut églement induire un phénomène de résistance systémique sur A. thaliana contre B. cinerea et les analyses de plantes mutées ont permis de determiner des parties des mécanismes impliqués dans l’ISR aini que des nouveaux mécanismes impliqués qui peuvent être induits par des microbes bénéfiques / In this thesis, the desert soil isolate, Saccharothrix algeriensis NRRL B-24137, has been evaluated for its bioactive properties towards the phytopathogenic fungus Botrytis cinerea, for its colonization of Vitis vinifera L., and Arabidopsis thaliana as well as to study the mechanisms of induced systemic resistance (ISR) towards B. cinerea. The results obtained allowed us firstly to show that Sa. algeriensis NRRL B-24137 can exhibit strong antifungal properties towards B. cinerea and that some metabolites can be responsible of this antifungal activity. Although these metabolites are still under consideration and that this study needs further works, we have demonstrated then the colonization properties of the desert soil isolate with grapevine plants. The results showed that the strain can form rhizospheric as well as endophytic subpopulations with grapevine plants (Cabernet Sauvignon cultivar graffed on 44-53 M rootstock) at early step of colonization. Then we have demonstrated that the beneficial strain could induce a systemic resistance towards B. cinerea. Although the mechanisms are not yet well understood, preliminary parts of this work demonstrated that the genes responsible of glucanase production, chitinase as well as inhibitor of polygalacturonase activity do not seems to be primed during the systemic resistance phenomenon. Finally we demonstrated that the interaction between Sa. algeriensis NRRL B-24137 and Arabidopsis thaliana plants results in a close association due also to a rhizo- and endophytic colonization of the model plant. The beneficial strain can also induce a systemic resistance in A. thaliana towards B. cinerea and analyzes of plant mutants have allowed to determine parts of the mechanisms involved in ISR as well as new mechanisms that could be trigerred by beneficial microbes