Bacillus amyloliquefaciens FZB42 ist ein frei lebendes Bakterium, das Pflanzenwurzeln besiedelt und das Pflanzenwachstum durch viele verschiedene Wirkmechanismen anregt. In dieser Arbeit wurden die molekularen Grundlagen dieser positiven Wirkungen, die dieses „Pflanzenwachstum fördernde Rhizobakterium“ (PGPR) auf seine Wirte ausübt, untersucht. Um den gegenseitigen Austausch von B. amyloliquefaciens und seinen Wirtspflanzen zu entschlüsseln, wurden umfangreiche Proteomstudien durchgeführt. Es wurden Referenzkarten der extrazellulären und zytosolischen Proteinfraktionen erstellt. Die größte Anzahl an ausgeschiedenen Proteinen konnte während der stationären Phase beobachtet werden. Die identifizierten extrazellulären Proteine gehören verschiedenen Funktionsklassen an, wobei die prominentesten Klassen am Kohlenhydrat-Abbau und den Transport von Molekülen durch die Zellwand beteiligt sind. Die zytosolischen Extrakte von Kulturen, die in 1C-Medium bzw. Mineralmedium angezogen wurden, und in der zweidimensionalen Gelelektrophorese (2 DE) aufgetrennt wurden, ergaben 461 und 245 verschiedene Protein-Einträge. Die erstellten Referenz-Karten wurden anschließend verwendet, um Proteine und Prozesse, in an der Interaktion mit Pflanzen beteiligt sind, zu identifizieren. Dafür wurden die Bakterien Wurzelexudaten von Mais (Zea mays L.) ausgesetzt. Die Proteine aus zwei Stämmen, denen die globalen Transkriptionsregulatoren (Degu, AbrB) und vier Sigma-Faktoren (SigB, SigM, SigV, und SigX) fehlen, wurden ebenfalls untersucht, um ihre Beteiligung an den bakteriellen Reaktionen auf die Wurzelausscheidungen zu analysieren. Zusammenfassend ist dies die erste Studie, die umfangreiche Proteomdaten von Gram-positiven PGPR präsentiert, wobei gleichzeitig die Veränderung der Expression von extrazellulären und zytoplasmatischen Proteinen, nach Zugabe von Wurzelexudaten, ausgewertet wurde. / Bacillus amyloliquefaciens strain FZB42 is a free-living bacterium that competitively colonizes plant roots and stimulates plant growth by many different modes of action. The molecular basis of singular beneficial effects that this Plant Growth-Promoting Rhizobacteria (PGPR) exert on their hosts have been studied. To decipher the molecular cross-talk of B. amyloliquefaciens and its’ host plants as a whole system, an extensive proteomic approach was performed. Reference maps of the extracellular and cytosolic protein fractions were established. The highest number of secreted proteins was observed during stationary growth phase. Identified extracellular proteins belong to different functional classes, with the most prominent classes involved in carbohydrate degradation and transportation of molecules across the cell wall. Cytosolic extracts obtained from cultures grown in 1C and minimal media subjected to the 2 Dimensional Electrophoresis (2 DE), revealed 461 and 245 different protein entries, respectively. Created reference maps were subsequently used to identify proteins and processes involved in the interaction with plants, prior to exposure of bacteria to maize (Zea mays L.) root exudates. The proteomics of two strains lacking expression of genes coding for global transcriptional regulators (degU, abrB) and four sigma factors (sigB, sigM, sigV, and sigX) were also inves-tigated, in order to analyse their involvement in bacterial responses to root exudates. In summary, this is the first study presenting comprehensive proteomics of Gram-positive PGPR, evaluating at the same time changes in protein expression caused by addition of root exudates at the extracellular and cytosolic level.

The inoculation with Azospirillum brasilense is one of the main strategies to supplement the inorganic inputs of nitrogen (N) and to increase the root development in maize. However, the beneficial inoculation effects are not always reached, which, in part, is due to genotypic variation in the plant host, resulting in different degrees of outcome. In this context, we aimed to study the genetic control and genomic prediction of maize traits related to the responsiveness to A. brasilense inoculation. For this, 118 maize hybrids were conducted under N stress and N stress plus A. brasilense treatments in controlled conditions over 2016 and 2017 seasons. We evaluated root and shoot traits and performed diallel analyses, association mapping, and genomic prediction methods considering 59,215 Single-Nucleotide Polymorphism (SNP) markers. Our results revealed a quantitative inheritance of the partnership-related maize traits, with both additive and non-additive genetic effects involved in the genetic control. Furthermore, several candidate genes were identified for the maize-A. brasilense association, especially with heterozygous (dis)advantage effects. In general, the prediction accuracies were higher mostly for the inoculated treatment compared to the non-inoculated. Finally, our findings enable a deeper understanding towards the genetic basis of the maize responsiveness to A. brasilense and may support plant breeders to establish selection strategies aiming the development of superior genotypes for this association. / A inoculação com Azospirillum brasilense é uma das principais estratégias para suplementar os insumos inorgânicos de nitrogênio (N) e aumentar o desenvolvimento radicular do milho. No entanto, os efeitos benéficos da inoculação nem sempre são alcançados, o que, em parte, é devido à variação genotípica da planta hospedeira, que ocasiona diferentes graus de resultados. Neste contexto, nosso objetivo foi estudar o controle genético e a predição genômica de caracteres de milho relacionados à responsividade para a inoculação com A brasilense. Para isso, 118 híbridos de milho foram conduzidos sob estresse de N e estresse de N mais A brasilense em condições controladas nos anos de 2016 e 2017. Nós avaliamos características de raiz e parte aérea e realizamos análises dialélicas, mapeamento associativo e métodos de predição genômica considerando 59.215 marcadores Single-Nucleotide Polymorphism (SNP). Nossos resultados revelaram uma herança quantitativa das características do milho relacionadas à essa parceria, com efeitos genéticos aditivos e não-aditivos envolvidos no controle genético. Além disso, vários genes candidatos foram encontrados para a associação milho-A brasilense, especialmente com efeitos de (des)vantagens de heterozigotos. Em geral, as acurácias de predição foram mais maiores principalmente para o tratamento inoculado em comparação ao não inoculado. Finalmente, nossos resultados possibilitam uma compreensão mais aprofundada das bases genéticas da responsividade do milho à A. brasilense e podem auxiliar os melhoristas de plantas a estabelecerem estratégias de seleção visando o desenvolvimento de genótipos superiores para essa associação.

Les bactéries promotrices de la croissance des plantes (PGPR) peuvent améliorer la performance et la tolérance des plantes lors de stress environnementaux. Arabidopsis thaliana est un modèle de choix pour étudier les mécanismes impliqués dans les interactions plante-bactéries. Nous avons analysé de multiples traits associés à la dynamique de croissance, au développement et la physiologie des végétaux afin d'évaluer les effets de l'inoculation par Phyllobacterium brassicacearum STM196, une PGPR isolée de la rhizosphère du colza, sur les réponses d'A. thaliana à des stress hydriques de différentes intensités. Grâce à des outils performants de phénotypage, nous avons développé une nouvelle approche d'analyse à haut-débit pour examiner l'implication de STM196 dans les stratégies de résistance des plantes au stress hydrique. Nos résultats montrent pour la première fois que les PGPR peuvent interférer dans les stratégies d'échappement des plantes grâce à des modifications de la croissance et du temps de floraison. De plus, STM196 induit une meilleure résistance au déficit hydrique modéré et une meilleure tolérance à la déshydratation sous une contrainte hydrique sévère. L'inoculation par STM196 peut ainsi représenter une valeur ajoutée aux stratégies de résistance intrinsèques aux plantes, ce qui est illustrée par sa remarquable capacité à promouvoir la survie et la production de biomasse végétale dans des environnements contrastés. Nos résultats soulignent l'importance des interactions plantes-bactéries dans les réponses des plantes à la sécheresse et offrent de nouvelles voies de recherches pour l'amélioration de la résistance à la sécheresse dans les cultures. / Plant growth promoting rhizobacteria (PGPR) can enhance plant performance and plant tolerance to environmental stresses. Arabidopsis thaliana is a useful organism to study the mechanisms involved in plant-PGPR interactions. We analyzed multiple plant traits related to growth dynamics, development and physiology in order to assess the effects of Phyllobacterium brassicacearum STM196 strain, isolated from the rhizosphere of oilseed rape, on Arabidopsis responses to well-defined soil water availability. Using powerful tools for phenotyping, we developed a new high-throughput analysis to examine the implication of STM196 on plant strategies to cope with water stress. Our results show for the first time that PGPR can interfere in escape strategies of plants through modifications in plant growth and flowering time. Moreover, STM196 induced a better resistance to moderate water deficit and a better tolerance to dehydration under a severe stress. Inoculation by STM196 can represent an added value to plant resistance strategies, as illustrated by its remarkable ability to promote plant survival and biomass production under contrasted environments. Our results highlight the importance of plant-bacteria interactions in plant responses to drought and provide a new avenue of investigations to improve drought resistance in crops.

Made available in DSpace on 2017-07-10T17:37:10Z (GMT). No. of bitstreams: 1 Sarah_Romano_Ortolan.pdf: 975526 bytes, checksum: d72ab61fc7bdc88b6ddbab0eb3a86e42 (MD5) Previous issue date: 2015-07-28 / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Wheat is considered the main cereal diet of the world population, but in recent years has achieved some gain in productivity of this culture despite having increased the use of nitrogen fertilizer. The use of plant growth promoting bacteria such as Herbaspirillum seropedicae SmR1 among others has been studied to obtain development of plants with less use of nitrogen fertilizers. However there is little information relating the effects of this interaction in plant development and grain yield. Objective of this study was to evaluate the carbon and nitrogen metabolism by certain enzymes, metabolites and indices related to the response to infectious stress on the wheat cultivars CD 104 and CD 120 in association with Herbaspirillum seropedicae bacteria. Two experiments were conducted. The experimental design was completely randomized with four replications in a 4x2. The first factor relates to the conditions inoculation with bacteria and/or nitrogen source in coverage are: control without inoculation with bacteria or added nitrogen fertilizer (C); application of nitrogen fertilizer (50 kg.ha-1) 30 days after sowing (N); inoculation with 106 cells of the bacterium H. seropedicae/seed at planting (Hs) and inoculation with bacteria combined with the application of nitrogen fertilizer (Hs + N) and the second factor refers to the phenological stages (tillering and booting). The results indicated that inoculation with H. seropedicae in wheat seeds of cv.s CD 104 and CD 120 in the two growth stages answered in relation to the indices related to stress with the involvement of enzymes of carbon and nitrogen metabolism. However prominent effect was not noticed to promote plant growth of wheat in late development, nor a deleterious effect of the bacterium for inoculation cv. CD 104 under the experimental conditions. For cv. CD 120 the differential effects indicate lower levels of stress and some level of association to positive effect on productivity when combined inoculation of bacteria to nitrogen fertilization. It was concluded that as well as pathogenic and stressors, H. seropedicae able to beneficially associate with wheat also provides similar interference pattern of carbon and nitrogen metabolism and stress levels / O trigo é considerado o principal cereal da dieta da população mundial, entretanto nos últimos anos tem se obtido pouco ganho de produtividade desta cultura apesar de se ter aumentado o uso de fertilizante nitrogenado. O uso de bactérias promotoras do crescimento vegetal, como Herbaspirillum seropedicae SmR1 entre outras, tem sido estudado para se obter desenvolvimento de plantas com menor uso de fertilizantes nitrogenados. Entretanto existem poucas informações que relacionam os efeitos desta interação no desenvolvimento da planta e de produtividade de grãos. Objetivo deste trabalho foi avaliar o metabolismo de carbono e nitrogênio através de algumas enzimas, metabólitos e índices relacionados à resposta ao estresse infeccioso em trigo das cultivares CD 104 e CD 120 em associação com a bactéria Herbaspirillum seropedicae em dois estádios fenológicos. Foram realizados dois experimentos. O delineamento experimental utilizado foi o inteiramente ao acaso, com 4 repetições, em esquema fatorial 4x2. O primeiro fator refere-se às condições de inoculação com bactéria e/ou fertilização nitrogenada em cobertura, sendo: controle, sem inoculação com bactéria ou adição de fertilizante nitrogenado (C); aplicação de fertilizante nitrogenado (50 kg.ha-1) após 30 dias da semeadura (N); inoculação de 106 células da bactéria H. seropedicae/semente na semeadura (Hs) e inoculação com a bactéria combinada com a aplicação de fertilizante nitrogenado (Hs + N) e o segundo fator refere-se aos estádios fenológicos (perfilhamento e emborrachamento). Os resultados indicaram que a inoculação com H. seropedicae em sementes de trigo das cv.s CD 104 e CD 120 nos dois estádios fenológicos responderam em relação aos índices relacionados ao estresse com envolvimento das enzimas do metabolismo de carbono e nitrogênio. Entretanto não foi percebido efeito proeminente de promoção do crescimento vegetal no final do desenvolvimento do trigo, tampouco efeito deletério da inoculação de bactéria para a cv. CD 104, nas condições experimentais. Para a cv. CD 120 os efeitos diferenciais indicam menor nível de estresse e algum nível de associação para efeito positivo na produtividade quando combinada a inoculação da bactéria com a fertilização nitrogenada. Foi possível concluir que assim como para agentes patogênicos e estressantes, a H. seropedicae, capaz de associar beneficamente com trigo também apresenta padrão semelhante de interferência do metabolismo de carbono e nitrogênio e índices de estresse

Symbioses between microbes and multicellular eukaryotes are found in all biomes, and encompass a spectrum of symbiotic lifestyles that includes parasitism and disease, commensalism, and mutually beneficial interdependent host-microbe relationships. Regardless of outcome, these symbiotic lifestyles are governed by a complex molecular "courtship" between microbe and potential host. This courtship is the primary determinant of the host range of a given microsymbiont. Host immunity poses a formidable barrier to the establishment of host-microbe relationships, and the majority of microbial suitors will be thwarted by it. Only by successfully "wooing" the host cell's immune defenses with the appropriate molecular signals can a microsymbiont successfully colonize its host. A strategy common to microsymbionts across the spectrum of symbiotic lifestyles and host organisms is the delivery of microbial-encoded effector proteins into the cytoplasm of host cells to manipulate the host cell's molecular machinery for the purposes of subverting host immunity. Bacteria, in particular, have adapted a number of secretion systems for this purpose. The most well-characterized of these is the type III secretion system (T3SS), a molecular apparatus that specializes in injecting type III effector (T3Es) proteins directly into host cells. The work in this thesis focuses on T3Es of plant-associated bacteria, with particular emphasis on mutualistic bacteria. We present evidence that collections of T3Es from Sinorhizobium fredii and Bradyrhizobium japonicum are, in stark contrast to those of phytopathogenic bacteria, in a co-evolutionary equilibrium with their hosts. This equilibrium is characterized by highly conserved T3E collections consisting of many "core" T3Es with little variation in nucleotide sequence. The T3Es of Mesorhizobium loti MAFF303099 suggest a completely different picture of the evolution of T3Es. MAFF303099 recently acquired its T3SS locus, and the work in this thesis provides an evolutionary snapshot of a mutualist that is innovating a T3E collection primarily through horizontal gene transfer. Collectively, this work represents the first comprehensive catalog of T3Es of rhizobia and, in the case of Sinorhizobium and Bradyrhizobium, the first evidence of purifying selection for T3Es. / Graduation date: 2012

Anaerobic digestion (AD) is becoming a widely adopted technology for conversion of organic waste and nutrient-rich fertiliser production due to its cost-effectiveness and sustainability. In this study, a batch experiment was conducted using five different types of food waste and cow dung (CD). No significant difference was observed among the four substrates that produced the highest methane (P<0.05). Based on the batch experiment results, two substrates were selected for semi-continuous digestion and the highest methane yield (67%) was obtained from co-digestion (CO). PCR-DGGE results revealed higher bacterial and archaeal diversity indices in CO as compared to mono-digestion of CD and mixed food waste. The high-throughput sequence analyses revealed that the Operational Taxonomic Units (OTUs) belonging to the phyla Bacteroidetes, followed by Firmicutes, Actinobacteria and Proteobacteria, were dominant in all treatments. The enhanced methane production in CO could be attributed to the neutral pH and partial shift of archaea from Methanosaeta to Methanosarcina. The digestate and fresh CD were screened for plant growth promoting bacteria (PGPB), nutrient and heavy metal content. The dung contained higher concentrations of heavy metals (P<0.05) and potential pathogens in comparison to the digestate. The use of digestate may, therefore, enhance soil fertility with minimal negative environmental effects. / School of Agriculture and Life Sciences / M. Sc. (Life Sciences)

Orientador: Orivaldo Arf / Resumo: Devido ao crescimento e a importância da utilização do milho segunda safra, pacotes tecnológicos que favoreçam o uso dessa nova época de cultivo para o milho são de extrema importância para a consolidação dessa safra. O manejo adequado do solo, a utilização de coberturas vegetais e considerando também os dados pouco consistentes na utilização da inoculação com bactérias promotoras de crescimento na cultura do milho, são práticas fundamentais para a busca de uma produção econômica e ambientalmente viável. Diante do exposto o objetivo desse trabalho foi o de avaliar o efeito das coberturas vegetais e dos modos de inoculação com Azospirillum brasilense nos diferentes sistemas de preparo do solo, no desenvolvimento e produtividade do milho segunda safra. O experimento foi desenvolvido nos anos agrícolas de 2013/14 e 2014/15 em área experimental da Fazenda de Ensino e Pesquisa pertencente à Faculdade de Engenharia de Ilha Solteira – UNESP, localizada no município de Selvíria – MS. O solo do local é do tipo LATOSSOLO VERMELHO-ESCURO Epi-eutrófico álico, textura argilosa, originalmente sob vegetação de cerrado. A precipitação média anual é de 1.313 mm e a temperatura média anual de 25°C. A área teve como cultura anterior a soja, e foi constituído por três subprojetos envolvendo coberturas vegetais (Crotalária spectabilis, Urochloa ruziziensis e C. spectabilis + U. ruziziensis), modos de inoculação com A. brasilense (testemunha - sem inoculação de A. brasilense, inoculação de A. bras... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: Due to the growth and importance of the use of second crop maize, technological packages that enhance the use of this new maize season are extremely important for this crop consolidation. Adequate soil management, cover crops and also the inconsistent data on the use of inoculation with growth promoting bacteria in maize, are fundamental practices for the search for an economically and environmentally viable production. This study animed to evaluate the effect of plant cover and inoculation methods with Azospirillum brasilense on different soil preparation systems, on the development and productivity of second crop maize. The experiment was developed in the agricultural years of 2013/14 and 2014/15 in an experimental area of the Education and Research Farm belonging to the Faculty of Engineering of Ilha Solteira - UNESP, located in the municipality of Selvíria -MS. The soil of the site is of the type RED-DARK LATOSOL Epi-eutrophic alic, clayey texture, originally under cerrado vegetation. The average precipitation cancels out is 1313 mm and the average annual temperature is 25 ° C. The area was previously cultivated with soybean, and was constituted by three subprojects involving plant cover crops (Crotalaria spectabilis, Urochloa ruziziensis and C. spectabilis + U. ruziziensis), inoculation methods with A. brasilense (control - without inoculation of A. brasilense , inoculation of A. brasilense on maize seeds, inoculation of A. brasilense on maize seed and foliar - V4 stage ... (Complete abstract click electronic access below) / Doutor

Submitted by Daiene Camila Dias Chaves Corsini (daieneagro@gmail.com) on 2018-11-09T18:57:12Z No. of bitstreams: 1 corsini_dcdc_dr_ilha.pdf: 3271631 bytes, checksum: 03bc61ace606b7d3a40171e912132c80 (MD5) / Approved for entry into archive by Cristina Alexandra de Godoy null (cristina@adm.feis.unesp.br) on 2018-11-09T19:15:19Z (GMT) No. of bitstreams: 1 corsini_dcdc_dr_ilha.pdf: 3271631 bytes, checksum: 03bc61ace606b7d3a40171e912132c80 (MD5) / Made available in DSpace on 2018-11-09T19:15:19Z (GMT). No. of bitstreams: 1 corsini_dcdc_dr_ilha.pdf: 3271631 bytes, checksum: 03bc61ace606b7d3a40171e912132c80 (MD5) Previous issue date: 2018-07-02 / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Devido ao crescimento e a importância da utilização do milho segunda safra, pacotes tecnológicos que favoreçam o uso dessa nova época de cultivo para o milho são de extrema importância para a consolidação dessa safra. O manejo adequado do solo, a utilização de coberturas vegetais e considerando também os dados pouco consistentes na utilização da inoculação com bactérias promotoras de crescimento na cultura do milho, são práticas fundamentais para a busca de uma produção econômica e ambientalmente viável. Diante do exposto o objetivo desse trabalho foi o de avaliar o efeito das coberturas vegetais e dos modos de inoculação com Azospirillum brasilense nos diferentes sistemas de preparo do solo, no desenvolvimento e produtividade do milho segunda safra. O experimento foi desenvolvido nos anos agrícolas de 2013/14 e 2014/15 em área experimental da Fazenda de Ensino e Pesquisa pertencente à Faculdade de Engenharia de Ilha Solteira – UNESP, localizada no município de Selvíria – MS. O solo do local é do tipo LATOSSOLO VERMELHO-ESCURO Epi-eutrófico álico, textura argilosa, originalmente sob vegetação de cerrado. A precipitação média anual é de 1.313 mm e a temperatura média anual de 25°C. A área teve como cultura anterior a soja, e foi constituído por três subprojetos envolvendo coberturas vegetais (Crotalária spectabilis, Urochloa ruziziensis e C. spectabilis + U. ruziziensis), modos de inoculação com A. brasilense (testemunha - sem inoculação de A. brasilense, inoculação de A. brasilense nas sementes do milho, inoculação de A. brasilense na semente do milho e via foliar - estádio V4 e inoculação de A. brasilense via foliar - estádio V4) em três sistemas de preparo de solo (plantio direto, cultivo mínimo e cultivo convencional). Os delineamentos experimentais foram de blocos casualizados em esquema de parcelas subdivididas em cada tipo de preparo do solo (subprojetos), sendo constituídos por três coberturas vegetais e quatro modos de inoculação com quatro repetições. Observou-se que no subprojeto com coberturas vegetais e modos de inoculação em sistema plantio direto, as coberturas vegetais obtiveram biomassa adequada para a manutenção de palha nesse sistema; a C. spectabilis obteve os maiores teores de nitrogênio (N) nas plantas e N acumulado, proporcionando as plantas de milho cultivadas após essa cobertura, maior altura de inserção de espigas; as plantas de milho cultivadas após a U. ruziziensis apresentou um maior população de Azospirillum por grama de solo. No cultivo mínimo, as coberturas vegetais obtiveram biomassa adequada para a manutenção de palha; para o teor de N, a espécie C. spectabilis apresentou maiores médias proporcionando as plantas de milho maior a altura de inserção de espiga, maior massa de plantas e maior população Azospirillum por grama de solo; a inoculação com A. brasilense via foliar proporcionou as plantas de milho maior massa seca de parte aérea; o modo de inoculação com A. brasilense proporcionou aumento da produtividade de grãos de milho quando essa foi feita nas sementes e via foliar. No sistema de preparo de solo convencional, as coberturas vegetais obtiveram biomassa adequada para a manutenção de palha, a massa seca de plantas de U. ruziziensis teve o maior valor médio e o maior acúmulo de N; a altura de plantas e altura de inserção de espigas foi maior quando houve a inoculação nas sementes de milho e via foliar com A. brasilense. / Due to the growth and importance of the use of second crop maize, technological packages that enhance the use of this new maize season are extremely important for this crop consolidation. Adequate soil management, cover crops and also the inconsistent data on the use of inoculation with growth promoting bacteria in maize, are fundamental practices for the search for an economically and environmentally viable production. This study animed to evaluate the effect of plant cover and inoculation methods with Azospirillum brasilense on different soil preparation systems, on the development and productivity of second crop maize. The experiment was developed in the agricultural years of 2013/14 and 2014/15 in an experimental area of the Education and Research Farm belonging to the Faculty of Engineering of Ilha Solteira - UNESP, located in the municipality of Selvíria -MS. The soil of the site is of the type RED-DARK LATOSOL Epi-eutrophic alic, clayey texture, originally under cerrado vegetation. The average precipitation cancels out is 1313 mm and the average annual temperature is 25 ° C. The area was previously cultivated with soybean, and was constituted by three subprojects involving plant cover crops (Crotalaria spectabilis, Urochloa ruziziensis and C. spectabilis + U. ruziziensis), inoculation methods with A. brasilense (control - without inoculation of A. brasilense , inoculation of A. brasilense on maize seeds, inoculation of A. brasilense on maize seed and foliar - V4 stage and inoculation of A. brasilense via foliar - V4 stage) in three soil tillage systems (no - tillage, no - tillage minimum and conventional cultivation). The experimental design was a randomized block design subdivided in each type of soil preparation (subprojects), consisting of three vegetation cover and four modes of inoculation with four replications. It was observed that in the subproject with vegetation cover and inoculation methods in no-tillage system, the vegetal cover obtained adequate biomass for the maintenance of straw in this system; C. spectabilis obtained the highest levels of nitrogen in the plants and N accumulated, providing the maize plants cultivated after this cover, greater height of spike insertion; maize plants grown after U. ruziziensis showed a larger population of Azospirillum per gram of soil. In the minimum cultivation, the vegetal coverings obtained adequate biomass for the maintenance of straw; for the N content, the species C. spectabilis showed higher mean values, giving the maize plants greater spike insertion height, larger plant mass and higher Azospirillum population per soil gram; the inoculation with A. brasilense via foliar provided the maize plants with the highest aerial part dry mass; the form of inoculation with A. brasilense provided an increase in the yield of maize grains when it was made in the seeds and foliar route. In the conventional tillage system, the plant cover obtained adequate biomass for the maintenance of straw, the dry mass of U. ruziziensis plants had the highest average value and the highest accumulation of N; plant height and spike insertion height were higher when inoculation was carried out on maize seeds and foliar route with A. brasilense. / FAPESP: 2013/26489-6

Überstände von Cyanobakterien sind ein vielversprechendes Produkt zur Förderung des Pflanzenwachstums, da sie alle Vorteile der freigesetzten bioaktiven Verbindungen, wie z. B. Phytohormone, enthalten, ohne die Zwänge der mikrobiellen Inokulationen. Es ist jedoch nur wenig darüber bekannt, wie Cyanobakterien die Reaktion der Pflanzen auf molekularer Ebene modulieren könnten. In dieser Studie wurde das in Chile heimische Gras Polypogon australis als Modell verwendet, um die Wirkung von Überständen aus Kulturen von sieben autochthonen Bodencyanobakterien zu untersuchen. Von diesen zeigten die Überstände der Kulturen von Trichormus sp. die beste wachstumsfördernde Wirkung auf P. australis. Die ICP-MS-Analyse ergab, dass die Überstände von Trichormus sp. eine ähnliche Nährstoffzusammensetzung aufwiesen wie das für das Wachstum der Cyanobakterien verwendete Medium BG-11, mit Ausnahme der Elemente P und Mn, die in der späten exponentiellen Phase der Kulturen verarmt waren. Dann wurden Überstände von Trichormus sp.-Kulturen, die in der späten exponentiellen Phase gesammelt wurden und die eine Menge von 32,7 pmol trans-Zeatin pro mg Chl-a enthielten, zur Bewertung der Reaktion von P. australis auf transkriptioneller Ebene verwendet. Ein BG-11-Medium, das frei von P und Mn war, wurde als Kontrolle verwendet. Ganzes Pflanzengewebe wurde 3 Stunden nach der Behandlung entnommen und für eine RNA-seq-Analyse verwendet. Die Ergebnisse zeigten, dass die Überstände von Trichormus sp. die Pflanzenreaktion hauptsächlich über die N- und Phytohormonwege modulierten, die in engem Zusammenhang mit dem C- und Lipidstoffwechsel stehen. Die behandelten Pflanzen wiesen 4 und 8 Tage nach der Anwendung größere Triebe auf als die Kontrollpflanzen, aber es wurden keine Unterschiede bei der Wurzellänge festgestellt. Dieser Phänotyp lässt sich durch die Induktion von Genen für die Gibberellin-Biosynthese in P. australis erklären, die durch andere Hormone wie Auxine, Brassinosteroide und Ethylen unterstützt wird. Andererseits wurde in mit P. australis behandelten Pflanzen eine induzierte systemische Resistenzreaktion beobachtet, die hauptsächlich durch einen Ethylen-Jasmonat-Crosstalk vermittelt wurde. Diese Arbeit unterstützt die Verwendung von Überständen als eine gute Option zur Förderung des Pflanzenwachstums.:Table of content Preliminary Page Resumen i Abstract ii Übersetzung iii DECLARATION ix 1. Introduction 1 1.1. Plant-growth promoting microorganisms 1 1.2. Soil cyanobacteria 2 1.3. Physiology of soil cyanobacteria 2 1.4. Cyanobacterial plant growth-promoting molecules 4 1.5. Plant response to bioactive compounds 6 1.6. Cyanobacterial supernatants 9 1.7. Polypogon australis as a plant study model 11 2. Methodology 13 2.1. Obtention of the cyanobacterial cultures 13 2.2. Supernatant collection from the cyanobacterial cultures 14 2.3. Cyanobacterial biomass quantification 15 2.3.1. Chlorophyll-a content 15 2.3.2. Biomass dry weight 15 2.3.3. Determination of the growth phases 15 2.4. Chemical characterization of the supernatants 16 2.4.1. Nitrate content 16 2.4.2. Total element content 16 2.4.3. Zeatin content 16 2.5. Preparation of the modified BG-11 medium (BG-11M medium) 17 2.6. Bioassays with cyanobacterial supernatants 18 2.6.1. Effect of supernatants of the 25 mL cultures on P. australis germination 18 2.6.2. Effect of supernatants of the 25 mL cultures on P. australis plants 18 2.6.3. Effect of supernatants of the 2,400 mL cultures on P. australis plants 19 2.7. Statistical analysis 19 2.8. Determination of transcriptional changes in P. australis. 20 2.8.1. Plant treatments and tissue collection 20 2.8.2. Total RNA extraction from plant tissue 20 2.8.3. DNA removal 20 2.8.4. mRNA sequencing, de novo assembly, and differential expression analysis 21 2.8.5. Contig annotation and functional classification 22 3. Results 24 3.1. Trichormus sp. cultures produce the highest biomass content 24 3.2. Trichormus sp. cultures have a low P content 25 3.3. Trichormus sp. supernatants have the best growth-promoting effect on the growth of P. australis 25 3.4. Supernatant nutrient content of Trichormus sp. cultures change through the growth phases 27 3.5. Supernatants used in the transcriptomic assay and BG-11M medium have a lower nutrient content than BG-11 medium 30 3.6. Trichormus sp. supernatants promote the growth of P. australis to a greater extent than the BG-11M medium 32 3.7. Trichormus sp. supernatants contain zeatin 33 3.8. Trichormus sp. supernatants modulated more P. australis genes than the BG-11M medium 34 3.9. Trichormus sp. supernatants regulate the gene expression of growth and defense responses in P. australis 37 4. Discussion 57 4.1. The plant-growth promoting effect of Thrichormus sp. supernatants 57 4.2. The role of P and Mn in the growth-promoting effect of Trichormus sp. supernatants 58 4.3. Modulation of P. australis N-metabolism by Trichormus sp. supernatants 59 4.4. P. australis nitrogen and carbon metabolism in response to Trichormus supernatants 63 4.5. P. australis phytohormone-metabolism modulated by Trichormus supernatants 64 4.6. The role of lipid metabolism in the response to Trichormus supernatants 67 4.7. P. australis defense response triggered by Trichormus supernatants 68 4.8. Phytohormone crosstalk and defense response in P. australis treated with Trichormus sp. supernatants 71 4.9. Perspectives and challenges for the biotechnological use of Trichormus sp. supernatants 73 5. Conclusion 76 Bibliographic references 77 Annexes 116 / Cyanobacterial supernatants are a promising plant growth-promoting product since they contain all the advantages of the released bioactive compounds, such as phytohormones, without the constraints of microbial inoculations. However, little is known about how cyanobacteria could modulate the plant response at a molecular level. In this research, the Chilean native grass, Polypogon australis, was used as a model for assaying the effect of supernatants obtained from cultures of seven autochthonous soil cyanobacteria. Of them, supernatants of Trichormus sp. cultures showed the best growth-promoting effects on P. australis. Analysis by ICP-MS showed that Trichormus sp. supernatants had a similar nutrient composition to the medium used for the cyanobacteria growth, BG-11, except for the elements P and Mn, which were depleted when the late exponential phase of the cultures was reached. Then, supernatants of Trichormus sp. cultures collected in the late exponential phase, which contained an amount of 32.7 pmol of trans-zeatin per mg of Chl-a, were employed for evaluating the P. australis response at a transcriptional level. A BG-11 medium free of P and Mn was utilized as a control. Whole plant tissue was collected 3 h-post treatment and used for an RNA-seq analysis. Results showed that Trichormus sp. supernatants modulated the plant response mainly by the N and phytohormones pathways, in close relation with C and lipid metabolism. Treated plants showed larger shoots than control plants 4 and 8 days after application, but no differences were observed in root length. This phenotype can be explained by the induction in P. australis of gibberellin biosynthesis genes, supported by other hormones such as auxins, brassinosteroids, and ethylene. On the other hand, an induced systemic resistance response was observed in P. australis-treated plants, mostly mediated by an ethylene-jasmonate crosstalk. This work supports the use of supernatants as a good plant growth-promoting option.:Table of content Preliminary Page Resumen i Abstract ii Übersetzung iii DECLARATION ix 1. Introduction 1 1.1. Plant-growth promoting microorganisms 1 1.2. Soil cyanobacteria 2 1.3. Physiology of soil cyanobacteria 2 1.4. Cyanobacterial plant growth-promoting molecules 4 1.5. Plant response to bioactive compounds 6 1.6. Cyanobacterial supernatants 9 1.7. Polypogon australis as a plant study model 11 2. Methodology 13 2.1. Obtention of the cyanobacterial cultures 13 2.2. Supernatant collection from the cyanobacterial cultures 14 2.3. Cyanobacterial biomass quantification 15 2.3.1. Chlorophyll-a content 15 2.3.2. Biomass dry weight 15 2.3.3. Determination of the growth phases 15 2.4. Chemical characterization of the supernatants 16 2.4.1. Nitrate content 16 2.4.2. Total element content 16 2.4.3. Zeatin content 16 2.5. Preparation of the modified BG-11 medium (BG-11M medium) 17 2.6. Bioassays with cyanobacterial supernatants 18 2.6.1. Effect of supernatants of the 25 mL cultures on P. australis germination 18 2.6.2. Effect of supernatants of the 25 mL cultures on P. australis plants 18 2.6.3. Effect of supernatants of the 2,400 mL cultures on P. australis plants 19 2.7. Statistical analysis 19 2.8. Determination of transcriptional changes in P. australis. 20 2.8.1. Plant treatments and tissue collection 20 2.8.2. Total RNA extraction from plant tissue 20 2.8.3. DNA removal 20 2.8.4. mRNA sequencing, de novo assembly, and differential expression analysis 21 2.8.5. Contig annotation and functional classification 22 3. Results 24 3.1. Trichormus sp. cultures produce the highest biomass content 24 3.2. Trichormus sp. cultures have a low P content 25 3.3. Trichormus sp. supernatants have the best growth-promoting effect on the growth of P. australis 25 3.4. Supernatant nutrient content of Trichormus sp. cultures change through the growth phases 27 3.5. Supernatants used in the transcriptomic assay and BG-11M medium have a lower nutrient content than BG-11 medium 30 3.6. Trichormus sp. supernatants promote the growth of P. australis to a greater extent than the BG-11M medium 32 3.7. Trichormus sp. supernatants contain zeatin 33 3.8. Trichormus sp. supernatants modulated more P. australis genes than the BG-11M medium 34 3.9. Trichormus sp. supernatants regulate the gene expression of growth and defense responses in P. australis 37 4. Discussion 57 4.1. The plant-growth promoting effect of Thrichormus sp. supernatants 57 4.2. The role of P and Mn in the growth-promoting effect of Trichormus sp. supernatants 58 4.3. Modulation of P. australis N-metabolism by Trichormus sp. supernatants 59 4.4. P. australis nitrogen and carbon metabolism in response to Trichormus supernatants 63 4.5. P. australis phytohormone-metabolism modulated by Trichormus supernatants 64 4.6. The role of lipid metabolism in the response to Trichormus supernatants 67 4.7. P. australis defense response triggered by Trichormus supernatants 68 4.8. Phytohormone crosstalk and defense response in P. australis treated with Trichormus sp. supernatants 71 4.9. Perspectives and challenges for the biotechnological use of Trichormus sp. supernatants 73 5. Conclusion 76 Bibliographic references 77 Annexes 116 / Los sobrenadantes de cianobacterias son prometedores productos promotores del crecimiento vegetal, pues contienen todas las ventajas de los compuestos bioactivos liberados, como fitohormonas, sin las limitaciones de las inoculaciones microbianas. Lamentablemente, poco se sabe sobre cómo las cianobacterias modularían la respuesta de las plantas a nivel molecular. En esta investigación, se utilizó la gramínea nativa chilena Polypogon australis como modelo para evaluar el efecto de sobrenadantes de cultivos de siete cianobacterias autóctonas de suelo. Los sobrenadantes de Trichormus sp. mostraron mejores efectos promotores del crecimiento de P. australis. Análisis mediante ICP-MS evidenciaron que estos sobrenadantes tenían un contenido nutricional similar al medio de crecimiento de las cianobacterias, BG-11, excepto por los elementos P y Mn, que se agotaron al alcanzarse la fase exponencial tardía de los cultivos. Para evaluar la respuesta de P. australis a nivel transcripcional, se emplearon sobrenadantes colectados en fase exponencial tardía de cultivos de Trichormus sp., que contenían una cantidad de 32,7 pmol de trans-zeatina por mg de Chl-a. Un medio BG-11 libre de P y Mn se utilizó como control. Tres horas después del tratamiento se recogió tejido de plantas completas y se le hizo un análisis de RNA-seq. Como resultado, los sobrenadantes principalmente modularon las vías de N y fitohormonas de la planta, en estrecha relación con los metabolismos de C y lípidos. Las plantas tratadas mostraron brotes más grandes que las plantas control, 4 y 8 días después de la aplicación, pero no se observaron diferencias en la longitud radicular. Este fenotipo puede explicarse por la inducción de biosíntesis de giberelina, apoyada por otras hormonas como auxinas, brasinoesteroides y etileno. Además, se observó una inducción de resistencia sistémica en las plantas tratadas, mediada por una interacción etileno-jasmonatos. Este trabajo corrobora el uso de sobrenadantes como una buena opción para promover el crecimiento de las plantas.:Table of content Preliminary Page Resumen i Abstract ii Übersetzung iii DECLARATION ix 1. Introduction 1 1.1. Plant-growth promoting microorganisms 1 1.2. Soil cyanobacteria 2 1.3. Physiology of soil cyanobacteria 2 1.4. Cyanobacterial plant growth-promoting molecules 4 1.5. Plant response to bioactive compounds 6 1.6. Cyanobacterial supernatants 9 1.7. Polypogon australis as a plant study model 11 2. Methodology 13 2.1. Obtention of the cyanobacterial cultures 13 2.2. Supernatant collection from the cyanobacterial cultures 14 2.3. Cyanobacterial biomass quantification 15 2.3.1. Chlorophyll-a content 15 2.3.2. Biomass dry weight 15 2.3.3. Determination of the growth phases 15 2.4. Chemical characterization of the supernatants 16 2.4.1. Nitrate content 16 2.4.2. Total element content 16 2.4.3. Zeatin content 16 2.5. Preparation of the modified BG-11 medium (BG-11M medium) 17 2.6. Bioassays with cyanobacterial supernatants 18 2.6.1. Effect of supernatants of the 25 mL cultures on P. australis germination 18 2.6.2. Effect of supernatants of the 25 mL cultures on P. australis plants 18 2.6.3. Effect of supernatants of the 2,400 mL cultures on P. australis plants 19 2.7. Statistical analysis 19 2.8. Determination of transcriptional changes in P. australis. 20 2.8.1. Plant treatments and tissue collection 20 2.8.2. Total RNA extraction from plant tissue 20 2.8.3. DNA removal 20 2.8.4. mRNA sequencing, de novo assembly, and differential expression analysis 21 2.8.5. Contig annotation and functional classification 22 3. Results 24 3.1. Trichormus sp. cultures produce the highest biomass content 24 3.2. Trichormus sp. cultures have a low P content 25 3.3. Trichormus sp. supernatants have the best growth-promoting effect on the growth of P. australis 25 3.4. Supernatant nutrient content of Trichormus sp. cultures change through the growth phases 27 3.5. Supernatants used in the transcriptomic assay and BG-11M medium have a lower nutrient content than BG-11 medium 30 3.6. Trichormus sp. supernatants promote the growth of P. australis to a greater extent than the BG-11M medium 32 3.7. Trichormus sp. supernatants contain zeatin 33 3.8. Trichormus sp. supernatants modulated more P. australis genes than the BG-11M medium 34 3.9. Trichormus sp. supernatants regulate the gene expression of growth and defense responses in P. australis 37 4. Discussion 57 4.1. The plant-growth promoting effect of Thrichormus sp. supernatants 57 4.2. The role of P and Mn in the growth-promoting effect of Trichormus sp. supernatants 58 4.3. Modulation of P. australis N-metabolism by Trichormus sp. supernatants 59 4.4. P. australis nitrogen and carbon metabolism in response to Trichormus supernatants 63 4.5. P. australis phytohormone-metabolism modulated by Trichormus supernatants 64 4.6. The role of lipid metabolism in the response to Trichormus supernatants 67 4.7. P. australis defense response triggered by Trichormus supernatants 68 4.8. Phytohormone crosstalk and defense response in P. australis treated with Trichormus sp. supernatants 71 4.9. Perspectives and challenges for the biotechnological use of Trichormus sp. supernatants 73 5. Conclusion 76 Bibliographic references 77 Annexes 116

Zur Aufklärung des Wirkungsmechanismus der toleranzerhöhenden Wirkung gegenüber Salinität des Pflanzenwurzeln besiedelnden PGPR Bacillus subtilis wurden bakterielle Stoffwechselprodukte der Stämme FZB24 und FZB41 bei der Testpflanze Tomate unter dem Einfluss von hohem Salzstress getestet. Das Kulturfiltrat mit der Gesamtheit der von B. subtilis produzierten Stoffwechselprodukte zeigte im axenischen Test zur Ermittlung des Wachstums nach 7-tägiger Behandlung der Sämlinge und nachfolgender Kultivierung unter Salzstress eine gewisse toleranzerhöhende Wirkung bei 0,1 %-Konzentration. Zur Produktaufschlüsselung wurde das Kulturfiltrat über Adsorberharz und HPLC fraktioniert. Diese Fraktionen, sowie die aus dem Kulturfiltrat nach 19 h Fermentation wurden ebenfalls bei Sämlingen axenisch getestet. Fraktionen mit verschiedenen Proteinen und Peptiden, die von B. subtilis produziert werden, zeigten teilweise eine konzentrationsabhängige Wirkung hinsichtlich der Wachstumsstimulierung und zugleich Toleranzerhöhung gegenüber Salzstress, weshalb nachfolgend ein Peptidextrakt aus B. subtilis einer Testung im axenischen System unterzogen wurde. Der Peptidextrakt zeigte gleichfalls eine erkennbare konzentrationsabhängige Wirkung. Mit gleichem Testsystem wurden Auxin-Präkursoren und Auxin selbst, die als Stoffwechselprodukte von B. subtilis nachgewiesen sind, sowohl als Wurzelbehandlung, wie auch als Blattbehandlung bei Sämlingen geprüft. Zusätzlich wurde die Wirkung der Auxine auf den Wassergehalt der Sämlinge unter Salzstress, sowie die Adventivwurzelbildung von Hypokotylsegmenten aus etiolierten Sämlingen in An- und Abwesenheit von Salinität getestet. Darüber hinaus wurde die Aufnahme und der Transport von Auxinen, ebenfalls bei Sprosssegmenten aus etiolierten Sämlingen in An- und Abwesenheit von Salinität geprüft. Schließlich wurde die Wirkung der Auxine auf das Wachstum und den Wassergehalt in einer Hydrokultur im Gewächshaus unter Salzstress ermittelt. Die Ergebnisse zeigen, dass namentlich Auxin-Präkursoren und z. T. Auxin als Stoffwechselprodukte von B. subtilis eine Erhöhung der Salzstresstoleranz bei der Testpflanze herbeiführen können, wenngleich die Wirkung auf die Salztoleranz sehr differenziert und unterschiedlich stark ausgeprägt war. Der vorhandene Effekt vor allem der Auxin-Präkursoren wird als offenbar bedeutendster Mechanismus für die wachstumsstimulierende und zugleich toleranzerhöhende Wirkung gegenüber Salinität des Rhizobakteriums bei Wurzelbesiedlung und Interaktion mit dem pflanzlichen Stoffwechsel diskutiert. / To find out the mode of tolerance increasing action against salinity of the plant root colonizing PGPR Bacillus subtilis, bacterial metabolites of the strains FZB24 and FZB41 were studied in the test plant tomato under the influence of high salinity. Because the culture filtrate with the whole range of produced metabolites by B. subtilis showed to a certain extent a tolerance increasing action at dilution of 0,1 % in axenic plant growth tests after 7 days treatment of seedlings and subsequent cultivation under salt stress, it has been fractionated with adsorber resin and HPLC. These fractions, as well as fractions from the culture filtrate after 19 h fermentation were tested also by seedlings in axenic culture. Fractions with different proteins and peptides, which were produced by B. subtilis, showed partly activities also depending of concentration with regard to the growth stimulation and at the same time tolerance increase against salt stress. Following also a peptide extract from B. subtilis was examined in the axenic plant test system, showing similarly a visible action depending of concentration. In the same test system there were tested further auxin precursors and auxin itself, which are known metabolites of B. subtilis, on seedlings both by root treatment and leaf treatment. Additionally was studied the action of auxins on the water content of the seedlings under salt stress, as well as on the adventitious root formation of hypokotyl segments from etiolated seedlings, in presence and absence of salinity. Finally it was studied the uptake and transport of auxins in segments of stems from etiolated seedlings in presence and absence of salinity. Lastly it was tested the action of auxins on plant growth and water content in a hydroponic cultivation under greenhouse conditions and salt stress. The results show that particularly auxin precursors and partly auxin as metabolites of B. subtilis can induce an increase in the salt stress tolerance of the test plant, although the action on the salt tolerance was differentiated and variable in its extent. The existing effect firstly of the auxin precursors is discussed as obviously main mechanism for the plant growth stimulating and at the same time tolerance increasing action of the rhizobacterium against salinity by root colonization and interaction with the plant metabolism.