O uso de bactérias promotoras de crescimento vegetal ou agentes de biocontrole como inoculantes agrícolas é uma alternativa importante e ecologicamente correta, com grandes benefícios na agricultura para substituir, ou ao menos suplementar, a excessiva utilização de fertilizantes e pesticidas. Neste trabalho avaliamos a capacidade de biocontrole e de competência rizosférica de três bactérias com características de promoção de crescimento vegetal (Plant growth promoting - PGP): Bacillus mycoides B38V, Paenibacillus riograndensis SBR5 e Burkholderia sp. 89. As três bactérias avaliadas apresentaram grande versatilidade na utilização de substratos, o que poderia lhes garantir uma vantagem competitiva no ambiente rizosférico. Porém, inconsistências foram observadas nos ensaios em câmara de crescimento, ou seja, as características de PGP e de biocontrole observadas in vitro não se refletiram em benefícios para a planta. A linhagem 89 destacou-se pela produção de um metabólito estável com ampla atividade contra fungos fitopatogênicos. Através de abordagens genômicas e de análises multilocus, descrevemos Burkholderia sp. 89 como uma nova espécie membro do complexo Burkholderia cepacia, denominada de B. catarinensis 89T. O sequenciamento de seu genoma, seguido de uma análise pela ferramenta AntiSMASH, revelou a presença de um agrupamento gênico de peptídeo sintetases não ribossomais (NRPS) relacionadas com a biossíntese do sideróforo ornibactina e um agrupamento híbrido NRPS-policetídeo sintetase responsável pela biossíntese do glicolipopeptideo cíclico com atividade antifúngica burkholdina. Como estratégia de purificação de metabólitos secundários foi utilizada a metodologia da mineração de genoma combinada com fracionamento guiado por bioensaios seguida de análises em espectrômetro de massas. Desta forma, purificamos com sucesso duas variantes de ornibactina, D e F (761 e 789 Da, respectivamente), e detectamos a variante ornibactina B (m/z= 733) e as moléculas sinalizadoras homoserina lactonas C6-HSL, 3OH-C8-HSL e C8-HSL. Análises de espectrometria de massas demonstraram a presença de um grupo de metabólitos com massas de 1240, 1254, 1268, 1216, 1244 e 1272 Da, que, provavelmente, são novas variantes do antifúngico burkoldina. Sendo assim, B. catarinensis 89T possui potencial biotecnológico com possíveis aplicações farmacêuticas e agronômicas para o biocontrole de fungos fitopatogênicos. / The use of plant growth promotion bacteria or biocontrol agents as agricultural inoculants is an important eco-friendly alternative to substitute, or at least supplement, the excessive use of fertilizers and pesticides. In this work, we evaluated the biocontrol potential and rhizosphere competence of three bacteria that had shown plant growth promotion (PGP) abilities: Bacillus mycoides B38V, Paenibacillus riograndensis SBR5 and Burkholderia sp. 89. All three bacteria presented great versatility in their substrate utilization, which could enable them to survive in a competitive rhizosphere environment. However, inconsistencies were observed in the greenhouse experiments, whereas their interesting abilities observed in vitro did not result in benefits to the plants. Strain 89 produces a stable metabolite with a wide range of antifungal activity. Genomic comparisons and multilocus sequence analysis revealed Burkholderia sp. 89 as a new species of the Burkholderia cepacia complex and we described it as B. catarinensis 89T. We sequenced its genome and analyzed it with the AntiSMASH tool. This in silico prediction revealed the presence of a nonribosomal peptide synthetase (NRPS) cluster, which is related to the production of the siderophore ornibactin. Moreover, a hybrid NRPS- polyketide synthetase cluster for the production of the antifungal cyclic glicolipopeptide burkholdin was also found. A genome mining combined with a bioassay-guided fractionation with further mass spectrometry analysis was applied for the purification of these compounds. This approach enabled us to purify and characterize two variants of the siderophore ornibactin, D and F (761 and 789 Da, respectively). Also, we could detect the variant ornibactin B (m/z= 733) and the quorum sensing molecules homoserine lactones C6-HSL, 3OH-C8-HSL and C8-HSL in the supernatant of B. catarinensis 89T. Mass spectrometry analysis showed the presence of a group of metabolites with the masses 1240, 1254, 1268, 1216, 1244 and 1272 Da, which are probably new variants of the antifungal metabolite burkoldin. Therefore, B. catarinensis 89T has a great biotechnological potential for the production of metabolites with pharmaceutical and agricultural applications for the biocontrol of phytopathogenic fungi.

Actinobactérias ocorrem em diversos ambientes e possuem grande potencial na produção de enzimas, antibióticos e fármacos. O presente trabalho teve como objetivo o isolamento, a identificação e a bioprospecção de actinobactérias da rizosfera de milho. Foram isoladas 60 linhagens de actinobactérias de plantas sadias de milho, cultivadas em diferentes regiões do Estado de São Paulo. Os isolados com maior atividade, CCMA30 e CCMA33, foram identificados por meio do seqüenciamento parcial do gene 16S rRNA como pertencentes à espécie Streptomyces griseorubiginosus, nestes isolados foi também detectada a presença de genes PKS.O fracionamento do extrato bruto da linhagem CCMA 33 revelou que a fração 7 (m/z 719-768) com atividade antifúngica, foi submetida a fragmentação (MS/MS), onde pelas buscas no dicionário de produtos naturais confirmou-se a presença de Julichromes Q 6-6, substância sintetizada por policetídeos e de mais duas Julichromes (Q 1-3 e Q 3-3). Estes resultados evidenciam a produção de biocompostos de interesse biotecnológico por actinobactérias da rizosfera. / Actinobacteria occur in different environments and have a great potential in producing enzymes, antibiotics and medical molecules. The present work aimed the isolation, identification and the bioprospection by rhizosphere actinobactérias. Sixty strains were isolated from healthy plants of maize, cultivated in different regions Socorro, Serra Negra and Ribeirão Preto. The two isolates with higher activity CCMA30 and CCMA33 were identified by the partial sequencing of the 16S rDNA gene as belonging to the species Streptomyces griseorubiginosus. In these isolates it was detected the presence of PKS genes. The fractioning of the crude extract of the strain CCMA 33 has revealed that fraction 7 (m/z 719-768) displayed antifungal activity and it was submitted to fragmentation (MS/MS), where by searches in the dictionary of natural products detected the presence of Julichromes Q6-6 and the presence of two other Julichromes (Q1-3 and Q3-3). These results highlight the production of secondary metabolites of biotechnological interest by rhizosphere actinobacteria.

A interação entre bactérias e plantas resulta na ocorrência de vários processos biológicos no ambiente e pode ser regulada por diferentes fatores. Considerando que um recíproco reconhecimento ocorre entre as espécies de bactérias e plantas, alterações nos fatores bióticos e abióticos interferem diretamente nesta interação levando a modificações na composição das comunidades bacterianas associadas às plantas. No presente trabalho foram avaliados diferentes fatores que estão diretamente relacionados a este assunto. Atualmente, a aplicação de técnicas de microbiologia molecular permite acessar alterações causadas nestas comunidades de maneira independente do cultivo bacteriano. Desta maneira, foi demonstrado que na rizosfera de plantas de tabaco, os diferentes estágios de desenvolvimento são os principais determinantes da composição da comunidade bacteriana em detrimento do genótipo das plantas, sejam estas transgênicas ou convencionais. Utilizando plantas transgênicas e convencionais de eucalipto de diferentes genótipos, foi verificado que diferentes plantas não transgênicas podem apresentar comunidades bacterianas mais distintas do que quando os clones não transgênicos são comparados com os transgênicos. No entanto, efeitos específicos podem ocorrer na interação bactéria-planta, como o observado pela inibição da população de Methylobacterium spp. em plantas transgênicas de eucalipto TR-15. Avaliando o efeito da inoculação de bactérias endofíticas na composição de comunidades bacterianas associadas à plantas de batata de diferentes cultivares, os resultados mostram que a inoculação de Pseudomonas putida altera a comunidade bacteriana de maneira similar a alteração da variedade da planta. Os demais isolados avaliados, classificados como Paenibacillus sp. e M. mesophilicum, causam menores alterações na composição das comunidades bacterianas. Adicionalmente foi demonstrado que a colonização das plantas de batata pelo endófito P. putida resulta em pequena alteração no perfil metabólico da planta hospedeira. Por fim, buscando melhores métodos de isolamento da comunidade bacteriana da rizosfera de batata, os resultados mostraram que a mimetização do ambiente pode resultar em melhor acesso da diversidade bacteriana por meio de isolamento e cultivo das espécies componentes desta comunidade. / The plant-bacteria interactions result in the occurrence of biological process in the environment and might be regulated by different factors. Considering that a reciprocal recognition occur amongst bacteria and plants species, shifts in biotic and abiotic factors interfere directly on these interactions, leading to modifications in the composition of bacterial communities to plants associated. In the present work different factors related to this subject were evaluated. Nowadays, the applications of techniques of molecular microbiology allow assessing the shifts caused on these communities by a culture independent approach. On this way, it was demonstrated that in rhizosphere of tobacco plants, different stages of plants development are main determinants of bacterial community composition rather than the plants genotypes, transgenic or not. Using plants transgenic or not, carrying different genotypes, it was verified that different non-transgenic plants could harbor bacterial communities more distinct than those observed in association with transgenic plants. However, specific effects could be observed like the inhibition of Methylobacterium spp. population in eucalyptus transgenic plants TR-15. Considering the effect of endophytic bacteria inoculation in the composition of bacterial communities associated to different cultivars of potato plants, the results show that inoculation of Pseudomonas putida causes similar shifts in the bacterial community similarly to those observed when different cultivars are considered. Other evaluated strains, classified as Paenibacillus sp. and M. mesophilicum, caused minor alterations in the composition of bacterial communities. In addition, it was demonstrated that plant colonization by endophytic P. putida results in small effects on the metabolic profile of host plant. At least, aiming better methodologies for bacteria isolation from potato rhizosphere, the results show that mimicking the natural environment could result in a better assessment of bacterial diversity by isolation of species present in this community.

A espécie Araucaria angustifolia, pertencente ao fragilizado Bioma Mata Atlântica, foi, durante décadas, uma das maiores fontes de madeira no Brasil. Essa espécie é de grande importância sendo fonte de alimento e matéria prima para a produção de móveis, polpa de celulose e vernizes. Devido à grande importância econômica e ambiental da araucária, estudos voltados à preservação e manejo da espécie se tornaram necessários. Este trabalho teve como finalidade isolar actinobactérias, presentes na rizosfera de araucária, antagônicos aos fungos Fusarium sp .e Armillaria sp. Estes patógenos são responsáveis por danos às raízes e sementes causando podridão e perda de mudas. Além disso, verificou-se também o efeito das actinobactérias sobre a germinação de esporos de Gigaspora rosea. Após a seleção dos melhores inibidores, identificação morfológica e seqüenciamento do gene 16S rRNA, verificou-se o efeito destes sobre o crescimento de Pinus taeda, na presença e na ausência do fungo ectomicorrízico (Suillus brevipes). Os isolados ainda foram avaliados quanto à produção de enzimas como quitinase, lipase, fosfatase, celulase, amilase e protease. Para o isolamento foram coletadas raízes de 15 árvores adultas presentes na mata nativa. Dez gramas de raízes frescas com resíduos de solo aderidos firmemente foram agitados por 30 minutos em solução salina 0,85 % padronizando como solo rizosférico aquele que soltou das raízes. Neste isolamento foram utilizadas duas metodologias: por plaqueamento e por separação física pela utilização de membrana de 0,45 micrômetros. Foram obtidos 33 possíveis actinobactérias. Os isolados foram testados quanto à capacidade antagônica pelo método de contato direto, inoculando-os a 30 mm de distância do fungo fitopatogênico Fusarium sp., em placas de petri contendo meio ISP2. Os halos de inibição foram medidos após 3, 5, 7 e 10 dias. De todos os isolados testados 6 mantiveram a inibição por 10 dias com manutenção de halos de inibição de até 4 mm contra um Fusarium sp. isolado de sementes de milho e 2 foram eficientes na inibição de Fusarium sp, patógeno de Pinus sp. A análise de inibição do fungo Armillaria sp, foi feito em meio líquido medindo o crescimento em mg/dia após 30 dias na presença de extratos de actinobactérias e também pela contagem de rizomorfas em placa de petri após 20 dias de incubação em contato direto com as actinobactérias. Verificou-se que de 28 isolados testados 24 foram capazes de inibir a produção de rizomorfas, destacando-se o isolado A43 que foi capaz de inibir ambos os fungos, Fusarium e Armillaria. Os esporos de Gigaspora rosea tiveram a taxa de germinação avaliada na presença de actinobactérias a partir da técnica de dupla camada. Todos os seis isolados testados foram capazes de estimular a germinação dos esporos desse fungo micorrizico. Nenhum dos seis isolados (A43, A43b, PNA, A64, A75 e A93) foi capaz de produzir fosfatases e lipases. Porém os isolados A93, A75, A64 e PNA produziram protease, quitinase e amilase. Em casa de vegetação, foi testado o efeito de seis isolados, na presença e não ausência de ectomicorriza (Suillus brevipes), sobre plântulas de Pinus taeda. Analisou-se o diâmetro, a altura, a massa seca da raiz e da parte aérea e também o fósforo da parte aérea. Destaca-se o isolado A43 que, quando na ausência de ectomicorriza, estimulou o desenvolvimento da planta em relação ao controletambém sem ectomicorriza, provocando aumento de 100 % para massa seca da parte aérea e da raiz. Os resultados encontrados neste trabalho poderão levar ao desenvolvimento de novas tecnologias, visando à identificação de novos metabólitos e novas técnicas de manejo, voltados ao controle de doenças de plantas, especialmente as espécies arbóreas. / The tree Araucaria angustifolia, belonging to the endangered Atlantic Forest biome, for many decades was the source of Brazilian wood. This species is also very important in providing food and feed, as well as raw material for joinery, cellulose pulp and varnish. Due to the economic and environmental importance of A. angustifolia, research projects involving the preservation and management of this species are becoming more urgent and necessary. The aim of this work was to isolate Araucaria rhizosphere actinobacteria with antagonic effects against the plant pathogens Fusarium sp. and Armillaria sp. These fungi cause root rot and seed damage, with the consequent loss of seedlings. Moreover, the effect of these actinobacteria on Gigaspora rosea spore germination was studied. After the selection of the best pathogen inhibitors, we also tested the effect of these microorganisms on Pinus taeda growth, in the presence or absence of the ectomycorrhizal fungus Suillus brevipes. The production of protease, chitinase, lipase, phosphatase, cellulase and amylase of these bacteria in culture media was also investigated. For the isolation of rhizosphere bacteria, we collected roots of 15 adult trees in a native forest. Ten grams of fresh roots with soil residues adhered to the surface were shaken in 0,85 % salt solution for 30 minutes. Two techniques were used, the dilution plate method and the coverage of the medium, utilizing a 0,45 µm membrane to separate these filamentous bacteria. About 33 actinobacteria were isolated. After isolation the actinobacteria were tested against the plant pathogenic fungi Fusarium spp., utilizing dual culture techniques and ISP2 medium. The inhibition halo was measured after 3, 5, 7 and 10 days. Six of our isolates maintained an inhibition zone measuring at least 4 mm against Fusarium sp. isolated from corn seed and 2 mm against the Fusarium, which causes root root of Pine trees. For the inhibition test of Armillaria, in liquid medium with the addition of culture extracts of actinobacteria, the growth in mg/day was measured after thirty days growth, and the number of rizomorphs produced in culture dishes after twenty days in dual culture with the actinobacteria was counted. Six bacteria proved to be antagonistic (A43, A43b, A64, PNA, A93 e A75), and only one had no effect. Possibly the elevation of the pH value played also a role in this situation. About 24 of 28 isolates inhibited the rizomorph production, especially the isolate A43 that showed a double antagonism against Fusarium and Armillaria. The dual layer test was used to investigate the reaction of the arbuscular mycorrhizal fungus Gigaspora rosea spore germination to the presence of actinobacteria. All the six actinobacteria stimulated the germination, but the germ tube did not grow straight forward as in the control. This result may indicate a negative effect against this arbuscular mycorrhizal fungus. None of the six isolates tested (A43, A43b, PNA, A64, A75 and A93) produced phosphatases and lipases, but A93, A75, A64 and PNA produced protease, amylase and chitinase. Isolates A43 and A43b did not produce any of the enzymes tested. This fact suggests that there is production of an antibiotic acting against the pathogenic fungi. Pinus taeda seedlings were grown under green-house conditions. After three months the stem diameter, shoot height, root and shoot dry weight and shoot phosphorus content were evaluated. Plants with ectomycorrhiza presented a significant growth promotion in comparison with the nonmycorrhizal ones. Among the actinobacteria in the absence of mycorrhiza only the isolate A43 produced a 100% growth enhancement in comparison with the control plant without ectomycorrhiza. The results presented in this dissertation could lead to the development of new technologies and new management techniques, with regard to the control of plant diseases, especially in tree species.

Face à une demande mondiale en bois en constante augmentation, les forêts plantées sont en rapide expansion, notamment au Brésil où les plantations d'eucalyptus pourraient couvrir 10 millions d'hectares d'ici 2020. Ces plantations hautement productives soulèvent des questions importantes au niveau (i) de leur durabilité dans un contexte de changement climatique et (i) de leur impact sur l’environnement, en particulier sur les cycles de l'eau, du carbone et des nutriments. La capacité des eucalyptus à développer un système racinaire très profond (<15 m) pour atteindre la nappe phréatique, pourrait jouer un rôle clé dans l’adaptation à des disponibilités plus faible en eau. Cependant, le rôle de ces racines fines profondes dans la nutrition des plantes a jusque-là été extrêmement peu documenté.Dans ce contexte, l'objectif général de cette thèse était d'évaluer l'influence de la profondeur et de la disponibilité de l’eau sur le fonctionnement des racines fines à travers l’étude de la rhizosphère. Pour cela, une plantation clonale d’Eucalyptus grandis de 5 ans a été étudiée au Brésil sous deux régimes hydriques contrastés : un traitement + W, recevant une pluviométrie normale a été comparé à un traitement –W dans lequel 37% des pluviolessivats sont exclus. Des essais ont été réalisés dans le but d'appliquer une technologie de laboratoire innovante au champ : les optodes, permettant la cartographie du pH rhizosphérique notamment. Les rhizodepôts libérés par les racines fines d’eucalyptus interférant avec le signal du capteur optique, nous n’avons pas pu obtenir de résultats interprétables. Cependant, des tests réalisés sur le pin nous laissent confiants quant à la possibilité d'utiliser ce système, en suivant quelques recommandations. Des analyses destructives ont été réalisées sur du sol rhizosphérique et non-rhizosphérique échantillonnés le long d'un profil de 4 m. Au niveau de la nutrition : une accumulation de potassium et de protons au sein de la rhizosphère a été observée, en particulier en dessous d’1 m (x3,0 et x1,1 pour K et H3O+ en + W, resp.) et en condition de réduction des précipitations (x7,0 et x1,4 pour K et H3O+ en –W, resp.). La répétition de ces mesures pendant la saison des pluies a confirmé l'enrichissement de protons et de K dans la rhizosphère, ce qui suggère que ces processus se produisent tout au long de l'année. Cependant, l’absence d’effet traitement durant la saison des pluies laisse suggérer une bonne résilience potentielle du système. La quantité de K amenée à la surface des racines fines par flux de masse, estimée à 2 kg de K ha-1 an-1, ne permet pas d’expliquer la quantité de potassium absorbé par les arbres, estimée à 17,5 kg de K ha-1 an-1, ni l'accumulation observée dans la rhizosphère. Une explication plus probable est l'altération des minéraux potassiques induite par le fonctionnement racinaire, et notamment l’acidification. La preuve de la possibilité d’association ectomycorhiziennes jusqu'à 4 m de profondeur renforce l'hypothèse d'un rôle clé des racines fines profondes dans la nutrition des plantes. Une concentration élevée en Al3+ a aussi été mesurée au sein de la rhizosphère (jusqu'à 12 mg kg-1). Au niveau du stockage du carbone : malgré la diminution exponentielle attendue de la concentration en C et N avec la profondeur (de 0,72 à 0,12 ‰ entre 0 et 4 m), nos résultats ont montré que plus de la moitié du stock de carbone contenu dans le sol non rhizosphérique était situé en dessous d’1 m. Une accumulation de C dans la rhizosphère a été mesurée, en particulier en profondeur (x1,4 en dessous d’1 m en + W) et en condition de réduction des pluies (x3,0 à 4 m en –W). Les mêmes tendances ont été observées pour N. L’effet rhizosphérique a été conservé pendant la saison des pluies, mais pas l'effet du traitement. Ce travail a confirmé que les racines fines profondes jouent un rôle clé, pour la nutrition des plantes et le stockage de carbone, en contexte de changement climatique particulièrement. / Due to the constant increase of the world demand for wood, the planted forests are in fast expansion notably in tropical countries such as Brazil where plantations of eucalypts, the most productive and spread out planted species, may reach 10 million ha by 2020. The expansion of these plantations on less fertile sites, combined with the context of climate change lead to important issues about (i) the sustainability of these plantations under more frequent and intense drought events and (i) the impact on the environment of these highly productive plantations with very short rotations (6 yr), particularly for nutrient, water and carbon cycles. Eucalypt trees are able to develop very deep root system (<15 m) to reach the water table, and this may play a key role to cope with decreasing soil water availability. However, the role of these deep fine roots in plant nutrition is dramatically under-documented. In this context, the general objective of this study was to evaluate the influence of soil depth combined with water availability on fine root functioning through characterization of the rhizosphere properties. For this purpose, a 5-yr-old clonal Eucalyptus grandis plantation was studied in Brazil in two contrasted water regimes: the +W treatment, receiving normal rainfall was compared with the –W treatment where 37% of the throughfall were excluded in order to mimick the future climate forecasted in the region. Exploratory tests were carried out for the introduction of an innovative lab technology under field conditions: the optodes, which allow mapping rhizosphere pH. The rhizodeposition of eucalypt fine roots interfered with the optical sensor signal and prevented us to get interpretable results. However, some tests on pine trees let us confident of the possibility of using our system for field studies at depth, using some recommendations. Destructive analyses of rhizosphere and bulk soil samples collected along a 4-m deep soil profile showed an effect of depth and rainfall reduction on rhizosphere pH, potassium concentration, rhizodeposition pattern and carbon storage capacity. Concerning nutrition issues, we found an accumulation of potassium and protons within the rhizosphere, especially below 1-m depth (x3.0 and x1.1 for K and H3O+ in +W, respectively) and in reduced rainfall conditions (x7.0 and x1.4 for K and H3O+ in –W, respectively). Repeating these measurements during rainy season confirmed the enrichment of protons and potassium within the rhizosphere, suggesting that these processes may occur all along the year but no treatment effect was observed anymore, pointing to a potential good resilience of the system. The amount of K brought to fine roots by mass flow was estimated to 2.0 kg K ha-1 yr-1 and could not explain the amount of potassium taken up by trees estimated to 17.5 kg K ha-1 yr-1 and the observed accumulation in the rhizosphere. A more likely explanation was the root-induced weathering of K-bearing minerals, partly related to rhizosphere acidification. Proof of ectomycorrhizal association down to 4-m depth further supported the hypothesis of a key role of deep fine roots in plant nutrition. High exchangeable Al3+ concentration was found within the rhizosphere (up to 12.0 mg kg-1). Concerning the carbon storage issue, despite an expected exponential decrease of C and N concentrations within the bulk soil with depth (0.72‰ to 0.12‰ from top soil to 4-m depth), our results showed that more than half of soil C stock within the bulk soil occurred below the first meter. An accumulation of C within the rhizosphere was found, especially at depth (x1.4 below 1 m in +W) and in reduced rainfall conditions (x3.0 at 4-m depth in –W). The same trends were found for N. The rhizosphere effect was conserved during rainy season but not the treatment effect. This work confirmed that deep fine roots play a key role, especially in the context of climate change, for plant nutrition and carbon storage.

Les plantes interagissent en permanence avec une grande diversité de microorganismes qu'elles sélectionnent entre autre au niveau de leurs racines. Certaines bactéries, qualifiées de PGPR (Plant Growth-Promoting Rhizobacteria), sont capables de stimuler la croissance et la santé de la plante, grâce à l'expression d'une large panoplie de propriétés phytobénéfiques. L'hypothèse actuelle serait que les PGPR possédant un nombre maximal de ces fonctions auraient un plus fort impact bénéfique sur le végétal. Toutefois, l'occurrence de ces PGPR multifonctions dans la rhizosphère n'est pas connue. De plus, les possibles interactions entre propriétés co-occurrentes au sein d'une même PGPR et la résultante de ces interactions sur la plante sont relativement peu documentées. Dans ce contexte, ce projet de thèse a eu comme objectif général de mieux comprendre la distribution des propriétés phytobénéfiques chez un groupe bactérien possédant un large éventail de ses propriétés, celui des Pseudomonas fluorescents et d'évaluer chez une PGPR modèle multifonction, Pseudomonas fluorescens F113, si des interactions fonctionnelles existent entre ces propriétés et déterminer leurs contributions respectives à l'effet phytostimulateur. L'étude d'environ 700 isolats de Pseudomonas provenant de la rhizosphère de deux cultivars de maïs et de sol nonrhizosphérique, a mis en évidence que ces cultivars sélectionnent majoritairement des Pseudomonas fluorescents (i) arborant un nombre réduit de propriétés phytobénéfiques (1 à 5 propriétés) et (ii) appartenant à des sous-groupes taxonomiques particuliers. Par ailleurs, l'étude de génomique comparative que nous avons menée au sein du groupe des Pseudomonas fluorescents souligne le lien entre phylogénie et les profils de propriétés phytobénéfiques possédées par les Pseudomonas. Enfin, chez P. fluorescens F113, les propriétés co-occurrentes ne contribuent pas de façon égale à l'effet observé sur la plante. L existence d'interactions entre ces propriétés co-occurrentes a également été mise en évidence. De ce réseau d'interactions, résultera un effet bénéfique observé sur la plante particulier. Ces résultats sont importants pour mieux comprendre la place et le rôle de ces PGPR multifonctions dans la rhizosphère. Ils permettent d'approfondir nos connaissances à propos du fonctionnement écologique des Pseudomonas fluorescents au sein du rhizomicrobiote / Plants are constantly interacting with a huge diversity of microorganisms, especially in the rhizosphere, where plant roots (through rhizodeposition) will select particular bacterial populations. Some bacteria, called PGPR (Plant Growth-Promoting Rhizobacteria), are able, in turn, to improve plant growth and health, through the expression of a wide range of plantbeneficial properties. The current hypothesis is that PGPR harboring a maximum number of these plant-beneficial properties would provide a better effect on plant. However, the occurrence of these multi-trait PGPR in the rhizosphere is unknown. In addition, the interaction between co-occurring plant-beneficial properties within the same PGPR and their resulting effects on plant are poorly documented. In this context, the aims of this thesis were to (i) determine the distribution of plant-beneficial properties in the fluorescent Pseudomonas bacterial group, known to harbor a wide range of these properties, and (ii) to evaluate in the PGPR model strain Pseudomonas fluorescens F113, if a crosstalk between plant-beneficial properties occurs and to determine the relative contribution of each co-occurring plantbeneficial properties. The study of 700 Pseudomonas isolates from the rhizosphere of two maize cultivars and from non-rhizosphere soils shows that cultivars mostly select fluorescent Pseudomonas (i) displaying few plant-beneficial properties (up to 5 properties) and (ii) belonging to particular taxonomic subgroups. Furthermore, the comparative genomic study we conducted within the fluorescent Pseudomonas group emphasizes the link between phylogeny and plant-beneficial profiles owned by Pseudomonas. Finally, in P. fluorescens F113, co-occurring plant-beneficial properties do not contribute equally to the observed effects on plant. The existence of crosstalks between these co-occurring plant-beneficial properties was also highlighted. This network of functional interactions may lead to specific effects on plant. These results are important for understanding the place and role of multi-trait PGPR in the rhizosphere. They help to deepen our understanding of the ecological functioning of fluorescent Pseudomonas among the rhizomicrobiote

Les Hydrocarbures Aromatiques polycycliques (HAP) sont des polluants organiques persistants, dont la faible disponibilité dans les sols historiquement contaminés limite leur biodégradation. La capacité des plantes à favoriser l’élimination de ces polluants par l’action des microorganismes associés à leur rhizosphère a été montrée (rhizodégradation). Toutefois les résultats sont variables, suggérant la variabilité spatio-temporelle des processus. Des études à différentes échelles de temps et d’espace, utilisant des microcosmes et des dispositifs in situ ont été menées pour préciser ces phénomènes. L’étude de la variabilité spatiale des HAP et de la diversité bactérienne a été menée au sein de deux rhizosphères contrastées (ray-grass et luzerne), à l’échelle centimétrique après 37 jours de culture sur un sol de friche industrielle. Les résultats ont montré une spatialisation de la teneur en sucre et du pH, de la diversité bactérienne et de l’abondance microbienne, spécifique de l’espèce végétale, mais sans structuration de la teneur en HAP. L’étude de la variabilité temporelle de ces processus a révélé la dissipation en 6 jours des polluants biodisponibles ainsi qu’un effet positif des plantes par rapport au sol nu sur la dissipation des HAP et sur l’expression des gènes de HAP-dioxygénase. Une étude à plus long terme (6 ans) et in situ a montré que le couvert végétal ralentit la dissipation des HAP et influencent fortement la diversité microbienne, tout comme certains paramètres édaphiques. L’ensemble de ces résultats montre l’importance de la biodisponibilité des HAP qui conditionne leur dissipation, et de la dynamique des communautés microbiennes dans la rhizosphère / Polycyclic Aromatic Hydrocarbons (PAH) are persistent organic pollutants in soil, whose degradation in aged-contaminated soil is limited by their low bioavailability. The ability of plants to promote pollutant dissipation through the action of rhizosphere microorganisms has been shown (rhizodegradation). However contrasted results were obtained suggesting spatio-temporal variability of processes. Different experiments, with different time and space scales, using microcosm and field trials were achieved to precise phenomena. Study of spatial variability of PAH and bacterial diversity were done in two-contrasted rhizospheres (ryegrass and alfalfa) at centimeter scale after 37 days of plant growth, on a wasteland aged contaminated soil Results showed spatial structuration of sugar content, pH, bacterial diversity and microbial density, depending on plant species, but no vertical gradient was observed for PAH concentration. Study of temporal variability of processes showed dissipation of bioavailable fraction of pollutant in just 6 days and in comparison with bare soil, a positive impact of plant was shown on PAH dissipation and on expression of PAH dioxygenase genes. A long-term study (over 6 years) in a field trial revealed that plant cover slowed down PAH dissipation and impacted bacterial and fungal diversity as edaphic parameters. All these results underlined the importance of PAH bioavailability for the dissipation process and of spatio-temporal dynamic of microbial community, in the rhizosphere

Le phosphore (P) est un élément nutritif essentiel au cycle de croissance des plantes. Du fait de leur forte rétention sur les phases solides, la concentration en ions phosphates (PO4) dans la solution du sol est généralement faible (Hinsinger, 2001). Ces dernières décennies la production agricole mondiale s'est accompagnée d'une augmentation massive de l'usage de fertilisants inorganiques (Tilman, 2002). Ces pratiques ne constituent pas une solution durable de par leurs impacts négatifs sur l'environnement et la raréfaction des ressources minières PO4 prévu ces prochaines décénies (Cordell et al., 2009). Le développement de systèmes de cultures associées céréale - légumineuse constitue une voie d'optimisation durable de la fertilité des sols vis-à-vis du P (e.g. Cassman et al. 1999 ; Betencourt et al., 2012). Cependant les variables contrôlant la disponibilité du P au sein de ces systèmes n'ont pas encore été clairement identifiées (e.g. Hinsinger et al., 2011 ; Li et al., 2010). Leur optimisation doit passer par une meilleure compréhension des processus et mécanismes rhizosphériques sous jacents à l'augmentation du P disponible. De récents travaux ont montré l'intérêt des modèles mécanistes pour l'amélioration de ces connaissances au sein de la rhizosphère du blé dur (e.g. Devau et al., 2010, 2011b). Cette approche repose sur une description thermodynamique (i.e. à l'équilibre) de l'adsorption des ions à la surface des minéraux et de la matière organique (Hiemstra et Van Riemsdijk, 1996 ; Kinniburgh et al., 1999).Dans ce contexte, l'objectif de mon travail de recherche était de développer et d'utiliser la modélisation mécaniste afin de déterminer les variables régulant la disponibilité du P au sein de la rhizosphère du pois chiche cultivé seul puis en association avec le blé dur. L'exsudation d'anions organique étant largement reconnu dans la rhizosphère du pois chiche (e.g. Veneklaas et al., 2003) des simulations in silico (i.e. prédictives) des effets de l'adsorption de citrate sur la disponibilité du P ont été préalablement réalisées. Ces dernières portaient sur des sols à propriétés contrastés (composition minéralogique, calcium échangeable, DOC, SOM, pH) afin d'identifier l'influence de ces variables sur le comportement du citrate et des ions PO4. Cette première étude a entre autre permis de mettre en évidence un mécanisme, jusqu'à lors inconnu, induisant une diminution du P disponible suite à de faibles apports en citrate. Les sorties du modèle ont permis d'expliquer ce résultat par une succession d'interactions électrostatiques impliquant respectivement le citrate (Cit3-), le calcium (Ca2+) et le P (PO43-) à la surface des minéraux argileux (Duputel et al., 2013a). Dans un second temps, des mesures ont permis de confirmer l'influence de ce mécanisme dans un chromic cambisol (Duputel et al., 2013b). Dans un dernier temps, en utilisant une approche combinant expérimentation sol – plante en rhizobox et modélisation mécaniste, mes recherches ont permis d'identifier et de hiérarchiser les processus et mécanismes régulant la disponibilité du P au sein de la rhizosphère du pois chiche et du blé dur cultivés seuls et en association (Duputel et al., 2013c,d). Ces résultats confirment la prédominance du mécanisme d'adsorption / désorption pour le contrôle de la disponibilité du P dans la rhizosphère. Les effets cumulés de trois processus racinaires permettent d'expliquer l'augmentation du P disponible mesurée dans la rhizosphère. Il s'agit du prélèvement en calcium, du flux de proton et de l'exsudation de citrate. Les effets des deux premiers processus racinaires prédominent. Le PO4 ainsi libéré dans la rhizosphère proviendrait essentiellement de l'illite, qui est une phase majeure pour la fixation de PO4 dans le sol étudié (i.e. luvisol). / Phosphorus (P) limits plant growth in many soils as phosphate (PO4) is naturally present in very low concentration in soil solutions (0.1 to 10 µM; Hinsinger 2001). Low soluble PO4 concentrations (i.e. available P) are the result of the very high adsorption affinity of PO4 for mineral surfaces and the low solubility of PO4-containing minerals (e.g. Shen et al., 2011). The use of PO4 fertilizers has permitted to increase P availability in soil and ultimately has contributed to massively increase crop yield the last decades (Tilman et al., 2002). Such practices are not sustainable nowadays because of the scarcity of phosphate rock and because of the environmental issues related to PO4 fertilization (e.g. Bumb and Baanante, 1996; Cordell et al., 2009). A way of research is the development of alternative strategy to better exploit soil P ressources, as the management of cereal – legume intercropping (e.g. Cassman et al. 1999 ; Betencourt et al., 2012). However the designation of variables that control P availability, under such a device, is difficult as the involved processes are strongly contextual (e.g. Hinsinger et al., 2011 ; Li et al., 2010). The managment of intercropping systems requires an acute knowledge about the processes and mechanisms that control soil P availability in a context of a P uptake facilitation phenomena. Recent studies used a new approach to better understand the complex interacting processes and controlling mechanisms that alter P availability in the rhizosphere of durum wheat (e.g. Devau et al., 2010, 2011b). It is based on the use of surface complexation modelling which offer a mechanistic description of the adsorption mechanism onto soil minerals and organic matter. (Hiemstra et Van Riemsdijk, 1996 ; Kinniburgh et al., 1999).The aim was to develop and use this approach in order to identify and rank the main involed processes and mechanisms that regulate P availability in the rhizosphere of chickpea sole-crop and chickpea – durum wheat intercropping. The release of organic anions is well known in the rhizosphere of chickpea. Therefore in silico (i.e. predictive) simulations of the effect of citrate adsorption on soil P availability were first carry out. The effect of several soil variables on the citrate efficiency were investigated (mineralogy, exchangeable Ca, DOC, pH, SOM). We found that citrate can either increase or decrease P availability in soil, depending mainly on the occurrence of 2:1 clay minerals and on the concentrations of citrate, adsorbed Ca, and soil organic carbon. Model output showed that the deleterious effect of citrate was due to electrostatics interactions between citrate, calciumand P onto mineral surface sites (Duputel et al., 2013a). Then a second experimental and modelling study confirmed that the release of citrate in soils and its subsequent adsorption onto minerals can produce the decrease of P availability (Duputel et al., 2013b). Finally, throught a combined approach (experiment - mechanistic modelling) my research works enable to identify and rank the processes and mechanisms that regulate P availability in the rhizosphere of intercropped species (Duputel et al., 2013c,d). Results underline the importance of adsorption mechanisms in our experimental conditions. Calcium uptake and soil acidification were the two main processes involved in the control of available P in the rhizosphere of the intercropped plants. Citrate release and P uptake were also involved in the variation of P availability but to a lower extent. Predominantly phosphate adsorbed onto illite was mobilized by such rhizosphere processes.

Le phosphore (P) est un nutriment majeur qui est souvent limitant pour la croissance des plantes dans les agro-écosystèmes mais le caractère non renouvelable des réserves en roches phosphatées rend urgente la nécessité de trouver une alternative aux fertilisants phosphatés et de mieux utiliser les ressources en P du sol. Dans ce contexte, l'objectif de ma thèse était d'élucider l'implication de processus rhizosphériques déterminant la disponibilité de P du sol dans des cultures associées céréale/légumineuse, et in fine l'acquisition de P par ces associations. Nous avons fait l'hypothèse que la facilitation de l'acquisition de P dans rhizosphère de ces cultures associées était la conséquence de processus microbiens, en lien avec des changements du carbone (C), de l'azote (N) et du P de la biomasse microbienne, ou de l'abondance de certains groupes microbiens spécifiques. Ma stratégie de recherche s'est donc focalisée sur les modifications de CNP de la biomasse microbienne et des communautés de microorganismes dans la rhizosphère de cultures associées relativement aux cultures pures correspondantes. Les effets de la fertilisation en P ou N et des modifications de pH en résultant en lien avec la fixation de N2 ont aussi été étudiés, à la fois au champ et en conditions contrôlées en microcosme de type rhizobox. Le blé dur et différentes espèces de légumineuses à graines ont été cultivées seules ou associées dans deux types de sols présentant des historiques de fertilisation différents. Dans une première expérience au champ avec un sol calcaire Méditerranéen présentant une forte disponibilité en P, nous avons montré que les cultures associées céréale/légumineuse pouvaient faciliter le recyclage de P en augmentant la biomasse microbienne et en modifiant sa stoechiométrie CNP dans la rhizosphère. Dans une seconde expérience au champ dans un essai de fertilisation P de longue durée, nous avons mis en évidence qu'une culture associée céréale/légumineuse pouvait augmenter le P de la biomasse microbienne à bas niveau de disponibilité en P et modifier les groupes microbiens susceptibles d'être impliqués dans le recyclage de P du sol. Enfin, nous avons conduit une expérience en rhizobox en conditions contrôlées avec le sol de cet essai de longue durée dans laquelle nous avons manipulé la disponibilité du nitrate, en vue d'évaluer les interactions des processus mentionnés plus haut et les modifications de pH induites par les racines dans la rhizosphère. En comparant la culture associée et les cultures pures correspondantes, nous avons obtenu des différences significatives de pH, de disponibilité en P, de CNP de la biomasse microbienne et de communautés de microorganismes dans la rhizosphère. Bien que ces modifications aient concerné un groupe de bactéries productrices de phytase, les relations causales entre les effets observés n'ont pu être établies. Une meilleure connaissance de tels processus devrait dans le futur nous permettre de définir des cultures associées performantes pour l'acquisition de P. / Phosphorus (P) is a major nutrient that is often limiting plant growth in agroecosystems but phosphate rocks being a finite resource, there is an urgent need to find alternatives to P fertilizers and to better use soil P resources. In this context, the aim of my thesis was to elucidate the implication of rhizosphere processes determining the availability of soil P in cereal/legume intercropping systems, and ultimately the acquisition of P by these intercrops. We hypothesized that the facilitation of P acquisition in the rhizosphere in such intercrops was the consequence of microbially mediated processes, as evidenced by shifts of microbial biomass carbon (C), nitrogen (N) and P, or of specific microbial groups. Thus, my research strategy focused on root-induced changes of microbial biomass CNP and community in the rhizosphere of intercrops relative to the same crops grown alone. The effects of P or N fertilization and consequent pH changes as related to N2-fixation were also investigated, either in field experiments or in the controlled conditions of rhizobox microcosms. Durum wheat and different grain legume species were grown as sole crops and intercropped on two types of soils with different fertilization histories. In a first field trial in a calcareous, Mediterranean soil with high P availability, we demonstrated that cereal-legumes intercrops could be used to facilitate P cycling through increasing the microbial biomass and altering its CNP stoichiometry in the rhizosphere. In a second field experiment in a long term P fertilizer trial, we evidenced that a cereal-legume intercrop could increase the microbial biomass P at low P availability and modify microbial groups possibly involved in soil P cycling. Finally, we designed a rhizobox experiment in controlled conditions with soils of this long term trial where we manipulated nitrate availability, in order to assess the interactions of the above-mentioned processes with root-induced pH changes in the rhizosphere. When comparing intercrops and sole crops, we obtained significant differences of soil pH, P availability and microbial biomass CNP and community in the rhizosphere. Although such changes concerned phytase-producing bacteria, the causal relationships between the observed effects still need to be established. A better knowledge of such processes shall help designing more P-efficient intercropping systems in the future.

Pour répondre aux besoins des populations humaines, l'agriculture est de plus en plus intensive, utilisant un très grand nombre d'engrais azotés pour augmenter les rendements. Ces engrais sont utilisés parce que l'azote est l'un des facteurs les plus important et limitant pour la croissance des plantes. L’azote sous forme de nitrate est soumis à des problématiques de pollutions pouvant affecter l’environnement ainsi que la santé humaine. Les défis de l'agriculture de demain sont donc de faire face à une population toujours plus nombreuse, tout en limitant l'impact sur notre environnement. C’est pour cela que la recherche et l’agriculture se questionnent de plus en plus sur l’utilisation d’autres produits comme les biostimulants ou des inhibiteurs, afin de limiter les intrants tout en conservant un taux de productivité viable. Une solution consisterait à agir sur les microorganismes du sol liés au cycle de l’azote afin de limiter les pertes des agrosystèmes en azote via le dégagement de gaz à effet de serre (N2O), de lessivage ou/et de volatilisation. En effet, dans les sols, le nitrate est également utilisé par les bactéries dites dénitrifiantes qui le réduisent en N2O (gaz à effet de serre) et N2, représentant alors une perte d’azote pour les cultures et une augmentation de la pollution atmosphérique. Par conséquent, les plantes sont en compétition directe avec ces bactéries pour l'assimilation du nitrate. Récemment, l’équipe encadrante de cette thèse a mis en évidence une stratégie développée par certaines plantes consistant en la production de métabolites secondaires : les procyanidines, qui inhibent la dénitrification des communautés microbiennes du sol. Les procyanidines ont la capacité d’inhiber, chez les bactéries dénitrifiantes, la première étape de la dénitrification transformant le nitrate en nitrite et ainsi d’empêcher l’utilisation du nitrate, sans toutefois exercer un effet antibactérien. Cette stratégie permet de conserver par conséquent le nitrate dans le sol, celui-ci pouvant alors être utilisé par les plantes pour leur nutrition et leur croissance. L’objectif principal de cette thèse a été d’évaluer l’effet de l’application de procyanidines exogènes à plusieurs concentrations en champs sur un modèle de plante d’intérêt économique, la laitue, ainsi que sur différents types de sols. Ces effets ont également été testés sur un autre modèle d’intérêt économique consommatrice d’azote, le céleri. Au cours de ces expérimentations, des mesures ont été effectuées sur (i) l’activité microbienne de dénitrification, (ii) les traits végétaux en lien avec la croissance et (iii) l’abondance des communautés bactériennes dénitrifiantes. Nos résultats montrent une induction d'un BDI en champs, une conservation du nitrate induisant à son tour une amélioration de la croissance des végétaux et une contre sélection par la plante des dénitrifiants. L’autre point abordé, plutôt fondamental mais qui a moins abouti faute de temps, consistait à mettre en évidence via l’utilisation de mutants d’Arabidopsis thaliana affectés dans la production des procyanidines ou surproduisant les procyanidines, un retour vers la plante de l’azote détourné suite au BDI / To meet the needs of human populations, agriculture is increasingly intensive, using a very large number of nitrogen fertilizers to increase yields. These fertilizers are used because nitrogen is one of the most important and limiting factors for plant growth. Nitrogen in the form of nitrate is subject to pollution problems that can affect the environment and human health. The challenges for tomorrow's agriculture are therefore to face an ever-increasing population, while limiting the impact on our environment. This is why research and agriculture are increasingly questioning the use of other products such as biostimulants or inhibitors, in order to limit inputs while maintaining a viable productivity rate. One solution would be to act on soil microorganisms linked to the nitrogen cycle in order to limit nitrogen losses from agrosystems through greenhouse gas (N2O) emissions, leaching and/or volatilization. Indeed, in soils, nitrate is also used by so-called denitrifying bacteria, which reduce it to N2O (greenhouse gases) and N2, representing a loss of nitrogen for crops and an increase in air pollution. As a result, plants compete directly with these bacteria for the assimilation of nitrate. Recently, the supervising team of this thesis has highlighted a strategy developed by some plants consisting of the production of secondary metabolites: procyanidins, which inhibit the denitrification of soil microbial communities. Procyanidins have the ability to inhibit the first step of denitrification in denitrifying bacteria, transforming nitrate into nitrite and thus preventing the use of nitrate, without however exerting an antibacterial effect. This strategy therefore preserves the nitrate in the soil, which can then be used by plants for their nutrition and growth. The main objective of this thesis was to evaluate the effect of the application of exogenous procyanidins at several field concentrations on a plant model of economic interest, lettuce, as well as on different soil types. These effects have also been tested on another model of economic interest that consumes nitrogen, celery. During these experiments, measurements were made on (i) microbial denitrification activity, (ii) plant traits related to growth and (iii) the abundance of denitrifying bacterial communities. Our results show an induction of a BDI in the field, a conservation of nitrate inducing in turn an improvement in plant growth and a counter-selection by the plant of denitrifiers. The other point addressed, which was rather fundamental but less successful due to a lack of time, was to highlight, via the use of Arabidopsis thaliana mutants affected in the production of procyanidins or overproducing procyanidins, a return to the plant of nitrogen diverted following the BDI