Increasing awareness to global climate change has drastically focused attention on finding solutions to reduce environmental impacts while still providing sufficient food for the increasing world population. Beneficial Nitrogen Fixing (BNF) microbes provide a possible solution by delivering biological nitrogen to plants resulting in reduced environmental impacts due to fertilizer runoff and eutrophication. One well studied model is that of Sinorhizobium meliloti and its legume host Medicago sativa (alfalfa), the fourth largest USA crop used for animal feed. Advancing research for this symbiosis model can provide solutions to enhance yield while minimizing environmental impacts. Chapter 2 focuses on the deviation of the S. meliloti chemotaxis system from the enteric paradigm. Quantitative immunoblots determined the cellular amounts of chemotaxis proteins. Overall, chemotaxis protein levels were approximately 10-fold lower in S. meliloti compared to B. subtilis and E. coli. Focusing on cellular stoichiometric ratios, S. meliloti generally exhibits drastically higher values for CheB, CheR, and CheY to the histidine kinase CheA monomer compared to E. coli and B. subtilis. Chapter 3 characterizes the role of McpX to quaternary ammonium compound (QAC) sensing. QACs are exuded by germinating alfalfa seeds. In vitro binding assays were performed to determine ligand binding characteristics. S. meliloti chemotaxis behavior to QACs was analyzed in in vivo capillary assays under real-time imaging. These studies strengthen our knowledge of the chemotaxis system in the symbiosis model of S. meliloti and alfalfa. The data can further be used to create a mathematical model of the dynamics of bacteria-host interaction. The results can be used to optimize chemotaxis to host plants to improve crop yield and protect watersheds. / Master of Science / Increasing awareness of global climate change has drastically focused attention on finding solutions to reduce environmental impacts while still providing sufficient food for the increasing world population. Beneficial Nitrogen Fixing (BNF) microbes may offer a feasible solution in reducing environmental impacts by supplying biological nitrogen to plants, which reduces fertilizer use and ultimately runoff. One-well studied beneficial microbial model is that of Sinorhizobium meliloti and its legume host Medicago sativa (alfalfa), the fourth most cultivated USA crop used for animal feed. Advancing research for this symbiosis model can provide solutions to enhance crop yield while diminishing environmental impacts.

Les Bradyrhizobium photosynthétiques sont capables d'induire la formation de nodules fixateurs d'azote chez certaines légumineuses du genre Aeschynomene. La découverte récente que certaines de ces souches ne possèdent pas les gènes canoniques nodABC indique l'existence d'un nouveau processus symbiotique rhizobium-légumineuse indépendant des facteurs Nod. L'objectif de ce travail de thèse a consisté à avancer dans la compréhension des mécanismes mis en jeu lors de cette nouvelle interaction. Dans un premier temps, à travers différentes approches cytologiques, le processus par lequel la bactérie infecte la plante en l'absence de facteurs Nod a été décrit. Dans un deuxième temps, afin de mettre en évidence les bases moléculaires de cette interaction, une banque de 15 000 mutants Tn5 de la souche ORS278 a été criblée sur plante. Ce criblage a permit l'identification de plus d'une centaine de gènes bactériens intervenant durant le processus symbiotique. Les résultats obtenus nous ont conduits à proposer un modèle dans lequel la mise en place de la symbiose Nod-indépendante impliquerait, d'une part, la synthèse bactérienne d'une cytokinine permettant le déclenchement de l'organogenèse nodulaire, et d'autre part, d'autres signaux bactériens intervenant dans l'étape de reconnaissance avec la plante hôte. Enfin, nous avons mis en place une technique de transformation génétique d'Aeschynomene et validé cet outil à travers l'étude de l'expression hétérologue de la noduline précoce MtENOD11. Il peut à présent être envisagé de conduire des études fonctionnelles sur Aeschynomene en vue de caractériser la voie de signalisation Nod-indépendante. / The photosynthetic Bradyrhizobium are able to induce the formation of nitrogen-fixing nodules in some legumes of the Aeschynomene genus. The recent discovery that some of these strains lack the canonical nodABC genes indicates the existence of a new symbiotic rhizobium-legume process that is independent of Nod factors. The aim of this work was to improve our understanding of the mechanisms involved in this new interaction. First, through various cytological approaches, the process by which the bacterium infects the plant in the absence of Nod factors has been described. Second, in order to decipher the molecular basis of this interaction, a library of 15,000 Tn5 mutants of the ORS278 strain was screened on plant. This screening allowed the identification of about one hundred bacterial genes involved in this symbiotic process. These results led us to propose a model in which the establishment of the Nod-independent symbiosis involves, on one han d, the synthesis of a bacterial cytokinin that triggers nodule organogenesis, and on the other hand, others bacterial signals that permit the recognition with the host plant. Finally, we developed a genetic transformation procedure of Aeschynomene and we validated this tool by studying the heterologous expression of the early nodulin MtENOD11. Now, functional studies on Aeschynomene are possible to permit the characterization of the Nod-independent signaling pathway.

L’interaction symbiotique légumineuses-rhizobium nécessite l'infection des racines de la plante par les bactéries et l’initiation de divisions cellulaires dans le cortex racinaire.Les cytokinines sont des hormones végétales agissant via une signalisation par phosphotransfert qui conduit à l’activation de Régulateurs de Réponse de type B (RRBs), des facteurs de transcription régulant l'expression des gènes de réponse primaire aux cytokinines. Une étude phylogénétique menée sur plusieurs espèces de légumineuses a révélé une expansion génique de la famille des RRBs et l’apparition de formes non-canoniques de ces facteurs de transcription. Chez Medicago truncatula,MtRRB3 est le RRB le plus fortement exprimé dans les racines et les nodosités et est impliqué dans la nodulation. En effet, les plantes dont l’expression de MtRRB3 a été réduite par ARNi ainsi que des mutants rrb3 présentent une diminution significative du nombre de nodosités formées. De plus, l’expression de gènes associés à la nodulation, tels que "Nodulation Signalling Pathway 2" (MtNSP2) et "Cell Cycle Switch 52A"(MtCCS52A), est réduite en réponse aux cytokinines dans ces mutants. Des fusions transcriptionnelles avec le rapporteur GUS montrent que MtRRB3, MtNSP2 et MtCCS52Aprésentent un profil d’expression spatiale largement chevauchant dans les racines et lesnodosités. Des expériences de ChIP-qPCR et de trans-activation en protoplastes indiquent par ailleurs que MtRRB3 peut respectivement interagir avec et activer les promoteurs des gènesMtNSP2 et MtCCS52A. Cette thèse a donc permis d’établir des mécanismes moléculaires impliqués dans les régulations transcriptionnelles médiées par les cytokinines lors de la mise en place des nodosités symbiotiques fixatrices d’azote. / The legume-rhizobium interaction requires the infection of plant roots by rhizobia and the initiation of cell divisions in the root cortex. Cytokinins, a class of plant hormones acts trough a phosphotranfert signalling leading to the activation of Type-B Response Regulators(RRBs) which are transcription factors regulating the expression of cytokinins primary response genes. Phylogenetic analyses carried out indifferent legume species genomes showed anexpansion of the RRB genes family associated toan increase in non-canonical RRBs. In Medicago truncatula nodules, MtRRB3 is the most expressed RRB in roots and nodules. MtRRB3 islinked to nodulation as MtRRB3 RNAi silencedplants as well as rrb3 mutants display asignificant decrease of nodule number. Inaddition, the expression of the nodulation related genes Nodulation Signalling Pathway 2(MtNSP2) and Cell Cycle Switch 52A(MtCCS52A) is reduced in response to cytokininsin rrb3 mutants. The expression pattern of apMtRRB3-GUS fusion overlaps with thepMtNSP2-GUS and pMtCCS52A-GUS fusions in roots and nodules. Finally, ChIP-qPCR and protoplast trans-activation experiments showed that MtRRB3 can respectively interacts with and activate MtNSP2 and MtCCS52A promoters. This thesis have thus established molecular mechanisms associated to transcriptional regulations mediated by cytokinins during the legume symbiotic nitrogen-fixing nodulation.

The study of root nodule symbiosis between nitrogen-fixing bacteria and leguminous plant species is important because of the ability to supplement fixed nitrogen fertilizers and increase plant growth in poor soils. Our group has isolated a mutant called nip in the model legume Medicago truncatula that is defective in nodule symbiosis. The nip mutant (numerous infections with polyphenolics) becomes infected by Sinorhizobium meliloti but then accumulates polyphenolic defense compounds in the nodule and fails to progress to a stage where nitrogen fixation can occur. Analysis of the transcriptome of nip roots prior to inoculation with rhizobia was undertaken using Affymetric Medicago Genome Array microarrays. The total RNA of 5-day old uninoculated seedlings was analyzed in triplicate to screen for the NIP gene based on downregulated transcript levels in the mutant as compared to wild type. Further microarray data was generated from 10 days post inoculation (dpi) nip and wild type plants. Analysis of the most highly downregulated transcripts revealed that the NIP gene was not identifiable based on transcript level. Putative gene function was assigned to transcripts with altered expression patterns in order to characterize the nip mutation phenotypically as inferred from the transcriptome. Functional analysis revealed a large number of chaperone proteins were highly expressed in the nip mutant, indicating high stress in the mutant prior to infection by rhizobia. Additionally, a database containing the information regarding the nip expression profile at both 0 days post inoculation (dpi) and 10 dpi were created for screening of candidate genes as predicted from sequence in the genomic region containing NIP.

Although plant growth-promoting rhizobacteria (PGPR) have shown tremendous potential to be used as inoculants for many agricultural crops, they may not survive severe environmental conditions in the field which could limit their large scale applications. Endophytic bacteria, which can be recovered from inside plant tissues such as roots, stems and leaves, might overcome this limitation due to their unique ecological niche inside plant roots where they are sheltered from external environmental disturbances. Some of these bacterial endophytes have beneficial effects on their host plants and stimulate plant growth or reduce disease symptoms, apparently through mechanisms that are similar to those proposed for PGPR. The objective of this study was to assess a collection of endophytic bacteria for PGPR traits and potential use to enhance the rhizobial-legume symbiosis. Forty isolates obtained from the roots of various plants were identified by fatty acid methyl ester (FAME) analysis, and 16S RNA gene sequencing analysis. The majority (i.e., 75%) were identified as Pseudomonas species. Many of these isolates were able to solubilize phosphate, produce indole-3-acetic acid (IAA), produce aminocyclopropane-1-carboxylic acid (ACC) deaminase, synthesize siderophores and show antagonistic activities against several soil-borne plant pathogenic fungi under in vitro conditions. Selected isolates were further evaluated for the ability to enhance plant growth and nodulation of alfalfa when co-inoculated with Sinorhizobium meliloti under growth chamber conditions using growth pouch and potted soil assays. Results revealed that P. putida strain EB EE 4-25, P. syringae strain EB XDE 4-48, and P. fluorescens strain EB EE 2-23 significantly increased shoot length, root length, enhanced nodulation and increased lateral root formation of alfalfa plants in growth pouch and potted soil assays when co-inoculated with S. meliloti strain P102 compared to plants inoculated with S. meliloti strain P102 alone. Results also suggested that expression of one or more of the mechanisms, such as solubilization of phosphate, production of IAA, production of siderophores, and ACC deaminase production might have played a role in the enhancement of the alfalfa- Sinorhizobium symbiosis. These results suggest that some endophytic bacterial strains may be useful as biofertilizers and/or biocontrol agents in sustainable agricultural practices.

Although plant growth-promoting rhizobacteria (PGPR) have shown tremendous potential to be used as inoculants for many agricultural crops, they may not survive severe environmental conditions in the field which could limit their large scale applications. Endophytic bacteria, which can be recovered from inside plant tissues such as roots, stems and leaves, might overcome this limitation due to their unique ecological niche inside plant roots where they are sheltered from external environmental disturbances. Some of these bacterial endophytes have beneficial effects on their host plants and stimulate plant growth or reduce disease symptoms, apparently through mechanisms that are similar to those proposed for PGPR. The objective of this study was to assess a collection of endophytic bacteria for PGPR traits and potential use to enhance the rhizobial-legume symbiosis. Forty isolates obtained from the roots of various plants were identified by fatty acid methyl ester (FAME) analysis, and 16S RNA gene sequencing analysis. The majority (i.e., 75%) were identified as Pseudomonas species. Many of these isolates were able to solubilize phosphate, produce indole-3-acetic acid (IAA), produce aminocyclopropane-1-carboxylic acid (ACC) deaminase, synthesize siderophores and show antagonistic activities against several soil-borne plant pathogenic fungi under in vitro conditions. Selected isolates were further evaluated for the ability to enhance plant growth and nodulation of alfalfa when co-inoculated with Sinorhizobium meliloti under growth chamber conditions using growth pouch and potted soil assays. Results revealed that P. putida strain EB EE 4-25, P. syringae strain EB XDE 4-48, and P. fluorescens strain EB EE 2-23 significantly increased shoot length, root length, enhanced nodulation and increased lateral root formation of alfalfa plants in growth pouch and potted soil assays when co-inoculated with S. meliloti strain P102 compared to plants inoculated with S. meliloti strain P102 alone. Results also suggested that expression of one or more of the mechanisms, such as solubilization of phosphate, production of IAA, production of siderophores, and ACC deaminase production might have played a role in the enhancement of the alfalfa- Sinorhizobium symbiosis. These results suggest that some endophytic bacterial strains may be useful as biofertilizers and/or biocontrol agents in sustainable agricultural practices.

Chemotaxis is the process by which motile bacteria navigate chemical gradients in order to position themselves in optimum environments for growth and metabolism. Sensory input from both the external environment and the internal cellular environment are sensed by chemotaxis transducers and transduced to a two-component system whose output interacts with the flagellum thereby regulating motility. Chemotaxis has been implicated in establishing the endosymbiotic relationship between the motile alpha-proteobacterium Rhizobium leguminosarum biovar viciae and its host Pisum sativa, the pea plant. An approach combing bioinformatical sequence analysis, molecular genetics, and behavioral analysis was used to characterize the chemotaxis system of R. leguminosarum and determine its contribution to this bacterium s lifestyle. A genome search revealed the presence of two chemotaxis gene clusters, che1 and che2. Homologs of each che cluster are major chemotaxis operons controlling flagellar motility in other bacterial species. For this reason we sought to determine the contribution of each che cluster to chemotaxis in R. leguminosarum. We found that while both che clusters contribute to the regulation of motility, che1 is the major che cluster controlling chemotaxis. Using competitive nodulation assays we determined that che1, but not che2, is essential for competitive nodulation. The major che cluster, che1, encodes a chemotaxis transducer, IcpA-Rl, with a globin coupled sensor domain. Chemotaxis transducers with a globin coupled sensor domain comprise a large class of proteins found in bacteria and archaea. These proteins have been shown to bind heme and sense oxygen and are therefore termed HemATs for heme-binding aerotaxis transducers. However, sequence analysis of IcpA-Rl reveals that it lacks the requisite amino acid residues for heme-binding and is therefore unlikely to sense oxygen. We present evidence that IcpA-Rl is likely an energy transducer and represents a novel function of the globin coupled sensor domain in sensing energy related parameters.

Perennial clovers are being evaluated for their potential to reduce groundwater levels in Australian cropping zones where many soils are considered too acidic for reliable lucerne nodulation. However, the release of effective inocula for perennial clovers into such areas where sub clover is the predominant legume, could potentially compromise nitrogen fixation from this valuable annual clover if the symbiosis between the new inoculants and sub clover is not optimal. Studies were therefore designed to increase our understanding of these symbiotic interactions to optimise the management of legume-rhizobia interactions to extend (rather than restrict) the use of legumes in new environments. To assist the understanding of interactions between clovers and their microsymbionts, a glasshouse-based study of the cross-inoculation characteristics of 38 strains of Rhizobium leguminosarum biovar trifolii (R. l. trifolii) associated with 38 genotypes of annual and perennial Trifolium spp. from world centres of diversity was undertaken. Rhizobial isolates and clovers were assembled from South and equatorial Africa, North and South America and the Euro-Mediterranean regions. There was substantial specificity amongst the African clovers for effective nodulation. No strain of rhizobia from the South American perennial T. polymorphum, or from the Ethiopian clovers, was able to nodulate sub clover effectively, whilst less than 33% of the 18 strains from these regions could form nodules with the less promiscuous Mediterranean annual T. glanduliferum. Seventy of 476 cross-inoculation treatments examined did not nodulate, whilst 81 treatments clearly demonstrated effective nodulation. The remainder of the crossinoculation pairings revealed only partially effective or ineffective nodulation. Two barriers to effective nodulation were identified from the cross- inoculation study: a geographic barrier representing the broad centres of clover diversity, across which few host- strain combinations were effective; and within each region, a significant barrier to effective nodulation between an isolate from an annual host on a perennial host, or vice versa. Clovers and their rhizobia from within the Euro-Mediterranean region of diversity were more able to overlap the annual/perennial barrier than genotypes from the other regions. The data indicate that it will be a substantial challenge to develop inocula for perennial clovers that do not adversely affect nitrogen fixation by sub clover and other annual clovers in commerce, especially if the perennial clovers originate from Africa or America. To investigate the management of legume-rhizobia interactions when introducing legumes into new environments, a study was initiated in Uruguay (Mediterranean annual clovers were introduced into a predominantly perennial clover setting) that could be considered opposite to the situation emerging within southern Australia (perennial clovers evaluated in a predominantly annual clover setting). The Uruguayan grasslands contain populations of indigenous R. l. trifolii that nodulate endemic T. polymorphum but form ineffective nodules on clovers originating from the Mediterranean region. Importantly in the Uruguayan setting, Government policy has facilitated the introduction of numerous varieties of annual Mediterranean clovers with the aim of improving overall winter production in their naturally managed grasslands. In an attempt to understand the rhizobial ecology of this scenario, a cross-row experiment was set-up in 1999 in a basaltic, acid soil in Glencoe, Uruguay, to follow the survival and symbiotic performance of nine exotic strains of R. l. trifolii. In this thesis I report on the ability of the introduced strains to compete for nodule occupancy of Mediterranean clover hosts and show the impacts of the introduced strains on the productivity of the indigenous Uruguayan clover, T. polymorphum. Of the introduced strains, WSM1325 was a superior inoculant and remained highly persistent and competitive in forming effective symbioses with the Mediterranean hosts, T. purpureum and T. repens, in the Uruguayan environment over a 3 year period. T. purpureum and T. repens, when inoculated with the introduced strains, did not nodulate with any indigenous R. l. trifolii as typed from nodules of T. polymorphum. Conversely, there were no nodules on the Uruguayan host T. polymorphum that contained the introduced R. l. trifolii. These results revealed that there were effective symbioses between strains of R. l. trifolii and clovers, even though the soil contained ineffective R. l. trifolii for all hosts. This represents the first reported example of selective nodulation for an effective symbiosis in situ with annual and perennial clovers in acid soils. This phenomenon raised the question of whether this was restricted to the particular edaphic scenario in Glencoe, Uruguay. Glasshouse-based experiments in Australia were conducted to further understand the selection phenomenon. Two strains were selected for comparisons; strain WSM1325 isolated from an annual clover in the Mediterranean and WSM2304 isolated from the perennial clover T. polymorphum in Uruguay, South America. Variables that may have been specific to Glencoe were investigated. Thus, the effect of cell density and strain ratio at the time of inoculation, as well as soil pH, were examined on the two hosts (T. purpureum and T. polymorphum). Each was exposed to the same effective and ineffective micro-symbionts. In co-inoculation experiments at a cell density of 104 cells mL-1, each host nodulated solely with its effective strain, even when this strain was out-numbered 100:1 by the ineffective strain. However, the selection process ceased when the effective strain was out-numbered 1000:1. At higher basal cell concentrations of 105 - 108 cells mL-1, selection for WSM1325 to form effective nodules on T. purpureum was evident, but was significantly reduced as the ratio of ineffective cells in the inoculum increased above 4-fold. These results indicate that the selection mechanism is highly dependent upon the basal rhizobial cell density. Soil pH did not significantly alter the process, which could not be simply explained by the rate of strain growth, or extent of nodulation. Greater precision was sought in the terminology applied to nodulation outcomes where legumes have a choice of micro-symbiotic partners from within the same species of root-nodule bacteria. The nominated preferred terms are “nonselective”, “exclusive”, and “selective” nodulation. In view of the difference in host range between WSM1325 and WSM2304 and the selective nodulation process, a preliminary investigation into the genetic backgrounds of WSM1325 and WSM2304 was conducted. A selected range of gene regions were amplified by PCR from each strain and sequenced. Comparative analysis of the nucleotide sequences revealed that although the 16S rRNA sequences were identical, the atpD, GSII and nodD sequences contained distinct differences revealing disparity between the pSym replicons and between the chromosomal replicons of these strains. Of the genes sequenced, the highest degree of divergence was noted for the symbiotic NodD protein products, which are known to be critical determinants in the nodulation of specific hosts. An examination of the nodD gene region of WSM1325 and WSM2304 revealed a further contrasting feature; the regulatory gene nodR was present in the nodD gene region of WSM1325 but absent in WSM2304. Since NodR is known to be required for adding highly unsaturated fatty acyl groups onto the Nod-factor backbone, I could now hypothesise that the nodulation incompatibility observed between Trifolium hosts and micro-symbionts obtained from different geographical locations may result from differences in Nod-factor decoration. With the full genome sequence of the two strains WSM1325 and WSM2304 soon to be available, the role of nodR and any link to the selection phenomenon described in this thesis can be addressed.

Made available in DSpace on 2014-06-11T19:32:54Z (GMT). No. of bitstreams: 0 Previous issue date: 2009-02-13Bitstream added on 2014-06-13T18:44:28Z : No. of bitstreams: 1 scaquitto_dc_dr_jabo.pdf: 429936 bytes, checksum: 3e636e555a2a2cd56aa2c19a9d5c4352 (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / O amendoim (Arachis hypogaea L.) faz parte da família Fabaceae, que é capaz de associar-se com bactérias do gênero Rhizobium. Em solos onde o amendoim é cultivado tem-se a presença de uma população autóctone desta bactéria. A necessidade de inoculação das sementes de amendoim em solos contendo população autóctone tem sido sempre discutida. Com o objetivo de avaliar o efeito da inoculação de sementes de amendoim cultivado em vasos contendo vermiculita e a interação existente entre microrganismo-planta e microrganismo-microrganismo, foi realizado um experimento sob cultivo em casa de vegetação, em vasos de 0,5 L, com o cultivar IAC 886 e a IAC Tatu-ST, com três repetições. Os tratamentos foram constituídos de aplicação dos isolados individualmente e combinados com a estirpe padrão tipo SEMIA 6144, Bradyrhizoibium sp., recomendada pelo Ministério da Agricultura e sem aplicação de inoculante, sem aplicação de qualquer isolado. Houve interação entre o tipo de plantio e a aplicação de inoculante para todas as variáveis analisadas. Para a massa de matéria seca da parte aérea houve uma diferença significativa. O variável número de nódulos e o teor de Nitrogênio também apresentam diferenças quanto à aplicação, sendo que, em todos os casos foram superiores ao controle sem aplicação com os isolados nativos. / Peanut (Arachis hypogaea L.) belonging to Fabacea family, is able of associating with bacteria from Rhizobium genus. In soils where peanut is grown, there is usually an autochthonous population of these bacterial, however, the need of inoculation in peanut seeds in soils containing autochthonous population has always been controversial subject. With the objective of evaluating the effects of peanut seeds inoculation cultivated in vases contend vermiculite and to verifying the interaction with plant regulators, an experiment was carried out in greenhouse conditions, with three repetitions containing Runner IAC 886 e IAC Tatu-ST cultivar. The treatments were constituted of application of the individually isolated and combined with the lineage standard Bradyrhizobium sp., SEMIA 6144, recommended by Bureau of the Agriculture. An interaction was observed between the type of plantation and the application of inoculants for all analysed variable. For the mass of dry matter of the aerial part it had a significant difference. The variable numbers of nodules and rate of Nitrogen also present differences related to the treatments, being that, in all the cases was greater to the control without application of the isolated natives.

Made available in DSpace on 2014-06-11T19:26:48Z (GMT). No. of bitstreams: 0 Previous issue date: 2002-08Bitstream added on 2014-06-13T19:55:07Z : No. of bitstreams: 1 herdani_ee_me_botfca.pdf: 252571 bytes, checksum: 741d513244366f02459406142b6b2359 (MD5) / O presente trabalho teve por objetivo estudar os efeitos, de seis níveis de água no solo, na nodulação da soja (Glycine max (L.) Merril). Foi instalado um experimento em casa de vegetação localizada junto à Área experimental do Departamento de Engenharia Rural, da Faculdade de Ciências Agronômicas (FCA) – Campus Botucatu – SP, UNESP, durante os meses de novembro a dezembro/2001, em solo Latossolo Vermelho Escuro, textura média. O delineamento experimental foi de blocos casualizados com tratamentos em esquema fatorial 2 X 6 (dois níveis de inoculação – com e sem inoculação e seis níveis de irrigação – 30 %, 40 %, 50 %, 60 %, 70 % e 80 % da capacidade de campo), com seis repetições e uma variedade (IAC –18) um tipo de solo. As diferentes unidades experimentais, foram formadas por vasos plásticos impermeáveis contendo uma planta em cada vaso, onde foram avaliados, na floração, quando o processo de fixação de N2 atinge o ponto máximo, número e massa seca dos nódulos. Na planta, foram avaliados, massa seca da parte aérea e das raízes e nitrogênio na planta, pode-se concluir que as plantas responderam positivamente ao aumento do nível de água no solo em relação ao desenvolvimento da parte aérea e radicular . Quando inoculadas apresentaram maior teor de nitrogênio da parte aérea. / The aiming of the work was study the efects of six soil moisture levels on soy nodulation (Glycine max (L.) Merril). A greenhouse experiment was installed at Department of Field Engineering of Faculdade de Ciências Agronômicas (FCA) in campus Botucatu-SP, UNESP, during november/2001 until december/2001, in na Oxisol medium texture. The experimental design was randomized blocks with 2 X 6 outlined treatments ( 2 inoculation levels- with and without inoculation and 6 irrigation levels: 30%, 40%, 50% 60%, 70% and 80% of field capacity), it was used 6 repetitions with IAC-18 variety. The different experimental units were formed by impermeable vases with one plant each. These vases were used for avaliation of, during flowering, N fixation (maximum rate) levels, number and dry mass of the nodules. It was analyzed as well, the dry matter and N levels of the aerial part of the plant and the root system. It could be concluded that there were, in all treatments, a plant positive response with the increment of water available in soil and a better development in inoculated plants.