Multiomic Approaches Reveal Hormonal Modulation and Nitrogen Uptake and Assimilation in the Initial Growth of Maize Inoculated with Herbaspirillum seropedicae.

Herbaspirillum seropedicae is an endophytic bacterium that can fix nitrogen and synthesize phytohormones, which can lead to a plant growth-promoting effect when used as a microbial inoculant. Studies focused on mechanisms of action are crucial for a better understanding of the bacteria-plant interaction and optimization of plant growth-promoting response. This work aims to understand the underlined mechanisms responsible for the early stimulatory growth effects of H. seropedicae inoculation in maize. To perform these studies, we combined transcriptomic and proteomic approaches with physiological analysis. The results obtained eight days after inoculation (d.a.i) showed increased root biomass (233 and 253%) and shoot biomass (249 and 264%), respectively, for the fresh and dry mass of maize-inoculated seedlings and increased green content and development. Omics data analysis, before a positive biostimulation phenotype (5 d.a.i.) revealed that inoculation increases N-uptake and N-assimilation machinery through differentially expressed nitrate transporters and amino acid pathways, as well carbon/nitrogen metabolism integration by the tricarboxylic acid cycle and the polyamine pathway. Additionally, phytohormone levels of root and shoot tissues increased in bacterium-inoculated-maize plants, leading to feedback regulation by the ubiquitin-proteasome system. The early biostimulatory effect of H. seropedicae partially results from hormonal modulation coupled with efficient nutrient uptake-assimilation and a boost in primary anabolic metabolism of carbon-nitrogen integrative pathways.

Engineering Posttranslational Regulation of Glutamine Synthetase for Controllable Ammonia Production in the Plant Symbiont Azospirillum brasilense.

Nitrogen requirements for modern agriculture far exceed the levels of bioavailable nitrogen in most arable soils. As a result, the addition of nitrogen fertilizer is necessary to sustain productivity and yields, especially for cereal crops, the planet's major calorie suppliers. Given the unsustainability of industrial fertilizer production and application, engineering biological nitrogen fixation directly at the roots of plants has been a grand challenge for biotechnology. Here, we designed and tested a potentially broadly applicable metabolic engineering strategy for the overproduction of ammonia in the diazotrophic symbiont Azospirillum brasilense. Our approach is based on an engineered unidirectional adenylyltransferase (uAT) that posttranslationally modifies and deactivates glutamine synthetase (GS), a key regulator of nitrogen metabolism in the cell. We show that this circuit can be controlled inducibly, and we leveraged the inherent self-contained nature of our posttranslational approach to demonstrate that multicopy redundancy can improve strain evolutionary stability. uAT-engineered Azospirillum is capable of producing ammonia at rates of up to 500 µM h-1 unit of OD600 (optical density at 600 nm)-1. We demonstrated that when grown in coculture with the model monocot Setaria viridis, these strains increase the biomass and chlorophyll content of plants up to 54% and 71%, respectively, relative to the wild type (WT). Furthermore, we rigorously demonstrated direct transfer of atmospheric nitrogen to extracellular ammonia and then plant biomass using isotopic labeling: after 14 days of cocultivation with engineered uAT strains, 9% of chlorophyll nitrogen in Setaria seedlings was derived from diazotrophically fixed dinitrogen, whereas no nitrogen was incorporated in plants cocultivated with WT controls. This rational design for tunable ammonia overproduction is modular and flexible, and we envision that it could be deployable in a consortium of nitrogen-fixing symbiotic diazotrophs for plant fertilization. IMPORTANCE Nitrogen is the most limiting nutrient in modern agriculture. Free-living diazotrophs, such as Azospirillum, are common colonizers of cereal grasses and have the ability to fix nitrogen but natively do not release excess ammonia. Here, we used a rational engineering approach to generate ammonia-excreting strains of Azospirillum. Our design features posttranslational control of highly conserved central metabolism, enabling tunability and flexibility of circuit placement. We found that our strains promote the growth and health of the model grass S. viridis and rigorously demonstrated that in comparison to WT controls, our engineered strains can transfer nitrogen from 15N2 gas to plant biomass. Unlike previously reported ammonia-producing mutants, our rationally designed approach easily lends itself to further engineering opportunities and has the potential to be broadly deployable.

Restoring degraded microbiome function with self-assembled communities.

The natural microbial functions of many soils are severely degraded. Current state-of-the-art technology to restore these functions is through the isolation, screening, formulation and application of microbial inoculants and synthetic consortia. These approaches have inconsistent success, in part due to the incompatibility between the biofertilizer, crop, climate, existing soil microbiome and physicochemical characteristics of the soils. Here, we review the current state of the art in biofertilization and identify two key deficiencies in current strategies: the difficulty in designing complex multispecies biofertilizers and the bottleneck in scaling the production of complex multispecies biofertilizers. To address the challenge of producing scalable, multispecies biofertilizers, we propose to merge ecological theory with bioprocess engineering to produce 'self-assembled communities' enriched for particular functional guilds and adapted to a target soil and host plant. Using the nitrogen problem as an anchor, we review relevant ecology (microbial, plant and environmental), as well as reactor design strategies and operational parameters for the production of functionally enriched self-assembled communities. The use of self-assembled communities for biofertilization addresses two major hurdles in microbiome engineering: the importance of enriching microbes indigenous to (and targeted for) a specific environment and the recognized potential benefits of microbial consortia over isolates (e.g. functional redundancy). The proposed community enrichment model could also be instrumental for other microbial functions such as phosphorus solubilization, plant growth promotion or disease suppression.

Nitrogen Fixation in Pozol, a Traditional Fermented Beverage.

Traditional fermentations have been widely studied from the microbiological point of view, but little is known from the functional perspective. In this work, nitrogen fixation by free-living nitrogen-fixing bacteria was conclusively demonstrated in pozol, a traditional Mayan beverage prepared with nixtamalized and fermented maize dough. Three aspects of nitrogen fixation were investigated to ensure that fixation actually happens in the dough: (i) the detection of acetylene reduction activity directly in the substrate, (ii) the presence of potential diazotrophs, and (iii) an in situ increase in acetylene reduction by inoculation with one of the microorganisms isolated from the dough. Three genera were identified by sequencing the 16S rRNA and nifH genes as Kosakonia, Klebsiella, and Enterobacter, and their ability to fix nitrogen was confirmed.IMPORTANCE Nitrogen-fixing bacteria are found in different niches, as symbionts in plants, in the intestinal microbiome of several insects, and as free-living microorganisms. Their use in agriculture for plant growth promotion via biological nitrogen fixation has been extensively reported. This work demonstrates the ecological and functional importance that these bacteria can have in food fermentations, reevaluating the presence of these genera as an element that enriches the nutritional value of the dough.

Diazotrophs Show Signs of Restoration in Amazon Rain Forest Soils with Ecosystem Rehabilitation.

Biological nitrogen fixation can be an important source of nitrogen in tropical forests that serve as a major CO2 sink. Extensive deforestation of the Amazon is known to influence microbial communities and the biogeochemical cycles they mediate. However, it is unknown how diazotrophs (nitrogen-fixing microorganisms) respond to deforestation and subsequent ecosystem conversion to agriculture, as well as whether they can recover in secondary forests that are established after agriculture is abandoned. To address these knowledge gaps, we combined a spatially explicit sampling approach with high-throughput sequencing of nifH genes. The main objectives were to assess the functional distance decay relationship of the diazotrophic bacterial community in a tropical forest ecosystem and to quantify the roles of various factors that drive the observed changes in the diazotrophic community structure. We observed an increase in local diazotrophic diversity (α-diversity) with a decrease in community turnover (ß-diversity), associated with a shift in diazotrophic community structure as a result of the forest-to-pasture conversion. Both diazotrophic community turnover and structure showed signs of recovery in secondary forests. Changes in the diazotrophic community were primarily driven by the change in land use rather than differences in geochemical characteristics or geographic distances. The diazotroph communities in secondary forests resembled those in primary forests, suggesting that at least partial recovery of diazotrophs is possible following agricultural abandonment.IMPORTANCE The Amazon region is a major tropical forest region that is being deforested at an alarming rate to create space for cattle ranching and agriculture. Diazotrophs (nitrogen-fixing microorganisms) play an important role in supplying soil N for plant growth in tropical forests. It is unknown how diazotrophs respond to deforestation and whether they can recover in secondary forests that establish after agriculture is abandoned. Using high-throughput sequencing of nifH genes, we characterized the response of diazotrophs' ß-diversity and identified major drivers of changes in diazotrophs from forest-to-pasture and pasture-to-secondary-forest conversions. Studying the impact of land use change on diazotrophs is important for a better understanding of the impact of deforestation on tropical forest ecosystem functioning, and our results on the potential recovery of diazotrophs in secondary forests imply the possible restoration of ecosystem functions in secondary forests.

Nitrogen fertilization and leaf spraying with Azospirillum brasilense in wheat: effects on mineral nutrition and yield / Adubação nitrogenada e pulverização foliar de Azospirillum brasilense em trigo: efeitos na nutrição mineral e produtividade

The use of Azospirillum brasilense has the potential to improve plant nitrogen (N) use efficiency, while a better understanding of alternative management practices with inoculation is necessary. The aim was to examine the effects of the leaf application of A. brasilense in association with nitrogen fertilization on the wheat crop. The experiment was conducted in Lidianópolis, Paraná, Brazil, in a completely randomized block design with four replications. The treatments included four doses of A. brasilense for leaf application (0, 200, 400, and 600 ml ha-1) and four doses of N (0, 40, 80, and 120 kg N ha-1). The nutritional content, yield components, quality, and yield of the wheat crop were evaluated. There was no interaction among the factors, nor did the treatments have an isolated effect on spike length, the number of spikelets per spike, spikes per m2, thousand grain weight, and test weight. However, doses of A. brasilense increased calcium and magnesium absorption at 283 and 380 ml ha-1, respectively. Similarly, nitrogen application increased the content of calcium, magnesium, and copper in the leaf at 61, 47, and 49 kg N ha-1, respectively. Nitrogen also increased the number of grains per spike and yield at 56 and 54 kg N ha-1, respectively. Yield correlated with the number of grains per spike and the manganese and copper content in the leaf.

O uso de Azospirillum brasilense apresenta potencial em melhorar a eficiência de uso do nitrogênio (N), sendo necessário melhor compreensão de formas alternativas de inoculação, visto que o tratamento químico de sementes pode comprometer a eficiência das bactérias. O objetivo foi avaliar os efeitos do A. brasilense aplicado via foliar associado a adubação nitrogenada na cultura do trigo. O experimento foi implantado em Lidianópolis, no estado do Paraná, Brasil, em blocos completos com tratamentos ao acaso e quatro repetições, sendo os tratamentos: quatro doses de A. brasilense via foliar (0, 200, 400 e 600 ml ha-1) e quatro doses de N (0, 40, 80 e 120 kg N ha-1). Foram avaliados os teores nutricionais, componentes de rendimento, qualidade e produtividade. Não houve interação entre os fatores, tampouco efeito isolado dos tratamentos para comprimento da espiga, número de espiguetas por espiga, espigas por m², massa de mil grãos e peso de hectolitro. Todavia as doses de A. brasilense, aumentaram a absorção de Ca e Mg até a dose de 283 e 380 ml ha-1, respectivamente. De modo similar, o N aplicado aumentou os teores foliares de cálcio, magnésio, além de cobre até a dose de 61, 47 e 49 kg ha-1 de N, respectivamente. O N também incrementou o número de grãos por espiga e a produtividade até a dose 56 e 54 kg N ha-1, respectivamente, porém a eficiência de uso

Nitrogen fertilization and leaf spraying with Azospirillum brasilense in wheat: effects on mineral nutrition and yield / Adubação nitrogenada e pulverização foliar de Azospirillum brasilense em trigo: efeitos na nutrição mineral e produtividade

The use of Azospirillum brasilense has the potential to improve plant nitrogen (N) use efficiency, while a better understanding of alternative management practices with inoculation is necessary. The aim was to examine the effects of the leaf application of A. brasilense in association with nitrogen fertilization on the wheat crop. The experiment was conducted in Lidianópolis, Paraná, Brazil, in a completely randomized block design with four replications. The treatments included four doses of A. brasilense for leaf application (0, 200, 400, and 600 ml ha-1) and four doses of N (0, 40, 80, and 120 kg N ha-1). The nutritional content, yield components, quality, and yield of the wheat crop were evaluated. There was no interaction among the factors, nor did the treatments have an isolated effect on spike length, the number of spikelets per spike, spikes per m2, thousand grain weight, and test weight. However, doses of A. brasilense increased calcium and magnesium absorption at 283 and 380 ml ha-1, respectively. Similarly, nitrogen application increased the content of calcium, magnesium, and copper in the leaf at 61, 47, and 49 kg N ha-1, respectively. Nitrogen also increased the number of grains per spike and yield at 56 and 54 kg N ha-1, respectively. Yield correlated with the number of grains per spike and the manganese and copper content in the leaf.(AU)

O uso de Azospirillum brasilense apresenta potencial em melhorar a eficiência de uso do nitrogênio (N), sendo necessário melhor compreensão de formas alternativas de inoculação, visto que o tratamento químico de sementes pode comprometer a eficiência das bactérias. O objetivo foi avaliar os efeitos do A. brasilense aplicado via foliar associado a adubação nitrogenada na cultura do trigo. O experimento foi implantado em Lidianópolis, no estado do Paraná, Brasil, em blocos completos com tratamentos ao acaso e quatro repetições, sendo os tratamentos: quatro doses de A. brasilense via foliar (0, 200, 400 e 600 ml ha-1) e quatro doses de N (0, 40, 80 e 120 kg N ha-1). Foram avaliados os teores nutricionais, componentes de rendimento, qualidade e produtividade. Não houve interação entre os fatores, tampouco efeito isolado dos tratamentos para comprimento da espiga, número de espiguetas por espiga, espigas por m², massa de mil grãos e peso de hectolitro. Todavia as doses de A. brasilense, aumentaram a absorção de Ca e Mg até a dose de 283 e 380 ml ha-1, respectivamente. De modo similar, o N aplicado aumentou os teores foliares de cálcio, magnésio, além de cobre até a dose de 61, 47 e 49 kg ha-1 de N, respectivamente. O N também incrementou o número de grãos por espiga e a produtividade até a dose 56 e 54 kg N ha-1, respectivamente, porém a eficiência de uso(AU)

Modulation of nitrogen metabolism of maize plants inoculated with Azospirillum brasilense and Herbaspirillum seropedicae.

Maize is highly responsive to the application of nitrogen to achieve high productivity. Inoculation with diazotrophic bacteria can improve plant growth with low N fertilization. The objective was to evaluate the inoculation of two species of diazotrophs on N metabolism in maize plants, in the presence of two concentrations of nitrogen in a hydroponic system. A factorial arrangement composed of two N levels (3.0 and 0.3 mM), with the presence of Hs-Herbaspirillum seropedicae, and Ab-Azospirillum brasilense or not. The parameters used were dry mass; N, P, and K accumulation; nitrate reductase activity; soluble fractions in roots and leaves. The inoculation altered the N metabolism and promoted greater development of maize plants, as well as a higher accumulation of P and K in the shoots. A more intensive process of N assimilation was evidenced when the plants were inoculated with H. seropedicae, leading to increased levels of NO3- and reduced N-amino, sugars, and NH4+ in leaves associated with high N level, opposite of A. brasilense.

Temporal assessment of microbial communities in soils of two contrasting mangroves

ABSTRACT Variations in microbial communities promoted by alterations in environmental conditions are reflected in similarities/differences both at taxonomic and functional levels. Here we used a natural gradient within mangroves from seashore to upland, to contrast the natural variability in bacteria, cyanobacteria and diazotroph assemblages in a pristine area compared to an oil polluted area along a timespan of three years, based on ARISA (bacteria and cyanobacteria) and nifH T-RFLP (diazotrophs) fingerprinting. The data presented herein indicated that changes in all the communities evaluated were mainly driven by the temporal effect in the contaminated area, while local effects were dominant on the pristine mangrove. A positive correlation of community structure between diazotrophs and cyanobacteria was observed, suggesting the functional importance of this phylum as nitrogen fixers in mangroves soils. Different ecological patterns explained the microbial behavior in the pristine and polluted mangroves. Stochastic models in the pristine mangrove indicate that there is not a specific environmental factor that determines the bacterial distribution, while cyanobacteria and diazotrophs better fitted in deterministic model in the same area. For the contaminated mangrove site, deterministic models better represented the variations in the communities, suggesting that the presence of oil might change the microbial ecological structures over time. Mangroves represent a unique environment threatened by global change, and this study contributed to the knowledge of the microbial distribution in such areas and its response on persistent contamination historic events.

Temporal assessment of microbial communities in soils of two contrasting mangroves

Variations in microbial communities promoted by alterations in environmental conditions are reflected in similarities/differences both at taxonomic and functional levels. Here we used a natural gradient within mangroves from seashore to upland, to contrast the natural variability in bacteria, cyanobacteria and diazotroph assemblages in a pristine area compared to an oil polluted area along a timespan of three years, based on ARISA (bacteria and cyanobacteria) and nifH T-RFLP (diazotrophs) fingerprinting. The data presented herein indicated that changes in all the communities evaluated were mainly driven by the temporal effect in the contaminated area, while local effects were dominant on the pristine mangrove. A positive correlation of community structure between diazotrophs and cyanobacteria was observed, suggesting the functional importance of this phylum as nitrogen fixers in mangroves soils. Different ecological patterns explained the microbial behavior in the pristine and polluted mangroves. Stochastic models in the pristine mangrove indicate that there is not a specific environmental factor that determines the bacterial distribution, while cyanobacteria and diazotrophs better fitted in deterministic model in the same area. For the contaminated mangrove site, deterministic models better represented the variations in the communities, suggesting that the presence of oil might change the microbial ecological structures over time. Mangroves represent a unique environment threatened by global change, and this study contributed to the knowledge of the microbial distribution in such areas and its response on persistent contamination historic events.(AU)

Temporal assessment of microbial communities in soils of two contrasting mangroves.

Variations in microbial communities promoted by alterations in environmental conditions are reflected in similarities/differences both at taxonomic and functional levels. Here we used a natural gradient within mangroves from seashore to upland, to contrast the natural variability in bacteria, cyanobacteria and diazotroph assemblages in a pristine area compared to an oil polluted area along a timespan of three years, based on ARISA (bacteria and cyanobacteria) and nifH T-RFLP (diazotrophs) fingerprinting. The data presented herein indicated that changes in all the communities evaluated were mainly driven by the temporal effect in the contaminated area, while local effects were dominant on the pristine mangrove. A positive correlation of community structure between diazotrophs and cyanobacteria was observed, suggesting the functional importance of this phylum as nitrogen fixers in mangroves soils. Different ecological patterns explained the microbial behavior in the pristine and polluted mangroves. Stochastic models in the pristine mangrove indicate that there is not a specific environmental factor that determines the bacterial distribution, while cyanobacteria and diazotrophs better fitted in deterministic model in the same area. For the contaminated mangrove site, deterministic models better represented the variations in the communities, suggesting that the presence of oil might change the microbial ecological structures over time. Mangroves represent a unique environment threatened by global change, and this study contributed to the knowledge of the microbial distribution in such areas and its response on persistent contamination historic events.

Development and nitrate reductase activity of sugarcane inoculated with five diazotrophic strains.

Diazotrophs are able to stimulate plant growth. This study aimed at evaluating the effect of inoculation of five diazotrophic strains on growth promotion and nitrate reductase (NR, EC 1.7.1.1) activity in sugarcane. An experiment was carried out from three stages of cultivation: sprouting, tubes, and in hydroponics. On the first two stages, seven treatments were adopted: uninoculated control; mixed inoculation with five strains; and individual inoculation with Gluconacetobacter diazotrophicus (Gd), Herbaspirillum rubrisubalbicans (Hr), Herbaspirillum seropedicae (Hs), Nitrospirillum amazonense (Na), and Paraburkholderia tropica (Pt). The four treatments showing the best performance were transferred to the hydroponic system for analysis of NR activity. Hs, Pt, and the mixture of all strains led to the highest seedling biomass in tubes, followed by Hr. In hydroponics, the mixture and the strain Hr had the highest growth-promoting effect. NR activity was influenced by inoculation only under low N supply conditions, with positive effect of Hr, Pt, and the mixture.

Plant Growth-Promoting Bacteria (PGPB): Isolation and Screening of PGP Activities.

Plant roots are associated with numerous and diverse types of beneficial and pathogenic microorganisms. Plant growth-promoting (rhizo)bacteria (PGPB or PGPR) are isolated from plants crops worldwide, and many of them are used as agricultural inoculants. Agricultural biofertilization and biocontrol of pathogens are eco-friendly alternatives to chemical usage and have less energy, environmental, and economic costs. PGPB isolation and evaluation are essentials steps for determining bacteria that are able to improve plant development and productivity. In this unit, we present protocols to isolate bacteria from soil and plant roots ("putative" diazotrophic and endospore-forming bacteria), as well to evaluate some of their beneficial characteristics for the promotion of plant growth (e.g., nitrogen fixation, production of indolic compounds and siderophores, phosphate solubilization, and 1-aminocyclopropane-1-carboxylate deaminase activity). © 2017 by John Wiley & Sons, Inc.

Lipopolysaccharides in diazotrophic bacteria.

Biological nitrogen fixation (BNF) is a process in which the atmospheric nitrogen (N2) is transformed into ammonia (NH3) by a select group of nitrogen-fixing organisms, or diazotrophic bacteria. In order to furnish the biologically useful nitrogen to plants, these bacteria must be in constant molecular communication with their host plants. Some of these molecular plant-microbe interactions are very specific, resulting in a symbiotic relationship between the diazotroph and the host. Others are found between associative diazotrophs and plants, resulting in plant infection and colonization of internal tissues. Independent of the type of ecological interaction, glycans, and glycoconjugates produced by these bacteria play an important role in the molecular communication prior and during colonization. Even though exopolysaccharides (EPS) and lipochitooligosaccharides (LCO) produced by diazotrophic bacteria and released onto the environment have their importance in the microbe-plant interaction, it is the lipopolysaccharides (LPS), anchored on the external membrane of these bacteria, that mediates the direct contact of the diazotroph with the host cells. These molecules are extremely variable among the several species of nitrogen fixing-bacteria, and there are evidences of the mechanisms of infection being closely related to their structure.

Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state / Caracterização molecular de bactérias fixadoras de nitrogênio isoladas de plantas brasileiras no estado de São Paulo

Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.

Quatorze linhagens de bactérias fixadoras de nitrogênio foram isoladas de diferentes espécies de plantas, incluindo cassava, milho e cana-de-açúcar, usando condições seletivas desprovidas de nitrogênio. A capacidade de fixar nitrogênio foi verificada por ensaio de redução de acetileno. Todas as linhagens fixadoras de nitrogênio testadas apresentaram hibridização positiva com sonda de gene nifH derivada de Azospirillum brasilense. As linhagens foram caracterizadas por RAPD, ARDRA e sequenciamento do gene 16S rDNA. As análises de RAPD revelaram 8 genótipos, as 6 linhagens restantes foram agrupadas em 3 grupos de RAPD, sugerindo uma origem clonal. ARDRA e seqüências de 16S rDNA foram alocadas em 13 grupos conhecidos de bactérias fixadoras de nitrogênio, incluindo organismos dos gêneros Azospirillum, Herbaspirillum, Pseudomonas e Enterobacteriaceae. Duas linhagens foram classificadas como Stenotrophomonas ssp. Os resultados da identificação molecular baseados em sequencias de 16S rDNA corroboram com dados obtidos em testes morfológicos e bioquímicos.

Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state.

Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.

Molecular characterization of nitrogen-fixing bacteria isolated from brazilian agricultural plants at São Paulo state

Fourteen strains of nitrogen-fixing bacteria were isolated from different agricultural plant species, including cassava, maize and sugarcane, using nitrogen-deprived selective isolation conditions. Ability to fix nitrogen was verified by the acetylene reduction assay. All potentially nitrogen-fixing strains tested showed positive hybridization signals with a nifH probe derived from Azospirillum brasilense. The strains were characterized by RAPD, ARDRA and 16S rDNA sequence analysis. RAPD analyses revealed 8 unique genotypes, the remaining 6 strains clustered into 3 RAPD groups, suggesting a clonal origin. ARDRA and 16S rDNA sequence analyses allowed the assignment of 13 strains to known groups of nitrogen-fixing bacteria, including organisms from the genera Azospirillum, Herbaspirillum, Pseudomonas and Enterobacteriaceae. Two strains were classified as Stenotrophomonas ssp. Molecular identification results from 16S rDNA analyses were also corroborated by morphological and biochemical data.

Quatorze linhagens de bactérias fixadoras de nitrogênio foram isoladas de diferentes espécies de plantas, incluindo cassava, milho e cana-de-açúcar, usando condições seletivas desprovidas de nitrogênio. A capacidade de fixar nitrogênio foi verificada por ensaio de redução de acetileno. Todas as linhagens fixadoras de nitrogênio testadas apresentaram hibridização positiva com sonda de gene nifH derivada de Azospirillum brasilense. As linhagens foram caracterizadas por RAPD, ARDRA e sequenciamento do gene 16S rDNA. As análises de RAPD revelaram 8 genótipos, as 6 linhagens restantes foram agrupadas em 3 grupos de RAPD, sugerindo uma origem clonal. ARDRA e seqüências de 16S rDNA foram alocadas em 13 grupos conhecidos de bactérias fixadoras de nitrogênio, incluindo organismos dos gêneros Azospirillum, Herbaspirillum, Pseudomonas e Enterobacteriaceae. Duas linhagens foram classificadas como Stenotrophomonas ssp. Os resultados da identificação molecular baseados em sequencias de 16S rDNA corroboram com dados obtidos em testes morfológicos e bioquímicos.