Nitrogen-fixation is an essential biochemical reaction involving the reduction of inert, atmospheric dinitrogen (N2) into biochemically accessible ammonia (NH3). Organisms that are capable of this process are collectively called “diazotrophs” and are ubiquitous in marine and terrestrial environments. Despite the wide distribution, little is known about the biological nature of the diverse groups of diazotrophs. This study was designed to address the influence of light and nutrients on nitrogen fixation and growth in several marine diazotrophic symbioses collectively termed “Diatom-Diazotroph Associations (DDAs).” The organisms of interest included the diatoms Hemiaulus haukii Grunow and Hemiaulus membranaceus Cleve, and their diazotrophic endosymbiont Richelia intracellularis Schmidt. The study included acetylene reduction assays, growth rate, and nutrient analysis experiments on both associations in order to better understand the similarities and differences within and between the two DDAs. The results indicate distinct differences in nitrogen fixation rates within and between the species. In the nitrogen addition experiment, the “no added nitrogen” treatment had the highest N2-fixation rate (N2-fixmax = 7.43 x 10-5 nmols N2 heterocyst-1min-1), followed by the added nitrate treatment (N2-fixmax = 6.49 x 10-5 nmols N2 heterocyst-1min-1) and the added ammonium treatment (N2-fixmax = 3.79 x 10-5 nmols N2 heterocyst-1min-1). The maximum growth rate occurred in the “added ammonium” treatment (0.42 divisions day-1), which had a higher percentage of asymbiotic cells than the two other treatments. The maximum recorded rate of N2-fixation for H. haukii was 7.43 x 10-5 nmol N2 heterocyst-1min-1 and the maximum value of N2-fixation for H. membranaceus was 1.88 x 10-4 nmol N2 heterocyst-1min-1. The maximum growth rate for H. haukii was 0.99 divisions day-1, and 1.06 divisions day-1 for H. membranaceus. Growth followed light saturation kinetics in H. haukii with a compensation light intensity (IC) of 10 µmol quanta m-2sec-1 and saturation light intensity (IK) of 100 µmol quanta m-2sec-1. H. haukii and H.membranaceus expressed light saturation kinetics in N2-fixation. N2-fixation was generally limited to the light period, with no evidence of a morning or evening enhancement. The DDAs grew solely on N2-fixation and did not use nitrate. This study contributes to current knowledge of DDAs and their role in global marine nitrogen fixation. / text

In the subtropical oligotrophic ocean, nitrogen fixation is an important source of new nitrogen (N) for supporting biological production. Previous studies have found that nitrogen-fixing Diatom-Diazotroph Associations (DDAs) are in high abundance in the intermediate salinity zone of large river plumes such as the Amazon and Mekong rivers, while Trichodesmium spp. becomes more abundant at higher salinities. This recurring pattern in the Amazon River plume suggests that strong salinity and nutrient gradients within the river plumes may lead to a cascade in diazotroph communities. I hypothesized that the Mississippi River, a major source of freshwater, nutrients and sediments to the northern Gulf of Mexico, creates a similar distribution of diazotroph communities. The relationship between large diazotrophs and salinity was examined in samples collected in July 2011 during a flood outflow from the Mississippi River. The dominant DDA, Hemiaulus spp. – Richelia spp., was at greatest abundance (≈31,000 cells L-1) west of the birdfoot delta on the periphery of the plume (≈29 salinity) where bottom water hypoxia was also observed. Trichodesmium spp., a cyanobacterium genus that occurs in both colonial and free trichome morphologies, was abundant at both high (≈35) salinities east of the delta reaching 20+ colonies L-1, as well as in the fresher (≈28) waters of the plume where it reached 3,500 trichomes L-1. Diazotroph distributions were separated east and west of the Mississippi River outflow, with DDAs being most abundant over bottom water hypoxic regions to the west and Trichodesmium spp. in high abundance to the east. The diazotroph – salinity gradient relationships present within the Amazon River plume were not present within the Gulf of Mexico. This study suggests that environmental factors other than salinity, such as nutrients or hypoxia, are influencing the distribution of diazotrophs around the Mississippi River plume. The seasonal hypoxia seen in the Gulf of Mexico with the co-occurring DDA increase could appear in other river systems. / text

Grandes esforços têm sido empreendidos para preservar recursos naturais e desenvolver sistemas de produção agrícola mais sustentáveis, reduzindo o uso de fertilizantes. Uma das alternativas ao uso de fertilizantes nitrogenados na agricultura são os inoculantes com bactérias diazotróficas. Os micro-organismos diazotróficos mais estudados para produção de inoculantes são os simbiontes noduladores de leguminosas. Para outros grupos de plantas, os diazotróficos mais utilizados são assimbióticos. Estudos conduzidos na Mata Atlântica e na Amazônia tem mostrado que a fixação biológica de nitrogêncio (FBN) assimbiótica associada a filosfera pode contribuir com aportes de N significativos, e que esses biomas podem abrigar uma grande diversidade de bactérias diazotróficas, sendo ambientes apropriados para prospectar diazotróficos eficientes para o uso na agricultura. O objetivo deste trabalho foi isolar novos genótipos de bactérias diazotróficas assimbióticas da filosfera e serapilheira de espécies arbóreas da floresta Amazônica, e estimar suas taxas de FBN e de síntese de ácido indolacético in vitro, bem como identificar taxonomicamente os isolados considerados mais promissores para uso biotecnológico. Bactérias diazotróficas de vida-livre foram isoladas da filosfera e serapilheira de três espécies arbóreas (Rinorea pubiflora, Amphirrhox longifolia e Chamaecrista xinguensis), e cultivadas em meio isento de N. Um total de 86 isolados foram obtidos. A taxa de fixação de nitrogênio foi estimada para todos os isolados, através do ensaio de redução de acetileno (ARA), pelo complexo nitrogenase. Para todos os isolados, também foi determinada a taxa de produção de ácido indolacético (AIA), na presença de L-triptofano. Os dados obtidos foram submetidos à análise de variância e as médias comparadas pelo teste de Scott-Knott e pelo teste de Duncan (p<0,05), através do software estatístico R. Os isolados que apresentaram as maiores taxas de redução de acetileno e de síntese de AIA foram identificados através do sequenciamento parcial do gene rRNA 16S. Cerca de 84,8% dos isolados apresentaram atividade de nitrogenase in vitro, e o teste estatístico desmonstrou não haver diferenças significativas entre as taxas de redução de acetileno entre os isolados, exceto o isolado 10RF, o qual apresentou uma taxa de redução de acetileno maior do que a dos demais isolados (p<0,05). Todos os isolados produziram AIA in vitro, e houve diferenças significativas na síntese de AIA entre os isolados, e quanto origem dos mesmos. Isolados bacterianos de Amphirrox longifolia sintetizaram mais AIA in vitro, em comparação aos isolados das outras duas espécies arbóreas. Cerca de 22% dos isolados apresentaram taxas de fixação de nitrogêncio maiores que 0.025ng N &mu;g-1 h-1, e 20% dos isolados produziram mais de 21,87 &mu;g/mL-1 de AIA. 21 isolados, considerados promissores para uso agrícola (fixadores de N e produtores de AIA), foram identificados através do sequenciamento do gene rRNA 16S, e afiliados aos gêneros Pseudomonas, Bacillus, Burkholderia, Citrobacter, Rhizobium e Stenotrophomonas. Aproximadamente 71 % dos isolados considerados promissores para promoverem o crescimento de plantas foram isolados da filosfera, indicando ser este um ambiente propício à prospecção de bactérias diazotróficas promotoras de crescimento de plantas. / Great efforts have been made to preserve natural resources and develop more sustainable agricultural production systems, reducing the use of fertilizers. One of the alternatives to the use of nitrogen fertilizers in agriculture is the use of inoculants with diazotrophic bacteria. The most studied diazotrophic microorganisms for the production of inoculants are nodule-forming symbionts of legumes. For other groups of plants, the most used diazotrophs are asymbiotic. Several studies in the Atlantic and Amazon forests have shown that the biological nitrogen fixation (BNF) associated with the phyllosphere may contribute with significant N inputs, and that these biomes can harbor a great diversity of diazotrophic bacteria, making them important environments for the prospection of efficient diazotrophs for agricultural use. The objective of this work was to isolate new genotypes of asymbiotic diazotrophic bacteria from the phyllosphere and litter of tree species of the Amazon forest, and to estimate their rates of BNF and indoleacetic acid biosynthesis in vitro, as well as to identify taxonomically the most promising isolates for biotechnological use. Asymbiotic diazotrophic bacteria were isolated from the phyllosphere and litter of three tree species (Rinorea pubiflora, Amphirrhox longifolia and Chamaecrista xinguensis), and cultivated in N-free medium. A total of 86 isolates were obtained. The nitrogen fixation rate was estimated for all isolates measuring the nitrogenase activity by the acetylene reduction assay (ARA). The rates of indoleacetic acid (IAA) production in the presence of L-tryptophan was also determined for all isolates. The data obtained were submitted to analysis of variance and the means were compared by the Scott-Knott test and the Duncan test (p<0.05) using the statistical software R. The isolates showing the highest rates of acetylene reduction and IAA biosynthesis were identified through the partial sequencing of the 16S rRNA gene. Approximately 84.8% of the isolates were positive for nitrogenase activity in vitro, and the statistical analyses showed that there were no significant differences between the acetylene reduction rates among the isolates, except the isolate 10RF, which showed a higher acetylene reduction rate than the other isolates (p<0.05). All isolates produced IAA in vitro, and there were significant differences in the biosynthesis rates among the isolates, as well as regarding the origin of the isolate. Bacterial isolates from Amphirrox longifolia synthesized higher amounts of IAA in vitro as compared to the isolates from the other two tree species. Approximately 22% of the isolates showed nitrogen fixation rates greater than 0.025 ng N &mu;g-1 h-1, and 20% of the isolates produced more than 21.87 &mu;g mL-1 of IAA. 21 isolates, considered promising for agricultural use (N-fixers and IAA producers), were identified by sequencing the 16S rRNA gene, and affiliated to the genera Pseudomonas, Bacillus, Burkholderia, Citrobacter, Rhizobium and Stenotrophomonas. Approximately 71% of the isolates considered promising to promote plant growth were isolated from the phyllosphere, suggesting that this environment is propitious to the prospection of diazotrophic plant growth promoting bacteria.

Submitted by Sandra Pereira (srpereira@ufrrj.br) on 2017-04-26T14:05:04Z No. of bitstreams: 1 2012 - Patricia Gon?alves Galv?o.pdf: 5289496 bytes, checksum: 8dcb41bc971793cc8b4cdf383401ade4 (MD5) / Made available in DSpace on 2017-04-26T14:05:05Z (GMT). No. of bitstreams: 1 2012 - Patricia Gon?alves Galv?o.pdf: 5289496 bytes, checksum: 8dcb41bc971793cc8b4cdf383401ade4 (MD5) Previous issue date: 2012-03-01 / Coordena??o de Aperfei?oamento de Pessoal de N?vel Superior - CAPES / The objective of this study was to evaluate the protein profile expression of G. diazotrophicus PAL5 and its defective mutant in the indole compound production (lao-) grown in the presence or absence of tryptophan through 2DE-PAGE technique. The spectrometric analysis allowed the identification of 24 differentially expressed proteins. The majority of the proteins down regulated in the wild type PAL5 cultivated with tryptophan as compared to the cultivation without the amino acid belonged to the category of transductional modification, protein turnover and chaperones. For the mutant lao- grown in the same conditions, the majority of the proteins that presented differential expression belonged to the category of production and conversion of energy. In addition, the majority of the protein differentially expressed in the mutant lao- as compared to the wild-type PAL5 strains belonged to carbohydrates metabolism and transport. On the other hand, no proteins related to the tryptophan biosynthesis were detected in any condition, possibly due to the low yield of the proteins during the spectrometric analysis. Furthermore, mutants lao- and nif- of G. diazotrophicus were used for inoculation of micropropagated sugarcane plants in order to determine the influence of auxins produced by the bacteria in the plant growth promotion in comparison with PAL5. The first experiment, carried out in hydroponic conditions for 10 days showed a significant inoculation effect of the wild type on plant shoot. The other experiment was conducted in a period of 120 days in pots containing sand:vermiculite substrate fertilized with 30 and 60 ppm with ammonium sulphate enriched with 15N. The plants were inoculated in vitro with the wild type and mutants lao- and nif-, and the results showed a visual difference in the roots inoculated with PAL5 that showed higher volume suggesting a higher number of secondary roots and root hairs. On the other hand, the plants inoculated with the lao- mutant were ticker and showed lower number of secondary roots and root hairs. The shoot biomass of plants inoculated with PAL5 was higher than those inoculated with the mutant strains for both N dose, however the difference was not significant. Plants grown with 60 kg N dose and inoculated with the mutants showed lower accumulation of dry shoot mass than plants inoculated with the wild type strain. In conclusion, the present study showed the occurrence of several differentially expressed proteins either in the wild type strain or in the mutant lao- grown in LGI-P with and without tryptophan. The role played by these proteins in the metabolism of the bacteria requires additional studies, including different growth conditions. In addition, the inoculation of micropropagated sugarcane plants suggested a hormonal effect of the bacteria mainly on the root development e consequently in the N use efficiency. However, the size of the pots may have limited the plant development, suggesting that new experiments should be carried out in more appropriated conditions to confirm the influence of the indol production and the BNF during the association of the G. diazotrophicus and sugarcane plants / Este estudo teve por objetivo avaliar o perfil de express?o de prote?nas de G. diazotrophicus PAL5 e seu mutante defectivo na produ??o de compostos ind?licos (lao-) cultivados na presen?a e aus?ncia de triptofano atrav?s da t?cnica de 2DE-PAGE. A an?lise por espectrometria de massa permitiu a identifica??o de 24 prote?nas diferencialmente expressas. A maioria das prote?nas com a express?o diminu?da em PAL5 cultivada em meio com triptofano em rela??o ao meio de cultivo sem esse amino?cido pertenceu ? categoria modifica??o p?s-traducional, turnover de prote?nas e chaperonas. No mutante lao- cultivado nas mesmas condi??es, a maioria das prote?nas que apresentaram express?o diferencial pertencia ? categoria produ??o e convers?o de energia. Em adi??o, a maioria das prote?nas que foram diferencialmente expressas no mutante lao- em compara??o com a estirpe selvagem PAL5 pertencia ? categoria metabolismo e transporte de carboidratos. Por outro lado, n?o foram observadas prote?nas relacionadas ? bioss?ntese de triptofano em nenhuma condi??o analisada possivelmente devido ao baixo rendimento das identifica??es por espectrometria. Al?m das an?lises dos perfis de prote?nas, os mutantes lao- e nif- de G. diazotrophicus foram inoculados em plantas de cana-de-a??car micropropagadas com o objetivo de determinar a influ?ncia das auxinas na promo??o do crescimento dessa cultura em compara??o com a estirpe selvagem PAL5. O primeiro experimento, conduzido em condi??es de hidroponia pelo per?odo de 10 dias, mostrou efeito significativo da inocula??o da estirpe selvagem na promo??o de crescimento da parte ?rea das plantas, enquanto que o mutante lao-, n?o diferiu estatisticamente do controle n?o inoculado. O outro experimento, foi conduzido por 120 dias em vasos com substrato areia:vermiculita contendo 30 ou 60 ppm de sulfato de am?nio enriquecido com 15N e as pl?ntulas foram inoculadas in vitro. Os resultados mostraram uma diferen?a visual nas ra?zes das plantas inoculadas com PAL5, que se mostraram mais volumosas, aparentando um n?mero mais elevado de ra?zes secund?rias e p?los radiculares. J? as plantas inoculadas com lao- apresentaram ra?zes mais grossas, com um n?mero muito reduzido de ramifica??es ou p?los radiculares. A biomassa seca da parte a?rea das plantas inoculadas com PAL5 foi superior ?quelas inoculadas com as estirpes mutantes para as duas doses de nitrog?nio, por?m essa diferen?a n?o foi significativa. N?o foram observadas evid?ncias de contribui??o da FBN, por?m as plantas inoculadas com PAL5 foram menos eficientes na recupera??o do N fertilizante. Em conclus?o, o presente estudo mostra a ocorr?ncia de diversas prote?nas diferencialmente expressas tanto na estirpe selvagem como em lao- quando crescidas na presen?a e aus?ncia do amino?cido triptofano. A defini??o do papel dessas prote?nas no metabolismo da bact?ria requer estudos adicionais, inclusive em diferentes condi??es de cultivo. Em adi??o, a inocula??o dessas bact?rias em plantas de cana-de-a??car mostrou o efeito hormonal da bact?ria no desenvolvimento das ra?zes e, por conseguinte na maior efici?ncia de uso do N aplicado. Entretanto, dado a limita??o de espa?o f?sico dos vasos para o desenvolvimento das plantas, sugere-se a realiza??o de novos experimentos, em condi??es mais apropriadas, para confirmar a influ?ncia da produ??o de ?ndoles e da FBN durante a associa??o da bact?ria com as plantas de cana-de-a??car.

Nitrogen fixation is an ecologically important microbial process that can contribute bioavailable combined N to habitats low in N. Shipworms, or wood-boring bivalves, host N2-fixing and cellulolytic symbiotic bacteria in gill bacteriocytes, which have been implicated as a necessary adaptation to an N-poor C-rich (wooden) diet. Shipworm symbionts are known to fix N within the gill habitat and newly fixed N is subsequently incorporated into non-symbiont containing host tissue. The presence of N2-fixation in gill bacteriocytes presents a conundrum because N2-fixation is tightly regulated by oxygen in most other diazotrophic microbes. Also, the direct evidence of new N being incorporated into the host tissue indicates that there are potentially complex nutrient cycles in this symbiosis, which have not been investigated. We used the cultivated symbiont Teredinibacter turnerae, which has been isolated from many shipworm species, as a model organism to elucidate controls on N2-fixation and N release in the shipworm symbiosis. Our results indicate that headspace oxygen concentration does not control biomass specific N2-fixation and respiration activity in T. turnerae, but it does influence the magnitude of the growth rate and timing of culture growth. Also, we examined the controls of oxygen on inorganic nutrient uptake rates, and documented a small amount of dissolved inorganic nitrogen release. While the N budget is only partially balanced, we provide indirect evidence for the allocation of fixed N to the excretion of exopolymeric substances and dissolved organic nitrogen; future studies that measure these additional N sinks are necessary to close the N budget. Although there are limitations of using pure cultures to investigate a complex symbiotic system, this study provides direct experimental evidence that T. turnerae has adaptations that are conducive to N2-fixation in gill bacteriocytes.

In the North Pacific Subtropical Gyre, a nearly-annual phytoplankton bloom forms near the subtropical front at ~30° N. Mixed communities of nitrogen-fixing diatom symbioses (diatom-diazotroph associations) increase 10²-10³ fold in these blooms. In July 2008 (31.46˚N 140.49˚W) and August 2009 (25.18 °N 154 °W), two blooms were sampled to determine diatom-diazotroph association species composition, physical, and chemical characteristics of the water column. In both 2008 and 2009, the dominant diatom-diazotroph association was the Hemiaulus hauckii-Richelia intracellularis symbiosis. The 2009 subtropical front bloom was missed; however, another bloom closer to Hawaii was sampled where diatom-diazotroph association abundance was 10-fold lower (10² cells Lˉ¹) than 2008 despite surface chlorophyll a values that were 3 times greater. Both blooms showed substantial changes in phytoplankton size structure with the >10 μm size chlorophyll a fraction increasing from 10 to 40 % in 2008. In the 2009 bloom, the non-symbiotic pennate diatom Mastogloia woodiana numerically dominated (>150,000 cells Lˉ¹) and formed aggregates that resulted in substantially higher % of netplankton chlorophyll a fractions. Summer open ocean blooms from the two years share a common trend of Hemiaulus dominance of the diatom-diazotroph association population and size structure changes. However, non-symbiotic species can dominate the overall bloom, and diatom-diazotroph association species may not be responsible for the chlorophyll a increase. These two years may represent different types of blooms or temporal changes within summer diatom blooms. The increased biomass in the larger-size fraction suggests these blooms are potential sites for carbon export from the surface layer. / text

Grandes esforços têm sido empreendidos para preservar recursos naturais e desenvolver sistemas de produção agrícola mais sustentáveis, reduzindo o uso de fertilizantes. Uma das alternativas ao uso de fertilizantes nitrogenados na agricultura são os inoculantes com bactérias diazotróficas. Os micro-organismos diazotróficos mais estudados para produção de inoculantes são os simbiontes noduladores de leguminosas. Para outros grupos de plantas, os diazotróficos mais utilizados são assimbióticos. Estudos conduzidos na Mata Atlântica e na Amazônia tem mostrado que a fixação biológica de nitrogêncio (FBN) assimbiótica associada a filosfera pode contribuir com aportes de N significativos, e que esses biomas podem abrigar uma grande diversidade de bactérias diazotróficas, sendo ambientes apropriados para prospectar diazotróficos eficientes para o uso na agricultura. O objetivo deste trabalho foi isolar novos genótipos de bactérias diazotróficas assimbióticas da filosfera e serapilheira de espécies arbóreas da floresta Amazônica, e estimar suas taxas de FBN e de síntese de ácido indolacético in vitro, bem como identificar taxonomicamente os isolados considerados mais promissores para uso biotecnológico. Bactérias diazotróficas de vida-livre foram isoladas da filosfera e serapilheira de três espécies arbóreas (Rinorea pubiflora, Amphirrhox longifolia e Chamaecrista xinguensis), e cultivadas em meio isento de N. Um total de 86 isolados foram obtidos. A taxa de fixação de nitrogênio foi estimada para todos os isolados, através do ensaio de redução de acetileno (ARA), pelo complexo nitrogenase. Para todos os isolados, também foi determinada a taxa de produção de ácido indolacético (AIA), na presença de L-triptofano. Os dados obtidos foram submetidos à análise de variância e as médias comparadas pelo teste de Scott-Knott e pelo teste de Duncan (p<0,05), através do software estatístico R. Os isolados que apresentaram as maiores taxas de redução de acetileno e de síntese de AIA foram identificados através do sequenciamento parcial do gene rRNA 16S. Cerca de 84,8% dos isolados apresentaram atividade de nitrogenase in vitro, e o teste estatístico desmonstrou não haver diferenças significativas entre as taxas de redução de acetileno entre os isolados, exceto o isolado 10RF, o qual apresentou uma taxa de redução de acetileno maior do que a dos demais isolados (p<0,05). Todos os isolados produziram AIA in vitro, e houve diferenças significativas na síntese de AIA entre os isolados, e quanto origem dos mesmos. Isolados bacterianos de Amphirrox longifolia sintetizaram mais AIA in vitro, em comparação aos isolados das outras duas espécies arbóreas. Cerca de 22% dos isolados apresentaram taxas de fixação de nitrogêncio maiores que 0.025ng N &mu;g-1 h-1, e 20% dos isolados produziram mais de 21,87 &mu;g/mL-1 de AIA. 21 isolados, considerados promissores para uso agrícola (fixadores de N e produtores de AIA), foram identificados através do sequenciamento do gene rRNA 16S, e afiliados aos gêneros Pseudomonas, Bacillus, Burkholderia, Citrobacter, Rhizobium e Stenotrophomonas. Aproximadamente 71 % dos isolados considerados promissores para promoverem o crescimento de plantas foram isolados da filosfera, indicando ser este um ambiente propício à prospecção de bactérias diazotróficas promotoras de crescimento de plantas. / Great efforts have been made to preserve natural resources and develop more sustainable agricultural production systems, reducing the use of fertilizers. One of the alternatives to the use of nitrogen fertilizers in agriculture is the use of inoculants with diazotrophic bacteria. The most studied diazotrophic microorganisms for the production of inoculants are nodule-forming symbionts of legumes. For other groups of plants, the most used diazotrophs are asymbiotic. Several studies in the Atlantic and Amazon forests have shown that the biological nitrogen fixation (BNF) associated with the phyllosphere may contribute with significant N inputs, and that these biomes can harbor a great diversity of diazotrophic bacteria, making them important environments for the prospection of efficient diazotrophs for agricultural use. The objective of this work was to isolate new genotypes of asymbiotic diazotrophic bacteria from the phyllosphere and litter of tree species of the Amazon forest, and to estimate their rates of BNF and indoleacetic acid biosynthesis in vitro, as well as to identify taxonomically the most promising isolates for biotechnological use. Asymbiotic diazotrophic bacteria were isolated from the phyllosphere and litter of three tree species (Rinorea pubiflora, Amphirrhox longifolia and Chamaecrista xinguensis), and cultivated in N-free medium. A total of 86 isolates were obtained. The nitrogen fixation rate was estimated for all isolates measuring the nitrogenase activity by the acetylene reduction assay (ARA). The rates of indoleacetic acid (IAA) production in the presence of L-tryptophan was also determined for all isolates. The data obtained were submitted to analysis of variance and the means were compared by the Scott-Knott test and the Duncan test (p<0.05) using the statistical software R. The isolates showing the highest rates of acetylene reduction and IAA biosynthesis were identified through the partial sequencing of the 16S rRNA gene. Approximately 84.8% of the isolates were positive for nitrogenase activity in vitro, and the statistical analyses showed that there were no significant differences between the acetylene reduction rates among the isolates, except the isolate 10RF, which showed a higher acetylene reduction rate than the other isolates (p<0.05). All isolates produced IAA in vitro, and there were significant differences in the biosynthesis rates among the isolates, as well as regarding the origin of the isolate. Bacterial isolates from Amphirrox longifolia synthesized higher amounts of IAA in vitro as compared to the isolates from the other two tree species. Approximately 22% of the isolates showed nitrogen fixation rates greater than 0.025 ng N &mu;g-1 h-1, and 20% of the isolates produced more than 21.87 &mu;g mL-1 of IAA. 21 isolates, considered promising for agricultural use (N-fixers and IAA producers), were identified by sequencing the 16S rRNA gene, and affiliated to the genera Pseudomonas, Bacillus, Burkholderia, Citrobacter, Rhizobium and Stenotrophomonas. Approximately 71% of the isolates considered promising to promote plant growth were isolated from the phyllosphere, suggesting that this environment is propitious to the prospection of diazotrophic plant growth promoting bacteria.

Marine primary productivity in large parts of the oceans is supported by nitrogen fixers (diazotrophs). Richelia, a genus of multiple closely related species of heterocystous filamentous cyanobacterial diazotrophs, are often found in symbioses with a few genera of diatoms. Several Richelia species: R. euintracellularis, R. intracellularis and R. rhizosoleniae, form stable host specific partnerships and these populations are important for the nitrogen (N) cycle, especially in the oligotrophic open oceans. In general, the gene nifH, which encodes the enzyme nitrogenase for N2 fixation, is used as a molecular marker for presence (DNA level) and activity (RNA level) of diazotrophs. However, evidence of cross-reactivity of the nifH assays between two of the Richelia species shows the risk of incorrect estimations of abundance and activity. Moreover, transcript abundance is rarely normalized to a housekeeping gene.  The aims of this work were to develop and assess new assays for the detection of three symbiotic Richelia species: R. euintracellularis, R. intracellularis and R. rhizosoleniae. These new assays targeted molecular markers that were indicative nutrient acquisition. For example, one assay targets a gene encoding the high affinity phosphate transporter (pstS), a second targets a gene involved in iron (Fe) transport (exbB) (indicator of P transport, Fe transport, respectively), and a third assays a housekeeping gene encoding Ribonuclease P protein (rnpA). All assays were used to estimate abundance and expression in lab and field based samples. The new assays have high species specificity as revealed by BlastN analyses and lab-based cross-hybridization assays. We applied the assays to samples from a lab-based experiment of the facultative symbiont R. rhizosoleniae RrhiSC01 in order to investigate the temporal dynamics of expression. This revealed periodic expression of nifH and pstS related to the photoperiod with higher expression in the early and late photoperiod, respectively. The peak of pstS expression in the late photoperiod is likely to support the high P requirement of both photosynthesis and N2 fixation. Given the temporal regulation of pstS (and nifH) expression shown here, one must consider these results when sampling and/or interpreting field studies. Moreover, a comparison of the Richelia draft genomes and environmental metagenomic assembled genomes (MAGs) show that gene copy number per genome of exbB and pstS varies between strains. Given that Richelia tend to live in low nutrient environments, including N, P, and often Fe, the increased copy number per genome for pstS and exbB could be suggestive/evidence of adaptation to constant nutrient limitation. In addition, the assays were applied to natural samples containing populations of R. intracellularis and R. euintracellularis collected in the North and South Atlantic Ocean from multiple depths (5-90 meters). The DNA based gene copy abundances showed higher estimates when nifH is used compared to rnpA, which is likely due to a higher degree of cross-reactivity in the nifH assays. Gene expression was present but low for all targets. However, expression of exbB and pstS was higher in surface water – where nutrients are expected to be depleted. Lastly, bulk fixation rates of N2 and carbon (C) showed that the activity was low and fixation rates did not increase with the addition of dissolved organic phosphate (DOP) (a 1:4 mixture of 2-aminoethylphosphonic acid and beta-glycerophosphate disodium salt hydrate). This could be due to patchiness and low abundance of diazotrophs and discrepancies between samples, seasonal variation or that the populations were not P limited. In summary, the new assays constitute a better option to interspecific detection of Richelia. However, more work is required to assess how the expression relates to limitation of nutrients and other external factors to better understand the activity of these biogeochemically important populations. / I stora delar av haven stöds marin primärproduktion av kvävefixerare (diazotrofer). Richelia är ett släkte med ett flertal närbesläktade arter av heterocysta, filamentösa och kvävefixerande cyanobakterier som lever i symbios med ett fåtal kiselalger. Dessa är viktiga för kvävecykeln, särskilt i de oligotrofa (kvävefattiga) öppna haven. Genen nifH som kodar för det kvävefixerande enzymet nitrogenas, brukar användas som en molekylär markör för att detektera diazotrofa populationer och dess genuttryck används som en indikator för kvävefixering. Det finns emellertid tecken på att nifH ger felaktiga uppskattningar av dessa populationers abundans och aktivitet. Dessutom, när nifH-uttryck används som ett mått på aktivitet är det endast kvävefixering man observerar. Andra faktorer som begränsar förekomsten av dessa organismer, t.ex. brist på järn eller fosfor, kan inte avläsas. Dessutom har uttrycket av nifH sällan normaliserats till en stabilt uttryckt gen vilket gör jämförelser svåra. Syftet med detta arbete var att utveckla och utvärdera nya kvantitativ realtidspolymeraskedjereaktion (qPCR)-analyser för specifik detektion av identitet, abundans och aktivitet av tre symbiotiska Richelia-arter: R. euintracellularis, R. intracellularis och R. rhizosoleniae. De nya analyserna är inriktade på generna som kodar för högaffinitetsfosfattransportören pstS och biopolymertransportproteinet exbB (indikator för P-transport respektive Fe-transport). Genen som kodar för ribonukleas P-protein, rnpA, en gen med stabilt uttryck, användes för att uppskatta abundans och för att normalisera uttrycket av de andra målgenerna.  BlastN-analyser och labb-baserade korshybridiseringsanalyser visar att de nya analyserna har hög artspecificitet. Vi tillämpade analyserna på en kultur av den fakultativa symbionten R. rhizosoleniae RrhiSC01 för att undersöka det periodiska uttrycksmönster av målgenerna kopplat till tid på dygnet. Resultatet visade på ett periodiskt uttryck av nifH och pstS relaterat till fotoperioden med högre uttryck i den tidiga, respektive sena fotoperioden.  Det ökade uttrycket av pstS i den sena fotoperioden stödjer sannolikt det höga P-kravet för både fotosyntes och N2-fixering. Den tidsmässiga regleringen av pstS-uttryck som visas här, behöver man ta hänsyn till vid provtagning och tolkning av fältstudier. ​​ Dessutom visar en jämförelse av Richelia-genom att genkopiantalet för exbB och pstS varierar mellan stammar av Richelia. Med tanke på att Richelia tenderar att leva i miljöer med låga nivåer av näringsämnen, inklusive kväve, fosfor och ofta järn, kan det ökade antalet kopior per genom av pstS och exbB ett tecken på anpassning till konstant näringsbegränsning. Analyserna tillämpades även på naturliga populationer av R. intracellularis och R. euintracellularis i norra och södra Atlanten. DNA-baserad estimering av abundans (genkopior) visar på en högre uppskattning när nifH används som markör jämfört med rnpA - troligtvis på grund av en högre grad av korsreaktivitet i nifH-analyserna. Genuttryck var närvarande men lågt för alla målgener i norra och södra Atlanten. Uttrycket av exbB och pstS var dock högre i ytvatten – där halter av näringsämnen förväntas vara låga. Slutligen var kväve- och kolfixering i planktonsamhället låg och ökade inte vid tillsats av fosfor, vilket kan bero på ojämn fördelning av celler, låg förekomst av diazotrofer och diskrepanser mellan prover, säsongsvariationer eller att populationerna inte var primärt begränsade av fosfor. Sammanfattningsvis utgör de nya analyserna ett bättre alternativ för specifik detektion av Richelia. Det krävs dock mer arbete för att bedöma hur uttrycket påverkas av  näringsbrist och yttre faktorer och för att bättre förstå aktiviteten hos dessa ovärderliga populationer.

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O desenvolvimento de tecnologias alternativas que visem aumentar a disponibilidade e eficiência do uso dos recursos minerais para as plantas aparecem como importante medida para se alcançar um crescimento sustentável da agricultura. Neste contexto, o suprimento de nitrogênio por meios biológicos, utilizando micro-organismos diazotróficos, dos quais os mais estudados são os rizóbios associados simbioticamente às leguminosas, é uma importante alternativa. Porém o sucesso atingido com essa simbiose em leguminosas ainda não foi alcançado em outras famílias de plantas, principalmente por limitações associadas à otimização das combinações planta-bactéria. Em gramíneas, diazotróficos endofíticos têm sido usados como inoculante, com resultados controversos. No entanto, é possível que micro-organismos diazotróficos epifíticos possam apresentar maior vantagem adaptativa devido à menor especificidade em sua relação com a planta, e serem mais eficientes em plantas não-leguminosas. O presente trabalho teve por objetivo avaliar o efeito de um inoculante produzido a partir de bactérias diazotróficas isoladas da filosfera no crescimento de milho e tomateiro. Ensaios foram realizados em condições de casa-de-vegetação, utilizando-se três diferentes isolados de bactérias diazotróficas encapsuladas em esferas de alginato sob diferentes níveis de adubação nitrogenada. A eficiência dos isolados em fixar nitrogênio foi avaliada através da atividade da nitrogenase. As populações de diazotróficos na rizosfera foram determinadas através da técnica do número mais provável (NMP) ao final do ensaio. Além disso, parâmetros como biomassa da parte aérea e das raízes foram analisados. Os dados foram submetidos à análise de variância, sendo as médias das variáveis comparadas pelo teste Duncan (p<0,05), utilizando-se o programa estatístico R. Plantas de milho, aos 60 dias após a inoculação com o isolado J8L, apresentaram matéria seca da raiz e atividade da nitrogenase 26 e 50 % superior ao controle não-inoculado, respectivamente. Plantas de tomateiro, 30 dias após a inoculação com o isolado E5L, apresentaram matéria seca da parte aérea e matéria seca da raiz 39 e 31 % superior ao controle não-inoculado, respectivamente. Parte dos efeitos observados no milho e tomateiro podem ser explicados pela atividade de fixação biológica de nitrogênio e produção de fitormônios, como auxina. A atividade da nitrogenase na rizosfera do milho, 60 dias após a inoculação foi maior no solo sem adição de N. O NMP não apresentou diferenças significativas e nos ensaios com milho e tomateiro, entre plantas inoculadas e não inoculadas, mostrando uma provável influência da comunidade diazotrófica nativa sobre a população final de diazotróficas analisada. De uma maneira geral, os isolados J8L e E5L proporcionaram ganhos significativos para as culturas do milho e tomateiro, mostrando potencial para uso como biofertilizantes. / The development of alternative technologies to increasing availability and use efficiency of mineral resources for plants is an important step for sustainable agricultural production. In this context, the supply of nitrogen by biological means, using diazotrophic microorganisms, of which the most studied are rhizobia symbiotically associated with legumes, is an important alternative. However, the efficiency of rhizobium-legume symbioses has not yet been achieved in other plant families, mainly due to limitations associated with the optimization of plant-bacterial combinations. In grasses, endophytic diazotrophs have been used as inoculant, with controversial results. However, it is possible that epiphytic diazotrophic microorganisms may have adaptive advantages over endophytes, due to the lower specificity in their associations with plants, and be more efficient in non-leguminous plants. The objective of the present work was to evaluate the effect of an inoculant produced with diazotrophic bacteria isolated from the phyllosphere on the growth of maize and tomato. Assays were carried out under greenhouse conditions using three isolates of diazotrophic bacteria encapsulated in alginate beads under different levels of nitrogen fertilization. The efficiency of the isolates in fixing nitrogen was evaluated determining nitrogenase activities. The populations of diazotrophic bacteria in the rhizosphere were determined by the most probable number (MPN) technique at the end of the experiment. In addition, parameters such as shoot and root biomass were analyzed. The data were submitted to the analysis of variance, and means were compared by the Duncan test (p<0.05), using the statistical program R. Maize plants at 60 days after inoculation with the J8L isolate presented root dry weight and nitrogenase activity in the rhizosphere 26 and 50% higher than the uninoculated control, respectively. Tomato plants, 30 days after inoculation with the E5L isolate, presented shoot and root dry weight 39 and 31% higher than the uninoculated control, respectively. Part of the effects observed in maize and tomato can be explained by the nitrogen fixation activity and auxin production. Nitrogenase activity in the maize rhizosphere, 60 days after inoculation was higher in the soil without N supply. The population of diazothrophic bacteria in the rhizosphere of inoculated and non-inoculated maize and tomato plants were not significantly different, showing a probable influence of the native diazotrophic community on the total diazotrophic population. In general, isolates J8L and E5L promoted growth of maize and tomato, showing potential for use as biofertilizers.