Seawater carbonate chemistry and combined physiological effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101 during experiments, 2010

Recent studies on the diazotrophic cyanobacterium Trichodesmium erythraeum(IMS101) showed that increasing CO2 partial pressure (pCO2) enhances N2 fixation and growth. Significant uncertainties remain as to the degree of the sensitivity to pCO2, its modification by other environmental factors, and underlying processes causing these responses. To address these questions, we examined the responses ofTrichodesmium IMS101 grown under a matrix of low and high levels of pCO2 (150 and 900 µatm) and irradiance (50 and 200 µmol photons m-2 s-1). Growth rates as well as cellular carbon and nitrogen contents increased with increasing pCO2 and light levels in the cultures. The pCO2-dependent stimulation in organic carbon and nitrogen production was highest under low light. High pCO2 stimulated rates of N2fixation and prolonged the duration, while high light affected maximum rates only. Gross photosynthesis increased with light but did not change with pCO2. HCO3- was identified as the predominant carbon source taken up in all treatments. Inorganic carbon uptake increased with light, but only gross CO2 uptake was enhanced under high pCO2. A comparison between carbon fluxes in vivo and those derived from 13C fractionation indicates high internal carbon cycling, especially in the low-pCO2treatment under high light. Light-dependent oxygen uptake was only detected underlow pCO2 combined with high light or when low-light-acclimated cells were exposed to high light, indicating that the Mehler reaction functions also as a photoprotective mechanism in Trichodesmium. Our data confirm the pronounced pCO2 effect on N2fixation and growth in Trichodesmium and further show a strong modulation of these effects by light intensity. We attribute these responses to changes in the allocation of photosynthetic energy between carbon acquisition and the assimilation of carbon and nitrogen under elevated pCO2. These findings are supported by a complementarystudy looking at photosynthetic fluorescence parameters of photosystem II, photosynthetic unit stoichiometry (photosystem I:photosystem II), and pool sizes of key proteins in carbon and nitrogen acquisition.

Experiment: Response of Nodularia spumigena to pCO2

The filamentous and diazotrophic cyanobacterium Nodularia spumigena plays a major role in the productivity of the Baltic Sea as it forms extensive blooms regularly. Under phosphorus limiting conditions Nodularia spumigena has a high enzyme affinity for dissolved organic phosphorus (DOP) by production and release of alkaline phosphatase. Additionally, it is able to degrade proteinaceous compounds by expressing the extracellular enzyme leucine aminopeptidase. As atmospheric CO2 concentrations are increasing, we expect marine phytoplankton to experience changes in several environmental parameters including pH, temperature, and nutrient availability. The aim of this study was to investigate the combined effect of CO2-induced changes in seawater carbonate chemistry and of phosphate deficiency on the exudation of organic matter, and its subsequent recycling by extracellular enzymes in a Nodularia spumigena culture. Batch cultures of Nodularia spumigena were grown for 15 days aerated with three different pCO2 levels corresponding to values from glacial periods to future values projected for the year 2100. Extracellular enzyme activities as well as changes in organic and inorganic compound concentrations were monitored. CO2 treatment-related effects were identified for cyanobacterial growth, which in turn was influencing exudation and recycling of organic matter by extracellular enzymes. Biomass production was increased by 56.5% and 90.7% in the medium and high pCO2 treatment, respectively, compared to the low pCO2 treatment and simultaneously increasing exudation. During the growth phase significantly more mucinous substances accumulated in the high pCO2 treatment reaching 363 µg Gum Xanthan eq /l compared to 269 µg Gum Xanthan eq /l in the low pCO2 treatment. However, cell-specific rates did not change. After phosphate depletion, the acquisition of P from DOP by alkaline phosphatase was significantly enhanced. Alkaline phosphatase activities were increased by factor 1.64 and 2.25, respectively, in the medium and high compared to the low pCO2 treatment. In conclusion, our results suggest that Nodularia spumigena can grow faster under elevated pCO2 by enhancing the recycling of organic matter to acquire nutrients.

Use of farmer-prioritized vertisol management options for enhanced green gram and tomato production in central Kenya

Green grams (Phaseolus aures L.) and tomato (Solanum lycopersicum L) are widely grown in the vertisols of the Mwea Irrigation Scheme alongside the rice fields. Green grams can fix nitrogen (biological nitrogen fixation) and are grown for its highly nutritious and curative seeds while tomatoes are grown for its fruit rich in fibres, minerals and vitamins. The two can be prepared separately or together in a variety of ways including raw salads and/or cooked/fried. They together form significant delicacies consumed with rice which is the major cash crop grown in the black cotton soils. The crops can grow well in warm conditions but tomato is fairly adaptable except under excessive humidity and temperatures that reduce yields. Socio-economic prioritization by the farming community and on-farm demonstrations of soil management options were instituted to demonstrate enhanced green gram and tomato production in vertisol soils of lower parts of Kirinyaga County (Mwea East and Mwea West districts). Drainage management was recognized by the farming community as the best option although a reduced number of farmers used drainage and furrows/ridges, manure, fertilizer and shifting options with reducing order of importance. Unavailability of labour and/or financial cost for instituting these management options were indicated as major hindrances to adopt the yield enhancing options. Labour force was contributed to mainly by the family alongside hiring (64.2%) although 28% and 5.2% respectively used hired or family labour alone. The female role in farming activities dominated while the male role was minimal especially at weeding. The youth role remained excessively insignificant and altogether absent at marketing. Despite the need for labour at earlier activities (especially when management options needed to be instituted) it was at the marketing stage that this force was directed. Soils were considered infertile by 60% but 40% indicated that their farms had adequate fertility. Analysis showed that ridging and application of farm yard manure and fertilizer improved fertility, crop growth and income considerably. Phosphate and zinc enhancement reduced alkalinity and sodicity. Green gram and tomato yields increased under ridges and farm yard manure application by 17-25% which significantly enhanced household income.

Inhibition of glutamine synthetase by phosphinothricin leads to transcriptome reprograming in root nodules of Medicago truncatula

Glutamine synthetase (GS) is a vital enzyme for the assimilation of ammonia into amino acids in higher plants. In legumes, GS plays a crucial role in the assimilation of the ammonium released by nitrogen-fixing bacteria in root nodules, constituting an important metabolic knob controlling the nitrogen (N) assimilatory pathways. To identify new regulators of nodule metabolism, we profiled the transcriptome of Medicago truncatula nodules impaired in N assimilation by specifically inhibiting GS activity using phosphinothricin (PPT). Global transcript expression of nodules collected before and after PPT addition (4, 8, and 24 h) was assessed using Affymetrix M. truncatula GeneChip arrays. Hundreds of genes were regulated at the three time points, illustrating the dramatic alterations in cell metabolism that are imposed on the nodules upon GS inhibition. The data indicate that GS inhibition triggers a fast plant defense response, induces premature nodule senescence, and promotes loss of root nodule identity. Consecutive metabolic changes were identified at the three time points analyzed. The results point to a fast repression of asparagine synthesis and of the glycolytic pathway and to the synthesis of glutamate via reactions alternative to the GS/GOGAT cycle. Several genes potentially involved in the molecular surveillance for internal organic N availability are identified and a number of transporters potentially important for nodule functioning are pinpointed. The data provided by this study contributes to the mapping of regulatory and metabolic networks involved in root nodule functioning and highlight candidate modulators for functional analysis.

Aplicação foliar de molibdênio em adubos verdes e o desempenho do feijoeiro em sucessão

The knowledge of molybdenum application in legumes on the availability of N, by BNF, increased enzymatic activity and the residual effect caused on crops growth and yield can contribute to the greater scientific understanding involved in green manure processes. The aim of this study was to evaluate the Mo application and the N from Crotalaria juncea and Canavalia ensiformis green manures on common bean performance. Were conducted field experiments for the crops succession system (green manures - common bean) and laboratory essays for the enzymatic activities. Green manure production was installed in a factorial arrangement 2 x 4, with two green manure legumes species, sunnhemp (Crotalaria juncea) and jack beans (Canavalia ensiformis), and four Mo doses (0, 40, 80, 120 g ha-1) in the form of sodium molybdate (Na2MoO4), foliar applied, in a randomized block design with four replicates. For succession crop (common bean) additional treatment was added, beans grown without any fertilization, following the same experimental design from the previous crop. The dry matter decomposition and the N mineralization of green manure were monitored through collection of residues over time, by using the litter bags method. In laboratory were carried out tests of nitrate reductase activity in green manures and common beans at 90 and 66 days after sowing, respectively. The sunnhemp responded linearly positively to the application of Mo as the dry matter and N accumulation. While the jack beans presented a negative quadratic response for dry matter and there was no adjustment of regression models to N. The jack beans showed a higher decomposition rate and N mineralization compared to sunnhemp. The half lives for decomposing 50% of dry matter on the soil was 123 and 104 days to sunnhemp and jack beans, respectively, and 50% of N present in the residues was mineralized at 93 and 85 days. In common bean, differed from the control for number of pods the dose of 40 g ha-1 of Mo in both species of green manures and the dose 80 g ha-1 of Mo in jack beans. For number of grains only in sunnhemp on the dose of 40 g ha-1 of Mo differ from the control. The nitrate reductase activity was influenced by developmental stage of green manure species. In common bean, the activity of nitrate reductase was up to three times higher than the dose 0 g ha-1 of Mo compared to treatment with application of Mo in both species. There was no effect of Mo doses or species of green manure on common bean yield.

Evaluación del efecto de la inoculación de Bacillus spp. con potencial fijador de nitrógeno en cultivos de Chlorella sorokiniana

El uso de microalgas y PGPBs como principio activo de problemas ambientales ha generado interés científico en los últimos años -- Entre las soluciones propuestas se encuentra el uso de los cocultivos de estos 2 tipos de microrganismos para la formulación de bioinsumos y la biorremedación -- El objetivo de este estudio se centró en evaluar el efecto de la inoculación de Bacillus spp. con potencial fijador de nitrógeno ambiental en cultivos de Chlorella sorokiniana a nivel de matraz -- Se encontró que 10 cepas PGPB, del banco de cepas del grupo CIBIOP de la Universidad EAFIT, probablemente fijaron nitrógeno debido a que crecen en medio NFb -- De las cuales, Bacillus subtilis EA-CB0575 promueve el crecimiento de la microalga Chlorella sorokiniana UTEX 1230, aumentando en un 94% la densidad celular y en 4.5 veces el tamaño de la microalga comparada su crecimiento individual -- Finalmente se usó el método ARA para evaluar fijación de nitrógeno, y se encontró que la bacteria fue capaz de reducir el acetileno en etileno cuando se siembra en medio NFb y BBM libre de nitrógeno, además cuando se cocultivo con la microalga, lo que indicaría que posiblemente la fijación de nitrógeno sea el método de promoción de crecimiento microalgar

Proteomic analysis of Rhizobium freirei PRF 81 responses to low pH.

Rhizobium freirei PRF 81 is employed in common bean commercial inoculants in Brazil, due to its outstanding efficiency in fixing nitrogen, competitiveness and tolerance to abiotic stresses. Among the environmental conditions faced by rhizobia in soils, acidity is perhaps the encountered most, especially in Brazil. So, we used proteomics based approaches to study the responses of PRF 81 to a low pH condition. R. freirei PRF 81 was grown in TY medium until exponential phase in two treatments: pH 6,8 and pH 4,8. Whole-cell proteins were extracted and separated by two-dimensional gel electrophoresis, using IPG-strips with pH range 4-7 and 12% polyacrilamide gels. The experiment was performed in triplicate. Protein spots were detected in the high-resolution digitized gel images and analyzed by Image Master 2D Platinum v 5.0 software. Relative volumes (%vol) of compared between the two conditions tested and were statistically evaluated (p ≤ 0.05). Even knowing that R. freirei PRF 81 can still grow in more acid conditions, pH 4.8 was chosen because didn´t affect significantly the bacterial growth kinetics, a factor that could compromise the analysis. Using a narrow pH range, the gel profiles displayed a better resolution and reprodutibility than using broader pH range. Spots were mostly concentrated between pH 5-7 and molecular masses between 17-95 kDa. From the six hundred well-defined spots analyzed, one hundred and sixty-three spots presented a significant change in % vol, indicating that the pH led to expressive changes in the proteome of R. freirei PRF 81. Of these, sixty-one were up-regulated and one hundred two was downregulated in pH 4.8 condition. Also, fourteen spots were only identified in the acid condition, while seven spots was exclusively detected in pH 6.8. Ninety-five differentially expressed spots and two exclusively detected in pH 4,8 were selected for Maldi-Tof identification. Together with the genome sequencing and the proteome analysis of heat stress, we will search for molecular determinants of PRF 81 related to capacity to adapt to stressful tropical conditions.

Uso de espectrometria de massa por ionização e dessorção a laser assistida por matriz : tempo de voo (MALDI-TOF MS) para formação de bibliotecas e caracterização da ocupação nodular em soja por estirpes de Bradyrhizobium

Dissertação (mestrado)—Universidade de Brasília, Instituto de Ciências Biológicas, Programa de Pós-Graduação em Biologia Microbiana, 2016.

Diversity of new root nodule bacteria from Erythrina velutina Willd., a native legume from the Caatinga dry forest (Northeastern Brazil).

This study aims to evaluate the phenotypical characteristics of bacterial isolates from mulungu (Erythrina velutina Willd.) nodules and determinate their Box-PCR fingerprinting. All bacteria were evaluated by the following phenotypic characteristics: growth rate, pH change, colony color and mucus production. The bacterial isolates able to re-nodulate the original host were also evaluated regarding its tolerance to increased salinity and different incubation temperatures, ability to growth using different carbon sources, intrinsic antibiotic resistance and ?in vitro? auxin biosynthesis. The molecular fingerprints were set up using the Box-PCR technique and the isolates were clustered by their profiles. Among the 22 bacterial isolates obtained, eight were able to re-nodulate the original host. Among the nodule inducing isolates, some were tolerant to 1% of NaCl and 39° C and all of them metabolized the maltose, fructose, glucose, sucrose and arabinose, were resistant to rifampicin and produced auxin. The bacteria showed low genetic similarity among them and reference strains, which indicates the great genetic variability of the isolates. The results of this work are the first reports about the bacterial isolates able to nodulate this species. A more deep study of these bacteria may reveal the existence of isolates tolerant to environmental stresses and suitable as a future mulungu inoculant.

Cultivares de cana-de-açúcar inoculada com bactérias diazotróficas em condições irrigadas no semiárido brasileiro.

A cultura da cana-de-açúcar constitui-se numa importante fonte bioenergética e a redução de custos em seu sistema produtivo torna-se cada vez mais relevante. A contribuição de bactérias diazotróficas, capazes de se associarem à planta e fornecerem nitrogênio pelo processo de fixação biológica é vantajoso, pois permite reduzir ou até mesmo suprimir o uso de fertilizantes nitrogenados. Assim, e ste trabalho objetivou avaliar as características de desenvolvimento de cultivares de cana-de-açucar inoculadas com bactérias fixadoras de nitrogênio, em condição irrigada no semiárido brasileiro. O estudo foi desenvolvido na Usina Agrovale S.A., Juazeiro-BA, em delineamento de blocos casualizados, sendo os tratamentos representados por seis cultivares de cana-de-açúcar: RB 012018; RB 012046; RB 72454; RB 867515; RB 92579 e RB 961003, cultivadas com e sem inoculação e dois tratamentos adicionais (RB867515 e RB 72454) adubados com 120 kg de N ha-1. As avaliações biométricas consistiram na contagem de perfilhos aos 30, 60 e 90 dias após o plantio em cana planta e 30, 60 e 90 dias após a colheita em cana soca de primeira folha. Avaliaram-se, ainda, a altura, o diâmetro do colmo e o número de colmos das plantas em cada período. Os resultados permitiram concluir que a inoculação com bactérias diazotróficas em cana planta é eficiente e promove aumento no número de perfilhos. A resposta das cultivares de cana-de-açúcar à inoculação com bactérias diazotróficas nos parâmetros número de colmos, diâmetro de colmos e altura das plantas depende da cultivar.

Agronomic efficiency of Bradyrhizobium in peanut under different environments in Brazilian Northeast.

Several legumes have natural ability to associate with nitrogen - fixing bacteria known as rhizobia. The efficiency of this association depends on the plant and bacterial genotype and the edaphoclimatic conditions. Peanut is a tropical legume able to associate with a wide range of rhizobia and the selection of efficient bacteria is important to increase the nitrogen fixation in this crop. In order to investigate the agronomic efficiency of two Bradyrhizobiumstrains, two peanut genotypes were used in field trails carried out in three environments located at Brazilian Northeast. The genotypes (BR1 and L7 Bege) were submitted to rhizobial inoculation (SEMIA 6144 or ESA 123, both Bradyrhizobium strains, and chemical nitrogen fertilization in randomized block design experiments. The following traits were analyzed: flowering (F), main axis height (MAH), number of nodules/plant (NN), number of pods/plant (NP) and weight of pods (WP). Differential responses were found in all to treatments to NN, NP and WP, in the three environments studied. Overall, ESA 123 showed good agronomic performance inducing higher pod production. The results supportthe evaluation of the Bradyrhizobium in further experiments aiming at its recommendation to commercial inoculants in Brazilian Northeast region.

Ocorrência de bactérias diazotroficas em diferentes genótipos de cana-de-açúcar.

O objetivo deste trabalho foi avaliar a localização e o numero de bactérias endolíticas em quatro genótipos de cana-de-açúcar e investigar sobre a possível existência de correlação com os resultados apresentados em trabalhos de quantificação da fixação biológica de nitrogênio (FBN). Fez-se um levantamento das bactérias diazotróficas presentes, e quantificou-se a população de Herbaspirillum spp. E Acetobacter diazotrophicus, em genótipos de cana-de-açúcar contrastantes quanto a capacidade de obter N da FBN. De acordo com o levantamento realizado neste trabalho, as bactérias estudadas (Azospirillum lipoferum, A. brasilense, A. amazonense, Herbaspirillum spp. e Acetobacter diazotrophicus) estavam presentes nos quatro genotipos avaliados e em todas as partes da planta, exceto A. amazonense, que nao foi isolado de amostras de folhas. A quantificaçãoo das bactérias Herbaspirillum spp. e A. diazotrophicus mostrou não haver diferenças significativas entre os genótipos, e que, geralmente, elas estão presentes em maior numero nas raízes. Enquanto Herbaspirillum spp. mantêm-se mais estável ao longo do ciclo da cultura, a população de A. diazotrophicus decresce com a aproximação do final do ciclo comercial. Pode-se sugerir que as diferenças entre as taxas de FBN encontradas nos diversos genótipos não e causada por diferenças na presença ou no numero das bactérias aqui estudadas The objective of this work was to find out the localization and number of endophytic bacteria in four sugar cane genotypes and investigate upon the possible existence of correlation to the results obtained in some studies about quantification of biological nitrogen fixation (BNF). A survey of the diazotrophic bacteria present in sugar cane genotypes differingin their capacity to obtain nitrogen through BNF was performed, and population of Herbaspirillum spp. and Acetobacter diazotrophicus was quantified. The bacteria tested in the survey were Azospirillum lipoferum, A. brasilense, A. amazonense, Herbaspirillum spp. and Acetobacter diazotrophicus. All these bacteria were present in the four genotypes and were found in all parts of the plants, except A. amazonense which was not isolated from leaf samples. The quantification of Herbaspirillum spp. and A. diazotrophicus showed that there were no significant differences among the sugar cane genotypes and, generally, the bacteria were in greater number in roots. While number of Herbaspirillum spp. remained stable during the life-cycle of the culture, the population of A. diazotrophicus suffer a decrease with the approach of the end of the commercial cycle. It is suggested that the differences in the rates of BNF found in sugar cane genotypes are not caused by differences in the presence or the number of the bacterial species studied here.

Ocorrência de micorrizas arbusculares e da bactéria diazotrofica Acetobacter diazotrophicus em cana-de-açúcar.

Foi avaliada a ocorrência e a distribuição de espécies de fungos micorrízicos arbusculares e A. diazotrophicus em plantios de cana-de-açúcar em diferentes tipos de manejo nos Estados do Rio de Janeiro e Pernambuco. Foram feitas 35 coletas de amostras de solo da rizosfera e de raízes de 14 variedades de cana-de-açúcar para extração de esporos e isolamento da bactéria. O numero de esporos variou de 18 a 2.070/ 100 mL de solo, e os maiores numero e diversidade de espécies foram verificados nos canaviais de Campos, RJ, especialmente naqueles que não adotam a queima de palhico. As espécies predominantes nas três localidades amostradas foram: Acaulospora sp., Scutellospora heterogama, Glomus etunicatum, Glomus occultum e Gigaspora margarita. A. diazotrophicus estava presente nas amostras de raízes colhidas em canaviais de Campos, com exceção de uma coleta de cana-de-açúcar plantada num solo usado como bacia de sedimentação de vinhaça. Não foi possível isolar essa bactéria a partir de esporos desinfestados dos FMAs nativos, apenas dos esporos lavados com agua estéril The occurrence and distribution of species of arbuscular mycorrhizae fungi and Acetobacter diazotrophicus in sugar cane (Saccharum officinarum) grown in different regimes of crop management in the States of Rio de Janeiro and Pernambuco were studied. Thirty five samples of the rhizosphere soil and roots were collected from 14 varieties of sugar cane for the extraction of spores and isolation of the bacterium. The number of spores varied from 18 to 2.070 per 100 mL of soil, and the greatest diversity of fungal species was found in the sugarcane fields of Campos (Rio de Janeiro State), especially in those where the sugarcane trash was not burned at harvest. The predominant species found in the three localities sampled were: Scutellospora heterogama, Glomus etunicatum, Glomus occultum, Glomus macrocarpum, Acaulospora sp. and Gigaspora margarita. A. diazotrophicus was present in almost all samples of root with the exception of one harvest of sugar cane taken from an area used for the sedimentation of vinasse (distillery waste). It was not possible to detect the bacterium from surface sterilised spores of native arbuscular mycorrhizal fungi (AMF), only from washed ones using sterile water.

Nodulation, gas exchanges and production of peanut cultivated with Bradyrhizobium in soils with different textures.

Nitrogen fertilization from biological source is an uncommon practice for peanut growers due to the limited results, mainly in environments with water restriction. In this study, the response of a commercial Bradyrhizobium was evaluated on the nodulation and production of peanuts grown in sandy and medium textured soils. Two experiments using different soils were carried out in the field during the dry season, in Campina Grande, Paraíba State, Brazil. Three peanut genotypes were submitted to the following treatments: 1-no nitrogen fertilization (control), 2- chemical fertilization (ammonium sulfate) and 3- inoculation with Bradyrhizobium [commercial strain BR 1405 (SEMIA 6144)]. A completely randomized 3x3 factorial design was adopted with five repetitions for both experiments. The evaluates variables were: height of the main stem, number of nodes/plant, root length, root dry weight, weight of pods/plant and number of pods/plant. In addition, gas exchanges were estimated using IRGA apparatus. Both genotypes (BRS Havana and L7 Bege) were benefited in relation to production due to an inoculation with SEMIA 6144. No physiological response was verified in genotypes or N-treatments to gas exchange, excepting for the Ci/Ca ratio in the medium textured soil experiment. BRS Havana showed low Ci/Ca ratio in Bradyrhizobium treatment, indicating that SEMIA 6144 improved the plants photosynthetic efficiency.