RESUMO
A group of Gram-negative plant-associated diazotrophic bacteria belonging to the genus Nitrospirillum was investigated, including both previously characterized and newly isolated strains from diverse regions and biomes, predominantly in Brazil. Phylogenetic analysis of 16S rRNA and recA genes revealed the formation of a distinct clade consisting of thirteen strains, separate from the formally recognized species N. amazonense (the closest species) and N. iridis. Comprehensive taxonomic analyses using the whole genomes of four strains (BR 11140T = AM 18T = Y-2T = DSM 2788T = ATCC 35120T, BR 11142T = AM 14T = Y-1T = DSM 2787T = ATCC 35119T, BR 11145 = CBAmC, and BR 12005) supported the division of these strains into two species: N. amazonense (BR 11142 T and BR 12005) and a newly proposed species (BR 11140 T and BR 11145), distinct from N. iridis. The phylogenomic analysis further confirmed the presence of the new Nitrospirillum species. Additionally, MALDI-TOF MS analysis of whole-cell mass spectra provided further evidence for the differentiation of the proposed Nitrospirillum species, separate from N. amazonense. Analysis of chemotaxonomy markers (i.e., genes involved in fatty acid synthesis, metabolism and elongation, phospholipid synthesis, and quinone synthesis) revealed that the new species highlights high similarity and evolutionary convergence with other Nitrospirillum species. This new species exhibited nitrogen fixation ability in vitro, it has similar NifHDK protein phylogeny position with the closest species, lacked denitrification capability, but possessed the nosZ gene, enabling N2O reduction, distinguishing it from the closest species. Despite being isolated from diverse geographic regions, soil types, and ecological niches, no significant phenotypic or physiological differences were observed between the proposed new species and N. amazonense. Based on these findings, a new species, Nitrospirillum viridazoti sp. nov., was classified, with the strain BR 11140T (DSM 2788T, ATCC 35120T) designated as the type strain.
Assuntos
Nitrogênio , Poaceae , Filogenia , RNA Ribossômico 16S/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: Many low- and-middle-income countries are disproportionately burdened by cervical cancer, resulting in high morbidity and mortality. HPV-DNA testing coupled with treatment with thermal ablation is a recommended screening and precancer treatment strategy, but not enough is known about how this can be effectively implemented in the context of integrated services. The (Scale Up Cervical Cancer Elimination by Secondary prevention Strategy, (SUCCESS) project is conducting a study to understand this approach, integrated into existing women's health services in Burkina Faso, Cote d'Ivoire, Guatemala, and the Philippines (2020-2024). METHODS: A hybrid effectiveness-implementation type III mixed-methods observational study design is used to assess feasibility, acceptability, and costs of integrated service delivery in 10 sites per country, selected considering urban/rural location, facility level, onsite/offsite laboratories, and health services type. In each country, a sample size of 2227 women aged 25-49 years will be enrolled with about 20% being women living with HIV. The primary outcome is proportion of HPV positive women completing precancer treatment, if eligible, within three months of screening. Data collection and analysis includes; facility and client exit surveys, key informant and client interviews, registries and project records extractions, and costing data analysis. Analysis includes descriptive statistics, context description, thematic analysis, and document analysis. Quantitative analyses will be stratified by participant's HIV status. DISCUSSION: Recruitment of study participants started in April 2022 (Burkina Faso and Côte d'Ivoire) and August 2022 (Guatemala and the Philippines). Enrolment targets for women screened, client exit, in-depth and key informant interviews conducted were reached in Burkina Faso and Cote d'Ivoire in November 2022. Guatemala and Philippines are expected to complete enrolment by June 2023. Follow-up of study Participants 12-months post-treatment is ongoing and is expected to be completed for all countries by August 2024. In LMICs, integrating cervical cancer secondary prevention services into other health services will likely require specific rather than incidental recruitment of women for screening. Reconfiguration of laboratory infrastructure and planning for sample management must be made well in advance to meet induced demand for screening. Trail Registration ClinicalTrials.Gov ID: NCT05133661 (24/11/2021).
Assuntos
Infecções por HIV , Infecções por Papillomavirus , Neoplasias do Colo do Útero , Humanos , Feminino , Masculino , Côte d'Ivoire/epidemiologia , Burkina Faso/epidemiologia , Neoplasias do Colo do Útero/diagnóstico , Neoplasias do Colo do Útero/prevenção & controle , Guatemala/epidemiologia , Filipinas/epidemiologia , Infecções por Papillomavirus/diagnóstico , Infecções por Papillomavirus/prevenção & controle , Infecções por HIV/prevenção & controle , Estudos Observacionais como AssuntoRESUMO
Endophytic diazotrophic plant growth-promoting bacteria Herbaspirillum rubrisubalbicans (HCC103), Herbaspirillum seropedicae (HRC54), Paraburkholderia tropica (Ppe8T), Gluconacetobacter diazotrophicus (Pal5T), and Nitrospirillum amazonense (CBAmC) have been used as inoculants for sugarcane. The genome sequences of these strains were used to design a set of specific primers for the real-time PCR (qPCR) assay. Primer specificity was confirmed by conventional PCR using the genomic DNAs of 25 related bacterial species and the five target strains. The qPCR assays were conducted using root and shoot samples from two sugarcane varieties (RB867515 and RB92579). These samples were collected both with and without inoculation, using the target strains specified in this study. The sugarcane plants were grown in a greenhouse, utilizing a substrate composed of sterile sand and vermiculite in a 2:1 ratio, for a duration of 55 days. The primers designed for this study successfully amplified target DNA fragments from each of the bacterial species, enabling their differentiation at the species level. The total bacterial population present in the sugarcane quantified using qPCR was on average 105.2 cells g-1 of fresh tissue. Across both evaluated varieties, it was observed that the population of inoculated bacteria tended to decrease over time and became more concentrated in the sugarcane roots compared to the aerial parts. The qPCR results suggest that both the host and the microbes influence the endophytic population and the bacterial number decreases with plant age.
Assuntos
Saccharum , Saccharum/microbiologia , Reação em Cadeia da Polimerase em Tempo RealRESUMO
An inoculant containing selected bacterial strains can be easily applied during the nursery please process, but in this case, substrate composition can affect its survival and, in consequence, bacterial colonization. The aim of this study was to evaluate the survival of five diazotrophic bacterial strains/species applied individually on 13 different substrates used in sugarcane seedling nurseries considering an active population higher than 105 cells g-1. In addition, one experiment was performed using two commercial substrates, coconut fiber, and Multiplant™, inoculated or not with a mixture of five bacterial strains to evaluate plant growth after 30 days. These strains are combined inoculants selected for sugarcane acting as plant growth promoters. Bacterial counts were determined every seven days using the Most Probable Number technique with four different semi-solid N-free media specific for each strain tested over 35 days. The survival order, independent of the substrate tested, was: Paraburkholderia tropica Pt-PPe8T > Nitrospirillum amazonense Na-CBAMc > Herbaspirillum seropedicae Hs-HRC54 = H. rubrisubalbicans Hr-HCC103 > Gluconacetobacter diazotrophicus Gd-PAL5T. All tested substrates influenced the bacterial survival, especially after 21 days of incubation. The population size can be partially controlled by the substrate pH and stimulated by the addition of slow-release fertilizer. Besides the differences in the bacterial population present in the two commercial substrates, plant growth was found to be stimulated by the inoculated bacteria, depending on the substrate and its sugarcane cultivar tested. The selection of a substrate used to produce new plantlets of sugarcane can contribute to bacterial survival and improve bacterial colonization.
Assuntos
Saccharum , Plântula , Saccharum/microbiologia , BactériasRESUMO
Strain Az39T of Azospirillum is a diazotrophic plant growth-promoting bacterium isolated in 1982 from the roots of wheat plants growing in Marcos Juárez, Córdoba, Argentina. It produces indole-3-acetic acid in the presence of l-tryptophan as a precursor, grows at 20-38 °C (optimal 38 °C), and the cells are curved or spiral-shaped, with diameters ranging from 0.5-0.9 to 1.8-2.2 µm. They contain C16â:â0, C18â:â0 and C18â:â1 ω7c/ω6c as the main fatty acids. Phylogenetic analysis of its 16S rRNA gene sequence confirmed that this strain belongs to the genus Azospirillum, showing a close relationship with Azospirillum baldaniorum Sp245T, Azospirillum brasilense Sp7T and Azospirillum formosense CC-Nfb-7T. Housekeeping gene analysis revealed that Az39T, together with five strains of the genus (Az19, REC3, BR 11975, MTCC4035 and MTCC4036), form a cluster apart from A. baldaniorum Sp245T, A. brasilense Sp7T and A. formosense CC-Nfb-7T. Average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Az39T and the aforementioned type strains revealed values below 96â%, the circumscription limit for the species delineation (ANI: 95.3, 94.1 and 94.0â%; dDDH: 62.9, 56.3 and 55.6â%). Furthermore, a phylogeny evaluation of the core proteome, including 809 common shared proteins, showed an independent grouping of Az39T, Az19, REC3, BR 11975, MTCC4035 and MTCC4036. The G+C content in the genomic DNA of these six strains varied from 68.3 to 68.5â%. Based on the combined phylogenetic, genomic and phenotypic characterization presented here, we consider that strain Az39T, along with strains Az19, REC3, BR 11975, MTCC4035 and MTCC4036, are members of a new Azospirillum species, for which the name Azospirillum argentinense sp. nov. is proposed. The type strain is Az39T (=LBPCV39T=BR 148428T=CCCT 22.01T).
Assuntos
Azospirillum brasilense , Azospirillum brasilense/genética , Técnicas de Tipagem Bacteriana , Composição de Bases , DNA Bacteriano/genética , Ácidos Graxos/química , Hibridização de Ácido Nucleico , Fosfolipídeos/análise , Filogenia , RNA Ribossômico 16S/genética , Análise de Sequência de DNA , Ubiquinona/análiseRESUMO
Little is known about the interaction between the inoculation of diazotrophic bacteria, the variety, and the substrate used in inoculated sugarcane seedling production. Bearing this in mind, the aim of this study was to evaluate the effects of diazotrophic bacteria inoculation, four sugarcane varieties and four different substrates on the growth and nitrogen accumulation of sugarcane seedlings. Parameters related to sprouting, height, and root growth, as well as nitrogen accumulation, were evaluated. The results indicate that inoculating sugarcane seedlings belonging to the following varieties: RB867515, RB92579, RB966928 and RB975932 with bacteria may increase sprouting speed index, height, root length, fresh and dry matter weight, as well as nitrogen accumulation, which has resulted in a more uniform stem diameter. However, the responses to inoculation differ from variety to variety and are also dependent on the substrate used with better results observed in the commercial substrate and the substrate mixtures containing a higher proportion of organic compost. Understanding the interaction between the variety and the substrate with the bacterial inoculation is essential to the success of the production of inoculated sugarcane seedlings.
Assuntos
Bactérias Fixadoras de Nitrogênio , Brotos de Planta/química , Fixação de Nitrogênio , Saccharum/crescimento & desenvolvimento , Substratos para Tratamento BiológicoRESUMO
Little is known about the interaction between the inoculation of diazotrophic bacteria, the variety, and the substrate used in inoculated sugarcane seedling production. Bearing this in mind, the aim of this study was to evaluate the effects of diazotrophic bacteria inoculation, four sugarcane varieties and four different substrates on the growth and nitrogen accumulation of sugarcane seedlings. Parameters related to sprouting, height, and root growth, as well as nitrogen accumulation, were evaluated. The results indicate that inoculating sugarcane seedlings belonging to the following varieties: RB867515, RB92579, RB966928 and RB975932 with bacteria may increase sprouting speed index, height, root length, fresh and dry matter weight, as well as nitrogen accumulation, which has resulted in a more uniform stem diameter. However, the responses to inoculation differ from variety to variety and are also dependent on the substrate used with better results observed in the commercial substrate and the substrate mixtures containing a higher proportion of organic compost. Understanding the interaction between the variety and the substrate with the bacterial inoculation is essential to the success of the production of inoculated sugarcane seedlings.(AU)
Assuntos
Fixação de Nitrogênio , Bactérias Fixadoras de Nitrogênio , Substratos para Tratamento Biológico , Brotos de Planta/química , Saccharum/crescimento & desenvolvimentoRESUMO
Azospirillum sp. strain Sp245T, originally identified as belonging to Azospirillum brasilense, is recognized as a plant-growth-promoting rhizobacterium due to its ability to fix atmospheric nitrogen and to produce plant-beneficial compounds. Azospirillum sp. Sp245T and other related strains were isolated from the root surfaces of different plants in Brazil. Cells are Gram-negative, curved or slightly curved rods, and motile with polar and lateral flagella. Their growth temperature varies between 20 to 38 °C and their carbon source utilization is similar to other Azospirillum species. A preliminary 16S rRNA sequence analysis showed that the new species is closely related to A. brasilense Sp7T and A. formosense CC-Nfb-7T. Housekeeping genes revealed that Azospirillum sp. Sp245T, BR 12001 and Vi22 form a separate cluster from strain A. formosense CC-Nfb-7T, and a group of strains closely related to A. brasilense Sp7T. Overall genome relatedness index (OGRI) analyses estimated based on average nucleotide identity (ANI) and digital DNA-DNA hybridization (dDDH) between Azospirillum sp. Sp245T and its close relatives to other Azospirillum species type strains, such as A. brasilense Sp7T and A. formosense CC-Nfb-7T , revealed values lower than the limit of species circumscription. Moreover, core-proteome phylogeny including 1079 common shared proteins showed the independent clusterization of A. brasilense Sp7T, A. formosense CC-Nfb-7T and Azospirillum sp. Sp245T, a finding that was corroborated by the genome clustering of OGRI values and housekeeping phylogenies. The DNA G+C content of the cluster of Sp245T was 68.4-68.6â%. Based on the phylogenetic, genomic, phenotypical and physiological analysis, we propose that strain Sp245T together with the strains Vi22 and BR12001 represent a novel species of the genus Azospirillum, for which the name Azospirillum baldaniorum sp. nov. is proposed. The type strain is Sp245T (=BR 11005T=IBPPM 219T) (GCF_007827915.1, GCF_000237365.1, and GCF_003119195.2).
Assuntos
Azospirillum brasilense/classificação , Azospirillum/classificação , Genoma Bacteriano , Filogenia , Técnicas de Tipagem Bacteriana , Composição de Bases , Brasil , DNA Bacteriano/genética , Flagelos/química , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
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.
Assuntos
Azospirillum brasilense/fisiologia , Herbaspirillum/fisiologia , Nitrogênio/metabolismo , Zea mays/metabolismo , Zea mays/microbiologia , Folhas de Planta/metabolismo , Raízes de Plantas/metabolismoRESUMO
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.
Assuntos
Inoculantes Agrícolas/fisiologia , Burkholderiaceae/fisiologia , Gluconacetobacter/fisiologia , Herbaspirillum/fisiologia , Nitrato Redutase/metabolismo , Proteínas de Plantas/metabolismo , Rhodospirillaceae/fisiologia , Saccharum/microbiologia , Fixação de Nitrogênio , Saccharum/enzimologia , Saccharum/crescimento & desenvolvimentoRESUMO
Root nodule bacteria were isolated from nodules on Mimosa pudica L. growing in neutral-alkaline soils from the Distrito Federal in central Brazil. The 16S rRNA gene sequence analysis of 10 strains placed them into the genus Rhizobium with the closest neighbouring species (each with 99 % similarity) being Rhizobium grahamii, Rhizobium cauense, Rhizobium mesoamericanum and Rhizobium tibeticum. This high similarity, however, was not confirmed by multi-locus sequence analysis (MLSA) using three housekeeping genes (recA, glnII and rpoB), which revealed R. mesoamericanum CCGE 501T to be the closest type strain (92 % sequence similarity or less). Chemotaxonomic data, including fatty acid profiles [with majority being C19 : 0 cyclo ω8c and summed feature 8 (C18 : 1ω7c/C18 : 1ω6c)], DNA G+C content (57.6 mol%), and carbon compound utilization patterns supported the placement of the novel strains in the genus Rhizobium. Results of average nucleotide identity (ANI) differentiated the novel strains from the closest species of the genus Rhizobium, R. mesoamericanum, R. grahamii and R. tibeticum with 89.0, 88.1 and 87.8 % similarity, respectively. The symbiotic genes essential for nodulation (nodC) and nitrogen fixation (nifH) were most similar (99-100 %) to those of R. mesoamericanum, another Mimosa-nodulating species. Based on the current data, these 10 strains represent a novel species of the genus Rhizobium for which the name Rhizobium altiplani sp. nov. is proposed. The type strain is BR 10423T (=HAMBI 3664T).
Assuntos
Mimosa/microbiologia , Filogenia , Rhizobium/classificação , Nódulos Radiculares de Plantas/microbiologia , Técnicas de Tipagem Bacteriana , Composição de Bases , Brasil , DNA Bacteriano/genética , Ácidos Graxos/química , Genes Bacterianos , Fixação de Nitrogênio , Hibridização de Ácido Nucleico , RNA Ribossômico 16S/genética , Rhizobium/genética , Rhizobium/isolamento & purificação , Análise de Sequência de DNA , Solo/química , SimbioseRESUMO
For centuries, the Acetobacteraceae is known as a family that harbors many species of organisms of biotechnological importance for industry. Nonetheless, since 1988 representatives of this family have also been described as nitrogen fixing bacteria able to plant growth promotion by a variety of mechanisms. Nitrogen fixation is a biological process that guarantees that the atmospheric N2 is incorporated into organic matter by several bacterial groups. Most representatives of this group, also known as diazotrophic, are generally associated with soil rhizosphere of many plants and also establishing a more specific association living inside roots, leaves, and others plants tissues as endophyte. Their roles as plant growth-promoting microorganisms are generally related to increase in plant biomass, phosphate and other mineral solubilization, and plant pathogen control. Here, we report many of these plant growth-promoting processes related to nitrogen fixing species already described in Acetobacteraceae family, especially Gluconacetobacter diazotrophicus and their importance to agriculture. In addition, a brief review of the state of art of the phylogenetics, main physiological and biochemical characteristics, molecular and functional genomic data of this group of Acetobacteraceae is presented.
Assuntos
Acetobacteraceae/genética , Acetobacteraceae/fisiologia , Agricultura , Gluconacetobacter/fisiologia , Fixação de Nitrogênio , Microbiologia do Solo , Biomassa , Endófitos , Filogenia , Folhas de Planta/microbiologia , Raízes de Plantas/microbiologia , RizosferaRESUMO
Brazilian sugarcane has been shown to obtain part of its nitrogen via biological nitrogen fixation (BNF). Recent reports, based on the culture independent sequencing of bacterial nifH complementary DNA (cDNA) from sugarcane tissues, have suggested that members of the Bradyrhizobium genus could play a role in sugarcane-associated BNF. Here we report on the isolation of Bradyrhizobium spp. isolates and a few other species from roots of sugarcane cultivar RB867515 by two cultivation strategies: direct isolation on culture media and capture of Bradyrhizobium spp. using the promiscuous legume Vigna unguiculata as trap-plant. Both strategies permitted the isolation of genetically diverse Bradyrhizobium spp. isolates, as concluded from enterobacterial repetitive intergenic consensus polymerase chain reaction (PCR) fingerprinting and 16S ribosomal RNA, nifH and nodC sequence analyses. Several isolates presented nifH phylotypes highly similar to nifH cDNA phylotypes detected in field-grown sugarcane by a culture-independent approach. Four isolates obtained by direct plate cultivation were unable to nodulate V. unguiculata and, based on PCR analysis, lacked a nodC gene homologue. Acetylene reduction assay showed in vitro nitrogenase activity for some Bradyrhizobium spp. isolates, suggesting that these bacteria do not require a nodule environment for BNF. Therefore, this study brings further evidence that Bradyrhizobium spp. may play a role in sugarcane-associated BNF under field conditions.
Assuntos
Bradyrhizobium/classificação , Bradyrhizobium/isolamento & purificação , Endófitos/classificação , Endófitos/isolamento & purificação , Saccharum/microbiologia , Proteínas de Bactérias/genética , Bradyrhizobium/genética , Bradyrhizobium/crescimento & desenvolvimento , Brasil , Análise por Conglomerados , Meios de Cultura/química , DNA Bacteriano/química , DNA Bacteriano/genética , DNA Ribossômico/química , DNA Ribossômico/genética , Endófitos/genética , Endófitos/crescimento & desenvolvimento , Técnicas Microbiológicas , Dados de Sequência Molecular , Tipagem Molecular , Nitrogenase/análise , Filogenia , Nodulação , Raízes de Plantas/microbiologia , RNA Ribossômico 16S/genética , Análise de Sequência de DNARESUMO
Gluconacetobacter diazotrophicus strain PAL5 is a nitrogen-fixing endophytic bacterium originally isolated from sugarcane and later on was found to colonize other plants such as rice, elephant grass, sweet potato, coffee, and pineapple. Currently, G. diazotrophicus has been considered a plant growth-promoting bacterium due to its characteristics of biological nitrogen fixation, phytohormone secretion, solubilization of mineral nutrients and antagonism to phytopathogens. Reverse transcription followed by quantitative real-time polymerase chain reaction (RT-qPCR) is a method applied for the quantification of nucleic acids because of its specificity and high sensitivity. However, the decision about the reference genes suitable for data validation is still a major issue, especially for nitrogen-fixing bacteria. To evaluate and identify suitable reference genes for gene expression normalization in the diazotrophic G. diazotrophicus, mRNA levels of fourteen candidate genes (rpoA, rpoC, recA, rpoD, fabD, gmk, recF, rho, ldhD, gyrB, gyrBC, dnaG, lpxC and 23SrRNA) and three target genes (matE, omp16 and sucA) were quantified by RT-qPCR after growing the bacteria in different carbon sources. The geNorm and Normfinder programs were used to calculate the expression stabilities. The analyses identified three genes, rho, 23SrRNA and rpoD, whose expressions were stable throughout the growth of strain PAL5 in the chosen carbon sources. In conclusion our results strongly suggest that these three genes are suitable to be used as reference genes for real-time RT-qPCR data normalization in G. diazotrophicus.
Assuntos
Carbono/metabolismo , Perfilação da Expressão Gênica/métodos , Gluconacetobacter/genética , Gluconacetobacter/metabolismo , Reação em Cadeia da Polimerase em Tempo Real/métodos , Padrões de Referência , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Perfilação da Expressão Gênica/normas , Genes Bacterianos , Gluconacetobacter/crescimento & desenvolvimento , Reação em Cadeia da Polimerase em Tempo Real/normasRESUMO
The species Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10(5) cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.
Assuntos
Anticorpos/análise , Gluconacetobacter/isolamento & purificação , Herbaspirillum/isolamento & purificação , Fixação de Nitrogênio , Saccharum , Ensaio de Imunoadsorção Enzimática , Técnicas Imunológicas , MétodosRESUMO
The species Gluconacetobacter diazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10(5) cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.
RESUMO
The species Gluconacetobacterdiazotrophicus, Herbaspirillum seropedicae and H. rubrisubalbicans are endophytic N2-fixing [diazotrophic] bacteria which colonise not only roots, but also the aerial tissue of sugar cane. However, the technique most commonly used to quantify the populations of these microbes in plants is by culturing serial dilutions of macerates of plant tissues in N free semi-solid media which are only semi-selective for the species/genera [the Most Probable Number (MPN) Technique] and each culture must be further subjected to several tests to identify the isolates at the species level. The use of species-specific polyclonal antibodies with the indirect ELISA (enzyme-linked immunosorbent assay) can be an alternative which is rapid and specific to quantify these populations of bacteria. This study was performed to investigate the viability of adapting the indirect ELISA technique to quantify individually the populations of these three species of diazotroph within the root and shoot tissues of sugarcane. The results showed that species-specific polyclonal antibodies could be obtained by purifying sera in protein-A columns which removed non-specific immuno-globulins. It was possible to quantify the three bacterial species in the Brazilian sugarcane variety SP 70-1143 in numbers above 10(5) cells per g fresh weight in roots, rhizomes and leaves. The numbers of the different bacterial species evaluated using the ELISA technique were found to be higher than when the same populations were evaluated using the MPN technique, reaching 1400 times greater for G. diazotrophicus and 225 times greater for Herbaspirillum spp. These results constitute the first quantification of Herbaspirillum using immunological techniques.
RESUMO
A polyphasic approach was applied to characterize 35 G. diazotrophicus isolates obtained from sugarcane varieties cultivated in Brazil. The isolates were analyzed by phenotypic (use of different carbon sources) and genotypic tests (ARDRA and RISARFLP techniques). Variability among the isolates was observed in relation to the carbon source use preference. Glucose and sucrose were used by all isolates in contrast to myo-inositol, galactose and ribose that were not metabolized. The results of the analysis showed the presence of two groups clustered at 68 percent of similarity. The genetic distance was higher when RISA-RFLP analysis was used. Analysis of 16S rDNA sequences from isolates showed that all of them belonged to the G. diazotrophicus species. Neither effect of the plant part nor sugarcane variety was observed during the cluster analysis. The observed metabolic and genetic variability will be helpful during the strain selection studies for sugarcane inoculation in association with sugarcane breeding programs.
Foi realizado a caracterização polifásica de 35 isolados obtidos de variedades de cana-de-açúcar cultivadas no Brasil, através de testes fenotípicos (uso de fontes diferentes de carbono) e genotípicos (técnicas de ARDRA e RISA-RFLP). Houve variação entre os isolados com relação à utilização de fontes de carbono. Glicose e sacarose foram usadas por todos isolados, diferentemente de mio-inositol, galactose e ribose que não foram metabolizados. Os resultados da análise polifásica dos dados confirmam a formação de dois grupos, que apresentaram 68 por cento de similaridade. Observou-se maior distância genética entre os isolados quando a técnica de RISA-RFLP foi aplicada. O sequênciamento da região 16S do rDNA mostrou que todos os isolados pertencem à espécie G. diazotrophicus. Não foi observado efeito da parte da planta ou variedade de cana-de-açúcar no agrupamento dos isolados. Em conjunto, esses resultados poderão auxiliar no estudo de seleção de estirpes para inoculação em cana-de-açúcar, orientando programas de melhoramento vegetal.
Assuntos
Variação Genética , Gluconacetobacter/genética , Gluconacetobacter/isolamento & purificação , Glucose/análise , Interações Ervas-Drogas , Técnicas In Vitro , Fixação de Nitrogênio , Fenótipo , Sacarose/análise , Métodos , Saccharum , Inoculações Seriadas , MétodosRESUMO
A resiliência é um fenômeno importante do desenvolvimento humano; no entanto, seu conceito encontra-se em fase de discussão, pois esse campo de pesquisa é recente na área das ciências humanas. O presente estudo teve como objetivo realizar um levantamento bibliográfico de pesquisas relacionadas à resiliência no período de 2000 a 2006. O método adotado compreendeu a análise documental, através do levantamento de pesquisas em bases de dados, com a utilização de palavras-chave. A análise dos dados consistiu na elaboração de categorias de análise tomando como base os resultados encontrados em pesquisas sobre resiliência. Os dados apontaram que há ênfase nas pesquisas básicas, prevalência de população de adultos nos estudos analisados e utilização da entrevista como principal instrumento. Os resultados também indicam que o conceito de resiliência está em construção; além disso, a resiliência tem relação direta com os fatores de risco e de proteção e, por último, que é necessário o desenvolvimento de programas de prevenção e intervenção.
Resilience is an important phenomenon in human development, however its concept is still discussed and defined because it is a relatively recent research field in the human sciences. The purpose of this study was to do a survey on the research related to resilience from 2000 to 2006. The adopted method consisted in the documental analysis of the surveys on data-base research using key words. The analysis of the data consisted in the separation of categories of analysis based on the findings in research about resilience. The data showed that there is an emphasis on basic research with the prevalence of adult population. They also showed that the interview was the main instrument of the survey. The results indicated that the concept of resilience is still in construction, that resilience has a direct relation with the risk and the protection factors and furthermore the results also showed that the development of prevention and intervention programs is necessary.
Assuntos
Humanos , Masculino , Feminino , Adaptação Psicológica , Psicologia Social , Fatores de Risco , Estresse Psicológico , SobrevidaRESUMO
A resiliência é um fenômeno importante do desenvolvimento humano; no entanto, seu conceito encontra-se em fase de discussão, pois esse campo de pesquisa é recente na área das ciências humanas. O presente estudo teve como objetivo realizar um levantamento bibliográfico de pesquisas relacionadas à resiliência no período de 2000 a 2006. O método adotado compreendeu a análise documental, através do levantamento de pesquisas em bases de dados, com a utilização de palavras-chave. A análise dos dados consistiu na elaboração de categorias de análise tomando como base os resultados encontrados em pesquisas sobre resiliência. Os dados apontaram que há ênfase nas pesquisas básicas, prevalência de população de adultos nos estudos analisados e utilização da entrevista como principal instrumento. Os resultados também indicam que o conceito de resiliência está em construção; além disso, a resiliência tem relação direta com os fatores de risco e de proteção e, por último, que é necessário o desenvolvimento de programas de prevenção e intervenção.(AU)
Resilience is an important phenomenon in human development, however its concept is still discussed and defined because it is a relatively recent research field in the human sciences. The purpose of this study was to do a survey on the research related to resilience from 2000 to 2006. The adopted method consisted in the documental analysis of the surveys on data-base research using key words. The analysis of the data consisted in the separation of categories of analysis based on the findings in research about resilience. The data showed that there is an emphasis on basic research with the prevalence of adult population. They also showed that the interview was the main instrument of the survey. The results indicated that the concept of resilience is still in construction, that resilience has a direct relation with the risk and the protection factors and furthermore the results also showed that the development of prevention and intervention programs is necessary.(AU)