Nitrospirillum viridazoti sp. nov., an Efficient Nitrogen-Fixing Species Isolated from Grasses.

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.

Designing and validation of specific primers for the quantitative detection of bacteria in sugarcane inoculant.

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.

Survival of five diazotrophic bacterial strains in different substrates used in sugarcane seedling propagation.

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.

Genome-based reclassification of Azospirillum brasilense Az39 as the type strain of Azospirillum argentinense sp. nov.

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 C16 : 0, C18 : 0 and C18 : 1 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 96 %, the circumscription limit for the species delineation (ANI: 95.3, 94.1 and 94.0 %; dDDH: 62.9, 56.3 and 55.6 %). 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 68.5 %. 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).

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

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

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

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

Nitrogen fixing bacteria in the family Acetobacteraceae and their role in agriculture.

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.

Endophytic Bradyrhizobium spp. isolates from sugarcane obtained through different culture strategies.

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.

Predominant nifH transcript phylotypes related to Rhizobium rosettiformans in field-grown sugarcane plants and in Norway spruce.

Although some sugarcane cultivars may benefit substantially from biological nitrogen fixation (BNF), the responsible bacteria have been not identified yet. Here, we examined the active diazotrophic bacterial community in sugarcane roots from Africa and America by reverse transcription (RT)-PCR using broad-range nifH-specific primers. Denaturing gradient gel electrophoresis (DGGE) profiles obtained from sugarcane showed a low diversity at all sample locations with one phylotype amounting up to 100% of the nifH transcripts. This major phylotype has 93.9-99.6% DNA identity to the partial nifH sequence from a strain affiliated with Rhizobium rosettiformans. In addition, nifH transcripts of this phylotype were also detected in spruce roots sampled in Germany, where they made up 91% of nifH transcripts detected. In contrast, in control soil or shoot samples two distinct nifH transcript sequences distantly related to nifH from Sulfurospirillum multivorans or Bradyrhizobium elkanii, respectively, were predominant. These results suggest that R. rosettiformans is involved in root-associated nitrogen fixation with sugarcane and spruce, plants that do not form root-nodule symbioses.

Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. in sugar cane (Saccharum spp) using differente polyclonal antibodies

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.

Quantification of natural populations of Gluconacetobacter diazotrophicus and Herbaspirillum spp. In sugar cane (Saccharum spp.) Using differente polyclonal antibodies.

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.

Response of micropropagated sugarcane varieties to inoculation with endophytic diazotrophic bacteria

Experimentos anteriores estimaram que a cana-de-açúcar pode obter até 60% do N acumulado via fixação biológica de nitrogênio (FBN). Neste trabalho, os efeitos da inoculação da mistura de cinco espécies de bactérias diazotróficas endofíticas foram testados em duas variedades de cana-de-açúcar micropropagadas, sob condições de campo. Após 17 meses de crescimento, a produtividade e a FBN apresentaram influência do genótipo vegetal e da localidade de cultivo. As maiores contribuições via FBN foram observadas no solo de menor fertilidade, para ambas variedades de cana-de-açúcar. Pequenos aumentos de produtividade foram observados para a variedade SP 701143 nos solos de baixa e média fertilidade. Por outro lado, a inoculação na variedade SP 813250 apresentou decréscimo de produtividade nos três tipos de solo testados.

Evaluation of the biological nitrogen fixation contribution in sugarcane plants originated from seeds and inoculated with nitrogen-fixing endophytes

A técnica de inoculação com bactérias diazotróficas endofíticas na cana-de-açúcar apresenta-se como uma prática alternativa para promover o crescimento vegetal menos dependente da adubação nitrogenada. Este trabalho teve como objetivo avaliar a contribuição da fixação biológica de nitrogênio (FBN) por diferentes estirpes de Herbaspirillum seropedicae e Gluconacetobacter diazotrophicus inoculadas em plantas de cana-de-açúcar originadas de semente. As sementes foram plantadas em vasos com solo não estéril, inoculadas com as diferentes bactérias e mantidas por 10 meses ao ar livre. As maiores contribuições da FBN ocorreram com a inoculação de estirpes Herbaspirillum seropedicae, e dependeram da espécie vegetal utilizada como testemunha. As raízes apresentaram-se como o órgão vegetal preferencial para o estabelecimento das espécies inoculadas.

Response of micropropagated sugarcane varieties to inoculation with endophytic diazotrophic bacteria

Previous studies estimated that sugarcane could obtain up to 60% of total nitrogen accumulated from BNF. Here a mixture of five endophytic diazotrophic strains was tested in a field trial, inoculated in two micropropagated sugarcane varieties and three locals, to determine the effects on commercial crop conditions. The sugarcane plantlets were inoculated in vitro, and after 17 months of growing in the field, the productivity and BNF contribution showed to be influenced by the plant genotype and soil type. The highest BNF contributions was observed in the poorest soil for both varieties. Smaller increases in productivity were observed for SP 701143 variety grown in soil with low or medium fertility. In contrast, a decrease in the stem productivity was observed in the SP 813250 variety grown in the three localities.

Experimentos anteriores estimaram que a cana-de-açúcar pode obter até 60% do N acumulado via fixação biológica de nitrogênio (FBN). Neste trabalho, os efeitos da inoculação da mistura de cinco espécies de bactérias diazotróficas endofíticas foram testados em duas variedades de cana-de-açúcar micropropagadas, sob condições de campo. Após 17 meses de crescimento, a produtividade e a FBN apresentaram influência do genótipo vegetal e da localidade de cultivo. As maiores contribuições via FBN foram observadas no solo de menor fertilidade, para ambas variedades de cana-de-açúcar. Pequenos aumentos de produtividade foram observados para a variedade SP 701143 nos solos de baixa e média fertilidade. Por outro lado, a inoculação na variedade SP 813250 apresentou decréscimo de produtividade nos três tipos de solo testados.

Evaluation of the biological nitrogen fixation contribution in sugarcane plants originated from seeds and inoculated with nitrogen-fixing endophytes

The inoculation technique with endophytic diazotrophic bacteria in sugarcane has been shown as an alternative practice to plant growth promotion. The aim of this work was to evaluate the biological nitrogen fixation (BNF) contribution by different strains of Herbaspirillum seropedicae and Gluconacetobacter diazotrophicus in sugarcane plant inoculated from seeds. The seeds were planted in pots filled with non-sterile soil, inoculated with the bacterial strains and grown 10 months outside of the greenhouse. The BNF contribution of the inoculated bacteria varied depending on the plant species used as a control. The highest BNF contribution as well as the highest populations of reisolated bacteria was observed with inoculation of H. seropedicae strains. The roots appeared to be the preferential tissues for the establishment of the inoculated species.

A técnica de inoculação com bactérias diazotróficas endofíticas na cana-de-açúcar apresenta-se como uma prática alternativa para promover o crescimento vegetal menos dependente da adubação nitrogenada. Este trabalho teve como objetivo avaliar a contribuição da fixação biológica de nitrogênio (FBN) por diferentes estirpes de Herbaspirillum seropedicae e Gluconacetobacter diazotrophicus inoculadas em plantas de cana-de-açúcar originadas de semente. As sementes foram plantadas em vasos com solo não estéril, inoculadas com as diferentes bactérias e mantidas por 10 meses ao ar livre. As maiores contribuições da FBN ocorreram com a inoculação de estirpes Herbaspirillum seropedicae, e dependeram da espécie vegetal utilizada como testemunha. As raízes apresentaram-se como o órgão vegetal preferencial para o estabelecimento das espécies inoculadas.