Bacterial soil community in a Brazilian sugarcane field

The assessment of bacterial communities in soil gives insight into microbial behavior under prevailing environmental conditions. In this context, we assessed the composition of soil bacterial communities in a Brazilian sugarcane experimental field. The experimental design encompassed plots containing common sugarcane (variety SP80-1842) and its transgenic form (IMI-1 - imazapyr herbicide resistant). Plants were grown in such field plots in a completely randomized design with three treatments, which addressed the factors transgene and imazapyr herbicide application. Soil samples were taken at three developmental stages during plant growth and analyzed using 16S ribosomal RNA (rRNA)-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) and clone libraries. PCR-DGGE fingerprints obtained for the total bacterial community and specific bacterial groups - Actinobacteria, Alphaproteobacteria and Betaproteobacteria - revealed that the structure of these assemblages did not differ over time and among treatments. Nevertheless, slight differences among 16S rRNA gene clone libraries constructed from each treatment could be observed at particular cut-off levels. Altogether, the libraries encompassed a total of eleven bacterial phyla and the candidate divisions TM7 and OP10. Clone sequences affiliated with the Proteobacteria, Actinobacteria, Firmicutes and Acidobacteria were, in this order, most abundant. Accurate phylogenetic analyses were performed for the phyla Acidobacteria and Verrucomicrobia, revealing the structures of these groups, which are still poorly understood as to their importance for soil functioning and sustainability under agricultural practices.

Achromobacter insolitus and Zoogloea ramigera associated with wheat plants (Triticum aestivum)

This study reports for the first time the presence of diazotrophic bacteria belonging to the genera Achromobacter and Zoogloea associated with wheat plants. These bacterial strains were identified by the analysis of 16S rDNA sequences. The bacterium IAC-AT-8 was identified as Azospirillum brasiliense, whereas isolates IAC-HT-11 and IAC-HT-12 were identified as Achromobacter insolitus and Zoogloea ramigera, respectively. A greenhouse experiment involving a non-sterilized soil was carried out with the aim to study the endophytic feature of these strains. After 40 days from inoculation, all the strains were in the inner of roots, but they were not detected in soil. In order to assess the location inside wheat plants, an experiment was conducted under axenic conditions. Fifteen days after inoculation, preparations of inoculated plants were observed by the scanning electron microscope, using the cryofracture technique, and by the transmission electron microscope. It was observed that all isolates were present on the external part of the roots and in the inner part at the elongation region, in cortex cells, but not in the endodermis or in the vascular bundle region. No colonizing bacterial cells were observed in wheat leaves.

The bacterial diversity in a Brazilian non-disturbed mangrove sediment

The bacterial diversity present in sediments of a well-preserved mangrove in Ilha do Cardoso, located in the extreme south of So Paulo State coastline, Brazil, was assessed using culture-independent molecular approaches (denaturing gradient gel electrophoresis (DGGE) and analysis of 166 sequences from a clone library). The data revealed a bacterial community dominated by Alphaproteobacteria (40.36% of clones), Gammaproteobacteria (19.28% of clones) and Acidobacteria (27.71% of clones), while minor components of the assemblage were affiliated to Betaproteobacteria, Deltaproteobacteria, Firmicutes, Actinobacteria and Bacteroidetes. The clustering and redundancy analysis (RDA) based on DGGE were used to determine factors that modulate the diversity of bacterial communities in mangroves, such as depth, seasonal fluctuations, and locations over a transect area from the sea to the land. Profiles of specific DGGE gels showed that both dominant (`universal` Bacteria and Alphaproteobacteria) and low-density bacterial communities (Betaproteobacteria and Actinobacteria) are responsive to shifts in environmental factors. The location within the mangrove was determinant for all fractions of the community studied, whereas season was significant for Bacteria, Alphaproteobacteria, and Betaproteobacteria and sample depth determined the diversity of Alphaproteobacteria and Actinobacteria.

Enrichment of autotrophic anaerobic ammonium-oxidizing consortia from various wastewaters

The option for biological nitrogen removal has recently been broadened with the description of simultaneous nitrification/denitrification, anaerobic ammonium oxidation (ANAMMOX) and the concept of CANON (completely autotrophic nitrogen removal over nitrite). An autotrophic anaerobic ammonium oxidation (AAAO) consortium was successfully selected and enriched from municipal treatment plant sludges in Sydney, Australia, but not from industrial coke-oven wastewater sludges. Chemolithoautotrophic basic salt (CLABS) medium was used in the selection of AAAO organisms and chloramphenicol was added to the initial stage of selection to eliminate denitrifiers. Two different temperatures, 37degreesC and 55degreesC, were used in the selection of mesophilic and thermophilic consortia, respectively. Thermophilic AAAO organisms were not selected at 55degreesC. Mesophilic AAAO activities, however, were evident in both batch and continuous cultures, whereby ammonium was consumed concurrently with a decrease of nitrite, giving a ratio of 1:1-1:1.3 in ammonium removal rate over nitrite consumption rate. A continuous-mode mesophilic fixed-bed reactor was established to enrich the AAAO consortium. After 1 year, biofilms, pinkish in color, had developed on the support media and side wall of the feed-line tubing. Ammonium and nitrite consumption increased from similar to15 mg to 60 mg d(-1) L-1 over a period of 243 days. Later, transmission electron microscopy (TEM) and fluorescence in situ hybridization (FISH) techniques revealed that the dominant cell type in the AAAO consortium had a similar morphology and 16S rDNA sequence homology to that of the recently described ANAMMOX organism, Brocadia anammoxidans.

Candidatus "Scalindua brodae", spec. nov., Candidatus "Scalindua wagneri", spec. nov., two new species of anaerobic ammonium oxidizing bacteria

Anaerobic ammonium oxidation (anammox) is both a promising process in wastewater treatment and a long overlooked microbial physiology that can contribute significantly to biological nitrogen cycling in the world's oceans. Anammox is mediated by a monophyletic group of bacteria that branches deeply in the Planctomycetales. Here we describe a new genus and species of anaerobic ammonium oxidizing planctomycetes, discovered in a wastewater treatment plant (wwtp) treating landfill leachate in Pitsea, UK. The biomass from this wwtp showed high anammox activity (5.0 +/- 0.5 nmol/mg protein/min) and produced hydrazine from hydroxylamine, one of the unique features of anammox bacteria. Eight new planctomycete 16S rRNA gene sequences were present in the 16S rRNA gene clone library generated from the biomass. Four of these were affiliated to known anammox 16S rRNA gene sequences, but branched much closer to the root of the planctomycete line of descent. Fluorescence in situ hybridization (FISH) with oligonucleotide probes specific for these new sequences showed that two species (belonging to the same genus) together made up > 99% of the planctomycete population which constituted 20% of the total microbial community. The identification of these organisms as typical anammox bacteria was confirmed with electron microscopy and lipid analysis. The new species, provisionally named Candidatus Scalindua brodae and Scalindua wagneri considerably extend the biodiversity of the anammox lineage on the 16S rRNA gene level, but otherwise resemble known anammox bacteria. Simultaneously, another new species of the same genus, Candidatus Scalindua sorokinii, was detected in the water column of the Black Sea, making this genus the most widespread of all anammox bacteria described so far.

Nitrifying microbial community analysis of nitrite accumulating biofilm reactor by fluorescence in situ hybridization

Biological nitrogen removal via nitrite pathway in wastewater treatment is very important especially in the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulations were carried out in a biofilm reactor. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.2 kg N/m3/d. Nitrite accumulation was achieved by the selective inhibition of nitrite oxidizers by free ammonia and oxygen limitation. Nitrite oxidation activity was recovered as soon as the inhibition factor was removed. Fluorescence in situ hybridization studies of the nitrite accumulating biofilm system have shown that genus Nitrosomonas which is specifically hybridized with probe NSM 156 was the dominant nitrifying bacteria while Nitrospira was less abundant than those of normal nitrification systems. Further FISH analysis showed that the combinations of Nitrosomonas and Nitrospira cells were identified as important populations of nitrifying bacteria in an autotrophic nitrifying biofilm system.

Nitrification of high strength ammonia wastewtaer treatment - process selection is the major factor.

Biological nitrogen removal via the nitrite pathway in wastewater treatment is very important in Saving the cost of aeration and as an electron donor for denitrification. Wastewater nitrification and nitrite accumulation were carried out in a biofilm airlift reactor with autotrophic nitrifying biofilm. The biofilm reactor showed almost complete nitrification and most of the oxidized ammonium was present as nitrite at the ammonium load of 1.5 to 3.5 kg N/m3.d. Nitrite accumulation was stably achieved by the selective inhibition of nitrite oxidizers with free ammonia and dissolved oxygen limitation. Stable 100% conversion to nitrite could also be achieved even under the absence of free ammonia inhibition on nitrite oxidizers. Batch ammonium oxidation and nitrite oxidation with nitrite accumulating nitrifying biofilm showed that nitrite Oxidation was completely inhibited when free ammonia is higher than 0.2 mg N/L. However, nitrite oxidation activity was recovered as soon as the free ammonia concentration was below the threshold level when dissolved oxygen concentration was not the limiting factor. Fluorescence in situ hybridization analysis of cryosectioned nitrite accumulating nitrifying biofilm showed that the β-subclass of Proteobacteria, where ammonia oxidizers belong, was distributed outside the biofilm whereas the α-subclass of Proteobacteria, where nitrite oxidizers belong, was found mainly in the inner part of the biofilm. It is likely that dissolved oxygen deficiency or limitation in the inner part of the nitrifying biofilm, where nitrite oxidizers exist, is responsible for the complete shut down of the nitrite oxidizers activity under the absence of free ammonia inhibition.

Culture-dependent characterization of cyanobacterial diversity in the intertidal zones of the Portuguese coast: A polyphasic study

Cyanobacteria are important primary producers, and many are able to fix atmospheric nitrogen playing a key role in the marine environment. However, not much is known about the diversity of cyanobacteria in Portuguese marine waters. This paper describes the diversity of 60 strains isolated from benthic habitats in 9 sites (intertidal zones) on the Portuguese South and West coasts. The strains were characterized by a morphological study (light and electron microscopy) and by a molecular characterization (partial 16S rRNA, nifH, nifK, mcyA, mcyE/ndaF, sxtI genes). The morphological analyses revealed 35 morphotypes (15 genera and 16 species) belonging to 4 cyanobacterial Orders/Subsections. The dominant groups among the isolates were the Oscillatoriales. There is a broad congruence between morphological and molecular assignments. The 16S rRNA gene sequences of 9 strains have less than 97% similarity compared to the sequences in the databases, revealing novel cyanobacterial diversity. Phylogenetic analysis, based on partial 16S rRNA gene sequences showed at least 12 clusters. One-third of the isolates are potential N2-fixers, as they exhibit heterocysts or the presence of nif genes was demonstrated by PCR. Additionally, no conventional freshwater toxins genes were detected by PCR screening.

Introdução, estabelecimento e adaptação de Bradirrizóbios simbiontes da soja em a solos brasileiros.

Rizóbios microssimbiontes da soja; Introdução de estirpes nos solos brasileiros; Adaptação das estirpes de B. japonicum / B. elkanii aos solos brasileiros; Competitividade das estirpes de B elkanii SEMIA 587 e 29 W; Competitividade das estirpes do sorogrupo SEMIA 566 de B. japonicum; Variabilidade nas estirpes de Bradyrhizobium após a introdução nos solos brasileiros; Transferência horizontal de genes entre estirpes inoculantes e rizóbios indígenas ou naturalizados nos solos brasileiros.

Genetic diversity and population structure in Vicia faba L. landraces and wild related species assessed by nuclear SSRs

Research Article

Biocontrol strain Pseudomonas fluorescens CHA0 and its genetically modified derivative with enhanced biocontrol capability exert comparable effects on the structure of a Sinorhizobium meliloti population in a gnotobiotic system

The impact of biocontrol strain Pseudomonas fluorescens CHA0 and of its genetically modified, antibiotic-overproducing derivative CHA0/pME3424 on a reconstructed population of the plant-beneficial Sinorhizobium meliloti bacteria was assessed in gnotobiotic systems. In sterile soil, the final density of the reconstructed S. meliloti population decreased by more than one order of magnitude in the presence of either of the Pseudomonas strains when compared to a control without addition of P. fluorescens. Moreover, there was a change in the proportion of each individual S. meliloti strain within the population. Plant tests also revealed changes in the nodulating S. meliloti population in the presence of strains CHA0 or CHA0/pME3424. In both treatments one S. meliloti strain, f43, was significantly reduced in its root nodule occupancy. Analysis of alfalfa yields showed a slight but statistically significant increase in shoot dry weight when strain CHA0 was added to the reconstructed S. meliloti population whereas no such effect was observed with CHA0/pME3424.

Peanut response to lime and molybdenum application in low pH soils

Liming acid soils is considered to assure the availability of Mo in crops. Additionally, in peanuts (Arachis hypogaea L.) the positive response to liming is associated to a better supply of Ca+2, Mo for the nitrogenase-complex activity, and other non-nitrogen fixing activities of the crop. This study was thus undertaken to assess the effect of lime, Mo, and the lime-Mo interaction on peanut crop, on an acid Ultisol at the Mococa Experimental Station, Instituto Agronômico, São Paulo State, Brazil, from 1987 to 1990. A randomized complete block design with four replications, in a 4 x 4 factorial arrangement, was used in the study. The factors included four lime rates (0, 2, 4, and 6 t ha-1) broadcast and incorporated into the soil, and Mo (0, 100, 200, and 300 g ha-1) as (NH4)2MoO4 applied as seed dressing. Lime was applied once at the beginning of the study while Mo was applied at every planting. Peanut seed cv 'tatu' was used. Significant increase in peanut kernel yield with liming was only evident in the absence of Mo, whereas the peanut response to Mo was observed in two out of the three harvests. A higher yield response (28 % increase) was found when Mo was applied without liming. Soil molybdenum availability, as indicated by plant leaf analysis, increased significantly when lime was applied. Molybdenum fertilization led to higher leaf N content, which in turn increased peanut yield in treatments with smaller lime doses.

Influence of fungicide seed treatment on soybean nodulation and grain yield

Biological N2 fixation is a major factor contributing to the increased competitiveness of Brazilian soybeans on the international market. However, the contribution of this process may be limited by adverse conditions to symbiotic bacteria, such as fungicide seed treatments. This study aimed to evaluate the effects of the fungicides carbendazim + thiram and carboxin + thiram on soybean nodulation, plant growth and grain yield. Two field experiments were carried out in the Cerrado region of the State of Roraima, in a soil with a low organic matter content and no soybean bradyrhizobia. In 2005, seeds were treated with fungicide carbendazim + thiram and commercial inoculants containing the Bradyrhizobium elkanii strains SEMIA 5019 and SEMIA 587 and B. japonicum strains SEMIA 5079 and SEMIA 5080. In 2006, soybean seeds were treated with the fungicides carbendazim + thiram or carboxin + thiram and inoculated separately with each one of the four strains. The plants were evaluated for number of nodules and dry weight, shoot dry weight and total N accumulated in shoots 35 days after plant emergence, while grain yield and N grain content were determined at harvest. Both fungicides reduced soybean nodulation, especially in the presence of B. elkanii strains. The fungicide carbendazim + thiram reduced nodulation by about 50 % and grain yield by more than 20 % (about 700 kg ha-1), in the treatment inoculated with of strain SEMIA 587.

In-furrow inoculation of soybean as alternative to fungicide and micronutrient seed treatment

Soybean is a major grain crop in Brazil, and yields can be considerably improved by inoculation with selected Bradyrhizobium strains. However, the incompatibility between inoculation and seed treatments with fungicides and micronutrients represents a major barrier to the achievement of high rates of biological N2 fixation. Inoculation practices that can alleviate the negative effects of agrochemicals must therefore be found and in-furrow inoculation seems to be an attractive alternative. This study reports the results of seven field experiments conducted in three growing seasons in Brazil; three in soils previously cropped with inoculated soybean (> 10(4) cells g-1 of soil of Bradyrhizobium), and four in areas where the crop was sown for the first time (< 10² cells g-1 of soil of Bradyrhizobium). The compatibility with fungicides and micronutrients was compared in seeds inoculated with peat or liquid inoculants, or treated with different doses of liquid inoculant in-furrow. In areas with established Bradyrhizobium populations, seed-applied agrochemicals did generally not affect nodulation, but also did not increase yields, while inoculation always increased N grain accumulation or yield, and N fertilizer decreased both nodulation and yield. Where soybean was sown for the first time, the seed treatment with agrochemicals affected nodulation when applied together with peat or liquid inoculant. In-furrow inoculation alleviated the effects of seed treatment with agrochemicals; the best performance was achieved with high Bradyrhizobium cell concentrations, with up to 2.5 million cells seed-1.

Diversity and efficiency of bradyrhizobium strains isolated from soil samples collected from around sesbania virgata roots using cowpea as trap species

The genetic diversity of ten Bradyrhizobium strains was evaluated for tolerance to high temperatures, to different salinity levels and for the efficiency of symbiosis with cowpea plants (Vigna unguiculata (L.) Walp.). Eight of these strains were isolated from nodules that appeared on cowpea after inoculation with suspensions of soil sampled from around the root system of Sesbania virgata (wand riverhemp) in ecosystems of South Minas Gerais. The other two strains used in our analyses as references, were from the Amazon and are currently recommended as cowpea inoculants. Genetic diversity was analyzed by amplifying repetitive DNA elements with the BOX primer, revealing high genetic diversity with each strain presenting a unique band profile. Leonard jar assays showed that the strains UFLA 03-30 and UFLA 03-38 had the highest N2-fixing potentials in symbiosis with cowpea. These strains had more shoot and nodule dry matter, more shoot N accumulation, and a higher relative efficiency than the strains recommended as inoculants. All strains grew in media of pH levels ranging from 4.0 to 9.0. The strains with the highest N2-fixing efficiencies in symbiosis with cowpea were also tolerant to the greatest number of antibiotics. However, these strains also had the lowest tolerance to high salt concentrations. All strains, with the exceptions of UFLA 03-84 and UFLA 03-37, tolerated temperatures of up to 40 ºC. The genetic and phenotypic characteristics of the eight strains isolated from soils of the same region were highly variable, as well as their symbiotic efficiencies, despite their common origin. This variability highlights the importance of including these tests in the selection of cowpea inoculant strains.