Genomic insights into organic acid production and plant growth promotion by different species of phosphate-solubilizing bacteria.

Bacteria can solubilize phosphorus (P) through the secretion of low-molecular-weight organic acids and acidification. However, the genes involved in the production of these organic acids are poorly understood. The objectives of this study were to verify the calcium phosphate solubilization and the production of low-molecular-weight organic acids by diverse genera of phosphate solubilizing bacterial strains (PSBS); to identify the genes related to the synthesis of the organic acids in the genomes of these strains and; to evaluate growth and nutrient accumulation of maize plants inoculated with PSBS and fertilized with Bayóvar rock phosphate. Genomic DNA was extracted for strain identification and annotation of genes related to the organic acids production. A greenhouse experiment was performed with five strains plus 150 mg dm- 3 P2O5 as Bayóvar rock phosphate (BRP) to assess phosphate solubilization contribution to maize growth and nutrition. Paraburkholderia fungorum UFLA 04-21 and Pseudomonas anuradhapurensis UFPI B5-8A solubilized over 60% of Ca phosphate and produced high amounts of citric/maleic and gluconic acids in vitro, respectively. Eleven organic acids were identified in total, although not all strains produced all acids. Besides, enzymes related to the organic acids production were found in all bacterial genomes. Plants inoculated with strains UFPI B5-6 (Enterobacter bugandensis), UFPI B5-8A, and UFLA 03-10 (Paenibacillus peoriae) accumulated more biomass than the plants fertilized with BRP only. Strains UFLA 03-10 and UFPI B5-8A increased the accumulation of most macronutrients, including P. Collectively, the results show that PSBS can increase maize growth and nutrient accumulation based on Bayóvar rock phosphate fertilization.

Rhizobacteria control damping-off and promote growth of lima bean with and without co-inoculation with Rhizobium tropici CIAT899.

Rhizoctonia solani compromises the production of lima bean, an alternative and low-input food source in many tropical regions. Inoculation of bacterial strains has been used, but research on their biocontrol and growth promotion potential on lima bean is scarce. The objective of this study was to evaluate the effects of inoculation with rhizobacterial strains of the genera Bacillus, Brevibacillus, Paenibacillus, Burkholderia, Pseudomonas, and Rhizobium in combination or not with N2-fixing Rhizobium tropici on the control of damping-off disease and growth promotion in lima bean plants. Greenhouse experiments were conducted to evaluate the inoculation with bacterial strains with biocontrol potential in combination or not with R. tropici in substrate infected with R. solani CML 1846. Growth promotion of these strains was also assessed. Strains of Brevibacillus (UFLA 02-286), Pseudomonas (UFLA 02-281 and UFLA 04-885), Rhizobium (UFLA 04-195), and Burkholderia (UFLA 04-227) co-inoculated with the strain CIAT 899 (Rhizobium tropici) were the most effective in controlling R. solani, reducing the disease incidence in 47-60% on lima bean. The promising strains used in the biocontrol assays were also responsive in promoting growth of lima bean under disease and sterile conditions. A positive synergistic effect of co-inoculation of different genera contributed to plant growth, and these outcomes are important first steps to improve lima bean production.

Polymeric formulations of liquid inoculants with rhizobia exopolysaccharides increase the survival and symbiotic efficiency of elite Bradyrhizobium strains.

We studied the survival of four elite strains of Bradyrhizobium in liquid inoculants with three formulations with exopolysaccharides extracted from other rhizobia genera, and the symbiotic efficiency of these elite strains with soybean and cowpea in a greenhouse. For that purpose, we verified the effectiveness of formulations for maintaining the cell viability of strains by counting the colony-forming units (CFU) per milliliter of the liquid inoculants with formulations after 90 days. For survival of the soybean inoculant strains, 29W and CPAC15, the largest number of CFU (> 1010 mL-1) after 90 days was observed in the PEPS formulation. For the cowpea inoculant strains, INPA3-11B and UFLA3-84, the REPS1 formulation had the largest number of CFU (> 1010 mL-1) after 90 days. The symbiotic efficiency of the PEPS formulation, followed by REPS2, was higher than that shown by the commercial inoculant in soybean. For cowpea, the three formulations with EPS, especially REPS1, showed symbiotic efficiency better than that of the commercial inoculant.

Ecological co-occurrence and soil physicochemical factors drive the archaeal community in Amazonian soils.

We evaluated the co-occurrence of archaeal taxonomic groups and soil physicochemical characteristics in relation to the structuring of the archaeal community in Amazonian soil under different land use systems. Soil samples were collected in primary forest (PF), secondary forest (SF), agricultural systems (AG) and cattle pastures (PA). Archaeal community composition was revealed based on high-throughput amplicon sequencing of the 16S rRNA gene. The results revealed co-occurrence of archaeal classes, with two groups formed: Thaumarchaeota classes, including South Africa Gold Mine-Group 1 (SAGMG-1), Crenarchaeotic group (SCG) and Crenarchaeota candidate division YNPFFA, with predominance in PF and SF; and Bathyarchaeota_unclassified, Methanomicrobia and Methanobacteria (Euryarchaeota) with the FHMa11 terrestrial group, with predominance in PA. The number of co-occurrences between groups was lower in SF, AG and PA (approximately 30%) than in PF. The qPCR analysis revealed that PF also had the largest number of archaeal representatives. Soil texture may be a limiting factor of interactions between groups since the most representative groups, SAGMG-1 and the SCG (over 20% in all sites), were positively associated with coarse sand, the soil factor most correlated with the groups (33% of the total). These results suggest that interactions between archaeal classes belonging to different phyla may be dependent on the number of individuals in the soil environment. In this context, differences in soil physical structure among the land use systems can reduce the representatives of key groups and consequently the co-occurrence of Archaea, which could compromise the natural dynamics of this complex environment.

Diversity and Efficiency of Rhizobia from a Revegetated Area and Hotspot-Phytophysiognomies Affected by Iron Mining as Indicators of Rehabilitation and Biotechnological Potential.

This study aimed to evaluate the resilience of phytophysiognomies under influence of iron mining by assessing the occurrence, diversity, and symbiotic efficiency of native communities of nitrogen-fixing bacteria that nodulate leguminous plants (rhizobia) in soils of an area revegetated with grass after iron mining activities and in the phytophysiognomies in adjacent areas (Canga, Atlantic Forest, Cerrado, and Eucalyptus-planted forest). Experiments for capturing rhizobia through two species of promiscuous plants, siratro (Macroptilium atropurpureum) and cowpea (Vigna unguiculata), were conducted in a greenhouse. The rhizobial strains isolated were characterized phenotypically, genetically (16S rRNA sequencing and BOX-PCR fingerprinting), and regard symbiotic efficiency of biological nitrogen fixation (BNF) compared to mineral nitrogen and reference strains. Cowpea captured a higher density of rhizobia than siratro did. However, most of the strains captured by siratro had greater symbiotic efficiency. The revegetated area proved to be the community most efficient in N2 fixation and was also the most diverse, whereas Canga was the least diverse. For the two trap species, the predominant genus captured in the revegetated area and in the phytophysiognomies was Bradyrhizobium. The greater symbiotic efficiency and the high genetic diversity of the rhizobial community in the revegetated area indicate the effectiveness of the soil rehabilitation process. The revegetated area and the phytophysiognomies proved to harbor strains with high biotechnological potential. Results indicate that the high functional redundancy of this group of bacteria contributes to the resilience of these phytophysiognomies and the revegetated area.

Bradyrhizobium campsiandrae sp. nov., a nitrogen-fixing bacterial strain isolated from a native leguminous tree from the Amazon adapted to flooded conditions.

The nitrogen-fixing bacterial strain UFLA 01-1174T was isolated from nodules of Campsiandra laurilifolia Benth. originating from the Amazon region, Brazil. Its taxonomic position was defined using a polyphasic approach. Analysis of the 16S rRNA gene placed the strain in the Bradyrhizobium genus, the closest species being B. guangdongense CCBAU 51649T and B. guangzhouense CCBAU 51670T, both with 99.8% similarity. Multilocus sequence analysis (MLSA) of recA, gyrB, glnII, rpoB, atpD, and dnaK indicated that UFLA 01-1174T is a new species, most closely related to B. stylosanthis BR 446T (94.4%) and B. manausense BR 3351T (93.7%). Average nucleotide identity (ANI) differentiated UFLA 01-1174T from the closest species with values lower than 90%. The G + C content in the DNA of UFLA 01-1174T is 63.6 mol%. Based on this data, we conclude that the strain represents a new species. The name proposed is Bradyrhizobium campsiandrae, with UFLA 01-1174T (= INPA 394BT = LMG 10099T) as type strain.

Chemical, physical, and biological attributes in soils affected by deposition of iron ore tailings from the Fundão Dam failure.

Monitoring degraded areas is essential for evaluation of the quality of the rehabilitation process. In this study, we evaluate how the physical and chemical characteristics of the mixture of iron ore tailings with the soil have affected the soil microbial biomass and activity in areas along the Gualaxo do Norte River after the Fundão Dam disaster. Composite soil samples were collected from areas that were impacted (I) and not impacted (NI) by the tailings. The following attributes were evaluated: chemical element content; soil density, porosity, and texture; microbial biomass carbon; basal respiration; and enzyme activity and density of microbial groups (bacteria, actinobacteria, fungi, arbuscular mycorrhizae, phosphate solubilizers, cellulolytic microorganisms, nitrifiers, ammonifiers, and diazotrophs). According to result, the deposition of tailings increased the pH and the soil available P, Cr, Fe, and Mn content and reduced organic matter. The physical and biological attributes were negatively affected, with increases in the silt content and density of the soil, and reduction in macroporosity and in the microbial biomass and activity of the soil (respiration and enzymes) in the impacted area. However, the impacted areas exhibited greater densities of some microbial groups (cellulolytic microorganisms, nitrifiers, and diazotrophic bacteria). Modifications in the organic matter and silt content are the main attributes associated with deposition of the tailings that affected soil microbial biomass and microbial activity. This may affect erosive conditions and the functionality of the ecosystem, indicating an imbalance in this environment. In contrast, the higher density of some microbial groups in the impacted areas show the high rehabilitation potential of these areas.

Bradyrhizobium uaiense sp. nov., a new highly efficient cowpea symbiont.

This study describes two Bradyrhizobium strains, UFLA03-164T and UFLA03-153, which share more than 99% sequence similarity of the 16S rRNA with the type strains of 15 species in this genus. The concatenation of three housekeeping genes (recA, gyrB, and dnaK) indicated that both strains formed a single clade separate from known Bradyrhizobium species. B. viridifuturi, represented by SEMIA 690T, is the closest neighboring species (96.2%). Low (< 92%) average nucleotide identity (ANI) was observed between strain UFLA03-164T and any of the closest species on the phylogenetic trees based on concatenated housekeeping genes. The DNA G+C content of UFLA03-164T is 63.25%. Phenotypic characteristics were determined for both UFLA strains. Based on the data, the two strains represent a new species for which the name Bradyrhizobium uaiense is proposed, with UFLA03-164T (= LMG 31509T) as type strain.

Bradyrhizobium brasilense sp. nov., a symbiotic nitrogen-fixing bacterium isolated from Brazilian tropical soils.

Four strains of rhizobia isolated from nodules of Vigna unguiculata (UFLA03-321T, UFLA03-320 and UFLA03-290) and Macroptilium atropurpureum (UFLA04-0212) in Brazilian soils were previously reported as a new group within the genus Bradyrhizobium. To determine their taxonomic position, these strains were characterized in this study using a polyphasic approach. The analysis of the 16S rRNA gene grouped the four strains with Bradyrhizobium pachyrhizi PAC48T. However, the concatenated sequence analysis of the two (recA and glnII) or three (atpD, gyrB and recA) housekeeping genes indicated that these strains represent a novel species of Bradyrhizobium, which is very closely related to B. pachyrhizi PAC48T and B. elkanii USDA 76T. Genomic relatedness analyses between the UFLA03-321T strain and B. elkanii USDA 76T and B. pachyrhizi PAC48T revealed an average nucleotide identity below 96% and values of estimated DNA-DNA hybridization below 70%, confirming that they represent genomically distinct species. Analysis of MALDI-TOF MS (Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry) profiles and phenotypic characteristics also allowed differentiation of the novel species from its two neighboring species. In phylogenetic analysis of nodC and nifH genes, UFLA03-321T exhibited maximum similarity with B. tropiciagri CNPSo 1112T. The data suggest that these four UFLA strains represent a novel species, for which the name Bradyrhizobium brasilense sp. nov. is proposed, with UFLA03-321T (=LMG 29353 =CBAS645) as type strain. G + C content in the DNA of UFLA03-321T is 63.9 mol %.

Bacteria isolated from soils of the western Amazon and from rehabilitated bauxite-mining areas have potential as plant growth promoters.

Several processes that promote plant growth were investigated in endophytic and symbiotic bacteria isolated from cowpea and siratro nodules and also in bacterial strains recommended for the inoculation of cowpea beans. The processes verified in 31 strains were: antagonism against phytopathogenic fungi, free-living biological nitrogen fixation, solubilization of insoluble phosphates and indole acetic acid (IAA) production. The resistance to antibiotics was also assessed. Sequencing of the partial 16S rRNA gene was performed and the strains were identified as belonging to different genera. Eight strains, including some identified as Burkholderia fungorum, fixed nitrogen in the free-living state. Eighteen strains exhibited potential to solubilize calcium phosphate, and 13 strains could solubilize aluminum phosphate. High levels of IAA production were recorded with L-tryptophan addition for the strain UFLA04-321 (42.3 µg mL⁻¹). Strains highly efficient in symbiosis with cowpea bean, including strains already approved as inoculants showed the ability to perform other processes that promote plant growth. Besides, these strains exhibited resistance to several antibiotics. The ability of the nitrogen-fixing bacteria to perform other processes and their adaptation to environmental conditions add value to these strains, which could lead to improved inoculants for plant growth and environmental quality.

The elite strain INPA03-11B approved as a cowpea inoculant in Brazil represents a new Bradyrhizobium species and it has high adaptability to stressful soil conditions.

The strain INPA03-11BT, isolated in the 1980s from nodules of Centrosema sp. collected in Manaus, Amazonas, Brazil, was approved by the Brazilian Ministry of Agriculture as a cowpea inoculant in 2004. Since then, several studies have been conducted regarding its phenotypic, genetic, and symbiotic characteristics under axenic and field conditions. Phenotypic features demonstrate its high adaptability to stressful soil conditions, such as tolerance to acidity, high temperatures, and 13 antibiotics, and, especially, its high symbiotic efficiency with cowpea and soybean, proven in the field. The nodC and nifH phylogenies placed the INPA strain in the same clade as the species B. macuxiense BR 10303T which was also isolated from the Amazon region. The sequencing of the 16S rRNA ribosomal gene and housekeeping genes, as well as BOX-PCR profiles, showed its potential as a new species, which was confirmed by a similarity percentage of 94.7% and 92.6% in Average Nucleotide Identity with the closest phylogenetically related species Bradyrhizobium tropiciagri CNPSo1112T and B. viridifuturi SEMIA690T, respectively. dDDH values between INPA03-11BT and both CNPSo 1112T and SEMIA690T were respectively 58.5% and 48.1%, which are much lower than the limit for species boundary (70%). Therefore, we propose the name Bradyrhizobium amazonense for INPA03-11BT (= BR3301 = SEMIA6463).

Microbiological attributes as indicators of soil quality in coffee growing systems in Southwest Bahia, Brazil.

Microorganisms are important indicators of soil quality due to their sensitivity to changes, reflecting the impacts caused by different land uses. The objective of this study was to evaluate the microbiological and physical-chemical attributes of the soil in areas cultivated with coffee under three different management systems (shaded coffee and full sun coffee with two spacings), as well as in adjacent areas under pasture and native forest, in Bahia, Brazil. The microbiological and physicochemical indicators evaluated were basal soil respiration (MBR), soil total organic carbon (TOC), microbial biomass carbon (MBC), metabolic quotient (qCO2), microbial quotient (qMic), enzyme activities (urease, acid phosphatase and fluorescein diacetate hydrolysis (FDA)). Physical and chemical indicators (particle size, texture, pH, P, K+, Ca2+, Mg2+, Al3+, and sum of bases) were also evaluated. Biological and chemical attributes were much more discriminative of study areas in the dry season. Microbial quotient (qMic) and metabolic quotient (qCO2) in the dry season showed that pasture is the most degraded land use. Conversely, nature forest and coffee with Grevillea were similar and were the best ones. In general, soil quality indicators were more sensitive to discriminate pasture and native forest from coffee systems, which, in turn, were not well discriminated among themselves.

Soil biological attributes in arsenic-contaminated gold mining sites after revegetation.

Recovery of arsenic contaminated areas is a challenge society faces throughout the world. Revegetation associated with microbial activity can play an essential role in this process. This work investigated biological attributes in a gold mining area with different arsenic contents at different sites under two types of extant revegetation associated with cover layers of the soil: BS, Brachiaria sp. and Stizolobium sp., and LEGS, Acacia crassicarpa, A. holosericea, A. mangium, Sesbania virgata, Albizia lebbeck and Pseudosamanea guachapele. References were also evaluated, comprising the following three sites: B1, weathered sulfide substrate without revegetation; BM, barren material after gold extraction and PRNH (private reserve of natural heritage), an uncontaminated forest site near the mining area. The organic and microbial biomass carbon contents and substrate-induced respiration rates for these sites from highest to lowest were: PRNH > LEGS > BS > B1 and BM. These attributes were negatively correlated with soluble and total arsenic concentration in the soil. The sites that have undergone revegetation (LEGS and BS) had higher densities of bacteria, fungi, phosphate solubilizers and ammonium oxidizers than the sites without vegetation. Principal component analysis showed that the LEGS site grouped with PRNH, indicating that the use of leguminous species associated with an uncontaminated soil cover layer contributed to the improvement of the biological attributes. With the exception of acid phosphatase, all the biological attributes were indicators of soil recovery, particularly the following: microbial carbon, substrate-induced respiration, density of culturable bacteria, fungi and actinobacteria, phosphate solubilizers and metabolic quotient.

Leguminous plants nodulated by selected strains of Cupriavidus necator grow in heavy metal contaminated soils amended with calcium silicate.

Increasing concern regarding mining area environmental contamination with heavy metals has resulted in an emphasis of current research on phytoremediation. The aim of the present study was to assess the efficiency of symbiotic Cupriavidus necator strains on different leguminous plants in soil contaminated with heavy metals following the application of inorganic materials. The application of limestone and calcium silicate induced a significant increase in soil pH, with reductions in zinc and cadmium availability of 99 and 94 %, respectively. In addition, improved nodulation of Mimosa caesalpiniaefolia, Leucaena leucocephala and Mimosa pudica in soil with different levels of contamination was observed. Significant increases in the nitrogen content of the aerial parts of the plant were observed upon nodulation of the root system of Leucaena leucocephala and Mimosa pudica by strain UFLA01-659 (36 and 40 g kg(-1)) and by strain UFLA02-71 in Mimosa caesalpiniaefolia (39 g kg(-1)). The alleviating effect of calcium silicate resulted in higher production of dry matter from the aerial part of the plant, an increase in nodule number and an increase in the nitrogen fixation rate. The results of the present study demonstrate that the combination of rhizobia, leguminous plants and calcium silicate may represent a key factor in the remediation of areas contaminated by heavy metals.

Genetic and symbiotic diversity of nitrogen-fixing bacteria isolated from agricultural soils in the western Amazon by using cowpea as the trap plant.

Cowpea is a legume of great agronomic importance that establishes symbiotic relationships with nitrogen-fixing bacteria. However, little is known about the genetic and symbiotic diversity of these bacteria in distinct ecosystems. Our study evaluated the genetic diversity and symbiotic efficiencies of 119 bacterial strains isolated from agriculture soils in the western Amazon using cowpea as a trap plant. These strains were clustered into 11 cultural groups according to growth rate and pH. The 57 nonnodulating strains were predominantly fast growing and acidifying, indicating a high incidence of endophytic strains in the nodules. The other 62 strains, authenticated as nodulating bacteria, exhibited various symbiotic efficiencies, with 68% of strains promoting a significant increase in shoot dry matter of cowpea compared with the control with no inoculation and low levels of mineral nitrogen. Fifty genotypes with 70% similarity and 21 genotypes with 30% similarity were obtained through repetitive DNA sequence (BOX element)-based PCR (BOX-PCR) clustering. The 16S rRNA gene sequencing of strains representative of BOX-PCR clusters showed a predominance of bacteria from the genus Bradyrhizobium but with high species diversity. Rhizobium, Burkholderia, and Achromobacter species were also identified. These results support observations of cowpea promiscuity and demonstrate the high symbiotic and genetic diversity of rhizobia species in areas under cultivation in the western Amazon.

Efficient nitrogen-fixing Rhizobium strains isolated from amazonian soils are highly tolerant to acidity and aluminium.

One of the most cultivated and consumed vegetables in Brazil is the common bean, Phaseolus vulgaris L. The symbiosis of this plant species with nitrogen-fixing bacteria that are adapted to the stresses commonly found in tropical soils can increase production. The aim of this study was to evaluate the symbiotic effectiveness of bacterial strains from soils under different land uses in the Amazon region. Further, rhizobia tolerance to acidity and aluminium and the involvement of some possible physiological mechanisms of such tolerance were also investigated. In assessing the efficiency of biological nitrogen fixation, inoculation with strains UFLA04-195, UFLA04-173 and UFLA04-202, belonging to the genus Rhizobium, resulted in greater plant growth, higher shoot nitrogen content and good nodulation compared to the inoculation with the strain CIAT 899 (R. tropici), and to the mineral nitrogen control or Burkholderia fungorum strains that nodulated or not bean plants. These efficient strains grew better at pH 5.0 than at pH 6.0 or pH 6.9; they also tolerated up to 1 mmol l(-1) of Al(3+) and showed an increased production of exopolysaccharides where the growing rates were less (pH 6.0 and pH 6.9). With respect to aluminium, the highest production of EPS produced greater tolerance to this element. Taken together, these results indicate that the strains evaluated in this study were tolerant to acidity and aluminium; they appeared to have developed resistance mechanisms such as EPS production and a resistant cell outer membrane (indicated by resistance to polymyxin and methyl violet). As these strains also gave increased yields of the host species, further studies on whether to recommend these strains as inoculants are already underway.

Rhizobia exopolysaccharides: promising biopolymers for use in the formulation of plant inoculants.

Inoculants with beneficial microorganisms comprise both selected strains and carriers that ensure a favorable microenvironment for cell survival and stability. Formulations of inoculants using synthetic polymers as carriers are common. However, only a few studies are available in the literature regarding the formulation of inoculants using natural biomolecules as carriers. Exopolysaccharides (EPS) are biomolecules produced by a vast array of microbial species, including symbiotic nitrogen-fixing bacteria, commonly known as rhizobia. EPS perform several functions, such as the protection against the deleterious effects of diverse environmental soil stresses. Two Rhizobium tropici strains and one Paraburkholderia strain were selected after semiquantitative analysis by scanning electron microscopy (SEM) of their EPS production in liquid YMA medium. Their EPS were characterized through a series of analytical techniques, aiming at their use in the formulation of plant inoculants. In addition, the effect of the carbon source on EPS yield was evaluated. Multi-stage fragmentation analysis showed the presence of xylose, glucose, galactose, galacturonic acid, and glucuronic acid in EPS chemical composition, which was confirmed by FT-IR spectra and 13C NMR spectroscopy. Thermal stability (thermogravimetric) was close to 270 °C and viscosity ranged from 120 to 1053.3 mPa.s. Surface morphology (SEM) was rough and irregular, with a cross-linked spongy matrix, which, together with the hydrophilic functional groups, confers water holding capacity. The present study showed that the three EPS have potential as microorganism carriers for formulation of microbial inoculants to be applied in plants.

Rhizobia and endophytic bacteria isolated from rainforest fragments within an iron ore mining site of the Eastern Brazilian Amazon.

The aim of the present study was to isolate and evaluate the diversity of rhizobial and endophytic bacterial strains from undisturbed native rainforests within an iron ore mining site of the Serra Norte de Carajás in the Eastern Brazilian Amazon region to assess their biotechnological utility in reclamation of areas. Experiments were conducted to capture strains from samples of the soil of these forests at the sites Arenito II, Noroeste II, and Sul IV using Macroptilium atropurpureum and Mimosa acutistipula var. ferrea as trap host plants. Only M. atropurpureum nodulated, and the different bacterial strains were isolated from its nodules. There was no difference in the number of nodules among the areas, but the Arenito II bacterial community was the most efficient, indicated by the aboveground biomass production and suitable shoot mass/root mass ratio. Fifty-two (52) bacterial isolates were obtained, distributed in five groups, including nodulating and endophytic bacteria: 32 from Arenito II, 12 from Noroeste II, and 8 from Sul IV. The nodulating Bradyrhizobium genus was common to the three areas, whereas Paraburkholderia was found only in Arenito II. The nodD1 gene was amplified in all the strains of both nodulating genera. Strains of the nodulating genus Methylobacterium were also isolated from the three areas; however, they did not nodulate the host of origin, and their nodD1 gene was not amplified. Endophytic strains were also isolated from the genera Paenibacillus, Pantoea, and Leifsonia in Arenito II, Leifsonia in Noroeste I, and Paenibacillus in Sul IV. The greater nodulation and rhizobial and endophytic bacterial diversity observed in Arenito II were probably due to the more suitable edaphic properties of the area. The isolated strains were incorporated in the collection of the Department of Soil Science of UFLA and will be investigated in relation to their symbiotic characteristics with native host plants, as well as their ability to perform other biological processes.

Classification of the inoculant strain of cowpea UFLA03-84 and of other strains from soils of the Amazon region as Bradyrhizobium viridifuturi (symbiovar tropici).

Cowpea (Vigna unguiculata L.) is a legume species that considerably benefits from inoculation with nitrogen fixing bacteria of the genus Bradyrhizobium. One of the strains recommended for inoculation in cowpea in Brazil is UFLA03-84 (Bradyrhizobium sp.). The aim of our study was to define the taxonomic position of the UFLA03-84 strain and of two other strains of Bradyrhizobium (UFLA03-144 and INPA237B), all belonging to the same phylogenetic group and isolated from soils of the Brazilian Amazon. Multilocus sequence analysis (MLSA) of the housekeeping genes atpD, gyrB, recA, and rpoB grouped (with similarity higher than 99%) the three strains with Bradyrhizobium viridifuturi SEMIA 690T. The analyses of average nucleotide identity and digital DNA-DNA hybridization supported classification of the group as Bradyrhizobium viridifuturi. The three strains exhibited similar behavior in relation to the most of the phenotypic characteristics evaluated. However, some characteristics exhibited variation, indicating phenotypic diversity within the species. Phylogenetic analysis of the nodC and nifH genes showed that the three strains are members of the same symbiovar (tropici) that contains type strains of Bradyrhizobium species coming from tropical soils (SEMIA 690TB. viridifuturi, CNPSo 1112TB. tropiciagri, CNPSo 2833TB. embrapense, and B. brasilense UFLA03-321T).

Tripartite symbiosis of Sophora tomentosa, rhizobia and arbuscular mycorhizal fungi.

Sophora tomentosa is a pantropical legume species with potential for recovery of areas degraded by salinization, and for stabilization of sand dunes. However, few studies on this species have been carried out, and none regarding its symbiotic relationship with beneficial soil microorganisms. Therefore, this study aimed to evaluate the diversity of nitrogen-fixing bacteria isolated from nodules of Sophora tomentosa, and to analyze the occurrence of colonization of arbuscular mycorrhizal fungi on the roots of this legume in seafront soil. Thus, seeds, root nodules, and soil from the rhizosphere of Sophora tomentosa were collected. From the soil samples, trap cultures with this species were established to extract spores and to evaluate arbuscular mycorhizal fungi colonization in legume roots, as well as to capture rhizobia. Rhizobia strains were isolated from nodules collected in the field or from the trap cultures. Representative isolates of the groups obtained in the similarity dendrogram, based on phenotypic characteristics, had their 16S rRNA genes sequenced. The legume species showed nodules with indeterminate growth, and reddish color, distributed throughout the root. Fifty-one strains of these nodules were isolated, of which 21 were classified in the genus Bacillus, Brevibacillus, Paenibacillus, Rhizobium and especially Sinorhizobium. Strains closely related to Sinorhizobium adhaerens were the predominant bacteria in nodules. The other genera found, with the exception of Rhizobium, are probably endophytic bacteria in the nodules. Arbuscular mycorrhizal fungi was observed colonizing the roots, but arbuscular mycorhizal fungi spores were not found in the trap cultures. Therefore Sophora tomentosa is associated with both arbuscular mycorhizal fungi and nodulating nitrogen-fixing bacteria.