Rhizobium Tropici Strain CIAT899 Research Articles

Impact of Fe + 2 ions on structural integrity of A0A6P1CI42_RHITR NifA protein from Rhizobium tropici strain CIAT 899

A0A6P1CI42_RHITR, a protein originating from Rhizobium tropici strain CIAT 899, has emerged as a key player in leguminous plant symbiosis and nitrogen fixation processes. Understanding the intricate details of its structure and function holds immense significance for unraveling the molecular mechanisms underlying its biological activities. In this study, we employed molecular modeling and molecular dynamics (MD) simulations to investigate the A0A6P1CI42_RHITR protein, with a specific emphasis on the influence of Fe-atoms, linker structural integrity, and conformational changes within the GAF domain. Our findings unveiled noteworthy conformational changes in the A0A6P1CI42_RHITR protein, particularly within the GAF domain, when Fe-atoms were present compared to its apo form. Significant conformational rearrangements after an initial 20 ns, accompanied by the opening of the ligand substrate accommodating loop in the GAF domain influenced by Fe-atoms was observed. At the residue level, the investigation revealed substantial activity variations in individual residues, particularly in those contributing to the GAF domain from positions 51 to 223. Intriguingly, the presence of Fe-atoms led to controlled movement of conserved cysteine residues at positions 467 and 472, indicating a correlation between interlinker domain motion and the activity of the GAF domain loop responsible for substrate accommodation. Moreover, in the presence of Fe-atoms, the distance between Cys467 and Cys472 residues was maintained, ensuring the overall structural integrity of the interdomain loop necessary for protein activation. Conversely, in the apo form, a sudden flip motion of cysteine residues’ thiol groups was observed, leading to a loss of structural integration. Overall, our study utilizing molecular modeling and MD simulations offers valuable insights into the structural dynamics and functional implications of the A0A6P1CI42_RHITR protein. Communicated by Ramaswamy H. Sarma

Influence of common bean genotypes and rhizobia interaction for nodulation and nitrogen fixation

Bacteria from the rhizobia group are able to associate symbiotically with bean crop, forming nodules in the root, in which the biological nitrogen-fixing process occurs. However, the efficiency of this process has been low and it can be attributed to genetic and environmental factors. Thus, the objective of this study was to evaluate the capacity of nodulation of local varieties and commercial common bean cultivars inoculated with a Rhizobium tropici strain used in commercial inoculants and rhizobia isolates from common bean root nodules. The experiment was carried out in a factorial scheme (2x4), in a randomized block design with four replicates. It was tested two local varieties and two commercial cultivars, inoculated with the reference Rhizobium tropici strain CIAT899 and the RBZ14 strain isolated from common bean nodules grown in soils of Southern Brazil, in adapted Leonard-type pots. The CIAT899 strain promoted either higher mass of viable nodules and higher nitrogen accumulation in the aerial part. The black group local variety showed better response than the cultivar TAA Dama for nodule viability, suggesting more efficiency for nodulation. The interaction between genotypes (local varieties and commercial cultivars) and bacteria showed the specificity of the complex symbiotic relationship of biological nitrogen fixation in common bean, requiring further studies of these interactions.

Co-inoculation of Rhizobium and Bradyrhizobium promotes growth and yield of common beans

Some strains of Bradyrhizobium benefit the biological N2 fixation (BNF) and growth of common beans (Phaseolus vulgaris L.) when co-inoculated with Rhizobium tropici under greenhouse conditions. Here, we evaluated the benefit of Bradyrhizobium diazoefficiens strain CPAC 7 co-inoculated with Rhizobium tropici strain CIAT 899 on common bean in the field. Three Bradyrhizobium strains were co-inoculated with R. tropici CIAT 899 in one experiment in the greenhouse and two in the field. The greenhouse experiment combined each of three Bradyrhizobium strains (29w, USDA 110, and CPAC 7) in co-inoculation with CIAT 899 in Leonard jars. Then, two of these strains, 29w and CPAC 7, were co-inoculated with Rhizobium on-seeds (1.2 × 106 CFU seed−1) or both on-seed and in-furrow (1.08 × 108 CFU m−1) in a field experiment. Finally, a second field experiment evaluated strain CPAC 7 at doses ranging from 1.5 × 105 to 1.2 × 106 CFU seed−1. Co-inoculation with strain CPAC 7 improved shoot biomass accumulation in Leonard jars. In the field, co-inoculation of CPAC 7 with CIAT 899 on-seed enhanced grain yield by 20% compared with inoculation of CIAT 899 only (p < 0.10). The percentage of N derived from the air (%Ndfa) was between 50 and 60%, with only a tendency towards co-inoculation benefit. Given that the %Ndfa of the inoculated plants was not statistically different between treatments, the increased grain yield was not entirely due to BNF but also due to an increase of soil N uptake. In the second field trial, larger doses of inoculum increased nodulation and root biomass, but significant differences in grain yield were not observed. Our results show that the Rhizobium-Bradyrhizobium co-inoculation can enhance growth and grain yield of common beans and indicate that a plant growth-promoting mechanism besides BNF may be involved in the beneficial co-inoculation effect. However, further adjustments are needed to ensure a better response under field conditions.

Nitrogen in Shoots, Number of Tillers, Biomass Yield and Nutritive Value of Zuri Guinea Grass Inoculated with Plant-Growth Promoting Bacteria

The objective of this study was to evaluate the effects of plant growth-promoting bacteria (PGPB) strains of Azospirillum brasilense, Pseudomonas fluorescens and Rhizobium tropici on biomass yield, number of tillers, nitrogen accumulation and nutritive value of shoots of Megathyrsus (syn. Panicum) maximus cultivar BRS Zuri (Zuri Guinea grass). For that, one experiment was performed for 14 months to evaluate inoculation and re-inoculation with Azospirillum brasilense strains Ab-V5 and Ab-V6, Pseudomonas fluorescens strain CCTB 03 and of co-inoculation with Rhizobium tropici strain CIAT 899 + A. brasilense strain Ab-V6 combined with N-fertilizer (100 kg of N ha-1). Shoot dry weight yield (SDWY), number of tillers (NT), total N concentration (TNC), total N uptake (TNU) and nutritive value of Zuri Guinea grass was evaluated for eight cuts, and inoculation increased all parameters. In the NT, the treatments inoculated with PGPB were superior to the positive non-inoculated control receiving N-fertilizer, by up to 36%. For the accumulated of SDWY the treatment re-inoculated with P. fluorescens CCTB 03 after each cut was statistically superior tin 7% the positive control. The PGPB when combined N-fertilizer also increased SDWY, NT, the relative chlorophyll index, TNC, total N uptake, neutral detergent fiber, acid detergent fiber, crude protein and in vitro digestibility dry matter of Zuri Guinea grass. The results indicate that PGPB can represent a sustainable alternative for reducing the use of N-fertilizers. The lower effects of re-inoculation with PGPB on the nutrition or yield of Zuri Guinea grass, demonstrating that the determination of the method of application and periodicity of inoculation still require investigation.

Effects of growth‐promoting bacteria on soybean root activity, plant development, and yield

AbstractRhizobia and other plant growth‐promoting rhizobacteria (PGPR) have been broadly used as inoculants in agriculture, resulting in morphofunctional improvements in roots and grain yield. This study was carried out during two cropping seasons under field and greenhouse conditions in Brazil to verify the effects of inoculation of two soybean cultivars with PGPR and secondary microbial metabolites (SMMs) on root activity and nodulation, plant development, and grain yield. Inoculation and co‐inoculation treatments consisted of Bradyrhizobium japonicum strain SEMIA 5079 and B. diazoefficiens strain SEMIA 5080 inoculated together, in combination with Bacillus subtilis strain QST 713, Azospirillum brasilense strains Ab‐V5 and Ab‐V6, and SMMs extracted from B. diazoefficiens strain USDA 110 and Rhizobium tropici strain CIAT 889. Root systems were evaluated by direct (optical reading) and indirect (rubidium nitrate application, 85RbNO3) methods. Increases of up to 1.6% in root diameter (0.01‐ to 0.5‐mm class), 28.5% in length, 19.7% in root volume, 17.8% in root surface area, 29% in the number of nodules, 27.2% in nodule dry weight, 13.5% in root dry weight, and 3.8% in shoot dry weight. Greater exploration and activity within and between rows following inoculation at up to 40 and 10 cm in depth, respectively, were observed in plants co‐inoculated with the standard inoculation (only Bradyrhizobium spp.) + SMMs + A. brasilense, resulting in a yield increase of 485 kg ha−1. The results emphasize the biotechnological potential of using secondary metabolites of rhizobia with inoculants containing rhizobia and PGPR to improve the growth and soybean yield in tropical conditions.

Regulation of hsnT, nodF and nodE genes in Rhizobium tropici CIAT 899 and their roles in the synthesis of Nod factors and in the symbiosis

Rhizobium tropici strain CIAT 899 possesses outstanding agronomic properties as it displays tolerance to environmental stresses, a broad host range and high effectiveness in fixing nitrogen with the common bean (Phaseolus vulgaris L.); in addition, it carries intriguing features such as five copies of the regulatory nodD gene, and the capacity to synthesize a variety of nodulation factors (NFs), even in a flavonoid-independent manner, when submitted to abiotic stresses. However, the roles of several nod genes of the repertoire of CIAT 899 remain to be determined. In this study, we obtained mutants for the hsnT, nodF and nodE genes of CIAT 899 and investigated their expression, NF structures and symbiotic properties. Either in the presence of the flavonoid apigenin, or of salt the expression of hsnT, nodF and nodE in wild-type CIAT 899 was highly up-regulated in comparison to the mutants of all five copies of nodD, indicating the roles that regulatory nodD genes play in the activation of hsnT, nodF and nodE; however, NodD1 was recognized as the main inducer. In total, 29 different NF structures were synthesized by wild-type CIAT 899 induced by apigenin, and 36 when induced by salt, being drastically reduced by mutations in hsnT, nodF and nodE, especially under osmotic stress, with specific changes related to each gene, indicating that the three genes participate in the synthesis of NFs. Mutations in hsnT, nodF and nodE affected differently symbiotic performance (nodule number and shoot dry weight), according to the host plant. Our results indicate that the expression of hsnT, nodF and nodE genes of CIAT 899 is mediated by nodD genes, and although these three genes do not belong to the main set of genes controlling nodulation, they contribute to the synthesis of NFs that will impact symbiotic performance and host specificity.

Response of common bean (Phaseolus vulgaris L.) to nitrogen, phosphorus and rhizobia inoculation across variable soils in Zimbabwe

Common bean is an important crop with potential to curb malnutrition in poor Sub-Saharan African populations. Yields of common bean (Phaseolus vulgaris L.) are, however poor, limited by low soil phosphorus (P), nitrogen (N) and poor biological N2-fixation. On-farm experiments were carried out to study the effect of N, P and rhizobia inoculation on common bean yield and yield components during the 2014/2015 and 2015/2016 cropping seasons in Eastern Zimbabwe. Experiments were conducted on five farmers’ fields located in two agroecologies; three fields were considered to be degraded with soil organic carbon (SOC) < 4 g kg−1 and available P < 6 mg kg−1, while the two non–degraded sites had SOC > 7 g kg−1 and available P > 15 mg kg−1. Two common bean varieties (Gloria and NUA45) were tested in a split-plot arranged in randomized complete block design. The main plot factor was the combination of N (0 and 40 kg ha−1) and P (0 and 20 kg ha−1), and the sub-plot factors were variety (Gloria and NUA 45) and inoculation with Rhizobium tropici strain CIAT899 (+/− inoculum). At planting, both N and P were applied at 20 kg ha−1, with an additional 20 kg ha−1 N top dressing applied at flowering. Analysis of variance indicated common bean did not respond to rhizobia inoculation (P > 0.05) whilst P significantly increased the number of nodules and active nodules per plant (P < 0.001), and grain yield. Application of 40 kg ha−1 N significantly increased the number of pods per plant, number of seeds per pod, and grain yields. A significant NP interaction was only observed on grain yield for non-degraded soils. Co-application of N and P in non-degraded sites increased grain yields from 0.27 to 1.48 Mg ha−1during the first season and from 0.37 to 2.09 Mg ha−1during the second season. On degraded sites, NP application resulted in uninspiring grain yield gains of 0.09 to 0.19 Mg ha−1 during the first season, and from 0.16 to 0.28 Mg ha−1 in the second season. In general, effects of N or P were not significantly different, suggesting that farmers could invest in either of these nutrients for increased common bean grain yields. Strategically, P investments would be more logical as residual P effects to rotational cereals improve overall cropping system performance. The response of common bean to inoculation in Zimbabwe still needs to be widely investigated for these and other varieties.

Co-inoculation of maize with Azospirillum brasilense and Rhizobium tropici as a strategy to mitigate salinity stress.

Plants are highly affected by salinity, but some plant growth-promoting bacteria (PGPB) may trigger induced systemic tolerance (IST), conferring protection against abiotic stresses. We investigated plant mechanisms under saline stress (170mM NaCl) when maize was singly or co-inoculated with Azospirillum brasilense strains Ab-V5 and Ab-V6 and Rhizobium tropici strain CIAT 899. Under greenhouse conditions, plants responded positively to inoculation and co-inoculation, but with differences between strains. Inoculation affected antioxidant enzymes that detoxify reactive oxygen species (ROS) - ascorbate peroxidase (APX), catalase (CAT) and superoxide dismutase (SOD) - mainly in leaves. Proline contents in leaves and roots and malondialdehyde (MDA) in leaves - plant-stress-marker molecules - were significantly reduced due to the inoculation, indicating reduced need for the synthesis of these molecules. Significant differences were attributed to inoculation in the expression of genes related to antioxidant activity, in general with upregulation of APX1, CAT1, SOD2 and SOD4 in leaves, and APX2 in roots. Pathogenesis-related genes PR1, prp2, prp4 and heat-shock protein hsp70 were downregulated in leaves and roots, indicating that inoculation with PGPB might reduce the need for this protection. Together the results indicate that inoculation with PGPB might provide protection from the negative effects of saline stress. However, differences were observed between strains, as A. brasilense Ab-V5 did not show salt tolerance, while the best inoculation treatments to mitigate saline stress were with Ab-V6 and co-inoculation with Ab-V6+CIAT 899. Inoculation with these strains may represent an effective strategy to mitigate salinity stress.

RNA-seq analysis of the Rhizobium tropici CIAT 899 transcriptome shows similarities in the activation patterns of symbiotic genes in the presence of apigenin and salt.

BackgroundRhizobium tropici strain CIAT 899 establishes effective symbioses with several legume species, including Phaseolus vulgaris and Leucaena leucocephala. This bacterium synthesizes a large variety of nodulation factors in response to nod-gene inducing flavonoids and, surprisingly, also under salt stress conditions. The aim of this study was to identify differentially expressed genes in the presence of both inducer molecules, and analyze the promoter regions located upstream of these genes.ResultsResults obtained by RNA-seq analyses of CIAT 899 induced with apigenin, a nod gene-inducing flavonoid for this strain, or salt allowed the identification of 19 and 790 differentially expressed genes, respectively. Fifteen of these genes were up-regulated in both conditions and were involved in the synthesis of both Nod factors and indole-3-acetic acid. Transcription of these genes was presumably activated through binding of at least one of the five NodD proteins present in this strain to specific nod box promoter sequences when the bacterium was induced by both apigenin and salt. Finally, under saline conditions, many other transcriptional responses were detected, including an increase in the transcription of genes involved in trehalose catabolism, chemotaxis and protein secretion, as well as ribosomal genes, and a decrease in the transcription of genes involved in transmembrane transport.ConclusionsTo our knowledge this is the first time that a transcriptomic study shows that salt stress induces the expression of nodulation genes in the absence of flavonoids. Thus, in the presence of both nodulation inducer molecules, apigenin and salt, R. tropici CIAT 899 up-regulated the same set of symbiotic genes. It could be possible that the increases in the transcription levels of several genes related to nodulation under saline conditions could represent a strategy to establish symbiosis under abiotic stressing conditions.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-016-2543-3) contains supplementary material, which is available to authorized users.

Agronomic and Economic Efficiency of Common-Bean Inoculation with Rhizobia and Mineral Nitrogen Fertilization

Management of biological nitrogen fixation in common bean still requires improvement. The objective of this study was to verify the compatibility of nitrogen fertilization with biological N2 fixation to increase common bean yield and profitability. Four field experiments were performed in four municipalities of Minas Gerais, Brazil, during the 2009 and 2010 winter crop season. The 2009 crop experiments were set up under a no-till system and the 2010 crop was conducted using conventional tillage. A randomized block experimental design was used with four replications and seven treatments combining application rates of mineral N (at sowing and/or topdressing) and seed inoculation with Rhizobium tropici strain CIAT899. Inoculation with 20 kg ha-1 N-urea at sowing and seed inoculation does not interfere with nodule dry matter and promotes yield comparable to that observed with 80 kg ha-1 N-urea with economic profitability in both no-till and conventional tillage systems. These results show the possibility of economic savings with respect to N fertilizers, but also a significant ecological contribution by avoiding problems associated with misuse of these fertilizers, such as eutrophication of waterways and denitrification.

Opening the "black box" of nodD3, nodD4 and nodD5 genes of Rhizobium tropici strain CIAT 899.

BackgroundTranscription of nodulation genes in rhizobial species is orchestrated by the regulatory nodD gene. Rhizobium tropici strain CIAT 899 is an intriguing species in possessing features such as broad host range, high tolerance of abiotic stresses and, especially, by carrying the highest known number of nodD genes—five—and the greatest diversity of Nod factors (lipochitooligosaccharides, LCOs). Here we shed light on the roles of the multiple nodD genes of CIAT 899 by reporting, for the first time, results obtained with nodD3, nodD4 and nodD5 mutants.MethodsThe three nodD mutants were built by insertion of Ω interposon. Nod factors were purified and identified by LC-MS/MS analyses. In addition, nodD1 and nodC relative gene expressions were measured by quantitative RT-PCR in the wt and derivative mutant strains. Phenotypic traits such as exopolysaccharide (EPS), lipopolysaccharide (LPS), swimming and swarming motilities, biofilm formation and indole acetid acid (IAA) production were also perfomed. All these experiments were carried out in presence of both inducers of CIAT 899, apigenin and salt. Finally, nodulation assays were evaluated in up to six different legumes, including common bean (Phaseolus vulgaris L.).ResultsPhenotypic and symbiotic properties, Nod factors and gene expression of nodD3, nodD4 and nodD5 mutants were compared with those of the wild-type (WT) CIAT 899, both in the presence and in the absence of the nod-gene-inducing molecule apigenin and of saline stress. No differences between the mutants and the WT were observed in exopolysaccharide (EPS) and lipopolysaccharide (LPS) profiles, motility, indole acetic acid (IAA) synthesis or biofilm production, either in the presence, or in the absence of inducers. Nodulation studies demonstrated the most complex regulatory system described so far, requiring from one (Leucaena leucocephala, Lotus burtii) to four (Lotus japonicus) nodD genes. Up to 38 different structures of Nod factors were detected, being higher under salt stress, except for the nodD5 mutant; in addition, a high number of structures was synthesized by the nodD4 mutant in the absence of any inducer. Probable activator (nodD3 and nodD5) or repressor roles (nodD4), possibly via nodD1 and/or nodD2, were attributed to the three nodD genes. Expression of nodC, nodD1 and each nodD studied by RT-qPCR confirmed that nodD3 is an activator of nodD1, both in the presence of apigenin and salt stress. In contrast, nodD4 might be an inducer with apigenin and a repressor under saline stress, whereas nodD5 was an inducer under both conditions.ConclusionsWe report for R. tropici CIAT 899 the most complex model of regulation of nodulation genes described so far. Five nodD genes performed different roles depending on the host plant and the inducing environment. Nodulation required from one to four nodD genes, depending on the host legume. nodD3 and nodD5 were identified as activators of the nodD1 gene, whereas, for the first time, it was shown that a regulatory nodD gene—nodD4—might act as repressor or inducer, depending on the inducing environment, giving support to the hypothesis that nodD roles go beyond nodulation, in terms of responses to abiotic stresses.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-2033-z) contains supplementary material, which is available to authorized users.

Regulatory nodD1 and nodD2 genes of Rhizobium tropici strain CIAT 899 and their roles in the early stages of molecular signaling and host-legume nodulation

BackgroundNodulation and symbiotic nitrogen fixation are mediated by several genes, both of the host legume and of the bacterium. The rhizobial regulatory nodD gene plays a critical role, orchestrating the transcription of the other nodulation genes. Rhizobium tropici strain CIAT 899 is an effective symbiont of several legumes—with an emphasis on common bean (Phaseolus vulgaris)—and is unusual in carrying multiple copies of nodD, the roles of which remain to be elucidated.ResultsPhenotypes, Nod factors and gene expression of nodD1 and nodD2 mutants of CIAT 899 were compared with those of the wild type strain, both in the presence and in the absence of the nod-gene-inducing molecules apigenin and salt (NaCl). Differences between the wild type and mutants were observed in swimming motility and IAA (indole acetic acid) synthesis. In the presence of both apigenin and salt, large numbers of Nod factors were detected in CIAT 899, with fewer detected in the mutants. nodC expression was lower in both mutants; differences in nodD1 and nodD2 expression were observed between the wild type and the mutants, with variation according to the inducing molecule, and with a major role of apigenin with nodD1 and of salt with nodD2. In the nodD1 mutant, nodulation was markedly reduced in common bean and abolished in leucaena (Leucaena leucocephala) and siratro (Macroptilium atropurpureum), whereas a mutation in nodD2 reduced nodulation in common bean, but not in the other two legumes.ConclusionOur proposed model considers that full nodulation of common bean by R. tropici requires both nodD1 and nodD2, whereas, in other legume species that might represent the original host, nodD1 plays the major role. In general, nodD2 is an activator of nod-gene transcription, but, in specific conditions, it can slightly repress nodD1. nodD1 and nodD2 play other roles beyond nodulation, such as swimming motility and IAA synthesis.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1458-8) contains supplementary material, which is available to authorized users.

Curing of a non-symbiotic plasmid of the Rhizobium tropici strain CIAT 899 affected nodule occupancy and competitiveness of the bacteria in symbiosis with common beans

Functional analyses of rhizobial plasmids have concentrated mainly on symbiotic plasmids (pSym) that carry genes required for nitrogen fixation and nodule formation. However, information on the other plasmids, termed cryptic or non-symbiotic, is still scarce. In this work, a collection of sequential cured derivatives with different combinations of plasmids was generated from the Rhizobium tropici strain CIAT 899 to study their functions in symbiosis with common bean plants. PCR analysis of the nif genes from all cured derivatives indicated that plasmid b is the symbiotic plasmid (pSym) while a and c are the cryptic plasmids. This genotype was confirmed by the presence of nodules in common bean plants inoculated with derivatives containing plasmid b. However, when plasmid a was missing in the derivative containing the pSym, nodules were formed but in lower numbers and were smaller in size. The derivative cured of plasmid a formed nodules with a large quantity of starch and crystals and showed a significant decrease in nitrogenase activity. In addition, the presence of bacteria in the intercellular spaces of the nodules was observed. A co-inoculation experiment involving both the wild-type CIAT 899 strain and derivative CIAT 899a suggested that the cryptic plasmid a contains genes involved in the competition for nodulation of Phaseolus vulgaris.

Effects of different salt concentrations and Rhizobium inoculation (native and Rhizobium tropici CIAT899) on growth of dry bean ( Phaseolus vulgaris L.)

Research was conducted to determine the effect of salinity on Rhizobium and growth of dry bean. A commercial cultivar (Akman 98) of dry bean ( Phaseolus vulgaris L.) was inoculated with Rhizobium tropici strain CIAT899 and native Rhizobium in solution culture with different salt concentrations (control, 5, 10, 20 and 40 mmol −1) added before inoculation. The results indicated that population of R. tropici strain CIAT899 and natural Rhizobium were decreased with salinity levels. However, the population count train CIAT899 was inversely proportional to salt concentration with high growth (8.0–9.0 × 10 5 cfu ml −1) at lower concentrations of control – 5 mmol −1 and low growth (4.0–6.0 × 10 5 cfu ml −1) at higher salt concentrations of 20 mmol −1. The plant root and shoot dry weight, chlorophyll content, plant height, root length, total nitrogen, symbiotic efficient and efficient rate were affected by salt stress in tested plant and both inoculations. Total nitrogen content decreased significantly at the highest level of salinity. It is important to decrease the soil salinity and to improve the yield of dry bean ( P. vulgaris L.) as well as the Rhizobium sp. associated with it.

Different and new Nod factors produced byRhizobium tropiciCIAT899 following Na+stress

The root nodule bacterium Rhizobium tropici strain CIAT899 is highly stress resistant. It grows under acid conditions, in large amounts of salt, and at high osmotic pressure. An earlier study reported a substantial qualitative and quantitative effect of acid stress on the biosynthesis of Nod factors. The aim of the present work was to investigate the effect of high salt (NaCl) concentrations, another common stress factor, on Nod factor production. For this purpose, thin-layer chromatography, HPLC and MS analyses were carried out. The expression of nodulation genes was also studied using a nodP:lacZ fusion. High concentrations of sodium enhanced nod gene expression and Nod factor biosynthesis. The effect is sodium specific because high potassium or chloride concentrations did not have this effect. Under salt stress conditions, 46 different Nod factors were identified in a CIAT899 culture, compared with 29 different Nod factors under control conditions. Only 15 Nod factor structures were common to both conditions. Under salt stress conditions, 14 different new Nod factor structures were identified that were not observed as being produced under neutral or acid conditions. The implications of our results are that stress has a great influence on Nod factor biosynthesis and that new, very interesting regulatory mechanisms, worth investigating, are involved in controlling Nod factor biosynthesis.

Sobrevivência e nodulação do Rhizobium tropici em sementes de feijão tratadas com fungicidas

O objetivo deste trabalho foi avaliar a sobrevivência e a capacidade de nodulação do Rhizobium tropici em sementes de feijão tratadas com fungicidas. O estudo foi realizado em laboratório e em casa de vegetação. Sementes de feijão (Phaseolus vulgaris) foram tratadas com seis diferentes fungicidas e inoculadas com Rhizobium tropici estirpe CIAT 899. A sobrevivência do Rhizobium nas sementes foi avaliada no tempo 0 e 24 horas após o tratamento com fungicidas e a inoculação, utilizando o método do número mais provável (NMP). A nodulação do feijão, em plantio realizado no tempo 0 e 24 horas após o tratamento com fungicidas, e a inoculação foram avaliadas em solo com população estabelecida de Rhizobium nativo aos trinta dias após a emergência das plantas. A sobrevivência do Rhizobium inoculado nas sementes foi prejudicada pela aplicação dos fungicidas avaliados. A nodulação do feijoeiro foi reduzida pela aplicação dos fungicidas, quando o plantio foi realizado 24 horas após o tratamento das sementes. Entretanto, foram encontrados nódulos em todos os tratamentos, evidenciando que houve nodulação do Rhizobium nativo do solo.

Improved survival of nutrient-starved cells of Rhizobium tropici CIAT899 in acid soil associated with high Al3+ and Mn2+ contents

The effect of nutrient starvation in Rhizobium tropici strain CIAT899 on stress resistance to Al3+ and Mn2+ and on survival in acid soil associated with high Al3+ and Mn2+ contents was examined. In the liquid culture medium, after starvation by the exhaustion of carbon, nitrogen, or phosphorus nutrients, CIAT899 survived at least 5 months with a slight loss of viability, indicating its ability to survive under starvation conditions. Upon re-suspension into a low phosphorus medium containing a high concentration of Al3+ (50 µM) or Mn2+ (500 µM), the carbon-starved cells showed an increased tolerance to Al3+ and Mn2+ compared with the non-starved cells. When inoculated to acid soils associated with high Al3+ and Mn2+ contents, the carbon-starved cells of CIAT899 showed an enhanced survival ability compared with the non-starved cells. These results suggest the advantage of the use of carbon-starved cells of Rhizobium as an inoculum to leguminous plants, especially in acid soil associated with high Al3+ and Mn2+ contents, in order to increase the nodulation by the inoculated strain through enhanced survival in soil.

Identification of a system that allows a Rhizobium tropici dctA mutant to grow on succinate, but not on other C4-dicarboxylates.

A defined insertion mutant of a gene encoding a homolog of the rhizobial C4-dicarboxylate permease (dctA) was constructed in Rhizobium tropici strain CIAT899. This mutant (GA1) was unable to grow on fumarate or malate; however, in contrast with other rhizobial dctA mutants, it retained a limited ability to grow on succinate with ammonia as a nitrogen source. Our results suggest the presence of a novel succinate-specific transport system in R. tropici. Biochemical characterization indicated that this alternative transport system in GAI is active and dependent on an energized membrane. It was also induced by succinate and aspartate, and was repressed by glucose and glycerol. Bean plants inoculated with GA1 showed a reduced nitrogen-fixing ability, achieving only 29% of the acetylene reduction activity determined in CIAT899 strain nodules, 33 days after inoculation. Also, bean plants inoculated with GA1 had reduced shoot dry weight compared with plants inoculated with the wild-type strain.

Combining ability for nodulation in common bean (Phaseolus vulgaris L.) genotypes from Andean and Middle American gene pools

Ten F1's obtained from crosses among five common bean genotypes of Andean (WAF 15, Mineiro Precoce and Batatinha) and Middle American (BAT 304 and Ouro) gene pools were assessed for their combining abilities for root nodulation with Rhizobium tropici strain CIAT 899. The plants were grown under controlled conditions and evaluated for number of nodules per plant (NN), nodule dry weight (NDW), mean nodule weight (MNW) and plant fresh weight (PFW). The subdivision of the treatment effects on the general (GCA) and specific combining effects (SCA) were performed according to Griffing's diallel analysis method 2, model 1. The analyses of variance and estimates of quadratic components showed that non-additive gene effects were more important in the expression of NN and PFW, whereas additive gene effect was predominant for NDW and MNW. A close association was observed between high number of nodules and GCA. Generally, crosses involving parents of different gene pools yielded hybrids with high positive estimates of SCA for all assessed traits. The genotypes of Andean origin WAF 15 and Mineiro Precoce are the most promising parents for breeding programs to increase NN and NDW in common beans.

A guaB mutant strain of Rhizobium tropici CIAT899 pleiotropically defective in thermal tolerance and symbiosis.

Rhizobium tropici strain CIAT899 displays a high intrinsic thermal tolerance, and had been used in this work to study the molecular basis of bacterial responses to high temperature. We generated a collection of R. tropici CIAT899 mutants affected in thermal tolerance using TnS-luxAB mutagenesis and described the characterization of a mutant strain, CIAT899-10T, that fails to grow under conditions of high temperature. Strain CIAT899-10T carries a single transposon insertion in a gene showing a high degree of similarity with the guaB gene of Escherichia coli and other organisms, encoding the enzyme inosine monophosphate dehydrogenase. The guaB strain CIAT899-10T does not require guanine for growth due to an alternative pathway via xanthine dehydrogenase and, phenotypically, in addition to the thermal sensitivity, the mutant is also defective in symbiosis with beans, forming nodules that lack rhizobial content. Guanine and its precursors restore wild-type tolerance to grow at high temperature. Our data show that, in R. tropici, the production of guanine via inosine monophosphate dehydrogenase is essential for growth at extreme temperatures and for effective nodulation.