Root Nodules Of Phaseolus Vulgaris Research Articles

Screening of stress tolerant bacterial strains possessing interesting multi-plant growth promoting traits isolated from root nodules of Phaseolus vulgaris L.

Rhizobia are soil bacteria known for their ability to nodulate and fix nitrogen with legumes. Some rhizobia also show different plant growth promoting characteristics (PGP); they can therefore be used as effective biofertilizers. Phaseolus vulgaris is considered a weak nitrogen fixer; therefore, screening experiments have been designed to select stress-tolerant effective bean rhizobia that have also PGP traits. Surveys of common bean fields in Morocco lead to the isolation of 113 bacteria inhabiting root nodules. Screening tests showed that 45 isolates exhibited positive phosphate solubilization, and auxin and siderophores production as well as high salt and pH tolerance. Correspondence analysis showed that salt tolerance and phosphate solubilization were origin related. Among the screened isolates, 18 were able to renodulate common bean but only 3 were highly effective with their host plant. Interestingly, phylogenetic analysis of 16S rRNA gene showed that the nodulating isolates clustering within the Rhizobium genus were represented by only two isolates related to R. miluonense and R. yanglingense while the remaining strains belonged to the Agrobacterium genus. Our results revealed also the co-existence of several endophytic bacteria with the symbiotic strains inside the nodules. These bacteria are stress tolerant and possess potential PGP traits, which indicate that they could stimulate common bean growth and contribute to N and P plants nutrition. • 45 nodule isolates out of 113 possess PGP traits and are highly salt and pH tolerant. • 3 highly effective strains obtained among18 that renodulate common bean. • 16S rDNA sequencing revealed that nodulating strains belong to Rhizobium and Agrobacterium genera. • Stress tolerant and PGP endophytic bacteria co-exist with rhizobia in the nodules. • Good candidates for common bean biofertilisation were selected.

Rhizobium chutanense sp. nov., isolated from root nodules of Phaseolus vulgaris in China.

Two Gram-stain-negative, rod-shaped bacterial strains (C5T and C16), isolated from root nodules of Phaseolus vulgaris L. in Jiangxi Province, PR China, were characterized by using a polyphasic taxonomical approach. The phylogenetic analysis of the 16S rRNA gene and three concatenated housekeeping genes (recA-glnII-atpD) revealed that C5T and C16 were members of the genus Rhizobium, yet were distinct from known species. The case for strain C5T representing a novel species was supported by genomic results. Pairwise digital DNA-DNA hybridization and average nucleotide identity values were much lower than the proposed and generally accepted species boundaries. The genome-based phylogenetic tree reconstructed by using the up-to-date bacterial core gene set consisting of 92 genes showed that the strains formed a monophyletic branch, further supporting this result. The symbiotic genes of nodC and nifH were identified in both strains; each could nodulate Phaseolus vulgaris and Glycine max but not Leucaena leucocephala, Pisum sativum or Medicago sativa plants. Major cellular fatty acids of C5T were summed feature 8 (C18 : 1 ω7c/C18 : 1 ω6c; 58.8 %), C18 : 1 ω7c 11-methyl (14.2 %) and C18 : 0 (8.1 %). The DNA G+C content of C5T was 61.4 mol%. Based on these genomic, chemotaxonomic and phenotypic characteristics, we propose a novel species: Rhizobium chutanense sp. nov. The type strain is C5T (=CCTCC AB 2018143T=LMG 30777T).

Transcriptome Analysis of Paraburkholderia phymatum under Nitrogen Starvation and during Symbiosis with Phaseolus Vulgaris.

Paraburkholderia phymatum belongs to the β-subclass of proteobacteria. It has recently been shown to be able to nodulate and fix nitrogen in symbiosis with several mimosoid and papilionoid legumes. In contrast to the symbiosis of legumes with α-proteobacteria, very little is known about the molecular determinants underlying the successful establishment of this mutualistic relationship with β-proteobacteria. In this study, we performed an RNA-sequencing (RNA-seq) analysis of free-living P. phymatum growing under nitrogen-replete and -limited conditions, the latter partially mimicking the situation in nitrogen-deprived soils. Among the genes upregulated under nitrogen limitation, we found genes involved in exopolysaccharides production and in motility, two traits relevant for plant root infection. Next, RNA-seq data of P. phymatum grown under free-living conditions and from symbiotic root nodules of Phaseolus vulgaris (common bean) were generated and compared. Among the genes highly upregulated during symbiosis, we identified—besides the nif gene cluster—an operon encoding a potential cytochrome o ubiquinol oxidase (Bphy_3646-49). Bean root nodules induced by a cyoB mutant strain showed reduced nitrogenase and nitrogen fixation abilities, suggesting an important role of the cytochrome for respiration inside the nodule. The analysis of mutant strains for the RNA polymerase transcription factor RpoN (σ54) and its activator NifA indicated that—similar to the situation in α-rhizobia—P. phymatum RpoN and NifA are key regulators during symbiosis with P. vulgaris.

Isolation and characterization of multi-potential Rhizobium strain ND2 and its plant growth-promoting activities under Cr(VI) stress

ABSTRACTSoil contaminated by chromium (Cr) is a major concern for sustainable agriculture. Considering this as a basis, the present study was designed to isolate Cr(VI)-reducing and plant growth-promoting bacterial strain from contaminated sampling sources. In this study, Rhizobium strain ND2 was isolated from the root nodules of Phaseolus vulgaris grown in leather industrial effluent contaminated soil. The strain ND2 exhibited strong resistance to different heavy metals and reduced 30 and 50 µg ml−1 concentrations of Cr(VI) completely after 80 and 120 h of incubation, respectively, as well as chromium adsorption and immobilization were confirmed by scanning electron microscopic equipped with energy X-ray spectroscopy. In addition, the strain produced 21.73 and 36.86 µg ml−1 of indole-3-acetic acid at 50 and 100 µg ml−1 of L-tryptophan supplimentations, respectively. Strain ND2 positively affected the exo-polysaccharide, ammonia, protease and catalase production and stimulated root length of various test crops under Cr(VI) stress. Moreover, Rhizobium strain ND2 has the potential to colonize the diverse agricultural crops. Thus, the present findings strongly suggested that the multipotential properties of ND2 could be exploited for bioremediation of contaminated sites with Cr(VI) as well as potential bio fertilizer for enhancing the agricultural productivity.

Homospermidine synthase contributes to salt tolerance in free-living Rhizobium tropici and in symbiosis with Phaseolus vulgaris

Background and aims Homospermidine is known to be the most abundant polyamine in root nodules of Phaseolus vulgaris induced by Rhizobium tropici. In addition, homospermidine is involved in the stress tolerance of fast-growing rhizobia and in the bacteroid protection from environmental changes. For that reason, in this work, we have investigated the role of the rhizobial homospermidine synthase in the response of P. vulgaris root nodules to salinity.

Rhizobium acidisoli sp. nov., isolated from root nodules of Phaseolus vulgaris in acid soils.

Two Gram-negative, aerobic, non-motile, rod-shaped bacterial strains, FH13T and FH23, representing a novel group of Rhizobium isolated from root nodules of Phaseolus vulgaris in Mexico, were studied by a polyphasic analysis. Phylogeny of 16S rRNA gene sequences revealed them to be members of the genus Rhizobium related most closely to 'Rhizobium anhuiense' CCBAU 23252 (99.7 % similarity), Rhizobium leguminosarum USDA 2370T (98.6 %), and Rhizobium sophorae CCBAU 03386T and others ( ≤ 98.3 %). In sequence analyses of the housekeeping genes recA, glnII and atpD, both strains formed a subclade distinct from all defined species of the genus Rhizobium at sequence similarities of 82.3-94.0 %, demonstrating that they represented a novel genomic species in the genus Rhizobium. Mean levels of DNA-DNA relatedness between the reference strain FH13T and the type strains of related species varied between 13.0 ± 2.0 and 52.1 ± 1.2 %. The DNA G+C content of strain FH13T was 63.5 mol% (Tm). The major cellular fatty acids were 16 : 0, 17 : 0 anteiso, 18 : 0, summed feature 2 (12 : 0 aldehyde/unknown 10.928) and summed feature 8 (18 : 1ω7c). The fatty acid 17 : 1ω5c was unique for this strain. Some phenotypic features, such as failure to utilize adonitol, l-arabinose, d-fructose and d-fucose, and ability to utilize d-galacturonic acid and itaconic acid as carbon source, could also be used to distinguish strain FH13T from the type strains of related species. Based upon these results, a novel species, Rhizobium acidisoli sp. nov., is proposed, with FH13T ( = CCBAU 101094T = HAMBI 3626T = LMG 28672T) as the type strain.

Inoculation of Phaseolus vulgaris with the nodule-endophyte Agrobacterium sp. 10C2 affects richness and structure of rhizosphere bacterial communities and enhances nodulation and growth.

Agrobacterium sp. 10C2 is a nonpathogenic and non-symbiotic nodule-endophyte strain isolated from root nodules of Phaseolus vulgaris. The effect of this strain on nodulation, plant growth and rhizosphere bacterial communities of P. vulgaris is investigated under seminatural conditions. Inoculation with strain 10C2 induced an increase in nodule number (+54%) and plant biomass (+16%). Grains also showed a significant increase in phosphorus (+53%), polyphenols (+217%), flavonoids (+62%) and total antioxidant capacity (+82%). The effect of strain 10C2 on bacterial communities was monitored using terminal restriction fragment length polymorphism of PCR-amplified 16S rRNA genes. When the initial soil was inoculated with strain 10C2 and left 15days, the Agrobacterium strain did not affect TRF richness but changed structure. When common bean was sown in these soils and cultivated during 75days, both TRF richness and structure were affected by strain 10C2. TRF richness increased in the rhizosphere soil, while it decreased in the bulk soil (root free). The taxonomic assignation of TRFs induced by strain 10C2 in the bean rhizosphere revealed the presence of four phyla (Firmicutes, Actinobacteria, Bacteroidetes and Proteobacteria) with a relative preponderance of Firmicutes, represented mainly by Bacillus species. Some of these taxa (i.e., Bacillus licheniformis, Bacillus pumilus, Bacillus senegalensis, Bacillus subtilis, Bacillus firmus and Paenibacillus koreensis) are particularly known for their plant growth-promoting potentialities. These results suggest that the beneficial effects of strain 10C2 observed on plant growth and grain quality are explained at least in part by the indirect effect through the promotion of beneficial microorganisms.

Occurrence of polyamines in root nodules of Phaseolus vulgaris in symbiosis with Rhizobium tropici in response to salt stress

Polyamines (PAs) are low molecular weight aliphatic compounds that have been shown to be an important part of plant responses to salt stress. For that reason in this work we have investigated the involvement of PAs in the response to salt stress in root nodules of Phaseolus vulgaris in symbiosis with Rhizobium tropici. The level and variety of PAs was higher in nodules, compared to leaves and roots, and in addition to the common PAs (putrescine, spermidine and spermine) we found homospermidine (Homspd) as the most abundant polyamine in nodules. UPLC-mass spectrometry analysis revealed the presence of 4-aminobutylcadaverine (4-ABcad), only described in nodules of Vigna angularis before. Indeed, the analysis of different nodular fractions revealed higher level of 4-ABcad, as well as Homspd, in bacteroids which indicate the production of these PAs by the bacteria in symbiosis. The genes involved in PAs biosynthesis in nodules displayed an induction under salt stress conditions which was not consistent with the decline of free PAs levels, probably due to the nitrogen limitations provoked by the nitrogenase activity depletion and/or the conversion of free PAs to theirs soluble conjugated forms, that seems to be one of the mechanisms involved in the regulation of PAs levels. On the contrary, cadaverine (Cad) and 4-ABcad concentrations augmented by the salinity, which might be due to their involvement in the response of bacteroids to hyper-osmotic conditions. In conclusion, the results shown in this work suggest the alteration of the bacteroidal metabolism towards the production of uncommon PAs such as 4-ABcad in the response to salt stress in legume root nodules.

Metabolic responses in root nodules of Phaseolus vulgaris and Vicia sativa exposed to the imazamox herbicide

Alterations on growth, amino acids metabolism and some antioxidant enzyme activities as result of imazamox treatment were examined in determinate and indeterminate nodules, formed by Phaseolus vulgaris and Vicia sativa, respectively. Young seedlings of both legumes were inoculated with their respective microsymbionts and grown under controlled conditions. At vegetative growth, plants were treated with imazamox (250μM) in the nutrient solution and harvested 7days after. Imazamox was mainly accumulated in V. sativa where concentrations were more than six fold higher than those detected in P. vulgaris. Nodule dry weight and total nitrogen content were reduced by the herbicide treatment: the highest decrease of nodule biomass (50%) and nitrogen content (40%) were registered in V. sativa and P. vulgaris, respectively. The concentration of branched-chain amino acids (BCAA) did not change in neither determinate nor indeterminate nodules even though the acetohydroxyacid synthase activity decreased in root and nodules of both symbioses with the herbicide application. Based on this last result and taking into account that total free amino acids increased in roots but not in nodules of common vetch, a possible BCAA translocation from root to nodule could occur. Our results suggest that the maintenance of BCAA balance in nodule become a priority for the plant in such conditions. The involvement of activities glutathione-S-transferase, guaiacol peroxidase and superoxide dismutase in the response of the symbioses to imazamox are also discussed.

Rhizobium azibense sp. nov., a nitrogen fixing bacterium isolated from root-nodules of Phaseolus vulgaris

Three microbial strains isolated from common beans, 23C2T (Tunisia), Gr42 (Spain) and IE4868 (Mexico), which have been identified previously as representing a genomic group closely related to Rhizobium gallicum, are further studied here. Their 16S rRNA genes showed 98.5-99% similarity with Rhizobium loessense CCBAU 7190BT, R. gallicum R602spT, Rhizobium mongolense USDA 1844T and Rhizobium yanglingense CCBAU 71623T. Phylogenetic analysis based on recA, atpD, dnaK and thrC sequences showed that the novel strains were closely related and could be distinguished from the four type strains of the closely related species. Strains 23C2T, Gr42 and IE4868 could be also differentiated from their closest phylogenetic neighbours by their phenotypic and physiological properties and their fatty acid contents. All three strains harboured symbiotic genes specific to biovar gallicum. Levels of DNA-DNA relatedness between strain 23C2T and the type strains of R. loessense, R. mongolense, R. gallicum and R. yanglingense ranged from 58.1 to 61.5%. The DNA G+C content of the genomic DNA of strain 23C2T was 59.52%. On the basis of these data, strains 23C2T, Gr42 and IE4868 were considered to represent a novel species of the genus Rhizobium for which the name Rhizobium azibense is proposed. Strain 23C2T (=CCBAU 101087T=HAMBI3541T) was designated as the type strain.

Fontibacillus phaseoli sp. nov. isolated from Phaseolus vulgaris nodules

A bacterial strain, designated BAPVE7BT, was isolated from root nodules of Phaseolus vulgaris in Spain. Phylogenetic analysis based on its 16S rRNA gene sequence placed the isolate into the genus Fontibacillus with Fontibacillus panacisegetis KCTC 13564T its closest relative with 97.1 % identity. The isolate was observed to be a Gram-positive, motile and sporulating rod. The catalase test was negative and oxidase was weak. The strain was found to reduce nitrate to nitrite and to produce β-galactosidase but the production of gelatinase, caseinase, urease, arginine dehydrolase, ornithine or lysine decarboxylase was negative. Acetoin production and aesculin hydrolysis were found to be positive. Growth was observed to be supported by many carbohydrates and organic acids as carbon source. MK-7 was identified as the predominant menaquinone and the major fatty acid (43.7 %) as anteiso-C15:0, as occurs in the other species of the genus Fontibacillus. Strain BAPVE7BT displayed a complex lipid profile consisting of diphosphatidylglycerol, phosphatidylglycerol, four glycolipids, four phospholipids, two lipids, two aminolipids and an aminophospholipid. Mesodiaminopimelic acid was detected in the peptidoglycan. The G+C content was determined to be 45.6 mol% (Tm). Phylogenetic, chemotaxonomic and phenotypic analyses showed that strain BAPVE7BT should be considered a new species of genus Fontibacillus, for which the name Fontibacillus phaseoli sp. nov. is proposed (type strain, LMG 27589T, CECT 8333T).

Oxidative stress in the root nodules of Phaseolus vulgaris is induced under conditions of phosphorus deficiency

One of the most adverse effects of phosphorus (P) deficiency on N2-fixing legumes is the generation of harmful active oxygen species which cause oxidative stress. And although oxidative stress has been widely studied in roots and shoots of various plant species, it has not yet sufficiently been documented in bean nodules so far. In this study, two recombinant inbred lines RIL115 (P-deficiency tolerant) and RIL147 (P-deficiency sensitive) of common bean and Concesa (local variety) were inoculated separately with the reference strain R. tropici CIAT899, RhM11 (R. gallicum) or RhM14 (R. tropici); two local strains of the Marrakesh region of Morocco. Nodulated plants were grown under semi-hydroponic conditions with sufficient or deficient P supply and analyzed for their oxidative responses at the flowering stage. The results indicated that P-deficiency decreased the growth of shoots (48 %) and nodules (32 %), particularly with RhM14 exhibiting the highest decrease (52 %) of nodulation. This constraint increased electrolyte leakage of nodules (40 %) as compared to leaves (20 %), especially for plants inoculated with RhM14 and CIAT899. Moreover, high H2O2 and malondialdehyde contents were noticed in P-deficient nodules of RhM14 and RhM11. These variations were associated with peroxidase activity stimulation in P-deficient nodules induced by CIAT899 and RhM14. In symbiosis with RIL115, these last strains exhibited the highest nodule phenol content. Overall, phenol content was mainly enhanced in P-deficient nodules (35 %) as compared to the leaves (16 %). It was concluded that the genotypes inoculated with CIAT899 and RhM11 are relatively P-deficiency tolerant combinations as compared to those inoculated with RhM14. Increase of oxidative stress in nodules rather than in leaves points to the need for further investigations of mechanisms that improve the root-nodule efficiency under adverse conditions.

Inoculation of Phaseolus vulgaris, Medicago laciniata and Medicago polymorpha with Agrobacterium sp. strain 10C2 may enhance nodulation and shoot dry weight but does not affect host range specificity

The aim of this work was to assess the effect of inoculation with the non-symbiotic Agrobacterium strain 10C2, previously isolated from root nodules of Phaseolus vulgaris, on nodulation, effectiveness and host range specificity. Two rhizobial strains, Ensifer meliloti 2011 and Ensifer medicae A321, were used in this study on the basis of their differential sensitivity to the in vitro antagonistic activity exercised by Agrobacterium sp. 10C2. Three host legumes, P. vulgaris, Medicago laciniata and M. polymorpha, were also selected for this experiment on the basis of their variable symbiotic specificity towards strains 2011 and A321. Cross inoculation experiments were conducted with or without co-inoculation with Agrobacterium sp. 10C2. Results showed that both rhizobial strains induced ineffective nodules on P. vulgaris. Nevertheless, inoculation with Agrobacterium sp. 10C2 slightly increased shoot dry weight with both strains and enhanced nodule number with strain 2011 only. Inoculation of M. polymorpha with strain 10C2 significantly increased the nodule number induced by E. medicae A321 but did not affect biomass production. However, inoculation of M. laciniata by strain 10C2 enhanced nodule number and shoot dry weight with strain 2011 only. Agrobacterium sp. 10C2 did not affect the non-inoculated control, nor the contrasted host range of both rhizobial strains towards M. polymorpha and M. laciniata. The potential negative effect of the in vitro antagonistic activity of strain 10C2 on reduction of nodule number or symbiotic effectiveness was not proved. No evidence of symbiotic gene transfer from the rhizobial strains to Agrobacterium sp. 10C2 was also proved. The effect of strain 10C2 is probably mediated by nodules since no result was observed in the non-inoculated control or when the rhizobial strain failed to induce nodules. It would be interesting to investigate the implications on nodule functioning, mainly the impact on nitrogenase activity, antioxidant enzymes and nodule senescence.

Genetic diversity and symbiotic effectiveness of rhizobia isolated from root nodules of Phaseolus vulgaris L. grown in soils of Iran

The common bean ( Phaseolus vulgaris L.) occupies more than 120,000 ha in Iran but information on the genetic structure and nitrogen fixation potential of the rhizobia nodulating this legume crop is scarce. Fifty-three bacterial isolates were sampled from root nodules of common bean cultivated in 12 different provinces of Iran. The genetic diversity of the isolates was analysed by PCR–RFLP of the 16S–23S rDNA internally transcribed spacer (ITS) region. At the similarity of 64%, common bean rhizobia were clustered into 10 groups, demonstrating a high degree of genetic variability among the isolates. Significant differences existed in the symbiotic potential of the isolates examined under greenhouse conditions. Ten isolates showed promising symbiotic characteristics in terms of shoot dry weight and symbiotic efficiency, and selected isolates revealed increased plant growth and yield under field conditions.

Systems biology of bacterial nitrogen fixation: High-throughput technology and its integrative description with constraint-based modeling

BackgroundBacterial nitrogen fixation is the biological process by which atmospheric nitrogen is uptaken by bacteroids located in plant root nodules and converted into ammonium through the enzymatic activity of nitrogenase. In practice, this biological process serves as a natural form of fertilization and its optimization has significant implications in sustainable agricultural programs. Currently, the advent of high-throughput technology supplies with valuable data that contribute to understanding the metabolic activity during bacterial nitrogen fixation. This undertaking is not trivial, and the development of computational methods useful in accomplishing an integrative, descriptive and predictive framework is a crucial issue to decoding the principles that regulated the metabolic activity of this biological process.ResultsIn this work we present a systems biology description of the metabolic activity in bacterial nitrogen fixation. This was accomplished by an integrative analysis involving high-throughput data and constraint-based modeling to characterize the metabolic activity in Rhizobium etli bacteroids located at the root nodules of Phaseolus vulgaris (bean plant). Proteome and transcriptome technologies led us to identify 415 proteins and 689 up-regulated genes that orchestrate this biological process. Taking into account these data, we: 1) extended the metabolic reconstruction reported for R. etli; 2) simulated the metabolic activity during symbiotic nitrogen fixation; and 3) evaluated the in silico results in terms of bacteria phenotype. Notably, constraint-based modeling simulated nitrogen fixation activity in such a way that 76.83% of the enzymes and 69.48% of the genes were experimentally justified. Finally, to further assess the predictive scope of the computational model, gene deletion analysis was carried out on nine metabolic enzymes. Our model concluded that an altered metabolic activity on these enzymes induced different effects in nitrogen fixation, all of these in qualitative agreement with observations made in R. etli and other Rhizobiaceas.ConclusionsIn this work we present a genome scale study of the metabolic activity in bacterial nitrogen fixation. This approach leads us to construct a computational model that serves as a guide for 1) integrating high-throughput data, 2) describing and predicting metabolic activity, and 3) designing experiments to explore the genotype-phenotype relationship in bacterial nitrogen fixation.

Rhizobium vallis sp. nov., isolated from nodules of three leguminous species

Four bacterial strains isolated from root nodules of Phaseolus vulgaris, Mimosa pudica and Indigofera spicata plants grown in the Yunnan province of China were identified as a lineage within the genus Rhizobium according to the analysis of 16S rRNA gene sequences, sharing most similarity with Rhizobium lusitanum P1-7(T) (99.1 % sequence similarity) and Rhizobium rhizogenes IAM 13570(T) (99.0 %). These strains also formed a distinctive group from the reference strains for defined species of the genus Rhizobium in a polyphasic approach, including the phylogenetic analyses of the 16S rRNA gene and housekeeping genes (recA, atpD, glnII), DNA-DNA hybridization, BOX-PCR fingerprinting, phenotypic characterization, SDS-PAGE of whole-cell proteins, and cellular fatty acid profiles. All the data obtained in this study suggested that these strains represent a novel species of the genus Rhizobium, for which the name Rhizobium vallis sp. nov. is proposed. The DNA G+C content (mol%) of this species varied between 60.9 and 61.2 (T(m)). The type strain of R. vallis sp. nov. is CCBAU 65647(T) ( = LMG 25295(T) =HAMBI 3073(T)), which has a DNA G+C content of 60.9 mol% and forms effective nodules on Phaseolus vulgaris.

Biosynthesis of compatible solutes in rhizobial strains isolated from Phaseolus vulgaris nodules in Tunisian fields

BackgroundAssociated with appropriate crop and soil management, inoculation of legumes with microbial biofertilizers can improve food legume yield and soil fertility and reduce pollution by inorganic fertilizers. Rhizospheric bacteria are subjected to osmotic stress imposed by drought and/or NaCl, two abiotic constraints frequently found in semi-arid lands. Osmostress response in bacteria involves the accumulation of small organic compounds called compatible solutes. Whereas most studies on rhizobial osmoadaptation have focussed on the model species Sinorhizobium meliloti, little is known on the osmoadaptive mechanisms used by native rhizobia, which are good sources of inoculants. In this work, we investigated the synthesis and accumulations of compatible solutes by four rhizobial strains isolated from root nodules of Phaseolus vulgaris in Tunisia, as well as by the reference strain Rhizobium tropici CIAT 899T.ResultsThe most NaCl-tolerant strain was A. tumefaciens 10c2, followed (in decreasing order) by R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3, R. etli 12a3 and R. gallicum bv. phaseoli 8a3. 13C- and 1H-NMR analyses showed that all Rhizobium strains synthesized trehalose whereas A. tumefaciens 10c2 synthesized mannosucrose. Glutamate synthesis was also observed in R. tropici CIAT 899, R. leguminosarum bv. phaseoli 31c3 and A. tumefaciens 10c2. When added as a carbon source, mannitol was also accumulated by all strains. Accumulation of trehalose in R. tropici CIAT 899 and of mannosucrose in A. tumefaciens 10c2 was osmoregulated, suggesting their involvement in osmotolerance. The phylogenetic analysis of the otsA gene, encoding the trehalose-6-phosphate synthase, suggested the existence of lateral transfer events. In vivo 13C labeling experiments together with genomic analysis led us to propose the uptake and conversion pathways of different carbon sources into trehalose. Collaterally, the β-1,2-cyclic glucan from R. tropici CIAT 899 was co-extracted with the cytoplasmic compatible solutes and its chemical structure was determined.ConclusionsThe soil bacteria analyzed in this work accumulated mainly disaccharides in response to NaCl stress. We could not find a direct correlation between the trehalose content of the rhizobial strains and their osmotolerance, suggesting that additional osmoadaptive mechanism should be operating in the most NaCl-tolerant strain R. tropici CIAT 899.

Rhizobium etli CE3 Bacteroid Lipopolysaccharides Are Structurally Similar but Not Identical to Those Produced by Cultured CE3 Bacteria

Rhizobium etli CE3 bacteroids were isolated from Phaseolus vulgaris root nodules. The lipopolysaccharide (LPS) from the bacteroids was purified and compared with the LPS from laboratory-cultured R. etli CE3 and from cultures grown in the presence of anthocyanin. Comparisons were made of the O-chain polysaccharide, the core oligosaccharide, and the lipid A. Although LPS from CE3 bacteria and bacteroids are structurally similar, it was found that bacteroid LPS had specific modifications to both the O-chain polysaccharide and lipid A portions of their LPS. Cultures grown with anthocyanin contained modifications only to the O-chain polysaccharide. The changes to the O-chain polysaccharide consisted of the addition of a single methyl group to the 2-position of a fucosyl residue in one of the five O-chain trisaccharide repeat units. This same change occurred for bacteria grown in the presence of anthocyanin. This methylation change correlated with the inability of bacteroid LPS and LPS from anthocyanin-containing cultures to bind the monoclonal antibody JIM28. The core oligosaccharide region of bacteroid LPS and from anthocyanin-grown cultures was identical to that of LPS from normal laboratory-cultured CE3. The lipid A from bacteroids consisted exclusively of a tetraacylated species compared with the presence of both tetra- and pentaacylated lipid A from laboratory cultures. Growth in the presence of anthocyanin did not affect the lipid A structure. Purified bacteroids that could resume growth were also found to be more sensitive to the cationic peptides, poly-l-lysine, polymyxin-B, and melittin.

Physiological implications of trehalase from Phaseolus vulgaris root nodules: partial purification and characterization

The purification and characterization of trehalase from common bean nodules as well as the role of this enzyme on growth, nodulation nitrogen fixation by examining the effects of the trehalase inhibitor validamycin A, was studied. Validamycin A did not affect plant and nodule mass, neither root trehalase and nitrogenase activity; however this treatment applied at the time of sowing increased nodule number about 16% and decreased nodule trehalase activity (16-fold) and the size of nodules. These results suggest that nodule trehalase activity of Phaseolus vulgaris could be involved in nodule formation and development. In addition, acid trehalase (EC 3.2.1.28) was purified from root nodules by fractionating ammonium sulfate, column chromatography on DEAE-sepharose and sephacryl S-300, and finally on native polyacrylamide gel electrophoresis. The purified homogeneous preparation of native acid trehalase exhibited a molecular mass of 42 and 45 kDa on SDS-PAGE. The enzyme has the optimum pH 3.9, K m of 0.109 mM, V max of 3630 nkat mg –1 protein and is relatively heat stable. Besides trehalose, it shows maximal activity with sucrose and maltose and, to a lesser degree melibiose, cellobiose and raffinose, and it does not hydrolyze on lactose and turanose. Acid trehalase was activated by Na +, Mn 2+, Mg 2+, Li +, Co 2+, K + and inhibited by Fe 3+, Hg + and EDTA.

Diverse bacteria isolated from root nodules of Phaseolus vulgaris and species within the genera Campylotropis and Cassia grown in China

Eighty bacterial isolates from root nodules of the leguminous plants Phaseolus vulgaris, Campylotropis spp. and Cassia spp. grown in China were classified into five groups by phenotypic analyses, SDS-PAGE of whole-cell proteins, PCR-based 16S rRNA gene restriction-fragment-length-polymorphism and sequencing. Thirty-three isolates from the three plant genera were identified as Agrobacterium tumefaciens because they are closely related to the type strain of A. tumefaciens. Fourteen isolates from P. vulgaris grown in Yunnan and Inner Mongolia were classified as R. leguminosarum bv. phaseoli based on their close relationship with the type strain in numerical taxonomy and in 16S rDNA phylogeny. Twenty-seven isolates from Campylotropis delavayi, P. vulgaris and four species of Cassia grown in the central zones of China were classified into three groups within the genus Bradyrhizobium. One of these three groups could be defined as Bradyrhizobium japonicum. Our results demonstrated that P. vulgaris and the species of Campylotropis and Cassia could form nodules with diverse rhizobia in Chinese soils, including novel lineages associated with P. vulgaris. These results also offered information about the convergent evolution between rhizobia and legumes since the rhizobial populations associated with P. vulgaris in Chinese soils were completely different from those in Mexico, the original cite of this plant. Some rhizobial species could be found in all of the three leguminous genera.