Strains Of Rhizobium Phaseoli Research Articles

The Cell Membrane of a Novel Rhizobium phaseoli Strain Is the Crucial Target for Aluminium Toxicity and Tolerance.

Soils with low pH and high aluminium (Al) contamination restrict common bean production, mainly due to adverse effects on rhizobia. We isolated a novel rhizobium strain, B3, from Kenyan soil which is more tolerant to Al stress than the widely used commercial strain CIAT899. B3 was resistant to 50 µM Al and recovered from 100 µM Al stress, while CIAT899 did not. Calcein labeling showed that less Al binds to the B3 membranes and less ATP and mScarlet-1 protein, a cytoplasmic marker, leaked out of B3 than CIAT899 cells in Al-containing media. Expression profiles showed that the primary targets of Al are genes involved in membrane biogenesis, metal ions binding and transport, carbohydrate, and amino acid metabolism and transport. The identified differentially expressed genes suggested that the intracellular γ-aminobutyric acid (GABA), glutathione (GSH), and amino acid levels, as well as the amount of the extracellular exopolysaccharide (EPS), might change during Al stress. Altered EPS levels could also influence biofilm formation. Therefore, these parameters were investigated in more detail. The GABA levels, extracellular EPS production, and biofilm formation increased, while GSH and amino acid level decreased. In conclusion, our comparative analysis identified genes that respond to Al stress in R. phaseoli. It appears that a large portion of the identified genes code for proteins stabilizing the plasma membrane. These genes might be helpful for future studies investigating the molecular basis of Al tolerance and the characterization of candidate rhizobial isolates that perform better in Al-contaminated soils than commercial strains.

Isolation and Characterization of High-Efficiency Rhizobia From Western Kenya Nodulating With Common Bean.

Common bean is one of the primary protein sources in third-world countries. They form nodules with nitrogen-fixing rhizobia, which have to be adapted to the local soils. Commercial rhizobial strains such as Rhizobium tropici CIAT899 are often used in agriculture. However, this strain failed to significantly increase the common bean yield in many places, including Kenya, due to the local soils’ low pH. We isolated two indigenous rhizobial strains from the nodules of common bean from two fields in Western Kenya that have never been exposed to commercial inocula. We then determined their ability to fix nitrogen in common beans, solubilize phosphorus, and produce indole acetic acid. In greenhouse experiments, common bean plants inoculated with two isolates, B3 and S2 in sterile vermiculite, performed better than those inoculated with CIAT899 or plants grown with nitrogen fertilizer alone. In contrast to CIAT899, both isolates grew in the media with pH 4.8. Furthermore, isolate B3 had higher phosphate solubilization ability and produced more indole acetic acid than the other two rhizobia. Genome analyses revealed that B3 and S2 are different strains of Rhizobium phaseoli. We recommend fieldwork studies in Kenyan soils to test the efficacy of the two isolates in the natural environment in an effort to produce inoculants specific for these soils.

Efficacy of rhizobacterial exopolysaccharides in improving plant growth, physiology, and soil properties.

The current study investigated the influence of exopolysaccharides (EPSs) producing plant growth-promoting rhizobacteria (PGPR) on the growth, physiology, and soil properties. The pre-isolated and compatible EPS producing PGPR strains were first screened based on improvement in soil aggregates in an incubation study. The screened strains (Rhizobium phaseoli strain Mn-6, Pseudomonas bathysetes strain LB5, and unidentified strain R2) were then employed in pot study for assessing improvements in maize growth, physiology, and soil properties. Eight treatments including T1 = control, T2 = Mn-6, T3 = R2, T4 = LB5, T5 = Mn-6 + R2, T6 = Mn-6 + LB5, T7 = R2 + LB5, and T8 = Mn-6 + R2 + LB5 were applied in completely randomized design (CRD) hexa replicated (half for root and half for soil, and yield attributes). The results depicted that among various treatments, the application of PGPR strain Mn-6 increased plant height, root length, root fresh and dry weight, root length density, SPAD value, leaf areas index, photosynthesis rate, transpiration, and stomatal conductance by 24, 79, 72, 90, 49, 35, 23, 21, 75, and 77%, respectively, compared with non-inoculated treatment. Similarly, significant improvement in maize yield and soil physical properties was also observed in response to the application of EPS-producing PGPR. Therefore, it is concluded that the application of EPS producing PGPR is an effective strategy to improve plant growth, physiology, yield, and soil physical properties. Moreover, EPS-producing PGPR should be exploited in field studies for their potential in improving plant growth and soil properties.

English

Common bean (Phaseolus vulgaris L.) is one of the most important and widely cultivated pulse crops in most developing countries. However, its cultivation is globally constrained mainly by low soil fertility and lack of improved agronomic practices. A field experiment was conducted at Hawassa University College of Agriculture, Hawassa Southern Ethiopia to determine the effect of N fertilization and Rhizobium phaseoli strain HB-429 inoculation on growth, nodulation, yield and yield components of common bean variety Hawassa Dume. The experiment was laid out as a randomized complete block design with three replications. Results showed significant increase in growth, nodulation, yield and yield components in plants inoculated with Rhizobium strain HB-429 over the control. Thus, from the results of this study, it can be concluded that Rhizobium inoculation with strain HB-429 is the best performing treatment to be recommended for profitable grain yield of common bean at the Hawassa and other similar areas. Key words: Common bean, grain yield, nitrogen, nodulation, Rhizobium inoculation

Characterization of Rhizobium grahamii extrachromosomal replicons and their transfer among rhizobia

BackgroundRhizobium grahamii belongs to a new phylogenetic group of rhizobia together with Rhizobium mesoamericanum and other species. R. grahamii has a broad-host-range that includes Leucaena leucocephala and Phaseolus vulgaris, although it is a poor competitor for P. vulgaris nodulation in the presence of Rhizobium etli or Rhizobium phaseoli strains. This work analyzed the genome sequence and transfer properties of R. grahamii plasmids.ResultsGenome sequence was obtained from R. grahamii CCGE502 type strain isolated from Dalea leporina in Mexico. The CCGE502 genome comprises one chromosome and two extrachromosomal replicons (ERs), pRgrCCGE502a and pRgrCCGE502b. Additionally, a plasmid integrated in the CCGE502 chromosome was found. The genomic comparison of ERs from this group showed that gene content is more variable than average nucleotide identity (ANI). Well conserved nod and nif genes were found in R. grahamii and R. mesoamericanum with some differences. R. phaseoli Ch24-10 genes expressed in bacterial cells in roots were found to be conserved in pRgrCCGE502b. Regarding conjugative transfer we were unable to transfer the R. grahamii CCGE502 symbiotic plasmid and its megaplasmid to other rhizobial hosts but we could transfer the symbiotic plasmid to Agrobacterium tumefaciens with transfer dependent on homoserine lactones.ConclusionVariable degrees of nucleotide identity and gene content conservation were found among the different R. grahamii CCGE502 replicons in comparison to R. mesoamericanum genomes. The extrachromosomal replicons from R. grahamii were more similar to those found in phylogenetically related Rhizobium species. However, limited similarities of R. grahamii CCGE502 symbiotic plasmid and megaplasmid were observed in other more distant Rhizobium species. The set of conserved genes in R. grahamii comprises some of those that are highly expressed in R. phaseoli on plant roots, suggesting that they play an important role in root colonization.

Inducing salt tolerance in mung bean through coinoculation with rhizobia and plant-growth-promoting rhizobacteria containing 1-aminocyclopropane-1-carboxylate deaminase

Twenty-five strains of plant-growth-promoting rhizobacteria (PGPR) containing 1-aminocyclopropane-1-carboxylate (ACC) deaminase and 10 strains of rhizobia were isolated from rhizosphere soil samples and nodules of mung bean. They were screened in separate trials under salt-stressed axenic conditions. The three most effective strains of PGPR (Mk1, Pseudomonas syringae ; Mk20, Pseudomonas fluorescens ; and Mk25, Pseudomonas fluorescens biotype G) and Rhizobium phaseoli strains M1, M6, and M9 were evaluated in coinoculation for their growth-promoting activity at three salinity levels (original, 4dS·m(-1), and 6 dS·m(-1)) under axenic conditions. The results showed that salinity stress significantly reduced plant growth but inoculation with PGPR containing ACC deaminase and rhizobia enhanced plant growth, thus reducing the inhibitory effect of salinity. However, their combined application was more effective under saline conditions, and the combination Mk20× M6 was the most efficient for improving seedling growth and nodulation. The effect of high ethylene concentrations on plant growth and the performance of these strains for reducing the negative impact of saline stress was also evaluated by conducting a classical triple-response bioassay. The intensity of the classical triple response decreased owing to inoculation with these strains, with the root and shoot lengths of inoculated mung bean seedlings increasing and stem diameter decreasing, which is a typical response to the dilution in a classical triple response bioassay. Thus, coinoculation with PGPR containing ACC deaminase and Rhizobium spp. could be a useful approach for inducing salt tolerance and thus improving growth and nodulation in mung bean under salt-affected conditions.

Substrate-Dependent Auxin Production by Rhizobium phaseoli Improves the Growth and Yield of Vigna radiata L. Under Salt Stress Conditions

Rhizobium phaseoli strains were isolated from the mung bean nodules, and, the most salt tolerant and high auxin producing rhizobial isolate N20 was evaluated in the presence and absence of L-tryptophan (L-TRP) for improving growth and yield of mung bean under saline conditions in a pot experiment. Mung bean seeds were inoculated with peat-based inoculum and NP fertilizers were applied at 30-60 kg ha-1, respectively. Results revealed that imposition of salinity reduced the growth and yield of mung bean. On the contrary, separate application of L-TRP and rhizobium appeared to mitigate the adverse effects of salt stress. However, their combined application produced more pronounced effects and increased the plant height (28.2%), number of nodules plant-1 (71.4%), plant biomass (61.2%), grain yield (65.3%) and grain nitrogen concentration (22.4%) compared with untreated control. The growth promotion effect might be due to higher auxin production in the rhizosphere and improved mineral uptake that reduced adverse effects of salinity. The results imply that supplementing rhizobium inoculation with L-TRP could be a useful approach for improving growth and yield of mung bean under salt stressed conditions.

Competition for nodule occupancy on Phaseolus vulgaris by Rhizobium etli and Rhizobium tropici strains can be efficiently monitored in an ultisol during the early stages of growth using a constitutive GUS gene fusion

The GUS gene fusion (pKW107) was transferred into 4 Rhizobium phaseoli strains (KIM5s, CIAT 895, CIAT 7202, CIAT 151) and 1 Rhizobium tropici strain (CIAT 899) in order to determine the nodulation competitiveness of these strains on common bean ( Phaseolus vulgaris) in soil. DNA was fingerprinted with the different mutants to verify correct integration of the glucuronidase gene in the strains' genomes. Co-inoculation trials in growth pouches proved that the GUS + mutant strains were unaltered in their nodulation capabilities. Plant studies in a non-sterile ultisol examined the value of GUS-marked Rhizobium strains for monitoring nodule occupancy under tropical conditions. At 14 and 21 days after planting (DAP), blue nodule color allowed consistently clear and reliable differentiation between nodules formed by GUS + rhizobia and indigenous soil rhizobia. At 30 DAP, however, expression of the GUS trait was inconsistent. Older nodules formed by the GUS + rhizobia were only a faint green after incubation in detection buffer, probably because of the restriction of gusA gene expression in the construct to free-living rhizobia. Nodules formed by GUS-harboring inocula strains were differentiated from ineffective green nodules formed by native rhizobia in 30 DAP nodules in microplates by crushing nodules before adding detection buffer. Data obtained by use of the GUS gene fusion were confirmed by data derived using the ELISA technique and antibiotic resistance markers. The GUS gene fusion was found to be a powerful tool to examine competitiveness of inocula strains in a tropical soil.

Compatibility and Coselection of Vesicular-Arbuscular Mycorrhizal Fungi and Rhizobia for Tropical Legumes

AbstractThe effect of dual inoculation on three local cultivars (Miss Kelly, Portland Red, Round Red) of red kidney bean (Phaseolus vulgaris, L.) with four strains of Rhizobium phaseoli (B36, B17, T2, and CIAT652) and three species of vesicular-arbuscular mycorrhizal (VAM) fungi (Glomus pallidum, Glomus aggregatum, and Sclerocystis microcarpa) was examined in sterilized and nonsterilized soil. Symbiotic efficiency (improved plant growth response and enhanced nitrogen (N) and phosphorus (P) nutrition) was dependent on the particular combination of Rhizobium strain, VAM fungus, and cultivar of kidney bean. Whereas rhizobial strains B36 and B17 paired with G. pallidum or G. aggregatum showed increased growth response of cv. Miss Kelly and Portland Red, rhizobial strain T2 paired with any of the three VAM fungi was the best compatible pairing for the cv. Round Red. It is suggested that even though dual inoculation significantly improves the growth response of the bean, the best pairings of VAM fungus and rhiz...

Development of Root Nodules in Phaseolus vulgaris Inoculated with Rhizobium and Mycorrhizal Fungi

Bean (Phaseolus vulgaris) was inoculated at planting with vesicular arbuscular mycorrhizal fungi (VAM) and Rhizobium phaseoli strain 127K44. Plants inoculated with both symbionts formed nodules in large clusters, as opposed to the single or linear groups of nodules that formed on plants inoculated only with Rhizobium. Light microscopic examination of root and nodule sections indicated that by 5 wk after inoculation, hyphal infection of the nodule cortex was abundant. Vesicles were observed in root and nodule tissue. Structures similar to degenerating arbuscules were seen in the central region of the nodule and in the nodule cortex. Intact arbuscules typical of VAM were not observed. Hyphae were also present in the central region of the nodule, particularly in regions where senescence of bacteria-filled cells had begun.

EVALUATION OF SEVERAL GROWING MEDIA FOR USE IN NITROGEN FIXATION RESEARCH

A wide range of methodologies, ranging from Leonard jar to growth pouch have been used to investigate the nitrogen fixation process in leguminous crops. The effectiveness of most of these research methods have been questioned. Problems encountered vary from difficulty in root separation to water log conditions. This study was undertaken to determine the effect of different growing media on nodule development and harvestability. Black and Red seed coat kidney bean were surface sterilized and inoculated with the Rhizobium phaseoli strain UMR 1899. Seeds were planted in 8.5 cm diameter sterile clay pots containing the respective growing medium. These growing media were sand, Promix GM, Promix BX, and fritted clay. The black seed coat kidney bean had higher germination rate under all media for all dates recorded. Black kidney bean grown in sand and fritted clay had plant heights significantly greater than ones grown in the other media on the third harvest date. Nodule activities as measured by shoot dry weight and nodule number were significantly higher in both beans grown in fritted clay than in other media. Promix GM plants with dry weight of .45g for the black bean and .32g for the red beans were the lowest. Nodule separation from the growing media was easiest when plants were grown in sand, however, this was not significantly different from that of plants grown in fritted clay.

Comparative study of siderophore‐like activity ofRhizobium phaseoliandPseudomonas fluorescens

Abstract Two strains of Rhizobium phaseoli (CP Mex. 46 and CP Mex. 3) and five of Pseudomonas fluorescens (A3, A5, A9, A10, and A12) were compared for their ability to inhibit the growth of six phytopathogenic bacterial strains on iron‐rich and iron‐deficient media. The strains CP Mex. 46 and CP Mex. 3 inhibited five and two of the phytopathogenic strains respectively, but only on the iron‐deficient medium. Each displayed their highest inhibitory activity against Xanthomonas campestris CBP 123. Similarly, Agrobacterium tumefaciens was inhibited by the strains A3, A5, A9, and X. campestris by A10 and A12. In all these cases it was inferred that the bacterial interference was mediated by competition for iron through excretion of siderophores. In addition Corynebactererium fscians was inhibited by A3; X. campestris pv phaseoli CBP 123 by A5 and A10; CBP 140 by A5, A9, and A10; and CBP 147 by A3 and A10; however, in these particular cases other mechanisms other than competition for iron may have been involved...

EVALUATION OF THE NITROGEN FIXING CAPABILITIES OF PHASEOLUS VULGARIS LINES, DEVELOPED FOR THE CARIBBEAN BASIN

Chemically fixed nitrogen is a costly import for Caribbean Basin Countries. Increased cost of fertilizer only serves to reduce crop yields in these areas. This greenhouse research was undertaken to evaluate the N2 fixing capabilities and yield potential of several Phaseolus vulgaris lines developed for use in Caribbean Basin countries. Ten common bean lines from breeding programs at the Universities of Puerto Rico and Wisconsin and two efficient Rhizobium phaseoli strains were used for the study. Plants treated with Rhizobium UMR 1899 and UMR 1632 had significantly higher stem and leaf dry weight than the control plants. Bean lines WBR 22-34, WBR 22-50, WBR 22-55, PR9056-98B and the cultivar Coxstone showed increased dry matter accumulation over that of the control plants. Plants treated with the Rhizobium strain UMR 1899 had the highest stem and leaf dry matter accumulation. Nodulation was significantly higher when plants were treated with UMR 1632. The lines WBR 22-34 and PR 9056-98B produced more nodules than the other lines used. Pod yield as measured by number of immature pods was highest for PR 9056-98B when inoculated with Rhizobium UMR 1899.

Stability of plasmids in Rhizobium phaseoli during culture

Genes governing symbiotic nitrogen fixation in rhizobia reside on symbiotic (Sym) plasmids. Additional plasmids with unknown functions (“cryptic” plasmids) usually accompany Sym plasmids in rhizobial cells. These cryptic plasmids are assumed to have beneficial roles in the soil environment. Little is known about the maintenance of cryptic plasmids during culture in the laboratory. We have examined the stabilities of both Sym and cryptic plasmids of three Rhizobium phaseoli strains cultured for 30, 60, 90 and 120 generations. Individual plasmids were labeled with Tn5-Mob, which codes for kanamycin resistance, to provide an initial means of detecting plasmid loss. Agarose gel-electrophoresis and hybridization with Tn5 probe were then used to identify alterations or loss of resistance. Kanamycin-sensitive isolates were infrequent and were due to either losses of TN5-Mob-labeled plasmids or deletions which included Tn5-Mob DNA. The three Sym plasmids and eight cryptic plasmids studied were highly stable.

Polysaccharides production by Rhizobium phaseoli and the typing of their excreted anionic polysaccharides.

The pattern of polysaccharide production amongst strains of Rhizobium phaseoli appear very varied: some strains produce anionic exopolysaccharides (EPS) as major polysaccharides (EPS) as major polymer without any other product, but most strains exhibit greater polysaccharide diversity. Apart from EPS they excrete capsular polysaccharides (CPS) and accumulate poly-beta-hydroxybutyric acid (PHB) and/or glycogen in their cells. The latter can then be used as C-sources for further synthesis of EPS and CPS. Some strains are only very poor producers or do not produce at all. Nine strains of R. phaseoli have been analysed and shown to possess the K-36 type of polysaccharide (EPS), as do strains of R. leguminosarum (6 strains) and R. trifolii (9 strains). Three strains of R. phaseoli have been found to possess the K-87 type of polysaccharide and types K-38 and K-44 polysaccharides have only been found in their own type strains.

Polysaccharides production byRhizobium phaseoliand the typing of their excreted anionic polysaccharides

The pattern of polysaccharide production amongst strains of Rhizobium phaseoli appear very varied: some strains produce anionic exopolysaccharides (EPS) as major polysaccharides (EPS) as major polymer without any other product, but most strains exhibit greater polysaccharide diversity. Apart from EPS they excrete capsular polysaccharides (CPS) and accumulate poly-beta-hydroxybutyric acid (PHB) and/or glycogen in their cells. The latter can then be used as C-sources for further synthesis of EPS and CPS. Some strains are only very poor producers or do not produce at all. Nine strains of R. phaseoli have been analysed and shown to possess the K-36 type of polysaccharide (EPS), as do strains of R. leguminosarum (6 strains) and R. trifolii (9 strains). Three strains of R. phaseoli have been found to possess the K-87 type of polysaccharide and types K-38 and K-44 polysaccharides have only been found in their own type strains.

Competitiveness and persistence of strains ofRhizobium phaseoli introduced into a Moroccan sandy soil

Sixteen strains ofRhizobium phaseoli were isolated from the Loukkos sandy soil and were compared with four selected strains ofR. phaseoli (CIAT 676, CIAT 57, Olivia, Viking 1) for their N2-fixing ability and for their serological affinities by means of the fluorescent antibody technique. Two indigenous strains were rated as highly effective, six as moderately effective, and eight were ineffective. Viking 1 was ranked as highly effective while the other inoculum strains were moderately effective. None of the 4 inoculum strains cross-reacted with the 16 indigenous strains. The indigenous strains were grouped into only two serogroups but showed a high degree of heterogeneity in regard to the strength of the immunofluorescence response. The inoculum strains outcompeted but did not eliminate the resident population for nodule occupancy on two French bean cultivars (Royalnel and Fetiche). The competitiveness, however, differed between inoculum strains. It was influenced by the host cultivar and by the host cultivar growth stage. Viking 1 was consistent in its high competitive ability whether it was inoculated singly or in a mixture with the other strains. It showed high persistence and formed more than 50% of the nodules 1 year after it was introduced. CIAT 57, Olivia, and CIAT 676 were mediocre in their persistence.

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Tolerance of high temperature and antibiotic resistance

Forty one strains ofRhizobium phaseoli were screened for the ability to multiply at high temperatures on yeast extract-mannitol agar. Most strains were tolerant of 30°C, eight strains were tolerant of 45°C and two of 47°C although the rate of multiplication was reduced at 45–47°C. The high temperature-tolerant strains were isolated from Kenyan soils and were fast-growing. Seven of the eight strains tolerant of 45–47°C lost their infectiveness after incubation at high temperature but four strains tolerant of 40°C remained infective after incubation at that temperature.

SelectingRhizobium phaseoli strains for use with beans (Phaseolus vulgaris L.) in Kenya: Infectiveness and tolerance of acidity and aluminium

Forty strains ofRhizobium phaseoli, isolated from Kenyan soils, were tested for infectiveness on common bean (Phaseolus vulgaris L.). 28 strains were infective and a cultivar × Rhizobium interaction was observed. 48 strains were screened for tolerance of acidity and Al in liquid culture. Assessment of visible turbidity after 14 days indicated 34 strains tolerant of pH 4.5 but none tolerant of pH 3.5. No strain was tolerant of 50 μM Al at pH 5.5. Three strains were tolerant of 20 μM Al at pH 5.5 and 10 μM Al at pH 4.5. Screening on a solid medium identified strains tolerant of 20 and 50 μM Al at pH 5.5 and 4.5 which were sensitive to these treatments in liquid culture. Those strains tolerant to 20 μM Al at pH 4.5 and 5.5 in liquid culture were correctly identified on the solid medium.

Reiterated DNA sequences in Rhizobium and Agrobacterium spp.

Repeated DNA sequences are a general characteristic of eucaryotic genomes. Although several examples of DNA reiteration have been found in procaryotic organisms, only in the case of the archaebacteria Halobacterium halobium and Halobacterium volcanii [C. Sapienza and W. F. Doolittle, Nature (London) 295:384-389, 1982], has DNA reiteration been reported as a common genomic feature. The genomes of two Rhizobium phaseoli strains, one Rhizobium meliloti strain, and one Agrobacterium tumefaciens strain were analyzed for the presence of repetitive DNA. Rhizobium and Agrobacterium spp. are closely related soil bacteria that interact with plants and that belong to the taxonomical family Rhizobiaceae. Rhizobium species establish a nitrogen-fixing symbiosis in the roots of legumes, whereas Agrobacterium species is a pathogen in different plants. The four strains revealed a large number of repeated DNA sequences. The family size was usually small, from 2 to 5 elements, but some presented more than 10 elements. Rhizobium and Agrobacterium spp. contain large plasmids in addition to the chromosomes. Analysis of the two Rhizobium strains indicated that DNA reiteration is not confined to the chromosome or to some plasmids but is a property of the whole genome.