Effect of Drought on the Growth and Survival of the Stress-Tolerant Bacterium <Emphasis Type="Italic">Rhizobium</Emphasis> sp. NBRI2505 <Emphasis Type="Italic">sesbania</Emphasis> and Its Drought-Sensitive Transposon Tn<Emphasis Type="Italic">5</Emphasis> Mutant

Studies were conducted to elucidate the nature of drought tolerance in the bacterium Rhizobium sp. NBRI2505 sesbania and its transposon Tn5 induced mutant to assess the role of salt, pH, and temperature stresses in contributing to drought tolerance, and to correlate drought tolerance and symbiotic effectiveness. Rhizobium sp. NBRI2505 sesbania tolerated yeast extract mannitol broth (YEB) containing 28% salt (NaCl; wt/vol) for up to 18 h of incubation at 30°C, survived a 2-h incubation in YEB at 65°C, and when subjected to drought stress, tolerated YEB containing 45% polyethylene glycol 6000 (PEG; wt/vol) for up to 5 days of incubation at 30°C. One drought-sensitive mutant Rhizobium sp. NBRI2505 sesbania T112 (T112) containing a single Tn5 insertion was selected after screening about 10,000 clones. T112 was specifically defective in its tolerance for drought: when subjected to drought stress, it tolerated YEB containing 45% PEG for up to 2 days of incubation at 30°C. T122 mutant was also more sensitive to the heat and desiccation stresses, compared with Rhizobium sp. NBRI2505 sesbania in the presence of 45% PEG. Our results demonstrated a positive effect of calcium on the survival of Rhizobium sp. sesbania under acidic stress conditions. The observed enhanced survival at pH 3 of Rhizobium sp. NBRI2505 sesbania and T112 in the presence of 5% CaCO₃ suggests the requirement of calcium for growth and survival, which may have an ecological significance in acidic soils. Mutant strain T112 produced ineffective symbiosis with the plant host in the presence of 2.5 and 5% PEG, indicating that drought tolerance is required for effective symbiosis. Received: 11 January 2002 / Accepted: 18 February 2002     

Effects of Salt and pH Stress on Temperature-Tolerant Rhizobium sp. NBRI330 Nodulating Prosopis juliflora

A study was conducted to examine the growth response of a rhizobial strain Rhizobium sp. NBRI330 isolated from root nodules of Prosopis juliflora growing in alkaline soil. The strain had the ability to nodulate P. juliflora. Nursery grown plants inoculated with Rhizobium sp. NBRI330 had 60.6% higher plant dry weight, as compared with uninoculated plants. The individual stress survival limit of a rhizobial strain Rhizobium sp. NBRI330 isolated from alkaline soil in a medium containing 32% (wt/vol) salt was 8 h, and at 55°C up to 3 h. The length of Rhizobium sp. NBRI330 in salt-stressed cells increased significantly to 3.04 μm from 1.75 μm of non-stressed control cells. On the contrary, the length of pH-stressed cells declined to 1.40 μm. Compared with non-stressed control rod-shaped cells, the shape of temperature-stressed cells changed to spherical, of 0.42 μm diameter. High temperature (45°C) was tolerated efficiently by Rhizobium sp. NBRI330 in the presence of salt at pH 12, as compared with pH 7. Received: 13 September 1999 / Accepted: 14 October 1999     

Factors affecting growth of Bdellovibrio on Rhizobium

The extent of decline in the population density of Rhizobium sp. exposed to Bdellovibrio was markedly reduced in the presence of montmorillonite, kaolinite or vermiculite but not by a soil clay fraction. Increasing levels of montmorillonite reduced the numbers of vibrios that appeared in a two-membered culture and allowed for greater survival of the rhizobia. Bdellovibrio and not Rhizobium sp. was retained when mixed with the three clay minerals, but no appreciable retention was evident with the soil clay fraction. Suspensions of colloidal soil organic matter protected the hosts from parasitism, although aqueous extracts of soil did not affect the relationship. Cells from old Rhizobium sp. cultures were attacked only after a lag phase, but rhizobia that had been stored were more rapidly lysed than cells tested immediately after removal from the growth medium. The possible significance of these findings to the survival of rhizobia in soils containing Bdellovibrio is discussed.     

Occurrence of Salt, pH, and Temperature-tolerant, Phosphate-solubilizing Bacteria in Alkaline Soils

An ecological survey was conducted to characterize 4800 bacterial strains isolated from the root-free soil, rhizosphere, and rhizoplane of Prosopis juliflora growing in alkaline soils. Of the 4800 bacteria, 857 strains were able to solubilize phosphate on plates. The incidence of phosphate-solubilizing bacteria (PSB) in the rhizoplane was highest, followed by rhizosphere and root-free soil. Eighteen bacterial strains out of 857 PSB were able to produce halo at 30°C in a plate assay in the presence of 5% salt (NaCl) and solubilize tricalcium phosphate in National Botanical Research Institute's phosphate growth medium (NBRIP) broth, in the presence of various salts, pHs, and temperatures. Among the various bacteria tested, NBRI4 and NBRI7 did not produced halo in a plate assay at 30°C in the absence of salt. Contrary to indirect measurement of phosphate solubilization by plate assay, the direct measurement of phosphate solubilization in NBRIP broth assay always resulted in reliable results. The phosphate solubilization ability of NBRI4 was higher than in the control in the presence of salts (NaCl, CaCl₂, and KCl) at 30°C. Phosphate solubilization further increased in the presence of salts at 37°C as compared with 30°C. At 37°C, CaCl₂ reduced phosphate solubilization ability of NBRI4 compared with the control. The results indicated the role of calcium salt in the phosphate solubilization ability of NBRI4. Received: 9 March 1999 / Accepted: 16 April 1999     

Characterization of high temperature-tolerant rhizobia isolated from Prosopis juliflora grown in alkaline soil

A method was developed for the fast screening and selection of high-temperature tolerant rhizobial strains from root nodules of Prosopis juliflora growing in alkaline soils. The high-temperature tolerant rhizobia were selected from 2,500 Rhizobium isolates with similar growth patterns on yeast mannitol agar plates after 72 h incubation at 30 and 45 degrees C, followed by a second screening at 47.5 degrees C. Seventeen high-temperature tolerant rhizobial strains having distinguishable protein band patterns were finally selected for further screening by subjecting them to temperature stress up to 60 degrees C in yeast mannitol broth for 6 h. The high-temperature tolerant strains were NBRI12, NBRI329, NBRI330, NBRI332, and NBRI133. Using this procedure, a large number of rhizobia from root nodules of P. juliflora were screened for high-temperature tolerance. The assimilation of several carbon sources, tolerance to high pH and salt stress, and ability to nodulate P. juliflora growing in a glasshouse and nursery of the strains were studied. All five isolates had higher plant dry weight in the range of 29.9 to 88.6% in comparison with uninoculated nursery-grown plants. It was demonstrated that it is possible to screen in nature for superior rhizobia exemplified by the isolation of temperature-tolerant strains, which established effective symbiosis with nursery-grown P. juliflora. These findings indicate a correlation between strain performance under in vitro stress in pure culture and strain behavior under symbiotic conditions. Pure culture evaluation may be a useful tool in search for Rhizobium strains better suited for soil environments where high temperature, pH, and salt stress constitutes a limitation for symbiotic biological nitrogen fixation.     

Demonstrating the potential of abiotic stress-tolerant Jeotgalicoccus huakuii NBRI 13E for plant growth promotion and salt stress amelioration

The present study aimed to demonstrate the potential of abiotic stress-tolerant Jeotgalicoccus huakuii NBRI 13E for plant growth promotion and salt stress amelioration. NBRI 13E was characterized for abiotic stress tolerance and plant growth-promoting (PGP) attributes under normal and salt stress conditions. Phylogenetic comparison of NBRI 13E was carried out with known species of the same genera based on 16S rRNA gene. Plant growth promotion and rhizosphere colonization studies were determined under greenhouse conditions using maize, tomato, and okra. Field experiment was also performed to assess the ability of NBRI 13E inoculation for improving growth and yield of maize crop in alkaline soil. NBRI 13E demonstrated abiotic stress tolerance and different PGP attributes under in vitro conditions. Phylogenetic and differential physiological analysis revealed considerable differences in NBRI 13E as compared with the reported species for Jeotgalicoccus genus. NBRI 13E colonizes in the rhizosphere of the tested crops, enhances plant growth, and ameliorates salt stress in a greenhouse experiment. Modulation in defense enzymes, chlorophyll, proline, and soluble sugar content in NBRI 13E-inoculated plants leads to mitigate the deleterious effect of salt stress. Furthermore, field evaluation of NBRI 13E inoculation using maize was carried out with recommended 50 and 100% chemical fertilizer controls, which resulted in significant enhancement of all vegetative parameters and total yield as compared to respective controls. Jeotgalicoccus huakuii NBRI 13E is reported for the first time for its ability to develop a bioinoculant formulation for stress amelioration and improved crop productivity.

     

Effect of High Temperature on <Emphasis Type="Italic">Pseudomonas putida</Emphasis> NBRI0987 Biofilm Formation and Expression of Stress Sigma Factor RpoS

Pseudomonas is an efficient plant growth–promoting rhizobacteria; however, among the limiting factors for its commercialization, tolerance for high temperature is the most critical one. After screening 2,500 Pseudomnas sp. strains, a high temperature tolerant–strain Pseudomonas putida NBRI0987 was isolated from the drought-exposed rhizosphere of chickpea (Cicer arietinum L. cv. Radhey), which was grown under rain-fed conditions. P. putida NBRI0987 tolerated a temperature of 40°C for ≤ 5 days. To the best of our knowledge, this is the first report of a Pseudomnas sp. demonstrating survival estimated by counting viable cells under such a high temperature. P. putida NBRI0987 colony-forming unit (CFU)/ml on day 10 in both the absence and presence of MgSO₄.7H₂O (MgSO₄) in combination with glycerol at 40°C were 0.0 and 1.7 × 10¹¹, respectively. MgSO₄ plus glycerol also enhanced the ability of P. putida NBRI0987 to tolerate high temperatures by inducing its ability to form biofilm. However, production of alginate was not critical for biofilm formation. The present study demonstrates overexpression of stress sigma factor σ ^S (RpoS) when P. putida NBRI0987 is grown under high-temperature stress at 40°C compared with 30°C. We present evidence, albeit indirect, that the adaptation of P. putida NBRI0987 to high temperatures is a complex multilevel regulatory process in which many different genes can be involved.     

Genetic diversity of fast-growing rhizobia that nodulate soybean ( Glycine max L. Merr)

The fast-growing Rhizobium sp. strain NGR234, isolated from Papua New Guinea, and 13 strains of Sinorhizobium fredii, isolated from China and Vietnam, were fingerprinted by means of RAPD, REP, ERIC and ARDRA. ERIC, REP and RAPD markers revealed a considerable genetic diversity among fast-growing rhizobia. Chinese isolates showed higher levels of diversity than those strains isolated from Vietnam. ARDRA analysis revealed three different genotypes among fast-growing rhizobia that nodulate soybean, even though all belonged to a subcluster that included Sinorhizobium saheli and Sinorhizobium meliloti. Among S. fredii rhizobia, two strains, SMH13 and HH303, might be representatives of other species of nitrogen-fixing organisms. Although restriction analysis of the nifD–nifK intergenic DNA fragment confirmed the unique nature of Rhizobium sp. strain NGR234, several similarities between Rhizobium sp. strain NGR234 and S. fredii USDA257, the ARDRA analysis and the full sequence of the 16S rDNA confirmed that NGR234 is a S. fredii strain. In addition, ARDRA analysis and the full sequence of the 16S rDNA suggested that two strains of rhizobia might be representatives of other species of rhizobia.     

Factors effecting chitinase activity of <Emphasis Type="Italic">Rhizobium</Emphasis> sp. from <Emphasis Type="Italic">Sesbania sesban</Emphasis>

Twenty six Rhizobium strains isolated from root nodules of Sesbania sesban were studied for chitinase activity on chitin agar plates. Among them, only 12 strains showed chitinase activity. The strain showing the highest chitinase activity was selected based on maximum clear zone/colony size ratio on chitin agar plates and chitinase activity in culture filtrate. The strain was identified as Rhizobium sp. which showed a high degree of similarity with Rhizobium radiobacter (= Agrobacterium radiobacter). The cultural and nutritional conditions were optimized for maximum chitinase activity. The Rhizobium sp. exhibited maximum chitinase activity after 36 h of incubation, at neutral pH. Among the different nutritional sources, arabinose and yeast extract were found to be good inducers for chitinase activity. Rhizobium sp. could degrade and utilize dead mycelia of Aspergillus flavus, Aspergillus niger, Curvularia lunata, Fusarium oxysporum and Fusarium udum.     

Studies on fast and slow growing Rhizobium spp. nodulating Cqjanus cajan and Cicer arietinum

Fast and slow growing Rhizobium spp. isolated from Cajanus cajan and Cicer arietinum were compared in terms of colony characteristics, utilisation of carbon sources, acid production, symbiotic effectiveness and nodulating competitiveness. Fast growing isolates from C. cajan and C. arietinum formed 3–6 mm diameter colonies on yeast-extract mannitol agar after 4 days and were unlike the slow growers which produced colonies of c. 1 mm diameter after 7–10 days at 28 °C. The fast growing Rhizobium spp. from C. cajan utilised a wider range of carbon sources than the slow growing isolates from this host. Fast and slow growing strains from C. arietinum were able to utilise most of the carbon sources tested suggesting that the slow growers possessed glycolytic pathways similar to those in other fast growing species of Rhizobium. In culture, slow growing isolates from C. cajan produced a near-neutral to alkaline reaction (pH 66·7-5) whereas the fast growers from this host and both fast and slow growing isolates from C. arietinum produced an acidic reaction (pH 4·4–5·6). These data are discussed in the context of Norris' (1965) evolutionary concept of the Leguminosae. Under glassshouse conditions, fast and slow growing strains isolated from C. cajan and C. arietinum were equally effective on their respective hosts. In competition with slow growing rhizobia, half of the fast growers formed more than 70% of the nodules on C. cajan grown in sand. In all but one instance similar results were obtained when plants were grown in soil. With C. arietinum grown in sand or soil, all fast growing isolates from this host formed more than 85% of the nodules in competition with slow growing strains.     

Phenotypic and genotypic characterisation of root nodule bacteria nodulating Millettia pinnata (L.) Panigrahi, a biodiesel tree

Aims

Milletia pinnata is a leguminous tropical tree that produces seed oil suitable for biodiesel and is targeted to be planted on marginal land associated with nitrogen poor soil. This study aimed to identify effective rhizobia species for M. pinnata.

Methods

Soil samples were collected from M. pinnata grown in Kununurra, Australia. Rhizobia were trapped, characterised and sequenced for 16S rRNA, atpD, dnaK and recA genes.

Results

Forty isolates tolerated pH 7 – 9, temperatures 29 – 37 °C, salinity below 1 % NaCl, and had optimal growth on mannitol, arabinose or glutamate as a single carbon source, a few grew on sucrose and none grew on lactose. Inoculation of isolates increased shoot dry weight of M. pinnata’s seedlings in nitrogen minus media. Slow-growing isolates were closely related to Bradyrhizobium yuanmingense, Bradyrhizobium sp. DOA10, Bradyrhizobium sp. ORS305 and B. liaoningense LMG 18230^T. The fast-growing isolates related to Rhizobium sp. 8211, R. miluonense CCBAU 41251^T, R miluonense CC-B-L1, Rhizobium sp. CCBAU 51330 and Rhizobium sp. 43015

Conclusions

Millettia pinnata was effectively nodulated by slow-growing isolates related to Bradyrhizobium yuanmingense, Bradyrhizobium sp. DOA10 Bradyrhizobium sp. ORS305, B. liaoningense LMG 18230^T and fast-growing isolates related Rhizobium sp. 8211, R. miluonense, Rhizobium sp. CCBAU 51330 and Rhizobium sp. 43015     

Response of Lotus rhizobia to acidity and aluminium in liquid culture and in soil

Measurements of multiplication in liquid culture indicated that fast-growing Lotus rhizobia (Rhizobium loti) were tolerant of acidity and aluminium (at least 50 μM A1 at pH 4.5). Slow-growing Lotus rhizobia (Bradyrhizobium sp. (Lotus)) were less tolerant of acidity but equally tolerant of A1. Both genera were able to nodulateLotus pedunculatus in an acid soil (pH 4.1 in 0.01M CaCl₂) and the slow-growing strains were more effective than the fast-growing strains in this soil over 30 days.     

Use of Two-Dimensional Polyacrylamide Gel Electrophoresis to Identify and Classify Rhizobium Strains

Fifty-seven strains of various Rhizobium species were analyzed by two-dimensional gel electrophoresis. Since the protein pattern on such gels is a reflection of the genetic background of the tested strains, similarities in pattern allowed us to estimate the relatedness between these strains. All group II rhizobia (slow growing) were closely related and were very distinct from group I rhizobia (fast growing). Rhizobium meliloti strains formed a distinct group. The collection of R. leguminosarum and R. trifolii strains together formed another distinct group. Although there were some similarities within the R. phaseoli, sesbania rhizobia, and lotus rhizobia, the members within these seemed much more diverse than the members of the above groups. The technique also is useful to determine whether two unknown strains are identical.     

Complete sequence of a Rhizobium plasmid carrying genes necessary for symbiotic association with the plant host

The soil bacteria rhizobia have the capacity to establish nitrogen-fixing symbiosis with their leguminous host plants. In most Rhizobium species the genes for nodule development and nitrogen fixation have been localized on large indigenous plasmids that are transmissible, allowing lateral transfer of symbiotic functions. A recent paper reports on the complete sequencing of the symbiotic plasmid pNGR234a from Rhizobium species NGR234⁽¹⁾, revealing not only putative new symbiotic genes but also possible mechanisms for evolution and lateral dispersal of symbiotic nitrogen-fixing abilities among rhizobia.     

Ecological Indicators of Native Rhizobia in Tropical Soils

The relationship between environment and abundance of rhizobia was described by determining the populations of root nodule bacteria at 14 diverse sites on the island of Maui. Mean annual rainfall at the sites ranged from 320 to 1,875 mm, elevation from 37 to 1,650 m, and soil pH from 4.6 to 7.9. Four different soil orders were represented in this study: inceptisols, mollisols, ultisols, and an oxisol. The rhizobial populations were determined by plant infection counts of five legumes (Trifolium repens, Medicago sativa, Vicia sativa, Leucaena leucocephala, and Macroptilium atropurpureum). Populations varied from 1.1 to 4.8 log₁₀ cells per g of soil. The most frequently occurring rhizobia were Bradyrhizobium spp., which were present at 13 of 14 sites with a maximum of 4.8 log₁₀ cells per g of soil. Rhizobium trifolii and R. leguminosarum occurred only at higher elevations. The presence of a particular Rhizobium or Bradyrhizobium sp. was correlated with the occurrence of its appropriate host legume. Total rhizobial populations were significantly correlated with mean annual rainfall, legume cover and shoot biomass, soil temperature, soil pH, and phosphorus retention. Regression models are presented which describe the relationship of legume hosts, soil climate, and soil fertility on native rhizobial populations.     

Short-term survival of rhizobia on arrowleaf clover seed sown at different depths

Seed of arrowleaf clover (Trifolium vesiculosum Savi) were inoculated with a streptomycin resistant mutant ofRhizobium leguminosarum biovartrifolii and planted on the surface of a Norwood fine sandy loam and at 10 and 25 mm depths. Populations of rhizobia declined from an excess of 10,000 seed⁻¹ immediately after inoculation to less than 100 within three to four days after sowing on the soil surface when water was the peat inoculant adhesive. Gum arabic as the adhesive promoted the survival of rhizobia. Populations of rhizobia on surface sown seed declined much more rapidly than on seed buried in soil. Although, the soil was nearly air dry, rhizobia on buried seed survived at populations exceeding 1,000 seed⁻¹. The maximum soil temperatures ranged between 21 and 36°C over the sampling time and did not seem to have a major influence on short term survival of rhizobia. Delayed germination of seed due to the higher temperature would indirectly influence the number of viable rhizobia present at germination.     

Degradation of aromatic compounds byRhizobium spp.

Summary The ability of rhizobia to utilize catechol, protocatechuic acid, salicylic acid, p-hydroxybenzoic acid and catechin was investigated. The degradation pathway of p-hydroxybenzoate byRhizobium japonicum, R. phaseoli, R. leguminosarum, R. trifolii andRhizobium sp. isolated from bean was also studied.R. leguminosarum, R. phaseoli andR. trifolii metabolized p-hydroxybenzoate to protocatechuate which was cleaved by protocatechuate 3,4-dioxygenasevia ortho pathway.R. japonicum degraded p-hydroxybenzoate to catechol which was cleaved by catechol 1,2-dioxygenase.Rhizobium sp., a bean isolate, dissimilatedp-hydroxybenzoate to salicylate. Salicylate was converted to gentisic acid prior to ring cleavage. The rhizobia convertedp-hydroxybenzoate to Rothera positive substance. Catechol and protocatechuic acid were directly cleaved by the species.R. japonicum converted catechin to protocatechuic acid.     

Effects of salt on rhizobia and bradyrhizobia: a review

Rhizobia and bradyrhizobia strains vary in their tolerance to salt-stress. Rhizobium strains (fast-growers) are more salt-tolerant than strains of Bradyrhizobium (slow-growers). However, salt-tolerance in both genera is dependent upon ionic species, pH value, temperature, carbon source and the presence of osmoprotectant solutes. The harmful effect of salts on growth of both genera can be attributed to the specific ion effect rather than the osmotic effect. The salt-tolerance of different strains of rhizobia and bradyrhizobia is not related to their ecological origin. Data for salt tolerance of 684 strains of rhizobia and bradyrhizobia were collected from many reports. Most of the reports confound the effects of salt and express the concentrations of salts in percentage (%), electrical conductivity (dS m^-1), molar concentration (m ) or osmotic pressure (MPa) regardless of their differences. All the published data were compiled and recalculated from the different expressions to their equivalent molar concentration (m ) of NaCl. A suggested classification of salt-tolerance of rhizobia and bradyrhizobia from the compiled data is presented.     

Novel associations between rhizobial populations and legume species within the genera Lathyrus and O xytropis grown in the temperate region of China

Fifty rhizobial isolates of Lathyrus and Oxytropis collected from northern regions of China were studied in their genotypic characterization based upon analyses of ARDRA, 16S-23S IGS PCR-RFLP, TP-RAPD, MLEE, sequences of 16S rDNA gene and housekeeping genes of atpD, recA and glnII. The results demonstrated that most of the Lathyrus rhizobia belonged to Rhizobium and most of the Oxytropis rhizobia belonged to Sinorhizobium. A novel group of Rhizobium sp. I and S. meliloti were identified as the main microsymbionts respectively associated with Lathyrus and Oxytropis species in the collection area, which were new associations between rhizobia and the mentioned hosts. This study also provides new evidence for biogeography of rhizobia. Supported by the National Program for Basic S&T Platform Construction (Grant No. 2005DKA21201-1), the National Natural Science Foundation of China (Grant No. 30670001), and the National Basic Research Program of China (Grant No. 2006CB100206)