Rhizobium Leguminosarum Biovar Trifolii Research Articles

Rhizobia Isolated from the Relict Legume Vavilovia formosa Represent a Genetically Specific Group within Rhizobium leguminosarum biovar viciae.

Twenty-two rhizobia strains isolated from three distinct populations (North Ossetia, Dagestan, and Armenia) of a relict legume Vavilovia formosa were analysed to determine their position within Rhizobium leguminosarum biovar viciae (Rlv). These bacteria are described as symbionts of four plant genera Pisum, Vicia, Lathyrus, and Lens from the Fabeae tribe, of which Vavilovia is considered to be closest to its last common ancestor (LCA). In contrast to biovar viciae, bacteria from Rhizobium leguminosarum biovar trifolii (Rlt) inoculate plants from the Trifolieae tribe. Comparison of house-keeping (hkg: 16S rRNA, glnII, gltA, and dnaK) and symbiotic (sym: nodA, nodC, nodD, and nifH) genes of the symbionts of V. formosa with those of other Rlv and Rlt strains reveals a significant group separation, which was most pronounced for sym genes. A remarkable feature of the strains isolated from V. formosa was the presence of the nodX gene, which was commonly found in Rlv strains isolated from Afghanistan pea genotypes. Tube testing of different strains on nine plant species, including all genera from the Fabeae tribe, demonstrated that the strains from V. formosa nodulated the same cross inoculation group as the other Rlv strains. Comparison of nucleotide similarity in sym genes suggested that their diversification within sym-biotypes of Rlv was elicited by host plants. Contrariwise, that of hkg genes could be caused by either local adaptation to soil niches or by genetic drift. Long-term ecological isolation, genetic separation, and the ancestral position of V. formosa suggested that symbionts of V. formosa could be responsible for preserving ancestral genotypes of the Rlv biovar.

Solubility Enhancement of Atrazine by Complexation with Cyclosophoraose Isolated from Rhizobium leguminosarum biovar trifolii TA-1.

Rhizobium leguminosarum biovar trifolii TA-1, a kind of soil bacteria, produces cyclosophoraoses (Cys). Cyclosophoraoses contain various ring sizes with degrees of polymerization ranging from 17 to 23. Atrazine is a hardly-soluble herbicide that contaminates soil and drinking water, and remains in soil for a long time. To remove this insoluble contaminant from aqueous solutions, we have enhanced the solubility of atrazine by complexation with Cys. The complex formation of Cys and atrazine was confirmed using 1H nuclear magnetic resonance (NMR), Fourier transform infrared (FT-IR) spectroscopy, differential scanning calorimetry (DSC), field emission scanning electron microscopy (FE-SEM), rotating frame nuclear overhauser spectroscopy (ROESY), and molecular modeling studies. The aqueous solubility of atrazine was enhanced 3.69-fold according to the added concentrations (20 mM) of Cys, compared to the 1.78-fold enhancements by β-cyclodextrin (β-CD). Cyclosophoraoses as an excellent solubility enhancer with long glucose chains that can effectively capture insoluble materials showed a potential application of microbial polysaccharides in the removal of hazardous hardly-soluble materials from aqueous solutions in the fields of biological and environmental industry.

Molecular diversity and phylogeny of indigenous Rhizobium leguminosarum strains associated with Trifolium repens plants in Romania.

The symbiotic nitrogen fixing legumes play an essential role in sustainable agriculture. White clover (Trifolium repens L.) is one of the most valuable perennial legumes in pastures and meadows of temperate regions. Despite its great agriculture and economic importance, there is no detailed available information on phylogenetic assignation and characterization of rhizobia associated with native white clover plants in South-Eastern Europe. In the present work, the diversity of indigenous white clover rhizobia originating in 11 different natural ecosystems in North-Eastern Romania were assessed by a polyphasic approach. Initial grouping showed that, 73 rhizobial isolates, representing seven distinct phenons were distributed into 12 genotypes, indicating a wide phenotypic and genotypic diversity among the isolates. To clarify their phylogeny, 44 representative strains were used in sequence analysis of 16S rRNA gene and IGS fragments, three housekeeping genes (atpD, glnII and recA) and two symbiosis-related genes (nodA and nifH). Multilocus sequence analysis (MLSA) phylogeny based on concatenated housekeeping genes delineated the clover isolates into five putative genospecies. Despite their diverse chromosomal backgrounds, test strains shared highly similar symbiotic genes closely related to Rhizobium leguminosarum biovar trifolii. Phylogenies inferred from housekeeping genes were incongruent with those of symbiotic genes, probably due to occurrence of lateral transfer events among native strains. This is the first polyphasic taxonomic study to report on the MLSA-based phylogenetic diversity of indigenous rhizobia nodulating white clover plants grown in various soil types in South-Eastern Europe. Our results provide valuable taxonomic data on native clover rhizobia and may increase the pool of genetic material to be used as biofertilizers.

Lasting effects of biosolids in agroecosystems

Lasting effects of biosolids in agroecosystems

Genotypic Differences in Root Hair Deformation and Subsequent Nodulation for Red Clover under Different Additions of Starter N Fertilization

AbstractRed clover cultivars, including diploid and tetraploid, are commonly used in legume‐based pasture mixtures. However, information on nodulation under different starter N regimens is limited. We hypothesized that there is genetic variability among different red clover cultivars for nodulation. A root hair deformation assay was conducted using three diploid (AC Christie, Tapani and CRS15) and three tetraploid (Tempus, CRS18 and CRS39) red clover cultivars by inoculating them with Rhizobium leguminosarum biovar trifolii. Nodulation and morphological characteristics of two selected red clover cultivars, AC Christie and Tempus, were determined under five starter N concentrations (0, 0.2, 0.4, 0.8 and 1.6 mg per plant). Inoculation with rhizobia increased root hair deformation with significant interaction across cultivars. Nodulation was delayed under high starter N concentrations, and genotypic differences were evident for days‐to‐nodule initiation. There was a positive quadratic response to starter N for AC Christie and a negative quadratic response for Tempus for nodulation. Tempus had more active nodules (92 %) than AC Christie (73 %). The genetic variability of red clover cultivars should be considered in N fixation studies and their response to availability of initial N.

Inclusion complexation of naproxen with cyclosophoraoses and succinylated cyclosophoraoses in different pH environments

Cyclosophoraoses [cyclic β-(1,2)-glucan, Cys] isolated from Rhizobium leguminosarum biovar trifolii TA-1 have unique structures and high solubility, which make it a potent solubilizer for host–guest inclusion complexation. Succinylated cyclosophorasoses (S-Cys) were also synthesized by chemically modifying isolated cyclosophoraoses. In ultraviolet-visible studies using naproxen (NAP), Cys was shown to form the most stable complexes with NAP (K 1:1 = 2457.9 M−1), which was followed by the negatively charged S-Cys (K 1:1 = 357.1 M−1) at pH 3.4. A further strong reduction in the complex stability constant was observed at pH 7.5. When the reduction in the stability constant was compared with other cyclic oligosaccharides (Cys; 119.2 M−1, CD; 14.48 M−1 and HP-CD; 6.75 M−1), S-Cys (K 1:1 = 5.6 M−1) was shown to have the highest decrease in stability constant. These results suggest that the S-Cys could regulate the efficiency of inclusion complexation at external pH values. NMR studies of complex formation between NAP and Cys also showed a different correlation pattern at pH 3.4 and 7.5. This difference in correlation demonstrates that the inclusion complexes between Cys and NAP formed as a result of the differential charge distribution of the carboxyl groups of NAP. The pH-dependent inclusion behavior of Cys for NAP was also evaluated using molecular docking simulations.

Heavy metal resistant Rhizobium leguminosarum biovar trifolii isolates: characterization and use in rhizoremediation of polluted soils

Heavy metal resistant Rhizobium leguminosarum biovar trifolii isolates: characterization and use in rhizoremediation of polluted soils

Biological activity of (Lipo)polysaccharides of the exopolysaccharide-deficient mutant Rt120 derived from Rhizobium leguminosarum bv. trifolii strain TA1

Lipopolysaccharides (LPS) from Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) and its mutant Rt120 in the pssBpssA intergenic region as well as degraded polysaccharides (DPS) derived from the LPS were elucidated in terms of their chemical composition and biological activities. The polysaccharide portions were examined by methylation analysis, MALDI-TOF mass spectrometry, and (1)H NMR spectroscopy. A high molecular mass carbohydrate fraction obtained from Rt120 DPS by Sephadex G-50 gel chromatography was composed mainly of L-rhamnose, 6-deoxy-L-talose, D-galactose, and D-galacturonic acid, whereas that from RtTA1 DPS contained L-fucose, 2-acetamido-2,6-dideoxy-D-glucose, D-galacturonic acid, 3-deoxy-3-methylaminofucose, D-glucose, D-glucuronic acid, and heptose. Relative intensities of the major (1)H NMR signals for O-acetyl and N-acetyl groups were 1 : 0.8 and 1 : 1.24 in DPS of Rt120 and RtTA1, respectively. The intact mutant LPS exhibited a twice higher lethal toxicity than the wild type LPS. A higher in vivo production of TNFα and IL-6 after induction of mice with Rt120 LPS correlated with the toxicity, although the mutant LPS induced the secretion of IL-1β and IFNγ more weakly than RtTA1 LPS. A polysaccharide obtained by gel chromatography on Bio-Gel P-4 of the high molecular mass material from Rt120 had a toxic effect on tumor HeLa cells but was inactive against the normal human skin fibroblast cell line. The polysaccharide from RtTA1 was inactive against either cell line. The potent inhibitory effect of the mutant DPS on tumor HeLa cells seems to be related with the differences in sugar composition.

Occurrence of plant growth-promoting traits in clover-nodulating rhizobia strains isolated from different soils in Rio Grande do Sul state

In the last decades, the use of plant growth-promoting rhizobacteria has become an alternative to improve crop production. Rhizobium leguminosarum biovar trifolii is one of the most promising rhizobacteria and is even used with non-legume plants. This study investigated in vitro the occurrence of plant growth-promoting characteristics in several indigenous R. leguminosarum biovar trifolii isolated from soils in the State of Rio Grande do Sul, Brazil. Isolates were obtained at 11 locations and evaluated for indoleacetic acid and siderophore production and inorganic phosphate solubilization. Ten isolates were also molecularly characterized and tested for antagonism against a phytopathogenic fungus and for plant growth promotion of rice seedlings. Of a total of 252 isolates, 59 produced indoleacetic acid, 20 produced siderophores and 107 solubilized phosphate. Some degree of antagonism against Verticillium sp. was observed in all tested isolates, reducing mycelial growth in culture broth. Isolate AGR-3 stood out for increasing root length of rice seedlings, while isolate ELD-18, besides increasing root length in comparison to the uninoculated control, also increased the germination speed index, shoot length, and seedling dry weight. These results confirm the potential of some strains of R. leguminosarum biovar trifolii as plant growth-promoting rhizobacteria.

Contributo para a melhoria de solos marginais destinados a pastagens pela aplicação de lama residual urbana, sem riscos ambientais

Sewage sludge (SS) application to soils reserved for pastures, still scarcely used in the country, often contributes to improve organic matter (O.M.) and some plant nutrient contents and to reduce the erosion risk, by increasing the soil vegetation cover. However, the occasional occurrence in SS of high levels of heavy metals, organic pollutant compounds, and pathogenic organisms restrict the SS rate to apply and makes indispensable their control in the soils to which they were applied. Also, the high nitrogen concentration often present in SS can inhibit the symbiotic rhizobium activity, with sequent damage in the leguminous species survival in grassland. In this work, dry matter yield, floristic composition, and copper (Cu) concentration in the plant biomass were evaluated, in two successive years of a field experiment with a sown pasture mixture, established in a poor Haplic Luvisol in the Mertola region. A biologically treated SS from Evora, rich in Cu, was applied. The evolution in the topsoil of the O.M., some plant nutrients, and aqua regia extractable Cu concentrations, was also examined for the same period. The magnitudes of the Rhizobium population (Rhizobium leguminosarum biovar trifolii) and of some indicators of faecal contamination (coliform bacteria and enterococcus) were evaluated too. Moreover, the evolution in the soil superficial layer of 11 polychlorinated biphenyls (PCBs), 13 organ chlorine pesticides, and 16 polycyclic aromatic hydrocarbons (PAHs) concentrations was examined for the 1st year following the SS application. The experiment, with a randomised block design, had three SS application rates (L0 = 0, L1 = 12, and L2 = 24 t/ha) as treatments and two replicates. The sown mixture consisted of Italian ryegrass, cocksfoot, five clover species, bird’s foot, and biserrula. Besides much higher biomass production, induced by higher soil available phosphorus concentration, the added SS neither polluted the soil with the analysed organic compounds, one year after application, nor increased the pathogenic microbial flora in two consecutive years. However, at the highest SS rate, soil aqua regia extractable Cu (0-10 cm depth) exceeded the maximum Portuguese legislated level in soil (100 mg kg-1), the rhizobium population was also reduced at the beginning of the 1st growing season, and legume percentage decreased at the 2nd growing season. The plant Cu concentrations were much lower than the maximum tolerable levels (25 mg kg-1) for the small ruminant’s diet, strongly suggesting that the SS application will not have damaging effects on the animal nutrition. Due to the undesirable effects of the L2 application rate, the L1 rate (12 t/ha) would be recommended for fertilizer purposes.

Enhancement of nodulation by some arid climate strains of Rhizobium leguminosarum biovar trifolii using protoplast fusion

Fourteen randomly clover indigenous nodulated Rhizobium strains were isolated from different locations in Saudi Arabia. They were identified as different strains of the genus Rhizobium leguminosarum biovar trifolii and characterized for their intrinsic antibiotic resistance against a range of antibiotics, nodulation capability and plasmid profiles. Results revealed the presence of high molecular weight plasmids (megaplasmids) in all the selected strains. Based on the ability for nodulation production, two weak strains (RtI1 and RtI2) and one efficient strain (RtA1) were selected for protoplast fusion and the numbers of nodules produced by the intra-specific protoplast fusion strains were investigated. Results clearly confirmed the effective role of the protoplast fusion in enhancing both nodulation production capacity of Rhizobium species and their range of antibiotic resistance. Protoplast fusion of the local Rhizobium species resulted in 1.93- to 5.67-fold increase in nodulation number compared to their parental strains, which was considered an excellent result concerning agricultural practices, especially the formation of nitrogen-fixing root nodules on legume crop plants. Protoplast fusion also produced fusants with a wide range of antibiotic resistance, another advantage added to the new strains against environmental stresses. In conclusion, protoplast fusion proved its efficiency as a tool for constructing a second generation of Rhizobia with much better characteristics for efficient applications in arid land.

Competitiveness of a native Rhizobium leguminosarum biovar trifolii strain for nodule occupancy is manifested during infection

The stages in the nodulation process that determined the competitiveness of R. leguminosarum bv. trifolii (Rlt) strain 20–15, which proved to be highly competitive for nodulation in Iceland fields tests over several years, is analysed. White clover (Trifolium repens L.) roots were inoculated with inoculum mixtures containing three strains (Rlt 20-15, Rlt 8-9 and Rlt 32-28) in different proportions and cell densities. Competitiveness in root colonization, formation of infection threads and nodule development was assessed for Rlt 20-15 and its weakest competitor, Rlt 32-28. ERIC-polymerase chain reaction (PCR) DNA fingerprinting was used to identify inoculated strains recovered from root surfaces and individual nodules. GFP or DsRed tagged strains were used to determine identity in root hairs and nodules. Both strains colonized the root equally at all inoculum ratios tested. But, Rlt 20-15 initiated significantly more infection threads and formed more nodules than Rlt 32-28. These results show that Rlt 20-15 expresses its nodulation competitiveness during infection, either at infection thread initiation or during successive growth in the infection threads. The data presented support earlier observations that this strain competed well in the field in spite of its inferior ability to survive in the soil.

Population size of indigenous Rhizobium leguminosarum biovar trifolii in long-term field experiments with sewage sludge cake, metal-amended liquid sludge or metal salts: Effects of zinc, copper and cadmium

There is conflicting evidence, and therefore continuing concern, as to whether metals in sewage sludge are deleterious to soil microbial processes and long-term agricultural productivity. Nine field experiments with sewage sludge cakes, three with metal-amended liquid sludges and three with inorganic metal salts were set up across Britain in 1994 to give individual metal dose–response treatments to try to answer this question. This study reports on the effects of Zn, Cu and Cd on the population size of Rhizobium leguminosarum biovar trifolii , a nitrogen fixing symbiont of white clover ( Trifolium repens ), in soils from these experiments over 11 years. Significant ( P < 0.05) reductions in indigenous rhizobial numbers occurred on the Zn metal dose–response treatments at eight of the sludge cake sites in 2005, but few consistent effects were evident on the Cu or Cd metal dose–response treatments during the 11-year monitoring period. The soil total Zn concentrations where effects occurred were near to the UK statutory limit of 300 mg kg −1 for soils receiving sewage sludge. No significant reductions occurred in any treatments on the metal-amended liquid sludge or inorganic metal salt experiments in which the metals would be expected to be in a more bioavailable form, even after 11 years. The effects in the sludge cake experiments were related consistently with soil total Zn, with no recovery to date. The reductions in clover rhizobial numbers in the sludge cake experiments were due to Zn effects on free-living rhizobia in the soil, with gradual die-off over a long time with increasing soil total Zn concentrations. Currently, no consistent adverse effects on rhizobia have been seen at the UK limits for Cu and Cd of 135 and 3 mg kg −1 , respectively.

Impact of Organic Fertilizers with and without Chemical Fertilizers on Soil Chemical Properties and the Establishment of Nitrogen-Fixing Bacteria in the Rhizosphere

Effects of organic fertilizers with and without the application of chemical fertilizers for seven years as part of a wheat-pearl millet cropping sequence on soil chemical properties and the establishment of nitrogen fixing bacteria in the rhizosphere were examined. The application of farmyard manure, poultry manure, and sugarcane filter cake alone or in combination with chemical fertilizers improved the soil organic C, total N, P, and K status. Larger populations of Azotobacter chroococcum and Rhizobium leguminosarum biovar trifolii in the rhizosphere of wheat and Egyptian clover respectively, were maintained in soils receiving organic fertilizers either alone or in combination with chemical fertilizers than in soils given chemical fertilizers alone.

Host–strain mediated selection for an effective nitrogen-fixing symbiosis between Trifolium spp. and Rhizobium leguminosarum biovar trifolii

We have previously reported significant strain–host incompatibility between the microsymbiont Rhizobium leguminosarum biovar trifolii and Trifolium spp. related to geographic and phenological barriers. Additionally, we have shown that an effective symbiosis between strains of R. l. trifolii and clover was established despite the soil harbouring ineffective R. l. trifolii capable of nodulating the host. We termed this “selection” for effective symbiosis. This paper reports glasshouse-based experiments to validate and further explore this in situ selection phenomenon. The effect of cell density and strain ratio at the time of inoculation, as well as soil pH, were investigated on two hosts (Trifolium purpureum and Trifolium polymorphum) that were each exposed to one microsymbiont capable and one incapable of N2 fixation. In co-inoculation experiments at a cell density of 104 cells/mL, each host nodulated solely with its effective strain, even when this strain was outnumbered 100-fold by the ineffective strain. However, the selection process ceased when the effective strain was outnumbered 1000-fold. At higher basal cell concentrations of 105–108 cells/mL, selection for WSM1325 to form effective nodulation on T. purpureum was evident, but was significantly reduced as the ratio of ineffective cells in the inoculum increased above 4-fold. The results indicate that the selection mechanism is highly dependent on the basal rhizobial cell density. Soil pH did not significantly alter the process, and both strains grew at similar rates and formed nodules at similar rates. A preliminary investigation into the genetic backgrounds of WSM1325 and WSM2304 revealed that although their 16S rRNA sequences were identical, they have considerable differences in their symbiotic and chromosomal replicons through examination of atpD, GSII and nodD sequences.

Application of physical and genetic map of Rhizobium leguminosarum bv. trifolii TA1 to comparison of three closely related rhizobial genomes

A combined physical and genetic map of Rhizobium leguminosarum biovar trifolii TA1 (RtTA1) genome was constructed and used in comparison of chromosomal organization with the closely related R. leguminosarum bv. viciae 3841 (Rlv) and Rhizobium etli CNF42 (Rhe). This approach allowed evaluation of chromosome and genome plasticity and provided important insights into R. leguminosarum lineage diversity. MssI, SmiI, PacI, and I-CeuI restriction endonucleases were chosen for the analysis, generating fragments with suitable size distributions for RtTA1 genome mapping. The fragments were assembled into a physical map using a combination of complementary methods, including multiple and partial digests of genomic DNA, hybridization with homologous gene probes, and cross-Southern hybridization. About 100 genetic markers were located on the RtTA1 restriction map. Comparison of genetic maps of RtTA1, Rlv, and Rhe revealed extensive chromosomal colinearity despite differences in the physical maps. The comparison provides bases for comprehensive analysis of the evolution of R. leguminosarum genome, indicating that, at least on the chromosomal level, no major rearrangements had occurred after the evolutionary divergence of R. leguminosarum biovars.

Effects of Cd, Zn, or both on soil microbial biomass and activity in a clay loam soil

We investigated Cd, Zn, and Cd + Zn toxicity to soil microbial biomass and activity, and indigenous Rhizobium leguminosarum biovar trifolii, in two near neutral pH clay loam soils, under long-term arable and grassland management, in a 6-month laboratory incubation, with a view to determining the causative metal. Both soils were amended with Cd- or Zn-enriched sewage sludge, to produce soils with total Cd concentrations at four times (12 mg Cd g−1 soil), and total Zn concentrations (300 mg Zn kg−1 soil) at the EU upper permitted limit. The additive effects of Cd plus Zn at these soil concentrations were also investigated. There were no significant differences in microbial biomass C (BC), biomass ninhydrin N (BN), ATP, or microbial respiration between the different treatments. Microbial metabolic quotient (defined as qCO2 = units of CO2–C evolved unit−1 biomass C unit−1 time) also did not differ significantly between treatments. However, the microbial maintenance energy (in this study defined as qCO2-to-μ ratio value, where μ is the growth rate) indicated that more energy was required for microbial synthesis in metal-rich sludge-treated soils (especially Zn) than in control sludge-treated soils. Indigenous R.leguminosarum bv. trifolii numbers were not significantly different between untreated and sludge-treated grassland soils after 24 weeks regardless of metal or metal concentrations. However, rhizobial numbers in the arable soils treated with metal-contaminated sludges decreased significantly (P Cd > Cd + Zn.

Genetic diversity of a natural population of Rhizobium leguminosarum biovar trifolii analysed from field nodules and by a plant infection technique

Abstract We compared trapping of Rhizobium leguminosarum biovar trifolii isolates under field soil and non-soil environmental conditions. Isolates were obtained from white clover ( Trifolium repens L.) and red clover ( T . pratense L.) grown directly in the field and from plants inoculated with soil from the same site using a plant infection technique. Isolates were identified by genomic polymerase chain reaction (PCR) fingerprinting using primers derived from the enterobacterial repetitive intergenic consensus (ERIC) sequences. The isolates trapped from soil dilutions in the laboratory included a number of major ERIC types that were not found in the field trapped nodules, suggesting that sampling of clover Rhizobium strains from soil dilutions may not be representative of the field nodulating population.

Arctic and subarctic soil populations of Rhizobium leguminosarum biovar trifolii nodulating three different clover species: Characterisation by diversity at chromosomal and symbiosis loci

Indigenous soil populations of Rhizobium leguminosarum biovar trifolii from Arctic and subarctic regions have been characterised with emphasis on chromosomal and symbiotic genes. Three clover species were used to trap rhizobia from soils along a latitudinal gradient from 78°N to 60°N in Norway. For the first time R. l. bv. trifolii was isolated from Svalbard at 78°N. Under the extreme conditions in the Arctic, rhizobia have survived as saprophytes and in symbiosis with clover legumes. The chromosomal diversity of the soil populations was mapped by rep-PCR. Separation of chromosomal types were strongly influenced by geographic origin. Symbiotic genes, the nodEF and nifDK IGS gene regions, were investigated by PCR-RFLP. The nifDK IGS were more conserved than the nodEF genes. Sym plasmids were widely distributed in different chromosomal types and across the latitudinal gradient.

Symbiotic and saprophytic survival of three unmarked Rhizobium leguminosarum biovar trifolii strains introduced into the field

The symbiotic and saprophytic persistence of three unmarked Rhizobium leguminosarum biovar trifolii (Rlt) strains introduced into a field site in Iceland were followed. This site was free of clover cultivation and initially devoid of clover-nodulating rhizobia as tested by nodulation studies. Nodule occupancy by strains was identified based on their distinct ERIC-polymerase chain reaction (PCR) DNA fingerprint patterns. The survival and persistence of the individual strains in soil were monitored by the quantitative real-time PCR (qRT-PCR) assay, targeting the host-specific nodE gene. The most dominant strain in the nodule population, Rlt 20-15, showed relatively less saprophytic survival ability and maintained high numbers only in the presence of the appropriate host plant. Conversely, the minor nodule occupant, Rlt 32-28, persisted in soil at a relatively higher abundance both in the presence of its host legumes and in the presence of a non-host grass. The qRT-PCR assay was successfully applied to quantify rhizobial strains directly in soil without culturing or nodulation. However, the assay demonstrated less sensitivity compared with the plant infection most-probable-number (MPN) method for estimating the population size of rhizobia in soil. The quantitative detection limit of our qRT-PCR assays was 1 x 10(3) cells per gram of soil, as opposed to the MPN test which has a detection limit of 10 cells per gram of soil.