Rhizobium Leguminosarum Bv Trifolii Research Articles

Microencapsulation of Rhizobium leguminosarum bv. trifolii with guar gum: Preliminary approach using spray drying

Rhizobium leguminosarum bv trifolii strains TA1 and CC275e have been widely used as effective nitrogen fixing strains for white clover in New Zealand, but rhizobia survival on seeds is usually poor due to different stress conditions. The aim of this study was to select one of those commercial strains grown in a solid carrier (core) and study the influence of the core:polymer ratio in a microencapsulation process by spray drying using guar gum as coating material. First, strains TA1 and CC275e grown on peat and diatomaceous earth were exposed to temperature and desiccation stress. Both strains were stable at 40 °C and completely died after five minutes at 80 °C, while CC275e was more stable than TA1 at 60 °C. TA1 and CC275e slightly decreased viability after six hours drying with either carriers, with no differences between strains. A central composite design was used to develop the microencapsulation process. Independent variables were: inlet temperature (130 °C) and feed flow rate (5 mL/min). Microparticles presented rhizobia loading in 107 CFU/g and mean particle size between 10 and 30 μm. Optimized process reached 50% yield and 107 CFU/g loading. Rhizobia viability dropped two logarithmic units during the microencapsulation/drying process, possibly due to the negative effects of dehydration and high outlet temperature (≈70 °C), suggesting the need to continue optimizing the process by improving the thermal profile in the drying chamber.

Nodulation competitiveness as a requisite for improved rhizobial inoculants of Trifolium pratense

Red clover (Trifolium pratense L.) is used in the improvement of grasslands in Uruguay and has been inoculated with commercial strain U204 of Rhizobium leguminosarum bv trifolii since 1970s. Native-naturalized rhizobia strains present in soil are the basis for selecting and developing new inoculants. With this aim, we evaluated the diversity of red clover rhizobia in Uruguayan red clover pastures both historically inoculated with U204 and non-inoculated ones. Thirty-eight different enterobacterial repetitive intergenic consensus (ERIC) PCR genomic fingerprints were identified, albeit surprisingly only one of 80 isolates showed an ERIC profile similar to U204. Under controlled conditions, red clover plants inoculated with one of the native isolates, strain 317, produced more biomass than those inoculated with the commercial U204. ERIC-PCR was also used to show that strain 317 competed for nodulation better than U204 in a field with previous history of inoculation. Moreover, both U204 and 317 were tagged with a gusA reporter gene and their competitiveness for nodulation assessed in various soil types. Again, strain 317 appeared more competitive than U204, particularly in soils with previous history of inoculation. Our results reinforce the long-known idea of assessing the actual needs of inoculation of legumes in different soils and suggest that the indigenous isolate 317 is an effective and competitive strain that can be used for development of a new red clover inoculant.

Application of CF effect, IAR and RAPD methods in evaluation or competitiveness between inoculated and indigenous strains of Rhizobium leguminosarum bv. trifolii

The paper evaluates the competitive ability of Rhizobium leguminosarum bv trifolii strain T49B1 used as inoculant for Trifolium repens L. (Viola) and indigenous population of rhizobia from chernozem, stagnosol and humic gleysol. Nodule bacteria were identified using calcofluor effects (CF) based on succinoglycan production, and confirmed by IAR patterns and RAPD fingerprinting (using SPH1 primer). T49B1 nodules occupancy level from 71.78% in pseudoglay to 89.56% in chernozem was detected. Inoculation by T49B1 strain showed it to be a very good competitor with the possibility to establish effective symbiosis and increase protein content compared to non-inoculated (control) plants.

Complete genome sequence of Rhizobium leguminosarum bv. trifolii strain WSM1325, an effective microsymbiont of annual Mediterranean clovers.

Rhizobium leguminosarum bv trifolii is a soil-inhabiting bacterium that has the capacity to be an effective nitrogen fixing microsymbiont of a diverse range of annual Trifolium (clover) species. Strain WSM1325 is an aerobic, motile, non-spore forming, Gram-negative rod isolated from root nodules collected in 1993 from the Greek Island of Serifos. WSM1325 is produced commercially in Australia as an inoculant for a broad range of annual clovers of Mediterranean origin due to its superior attributes of saprophytic competence, nitrogen fixation and acid-tolerance. Here we describe the basic features of this organism, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a microsymbiont of annual clovers. We reveal that its genome size is 7,418,122 bp encoding 7,232 protein-coding genes and 61 RNA-only encoding genes. This multipartite genome contains 6 distinct replicons; a chromosome of size 4,767,043 bp and 5 plasmids of size 828,924 bp, 660,973 bp, 516,088 bp, 350,312 bp and 294,782 bp.

Comparative Analysis of Rice Transformation Using Agrobacterium tumefaciens and Rhyzobium leguminosarum

This study was aimed to study the effectiveness of Rhizobium transformation system compared to the most widely used Agrobacterium mediated transformation system on three rice cultivars, Ciherang (Indica), Nipponbare (Japonica), and Rojolele (Javanica). Six day old calli induced from immature embryos were inoculated with Rhizobium leguminosarum bv trifolii ANU845 and Agrobacterium tumefaciens LBA288 that harbored with vector pCAMBIA 5106. This plasmid contained a minimum set of transfer machinery genes and had a gusplus and an hptII gene driven by 35S CaMV promoter in the T-DNA. The results showed that the transformation frequencies (number of PCR positive plants per number of calli inoculated) ranging from 0 to 12.05 % depend on the genotype and transfer agent used. The highest transformation frequency (12.05%) was obtained in Ciherang transformed with R. leguminosarum. Most of the transgenic rice obtained by Rhizobium transformation were normal in morphology and fertile similar to those obtained by Agrobacterium transformation. Integration, expression and inheritance of transgenes were demonstrated by molecular and genetic analysis in T0 and T1 generations. Key words : Rhizobium leguminosarum, immature embryos, Agrobacterium tumefaciens

Complete genome sequence of Rhizobium leguminosarum bv trifolii strain WSM2304, an effective microsymbiont of the South American clover Trifolium polymorphum.

Rhizobium leguminosarum bv trifolii is the effective nitrogen fixing microsymbiont of a diverse range of annual and perennial Trifolium (clover) species. Strain WSM2304 is an aerobic, motile, non-spore forming, Gram-negative rod, isolated from Trifolium polymorphum in Uruguay in 1998. This microsymbiont predominated in the perennial grasslands of Glencoe Research Station, in Uruguay, to competitively nodulate its host, and fix atmospheric nitrogen. Here we describe the basic features of WSM2304, together with the complete genome sequence, and annotation. This is the first completed genome sequence for a nitrogen fixing microsymbiont of a clover species from the American center of origin. We reveal that its genome size is 6,872,702 bp encoding 6,643 protein-coding genes and 62 RNA only encoding genes. This multipartite genome was found to contain 5 distinct replicons; a chromosome of size 4,537,948 bp and four circular plasmids of size 1,266,105 bp, 501,946 bp, 308,747 bp and 257,956 bp.

Competition for nodule occupancy between introduced and native strains of Rhizobium leguminosarum biovar trifolii

The aim of this work was to evaluate the competitive ability between Rhizobium leguminosarum bv trifolii strain U204 used as commercial inoculants in Uruguay for Trifolium repens L. and Trifolium pratense L. and two native strains isolated from inoculated pastures of T. pratense. T126 is an efficient nitrogen fixer and a melanin producer strain; T70 is inefficient and a melanin non-producer strain; and U204 is very efficient in both hosts but is a melanin non-producer strain. Competitiveness between the strains was determined in experiments in pots and in growth pouches under controlled conditions. In the last experiment, we evaluated pH of plant nutrient solution and inoculum ratios. Plant dry weight was determined, and the identification of nodule bacteria was done using melanin production and DNA fingerprinting (GTG5-PCR). The U204 symbiotic efficiency was not affected by the co-inoculation with the others two native strains. The T70 strain was a poor competitor when was co-inoculated with one of the effective strains in both experiments. Our results confirmed a “selective nodulation” because an effective symbiosis occurred preferentially over an ineffective one in Trifolium species. The native effective strain competed with U204 for nodule formation in both clovers species, but the nodule occupancy depended on the inoculum ratio. The pH of nutritive solution did not affect competition ability of the studied strains. It may be possible to isolate efficient, competitive, and genetically different native rhizobial strains to be used as inoculant strains for clover pastures in Uruguay. Both (GTG)5-PCR and melanin production were useful methods to identify nodulating bacteria in competition studies.

Overproduction of the rhizobial adhesin RapA1 increases competitiveness for nodulation

Competition for nodulation is a complex problem where bacterial adhesins, which are required for root colonization, may play a role. However, the possible influence of adhesins on competitiveness was scarcely studied. In this work, the Rhizobium leguminosarum bv trifolii adhesion protein RapA1 was overproduced from a pHC60-derived plasmid and expressed in R200 strain. When an overproducing strain and a control-carrying empty vector were co-inoculated on clover plants, a positive effect of RapA1 on competition for nodule occupation was observed. Therefore, optimization of RapA1 expression may be considered for improvement of rhizobial competitiveness.

Phenotypic and Genetic Diversity of Rhizobia Isolated from Nodules of Clover Grown in a Zinc and Cadmium Contaminated Soil

Metal contamination can affect the diversity of microbes living in soil. We compared phenotypic and genetic characteristics of Rhizobium leguminosarum bv trifolii isolated from clovers (Trifolium pratense) found at a metal contaminated and a control site, or isolated from nodules of clovers used in most probable number (MPN) determinations of rhizobia in the metal contaminated soil. Analysis of variations in genetic patterns using BOX‐PCR was used for genetic characterization. Zinc and Cd tolerance of each isolate was also determined. Rhizobia isolated from the control soil compared with the metal contaminated soil differed both genetically and phenotypically. Genetic diversity, measured by AMOVA, was high and not related to the presence or the absence of high metal concentrations. Isolates originating from the metal contaminated soil were more tolerant to both Cd (minimal inhibitory concentration [MIC50] = 2.5 μM) and Zn (MIC50 = 92.9 μM) compared with those observed for isolates of control soil (Cd MIC50 = 2.0 μM and Zn MIC50 = 41.9 μM). Rhizobia originating from the metal contaminated soil expressed a higher number of metal phenotypes (9) compared with isolates of control soils (6). Slow rates of metal accumulation over the years favored an adaptation of the rhizobia to the metal rather than elimination of metal sensitive organisms and the selection of a few preexisting metal tolerant organisms.

Physiological responses to acid stress of an acid-soil tolerant and an acid-soil sensitive strain of Rhizobium leguminosarum biovar trifolii

Physiological responses to acid stress in two strains of Rhizobium leguminosarum bv trifolii of differing acid-soil tolerance were compared. Acidity affected the size and morphology of the acid-tolerant strain, WSM409, but not of the acid-sensitive strain, TA1. Acid grown cells of WSM409 and TA1 had less cell-associated Ca and Mg and more P than cells grown at pH 7.0. Potassium content was lower in acid grown cells; WSM409 was less affected by pH than that in TA1. WSM409 was more tolerant of pH shock at pH 3.5 when grown at pH 4.8 than when grown at pH 7.0. TA1 was more sensitive to pH shock when grown at pH 4.8 than when grown at pH 7.0. WSM409 shows a characteristic adaptive acid tolerance response, whereas TA1 shows an acid sensitive response.

Persistence of introduced strains of Rhizobium leguminosarum bv trifolii in acidic soils of north-eastern Victoria

Summary. A 5-year study was undertaken to establish if introduced rhizobia with higher tolerance to Al than the current inoculant Rhizobium can persist and continue nodulating subterranean clover (Trifolium subterraneum L.) in acidic soils. Two Rhizobium leguminosarum bv trifolii strains were introduced as seed inoculants with subterranean clover at 2 acidic sites (pHCa 4.1 and pHCa 4.3), where lime and gypsum had been applied as soil amendments. Strain NA3001 was selected for its tolerance to high Al concentrations when grown on an agar medium and WU95, which is a widely used commercial inoculant strain, for its relatively poor tolerance to Al when grown on agar. Liming the soil increased its pH and reduced the concentration of extractable Al at both sites. In the year the subterranean clover was sown, strain WU95 had nodule occupancy of 20–49%, decreasing with time to 4–7% after 5 seasons (1991–95). The nodule occupancy of strain NA3001 was initially lower than strain WU95 (14–16%), but its occupancy did not vary with time (significant strain x time interactions, P<0.05). These data indicate that the acid-tolerant strain NA3001 has the potential to persist in these strongly acidic soils and, despite the presence of high background populations of naturalised rhizobia, to continue initiating nodulation. The use of soil amendments (lime and gypsum) to increase pH and reduce soluble Al concentrations did not affect the nodule occupancy of either NA3001 or WU95 with time, nor did it slow the rate of decline in nodule occupancy of WU95.

The use of GUS-reporter gene to study the effect of Azospirillum-Rhizobium coinoculation on nodulation of white clover

The gusA-marked Azospirillum lipoferum T1371, constructed by inserting transposon mTn5SSgusA20 from Escherichia coli S17-1 λ-pir into the genome of Azospirillum lipoferum 137, was used to evaluate its effect on nodulation of white clover with and without Rhizobium inoculation. When inoculated alone, Azospirillum colonized the tap roots, secondary roots and root hairs. The combined inoculation of white clover with Rhizobium leguminosarum bv trifolii and A. lipoferum enhanced the number of nodules by 2–3 times from 5 to 20 days after inoculation (DAI). The combined inoculation also enhanced acetylene reduction activity by 2.3–2.7 times at 20 DAI. Moreover, Azospirillum was observed colonizing the tap root, root hairs and sites near or on the nodules. These results suggest that the formation of additional infection sites by A. lipoferum, with a combined inoculation, may be the mechanism that will enhance nodulation and nitrogen fixation of white clover.

Structure and role in symbiosis of the exoB gene of Rhizobium leguminosarum bv trifolii.

The Rhizobium leguminosarum bv trifolii exoB gene has been isolated by heterologous complementation of an exoB mutant of R. meliloti. We have cloned a chromosomal DNA fragment from the R. leguminosarum bv trifolii genome that contains an open reading frame of 981 bp showing 80% identity at the amino acid level to the UDP-glucose 4-epimerase of R. meliloti. This enzyme produces UDP-galactose, the donor of galactosyl residues for the lipid-linked oligosaccharide repeat units of various heteropolysaccharides of rhizobia. An R. leguminosarum bv trifolii exoB disruption mutant differed from the wild type in the structure of both the acidic exopolysaccharide and the lipopolysaccharide. The acidic exopolysaccharide made by our wild-type strain is similar to the Type 2 exopolysaccharide made by other R. leguminosarum bv trifolii wild types. The exopolysaccharide made by the exoB mutant lacked the galactose residue and the substitutions attached to it. The exoB mutant induced the development of abnormal root nodules and was almost completely unable to invade plant cells. Our results stress the importance of exoB in the Rhizobium-plant interaction.

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 microM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2-5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 microM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 microM added iron) during the stationary phase of growth.

Competition between a native isolate of Rhizobium leguminosarum bv trifolii and two commercial inoculant strains for nodulation of clover

A naturalized rhizobial isolate from nodules obtained from persistent clover pastures was evaluated for its ability to form effective nodules in the presence of two commercial inoculant strains Rhizobium leguminosarum bv trifolii U28 and WU290 recommended for white clover and subterranean clover, respectively. The competition experiment was performed in tubes, and indirect ELISA was used for identification of rhizobia present in the nodules. The ratios between the number of nodules containing only the naturalized isolate (PA8) and the number of nodules containing only the U28 strain were directly proportional to their respective inoculum densities when white clover was used as host plant. However, when the competition assay was done with different mixtures of PA8 and WU290, no WU290 bacteria were identified in subterranean clover nodules. The symbiotic performance of PA8, together with its persistence and competitive ability, suggests that naturalized strains may be useful in attempting to establish and maintain high-yielding pastures in Uruguay.