Rhizobium Meliloti Strain Research Articles

Microscopic studies of the phytoplasma infection effect on the symbiotic system Medicago sativa – Rhizobium meliloti in simulated conditions

Aim. Aim of the study was to microscopically investigate the interaction of Acholeplasma laidlawii var. granulum 118 with the target plant Medicago sativa, as well as to determine the role of Rhizobium meliloti strains with natural and defective polysaccharides in the plant’s resistance occurrence. Methods. Cultivation of plants and their inoculation with bacteria was performed under conditions of microvegetation experiment. The study of the ultrastructure of alfalfa roots and nodules was carried out with both light and electron microscopy. Results. The rhizobial mutant strain, defective in the synthesis of lipopolysaccharides, more often formed atypical nodules on M. sativa, which aged faster. In the variant with double inoculation of alfalfa with rhizobia together with acholeplasma changes in the morphology of the lateral roots of plants, as well as deformation of the nodules were observed. Conclusions. Results of this study indicate not only the ability of phytoplasmas to penetrate the root system and migrate to plant aboveground organs, but also demonstrate the possibility of their presence in the nodules formed by rhizobia.
 Keywords: Mollicutes, acholeplasma, rhizobia, mutant, lipopolysaccharides.

Can differences of nitrogen nutrition level among Medicago truncatula genotypes be assessed non-destructively? Probing with a recombinant inbred lines population

The international consensus on Medicago truncatula as a model system has lead to the development of powerful approaches for dissecting the genetic and molecular bases of legume nitrogen nutrition. However, such approaches now come up against a poor knowledge of the phenotypic traits that should be used for the large-scale screening of the genotypic variability associated with nitrogen nutrition. This issue was unravelled in a previous report, in which an ecophysiological approach allowed a better understanding of the relationships between plant nitrogen nutrition and plant growth traits, for the model symbiotic association between M. truncatula cv. Jemalong and Rhizobium meliloti strain 2001. From this analysis, phenotypic traits were identified as potentially relevant for the large-scale screening of the genotypic variability. Here, by the phenotyping of a recombinant inbred lines population, we show that the proposed methodology provides a valuable support for assisting the detection of genetic variants affected for nitrogen uptake. Especially, the relative expansion rate of plant leaf area is identified as a good proxy for ranking genotypes according to their ability to uptake nitrogen in given environmental conditions. As leaf area can be measured non-destructively, such finding should pave the way for a more efficient evaluation of the genetic variability.Addendum to: Moreau D, Voisin AS, Salon C, Munier-Jolain N. The model symbiotic association between Medicago truncatula cv. Jemalong and Rhizobium meliloti strain 2001 leads to N-stressed plants when symbiotic N2 fixation is the main N source for plant growth. J Exp Bot 2008; 59:3509-22; PMID: 18703494; DOI:10.1093/jxb/ern203.

The model symbiotic association between Medicago truncatula cv. Jemalong and Rhizobium meliloti strain 2011 leads to N-stressed plants when symbiotic N2 fixation is the main N source for plant growth

A better knowledge of the nitrogen nutrition of Medicago truncatula at the whole plant level and its modulation by environmental factors is a crucial step to reach a complete understanding of legume nitrogen nutrition. This study was based on the symbiotic system that is the most commonly used by the research community (M. truncatula cv. Jemalong A17 x Rhizobium meliloti strain 2011). Plant nitrogen nutrition was analysed in relation to carbon nutrition, under a range of nitrate concentrations in the nutrient solution and different light conditions. This study shows that this 'model symbiotic association' does not allow the plant to meet its nitrogen requirements, when dinitrogen fixation is the main nitrogen source for plant growth. A strong interaction between nitrogen and carbon nutrition was shown: when plant nitrogen requirements were not sustained, plant leaf area was much affected whereas photosynthesis per unit leaf area remained relatively stable. Both total nitrogen uptake and leaf area increased with increasing nitrate concentration in the nutrient solution; the magnitude of these responses varied according to the light conditions. Interestingly, the plant nitrogen nutrition level remained nearly unaffected by the light conditions. The observed nitrogen-limitation in this 'model symbiotic association' is an important finding for the research community. Based on practical recommendations regarding both the experimental conditions and the phenotypic traits to consider, a methodological framework was proposed to (i) help genomicists to assess plant nitrogen nutrition better, and (ii) assist in the detection of new genetic variants affected for nitrogen uptake in large-scale phenotyping studies.

Colonization and Nitrogenase Activity of Triticum aestivum (cv. Baccross and Mahdavi) to the Dual Inoculation with Azospirillum brasilense and Rhizobium meliloti plus 2,4-D

The potential enhancement of root colonization and nitrogenase activity of wheat cultivars (Baccross and Mahdavi) was studied with application of two Azospirillum brasilense strains (native and Sp7) co-inoculated with two Rhizobium meliloti strains (native and DSMZ 30135). The results indicated that the colonization was different due to the strains and cultivars of wheat were used. Native A. brasilense colonized wheat root better than Sp7 strain. However, Baccross cv. reacted better with native Azospirillum compared to Mahdavi cv. which reacted better with Sp7. When plants inoculated with dual inoculants (SP7 with standard Rhizobium), the colonization of Azospirillum were increased significantly (from 1.67 x 10(5) to 22 x 10(5) cfu g(-1) FW for Baccras cv. and 3.67 x 10(5) to 26 x 10(5) cfu g(-1) FW for Mahdavi cultivar). When the standard Rhizobium as co-inoculants changed to the native Rhizobium, the colonization of Azospirillum was higher when compared to the single inoculants but was almost the same when compared to the standard Rhizobium. When the standard or native strains of Rhizobium used as single inoculation of wheat roots, the number of Rhizobium in the wheat roots were not changed significantly. However, when plants co-inoculated with Rhizobium and Azospirillum, the colonization of Rhizobium was increased. Co-inoculation of standard strain of R. melilot with A. brasilense Sp7 showed that the colonization of Rhizobium were increased from 0.67 x 10(5) to 21 x 10(5) cfu g(-1) FW for Baccross cv. and 0.33 x 10(5) to 18 x 10(5) cfu g(-1) FW for Mahdavi cv. This behavior was the same when inoculation of Rhizobium was happened with the native one. In dual inoculation, the highest nitrogenase activity was measured in combination of the local strains (native A. brasilense with the native R. meliloti) and the lower one belongs to the combination of standard strains (Sp7 with standard R. meliloti). The difference in nirtogenase activity for different cultivars of wheat with Sp7 and standard Rhizobium is not significant but the difference for Sp7 strain plus native Rhizobium is significant (p > 0.05). However, the differences were not significant (p < 0.05) for nitrogenase activity in bacterial tubes, the difference for nitrogenase activity of co-inoculated plants with combination of Sp7 and Rhizobium either standard or native were significantly different.

Effect ofRhizobium inoculation and nitrogen fertilization on alfalfa under irrigation

Abstract Inoculation with Rhizobium meliloti and nitrogen fertilization on alfalfa increasedthe plant density, forage fresh yield and the protein content, and decreased the crude fibrecontent of alfalfa. Keywords: alfalfa, forage yield, nitrogen fixation, Rhizobium . Introduction Alfalfa is the most important cultivated forage legume in the Sudan, where it is grown underirrigation usually without fertilizers. Elsewhere the effect of nitrogen fertilization has varied:for example Giroux (1981) found that 40kg N/ha before sowing significantly increased theyield on a silty clay loam, but not on clay. The effect of Rhizobium inoculation also varies(Kots et al. 1995; Provorov and Tikhonovich 2003). This study concerned the effect of Rhizobium inoculation on growth, symbiosis, quality and forage yield, and the performanceof alfalfa under increasing doses of nitrogen. Materials and methods Rhizobium meliloti strain ENRRI A12 was supplied by the National Centre for Research,Khartoum. Broth culture (400 ml of 1 × 10

Simultaneous and comparative assessment of parent and mutant strain of Rhizobium meliloti for nutrient limitation and enhanced polyhydroxyalkanoate (PHA) production using optimization studies

Nutrient limitation conditions, optimization and comparison of polyhydroxyalkanoate (PHA) yields and biomass production by parent and mutant strains of Rhizobium meliloti were investigated. Complex interactions among concentrations of sucrose (5–55 g/l), urea (0.05–0.65 g/l) inoculum (10–250 ml/l) and K 2HPO 4 (0.5–2 g/l), were studied using central composite rotatable design (CCRD) experiments. Phosphate-limiting medium (0.33 g K 2HPO 4/l) in the presence of excess carbon (sucrose 42.5 g/l) results in more production of PHA (2.2 g/l) in the parent strain. In comparison, the mutant strain required moderate levels of sucrose (30 g/l), along with excess of phosphate (1 g/l) for high PHA content of cell biomass (80%) and PHA yield (3.3 g/l). Optimised PHA production (biomass 4.8 g/l and PHA 3.09 g/l) by the parent strain occurred at: sucrose 51.58 g/l, urea 0.65 g/l, K 2HPO 4 0.48 g/l and inoculum 10 ml/l. In the mutant strain, higher yields of biomass (9.05 g/l) and PHA (5.66 g/l) were obtained in Optimised medium containing: sucrose 55 g/l, urea 0.65 g/l, K 2HPO 4 1.0 g/l and inoculum 150.58 ml/l.

Remedial inoculation of Rhizobium meliloti strains and nodule occupancy on Medicago rigidula (L.) All. and M. truncatula Gaertn

Because post-planting inoculation of soil with Rhizobium sp. is often practiced when seed inoculation fails, the potential of delayed (remedial) root inoculation as a feasible agricultural practice for two annual pasture legumes was investigated. Roots of Medicago rigidula International Center for Agricultural Research in the Dry Areas (ICARDA) sel.716, and Medicago truncatula cv. Jemalong, were pre-exposed to Rhizobium meliloti strains. Subsequently, roots were exposed to a secondary inoculum after 6, 48, and 168 h (8 days) to simulate remedial inoculation. Subsequent establishment of the secondary inoculum strains in the nodules was investigated. Combinations of highly effective (capacity for nitrogen fixation) and host compatible strains (ICARDA M15 and M28) for both hosts, and effective–ineffective strains (M28 and M34) for M. rigidula and M. truncatula, respectively, were used to determine the proportional nodule occupancy responses based on their differential resistance to spectinomycin and streptomycin, and in the case of M34, on nodule characteristics. When equal populations of M28 and M15 were applied immediately after inoculation, nodule population differences were not significant in M. rigidula but were significantly greater ( P≤0.01) for strain M28 in M. truncatula. M. trunculata plants, pre-exposed to M15 and receiving the ineffective M34 strain as a secondary inoculant, showed that predisposition of the plant to accept the primary inoculant was not a factor in nodule occupancy success. Nodule occupancy of the primary inoculant (M15) was suppressed significantly ( P≤0.01) by M34. This was not the case for all other combinations of effective strains in both legumes. Results indicate that the early events in the nodulation process of annual Medicago spp. coupled with host-specificity factors are perhaps the most critical for competition among R. meliloti strains for nodule occupancy. We conclude that remedial R. meliloti inoculation applied after the seed has been planted and colonized by soil bacteria may be of little benefit to legume growers.

The application of isotopic ( 32 P and 15 N) dilution techniques to evaluate the interactive effect of phosphate-solubilizing rhizobacteria, mycorrhizal fungi and Rhizobium to improve the agronomic efficiency of rock phosphate for legume crops

A pot experiment was designed to evaluate the interactive effects of multifunctional microbial inoculation treatments and rock phosphate (RP) application on N and P uptake by alfalfa through the use of 15N and 32P isotopic dilution approaches. The microbial inocula consisted of a wild type (WT) Rhizobium meliloti strain, the arbuscular mycorrhizal (AM) fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe, and a phosphate solubilizing rhizobacterium (Enterobacter sp.). Inoculated microorganisms were established in the root tissues and/or in the rhizosphere soil of alfalfa plants (Medicago sativa L.). Improvements in N and P accumulation in alfalfa corroborate beneficial effects of Rhizobium and AM interactions. Inoculation with selected rhizobacteria improved the AM effect on N or P accumulation in both the RP-added soil and in the non RP-amended controls. Measurements of the 15N/14N ratio in plant shoots indicate an enhancement of the N2 fixation rates in Rhizobium-inoculated AM-plants, over that achieved by Rhizobium in non-mycorrhizal plants. Whether or not RP was added, AM-inoculated plants showed a lower specific activity (32P/31P) than did their comparable non-mycorrhizal controls, suggesting that the plant was using otherwise unavailable P sources. The phosphate-solubilizing, AM-associated, microbiota could in fact release phosphate ions, either from the added RP or from the indigenous ``less-available'' soil phosphate. A low Ca concentrations in the test soil may have benefited P solubilization. Under field conditions, the inoculation with AM fungi significantly increased plant biomass and N and P accumulation in plant tissues. Phosphate-solubilizing rhizobacteria improved mycorrhizal responses in soil dually receiving RP and organic matter amendments. Organic matter addition favoured RP solubilization. This, together with a tailored microbial inoculation, increased the agronomic efficiency of RP in the test soil that was Ca deficient at neutral pH.

Growth and nitrogen fixation of annual Medicago-Rhizobium associations during winter in Mediterranean region

Low winter temperature in Mediterranean rangelands delays the growth of most pasture and forage plants and, in particular, annual Medicago (medics). Finding proper associations of medic- Rhizobium, capable to grow and fix nitrogen at low temperature, is necessary to extend the grazing period. In this paper, we studied the performance of three Medicago cultivars: M. aculeata accessions 80 (A) and 5099 (B) and M. rigidula accession 716 (R), grown in the field with three different nitrogen sources. The first treatment (N) was obtained through fertilization, while the second (I) was ensured by inoculation with different strains of Rhizobium meliloti (M620, M508 and BZI). A third treatment (C) was used as a control, neither inoculated nor fertilized. The trial was repeated for 2 consecutive years (1998 and 1999) at ICARDA research station (Aleppo, Syria). Plant development, dry matter production and nodule initiation were investigated. The percentage of fixation-derived shoot nitrogen was also quantified using 15N dilution technique. Our results indicate a genotypic variation in low-temperature tolerance of annual medics. Accession A had a slow growth pattern with low dry matter production compared with B and R over the two seasons. In the two seasons and at the early growth stages, Rhizobium strain played a key role in plant growth, where both inoculated and fertilized plants of accession R had a comparable dry matter. Furthermore, the proportion of nitrogen derived from fixation in A was very low, which was reflected, in turn, on the total amount of fixed nitrogen. The proportion of fixed nitrogen of the association R-BZI was high for the 2 years, where it reached 72 and 87% of the total N in the plant, respectively. It is suggested that associations differed in their response to low winter temperature. Among those we studied, M. rigidula accession 716, inoculated with the strain BZI, proved to be the most adapted association to our environment. In addition, M. aculeata accession 5099 seems to be promising for winter growth, but it needs to be inoculated with a strain more adapted to low temperature.

Ribosomal protein gene cluster analysis in eubacterium genomics: homology between Sinorhizobium meliloti strain 1021 and Bacillus subtilis

The first whole genome sequence of a symbiotic soil bacterium, Sinorhizobium meliloti (formely named Rhizobium meliloti) strain 1021, is due in 2001. As an active participant in the European and North American consortium that has completed this work, our group has sequenced a region on the chromosome containing clusters rpoBC, str, S10, spc and alpha corresponding to 30 protein genes. The structural organization and function of these genes were compared with those of orthologs in another 15 complete eubacterial genomes available in databases. This study, involving the DNA and amino acid sequences as well as the organization of the whole region (gene order, cluster order, etc.), has shown that the phylogenetic tree resulting from a comparison of the amino acid sequence is rather similar to that derived from 16S rRNA sequence data. However, the tree achieved by aligning DNA sequences groups the organisms with a high GC content (>60% GC), while that based on a comparison of gene cluster orientation and organization reveals a greater level of correspondence between the alpha-proteobacteria S.meliloti and the firmicute Bacillus subtilis.

Delayed inoculation and competition of nitrogen-fixing strains in Medicago noeana (Boiss.) and Medicago polymorpha (L.)

Seed inoculation is frequently essential for annual Medicago establishment in dryland farming systems, particularly in Mediterranean-type environments. As post-planting soil inoculation is often practised when seed inoculation fails, the effect of delayed inoculation was investigated. Roots of Medicago noeana ICARDA sel. 1938, and Medicago polymorpha cv. Circle Valley, were pre-exposed to Rhizobium meliloti strains. Subsequently, roots were exposed to a secondary inoculum after 6, 48 and 168 h to simulate delayed inoculation, and subsequent establishment of other strains in the nodules were investigated. Combinations of highly effective and host compatible strains (M29 and M15) and effective–ineffective strains (M29 and M33) were used to evaluate proportional nodulation responses. Plants were harvested after 6 weeks of growth under environmentally controlled conditions. Nodules were assessed for distribution in the root system and for occupancy based on their differential resistance to spectinomycin and streptomycin, and, in the case of M33, on nodule characteristics. The strain M29 was a better competitor than M15 when applied in equal density to M. polymorpha, at zero time. When forming nodules with M. noeana, M15 was equally competitive under the same conditions. With M29 as the primary inoculum, and M15 inoculation delayed for 6, 48 and 168 h, the incidence of M29 nodules increased on M. noeana from 55% (at zero time) to 83, 80, and 95% and, on M. polymorpha, from 72% (at zero time) to 80, 90, and 97% for the three inoculation time delays, respectively. Conversely, strain M15 dominated nodule production at all time intervals when used as primary inoculant on both hosts. The percentage of total nodulation by M33, applied at the three later inoculation times, was markedly lower (21, 2 and 0%, respectively) when M. polymorpha was pre-exposed to M29. This suggested a host preference for M29, even if applied as a late inoculum. Pre-exposure of 2-day-old M. noeana seedlings to the ineffective strain M33 as the primary inoculant resulted in nodule number increases ( P≤0.01) as compared with M29. Nonetheless, when M29 was the primary inoculum, M33 was able to produce significantly fewer nodules than its competitor when applied at the 6 and 48 h time delays. Results indicate that the early events in the nodulation process of annual medics coupled with host-specificity factors are perhaps the most critical for competition among R. meliloti strains for nodule formation. Therefore, remedial inoculation after the seed has been planted may be of little benefit.

Cell surface properties of rhizobial strains isolated from soils contaminated with hydrocarbons: hydrophobicity and adhesion to sandy soil

Ten strains of Rhizobium meliloti and two non-rhizobial strains, Escherichia coli JM105 and Comamonas testosteroni B-356, were studied for their behaviour in soil and water matrices by determining their hydrophobicity, as measured by bacterial adherence to n-octane, and adhesion, as measured by their retention on sandy soil particles. The hydrophobicity values for the rhizobial strains were similar to that for E. coli (around 15%), whereas C. testosteroni, which belongs to the Pseudomonads group, showed a significantly higher level (33%). Both non-rhizobial strains, however, showed higher levels of adhesion, (85% and 78%, respectively) than did the rhizobial strains (≈55%). The cell surface properties of the rhizobial strains were influenced by the composition of the growth media used, being significantly higher with nutrient-rich media. The nature or level of contaminants in the soil from which these strains were originally isolated did not, however, influence these properties. These characteristics are important in determining the fate of bacteria in the unsaturated subsurface soil environment because of their influence on the vertical transport, distribution and survival. Information about these characteristics will be particularly useful in choosing strains for agricultural applications (e.g., as biofertilizers or biocontrol agents) or in situ soil environmental operations (e.g., bioaugmentation for bioremediation of pollutants).

Influence of arbuscular-mycorrhizal fungi, Rhizobium meliloti strains and PGPR inoculation on the growth of Medicago arborea used as model legume for re-vegetation and biological reactivation in a semi-arid mediterranean area

Medicago arborea can be used for re-vegetationpurposes under semiarid conditions. These woody legumes have the ability toforman association with arbuscular mycorrhizal (AM) fungi and rhizobial bacteria,which can be maximised by microorganisms producing certain stimulatingmetabolites acting as plant growth promoting rhizobacteria (PGPR). The effectsof single and combined inoculations using microorganisms with different andinteractive metabolic capacities, namely three Glomusspecies, two Rhizobium meliloti strains (a wild type, WTand its genetically modified derivative GM) and a plant growth promotingrhizobacterium, (PGPR), were evaluated. All three inoculated AM fungi affectedMedicago growth in different ways. Differences weremaintained when soil was co-inoculated with each of the rhizobial strains (WTorGM) and the PGPR. Mycorrhizal fungi were effective in all cases, but the PGPRonly affected plant growth specific microbial situations. PGPR increased growthof G. mosseae-colonised plants associated withRhizobium WT strain by 36% and those infected byG. deserticola when associated with the rhizobial GMstrainby 40%. The most efficient microbial treatments involved mycorrhizalinoculation, which was an indication of the AM dependency of this plantspecies.Moreover, PGPR inoculation was only effective when associated with specificmycorrhizal endophytes (G. mosseae plus WT andG.deserticola plus GM rhizobial strain). The reduced root/shoot (R/S)ratio resulting from PGPR inoculation, was an indication of more effective rootfunction in treated plants. AM colonisation and nodule formation wereunaffectedby the type of AM fungus or bacteria (rhizobial strain and/or PGPR). AM fromnatural soil were less infective and effective than those from the collection.The results supported the existence of selective microbial interactionsaffecting plant performance. The indigenous AM fungi appeared to be ineffectiveand M. arborea behaved as though it was highly dependentonAM colonisation, which implied that it must have a mycorrhizal association toreach maximum growth in the stressed conditions tested. Optimum growth ofmycorrhizal M. arborea plants was associated with specificmicrobial groups, accounting for a 355% increase in growth overnodulatedcontrol plants. The beneficial effect of PGPR in increasing the growth of awoody legume, such as M. arborea under stress, was onlyobserved with co-inoculation of specific AM endophytes. As a result of theinteraction, only shoot biomass was enhanced, but not as a consequence ofenhancing of the colonising abilities of the endophytes. The growthstimulation,occurring as a consequence of selected microbial groups, may be critical anddecisive for the successful establishment of plants under Mediterraneanclimaticand soil conditions.

Cloning and Characterization of the Gene for Phosphatidylcholine Synthase

Phosphatidylcholine (PC) is the major membrane-forming phospholipid in eukaryotes and can be synthesized by either of two pathways, the CDP-choline pathway or the methylation pathway. In prokaryotes only the methylation pathway was thought to occur. Recently, however, we could demonstrate (de Rudder, K. E. E., Sohlenkamp, C., and Geiger, O. (1999) J. Biol. Chem. 274, 20011-20016) that a second pathway for phosphatidylcholine biosynthesis exists in Sinorhizobium (Rhizobium) meliloti involving a novel enzymatic activity, phosphatidylcholine synthase, that condenses choline and CDP-diacylglyceride in one step to form PC and CMP. Using a colony autoradiography method we have isolated mutants of S. meliloti deficient in phosphatidylcholine synthase and which are no longer able to incorporate radiolabeled choline into PC. Complementation of such mutants with a sinorhizobial cosmid gene bank, subcloning of the complementing fragment, and sequencing of the subclone led to the identification of a gene coding for a presumptive CDP-alcohol phosphatidyltransferase. Amplification of this gene and its expression in Escherichia coli demonstrates that it codes for phosphatidylcholine synthase. Genomes of some pathogens (Pseudomonas aeruginosa and Borrelia burgdorferi) contain genes similar to the sinorhizobial gene (pcs) for phosphatidylcholine synthase. Although pcs-deficient S. meliloti knock-out mutants show wild type-like growth and lipid composition, they are unable to perform rapid PC biosynthesis that normally is achieved via the phosphatidylcholine synthase pathway in S. meliloti wild type.

Accumulation of ppGpp in symbiotic and free-living nitrogen-fixing bacteria following amino acid starvation

Following amino acid or ammonium starvation, ppGpp is accumulated by Rhizobium meliloti strain 1021 but not by R. meliloti strain 41 or Rhizobium tropici. Azorhizobium caulinodans ORS571 produced ppGpp following amino acid deprivation; however, the free-living nitrogen-fixing bacteria Azotobacter vinelandii and Azomonas agilis did not produce ppGpp. Western blot analysis using anti-RelA antibody demonstrated that R. meliloti strain 1021, Azotobacter vinelandii and Azorhizobium caulinodans cross-reacted under conditions that detected RelA in Escherichia coli CF1648. Cross-reaction was not observed in R. meliloti strain 41, R. tropici, or Azomonas agilis. All strains that accumulated ppGpp also produced high intracellular levels of ATP.

Variation of Local Populations of Medicago arabica (L.) Huds. and Strains of Rhizobium meliloti and their Symbiotic Compatibility in Nitrogen Fixation Capacity

Variation of Local Populations of Medicago arabica (L.) Huds. and Strains of Rhizobium meliloti and their Symbiotic Compatibility in Nitrogen Fixation Capacity

Effect of amaranth on the growth of Rhizobium and Bradyrhizobium strains

The growth rate of different strains of Bradyrhizobium and Rhizobium was studied in media containing amaranth seed meal instead of yeast extract. Results obtained in erlenmeyer flasks and stirred fermenters show that both Bradyrhizobium japonicum strains E109, E110, 5019, 587 and Rhizobium melilotistrains B36, B323, B399, Lq22, Lq42, Lq51 and U322, grow satisfactorily in amaranth seed meal medium. Cell count obtained for the strains tested was greater than 4 × 1010 viable cells.ml−1. Amaranth seed meal (4 g.l−1) is a suitable component for culture media that can be used instead of yeast extract.

Localization of oleosomes in the root nodules of alfalfa (Medicago sativa)

Histological studies of rhizobia-induced nodules and spontaneous pseudonodules of alfalfa were undertaken to reveal the distribution pattern of oleosomes. Alfalfa (Medicago sativa L. cv. 'Algonquin') seedlings were inoculated with Rhizobium meliloti (strain Balsac), and grown in controlled conditions. Seedlings were also grown aseptically to obtain spontaneous pseudonodules. Nodule slices were fixed in a paraformaldehyde-glutaraldehyde mixture and processed for embedding in Spurr's medium after OsO4 treatment and en bloc staining, with p-phenylenediamine (pPD) in 70% ethanol, for oleosomes. As a control, hexane extraction of lipids was employed. In pseudonodules, oleosomes were present in the vascular parenchyma, the meristem region and in the five to seven layers of cortical cells adjacent to the vascular tissue. The allocation of energy stored as triacylglycerides, in the form of oleosomes, and starch indicates that pseudonodules are a sink for carbon, possibly functioning as storage organs. In mature nodules, oleosomes were distinguished clearly in nodule parenchyma but were absent in infected cells. Young nodules were devoid of oleosomes. The prevalence of oleosomes in the mature nodules may indicate that the triacylglycerides are stored for overwintering, as alfalfa nodules are known to be perennial. The oleosomes in root nodules of alfalfa do not appear to be directly associated with nitrogen fixation per se.Key words: oleosomes, root nodules, Medicago sativa, Rhizobium, pseudonodules.

The use of isotopic dilution techniques to evaluate the interactive effects of Rhizobium genotype, mycorrhizal fungi, phosphate-solubilizing rhizobacteria and rock phosphate on nitrogen and phosphorus acquisition by Medicago sativa.

A pot experiment was designed to evaluate the interactive effects of multiple microbial inoculation treatments and rock phosphate (RP) application on N and P acquisition by alfalfa plants using 15 N and 32 P isotopes. The microbial inocula consisted of a wild type (WT) Rhizobium meliloti strain, its genetically modified (GM) derivative, which had an enhanced competitiveness, the arbuscular mycorrhizal (AM) fungus Glomus mosseae (Nicol. and Gerd.) Gerd. and Trappe, and a phosphate-solubilizing rhizobacterium (Enterobacter sp.). Inoculated micro-organisms became established in the root tissues and/or in the rhizosphere soil of alfalfa plants (Medicago sativa L.). The GM Rhizobium strain did not interfere with AM formation. Inoculated phosphate-solubilizing rhizobacteria established in the alfalfa rhizosphere, but the level of establishment was lower where the natural population of phosphate-solubilizing bacteria was stimulated by AM inoculation and RP application. The stimulation of these indigenous bacteria was also greater in the rhizosphere of alfalfa nodulated by the GM Rhizobium. Improvements in N and P accumulation in alfalfa corroborate beneficial effects of the improved GM Rhizobium on AM performance, in RP-amended plants. Inoculation with Enterobacter did not improve the AM effect on N or P accumulation in the RP-added soil, but it did in the non RP-amended controls. Measurements of the 15 N∶14 N ratio in plant shoots indicated enhanced N2 fixation rates in Rhizobium-inoculated AM-plants, over that achieved by the same Rhizobium strain in non-mycorrhizal plants. Regardless of the Rhizobium strain and of whether or not RP was added, AM-inoculated plants showed a lower specific activity (32 P∶31 P) than did their comparable non-mycorrhizal controls, suggesting that the plant was using otherwise unavailable P sources. The phosphate-solubilizing, AM-associated, microbiota could in fact release phosphate ions, either from the added RP or from the indigenous 'less-available' phosphate. Deficiency in Ca concentration in soil solution in the neutral test soil might benefit P solubilization. The proportion of plant P derived either from the labelled soil P (labile P pool) or from RP was similar for AM inoculated and non-mycorrhizal controls (without Enterobacter inoculation) for each Rhizobium strain, but the total P uptake, regardless of the P source, was far higher in AM-plants. Enterobacter inoculation seems to improve the use of RP in the rhizosphere of non-mycorrhizal plants inoculated with the WT Rhizobium.

Genotypic diversity of Sinorhizobium (formerly Rhizobium) meliloti strains isolated directly from a soil and from nodules of alfalfa ( Medicago sativa) grown in the same soil

The genetic structure of a population of Sinorhizobium meliloti (formerly Rhizobium meliloti) isolated from nodules of alfalfa ( Medicago sativa cv. Europe) or isolated directly from the soil using an elective medium and colony hybridization was assessed by three molecular methods. Sixty-seven S. meliloti isolates were obtained from alfalfa nodules and 61 directly from soil. Plasmid profiles of S. meliloti isolates were analyzed as well as insertion sequence (IS) fingerprints using probes corresponding to the two indigenous IS elements: IS Rm1 and IS Rm5. The presence of a gene involved in nodule forming efficiency ( nfeA gene) was also determined by PCR using specific primers, among S. meliloti isolates. Plasmid profile analysis revealed the presence of 23 distinct plasmid types among the 128 isolates tested and the distribution of plasmid types was significantly different between isolates obtained from nodules and isolates obtained from soil. The distributions of plasmid types among S. meliloti nodule isolates were similar to those in a collection of nodule isolates obtained from the same field 8 years previously. We have thus demonstrated that the distribution of plasmid types in alfalfa nodules has not varied over long periods in the absence of the host plant. IS fingerprinting was more discriminative than plasmid profiling as a total of 65 distinct IS genotypes were found among the same isolates. The distribution of IS genotypes was different between isolates obtained from nodules and those isolated from soil. These results suggest that sampling S. meliloti populations using the host plant may not be representative of existing soil populations. Genes like nfeA were detected in neither the nodule nor soil isolates. The dominant plasmid type in nodule isolates (30% of the population), carrying a large plasmid of 230 kb, is poorly represented among soil isolates (8%). This result suggests that this plasmid might be involved in the competitiveness of S. meliloti strains.