Influence of soil pH on the nitrate-reducing microbial populations and their potential to reduce nitrate to NO and N2O

Abstract After the introduction of Rhizobium leguminosarum biovar trifolii into natural loamy sand and silt loam, bacterial numbers increased only directly after inoculation. Thereafter, bacterial numbers decreased until an equilibrium was reached. This decrease was exponential on a log scale and could be described by the function Y = A + B − R ', where Y is the log number of rhizobial cells at time: T ; A represents the lgo of the final population size; B is the difference between the log (initial number of bacteria) and A ; R is the daily reduction factor of Y−A and t is time in days after inoculation. The final population sizes increased with increasing inoculum densities (10⁴−10⁸ bacteria/g soil). In sterilized soil, however, the populations increased up to an equilibrium, which was not affected by the inoculum density.
The final population sizes were higher in silt loam than in loamy sand in natural, as well as in sterilized soil. The final population size was reached earlier in natural silt loam than in loamy sand. Also the growth rate in sterilized soil was higher in silt loam than in loamy sand. The growth rate of low inoculum densities in silt loam was exponential and approximately the same as in yeast extract mannitol broth. The growth rate in loamy sand could be improved by incresing the bulk density of the soil from 1.0 to 1.4 g/cm³.     

Effect of cadmium, chromium and copper on symbiotic and free-living Rhizobium leguminosarum biovar trifolii

Abstract Plasmid-minus derivatives of Rhizobium leguminosarum biovar trifolii have been isolated. Cured strains lacked symbiotic properties, however they showed increased heavy metal resistance. In the presence of 70 ppm chromium the parent strain, unlike cured derivatives, is unable to grow explanta but can nevertheless nodulate clover.
We propose that rhizobia can circumvent exposure to the heavy metal by entering the plant roots.
Acetylene reduction tests showed that nodulated plants, grown in the presence of 10 ppm of chromium, had an increased nitrogenase activity compared to the control plants.     

Symbiotic growth of indigenons white clover (Trifolium repens) with local Rhizobium leguminosarum biovar trifolii

To study a possible adaptation of the symbiosis between white clover (Trifolium repens L.) and Rhizobium leguminosarum biovar trifolii with regard to light and temperature at northern latitudes, local seed populations of white clover and isolates of R. leguminosarum biovar trifolii from 3 different latitudes in Norway, 58°48'N, 67°20'N and 69°22'N, were used. The commercial cultivar Undrom was used as a reference plant. The experiments were done at 18 and 9°C under controlled conditions in a phytotron during the natural growing season at 69° 39'N. Growth of the plants was evaluated by number and size of leaves, dry matter production and total N-content. At 18°C the white clover plants were harvested twice while at 9°C there was only one growth period. The results from first harvest at 18°C and total growth at 9°C, showed that white clover populations from northern Norway had a lower growth potential than the population from the south and cv. Undrom. This difference was not apparent in the second growth period at 18°C. Growth of the plants from seeds to first harvest was enhanced by mineral nitrogen compared to plants dependent on Rhizobium only. However, after a second growth period dry weight and total nitrogen content of the plants with nitrogen fixation were comparable to the plants receiving mineral nitrogen. Statistical analysis showed that the most important factor for the variation in dry matter production was the plant population. Within the populations at 9°C and at first harvest at 18°C, there were no significant differences in dry matter production with different Rhizobium inoculum. In the second growth period at 18°C, different inoculum gave significantly different amount of dry matter within a population. The results showed a significant interaction between plant population and Rhizobium inoculum, and the results indicated that plants from the north gave higher yield when nodulated by Rhizobium from the north than from the south.     

Rhizobial polysaccharide-degrading enzymes from roots of legumes

The surface structure and chemistry of symbiotic bacteria from the genus Rhizobium are probably important for the outcome of the infection of legume hosts. Exopolysaccharide, capsular polysaccharide, lipopolysaccharide and a low-molecular-weight polysaccharide were isolated from R. trifolii UTC 110-1 and R. leguminosarum UTC 114-5 and partially characterized. No or only minor differences in sugar composition could be found for the corresponding fractions from the two organisms. A general method to measure low activities of polymer-degrading enzymes was developed, and used to determine enzyme activities in root extracts of Trifolium repens L. cv. Lena and Pisum xativiini L. cv. Little Marvel against the isolated rhizobial polysaccharides. An enzyme preparation from T. repens partially degraded all polysaccharides isolated from its symbiont R. trifolii while polysaccharides from R. leguminosarum , symbiont of P. sativum , were degraded to a much lesser extent. Correspondingly, an enzyme preparation from P. sativum degraded all polysaccharides isolated from both its symbiont R. leguminosarum and its non-symbiont R. trifolii. The amount of symbiont polysaccharides degraded was larger than the amount of polysaccharides degraded from the non-symbiont R. trifolii.     

Neighbourhood effect of genotypes of Rhizobium leguminosarum biovar trifolii, Trifolium repens and Lolium perenne

 Ryegrass, white clover and Rhizobium isolated from the corresponding clover nodules, were harvested from a natural pasture in the Massif Central mountains (France). The specificity between Lolium, Trifolium and Rhizobium, and the genetic diversity of Rhizobium were examined. This study showed that: 1) Natural neighbouring combinations of white clover and ryegrass, re-planted together in pots, accumulated a higher biomass than non-neighbouring ones. This increase of mass is higher in the presence of the native strain of Rhizobium. 2) When white clover was inoculated with a mixture of Rhizobium strains, nodules were more often formed by its native strain. 3) The genetical diversity of the Rhizobium leguminosarum biovar trifolii was very high, as revealed by electrophoresis of esterases on seven substrates. These results support the hypothesis that there is a co-adaptation between white clover, ryegrass and Rhizobium Received: 25 March 1996 / Accepted: 13 September 1996     

Comparison of geographically distant populations of Rhizobium isolated from root nodules of Phaseolus vulgaris

Seventy-two rhizobial strains were isolated from the root nodules of french beans ( Phaseolus vulgaris ). They were sampled from two geographically distant field populations and 18 additional different sites in France. They were characterized by a) plasmid profiles, (b) RFLP analysis of total cellular DNA using various chromosomal and symbiotic gene probes (including nif H from Rhizobium etli bv. phaseoli ) and c) their ability to nodulate a potential alternative host, L. leucocephala. Over half of the isolates were ascribed to Rhizobium leguminosarum bv. phaseoli on the basis of the hybridization analysis, the possession of multiple copies of nif H and their inability to nodulate L. leucocephala. The remaining isolates belonged to 2 groups which were shown to be genomically distinct from R. leguminosarum bv. phaseoli, R. etli bv. phaseoli and R. tropici. Most members of these two groups shared with R. tropici the ability to nodulate L. leucocephala and, for isolates of only one of these groups, the presence of one copy of nif H. Members of each of the 3 taxa were widely distributed in France and circumstantial evidence of pSym transfer between them was shown. R. leguminosarum bv. phaseoli and one of the two novel groups co-occurred within the two geographically distant populations. Individual genotypes were conserved between them. The finding of a third taxon at various other locations indicated additional diversity among rhizobia nodulating beans.     

Nodulation of Trifolium repens at low pH by single and paired strains of Rhizobium leguminosarum biovar trifolii

Detailed individual nodulation profiles were obtained for five strains of Rhizobium leguminosarum biovar trifolii inoculated onto roots of Trifolium repens seedlings growing on an agar medium of pH 4.5. The time of appearance and the location of every nodule were noted for a period of 10 days after inoculation. Using these nodulation frequency profiles, pairings of strains were identified and six mixed-strain inoculation (1:1 ratio) experiments were subsequently performed at pH 4.5. Results from the mixed-inoculum experiments showed that the performance of a Rhizobium strain in single culture could not be reliably used to predict the outcome of a paired-inoculation study and that some seedlings were exclusively nodulated by rhizobia that performed poorly at low pH in single-culture inoculations. Received: 26 November 1996 / Accepted: 18 April 1997     

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 trifoliiexoB 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. Received: 31 May 1996 / Accepted: 18 December 1996     

Elevated atmospheric CO2 alters microbial population structure in a pasture ecosystem

An increase in concentration of atmospheric CO₂ is one major factor influencing global climate change. Among the consequences of such an increase is the stimulation of plant growth and productivity. Below‐ground microbial processes are also likely to be affected indirectly by rising atmospheric CO₂ levels, through increased root growth and rhizodeposition rates. Because changes in microbial community composition might have an impact on symbiotic interactions with plants, the response of root nodule symbionts to elevated atmospheric CO₂ was investigated. In this study we determined the genetic structure of 120 Rhizobium leguminosarum bv. trifolii isolates from white clover plants exposed to ambient (350 μmol mol^?1) or elevated (600 μmol mol^?1) atmospheric CO₂ concentrations in the Swiss FACE (Free‐Air‐Carbon‐Dioxide‐Enrichment) facility. Polymerase Chain Reaction (PCR) fingerprinting of genomic DNA showed that the isolates from plants grown under elevated CO₂ were genetically different from those isolates obtained from plants grown under ambient conditions. Moreover, there was a 17% increase in nodule occupancy under conditions of elevated atmospheric CO₂ when strains of R. leguminosarum bv. trifolii isolated from plots exposed to CO₂ enrichment were evaluated for their ability to compete for nodulation with those strains isolated from ambient conditions. These results indicate that a shift in the community composition of R. leguminosarum bv. trifolii occurred as a result of an increased atmospheric CO₂ concentration, and that elevated atmospheric CO₂ affects the competitive ability of root nodule symbionts, most likely leading to a selection of these particular strains to nodulate white clover.     

Plant phenology and genetic variability in root and nodule development strongly influence genetic structuring of Rhizobium leguminosarum biovar viciae populations nodulating pea

The symbiotic relationships between legumes and their nitrogen (N(2))-fixing bacterial partners (rhizobia) vary in effectiveness to promote plant growth according to both bacterial and legume genotype. To assess the selective effect of host plant on its microsymbionts, the influence of the pea (Pisum sativum) genotype on the relative nodulation success of Rhizobium leguminosarum biovar viciae (Rlv) genotypes from the soil populations during plant development has been investigated. Five pea lines were chosen for their genetic variability in root and nodule development. Genetic structure and diversity of Rlv populations sampled from nodules were estimated by molecular typing with a marker of the genomic background (rDNA intergenic spacer) and a nodulation gene marker (nodD region). Differences were found among Rlv populations related to pea genetic background but also to modification of plant development caused by single gene mutation. The growth stage of the host plant also influenced structuring of populations. A particular nodulation genotype formed the majority of nodules during the reproductive stage. Overall, modification in root and nodule development appears to strongly influence the capacity of particular rhizobial genotypes to form nodules.     

The construction of recA–deficient Rhizobium meliloti and R. leguminosarum strains marked with gusA or luc cassettes for use in risk–assessment studies

A vector system was developed employing the recA genes of Rhizobium meliloti and Rhizobium leguminosarum biovar. viciae as target sequences for the stable genomic integration of foreign DNA. The plasmid vectors can be used either as integration vectors (single cross–over), or as gene replacement vectors (double cross–over). Gene replacement results in the antibiotic–marker–free integration of cloned DNA into the recA genes of R. meliloti and R. leguminosarum bv. viciae. Consequently, the recombinant strains become recombination deficient (RecA^-). The expression of integrated genes is under the control of the neomycin phosphotransferase II (nptll) promoter of transposon Tn5. The system was used to construct recA mutant strains of R. meliloti and R. leguminosarum by. viciae, carrying the Escherichia coli gusA gene encoding β–glucuronidase as well as the firefly (Photinus pyralis) luc gene encoding luciferase as marker genes. The GUS activity in the constructed strains was found to be absolutely stable over more than 100 generations of non–selective growth in liquid culture. The stability was also confirmed in root–nodule passages. In addition, the potential use of the luc gene as a stable genetic marker in the unequivocal identification of tagged strains among indigenous microbes in non–sterile soil was demonstrated. It is proposed to use bioluminescent recA mutants as model organisms in risk assessment studies with genetically engineered Rhizobium strains.     

Isolation of root hairs from seedlings of Pisum sativum. Identification of root hair specific proteins by in situ labeling

A procedure was developed which allows the large-scale isolation of root hairs from seedlings of Pisum sativum . L. cvs. Kleine Rheinländerin and Rosa Krone. The method may yield up to 50 g fresh weight of root hairs per 3.10⁴ seedlings. In a modified form considerable amounts of root hair material may be harvested, even after incubation of the roots in aqueous solutions. Thus, detailed biochemical studies on the root hair system have become feasible.
The occurrence of specific proteins in membrane fractions of P. sativum root hairs was demonstrated as follows: Incubation of root hairs in situ with 3-azidonaphthalene-2,7-disulfonate – a strongly anionic, photoactivated fluorescent marker – followed by gel electrophoresis of membrane fractions showed the presence of root-hair specific proteins which, since the system was intact, suggests that they are on the outer surface of the cells.     

Natural endophytic association between Rhizobium leguminosarum bv. trifolii and rice roots and assessment of its potential to promote rice growth

For over 7 centuries, production of rice (Oryza sativa L.) in Egypt has benefited from rotation with Egyptian berseem clover (Trifolium alexandrinum). The nitrogen supplied by this rotation replaces 25- 33% of the recommended rate of fertilizer-N application for rice production. This benefit to the rice cannot be explained solely by an increased availability of fixed N through mineralization of N- rich clover crop residues. Since rice normally supports a diverse microbial community of internal root colonists, we have examined the possibility that the clover symbiont, Rhizobium leguminosarum bv. trifolii colonizes rice roots endophytically in fields where these crops are rotated, and if so, whether this novel plant-microbe association benefits rice growth. MPN plant infection studies were performed on macerates of surface-sterilized rice roots inoculated on T. alexandrinum as the legume trap host. The results indicated that the root interior of rice grown in fields rotated with clover in the Nile Delta contained 10⁶ clover-nodulating rhizobial endophytes g fresh weight of root. Plant tests plus microscopical, cultural, biochemical, and molecular structure studies indicated that the numerically dominant isolates of clover-nodulating rice endophytes represent 3 – 4 authentic strains of R. leguminosarum bv. trifolii that were Nod Fix on berseem clover. Pure cultures of selected strains were able to colonize the interior of rice roots grown under gnotobiotic conditions. These rice endophytes were reisolated from surface-sterilized roots and shown by molecular methods to be the same as the original inoculant strains, thus verifying Koch's postulates. Two endophytic strains of R. leguminosarum bv. trifolii significantly increased shoot and root growth of rice in growth chamber experiments, and grain yield plus agronomic fertilizer N-use efficiency of Giza-175 hybrid rice in a field inoculation experiment conducted in the Nile Delta. Thus, fields where rice has been grown in rotation with clover since antiquity contain Fix strains of R. leguminosarum bv. trifolii that naturally colonize the rice root interior, and these true rhizobial endophytes have the potential to promote rice growth and productivity under laboratory and field conditions.     

In vivo expression technology (IVET) selection of genes of Rhizobium leguminosarum biovar viciae A34 expressed in the rhizosphere

IVET was used to identify genes that are specifically expressed in the rhizosphere of the pea-nodulating bacterium Rhizobium leguminosarum A34. A library of R. leguminosarum A34 cloned in the integration vector pIE1, with inserts upstream of a promoter-less purN:gfp:gusA, was conjugated into purN host RU2249 and recombined into the genome. After removal of colonies that expressed the reporter genes of the vector under laboratory conditions, the library was inoculated into a nonsterile pea rhizosphere. The key result is that 29 rhizosphere-induced loci were identified. Sequence analysis of these clones showed that a wide variety of R. leguminosarum A34 genes are expressed specifically in the rhizosphere including those encoding proteins involved in environmental sensing, control of gene expression, metabolic reactions and membrane transport. These genes are likely to be important for survival and colonization of the pea rhizosphere.     

The glnB gene of Rhizobium leguminosarum biovar viceae

Abstract An open-reading frame (ORF111) upstream of the glutamine synthetase I structural gene ( glnA ) in Rhizobium leguminosarum biovar viceae encodes a protein which is highly homologous to the P_II protein (encoded by glnB ) of enteric bacteria. ORF111 was cloned in a number of different plasmid vectors and shown to complement a K. pneumoniae glnB mutant. We propose that ORF111 encodes the P_II protein of R. leguminosarum and that it should be designated glnB .     

Construction and characterization of a Rhizobium leguminosarum biovar viciae strain designed to assess horizontal gene transfer in the environment

Abstract An integration vector was developed which inserts cloned DNA in a non-essential site of the Rhizobium leguminosarum biovar viciae chromosome. The expression of integrated genes is under the control of the constitutive neomycin phosphotransferase II ( npt II) promotor of transposon Tn5. The design of the vector ensures that loss of vector sequences can be detected, enabling selection of progeny containing only the requisite DNA. The newly constructed vector was employed to insert the Escherichia coli gusA gene conferring GUS activity into R. leguminosarum bv. viciae strain LRS39401 which is cured of its symbiotic plasmid (pSym). One GUS-positive transconjugant, strain CT0370, was shown to have lost all vector sequences. Conjugal transfer of pSym2004 (a Tn5-tagged derivative of symbiotic plasmid pRL1JI, which specifies pea nodulation and symbiotic nitrogen fixation) to CT0370, restored the GUS-positive strain's symbiotic proficiency. Strain CT0370 is presently being used in a field release experiment in order to assess the extent of pSym transfer in a natural R. leguminosarum bv. viciae population under environmental conditions.