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.     

Diversity and specificity of Rhizobium leguminosarum biovar viciae on wild and cultivated legumes

The symbiotic partnerships between legumes and their root-nodule bacteria (rhizobia) vary widely in their degree of specificity, but the underlying reasons are not understood. To assess the potential for host-range evolution, we have investigated microheterogeneity among the shared symbionts of a group of related legume species. Host specificity and genetic diversity were characterized for a soil population of Rhizobium leguminosarum biovar viciae (Rlv) sampled using six wild Vicia and Lathyrus species and the crop plants pea (Pisum sativum) and broad bean (Vicia faba). Genetic variation among 625 isolates was assessed by restriction fragment length polymorphism (RFLP) of loci on the chromosome (ribosomal gene spacer) and symbiosis plasmid (nodD region). Broad bean strongly favoured a particular symbiotic genotype that formed a distinct phylogenetic subgroup of Rlv nodulation genotypes but was associated with a range of chromosomal backgrounds. Host range tests of 80 isolates demonstrated that only 34% of isolates were able to nodulate V. faba. By contrast, 89% were able to nodulate all the local wild hosts tested, so high genetic diversity of the rhizobial population cannot be ascribed directly to the diversity of host species at the site. Overall the picture is of a population of symbionts that is diversified by plasmid transfer and shared fairly indiscriminately by local wild legume hosts. The crop species are less promiscuous in their interaction with symbionts than the wild legumes.     

Hydrogenase genes are uncommon and highly conserved in Rhizobium leguminosarum bv. viciae

A screening for hydrogen uptake (hup) genes in Rhizobium leguminosarum bv. viciae isolates from different locations within Spain identified no Hup+ strains, confirming the scarcity of the Hup trait in R. leguminosarum. However, five new Hup+ strains were isolated from Ni-rich soils from Italy and Germany. The hup gene variability was studied in these strains and in six available strains isolated from North America. Sequence analysis of three regions within the hup cluster showed an unusually high conservation among strains, with only 0.5-0.6% polymorphic sites, suggesting that R. leguminosarum acquired hup genes de novo in a very recent event.     

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.     

Effects of age, supra-ambient oxygen and repeated assays on acetylene reduction and root respiration in pea

An open flow-through gas system was used to investigate the effect of plant age on nitrogenase activity in relation to root respiration (measured as CO₂ release) and supra-ambient O₂ levels in 24- to 51-day-old, nodulated Pisum sativum L. cv. Bodil. The effect of assaying plants repeatedly was also studied. The respiratory efficiency of nitrogenase [mol CO₂ (mol C₂H₄)⁻¹] and the relative decline in nitrogenase (EC 1.7.99.2) activity in response to introduction of C₂H₂ in the gas stream were unaffected by plant age. In contrast, the nitrogenase-linked respiration as a proportion of total root respiration increased with time. Accordingly, the specific respiration linked-to growth and maintenace of the noduled root system decreased with time. C₂H₂ reduction and root respiration were increased by supra-ambient O₂ levels, but the tolerance to high O₂ concentrations seemed to decrease with plant age. Repeated C₂H₂ assays on the same plants decreased their rate of growth and N accumulation: in addition, nitrogenase activity and root respiration were somewhat negatively affected. The results indicate that results from experiments with plants of different ages cannot always be directly compared, and that repeated C₂H₂ assays on the same plants should be applied with caution in physiological work.     

Characteristics of nitrate uptake into seedlings of pea (Pisum sativum L. cv. Feltham First). Changes in net NO−3 uptake following inoculation with Rhizobium and growth in low nitrate concentrations

Abstract Nitrate uptake into intact pea seedlings (Pisum sativum L. cv. Feltham First) grown in hydroponic culture has been investigated. Following inoculation with Rhizobium leguminosarum a twofold increase in net nitrate uptake was observed. Changes in morphological characteristics following inoculation were found to decrease the effective area available for absorption. There was a two-fold decrease in net nitrate uptake into intact seedlings grown in the presence of N compared with N free media. In the former case net nitrate uptake appeared to stall at regular intervals. In both cases only the initial rates of nitrate uptake were found to be responsive to the external nitrate concentration. The results are discussed in terms of current models for the regulation of NO^?₃ uptake by higher plants.     

Diversity and stability of plasmid transfer in isolates from a single field population of Rhizobium leguminosarum bv. viciae

Abstract Isolates of R. leguminosarum bv. viciae from pea and lentil nodules taken at one field site in France were tested in the laboratory for their ability to donate and receive plasmids by conjugation. Five isolates of 20 tested as donors were found to be capable of donating a plasmid which restored the ability to nodulate V. sativa to an isolate which had spontaneously lost this ability. Of 16 isolates tested as recipients all were found to be competent to receive one or more Tn5-labelled test plasmids at a frequency that varied widely (10⁻⁹− 10⁻³ per recipient) dependent upon both the recipient and the plasmid transferred. Three distinct plasmids carrying genes essential for symbiotic functions (pSym) were consistently shown to be transferred at a lower frequency than a cryptic plasmid. Collectively, these results indicate a significant potential for plasmid transfer within the natural soil population. During this work, several independent derivatives were obtained which contained two bv. viciae pSym. These plasmids usually appeared to be compatible together in cells ex planta, but the one acquired in matings was apparently frequently lost (10⁻² per cell) in nodules of V. sativa . Hybrid derivatives containing bv. viciae and bv. phaseoli pSym, apparently retained both plasmids in nodules when P. vulgaris was the host plant but lost the bv. phaseoli pSym at high frequency (4 × 10⁻¹ per cell) in nodules of V. sativa . Structural rearrangements among the plasmids of these transconjugants were also detected in cells recovered from nodules.     

Plasmid profiles and restriction fragment length polymorphism of Rhizobium leguminosarum bv. viciae in field populations

Abstract 56 isolates of Rhizobium leguminosarum biovar viciae from one field were characterized by analysis of plasmid profile, total DNA restriction pattern and restriction fragment length polymorphism (RFLP) of 2 chromosomal regions and of symbiotic (Sym) plasmid. Different levels of similarity exist in patterns generated by the different techniques. At the level of partial similarity these techniques give comparable results for more than 80% of the isolates, with the exception of RFLP profiling with the Sym probe. Analysis at this level allows the grouping of the isolates that have most of their non-Sym genome similarly organized. At the level of total similarity, the techniques are no more equivalent and provide complementary information on possible evolution of the different elements of the genome identified by each specific technique. The non-Sym plasmids defining classes were strongly associated with specific chromosomal backgrounds. In contrast, variations in Sym plasmids were not related with variations in the remaining genome. Host range towards chromosomes was variable among the Sym plasmids, which may reflect plasmid transfer between strains.     

Rhizobium strain effects on pea: The relation between nitrogen accumulation, phosphoenolpyruvate carboxylase activity in nodules and asparagine in root bleeding sap

Pisum sativum L. cv. Bodil was infected with various strains of Rhizobium leguminosarum (R501, 128c53, B155, 18a or 1044). The Rhizobium genotype influenced the activity of the plant enzyme phosphoenoipyruvate (PEP) carboxylase (EC 4.1.1.31), and the assimilation of fixed N in the root nodules. The specific activity of nodule PEP carboxylase was lowest in the symbioses, which accumulated the least total N (R501 and 128c53). The root bleeding sap of the less effective symbioses contained a lower proportion of asparagine and a higher proportion of glutamine than the more effective symbioses (B155,18a and 1044). The N yield of the symbioses was related neither to the net respiratory CO₂ evolution of the root system nor to the nitrogenase linked nodule respiration. The lower yielding symbioses accumulated a larger proportion of the fixed N in the nodules due to a higher proportion of total dry weight contained in the nodule tissue. However, the concentration of soluble protein in the nodules of the lower-yielding symbioses was lower than that recorded for the higher yileding symbioses. The effect of the Rhizobium strains on N yield was maintained at maturity, and reflected in seed yields.     

A hierarchical analysis of population genetic structure in Rhizobium leguminosarum bv. trifolii

Little is known about the population processes that shape the genetic diversity in natural populations of rhizobia. A sample of 912 Rhizobium leguminosarum biovar trifolii isolates were collected from naturalized red clover populations ( Trifolium pratense ) and analyzed for 15 allozyme loci to determine the levels and distribution of genetic diversity. Hierarchical analyses compared different sampling levels, geographical separation, and temporal separation. Total genetic diversity across all isolates was H = 0.426, with 57.6% of the total diversity found among isolates obtained from individual red clover plants. Relatively low genetic differentiation among populations and high differentiation among plants within populations was observed; this suggests that gene flow and founder effect act differently at geographical and local scales. Significant differences were observed in (i) allele frequencies among populations and among plants within populations, and (ii) the frequency distribution of the most widespread and the most abundant strains. When multilocus linkage disequilibrium was calculated, significant levels of disequilibrium were observed in the total sample and in three of the eight populations.     

Effect of Seed Treatment with Rhizobium leguminosarum on Pythium Damping-off, Seedling Height, Root Nodulation, Root Biomass, Shoot Biomass, and Seed Yield of Pea and Lentil

Field experiments were conducted in 2004 and 2005 to determine the effects of seed treatment with Rhizobium leguminosarum bv. viceae on damping‐off, seedling height, root nodule mass, root biomass, shoot biomass and seed yield of pea and lentil in a field naturally infested with Pythium spp. Compared with the untreated controls, treatment of pea seeds with R. leguminosarum bv. viceae strains R12, R20 or R21 significantly (P < 0.05) reduced incidence of damping‐off, promoted seedling growth and increased root nodule mass, root biomass and shoot biomass. Seed treatments with R12 or R21 also resulted in a significant (P < 0.05) increase in seed yield of pea. The strain R21 was most effective among the four strains of R. leguminosarum bv. viceae tested in peas. Although, the level of disease control by strain R21 was similar to seed treatment with the fungicide Thiram^TM, R21 was more effective in enhancing root nodule production and promoting plant growth. For lentil, treatment of seeds with R. leguminosarum bv. viceae strains R12 or R21 significantly (P < 0.05) reduced incidence of damping‐off compared with the untreated control. All of the four strains of R. leguminosarum bv. viceae tested increased lentil seedling height, root nodule mass and shoot biomass, and all except R20 increased root biomass. Seed yield was higher for the treatments of R12 and R21. The strain R12 was most effective among the four strains of R. leguminosarum bv. viceae tested in lentil. Although, strain R12 was as effective as Thiram^TM for control of damping‐off of lentil, it was more effective than Thiram^TM for the production of root nodules and promotion of plant growth. The study concludes that seed treatment with R. leguminosarum bv. viceae is effective in control of Pythium damping‐off of pea and lentil and that the efficacy of control is strain specific, strain R21 for control of the disease on pea and strain R12 for control of the disease on lentil.     

Nitrate reductase activity in nodules of pea inoculated with hydrogenase positive and hydrogenase negative strains of Rhizobium leguminosarum

The nitrate reductase (NR, EC 1.6.6.1) activity in root nodules formed by hydrogenase positive (Hup⁺) and hydrogenase negative (Hup⁻) Rhizobium leguminosarum strains was examined in symbioses with the pea cultivar Alaska ( Pisum sativum L.), Rates of activity were determined by the in vivo assay in nodules from plants that were only N₂-dependent or grown in the presence of 2 m M KNO₃. The rates varied widely among strains, regardless of the Hup phenotype of the R. leguminosarum strain used for inoculation, but the overall results indicated that nodules formed by Hup⁻ strains accumulated more nitrite in the incubation medium than did those with Hup⁻ phenotypes. Total plant dry weight and reduced nitrogen content of pea plants grown in the presence of 2 m M KNO₃ and inoculated with single Hup⁺ and Hup⁻ R. leguminosarum strains were statistically different among some strains. These observations suggest that the possible advantages derived from the presence of the Hup system on whole plant growth may be counteracted by the higher rates of NR activity in the Hup⁻ strains in the R. leguminosarum -pea symbiosis.     

Population level processes in Rhizobium leguminosarum bv. trifolii: the role of founder effects

The importance of genotype-specific selection between host and symbiont, founder effect, and clonal reproduction in Rhizobia leguminosarum biovar trifolii populations is relatively unknown. A field experiment was conducted to sample 1268 isolates of R. l. bv. trifolii from four genotypically distinct Trifolium pratense plants for allozyme variation at nine loci. Genetic and genotypic variation, population genetic substructure, and linkage disequilibrium were estimated. Of the 1268 isolates 188 genotypically distinct strains (electrophoretic types or ETs) were identified with an average of 11.04 different ETs per plant. Total genetic diversity in the plot was 0.346 and most of the variation was found within plants (= 80%). Our data suggests that genotype-specific selection between the rhizobia and the four host-plant genotypes tested does not influence local population structure, but evidence of founder effect was present. Significant linkage disequilibrium was observed and is most likely due to the clonal reproduction of R. l. bv. trifolii.     

The Involvement of Endogenous Phenolic Compounds in the Response of Pea Seedling Roots to Inoculation with Rhizobium leguminosarum at Various Temperatures

Endogenous phenolic compounds (PC) affecting Rhizobium leguminosarum bv. viceae propagation were isolated from the roots of etiolated pea (Pisum sativum L.) seedlings before and within one or two day after inoculation. It was established that, during the first day after inoculation, PC-induced stimulation of bacterial growth in roots was replaced by its inhibition, which was somewhat more pronounced at 8°C. The ratio between PC fractions was also changed during the first day after inoculation, especially strongly at low temperature; and this was evidently the cause for Rhizobium growth inhibition in root cells.     

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.     

Plasmids and saprophytic growth of Rhizobium leguminosarum bv. trifolii W14-2 in soil

Abstract: The role of plasmids in the saprophytic growth of Rhizobium is mostly unknown. Plasmid-cured and complemented derivatives of R. leguminosarum bv. trifolii strain W14-2 were used to investigate the role of plasmids in the growth of this strain in sterile soil incubated under favorable moisture and temperature conditions. Strain W14-2 contains four plasmids ( a,b,c,d ). Absence of single plasmids in plasmid-cured derivatives generally did not reduce growth in soil when compared to the wild-type but absence of plasmid a delayed growth. Derivatives were unable to grow in soil when only plasmids a or d were present in cells. When only plasmids b or c were present, growth was delayed and the final population in 7 days was approximately 10% of the wild-type population. When the wild-type was co-inoculated at equal population into soil with derivatives lacking plasmids a , c , or d, the population of the wild-type at 7 days incubation was approximately 10 times larger than those of the derivatives. Elimination of only plasmid b did not reduce the ability of the strain to grow in soil when competing with the wild-type. Plasmids were involved in saprophytic growth of strain W14-2 in soil and may be important to the ecology of Rhizobium .