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.     

Rhizobium leguminosarum bv. viciae genotypes interact with pea plants in developmental responses of nodules, roots and shoots

The variability of the developmental responses of two contrasting cultivars of pea (Pisum sativum) was studied in relation to the genetic diversity of their nitrogen-fixing symbiont Rhizobium leguminosarum bv. viciae. A sample of 42 strains of pea rhizobia was chosen to represent 17 genotypes predominating in indigenous rhizobial populations, the genotypes being defined by the combination of haplotypes characterized with rDNA intergenic spacer and nodD gene regions as markers. We found contrasting effects of the bacterial genotype, especially the nod gene type, on the development of nodules, roots and shoots. A bacterial nod gene type was identified that induced very large, branched nodules, smaller nodule numbers, high nodule biomass, but reduced root and aerial part development. The plants associated with this genotype accumulated less N in shoots, but N concentration in leaves was not affected. The results suggest that the plant could not control nodule development sustaining the energy demand for nodule functioning and its optimal growth. The molecular and physiological mechanisms that may be involved are discussed.     

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.     

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.     

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.     

Effects of elevated oxygen tensions on acetylene reduction in Alnus incana-Frankia symbioses

An open flow-through gas system was used to determine the effect of C₂H₂ and elevated O₂ on acetylene reduction activity (ARA) and respiration of the intact, potted root system of Alnus incana (L.) Moench in symbiosis with Frankia Avcll or with a local source of Frankia . Both symbiotic systems responded to C₂H₂ by an immediate plateau range in ARA. The Plateau in ARA was in some cases followed by a decline of less extent than reported for many legumes. A concurrent decline in net respiration of the root system was on average 8% of the CO₂ efflux prior to C₂H₂ introduction.
Respiration of the root systems in both symbioses responded to elevated oxygen levels in the 10 kPa C₂H₂ atmosphere by an increase of up to 17% of the net respiration prior to C₂H₂ introduction in 21 kPa O₂. In contrast, the elevated oxygen levels resulted in an immediate drop in ARA followed by a minor increase to a stable level lower than that at the preceding, lower oxygen tension. The symbiosis with the local Frankia had lost all ARA when the partial pressure of O₂ exceeded 50 kPa, whereas the symbiosis with Avcll still had some activity at 80 kPa O₂. This difference in tolerance of elevated O₂ clearly shows that the oxygen exclusion mechanisms may be controlled by the microsymbiont in Alnus-Frankia symbioses. The symbiotic systems recovered ARA to a similar extent when returned from elevated O₂ levels to 21 kPa O₂.     

Genetic variability in nodulation and root growth affects nitrogen fixation and accumulation in pea

BACKGROUND AND AIMS: Legume nitrogen is derived from two different sources, symbiotically fixed atmospheric N(2) and soil N. The effect of genetic variability of root and nodule establishment on N acquisition and seed protein yield was investigated under field conditions in pea (Pisum sativum). In addition, these parameters were related to the variability in preference for rhizobial genotypes. METHODS: Five different spring pea lines (two hypernodulating mutants and three cultivars), previously identified in artificial conditions as contrasted for both root and nodule development, were characterized under field conditions. Root and nodule establishment was examined from the four-leaf stage up to the beginning of seed filling and was related to the patterns of shoot dry matter and nitrogen accumulation. The genetic structure of rhizobial populations associated with the pea lines was obtained by analysis of nodule samples. The fraction of nitrogen derived from symbiotic fixation was estimated at the beginning of seed filling and at physiological maturity, when seed protein content and yield were determined. KEY RESULTS: The hypernodulating mutants established nodules earlier and maintained them longer than was the case for the three cultivars, whereas their root development and nitrogen accumulation were lower. The seed protein yield was higher in 'Athos' and 'Austin', the two cultivars with increased root development, consistent with their higher N absorption during seed filling. CONCLUSION: The hypernodulating mutants did not accumulate more nitrogen, probably due to the C cost for nodulation being higher than for root development. Enhancing exogenous nitrogen supply at the end of the growth cycle, by increasing the potential for root N uptake from soil, seems a good option for improving pea seed filling.     

The effects of soybean cultivar,rhizobium strain and nitrate on plant growth,nodule mass and acetylene reduction rate

Summary The effects of twelve strains ofBradyrhizobium japonicum and ten cultivars of soybean (Glycine max (L.) Merr.) on plant and nodule weights, and acetylene reduction rates (33 to 41 days) were measured in the presence and absence of 6mM nitrate. No interactions between strains and cultivars were observed. Strain by nitrate interactions were found for plant and nodule weights, and acetylene reduction rates per gram of nodule. Cultivar by nitrate interactions were found for nodule weights, acetylene reduction rates per plant and per gram of nodule. Blackhawk with all strains, and all cultivars with strains 110 and CB 1809, seemed to be able to grow as well in the absence of nitrate (utilizing nodule fixation) as in its presence. The problems of identifying strains and cultivars with especially good nitrogen fixing ability in the presence of nitrate are discussed.     

Effect of nitrate on nitrogen fixation and nodule carbohydrate and organic acid concentrations in pea mutants deficient in nitrate reductase

Nitrate inhibits symbiotic N₂ fixation and a number of hypotheses concerned with NO₃⁻ assimilation have been suggested to explain this inhibition. These hypotheses were tested using a pea ( Pisum sativum L. cv. Juneau) with normal nitrate reductase NR; (EC 1,6,6,4) activity and two mutants of cv. Juneau, A317 and A334, with impaired NR activity. The plants were inoculated with three strains of Rhizobium leguminosarum and grown for 3 weeks in N-free medium, followed by 1 week in medium supplemented with 0, 5 or 10 m M KNO₃ before harvesting. NO₃ was taken up at comparable rates by the parent and the mutants and accumulated in leaf and stem tissue of the latter. Acetylene reduction rates were inhibited similarly in both the parent and mutants in the presence of KNO₃ but there were differences among rhizobial strains. Starch concentration of the nodules decreased by 46% in the presence of KNO₃ and there were differences among rhizobial strains but not among pea genotypes. Malate and succinate accumulated in nodules in the presence of KNO₃. These data are not consistent with the photosynthate deprivation hypothesis as a primary mechanism for NO₃ inhibition of N₂ fixation since NO₃ affected the nodule carbohydrate composition of all three pea genotypes in a similar manner. The lack of correlation between NR activity and NO₃ inhibition of N₂ fixation suggests that NO₃ assimilation may be only indirectly involved in the inhibition phenomenon.     

Beneficial interaction between photosynthesis and respiration in mesophyll protoplasts of pea during short light-dark cycles

The respiratory uptake or photosynthetic evolution of oxygen by mesophyll protoplasts of pea ( Pisum sativum L. cv. Arkel) were monitored during successive short. (3–5 min) cycles of darkness and illumination. The rate of respiration was nearly doubled after 3–4 short periods of illumination while there was a 15–20% enhancement in photosynthesis with cycles of illumination and darkness preceding illumination. Such interaction between photosynthesis and respiration was statistically significant when bicarbonate was present in the reaction medium. The inhibitors of photosynthesis [3(3,4–dichlorophenyl)-l,l-dimethylurea (DCMU), glyceraldehyde] decreased respiration after periods of illumination, whereas inhibitors of respiratory electron transport (Rotenone, antimycin A, NaN₃) suppressed photosynthesis, as well. We suggest that a rapid beneficial interaction exists between photosynthesis and respiration in protoplasts, even during short cycles of light and darkness.     

First report of non-mycorrhizal plant mutants (Myc) obtained in pea (Pisum sativum L.) and fababean (Vicia faba L.)

Genetic resistance to vesicular-arbuscular (VA) mycorrhiza formation has been obtained in spontaneous or chemically induced mutants of two mycorrhiza-forming species (Pisum sativum L. and Vicia faba L.). The eight mutants, termed myc⁻, are characterized by aborted infections limited to one or two host cells. Expression of the myc⁻ character is associated with that of the nod⁻ character in both legumes, and is likewise under recessive genetic control. Preliminary analysis of the genetic behaviour of the myc⁻ mutants in diallel crosses has shown that at least three genes are involved in VA mycorrhiza infection.     

The effect of butyrate on the cell cycle in root meristems of Pisum sativum

Sodium butyrate at 5 mM in aerated White's medium reduced the mitotic index in root meristems of seedlings of Pisum sativum to < 1% after 12 h. This effect was lessened as the butyrate concentrations were lowered. The fraction of the root meristem nuclei in G2 increased to ~ 70% after 12 h in butyrate. After 12 h exposure to butyrate, seedlings transferred lo medium without butyrate gradually re-established their normal root meristem mitotic pattern, with a burst of mitosis at 10 h after the transfer. Even a brief exposure to butyrate inhibited DNA synthesis, and nuclei released from butyrate exposure were still unable to resume normal DNA synthesis even after 12 h. This information suggests that butyrate halts progression through the cell cycle by arresting meristem nuclei in G2 and inhibiting DNA synthesis.     

Maximum axial root growth pressure in pea seedlings: effects of measurement techniques and cultivars

Values of the maximum axial growth pressure (σ_max) of seedling pea (Pisum sativum L.) roots reported in the literature, obtained using different apparatuses and cultivars, range from 0.3 MPa to 1.3 MPa. To investigate possible reasons for this large range, we studied the effect of apparatus and cultivar on measurements of σ_max in peas. We describe four types of apparatus which can be used to measure axial root growth force and hence σ_max, and used them to measure σ_max in seedling pea roots using cultivar Meteor. Two of these apparatuses were also used to compare σ_max for three pea cultivars (Helka, Meteor and Greenfeast). Both cultivar and apparatus significantly affected σ_max , but there were greater differences between apparatuses than between the three cultivars. Estimating root cross-sectional area from the diameter of cross-sections, rather than from in situ measurements (i.e. measurements made with the root still in place in the apparatus) may explain these differences. This revised version was published online in June 2006 with corrections to the Cover Date.     

Light-enhanced dark respiration in leaves and mesophyll protoplasts of pea in relation to photorespiration,respiration and some metabolites content

The respiration rate of leaves and mesophyll protoplasts of pea (Pisum sativum L.), from plants which were previously kept in darkness for 24 h was doubled following a period of photosynthesis at ambient level of O₂ (21 %), whereas the low level of O₂ (1 % and 4 % for leaves and protoplasts, respectively) reduced this light-enhanced dark respiration (LEDR) to the rate as noted before the illumination. Similarly to respiration rate, the oxygen at used concentrations had no effect on the ATP/ADP ratio in the dark-treated leaves. However, the ATP/ADP ratio in leaves photosynthesizing at 21 % O₂ was higher (up to 40 %, dependence on CO₂ concentration in the range 40–1600 1 dm⁻³) than in those photosynthesizing at 1 % O₂ or darkened at air (21 % O₂). Also, at 1 % O₂ the accumulation of malate was suppressed (by about 40 %), to a value noted for leaves darkened at 21 % O₂. The dark-treatment of leaves reduced the ability of isolated mitochondria to oxidize glycine (by about twofold) and succinate, but not malate. Mitochondria from both the light- and dark-treated leaves did not differ in qualitative composition of free amino acids, however, there were significant quantitative differences especially with respect to aspartate, alanine, glutamate and major intermediates of the photorespiratory pathway (glycine, serine). Our results suggest that accumulation of photorespiratory and respiratory metabolites in pea leaves during photosynthesis at 1 % O₂ is reduced, hence the suppression of postillumination respiration rate.     

Gas-exchange activity,carbohydrate status,and protein turnover in root nodule subpopulations of field pea (Pisum sativum L. cv. Century)

Root nodule ontogeny was followed in different parts of the root system of field peas (Pisum sativum L. cv. Century) to investigate the contribution to total nitrogen fixation by different nodule subpopulations. Seed-inoculated plants were grown to maturity in controlled-environment growth chambers. In a flow-through system nitrogenase activity (H₂-evolution in air) and nodulated-root respiration (net CO₂-evolution) were measured weekly or biweekly in different parts (top and mid) of the root system. Root nodule extracts were assayed for total soluble cytosolic protein, total heme, proteolytic capacity (at pH 7.0), soluble carbohydrates and starch. Total nitrogenase activity and nodule respiration were higher in the top zone, which was explained by differences in root and nodule mass. Nodule specific nitrogenase activity was similar in both zones, and gradually declined throughout the experiment. No differences were found between nodule subpopulations in the dry-matter specific concentrations of glucose, fructose, sucrose or starch. Neither did nodule concentrations of protein or leghemoglobin differ between the zones. Throughout reproductive growth, no decline was found in total or nodule specific nitrogenase activity, in any of the nodule subpopulations. Growth of the root nodules continued throughout the experiment, though growth of shoot and roots had ceased. The data gives no support for carbohydrate limitation in root nodules during pod-filling, since nodule respiration remained high, the concentration of soluble carbohydrates increased significantly, and the amount of starch was not reduced. We conclude that when this symbiosis is grown under controlled conditions, nitrogenase activity in nodules sub-sampled from the crown part of the root system is representative for the whole nodule population.     

Neutral growth inhibitors in light-grown dwarf pea shoots –separation, identification and growth regulation

To correlate endogenous growth inhibitors of dwarf pea to its dwarfism a thorough search of growth inhibitors was made in the neutral fraction of an acetone extract from the shoots of light-grown seedlings of Pisum sativum L. cvs Progress No 9 and Alaska. From both cultivars six inhibitors were separated and named A-1, A-2, A-3, B-1, B-2 and B-3 based on their order of elution from the silica gel column. Their contents as determined by cress root bioassay were: in cv. Progress 0.40, 16.5, 6.36, 1.02, 0.11 and 0.10, and in cv. Alaska 0.33, 2.35, 3.51, 0.95, 0.10 and 0.09 cress units (g fresh weight)⁻¹. Their contributions to growth regulation of the relevant pea plants were estimated as the products of the above-stated contents times the ratios of the specific activities of each standard sample in the cress roots and the relevant pea cultivars, and they were; in cv. Progress A-2, 5.44 and A-3, 2.10, and in cv. Alaska A-2, 0.68 and A-3, 0.88, those of A-2 and A-3 constituting more than 90% of the total contribution of the six inhibitors in either cultivar. The great differences in the content and contribution to growth regulation of A-2 and A-3 between the two cultivars suggest that the higher contents of and greater responsiveness to the two inhibitors in cv. Progress may be causes of dwarfism of this cultivar. A-l and A-3 were spectroscopically identified with pisatin and a mixture of cis, trans– , and trans, trans xanthoxins.