Relative energy efficiency of nitrogen fixation by nodules of chickpeas (Cicer arietinum L.) produced by different strains ofRhizobium

Hydrogen evolution from a nitrogenase-catalyzed reaction in nodules has been proposed as one of the main causes of inefficiency in theRhizobium-legume symbiosis. We have evaluated the energy efficiency of the nitrogen fixation by chickpeas as affected by specific strains ofRhizobium. All seventeen strains tested produced nodules that evolved amounts of hydrogen ranging from 2.4 to 7.5 μmol/h×g fresh nodule weight. The equivalent energy losses represented from 31% to 58% of the nitrogenase activity estimated as acetylene reduction. No hydrogen uptake hydrogenase activity was detected in any of the bacteroid suspensions from nodules produced by the strains examined. The need to select strains of chickpea rhizobia with higher energy efficiency to improve productivity is emphasized.     

Nitrate Effect on Nitrogen Fixation (Acetylene Reduction): ACTIVITIES OF LEGUME ROOT NODULES INDUCED BY RHIZOBIA WITH VARIED NITRATE REDUCTASE ACTIVITIES

The effect of nitrate on symbiotic nitrogen fixation by root nodules of cowpea (Vigna unguiculata L., Walp., cv. California Blackeye) and lupine (Lupinus augustifolius L., cv. Frost) plants inoculated with nitrate reductase-expressing and nitrate reductase-nonexpressing Rhizobium strains were examined. Nitrate reductase of Rhizobium bacteroids in the nodules of cowpea and lupine reduced nitrate to nitrite. Both cowpea and lupine nodules accumulated nitrite when grown in the presence of 15 millimolar nitrate and induced by Rhizobium strains which express nitrate reductase activity (Rhizobium sp. 32H1 and 127E15). The nitrogen fixation (acetylene reduction) activities of cowpea and lupine nodules were inhibited by nitrate whether the nodules were induced by Rhizobium strains that express (Rhizobium sp. 32H1 and 127E15) or do not express (Rhizobium sp. 127E14 and R. lupini ATCC 10318) nitrate reductase activity. These findings indicate that nitrite, the product of bacteroid nitrate reductase, may not play a role in the inhibitory effect of nitrate on nitrogen fixation activities of legume root nodules. However, the degree of inhibition on the fixation activity by nitrate varied in different legume-Rhizobium combinations.     

Hydrogen uptake by Robinia pseudoacacia nodules

Summary Hydrogen uptake is thought to increase the efficiency of nitrogen fixation by recycling H₂ produced by nitrogenase that would otherwise be lost by diffusion. Here we demonstrate the capacity of eight Rhizobium strains to take up molecular hydrogen. Uptake by nodule homogenates from Robinia pseudoacacia was measured amperometrically under nitrogenase repression. Markedly lower activities were found than in soybean nodules. In addition hydrogenase activity was detected by the ability of bacteroids to reduce methylene blue in the presence of hydrogen. It was demonstrated that hydrogenase structural genes are present in the black locust symbiont, Rhizobium sp. strain R1, using hybridization with a plasmid, which contained hydrogenase genes from R. leguminosarum bv. viceae.     

Selection of symbiotically energy efficient strains of Rhizobium japonicum by their ability to induce a H2-uptake hydrogenase in the free-living state

The capacity of inducing a H₂-uptake hydrogenase in free-living cultures was examined in 21 strains of Rhizobium japonicum. Four strains were found to take up H₂ at rapid rates after 3 days of growth on agar slants inside sealed vials provided with an atmosphere of 5% H₂ in air. Soybean nodules from these strains lost little or no H₂ in air and their bacteroids oxidized H₂ at rates that were similar to those observed in free-living cultures. In contrast, three randomly chosen strains of R. japonicum that showed no H₂-uptake capacity in free-living state produced nodules which lost large amounts of H₂ and the corresponding bacteroids had no hydrogenase activity. A screening procedure is described for the selection of Rhizobium strains producing high energy-efficient nodules based on a test of their ability to induce a H₂-uptake hydrogenase in asymbiotic conditions.     

蚕豆根瘤菌接种效应研究

为发掘和利用青海冷凉地区蚕豆优良的根瘤菌种质资源,确定根瘤菌的接种效应。将分离、纯化、分子鉴定的16株蚕豆根瘤菌通过盆栽回接试验的方法进行筛选。结果表明,筛选出6株根瘤菌,它们与青海13号蚕豆共生匹配效果较好,共生固氮能力强,促进蚕豆生长效果明显。     

Relative efficiency of nitrogen fixation and the occurrence of hydrogenase in pea root nodules

Summary The apparent K_m(hydrogen) for uptake of hydrogen by pea root nodules was determined. This enabled the concentration gradient necessary for the evolution of hydrogen to be calculated for nodules with no hydrogenase activity. This indicated that hydrogen inhibition of nitrogenase is not likely to be the cause of the low relative efficiency of legume root nodules. The factors that affect electron allocation between protons and nitrogen in nitrogenase are reviewed and it is concluded that there must be some as yet unknown factor that affects electron distribution inRhizobium nitrogenase. One possibility is put forward and considered. A strain ofRhizobium was used that was found to possess hydrogenase activity in combination with pea variety Feltham First but not with variety Meteor. The control of this enzyme is briefly discussed.     

Nodule-specific host proteins in effective and ineffective root nodules of Pisum sativum

Nodule-specific root proteins – so called nodulins – were identified in root nodules of pea plants by an immunological assay. Nodulin patterns were examined at different stages of nodule development. About 30 nodulins were detectable during development. Some were preferentially synthesized before nitrogen fixation started, whereas the majority were synthesized concomitantly with leghaemoglobin. Some of the nodulins were located within the peribacteroid membrane. Ineffective Rhizobium strains (a natural nod⁺fix^- and a pop ^-fix^-) appeared to be useful in studying the expression of nodulin genes. Synthesis of some nodulins was repressed in ineffective root nodules, indicating that nodulins are essential for the establishment of nitrogen fixation. In both types of ineffective root nodules, leghaemoglobin synthesis was not completely repressed. Low amounts of leghaemoglobin were always detected in young ineffective root nodules whereas in old nodules no leghaemoglobin was present.     

Interaction between alfalfa cultivars and Rhizobium strains for nitrogen Fixation

Summary Two experiments were conducted in the greenhouse to study the interaction between alfalfa cultivars (Medicago sativa L. and M. falcata L.) and strains of Rhizobium meliloti Dang. for acetylene reduction rate, plant height and dry weights of shoot, root and whole plant. Fifteen alfalfa cultivars were inoculated with 10 strains of Rhizobium in Experiment I. Variance component analysis revealed that more than 30% of the total variance was due to alfalfa cultivars for acetylene reduction rate and 26% was accounted for by Rhizobium strains. More than 36% of the total variation was attributed to the interaction between alfalfa cultivars and Rhizobium strains for this character. Twenty-five host cultivars and 11 Rhizobium strains were included in Experiment II. The results also showed that the interaction of alfalfa cultivars and Rhizobium strains contributed the largest portion of the total variation for dry weights of shoot, root and whole plant and acetylene reduction rate. The results clearly demonstrated that the non-additive effects were the major component of variation for these characters associated with nitrogen fixation in alfalfa. Therefore, an effective way of improving nitrogen fixation in alfalfa is to select for a favourable combination of specific Rhizobium strains and alfalfa cultivars.     

Hydrogen production and uptake by pea nodules as affected by strains of Rhizobium leguminosarum

Hydrogen evolution from root nodules has been reported to make N₂ fixation by some legume-Rhizobium symbiotic systems inefficient. We have surveyed the extent of H₂ evolution and estimated relative efficiencies of nodules of Austrian winter peas formed by 15 strains of R. leguminosarum. Their rates of H₂ evolution in air were about 30% of the rates of H₂ evolution under an atmosphere in which N₂ was replaced by Ar. Relative efficiency values based on C₂H₂ reduction rates ranged from 0.55 to 0.80. With some of the strains, hydrogenase activities were demonstrated in intact nodules and in bacteroids, but the levels of activity were insufficient to recycle all the H₂ evolved by the nitrogenase system. In both intact nodules and bacteroids the hydrogenase is less sensitive to O₂ damage than the nitrogenase system, so H₂ uptake capacity was observed in intact nodules by suppressing the nitrogenase-dependent H₂ evolution with an atmosphere containing a high O₂ concentration, and in bacteroids by using aerobically prepared bacteroid suspensions. The hydrogenase activity of both was dependent on O₂ consumption. A K _mfor H₂ of near 4 M was determined in suspension of bacteroids from nodules formed by strains 128C53 and 128C56.     

Hydrogen Oxidation by the Host-Controlled Uptake Hydrogenase Phenotype of Bradyrhizobium japonicum in Symbiosis with Soybean Host Plants

Symbioses between uptake hydrogenase host-regulated (Hup-hr) phenotypes of Bradyrhizobium japonicum and exotic, agronomically unadapted soybean germ plasm were examined for expression of uptake hydrogenase activity. Determinations for hydrogen evolution and uptake hydrogenase activity identified five plant introduction (PI) lines which formed hydrogen-oxidizing symbioses with strains USDA 61 and PA3 6c. Hup-hr strains belonging to serogroup 94 expressed uptake hydrogenase activity in symbioses with PI 181696 and PI 219655 at rates sufficient to prevent hydrogen from escaping the nodules. The identification of soybean germ plasm forming hydrogen-oxidizing symbioses with Hup-hr bradyrhizobia potentially has implications for enhancing nitrogen fixation efficiency in soybean production.     

Studies on symbiotic nitrogen fixation by a new strain of tetraploid red clover (UO36)

Summary Some characteristics of the symbiotic nitrogen fixation by tetraploid red clover (UO36) have been studied in laboratory experiments. Cross-inoculations were made on tetraploid and diploid clover plants usingRhizobium isolates from both types of clover. Nodulation activity and nitrogen fixation were determined. No sign of specific strains necessary for optimal fixation of nitrogen by theUO36-symbiosis was observed in comparison to the diploid clover strain.Rhizobium cultures effective in connection with cultivation of diploid clover will probably also be effective when cultivating tetraploid clover strains.     

The inheritance of preference for strains of Rhizobium trifolii by white clover (Trifolium repents)

The preference or selection of Rhizobium strains by both parental and F₁ white clover plants was studied using antibiotic resistant mutants of Rhizobium trifolii. Highly significant differences were found between crosses in their preference for 75 str. Progenies from crosses between plants in which the preference for 75 str was 90–100% had 85% of the nodules inhabited by that strain. Progenies of crosses between plants in which the preference for 75 str was less than 50% had only 13% of their nodules inhabited by 75 str. Intermediate values were found for progenies from other crosses. The inheritance for this particular character was clearly additive and without any dominance or maternal effect. The results are discussed from the point of view of improving symbiotic nitrogen fixation by breeding white clover for uniform preference for effective strains of R. trifolii.     

Rhizobium nod genes are involved in the induction of two early nodulin genes in Vicia sativa root nodules

Nodulin gene expresison was studied in Vicia sativa (common vetch) root nodules induced by several Rhizobium and Agrobacterium strains. An Agrobacterium transconjugant containing a R. leguminosarum symplasmid instead of its Ti-plasmid, that was previously shown to form empty nodules on pea, induced nodules on Vicia roots in which nodule cells were infected with bacteria. In the Vicia nodules induced by this transconjugant, two so-called early nodulin genes were found to be expressed, whereas in the nodules formed on pea the expression of only one early nodulin gene was detected. In both cases the majority of the nodulin genes was not expressed.Apparently, an intracellular location of the bacteria is not sufficient for the induction of the majority of the nodulin genes. All nodulin genes were expressed in nodules induced by cured Rhizobium strains containing cosmid clones that have a 10 kb nod region of the sym-plasmid in common. Since in tumours no nodulin gene expression was found at all, the Agrobacterium chromosome does not contribute to the induction of nodulin genes. Therefore it is concluded that the signal for the induction of the expression of the two Vicia early nodulin genes is encoded by the nod-region, and the signal involved in the induction of all other nodulin genes has to be located outside the sym-plasmid, on the Rhizobium chromosome. The apparent difference in early nodulin gene expression between pea and Vicia is discussed in the light of the usefulness of Agrobacterium transconjugants in the study of nodulin gene expression.     

Effect of dinitroaniline herbicides on rhizobia, nodulation and N2(C2H2) fixation of four groundnut cultivars

Field, greenhouse and laboratory investigations were conducted to determine the effect of four dinitroaniline herbicides on rhizobia, nodulation and nitrogen fixation of four groundnut cultivars. Benefin, dinitramine and nitralin used at recommended levels decreased nodule dry weight, nitrogenase activity and total nitrogen of groundnut tops and pod yield in three cultivars Kadiri 71-1, Kadiri-2, ICGS-11 and not for a fourth cultivar, Kadiri-3 of groundnut (Arachis hypogaea L.), but fluchloralin used at the recommended level increased the nodulation rate, nitrogenase activity and total nitrogen of groundnut tops and pod yield compared to untreated plants. Studies were conducted in vitro to determine the relative toxicity of the herbicides on four Rhizobium strains isolated from the nodules of four cultivars of groundnut. It was found that various strains of rhizobia differ in their sensitivity to different rates of the herbicides tested. Carbon dioxide exchange rate (CER) of all the cultivars which received herbicide treatment was measured at different time intervals to determine the relationship between photosynthesis and inhibition of nodulation. The lack of adverse effect on the CER of four cultivars when treated at recommended concentrations indicated that nitrogen fixation was affected in cultivars Kadiri 71-1, Kadiri-2 and ICGS-11 due to inhibition of nodulation.     

Loss‐of‐function of ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (APN1) in Lotus japonicus restricts efficient nitrogen‐fixing symbiosis with specific Mesorhizobium loti strains

The nitrogen‐fixing symbiosis of legumes and Rhizobium bacteria is established by complex interactions between the two symbiotic partners. Legume Fix^– mutants form apparently normal nodules with endosymbiotic rhizobia but fail to induce rhizobial nitrogen fixation. These mutants are useful for identifying the legume genes involved in the interactions essential for symbiotic nitrogen fixation. We describe here a Fix^– mutant of Lotus japonicus, apn1, which showed a very specific symbiotic phenotype. It formed ineffective nodules when inoculated with the Mesorhizobium loti strain TONO. In these nodules, infected cells disintegrated and successively became necrotic, indicating premature senescence typical of Fix^– mutants. However, it formed effective nodules when inoculated with the M. loti strain MAFF303099. Among nine different M. loti strains tested, four formed ineffective nodules and five formed effective nodules on apn1 roots. The identified causal gene, ASPARTIC PEPTIDASE NODULE‐INDUCED 1 (LjAPN1), encodes a nepenthesin‐type aspartic peptidase. The well characterized Arabidopsis aspartic peptidase CDR1 could complement the strain‐specific Fix^– phenotype of apn1. LjAPN1 is a typical late nodulin; its gene expression was exclusively induced during nodule development. LjAPN1 was most abundantly expressed in the infected cells in the nodules. Our findings indicate that LjAPN1 is required for the development and persistence of functional (nitrogen‐fixing) symbiosis in a rhizobial strain‐dependent manner, and thus determines compatibility between M. loti and L. japonicus at the level of nitrogen fixation.     

Nitrogen fixation and poly-β-hydroxybutyric acid content in bacteroids ofRhizobium lupini andRhizobium leguminosarum

Summary Experiments carried out in a sand culture have demonstrated that during the growth ofVicia faba andLupinus luteus inoculated with effective strains of Rhizobium, and when the behaviour of bacteroids isolated from nodules ofLupinus luteus, Pisum sativum andVicia faba which had been inoculated with effective and ineffective strains and when comparisons were made between bacteriods isolated from effective nodules ofVicia faba andLupinus luteus either at midday or at midnight there is a reverse correlation between the intensity of nitrogen fixation and respiration, on the one hand, and the content of poly--hydroxybutyric acid (PHB), on the other. This evidence suggests an important role played by PHB in the supply of symbiotic fixation with energy and carbon substrates.Glucose and -hydroxybutyrate were the best substrates for PHB synthesis in the suspension of bacteroids of an effective strain ofR. lupini at all stages of plant growth. At the stage of active nitrogen fixation (flowering) PHB was actively synthesized in the presence of succinate. In the absence of exogenous substrates the polymer degraded, the process being enhanced in the presence of ammonium ions. When ammonium was added together with glucose, PHB synthesis did not occur and at the flowering stage the polymer broke down particularly rapidly. re]19760505     

Rhizobium strains expressing uptake hydrogenase in different host species of cowpea miscellany

Uptake hydrogenase activity in nodules of green gram (Vigna radiata (L.) (Wilczek)), black gram (Vigna mungo (L.) (Hepper)), cowpea (Vigna unguiculata (L.) and cluster bean (Cyamopsis tetragonoloba (L.) (Taub.)), formed with two Hup⁺ (S24 and CT2014) and one Hup⁻ (M11)Rhizobium strains, was determined at different levels of external H2 in air atmosphere. Nodules of all the 4 host species formed by inoculation with strains S24 and CT2014, showed H2 uptake but not those formed with strain M11. H₂ uptake rates were higher in 1 and 2% H₂ in air atmosphere (v/v) than at 5 or 10% levels in all the host species. Variations in the relative rates of H₂ uptake were observed both, due to host species as well as due toRhizobium strains. However, no host dependent complete repression of the expression of H₂ uptake activity was observed in nodules of any of the host species formed with Hup⁺ strains.     

Determination of Hydrogenase in Free-living Cultures of Rhizobium japonicum and Energy Efficiency of Soybean Nodules

A sensitive tritium exchange assay was applied to the Rhizobium system for measuring the expression of uptake hydrogenase in free-living cultures of Rhizobium japonicum. Hydrogenase was detected about 45 hours after inoculation of cultures maintained under microaerophilic conditions (about 0.1% O₂). The tritium exchange assay was used to screen a variety of different strains of R. japonicum (including major production strains) with the findings that about 30% of the strains expressed hydrogenase activity with identical results being observed using an alternative assay based on uptake of H₂. The relative efficiency of intact soybean nodules inoculated with 10 different rhizobial strains gave results identical to those obtained using free-living cultures. The tritium exchange assay provides an easy, quick, and accurate assessment of H₂ uptake efficiency of intact nodules.     

Host specificity of Rhizobium strains isolated from nitrogen-fixing trees and nitrogenase activities of strain GRH2 in symbiosis with Prosopis chilensis

Host plant specificity was examined in symbiosis between Rhizobium strains isolated from legume-tree root nodules and herbaceous or woody legumes from which they were isolated. Strain GRH2 isolated from Acacia cyanophylla formed effective nodules on Acacia, Prosopis and Medicago sativa as well. Nitrogenase activity, measured as acetylene reduction, of strain GRH2 in symbiosis with Prosopis chilensis was the highest (P 0.05) among the tropical legumes studied and was similar to those found for other associations involving herbaceous legumes. Relative efficiency of nitrogenase varied from 0.3 to near 1 during the light time of the photoperiod. However no hydrogen uptake activity was detected by the amperometric method used. Rhizobium strains GRH3, GRH5 and GRH9 isolated from A. melanoxylon, P. chilensis and Sophora microphylla, respectively, also showed a very low host-range specificity. All isolates were infective and effective on at least one of the herbaceous legumes tested. These data demonstrate the lack of specificity of Rhizobium strains isolated from nitrogen-fixing tree root nodules and that these strains can form effective nodules on herbaceous legumes.     

Effects of disease resistance genes on Rhizobium symbiosis in an annual legume

Summary The evolution of disease resistance in plants may be constrained if genes conferring resistance to pathogens interfere with plant responses toward other, nonpathogenic organisms. To test for such effects, we compared symbiotic nitrogen fixation in Amphicarpaea bracteata plants that differed at a major locus controlling resistance to the pathogen Synchytrium decipiens. Both resistant and susceptible plant genotypes nodulated successfully and grew significantly better in the presence of Rhizobium, although growth enhancement by Rhizobium was altered by different levels of nitrate fertilization. Plants homozygous for disease resistance achieved 2% higher growth than susceptible homozygotes across all treatments, but this difference was not significant. Resistant and susceptible plant genotypes did not differ in the mean number of nodules formed per plant or in nodule diameter. However, there was highly significant variation among replicate families within each disease resistance category for both nodulation characteristics. These results imply that genetic variation exists among A. bracteata plants both for diease resistance and for traits affecting symbiotic nitrogen fixation. However, there were no evident pleiotropic effects of disease resistance genes on the plant-Rhizobium symbiosis.