Legume Symbiosis Research Articles

Natural occurrence of mycotoxins in broad bean (Vicia faba l.) seeds and their effect on Rhizobium‐legume symbiosis

AbstractSeeds of faba bean cultivar Giza 3 were screened for natural presence of mycotoxins. Eleven out of 100 samples were contaminated. Aflatoxins B1 and B2 were found in 7 samples while aflatoxins B1, B2, G1 and G2 and ochratoxin A were each detected twice in separate samples. Mycotoxins at concentrations of 100 or 200 μg kg−1 soil significantly decreased nodule number, nodule fresh weight and total nitrogenase activity, leading to reductions in dry matter accumulation and nitrogen yield of the bean. Mycotoxins also suppressed specific nitrogenase activity. NADH‐dependent glutamate dehydrogenase (NADH‐GDH) as well as glutamate synthase (NADH‐GOGAT) activities. In addition, mycotoxins inhibited synthesis of leghaemoglobin, carbohydrated and protein in the nodule cytosol. Of the mycotoxins tested, aflatoxin B1 was the most toxic. The decline in nitrogenase activity and total N concentration in the plants could be attributed to mycotoxins interference with normal nodule physiology and function.

Perennial forage legume growth in acidic soils from the major series of the Appalachian Hill‐Lands

Abstract The lack of highly effective forage legume symbioses is thought to be a major constraint to the renovation of acidic soils of the Appalachian region for pasture use. A growth chamber experiment was initiated with 14 fresh soils, each of a different series, from diverse sites within the region to determine the need for rhizobial inoculation, and the soil chemical properties limiting growth, of alfalfa (Medicago sativa L.), red clover (Trifolium pratense L.), white clover (Trifolium repens L.), and birdsfoot trefoil (Lotus corniculatus L.). Effective, naturalized populations of rhizobia producing acceptable (>50% maximum) shoot growth were found in few of the soils. Commercial rhizobial mixtures were effective in producing similarly acceptable growth in three soils for alfalfa and red clover, but only in one soil for white clover and birdsfoot trefoil. Soil rankings based on asymbiotic shoot growth were similar for all species, particularly when only the better soils were considered, and changed li...

Molecular mechanism of host specificity in legume-rhizobium symbiosis

Rhizobium - legume symbiosis is a highly specific interaction between the two partners. Host specificity is evident at early stages of infection and results from multiple interactions involving signalling among bacteria and host plants. Host specific plant signals (flavanoids) convert the NodD protein to an active form and its binding with nod box initiates the transcription of inducible nod operons. Common nod genes ( nodABC) code for an extracellular mitogenic Nod factor which is required for nodule organogenesis. Host specific genes ( hsn) modify the Nod factor to induce root hair deformation on specific hosts. The structure of Nod factor controls host range distinction between species and biovars of rhizobia. Interactions of lectins and Exopolysaccharide/Lipopolysaccharide result in host specific attachment of Rhizobium and its subsequent invasion. Change in Expopolysaccharide structure by the transfer of hsn genes enables the Rhizobium to bind with heterologous host lectins. Conversely, changes in root lectins via gene manipulation enables the heterologous rhizobia to bind and initiate nodulation on heterologous hosts. Finally, host specific signals are required to initiate nitrogen fixation in nodules that are formed.

Characterization of structural defects in the lipopolysaccharides of symbiotically impaired Rhizobium leguminosarum biovar viciae VF-39 mutants

The lipopolysaccharides (LPS) of a wild type strain of Rhizobium leguminosarum biovar viciae (strain VF-39) and two symbiotically defective Tn5 mutants (VF-39-32 and VF-39-86) have been studied. The LPS of the mutants reflected impaired synthesis of the O-antigen. In the LPS of one mutant, the core tetrasaccharide was lacking and in that of the other it was truncated to a disaccharide containing mannose and 3-deoxy- d- manno-oct-2-ulosonic acid (KdO). The latter mutant also synthesized an unusual carbohydrate component containing mannose, galactose, and an unidentified saccharide. The lipid A composition was similar to that found in other strains of R. leguminosarum biovar viciae. The O-antigen of the wild-type bacterium contained 2- O-methylfucose, fucose, 3,6-dideoxy-3-(methylamino)hexose, glucose, 2-amino-2,6-dideoxyhexose, and heptose. This study clearly defines a role for the bacterial LPS in the proper functioning of the Rhizobium legume symbiosis.

Structure and growth of infection threads in the legume symbiosis with Rhizobium leguminosarum

SummarySpecific antibodies and enzyme–gold probes were used to study the structure and development of infection threads in nodules induced by Rhizobium leguminosarum on the roots of Vicia, Pisum and Phaseolus. In Pisum nodules, the tubular infection thread wall contains polysaccharides antigenically similar to those of the cell wall, including cellulose, xyloglucan, methyl‐esterified pectin and non‐esterified pectin, but none of these wall components is present around the infection droplet structures from which bacteria are internalized by plant plasma membrane. As reported previously for pea nodules, the luminal matrix of infection threads and infection droplets contains a plant glycoprotein; this glycoprotein is also secreted by infected and uninfected cortical cells of a Vicia root at the earliest stages of nodule initiation. Synthesis of a transcellular infection thread apparently involves reorganized deposition of components normally targeted to the cell wall, and infection thread growth is orientated anticlinally through the outer cortex in the same plane observed for the deposition of new cell walls following mitosis. Both the development of infection threads in the outer cortex and the initiation of cell division in the inner cortex are preceded by a similar process of cell reactivation involving centralization of nuclei and the development of anticlinal transvacuolar strands. It is therefore suggested that the two Rhizobium‐induced processes of infection thread growth and cortical cell division may both be consequences of a similar plant cell response in the inner and outer root cortex, respectively. Phaseolus nodules contained only short intracellular infection structures which terminated within individual cells and contained no luminal matrix material. The differences in infection thread structure between Pisum and Phaseolus nodules may reflect differences in ontogeny between “indeterminate” and “determinate” nodule meristems.

Two C4-dicarboxylate transport systems in Rhizobium sp. NGR234: rhizobial dicarboxylate transport is essential for nitrogen fixation in tropical legume symbioses.

To investigate the role of dicarboxylate transport in nitrogen-fixing symbioses between Rhizobium and tropical legumes, we made a molecular genetic analysis of the bacterial transport system in Rhizobium sp. NGR234. This braod host range strain fixes nitrogen in association with evolutionarily divergent legumes. Two dicarboxylate transport systems were cloned from Rhizobium NGR234. One locus was chromosomally located, whereas the other was carried on the symbiotic plasmid (pSym) and contained a dctA carrier protein gene, which was analyzed in detail. Although the DNA and derived amino acid sequences of the structural gene were substantially homologous to that of R. meliloti, its promoter sequences was quite distinct, and the upstream sequence also exhibited no homology to dctB, which is found at this position in R. meliloti. A site-directed internal deletion mutant in dctA of NGR234 exhibited a (unique) exclusively symbiotic phenotype that could grow on dicarboxylates ex planta, but could not fix nitrogen in planta. This phenotype was found for tested host plants of NGR234 with either determinate- or indeterminate-type nodules, confirming for the first time that symbiosis-specific uptake of dicarboxylates is a prerequisite for nitrogen fixation in tropical legume symbioses.

A model of nitrogen flow by malonamate in [formula omitted]-soybean symbiosis

Two types of novel malonamidases were found in soybean nodules. One (E1) catalyzes the formation of malonamate from malonate and its hydrolysis to ammonia, whereas the other (E2) acts mainly on the hydrolysis of malonamate. E1 and E2 were found in bacteroids, but only E2 was found in the plant cytosol of the nodule. The substrate requirements of E1 and E2 were highly specific for malonate and malonamate, respectively. From these and other results reported previously, we propose that malonamate plays an important role as a nitrogen carrier in the Rhizobium legume symbiosis.

Efficiency of crop inoculation with endomycorrhizal fungi

Soddy-podzolic soils of the Soviet Union contain spores of endomycorrhizal fungi, wich infect major agricultural crops to form vesicular-arbuscular mycorrhizae. Efficient endomycorrhizal fungi pertaining to the genus Glomus have been isolated from plant roots. Inoculation of these fungi on to oat, barley, vetch, clover, potato, and onion enhanced the yield and the phosphorus content. Triple symbiosis of legumes with endomycorrhizal fungi and Rhizobium increased the plant nitrogen content.

A chromosomal genetic map of Rhizobium sp. NGR234 generated with Tn5-Mob

Rhizobium sp. NGR234 in a fast-growing Rhizobium strain with a broad host range. The location and role of chromosomal genes involved in cellular metabolism or in the legume symbioses is unknown. We isolated a series of auxotrophic and antibiotic resistant mutants of NGR234 and utilized a chromosome mobilization system based on Tn5-Mob and pJB3JI; Tn5-Mob donor strains behaved like Hfr strains, transferring the chromosome polarly at high frequency from a fixed point of insertion. The use of four different strains with Tn5-Mob located at different nutritional loci in crosses with double auxotrophic recipients, allowed us to build up a circular linkage map of NGR234 based on relative recombination frequencies. Also, symbiotically important genes identified by site-directed mutagenesis, such as hemA and ntrA, could be located and mapped on the chromosome.

Root lectin as a determinant of host–plant specificity in the Rhizobium–legume symbiosis

THE induction of nitrogen-fixing nodules in legume roots by soil bacteria from the genera Rhizobium and Bradyrhizobium is host-plant-specific. This specificity is expressed at an early stage of the infection process and results from multiple interactions between bacterial and plant products. Among these, it has been suggested that root lectin recognized by bacterial receptor molecules is an important determinant of host specificity1,2. Lectins are carbohydrate-binding proteins and it is known that legumes belonging to different cross-inoculation groups produce lectins with different sugar-binding specificities3. We have tested this suggestion by introducing the pea lectin gene into white clover roots using Agrobacterium rhizogenes as a vector. The 'hairy' clover roots that result can be nodulated by a Rhizobium strain usually specific for plants from the pea cross-inoculation group.

Advances in the utilization of rhizobia in arid environments

Abstract There are numerous poorly known and unexploited rhizobia‐legume symbioses in arid ecosystems. These symbiotic associations have a considerable potential as sources for food, fodder, and energy. Expanded acquisition of information is needed on rhizobial strain specificities, salt and drought resistance, and on inoculation needs and applicable technologies for arid lands. Development of appropriate technologies for maximal protein recovery and for production of legume biomass as an energy source should be implemented. An important aspect is the utilization of molecular genetics for the development of economically improved legume symbioses.

Bacteroids in theRhizobium-Legume Symbiosis Inhabit a Plant Internal Lytic Compartment: Implications for other Microbial Endosymbioses

Bacteroids in the<i>Rhizobium</i>-Legume Symbiosis Inhabit a Plant Internal Lytic Compartment: Implications for other Microbial Endosymbioses

Cloning of hemA from Rhizobium sp. NGR234 and symbiotic phenotype of a gene-directed mutant in diverse legume genera

The hemA gene which encodes δ-aminolaevulinic acid synthase (ALAS), was cloned and characterized from the broad host-range Rhizobium strain NGR234. A cosmid, identified by hybridization with the cloned gene of R. meliloti and complementation of an R. meliloti hemA mutant, was subcloned to yield a 5.5 kb fragment containing the entire NGR234 gene. A physical-genetic map was made and the Ω interposon was introduced into a single EcoRI site which bisects the gene. The mutated gene was homogenotized into NGR234 to generate a hemA mutant, with a view to evaluating the role of rhizobial bacteroid ALAS activity for a wide variety of legume symbioses. The mutant strain formed an ineffective (Fix−) symbiosis with all tested host plants. These included tropical legumes that produce either indeterminate (Leucaena) or determinate (Desmodium, Macroptilium, Lablab, Vigna) root nodules.

Short-term effects of nitrate on nitrate reductase activity and symbiotic dinitrogen fixation in faba bean and pea

The effects of a short-term supply of combined nitrogen (N) on the Rhizobium–legume symbiosis were studied in faba bean (Vicia faba L.) and pea (Pisum sativum L.) nodulated by R. leguminosarum biovar viceae isolate 175F5 or 175F19 and subjected to increasing levels of [Formula: see text] (0, 5, 10, or 20 mol∙m−3) from 28 to 36 days after planting. Trends in N2-fixing activity (acetylene reduction) showed that faba bean was more tolerant to [Formula: see text] than pea with isolate 175F5 but not with isolate 175F19. Nitrite reductase activities in the leaf, root, and nodule cytosol fractions were severalfold higher than nitrate reductase activities for both hosts. The levels of these enzymes in the nodule cytosol, the absence of bacteroid nitrate reductase and the lack of induction of bacteroid nitrite reductase in response to [Formula: see text] addition are consistent with the lack of [Formula: see text] accumulation in nodules of both hosts. Therefore, it is unlikely that the inhibitory effects of [Formula: see text] on N2 fixation are due to [Formula: see text] inhibition of nitrogenase. The relative levels of nitrate reductase activity in the root and nodule cytosol fractions were, respectively, higher and lower in the two faba bean symbioses ([Formula: see text] tolerant) and in the pea–175F5 symbiosis ([Formula: see text] sensitive) than in the pea–175F19 symbiosis ([Formula: see text] tolerant).

Role of the Nodulation Restrictive Allele Rj4, in Soybean Evolution

Summary The symbiosis of legumes with rhizobia has been subject to the selection pressures of 200 million years of evolution. Evidence from soybean of ecogeographic distribution of nodulation compatibilities with specific Rhizobium strains indicates that the coevolution of host and microsymbiont has played a role in the evolution of compatibilities. Some strains of bradyrhizobia that nodulate soybean also produce a substance called rhizobitoxine that interferes with chlorophyll synthesis. The Rj 4 allele in soybean conditions an ineffective nodulation response with specific strains of bradyrhizobia while the rj 4 allele permits normal nodulation with these strains. Two near isogenic soybean lines (99.95 % genetically identical) differing specifically in the Rj 4 vs. 774 alleles have been synthesized and used to test an array of chlorosis-inducing and non-chlorosis-inducing bradyrhizobia for response with the Rj 4 allele. The Rj 4 allele was found to interdict nodulation with 33.3 % of the chlorosis inducing rhizobia and only 2.7 % of the nonchlorosis inducing rhizobia. Thus, the Rj 4 allele may have a positive value to the host plant in areas with significant populations of chlorosis-inducing rhizobia by protecting the plant from nodulation with some of these rhizobia. The frequency of ineffective nodulation with the chlorosis-inducing strain USDA61 shows a progressive loss with domestication from the wild species Glycine soja (63 %) to the Asiatic plant introductions of G. max (26 %), and with selection for agronomic types in North America as demonstrated by the preliminary test lines (13.7 %) and the more highly selected uniform test lines (8.5 %). This may be an example of the loss of rhizobial specificity in a plant species occurring in historic time.

Effects of continuous combined nitrogen supply on symbiotic dinitrogen fixation of faba bean and pea inoculated with different rhizobial isolates

Combined nitrogen (N) has adverse effects on virtually all stages of the Rhizobium–legume symbiosis. Tolerance to combined N varies among legume hosts and rhizobial isolates, but the contribution of each symbiotic partner is not well established. The effects of combined N were studied in faba bean (Vicia faba L.) and pea (Pisum sativum L.), using the same Rhizobium leguminosarum isolates for both hosts. In one experiment, faba bean and pea were inoculated individually with four rhizobial isolates and grown for 28 days in the continuous presence of 0, 2.5, 5.0, or7.5 mol m−3 NH4NO3. For each isolate, faba bean was consistently more tolerant to combined N than pea as shown by significantly smaller rates of decrease in N2-fixing activity (acetylene reduction) in faba bean than in pea. The results were substantiated by those of a similar experiment in which increasing levels of 15N-labeled [Formula: see text] (5, 10, or 15 mol m−3) were supplied continuously to faba bean and pea inoculated individually with two rhizobial isolates. Comparisons of the different symbioses based on the proportion of total plant N derived from N2 fixation confirmed the conclusions reached using acetylene reduction activities.

Response of Rhizobium leguminosarum isolates to different forms of inorganic nitrogen during nodule development in pea ( Pisum sativum L.)

Combined nitrogen inhibits nodule development and nitrogen fixation in Rhizobium -legume symbioses. Isolates of R. leguminosarum which had been shown to vary in symbiotic effectiveness in the presence of NN 4NO 3 were used as inoculum for peas grown with NH 4NO 3 KNO 3 or NH 4Cl and harvested between 14 and 24 days after planting. Plants grown in 2m m NH 4NO 3 showed inhibition of nodule development and function 17 days after seeding but there were no significant differences (SDs) between isolates of high and low effectiveness. In the presence of 5 m M NH 4NO 3 nodule development and C 2H 2 reduction were severely inhibited from 14 days onwards. Plants inoculated with isolates of high effectiveness had more tap-root nodules, higher C 2H 2 reduction rates and higher leghemoglobin content than those inoculated with isolates of low effectiveness. In plants grown with 5 m m KNO 3 or 5 m m NN 4Cl, NO 3 − had the major inhibitory effect on nodulation and nodule development, but NH 4 + began to show inhibitory effects on nodule development after 17 days. There were no SDs between isolates of low and high effectiveness at these concentrations of NO 3 − and NH 4 +. Differences in symbiotic response of R. leguminosarum to combined nitrogen are small and appear during nodule development rather than at nodule initiation.

The Rhizobium ? legume symbiosis: observation of root infection by bright-field microscopy after staining with methylene blue

Staining of infected legume roots with 0.01% methylene blue facilitated the observation of the initial steps of the Rhizobium-legume symbiosis. It allowed particularly the visualization by bright-field microscopy of infection threads in the root hairs and the root cortex of the host plant.

Simulation of marked root hair curling in Rhizobium‐legume symbiosis

The soil bacterium Rhizobium infects its leguminous host plants in temperate regions of the world mostly by way of the growing root hairs. Root hair curling is a prerequisite for root hair infection, although sidelong root hair infections occasionally have been observed. The processes underlying Rhizobium‐induced root hair curling are unknown.Computer simulation of root hair growth indicates that one‐sided tip growth inhibition by Rhizobium can result in root hair curling when three conditions are simultaneously fulfilled: 1) rhizobial growth inhibition is strong enough to prevent removal out of the tip growth range: 2) root hair surface growth between the attached Rhizobium and the root hair top is inhibited; 3) rhizobial growth inhibition is limited to one side of the root hair.The results predict that root hair curling by stimulation of tip growth is improbable. This study accentuates the need for information about the growth processes contributing to tip growth in leguminous root hairs.

Acetylene reduction and hydrogen evolution by nitrogenase in a Rhizobium–legume symbiosis

The relationship between acetylene reduction and hydrogen evolution in air was examined in peas (Pisum sativum L. cv. Alaska) inoculated with Rhizobium leguminosarum strain 3740, which lacked uptake hydrogenase activity (Hup−). In the absence of a system for recycling hydrogen, changes in the relative efficiency of N2 fixation (RE), which is defined as RE = 1 − (hydrogen evolved in air)/(acetylene reduced), presumably reflect an altered capacity of nitrogenase to allocate electrons between protons and N2. The RE of plants grown without combined nitrogen declined during the vegetative phase of growth and increased after flowering. Continuous high light or elevated CO2 conditions in the absence of nitrate maintained throughout ontogeny accentuated decreases in RE. When nitrate was present in the growth medium declines in RE during the vegetative phase were lessened. These results are consistent with the concept that the electron allocation coefficient of nitrogenase varies in the absence of uptake hydrogenase activity.