The effect of inoculation with <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> on the contents of cytoplasmic protein and free amino acids in the roots of pea seedlings

The changes in the contents of protein and free amino acids in pea plants inoculated with Rhizobium leguminosarum were studied taking into account the susceptibility of roots to root nodule bacteria. The content of cytoplasmic protein during infection increased in the actively growing root zone (0–5 mm) and decreased in the root zones susceptible to rhizobia (5–20 mm from the root tip). The quantitative composition of free amino acids changed essentially upon inoculation of pea seedlings with R. leguminosarum.     

Genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine <Emphasis Type="Italic">Lathyrus vernus</Emphasis> (L.) Bernh.

The genetic diversity and phylogeny of root nodule bacteria entering into symbiosis with bitter peavine Lathyrus vernus (L.) Bernh. (Fabaceae) growing in various regions of the Republic of Bashkortostan were studied. RAPD analysis revealed a high degree of polymorphism of the DNA of the isolated strains giving evidence of the heterogeneity of the microorganisms in question. The study of the phylogeny of microsymbionts based on comparative analysis of the nucleotide sequences of 16S rRNA genes showed that the bacteria isolated from the plant nodules of L. vernus growing on the territory of Ufa and Beloretsk raions belonged to the species Rhizobium leguminosarum, whereas the microsymbionts of L. vernus growing on the territory of Tatyshly raion belonged to the species Rhizobium tropici,@ except for several strains of Rhizobium leguminosarum     

Auxin and nitric oxide control indeterminate nodule formation

Background  

Rhizobia symbionts elicit root nodule formation in leguminous plants. Nodule development requires local accumulation of auxin. Both plants and rhizobia synthesise auxin. We have addressed the effects of bacterial auxin (IAA) on nodulation by using Sinorhizobium meliloti and Rhizobium leguminosarum bacteria genetically engineered for increased auxin synthesis.     

Rhizoplane colonization of pea seedlings by Rhizobium leguminosarum and a deleterious root colonizing Pseudomonas sp. and effects on plant growth

Some pseudomonads produce a toxin that specifically inhibits winter wheat (Triticum aestivum L.) root growth and the growth of several microorganisms. The toxin does not inhibit pea (Pisum sativum) root growth, but the organisms are aggressive root colonizers and their effect on Rhizobium leguminosarum growth, colonization, and nodulation of peas was not known. Peas were grown in Leonard jars in the greenhouse. Pea roots were inoculated with R. leguminosarum, a toxin-producing Pseudomonas sp., both, or neither (control). The Pseudomonas sp. colonized pea roots more rapidly and in greater number than R. leguminosarum after ten days. In the presence of the Pseudomonas sp., the R. leguminosarum population on the rhizoplane was less at ten days. When the roots were inoculated with both R. leguminosarum and Pseudomonas sp., the number of nodules were greater than when R. leguminosarum was inoculated alone, but nodule dry weight and pea shoot biomass were similar to plants inoculated with only R. leguminosarum. Although these results need confirmation with non-sterile soil and field studies, these preliminary results indicate that peas will not be affected by wheat root-inhibitory rhizobacteria.     

New taxonomic markers for identification of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> and discrimination between closely related species

Rhizobia, producing species-specific exopolysaccharides (EPSs), comprise a very diverse group of soil bacteria that are able to establish nitrogen-fixing symbioses with legumes. Based on the sequences of R. leguminosarum EPS synthesis genes, a sensitive and reliable PCR-based method for identification and subsequent discrimination between Rhizobium species has been developed and tested. For identification of R. leguminosarum, primer sets I–III complementary to sequences of rosR, pssA and pssY genes were proposed. Further sets of primers (IV–VII) were designed for discrimination between R. leguminosarum biovars. The usefulness of the method was examined using a wide range of R. leguminosarum strains isolated from different host plants nodules originating from different regions of Poland. We demonstrate a high discriminating power of primer sets I–III that allow distinguishing R. leguminosarum and two closely related species, R. etli and R. gallicum. This new approach is applicable to identification of R. leguminosarum strains, originating from nodules or soil, where many other closely related bacteria are expected to be present. Based on the nucleotide sequence of rosR and pssA genes, phylogenetic relationships of selected R. leguminosarum isolates were determined. Our results indicate that both rosR and pssA might be useful markers to differentiate and define relationships within a group of R. leguminosarum strains.     

The actinobacterium <Emphasis Type="Italic">Microbacterium</Emphasis> sp. 16SH accepts pBBR1-based pPROBE vectors,forms biofilms,invades roots,and fixes N<Subscript>2</Subscript> associated with micropropagated sugarcane plants

Members of the genus Microbacterium lineage of Gram-positive actinobacteria are increasingly being reported to display significant traits associated with environmental biotechnology and bioengineering. 16SH is a nitrogen-fixing bacterial strain isolated from a surface-sterilized stem of sugarcane grown in Guangxi, China. Analysis of 16S rRNA gene sequences revealed that 16SH belonged to the genus Microbacterium. pPROBE-pTet^r plasmids were constructed by cloning the promoter region of the Tet ^r gene into the promoterless pPROBE-AT, -OT, and -TT vectors derived from the pBBR1 plasmid that has a broad host range of Gram-negative bacteria and sequence similarities to plasmids from Gram-positive bacteria. The pPROBE-pTet^r plasmids expressed the gfp reporter gene and were stably maintained in 16SH cells without antibiotic selection in free-living state and in planta. Confocal microscopy on intact roots of micropropagated sugarcane plantlets showed that gfp-tagged 16SH cells formed biofilms on root maturation and elongation zones but not on root meristem zones and root caps, and colonized in intercellular spaces of root cortices. Inoculation of 16SH significantly increased biomass and nitrogen content of micropropagated sugarcane seedlings grown with a nitrogen fertilization of 6.3 mg N/kg soil. ¹⁵ N isotope dilution assays demonstrated that biological nitrogen fixation contributed to this plant growth promotion. This study for the first time demonstrated that the pBBR1-based pPROBE plasmids provided an efficient genetic transfer system for a Gram-positive Microbacterium strain, and that a nitrogen-fixing Microbacterium endophyte colonized in intact host plants and fixed N₂ associated with the host plants.     

Characterization of Novel Plant Growth Promoting Endophytic Bacterium <Emphasis Type="Italic">Achromobacter xylosoxidans</Emphasis> from Wheat Plant

Nine diazotrophic bacteria were isolated from surface-sterilized roots and culms of wheat variety Malviya-234, which is grown with very low or no inputs of nitrogen fertilizer. Out of the nine bacteria, four showed indole acetic acid (IAA) production, and five were positive for P solubilization. One isolate, WM234C-3, showed appreciable level of nitrogenase activity, IAA production, and P solubilization ability, and was further characterized with a view to exploiting its plant growth promoting activity. Based on 16S rDNA sequence analysis, this isolate was identified as Achromobacter xylosoxidans. Diazotrophic nature of this particular isolate was confirmed by Western blot analysis of dinitrogenase reductase and amplification of nifH. Analysis of the nifH sequence showed close homology with typical diazotrophic bacteria. Endophytic nature and cross-infection ability of WM234C-3 were tested by molecular tagging with gusA fused to a constitutive promoter followed by inoculation onto rice seedlings in axenic conditions. At 21 days after inoculation, the roots showed blue staining, the most intense color being at the emergence of lateral roots and root tips. Microscopic observation confirmed colonization of gus-tagged WM234C-3 in the intercellular spaces of cortical as well as vascular zones of roots. Inoculation of gus-tagged WM234C-3 to rice plants resulted in significant increase in root/shoot length, fresh weight, and chlorophyll a content. Plant growth promoting features coupled with cross-infection ability suggest that this endophytic bacterium may be exploited as agricultural agent for various crops after a thorough and critical pathogenicity test.     

Localization of acid phosphatase activity in the apoplast of pea (Pisum sativum L.) root nodules grown under phosphorus deficiency

ACPase activity was localized in the apoplast of pea root nodules under phosphorus deficiency. Pea plants (Pisum sativum L. cv. Sze ciotygodniowy) where inoculated with Rhizobium leguminosarum bv. viciae 248 and were cultured on nitrogen-free medium with phosphate (−N/+P) or phosphate-deficient (−N/−P) one. In comparison with control nodules, P-deficient nodules showed the increase of ACPase activity in plant cell walls and the infection threads. The increase in bacterial ACPase activity under P-deficiency may reflect higher demand for inorganic phosphorus that is necessary for bacteria multiplication within the infection threads. The increase of ACPase activity in nodule apoplast under P stress may enlarge the availability of phosphate for plant and bacteria.     

Effects of boron and calcium nutrition on the establishment of the Rhizobium leguminosarum–pea (Pisum sativum) symbiosis and nodule development under salt stress

The effects of modifying boron (B) and calcium (Ca²⁺) concentrations on the establishment and development of rhizobial symbiosis in Pisum sativum plants grown under salt stress were investigated. Salinity almost completely inhibited the nodulation of pea plants by Rhizobium leguminosarum bv. viciae 3841. This effect was prevented by addition of Ca²⁺ during plant growth. The capacity of root exudates derived from salt‐treated plants to induce Rhizobium nod genes was not significantly decreased. However, bacterial adsorption to roots was highly inhibited in plants grown with 75 mM NaCl. Moreover, R. leguminosarum 3841 did not grow in minimal media containing such salt concentration. High Ca²⁺ levels enhanced both rhizobial growth and adsorption to roots, and increased nodule number in the presence of high salt. Nevertheless, the nodules developed were not functional unless the B concentration was also increased. Because B has a strong effect on infection and cell invasion, these processes were investigated by fluorescence microscopy in pea nodules harbouring a R. leguminosarum strain that expresses green fluorescent protein. Salt‐stressed plants had empty nodules and only those treated with high B and high Ca²⁺ developed infection threads and exhibited enhanced cell and tissue invasion by Rhizobium. Overall, the results indicate that Ca²⁺ promotes nodulation and B nodule development leading to an increase of salt tolerance of nodulated legumes.     

The effect of biotic and physical factors on the competitive ability of <Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis>

Background  

Rhizobium leguminosarum bv. viciae (Rlv) is a soil bacterium which can form nitrogen-fixing symbiotic relationships with leguminous plants. Numerous rhizobial strains found in soils compete with each other. Competition can occur both during the saprophytic growth phase in the rhizosphere and inside plant tissues, during the symbiotic phase. Competition is important as it may affect the composition of rhizobial populations present in the soil and in the root nodules of plants.     

Glutathione in adaptation of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> to cadmium stress

The role of glutathione (GSH) in the adaptation of wild type Arabidopsis thaliana plants to Cd stress was investigated. The nutrient solution (control or containing 50 or 100 μM Cd) was supplemented with buthionine sulfoximine (BSO; 50, 100, 500 μM, to decrease the GSH content in plants) or GSH (50, 100, 500 μM, to increase its content in plants) in order to find how GSH content could regulate Cd stress responses. BSO application did not influence plant biomass, while exogenous GSH (especially 500 μM) reduced root biomass. BSO (500μM) in combination with Cd (100 μM) increased Cd toxicity on root growth (by over 50 %), most probably due to reduced GSH content and phytochelatin (PC) accumulation (by over 96 %). On the other hand, combination of exogenous GSH (500 μM) with Cd (100 μM) was also more toxic to plants than Cd alone despite a significant increase in GSH and PC accumulation (up to 2.7 fold in the roots). This fact could indicate that the natural content of endogenous GSH in wild type A. thaliana plants is sufficient for Cd-tolerance. A decrease in this GSH content led to decreased Cd-tolerance of the plants but an increase in GSH content did not enhance Cd-tolerance, and it showed even toxic effect on the plants.     

Relationship between boron and calcium in the N2-fixing legume–rhizobia symbiosis

Because boron (B) and calcium (Ca²⁺) seem to have a strong effect on legume nodulation and nitrogen fixation, rhizobial symbiosis with leguminous plants, grown under varying concentrations of both nutrients, was investigated. The study of early pre‐infection events included the capacity of root exudates to induce nod genes, and the degree of adsorption of bacteria to the root surface. Both phenomena were inhibited by B deficiency, and increased by addition of Ca²⁺, resulting in an increase of the number of nodules. The infection and invasion steps were investigated by fluorescence microscopy in pea nodules harbouring a Rhizobium leguminosarum strain that constitutively expresses green fluorescent protein. High Ca²⁺ enhanced cell and tissue invasion by Rhizobium, which was highly inhibited after B deficiency. This was combined with an increased B concentration in nodules of plants grown on B‐free medium and supplemented with high Ca²⁺ concentrations, and that can be attributed to an increased B import to the nodules. Histological examination of indeterminate (pea) and determinate (bean) nodules showed an altered nodule anatomy at low B content of the tissue. The moderate increase in nodular B due to additional Ca²⁺ was not sufficient to prevent the abnormal cell wall structure and the aberrant distribution of pectin polysaccharides in B‐deficient treatments. Overall results indicate that the development of the symbiosis depends of the concentration of B and Ca²⁺, and that both nutrients are essential for nodule structure and function.     

Potential of efficient and resistant plant growth‐promoting rhizobacteria in lead uptake and plant defence stimulation in Lathyrus sativus under lead stress

• The ability of plant growth‐promoting rhizobacteria (PGPR) to enhance Lathyrus sativus tolerance to lead (Pb) stress was investigated.
• Ten consortia formed by mixing four efficient and Pb‐resistant PGPR strains were assessed for their beneficial effect in improving Pb (0.5 mM) uptake and in inducing the host defence system of L. sativus under hydroponic conditions based on various physiological and biochemical parameters.
• Lead stress significantly decreased shoot (SDW) and root (RDW) dry weight, but PGPR inoculation improved both dry weights, with highest increases in SDW and RDW of plants inoculated with I5 (R. leguminosarum (M5) + P. fluorescens (K23) + Luteibacter sp. + Variovorax sp.) and I9 (R. leguminosarum (M5) + Variovorax sp. + Luteibacter sp. + S. meliloti) by 151% and 94%, respectively. Additionally, inoculation significantly enhanced both chlorophyll and soluble sugar content, mainly in I5 inoculated leaves by 238% and 71%, respectively, despite the fact that Pb decreased these parameters. We also found that PGPR inoculation helps to reduce oxidative damage and enhances antioxidant enzyme activity, phenolic compound biosynthesis, carotenoids and proline content. PGPR inoculation increased Pb uptake in L. sativus, with highest increase in shoots of plants inoculated with I5 and I7, and in roots and nodules of plants inoculated with I1. Moreover, PGPR inoculation enhanced mineral homeostasis for Ca, Cu and Zn under Pb stress, mainly in plants inoculated with I1, I5, I7 and I9.
• Results of our study suggest the potential of efficient and Pb‐resistant PGPR in alleviating harmful effects of metal stress via activation of various defence mechanisms and enhancing Pb uptake that promotes tolerance of L. sativus to Pb stress.

     

Siderophore and organic acid production in root nodule bacteria

Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 M added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H1, B. japonicum USDA110 and CB1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TAL1000, B. japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2–5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron.All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 M added iron).Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TAL1000, R. leguminosarum bv viciae MNF300 and B. japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 M added iron) during the stationary phase of growth.Abbreviations CAS chrome azurol S - HDTMA hexadecyltrime-thylammonium bromide     

Effect of nutrient medium pH on symbiotic nitrogen fixation byRhizobium leguminosarum andPisum sativum

Summary Experiments were performed to measure the pH-sensitive steps in nodulation and symbiotic fixation byPisum sativum and isolate RP-212-1 ofRhizobium leguminosarum. An aeroponic system with rigorous pH control was used to obtain numerous effective nodules. After exposure to various pH levels, the following responses were measured: (1) legume root growth and development, (2) survival and growth rate of a single effective bacterial isolate, (3) degree of nodulation, (4) rate of nitrogen fixation, (5) plant biomass, and (6) nitrogen content of plants. Both bacterial growth and root development were adequate at all pH levels from 4.4 to 6.6, but efficient nodulation and nitrogen fixation did not occur at pH 4.8 and below. The processes required for symbiosis were about 10 times as sensitive to acidity as either bacterial growth or root growth alone. Nodulation was the most acid-sensitive step.     

Use of Rhizobia in the Control of Root Rot Diseases of Sunflower, Okra, Soybean and Mungbean

Biocontrol potential of Rhizobium and Bradyrbizobium against soilborne root infecting fungi was tested. In vitro tests Rhizobium meliloti inhibited growth of Macrophomina phaseolina, Rhizoctonia solani and Fusarium solani while Bradyrhizobium japonicum inhibited M. phaseolina and R. solani producing zones of inhibition. In field R. meliloti, R. leguminosarum and B. japonicum used either as seed dressing or as soil drench reduced infection of M. phaseolina, R. solani and Fusarium spp., in both leguminous (soybean, mungbean) and non-leguminous (sunflower and okra) plants.     

Comparative Characteristics of Carbohydrate Binding by Lectins from Broad Bean,Pea, Common Vetch,and Lentil Seeds

Lectins from the seeds of broad bean (Vicia faba L.), pea (Pisum sativum L.), common vetch (V. sativa L.), and lentil (Lens culinaris Medik.) were isolated and purified by affinity chromatography. The hemagglutinating activity of lectins was most effectively inhibited by methyl--D-mannopyranoside, trehalose, and D-mannose. Other carbohydrate haptens, such as methyl--D-glucopyranoside, maltose, and alginic and D-glucuronic acids were less effective. Two lectins obtained from different lentil cultivars, unlike other lectins, had a relatively high affinity for melecitose, N-acetyl-D-glucosamine, L-sorbose, and sucrose. Furthermore, these lectins interacted with soluble starch. All the lectins examined had similar, but not identical, carbohydrate-binding properties. Because of their similar D-mannose/D-glucose specificity, these lectins interacted with lipopolysaccharides and exopolysaccharides of Rhizobium leguminosarum bv. viciae, root nodule bacteria that infect broad-bean, pea, common-vetch, and lentil plants with the formation of nitrogen-fixing symbiosis. However, owing to individual distinctions of carbohydrate-binding properties, these lectins showed a higher affinity for the polysaccharides of those microsymbionts within the R. leguminosarum bv. viciae species that were better specialized towards one or the other host plant from the cross inoculation group of legumes.     

Cytokinin Content during Early Stages of Legume-Rhizobial symbiosis and effect of hypothermia

The effects of pea (Pisum sativum L.) seedling inoculation with nodule bacteria (Rhizobium leguminosarum bv. viceae) and hypothermia on the content of cytokinins (Ck) in seedling roots during 8 days after external factor action were studied. As soon as a day after inoculation with Rh. leguninosarum, the content of Ck in the seedling roots increased, especially in the root zone most susceptible to infection (0?C20 mm from the tip); this not only affected growth of the whole roots but also determined the initiation of nodule primordia. Hypothermia (8°C) reduced the content of CK as soon as in a day. After 5 and 8 days of hypothermia, the content of Ck in inoculated plants was approximately twice higher than in non-inoculated plant roots.     

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.     

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.