Agronomical parameters of host and non-host legumes inoculated with Melilotus indicus-isolated rhizobial strains in desert unreclaimed soil.

In a search for identification of rhizobial strains with superior N2-fixation efficiency and improved plant agronomic characteristics upon inoculation, four strains, 4.21, 9.17, 11.2 and 14.1, isolated from root nodules of wild-grown Melilotus indicus have been used to inoculate field-grown common bean, pea, cowpea and fenugreek plants. Uninoculated plants and those inoculated with host-specific commercial inoculants were used as a control. The root length, shoot height, shoot dry weight and root dry weight and the grain yield of the plants were determined after harvest. The content of N, organic C and carbohydrates content of the grain were also recorded. The inoculation with the strains 4.21 and 14.1 increased the grain yield of the fenugreek compared both with the uninoculated plants and those inoculated with the commercial strain ARC-1. The grain yield of the common bean treated with the strains 9.17 and 14.1 was also higher than that of the uninoculated and the commercial strains ARC-301. In contrast, none of the strains increased the grain yield of the pea and cowpea plants compared to the commercial strains ARC-201 and ARC-169, respectively. Significant increases of some agronomical parameters were observed in some plant-bacterium couples, albeit nodulation was not observed. It is possible that the positive effects of rhizobial inoculation on the agronomical parameters of the non-nodule forming legumes could be due to plant growth promotion characteristic of the strains used for inoculation. Analysis of the phylogeny of the almost complete 16S rRNA sequence of the rhizobial inoculants revealed that the strains 4.21 and 9.17 clustered together with R. skierniewicense and R. rosettiformans, respectively, and that the strains 11.2 and 14.1 grouped with E. meliloti. All the four strains produced IAA, and showed biocontrol activity against Rhizotocnia solani, Fusarium oxysporum, Pythium ultimum, Alternaria alternata and Sclerotonia rolsfi, albeit to a different extent.

[Sinorhizobium meliloti strains screening for efficient bactarization of Melilotus albus Medik].

The data presents about analytical selection of root nodule bacteria of Melilotus to obtain bacterial fertilizer under sweet clover, presowing inoculation of it seeds and form a legume-rhizobial effective symbiosis. From natural melilot population a number of new strains had been allocated, inoculation of them was contributed to an increase of height. biomass Melilotus albus Medik., and nitrogenase activity in comparison to the influence of the existing production strains. The identification of most effective strains Sinorhizobium meliloti had been determined.

The Plasmid Mobilome of the Model Plant-Symbiont Sinorhizobium meliloti: Coming up with New Questions and Answers.

Rhizobia are Gram-negative Alpha- and Betaproteobacteria living in the underground which have the ability to associate with legumes for the establishment of nitrogen-fixing symbioses. Sinorhizobium meliloti in particular-the symbiont of Medicago, Melilotus, and Trigonella spp.-has for the past decades served as a model organism for investigating, at the molecular level, the biology, biochemistry, and genetics of a free-living and symbiotic soil bacterium of agricultural relevance. To date, the genomes of seven different S. meliloti strains have been fully sequenced and annotated, and several other draft genomic sequences are also available. The vast amount of plasmid DNA that S. meliloti frequently bears (up to 45% of its total genome), the conjugative ability of some of those plasmids, and the extent of the plasmid diversity has provided researchers with an extraordinary system to investigate functional and structural plasmid molecular biology within the evolutionary context surrounding a plant-associated model bacterium. Current evidence indicates that the plasmid mobilome in S. meliloti is composed of replicons varying greatly in size and having diverse conjugative systems and properties along with different evolutionary stabilities and biological roles. While plasmids carrying symbiotic functions (pSyms) are known to have high structural stability (approaching that of chromosomes), the remaining plasmid mobilome (referred to as the non-pSym, functionally cryptic, or accessory compartment) has been shown to possess remarkable diversity and to be highly active in conjugation. In light of the modern genomic and current biochemical data on the plasmids of S. meliloti, the current article revises their main structural components, their transfer and regulatory mechanisms, and their potential as vehicles in shaping the evolution of the rhizobial genome.

Characterization of the twin-arginine transport secretome in Sinorhizobium meliloti and evidence for host-dependent phenotypes.

The twin-arginine transport (Tat) system is essential for cell viability in Sinorhizobium meliloti and may play a role during the development of root nodules. Utilizing an in vivo recombination strategy, we have constructed 28 strains that contain deletions in predicted Tat substrates. Testing of these mutations for symbiotic proficiency on the plant hosts alfalfa and sweet clover shows that some of these mutations affect associations with these hosts differentially.

Arbuscular mycorrhizal fungi on growth, nutrient status, and total antioxidant activity of Melilotus albus during phytoremediation of a diesel-contaminated substrate.

This research evaluated the effects of arbuscular mycorrhizal fungi (AMF) on growth, nutritional status, total antioxidant activity (AOX), total soluble phenolics content (TPC), and total nitrate reductase activity (NRA) of leaves and roots of Melilotus albus Medik planted in diesel-contaminated sand (7500 mg kg(-1)). Seedlings of Melilotus either Non inoculated (Non-AMF) or pre-inoculated plants (AMF) with the AMF-inoculum Glomus Zac-19 were transplanted to non-contaminated or contaminated sand. After 60 days, diesel significantly reduced plant growth. AMF- plants had no significant greater (64% and 89%, respectively) shoot and leaf dry weight than Non-AMF plants, but AMF plants had lower specific leaf area. AMF-plants had significantly greater content of microelements than non-AMF plants. Regardless diesel contamination, the total AOX and TPC were significantly higher in leaves when compared to roots; in contrast, NRA was higher in roots than leaves. Diesel increased total AOX of leaves, but AMF-plants had significantly lower AOX than non-AMF plants. In contrast, roots of AMF-plants had significantly higher AOX but lower NRA than non-AMF plants. AMF-colonization in roots detected via the fungal alkaline phosphatase activity was significantly reduced by the presence of diesel. AMF-inoculation alleviated diesel toxicity on M. albus by enhancing plant biomass, nutrient content, and AOX activity. In addition, AMF-plants significantly contributed in higher degradation of total petroleum hydrocarbons when compared to non-AMF-plants.

Isolation and characterization of pyrene metabolizing microbial consortia from the plant rhizoplane.

Most research on the ecology of PAH degrading bacteria in the rhizosphere has focused on individual strains that grow on specific PAHs. Thus, there are fundamental questions as to importance of microbial consortia for PAH degradation in the plant rhizosphere. The study reported here characterized cultivable pyrene degrading rhizoplane microbial communities from two different plant species using a root printing technique on agar plates. Colonies were revealed by formation of clearing zones on medium containing a thin film of pyrene on the surface of a mineral nutrient agar. Prints of the rhizoplane colonies were obtained from roots of Melilotus officinalis (sweet yellow clover) and Andropogon gerardii (big bluestem) plants. Phylogenetic characterizations of selected pyrene degrading colonies were assessed by PCR-DGGE and DNA sequencing. Results showed that different populations of cultivable pyrene degraders were obtained from representative consortia that were examined. Many of the PAH degrading consortia consisted of mixtures of bacterial species that were unable to degrade pyrene by themselves. While this study focused on culturable PAH degraders, the results suggest that pyrene degradation in the rhizosphere commonly involves the activity of bacterial consortia in which various species of bacteria interact to achieve PAH degradation.

Ensifer, Phyllobacterium and Rhizobium species occupy nodules of Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a Canadian site without a history of cultivation.

Phage-resistant and -susceptible bacteria from nodules of alfalfa and sweet clover, grown at a site without a known history of cultivation, were identified as diverse genotypes of Ensifer, Rhizobium and Phyllobacterium species based on sequence analysis of ribosomal (16S and 23S rRNA) and protein-encoding (atpD and recA) genes, Southern hybridization/RFLP and a range of phenotypic characteristics. Among phage-resistant bacteria, one genotype of Rhizobium sp. predominated on alfalfa (frequency approximately 68 %) but was recovered infrequently ( approximately 1 %) from sweet clover. A second genotype was isolated infrequently only from alfalfa. These genotypes fixed nitrogen poorly in association with sweet clover and Phaseolus vulgaris, but were moderately effective with alfalfa. They produced a near-neutral reaction on mineral salts agar containing mannitol, which is atypical of the genus Rhizobium. A single isolate of Ensifer sp. and two of Phyllobacterium sp. were recovered only from sweet clover. All were highly resistant to multiple antibiotics. Phylogenetic analysis indicated that Ensifer sp. strain T173 is closely related to, but separate from, the non-symbiotic species 'Sinorhizobium morelense'. Strain T173 is unique in that it possesses a 175 kb symbiotic plasmid and elicits ineffective nodules on alfalfa, sweet clover, Medicago lupulina and Macroptilium atropurpureum. The two Phyllobacterium spp. were non-symbiotic and probably represent bacterial opportunists. Three genotypes of E. meliloti that were symbiotically effective with alfalfa and sweet clover were encountered infrequently. Among phage-susceptible isolates, two genotypes of E. medicae were encountered infrequently and were highly effective with alfalfa, sweet clover and Medicago polymorpha. The ecological and practical implications of the findings are discussed.

Rhizobia from Lanzarote, the Canary Islands, that nodulate Phaseolus vulgaris have characteristics in common with Sinorhizobium meliloti isolates from mainland Spain.

The stable, low-molecular-weight (LMW) RNA fractions of several rhizobial isolates of Phaseolus vulgaris grown in the soil of Lanzarote, an island of the Canary Islands, were identical to a less-common pattern found within Sinorhizobium meliloti (assigned to group II) obtained from nodules of alfalfa and alfalfa-related legumes grown in northern Spain. The P. vulgaris isolates and the group II LMW RNA S. meliloti isolates also were distinguishable in that both had two conserved inserts of 20 and 46 bp in the 16S-23S internal transcribed spacer region that were not present in other strains of S. meliloti. The isolates from P. vulgaris nodulated bean but not Medicago sativa, while those recovered from Medicago, Melilotus, and Trigonella spp. nodulated both host legumes. The bean isolates also were distinguished from those of Medicago, Melilotus, and Trigonella spp. by nodC sequence analysis. The nodC sequences of the bean isolates were most similar to those reported for S. meliloti bv. mediterranense and Sinorhizobium fredii bv. mediterranense (GenBank accession numbers DQ333891 and AF217267, respectively). None of the evidence placed the bean isolates from Lanzarote in the genus Rhizobium, which perhaps is inconsistent with seed-borne transmission of Rhizobium etli from the Americas to the Canaries as an explanation for the presence of bean-nodulating rhizobia in soils of Lanzarote.

[Recombinant expression of an autoinducer synthase of Sinorhizobium sp.1128 in Escherichia coli].

OBJECTIVE: To explore the roles of quorum sensing system in establishing symbiosis between bacterium Sinorhizobium sp.1128 and its plant host Melilotus suaveolens Ledeb. METHODS: According to homologous analysis, we designed primers to amplify the autoinducer synthase encoding genes in Sinorhizobium sp.1128 according to Sinorhizobium medicae WSM419 genome sequences. The autoinducer synthase encoding genes were cloned into the expression vector of pYC12 and expressed in E. coli DH5alpha. Thin-layer chromatography (TLC) assay was used to study their roles in autoinducer production. A duplicated inactivation of the gene was used to explore its function in plant nodulation. RESULTS: Homologous analysis showed that at least three annotated acylated homoserine lactone (AHL) synthase genes existed in Sinorhizobium medicae WSM419 genome. We cloned these three autoinducer synthase genes in Sinorhizobium sp.1128. One of these genes named traI2 was over expressed in E. coli DH5alpha. At least two different AHLs were produced by the recombinant strain. Disruption of traI2 reduced both the autoinducers (AI) activities and AHL production by TLC detection. Furthermore, the complementation of traI2 reverted the phenotype of AI activities. These findings demonstrate that traI2 was responsible for AI synthesis in Sinorhizobium sp.1128. More important, the traI2 deficient strains were defective in nodule formation on their host plant. CONCLUSION: The quorum sensing circuits in Sinorhizobium sp.1128 may play an important role in symbiosis between plant and bacterium.

Endophytic occupation of root nodules and roots of Melilotus dentatus by Agrobacterium tumefaciens.

Agrobacterium strains have been frequently isolated from the root nodules of different legumes. Various possible mechanisms have been proposed to explain the existence of these bacteria in nodules, but there is no sufficient experimental evidence to support the estimations. In this work, we proved that the Agrobacterium strain CCBAU 81181, which was originally isolated from the root nodules of Onobrychis viciaefolia, and a symbiotic strain of Sinorhizobium meliloti CCBAU 10062 could coinhabit the root nodules of Melilotus dentatus. Analyses were performed by using a fluorescence marker, reisolation of bacteria from nodules, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of whole cellular proteins, and polymerase chain reaction amplification of symbiotic genes. The inoculation of A. tumefaciens CCBAU 81181 did not affect the growth and nodulation of plants. CCBAU 81181 and 24 other Agrobacterium strains isolated from nodules were incapable of nodulating on their original or alternative host and 22 strains of these strains were endophytes in the roots and stems of their hosts. Also, the tumor-inducing A. tumefaciens strains IAM 13129(T) and C58 were found capable of entering the roots of Glycyrrhiza pallidiflora, but did not cause pathogenic symptoms. With these results, we conclude that A. tumefaciens strains could be endophytic bacteria in the roots, stems, and root nodules. This finding partially explains why Agrobacterium strains were frequently isolated from the surface-sterilized nodules.

The expression of MaEXP1, a Melilotus alba expansin gene, is upregulated during the sweetclover-Sinorhizobium meliloti interaction.

Expansins are a highly conserved group of cell wall-localized proteins that appear to mediate changes in cell wall plasticity during cell expansion or differentiation. The accumulation of expansin protein or the mRNA for specific expansin gene family members has been correlated with the growth of various plant organs. Because expansin proteins are closely associated with plant cell wall expansion, and as part of a larger study to determine the role of different gene products in the legume-Rhizobium spp. symbiosis, we investigated whether a Melilotus alba (white sweetclover) expansin gene is expressed during nodule development. A cDNA fragment encoding an expansin gene (EXP) was isolated from Sinorhizobium meliloti-inoculated sweetclover root RNA by reverse-transcriptase polymerase chain reaction using degenerate primers, and a full-length sweetclover expansin sequence (MaEXP1) was obtained using 5' and 3' rapid amplification of cDNA end cloning. The predicted amino acid of the sweetclover expansin is highly conserved with the various alpha-expansins in the GenBank database. MaEXP1 contains a series of eight cysteines and four tryptophans that are conserved in the alpha-expansin protein family. Northern analysis and whole-mount in situ hybridization analyses indicate that MaEXP1 mRNA expression is enhanced in roots within hours after inoculation with S. meliloti and in nodules. Western and immunolocalization studies using a cucumber expansin antibody demonstrated that a cross-reacting protein accumulated in the expanding cells of the nodule.

Temporal effects on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa and Melilotus alba.

An assessment was made of the impact of temporal separation on the composition of a population of Sinorhizobium meliloti associated with Medicago sativa (alfalfa) and Melilotus alba (sweet clover) grown at a single site that had no known history of alfalfa cultivation. Root nodules were sampled on six occasions over two seasons, and a total of 1620 isolates of S. meliloti were characterized on the basis of phage sensitivity using 16 typing phages. Plant infection tests indicated that symbiotic S. meliloti were deficient in the soil at the time of planting and that these bacteria were present at low density during the first season (<10(2)/g of soil); in the second season numbers increased markedly to about 10(5)/g of soil. Overall, 37 and 51 phage types, respectively, were encountered among the nodule isolates from M. sativa and M. alba. The data indicate significant temporal shifts in the frequency and diversity of types associated with the two legume species. Apparent temporal variation with respect to the frequency of types appeared largely unpredictable and was not attributable to any one sampling time. The results indicate an apparent reduction in phenotypic diversity over the course of the experiment. Differential host plant selection of specific types with respect to nodule occupancy was indicated by significant interactions between legume species and either the frequency or diversity of phage types. Isolates from M. sativa that were resistant to lysis by all typing phages (type 14) were unusual in that they were predominant on this host at all sampling times (between 53% and 82% nodule occupancy) and were relatively homogeneous on the basis of DNA hybridization with 98% of the isolates analysed sharing the same nod EFG hybridization profile. In contrast, those isolates from M. alba comprising type 14 were encountered at low total frequency (2%) and were genetically heterogeneous on the basis of Southern hybridization. The implications of the observed temporal and host plant variation for ecological studies are discussed.