Construction of a Symbiotically Effective Strain of Rhizobium leguminosarum bv. trifolii with Increased Nodulation Competitiveness

Genes involved in nodulation competitiveness (tfx) were inserted by marker exchange into the genome of the effective strain Rhizobium leguminosarum bv. trifolii TA1. Isogenic strains of TA1 were constructed which differed only in their ability to produce trifolitoxin, an antirhizobial peptide. Trifolitoxin production by the ineffective strain R. leguminosarum bv. trifolii T24 limited nodulation of clover roots by trifolitoxin-sensitive strains of R. leguminosarum bv. trifolii. The trifolitoxin-producing exconjugant TA1::10-15 was very competitive for nodulation on clover roots when coinoculated with a trifolitoxin-sensitive reference strain. The nonproducing exconjugant TA1::12-10 was not competitive for nodule occupancy when coinoculated with the reference strain. Tetracycline sensitivity and Southern analysis confirmed the loss of vector DNA in the exconjugants. Trifolitoxin production by TA1::10-15 was stable in the absence of selection pressure. Transfer of tfx to TA1 did not affect nodule number or nitrogenase activity. These experiments represent the first stable genetic transfer of genes involved in nodulation competitiveness to a symbiotically effective Rhizobium strain.     

Trifolitoxin Production and Nodulation Are Necessary for the Expression of Superior Nodulation Competitiveness by Rhizobium leguminosarum bv. trifolii Strain T24 on Clover

Rhizobium leguminosarum bv. trifolii T24 is ineffective in symbiotic nitrogen fixation, produces a potent antibiotic (referred to here as trifolitoxin) that is bacteriostatic to certain Rhizobium strains, and is very competitive for clover root nodulation (EA Schwinghamer, RP Belkengren 1968 Arch Mikrobiol 64: 130-145). The primary objective of this work was to demonstrate the roles of nodulation and trifolitoxin production in the expression of nodulation competitiveness by T24. Unlike wildtype T24, transposon mutants of T24 lacking trifolitoxin production were unable to decrease clover nodulation by an effective, trifolitoxin-sensitive strain of R. leguminosarum bv. trifolii. A non-nodulating transposon mutant of T24 prevented clover nodulation by a trifolitoxin-sensitive R. leguminosarum bv. trifolii when co-inoculated with a T24 mutant lacking trifolitoxin production. Neither mutant alone prevented nodulation by the trifolitoxin-sensitive strain. These results demonstrate that trifolitoxin production and nodulation are required for the expression of nodulation competitiveness by strain T24. A trifolitoxin-sensitive strain of R. meliloti did not nodulate alfalfa when co-inoculated with T24 and a trifolitoxin-resistant strain of R. meliloti. Thus, a trifolitoxin-producing strain was useful in regulating nodule occupancy on a legume host other than clover. Trifolitoxin production was constitutive in both minimal and enriched media. Trifolitoxin was found to inhibit the growth of 95% of all strains of R. leguminosarum bvs. trifolii, viceae, and phaseoli tested. Strains of all 13 biotypes of R. leguminosarum bv. trifolii were inhibited by trifolitoxin. Three strains of R. fredii were also inhibited. Strain T24 ineffectively nodulated 46 clover species, did not nodulate Trifolium ambiguum, and induced partially effective nodules on Trifolium micranthum. Since T24 produced partially effective nodules on T. micranthum and since a trifolitoxin-minus mutant of T24 induced ineffective nodules, trifolitoxin production is not the cause of the symbiotic ineffectiveness of T24.     

Identifying abnormalities in symbiotic development between Trifolium spp. and Rhizobium leguminosarum bv. trifolii leading to sub-optimal and ineffective nodule phenotypes

Background and Aims

Legumes overcome nitrogen limitations by entering into a mutualistic symbiosis with N₂-fixing bacteria (rhizobia). Fully compatible associations (effective) between Trifolium spp. and Rhizobium leguminosarum bv. trifolii result from successful recognition of symbiotic partners in the rhizosphere, root hair infection and the formation of nodules where N₂-fixing bacteroids reside. Poorly compatible associations can result in root nodule formation with minimal (sub-optimal) or no (ineffective) N₂-fixation. Despite the abundance and persistence of strains in agricultural soils which are poorly compatible with the commercially grown clover species, little is known of how and why they fail symbiotically. The aims of this research were to determine the morphological aberrations occurring in sub-optimal and ineffective clover nodules and to determine whether reduced bacteroid numbers or reduced N₂-fixing activity is the main cause for the Sub-optimal phenotype.

Methods

Symbiotic effectiveness of four Trifolium hosts with each of four R. leguminosarum bv. trifolii strains was assessed by analysis of plant yields and nitrogen content; nodule yields, abundance, morphology and internal structure; and bacteroid cytology, quantity and activity.

Key Results

Effective nodules (Nodule Function 83–100 %) contained four developmental zones and N₂-fixing bacteroids. In contrast, Sub-optimal nodules of the same age (Nodule Function 24–57 %) carried prematurely senescing bacteroids and a small bacteroid pool resulting in reduced shoot N. Ineffective-differentiated nodules carried bacteroids aborted at stage 2 or 3 in differentiation. In contrast, bacteroids were not observed in Ineffective-vegetative nodules despite the presence of bacteria within infection threads.

Conclusions

Three major responses to N₂-fixation incompatibility between Trifolium spp. and R. l. trifolii strains were found: failed bacterial endocytosis from infection threads into plant cortical cells, bacteroid differentiation aborted prematurely, and a reduced pool of functional bacteroids which underwent premature senescence. We discuss possible underlying genetic causes of these developmental abnormalities and consider impacts on N₂-fixation of clovers.     

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.     

Microscopic analysis of the effect ofRhizobium leguminosarum biovartrifolii host specific nodulation genes in the infection of white clovers

Summary A microscopic assessment is presented of the comparative infection capacity of wild-type and hybrid strains ofRhizobium leguminosarum bv.viciae withR. l. bv.trifolii strain ANU 843 on white clover seedlings. TheR. l. bv.viciae hybrid strains contained defined DNA segments coding for different combinations ofR. l. bv.trifolii host-specific nodulation genes. White clover plants were examined over a 72 h period to assessRhizobium infectivity, the morphological changes in root hair growth; colonisation ability of rhizobia; infection thread initiation and the ability to induce cortical cell division.R. l. bv.viciae strain 300 induced root hair curling more slowly than strain ANU 843 or any of the hybrid strain 300 bacteria, and when curling had taken place, there was poorer colonization by strain 300 within the folded hair cell, no evidence of infection thread formation and only limited cortical cell division 72 h after inoculation. The addition of the host-specific nodulation genes ofR. l. bv.trifolii to strain 300 was necessary to induce infection threads and establish a normal pattern of nodulation of the roots of white clovers.     

Genome sequence of the Trifolium rueppellianum -nodulating Rhizobium leguminosarum bv. trifolii strain WSM2012.

Rhizobium leguminosarum bv. trifolii WSM2012 (syn. MAR1468) is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from an ineffective root nodule recovered from the roots of the annual clover Trifolium rueppellianum Fresen growing in Ethiopia. WSM2012 has a narrow, specialized host range for N₂-fixation. Here we describe the features of R. leguminosarum bv. trifolii strain WSM2012, together with genome sequence information and annotation. The 7,180,565 bp high-quality-draft genome is arranged into 6 scaffolds of 68 contigs, contains 7,080 protein-coding genes and 86 RNA-only encoding genes, and is one of 20 rhizobial genomes sequenced as part of the DOE Joint Genome Institute 2010 Community Sequencing Program.     

Sequence Analysis of Hypothetical Lysine Exporter Genes of Rhizobium leguminosarum bv. trifolii from Calamine Old Waste Heaps and Their Evolutionary History

The aim of this study was to identify heavy metal detoxification system in Rhizobium leguminosarum bv. trifolii isolated from Trifolium repens inhabiting old (70–100 years) Zn–Pb waste heaps in Poland by PCR reaction with czcD1 and czcD2 primers. By sequence analysis, four different genotypes of obtained amplicons were identified among eight examined isolates. Their sequence similarity ranged 91–99 %. They indicated the highest sequence identity to the hypothetical lysine exporter gene of R. leguminosarum bv. trifolii WSM1325 (91–97 %) and 76–81 % sequence similarity to hypothetical lysine exporter genes of R. leguminosarum bv. trifolii WSM2304 and R. etli CFN42 and CIAT652. On phylogenetic tree of obtained amplicons, all four studied R. leguminosarum bv. trifolii genotypes formed common monophyletic cluster with R. leguminosarum bv. trifolii WSM1325 at 100 % bootstrap support showing that all four amplicons obtained in PCR with czcD1 and czcD2 primers are fragments of hypothetical lysine exporter gene (lysE). We also suggest that Lys efflux exporter may participate in heavy metal transport out of R. leguminosarum bv. trifolii cells.     

Elevated atmospheric CO2 alters microbial population structure in a pasture ecosystem

An increase in concentration of atmospheric CO₂ is one major factor influencing global climate change. Among the consequences of such an increase is the stimulation of plant growth and productivity. Below‐ground microbial processes are also likely to be affected indirectly by rising atmospheric CO₂ levels, through increased root growth and rhizodeposition rates. Because changes in microbial community composition might have an impact on symbiotic interactions with plants, the response of root nodule symbionts to elevated atmospheric CO₂ was investigated. In this study we determined the genetic structure of 120 Rhizobium leguminosarum bv. trifolii isolates from white clover plants exposed to ambient (350 μmol mol^?1) or elevated (600 μmol mol^?1) atmospheric CO₂ concentrations in the Swiss FACE (Free‐Air‐Carbon‐Dioxide‐Enrichment) facility. Polymerase Chain Reaction (PCR) fingerprinting of genomic DNA showed that the isolates from plants grown under elevated CO₂ were genetically different from those isolates obtained from plants grown under ambient conditions. Moreover, there was a 17% increase in nodule occupancy under conditions of elevated atmospheric CO₂ when strains of R. leguminosarum bv. trifolii isolated from plots exposed to CO₂ enrichment were evaluated for their ability to compete for nodulation with those strains isolated from ambient conditions. These results indicate that a shift in the community composition of R. leguminosarum bv. trifolii occurred as a result of an increased atmospheric CO₂ concentration, and that elevated atmospheric CO₂ affects the competitive ability of root nodule symbionts, most likely leading to a selection of these particular strains to nodulate white clover.     

Subnanomolar concentrations of membrane chitolipooligosaccharides from Rhizobium leguminosarum biovar trifolii are fully capable of eliciting symbiosis-related responses on white clover

Axenic seedling bioassays were performed on white clover, vetch, and alfalfa to assess the variety and dose responses of biological activities exhibited by membrane chitolipooligosaccharides (CLOSs) from wild type Rhizobium leguminosarum bv. trifolii ANU843. Subnanomolar concentrations of CLOSs induced deformation of root hairs (Had) and increased the number of foci of cortical cell divisions (Ccd) in white clover, some of which developed into nodule meristems. In contrast, ANU843 CLOSs were unable to induce Had in alfalfa and required a 10⁴-fold higher threshold concentration to induce this response in vetch. Also, ANU843 CLOSs were not mitogenic on either of these non-host legumes. In addition, CLOS action also increased chitinase activity in white clover root exudate. Thus, the membrane CLOSs from wild type R. leguminosarum bv. trifolii are fully capable of eliciting various symbiosis-related responses in white clover in the same concentration range as extracellular CLOSs of other rhizobia on their respective legume hosts. These results and our earlier studies indicate that membrane CLOSs represent one of many different classes of bioactive metabolites made by R. leguminosarum bv. trifolii which elicit more intense symbiosis-related responses in white clover than in other legumes. Therefore, CLOSs evidently play an important role in symbiotic development, but they may not be the sole determinant of host-range in the Rhizobium-clover symbiosis.Abbreviations Ccd cortical cell division - CLOS chitolipooligosaccharide - Had root hair deformation     

<Emphasis Type="Italic">Rhizobium leguminosarum</Emphasis> bv. <Emphasis Type="Italic">trifolii rosR</Emphasis> is required for interaction with clover,biofilm formation and adaptation to the environment

Background  

Rhizobium leguminosarum bv. trifolii is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Trifolium spp. Bacterial surface polysaccharides are crucial for establishment of a successful symbiosis with legumes that form indeterminate-type nodules, such as Trifolium, Pisum, Vicia, and Medicago spp. and aid the bacterium in withstanding osmotic and other environmental stresses. Recently, the R. leguminosarum bv. trifolii RosR regulatory protein which controls exopolysaccharide production has been identified and characterized.     

Isolation of Rhizobium leguminosarum (biovar trifolii) Strains from Ethiopian Soils and Symbiotic Effectiveness on African Annual Clover Species

Strains of Rhizobium leguminosarum (biovar trifolii) isolated from two Ethiopian soils or obtained from a commercial source were evaluated for symbiotic effectiveness on five African annual clover species. Numerous Rhizobium trifolii strains that exhibited varying levels of symbiotic effectiveness were isolated from both soils (a nitosol and a vertisol), and it was possible to identify strains that were highly effective for each clover species. The soil isolates were, as a group, superior to the strains from the commercial source. Several R. trifolii strains were found to be effective on more than one clover species, and there appeared to be at least two and possibly three distinct cross-inoculation effectiveness groups.     

Enhancement of symbiotic nitrogen fixation by vitamin-secreting fluorescent Pseudomonas

Fluorescent Pseudomonas sp. strain 267 promotes growth of nodulated clover plants under gnotobiotic conditions. In the growth conditions (60 M FeCl₃), the production of siderophores of the pseudobactin-pyoverdin group was repressed. Plant growth enhancement results from secretion of B vitamins by Pseudomonas sp. strain 267. This was proven by stimulation of clover growth by naturally auxotrophic strains of Rhizobium leguminosarum bv. trifolii and marker strains E. coli thi^- and R. meliloti pan^- in the presence of the supernatant of Pseudomonas sp. strain 267. The addition of vitamins to the plant medium increased symbiotic nitrogen fixation by the clover plants.     

Genetic structure of the introduced and local populations of <Emphasis Type="Italic">Rhizobioum leguminosarum</Emphasis> in plant-soil systems

Comparative study of Rhizobium leguminosarum populations formed under the conditions of the Srednii Island (White Sea) demonstrated the introduced clover rhizobia (R. l. bv. trifolii) to be more variable than the aboriginal vetch/vetchling rhizobia (R. l. bv. viceae) in the chromosomal IGS locus, while being less variable in the plasmid-located symbiotic genes nodD and nifH. The analysis of these genes revealed the most pronounced differences between the clover and vetch/vetchling rhizobia populations. These differences, together with the results of ERIC-fingerprinting, indicated that the evolution of the clover rhizobia was mainly linked with the adaptation to local soil environment, and the evolution of the vetch/vetchling rhizobia, to the adaptation to various species of the host plants. High panmixia of R. leguminosarum population suggests its evolution to be based on the combinatory variability associated with the transfer of Sym-plasmids between R. l. bv. trifolii and R. l. bv. viceae, as well as with genomic rearrangements in the resulting recombinants.     

Rhizobium pisi sv. trifolii K3.22 harboring nod genes of the Rhizobium leguminosarum sv. trifolii cluster

The taxonomic status of the Rhizobium sp. K3.22 clover nodule isolate was studied by multilocus sequence analysis (MLSA) of 16S rRNA and six housekeeping chromosomal genes, as well as by a subsequent phylogenic analysis. The results revealed full congruence with the Rhizobium pisi DSM 30132^T core genes, thus supporting the same taxonomic position for both strains. However, the K3.22 plasmid symbiosis nod genes demonstrated high sequence similarity to Rhizobium leguminosarum sv. trifolii, whereas the R. pisi DSM 30132^Tnod genes were most similar to R. leguminosarum sv. viciae. The strains differed in the host range nodulation specificity, since strain K3.22 effectively nodulated red and white clover but not vetch, in contrast to R. pisi DSM 30132^T, which effectively nodulated vetch but was not able to nodulate clover. Both strains had the ability to form nodules on pea and bean but they differed in bean cultivar specificity. The R. pisi K3.22 and DSM 30132^T strains might provide evidence for the transfer of R. leguminosarum sv. trifolii and sv. viciae symbiotic plasmids occurring in natural soil populations.     

Expression by Soil Bacteria of Nodulation Genes from Rhizobium leguminosarum biovar trifolii

Gram-negative, rod-shaped bacteria from the soil of white clover-ryegrass pastures were screened for their ability to nodulate white clover (Trifolium repens) cultivar Grasslands Huia and for DNA homology with genomic DNA from Rhizobium leguminosarum biovar trifolii ICMP2668 (NZP582). Of these strains, 3.2% were able to hybridize with strain ICMP2668 and nodulate white clover and approximately 19% hybridized but were unable to nodulate. Strains which nodulated but did not hybridize with strain ICMP2668 were not detected. DNA from R. leguminosarum biovar trifolii (strain PN165) cured of its symbiotic (Sym) plasmid and a specific nod probe were used to show that the relationship observed was usually due to chromosomal homology. Plasmid pPN1, a cointegrate of the broad-host-range plasmid R68.45 and a symbiotic plasmid pRtr514a, was transferred by conjugation to representative strains of nonnodulating, gram-negative, rod-shaped soil bacteria. Transconjugants which formed nodules were obtained from 6 of 18 (33%) strains whose DNA hybridized with that of PN165 and 1 of 9 (11%) strains containing DNA which did not hybridize with that of PN165. The presence and location of R68.45 and nod genes was confirmed in transconjugants from three of the strains which formed nodules. Similarly, a pLAFR1 cosmid containing nod genes from a derivative of R. leguminosarum biovar trifolii NZP514 formed nodules when transferred to soil bacteria.     

Arsenic methylation by a genetically engineered <Emphasis Type="Italic">Rhizobium</Emphasis>-legume symbiont

Background and aims

Arsenic (As) is one of the most widespread environmental contaminants. The aim of our study was to test a novel bioremediation system based on the symbiosis between leguminous plant and genetically engineered rhizobia.

Methods

The arsenite [As(III)] S-adenosylmethionine methyltransferase gene (CrarsM) from the alga Chlamydomonas reinhardtii was inserted into the chromosome of Rhizobium leguminosarum bv. trifolii strain R3. The As methylation ability of the recombinant Rhizobium was tested under free living conditions and in symbiosis with red clover plants. Arsenic speciation was determined using high-performance liquid chromatography-inductively coupled plasma mass spectrometry.

Results

Under free-living conditions, CrarsM-recombinant R. leguminosarum gained the ability to methylate As(III) to methylated arsenicals, including methylarsenate [MAs(V)], dimethylarsenate [DMAs(V)] and trimethylarsine oxide [TMAs(V)O]. Red clover plants were inoculated with either control (non-recombinant) or CrarsM-recombinant R. leguminosarum and exposed to 5 or 10 μM arsenite. No methylated As species were detected in red clover plants inoculated with control R. leguminosarum. In contrast, all three methylated species were detected in both the nodules and the shoots when the recombinant Rhizobium established symbiosis with red clover, accounting for 74.7–75.1% and 29.1–42.4% of the total As in the two plant tissues, respectively. The recombinant symbiont also volatilized small amounts of As.

Conclusions

The present study demonstrates that engineered rhizobia expressing an algal arsM gene can methylate and volatilize As, providing a proof of concept for potential future use of legume-rhizobia symbionts for As bioremediation.

     

Selection and characterization of Spanish Trifolium-nodulating rhizobia for pasture inoculation

Identification of elite nitrogen-fixing rhizobia strains is a continuous and never ending effort, since new legume species can be cultivated in different agro systems or are introduced into new areas. This current study reports on the taxonomic affiliation and symbiotic proficiency of nine strains of Trifolium-nodulating rhizobia isolated from different pasture areas in Spain, as well as three Rhizobium leguminosarum bv. trifolii reference strains, on eleven Trifolium species. Based on 16S rRNA gene sequences the strains belonged to the R. leguminosarum species complex. Additional phylogenetic analyses of the housekeeping genes recA, atpD and rpoB showed the strains were closely related to the species R. leguminosarum, R. laguerreae, R. indicum, R. ruizarguesonis or R. acidisoli. In addition, three strains had no clear affiliation and could represent putative new species, although two of the reference strains were positioned close to R. ruizarguesonis. nodC gene phylogeny allowed the discrimination between strains isolated from annual or perennial Trifolium species and placed all of them in the symbiovar trifolii. Neither geographic origin nor host-plant species could be correlated with the taxonomic affiliation of the strains and a high degree of phenotypic diversity was found among this set of strains. The strong interaction of plant species with the rhizobial strains found for biological nitrogen fixation (BNF) was noteworthy, and allowed the identification of rhizobial strains with a maximum proficiency for certain trefoil species. Several strains showed high BNF potential with a wide range of clover species, which made them valuable strains for inoculant manufacturers and they would be particularly useful for inoculation of seed mixtures in natural or cultivated pastures.