Trifolii Strain Research Articles

A potential trade-off between reproduction and enhancement of thermotolerance in Liriomyza trifolii populations driven by thermal acclimation

The invasive pest, Liriomyza trifolii, poses a significant threat to ornamental and vegetable plants. It spreads rapidly and causes large-scale outbreaks with pronounced thermotolerance. In this study, we developed L. trifolii strains adapted to high temperatures (strains designated 35 and 40); these were generated from a susceptible strain (designated S) by long-term thermal acclimation to 35 °C and 40 °C, respectively. Age-stage, two-sex life tables, thermal preferences, critical thermal limits, knockdown behaviors, eclosion and survival rates as well as expression of genes encoding heat shock proteins (Hsps) were compared for the three strains. Our findings indicated that the thermotolerance of L. trifolii was enhanced after long-term thermal acclimation, which suggested an adaptive plastic response to thermal stress. A trade-off between reproduction and thermotolerance was observed under thermal stress, potentially improving survival of the population and fostering adaptionary changes. Acclimation at 35 °C improved reproductive performance and population density of L. trifolii, particularly by enhancing the fecundity of female adults and accelerating the speed of development. Although the 40 strain exhibited the highest developmental speed and greater thermotolerance, it incurred a larger reproductive cost. This study provides a theoretical framework for monitoring and controlling leafminers and understanding their evolutionary adaptation to environmental changes.

Adaptation of Rhizobium leguminosarum sv. trifolii strains to low temperature stress in both free-living stage and during symbiosis with clover

Legume-rhizobial symbiosis plays an important role in agriculture and ecological restoration. This process occurs within special new structures, called nodules, formed mainly on legume roots. Soil bacteria, commonly known as rhizobia, fix atmospheric dinitrogen, converting it into a form that can be assimilated by plants. Various environmental factors, including a low temperature, have an impact on the symbiotic efficiency. Nevertheless, the effect of temperature on the phenotypic and symbiotic traits of rhizobia has not been determined in detail to date. Therefore, in this study, the influence of temperature on different cell surface and symbiotic properties of rhizobia was estimated. In total, 31 Rhizobium leguminosarum sv. trifolii strains isolated from root nodules of red clover plants growing in the subpolar and temperate climate regions, which essentially differ in year and day temperature profiles, were chosen for this analysis. Our results showed that temperature has a significant effect on several surface properties of rhizobial cells, such as hydrophobicity, aggregation, and motility. Low temperature also stimulated EPS synthesis and biofilm formation in R. leguminosarum sv. trifolii. This extracellular polysaccharide is known to play an important protective role against different environmental stresses. The strains produced large amounts of EPS under tested temperature conditions that facilitated adherence of rhizobial cells to different surfaces. The high adaptability of these strains to cold stress was also confirmed during symbiosis. Irrespective of their climatic origin, the strains proved to be highly effective in attachment to legume roots and were efficient microsymbionts of clover plants. However, some diversity in the response to low temperature stress was found among the strains. Among them, M16 and R137 proved to be highly competitive and efficient in nodule occupancy and biomass production; thus, they can be potential yield-enhancing inoculants of legumes.

Symbiotic efficiency of Rhizobium leguminosarum sv. trifolii strains originating from the subpolar and temperate climate regions

Red clover (Trifolium pratense L.) is a forage legume cultivated worldwide. This plant is capable of establishing a nitrogen-fixing symbiosis with Rhizobium leguminosarum symbiovar trifolii strains. To date, no comparative analysis of the symbiotic properties and heterogeneity of T. pratense microsymbionts derived from two distinct geographic regions has been performed. In this study, the symbiotic properties of strains originating from the subpolar and temperate climate zones in a wide range of temperatures (10–25 °C) have been characterized. Our results indicate that all the studied T. pratense microsymbionts from two geographic regions were highly efficient in host plant nodulation and nitrogen fixation in a wide range of temperatures. However, some differences between the populations and between the strains within the individual population examined were observed. Based on the nodC and nifH sequences, the symbiotic diversity of the strains was estimated. In general, 13 alleles for nodC and for nifH were identified. Moreover, 21 and 61 polymorphic sites in the nodC and nifH sequences were found, respectively, indicating that the latter gene shows higher heterogeneity than the former one. Among the nodC and nifH alleles, three genotypes (I–III) were the most frequent, whereas the other alleles (IV–XIII) proved to be unique for the individual strains. Based on the nodC and nifH allele types, 20 nodC-nifH genotypes were identified. Among them, the most frequent were three genotypes marked as A (6 strains), B (5 strains), and C (3 strains). Type A was exclusively found in the temperate strains, whereas types B and C were identified in the subpolar strains. The remaining 17 genotypes were found in single strains. In conclusion, our data indicate that R. leguminosarum sv. trifolii strains derived from two climatic zones show a high diversity with respect to the symbiotic efficiency and heterogeneity. However, some of the R. leguminosarum sv. trifolii strains exhibit very good symbiotic potential in the wide range of the temperatures tested; hence, they may be used in the future for improvement of legume crop production.

Genomic Insights into the Symbiotic and Plant Growth-Promoting Traits of "Candidatus Phyllobacterium onerii" sp. nov. Isolated from Endemic Astragalus flavescens.

A novel strain of Gram-negative, rod-shaped aerobic bacteria, identified as IY22, was isolated from the root nodules of Astragalus flavescens. The analysis of the 16S rDNA and recA (recombinase A) gene sequences indicated that the strain belongs to the genus Phyllobacterium. During the phylogenetic analysis, it was found that strain IY22 is closely related to P. trifolii strain PETP02T and P. bourgognense strain STM 201T. The genome of IY22 was determined to be 6,010,116 base pairs long with a DNA G+C ratio of 56.37 mol%. The average nucleotide identity (ANI) values showed a range from 91.7% to 93.6% when compared to its close relatives. Moreover, IY22 and related strains had digital DNA-DNA hybridization (dDDH) values ranging from 16.9% to 54.70%. Multiple genes (including nodACDSNZ, nifH/frxC, nifUS, fixABCJ, and sufABCDES) associated with symbiotic nitrogen fixation have been detected in strain IY22. Furthermore, this strain features genes that contribute to improving plant growth in various demanding environments. This study reports the first evidence of an association between A. flavescens and a rhizobial species. Native high-altitude legumes are a potential source of new rhizobia, and we believe that they act as a form of insurance for biodiversity against the threats of desertification and drought.

Comparative genomic analysis of diverse rhizobia and effective nitrogen-fixing clover-nodulating Rhizobium strains adapted to Egyptian dry ecosystems

Rhizobium strains that nodulate Egyptian winter clover belong to two symbiotic species, Rhizobium aegyptiacum sv. trifolii and Rhizobium bangladeshense sv. trifolii. Rhizobia from Egyptian clover are often tolerant of extreme environmental conditions and are generally poorly characterized, despite the importance of this crop species. Here, we report on the comparative genomic analysis of four stress-tolerant, highly effective nitrogen-fixing, Egyptian-clover-nodulating rhizobia, R. aegyptiacum sv. trifolii strain Rhiz950 and Rhizobium bangladeshense sv. trifolii strains Rhiz1002, Rhiz1017 and Rhiz1024, as well as other diverse Rhizobium strains. While Rhizobium bangladeshense sv. trifolii strains Rhiz1002, Rhiz1017, and Rhiz1024 had similar genome sizes of around 6.6 Mb, the salt tolerant strain Rhiz950 had a slightly larger genome size of 7.4 Mb. All four strains lacked genes responsible for methylation and sulfation of nod factors (nodS and nodHPQ). They also lacked nodZ, and instead had the functionally-equivalent nod factor fucosylation gene nolK. No strains contained homocitrate synthase (encoded by nifV) that is essential for free-living nitrogen fixing by diazotrophs. A number of genes unique to the Egyptian clover strains and to other rhizobia are potential candidates for variation in stress tolerance and host-range.

Exopolysaccharide Carbohydrate Structure and Biofilm Formation by Rhizobium leguminosarum bv. trifolii Strains Inhabiting Nodules of Trifoliumrepens Growing on an Old Zn-Pb-Cd-Polluted Waste Heap Area.

Heavy metals polluting the 100-year-old waste heap in Bolesław (Poland) are acting as a natural selection factor and may contribute to adaptations of organisms living in this area, including Trifolium repens and its root nodule microsymbionts—rhizobia. Exopolysaccharides (EPS), exuded extracellularly and associated with bacterial cell walls, possess variable structures depending on environmental conditions; they can bind metals and are involved in biofilm formation. In order to examine the effects of long-term exposure to metal pollution on EPS structure and biofilm formation of rhizobia, Rhizobium leguminosarum bv. trifolii strains originating from the waste heap area and a non-polluted reference site were investigated for the characteristics of the sugar fraction of their EPS using gas chromatography mass-spectrometry and also for biofilm formation and structural characteristics using confocal laser scanning microscopy under control conditions as well as when exposed to toxic concentrations of zinc, lead, and cadmium. Significant differences in EPS structure, biofilm thickness, and ratio of living/dead bacteria in the biofilm were found between strains originating from the waste heap and from the reference site, both without exposure to metals and under metal exposure. Received results indicate that studied rhizobia can be assumed as potentially useful in remediation processes.

Complete Genome Sequences of Trifolium spp. Inoculant Strains Rhizobium leguminosarum sv. trifolii TA1 and CC275e: Resources for Genomic Study of the Rhizobium-Trifolium Symbiosis.

Rhizobium leguminosarum symbiovar trifolii strains TA1 and CC275e are nitrogen-fixing microsymbionts of Trifolium spp. and have been used as commercial inoculant strains for clovers in pastoral agriculture in Australia and New Zealand. Here we present the complete genome sequences of both strains, resolving their multipartite genome structures and allowing for future studies using genomic approaches.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY 4.0 International license.

Rhizobium leguminosarum bv. trifolii NodD2 Enhances Competitive Nodule Colonization in the Clover-Rhizobium Symbiosis.

Establishment of the symbiotic relationship that develops between rhizobia and their legume hosts is contingent upon an interkingdom signal exchange. In response to host legume flavonoids, NodD proteins from compatible rhizobia activate expression of nodulation genes that produce lipochitin oligosaccharide signaling molecules known as Nod factors. Root nodule formation commences upon legume recognition of compatible Nod factor. Rhizobium leguminosarum was previously considered to contain one copy of nodD; here, we show that some strains of the Trifolium (clover) microsymbiont R. leguminosarum bv. trifolii contain a second copy designated nodD2. nodD2 genes were present in 8 out of 13 strains of R. leguminosarum bv. trifolii, but were absent from the genomes of 16 R. leguminosarum bv. viciae strains. Analysis of single and double nodD1 and nodD2 mutants in R. leguminosarum bv. trifolii strain TA1 revealed that NodD2 was functional and enhanced nodule colonization competitiveness. However, NodD1 showed significantly greater capacity to induce nod gene expression and infection thread formation. Clover species are either annual or perennial and this phenological distinction is rarely crossed by individual R. leguminosarum bv. trifolii microsymbionts for effective symbiosis. Of 13 strains with genome sequences available, 7 of the 8 effective microsymbionts of perennial hosts contained nodD2, whereas the 3 microsymbionts of annual hosts did not. We hypothesize that NodD2 inducer recognition differs from NodD1, and NodD2 functions to enhance competition and effective symbiosis, which may discriminate in favor of perennial hosts.IMPORTANCE Establishment of the rhizobium-legume symbiosis requires a highly specific and complex signal exchange between both participants. Rhizobia perceive legume flavonoid compounds through LysR-type NodD regulators. Often, rhizobia encode multiple copies of nodD, which is one determinant of host specificity. In some species of rhizobia, the presence of multiple copies of NodD extends their symbiotic host-range. Here, we identified and characterized a second copy of nodD present in some strains of the clover microsymbiont Rhizobium leguminosarum bv. trifolii. The second nodD gene contributed to the competitive ability of the strain on white clover, an important forage legume. A screen for strains containing nodD2 could be utilized as one criterion to select strains with enhanced competitive ability for use as inoculants for pasture production.

Symbiosis between Rhizobium leguminosarum bv. trifolii strain TA1 and a white clover cultivar benefits clover tolerance to cadmium toxicity

ABSTRACT New Zealand pastoral productivity relies heavily on biological nitrogen (N) fixation from rhizobia bacteria. Cadmium (Cd), present in pasture soils as an impurity in phosphate fertilisers, may have toxic effects on both plant growth and rhizobia activity. Effects of Cd on growth of seedlings of a white clover cultivar ‘Grasslands Tribute’, in the absence and presence of the N2-fixing Rhizobium leguminosarum bv. trifolii strain TA1, were assessed in vermiculite microcosm assays. The lowest observable effect concentrations resulting in significantly lower white clover shoot weights than those grown without Cd were 0.07 mg Cd kg−1 (measured as Ca(NO3)2-extractable Cd, Cdextractable) in the absence of Rhizobium, and 2.33 mg Cdextractable kg−1 for Rhizobium-inoculated seedlings. Shoot N content was unaffected in white clover exposed to <5 mg Cdextractable kg−1. The results indicate symbiosis with Rhizobium strain TA1 conferred tolerance to white clover ‘Grasslands Tribute’ against Cd toxicity, and that this symbiosis is unlikely to be adversely affected by Cd concentrations reported (<0.003 mg Cdextractable kg−1) in New Zealand pasture soils.

Microencapsulation of Rhizobium leguminosarum bv. trifolii with guar gum: Preliminary approach using spray drying

Rhizobium leguminosarum bv trifolii strains TA1 and CC275e have been widely used as effective nitrogen fixing strains for white clover in New Zealand, but rhizobia survival on seeds is usually poor due to different stress conditions. The aim of this study was to select one of those commercial strains grown in a solid carrier (core) and study the influence of the core:polymer ratio in a microencapsulation process by spray drying using guar gum as coating material. First, strains TA1 and CC275e grown on peat and diatomaceous earth were exposed to temperature and desiccation stress. Both strains were stable at 40 °C and completely died after five minutes at 80 °C, while CC275e was more stable than TA1 at 60 °C. TA1 and CC275e slightly decreased viability after six hours drying with either carriers, with no differences between strains. A central composite design was used to develop the microencapsulation process. Independent variables were: inlet temperature (130 °C) and feed flow rate (5 mL/min). Microparticles presented rhizobia loading in 107 CFU/g and mean particle size between 10 and 30 μm. Optimized process reached 50% yield and 107 CFU/g loading. Rhizobia viability dropped two logarithmic units during the microencapsulation/drying process, possibly due to the negative effects of dehydration and high outlet temperature (≈70 °C), suggesting the need to continue optimizing the process by improving the thermal profile in the drying chamber.

Selection of Competitive and Efficient Rhizobia Strains for White Clover.

The practice of inoculating forage legumes with rhizobia strains is widespread. It is assumed that the inoculated strain determines the performance of the symbiosis and nitrogen fixation rates. However, native-naturalized strains can be competitive, and actual nodule occupancy is often scarcely investigated. In consequence, failures in establishment, and low productivity attributed to poor performance of the inoculant may merely reflect the absence of the inoculated strain in the nodules. This study lays out a strategy followed for selecting a Rhizobium leguminosarum sv. trifolii strain for white clover (Trifolium repens) with competitive nodule occupancy. First, the competitiveness of native-naturalized rhizobia strains selected for their efficiency to fix N2 in clover and tagged with gusA was evaluated in controlled conditions with different soils. Second, three of these experimental strains with superior nodule occupancy plus the currently recommended commercial inoculant, an introduced strain, were tested in the field in 2 years and at two sites. Plant establishment, herbage productivity, fixation of atmospheric N2 (15N natural abundance), and nodule occupancy (ERIC-PCR genomic fingerprinting) were measured. In both years and sites, nodule occupancy of the native-naturalized experimental strains was either higher or similar to that of the commercial inoculant in both primary and secondary roots. The difference was even greater in stolon roots nodules, where nodule occupancy of the native-naturalized experimental strains was at least five times greater. The amount of N fixed per unit plant mass was consistently higher with native-naturalized experimental strains, although the proportion of N derived from atmospheric fixation was similar for all strains. Plant establishment and herbage production, as well as clover contribution in oversown native grasslands, were either similar or higher in white clover inoculated with the native-naturalized experimental strains. These results support the use of our implemented strategy for developing a competitive inoculant from native-naturalized strains.

Phylogenetic relationships among introduced and autochthonous rhizobia nodulating Trifolium spp. in Uruguayan soils

Trifolium pratense (red clover) and T. repens (white clover) inoculated with Rhizobium leguminosarum sv. trifolii strain U204 are used to improve pastures in Uruguay. Although inoculation is a common practice in the country, there has been a progressive abandonment by farmers because they do not always see the benefits. The presence of efficient rhizobial populations may be one of the reasons why a response to inoculation is not always observed. We studied the identities and phylogenetic relationships among the clover rhizobia populating Uruguayan soils by analysis of 16S rRNA genes, ITS sequences and housekeeping (atpD, glnII, recA, rpoB) and symbiotic (nodA, nodC, nifH) genes. Strains like P3 and P33, isolated from the endemic Trifolium polymorphum, are inefficient in red and white clover. These strains clustered separately from the symbionts of these clovers. Bacteria which are efficient in red and white clovers clustered in either of two groups: one, represented by isolate 249, are likely naturalized rhizobia derived from the inoculant U204; and other included strains like U317 and N2, which share symbiotic genes with the inoculant. We hypothesize that these strains may be derived from native strains that acquired symbiotic gene from inoculant strains. These types of rhizobia are strong candidates for a second generation of clover inoculants for Uruguay.

Mutation in the pssZ Gene Negatively Impacts Exopolysaccharide Synthesis, Surface Properties, and Symbiosis of Rhizobium leguminosarum bv. trifolii with Clover.

Rhizobium leguminosarum bv. trifolii is a soil bacterium capable of establishing a nitrogen-fixing symbiosis with clover plants (Trifolium spp.). This bacterium secretes large amounts of acidic exopolysaccharide (EPS), which plays an essential role in the symbiotic interaction with the host plant. This polymer is biosynthesized by a multi-enzymatic complex located in the bacterial inner membrane, whose components are encoded by a large chromosomal gene cluster, called Pss-I. In this study, we characterize R. leguminosarum bv. trifolii strain Rt297 that harbors a Tn5 transposon insertion located in the pssZ gene from the Pss-I region. This gene codes for a protein that shares high identity with bacterial serine/threonine protein phosphatases. We demonstrated that the pssZ mutation causes pleiotropic effects in rhizobial cells. Strain Rt297 exhibited several physiological and symbiotic defects, such as lack of EPS production, reduced growth kinetics and motility, altered cell-surface properties, and failure to infect the host plant. These data indicate that the protein encoded by the pssZ gene is indispensable for EPS synthesis, but also required for proper functioning of R. leguminosarum bv. trifolii cells.

TYPE VB AND VI SECRETION SYSTEMS AS COMPETITION AGENTS OF GRAM-NEGATIVE BACTERIA

Abstract Bacterial competition, defined as a local neighbour interactions, can lead to competitors coexistence, bacterial community self-organization or as travelling waves of species dominance in ecological niches. Bacteria have developed many mechanisms to communicate and compete. Kin discrimination mechanisms in bacterial populations allow species to distinguish a friend from a foe in bacterial environment. Type Vb and VI secretion systems (TVIbSS and TVISS) play crucial role in this phenomenon. A contact-dependent growth inhibition (CDI), primarily found in Escherichia coli strains, utilities CdiB/CdiA protein of type Vb secretion system, described also as two-partner secretion (TPS) system, to inhibit growth of non-kin strains, where cell contact is required. Presence of an intracellular small immunity protein (CdiI) protects E. coli cells from autoinhibition. Other bacterial competition system, primarily found in nodulation process of Rhizobium leguminosarum bv. Trifolii strain, engages type VI secretion system. The structure of TVISS is more complicated and comprises the series of proteins with structural homology to bacteriophage tail proteins and membrane proteins which builds the core of the system (Tss proteins). Meanwhile, other proteins of the TVISS was described as associated proteins (Tag proteins). Important proteins for TVISS are haemolysin coregulated protein (Hcp) which has hexameric, tubular structure and VgrG protein (valine-glycine repeat G) which play a dual role in the process: as a chaperone protein in secretion of effector toxin or/and as a secreted toxin itself. Despite the structural differences of both secretion systems they show functional homology in competition phenomenon and govern the social life of bacterial community. 1. Introduction. 2. Contact-dependent growth inhibition. 2.1. Structure o CDI machinery. 2.2. Effectors of CDI system. 3. Type VI secretion system. 3.1. Structure of type VI secretion system. 3.2. Effectors of type VI secretion system. 4. Membership to polymorphic toxins system. 5. Role of the systems in bacterial biology. 6. Conclusions

Host-dependent symbiotic efficiency of Rhizobium leguminosarum bv. trifolii strains isolated from nodules of Trifolium rubens

Trifolium rubens L., commonly known as the red feather clover, is capable of symbiotic interactions with rhizobia. Up to now, no specific symbionts of T. rubens and their symbiotic compatibility with Trifolium spp. have been described. We characterized the genomic diversity of T. rubens symbionts by analyses of plasmid profiles and BOX–PCR. The phylogeny of T. rubens isolates was inferred based on the nucleotide sequences of 16S rRNA and two core genes (atpD, recA). The nodC phylogeny allowed classification of rhizobia nodulating T. rubens as Rhizobium leguminosarum symbiovar trifolii (Rlt). The symbiotic efficiency of the Rlt isolates was determined on four clover species: T. rubens, T. pratense, T. repens and T. resupinatum. We determined that Rlt strains formed mostly inefficient symbiosis with their native host plant T. rubens and weakly effective (sub-optimal) symbiosis with T. repens and T. pratense. The same Rlt strains were fully compatible in the symbiosis with T. resupinatum. T. rubens did not exhibit strict selectivity in regard to the symbionts and rhizobia closely related to Rhizobium grahamii, Rhizobium galegae and Agrobacterium radiobacter, which did not nodulate Trifolium spp., were found amongst T. rubens nodule isolates.

Arsenic methylation by a genetically engineered Rhizobium-legume symbiont.

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. 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. 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. 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.

Surface Properties of Wild-Type Rhizobium leguminosarum bv. trifolii Strain 24.2 and Its Derivatives with Different Extracellular Polysaccharide Content.

Rhizobium leguminosarum bv. trifolii is a soil bacterium able to establish symbiosis with agriculturally important legumes, i.e., clover plants (Trifolium spp.). Cell surface properties of rhizobia play an essential role in their interaction with both biotic and abiotic surfaces. Physicochemical properties of bacterial cells are underpinned by the chemical composition of their envelope surrounding the cells, and depend on various environmental conditions. In this study, we performed a comprehensive characterization of cell surface properties of a wild-type R. leguminosarum bv. trifolii strain 24.2 and its derivatives producing various levels of exopolysaccharide (EPS), namely, pssA mutant Rt5819 deficient in EPS synthesis, rosR mutant Rt2472 producing diminished amounts of this polysaccharide, and two EPS-overproducing strains, Rt24.2(pBA1) and Rt24.2(pBR1), under different growth conditions (medium type, bacterial culture age, cell viability, and pH). We established that EPS plays an essential role in the electrophoretic mobility of rhizobial cells, and that higher amounts of EPS produced resulted in greater negative electrophoretic mobility and higher acidity (lower pKapp,av) of the bacterial cell surface. From the tested strains, the electrophoretic mobility was lowest in EPS-deficient pssA mutant. Moreover, EPS produced by rhizobial strains resulted not only in an increase of negative surface charge but also in increased hydrophobicity of bacterial cell surface. This was determined by measurements of water contact angle, surface free energy, and free energy of bacterial surface–water–bacterial surface interaction. Electrophoretic mobility of the studied strains was also affected by the structure of the bacterial population (i.e., live/dead cell ratio), medium composition (ionic strength and mono- and divalent cation concentrations), and pH.

Production of exopolysaccharide by Rhizobium leguminosarum bv. trifolii and its role in bacterial attachment and surface properties

The acidic exopolysaccharide (EPS) produced by Rhizobium leguminosarum bv. trifolii is required for the establishment of effective symbiosis with compatible host plants (Trifolium spp.). In the rhizobium-legume interaction, early stages of root infection and nodule development have been well studied from a genetic standpoint. However, factors important for colonization of several surfaces by rhizobia, including soil particles and roots, have not yet been thoroughly investigated. The aim of this study was establishing of environmental factors affecting production of EPS by R. leguminosarum bv. trifolii strain 24.2 and the role of this polysaccharide in bacterial surface properties and attachment ability. Besides the wild-type strain, its derivatives differing in the level of EPS produced were used to these analyses. The ability of attachment to abiotic and biotic surfaces of these strains were established using CFU counting experiments. Three-dimensional structure and other parameters of biofilms formed were characterized in confocal laser scanning microscopy. Electrokinetic (zeta) potential of rhizobial cells were determined using Laser Doppler Velocimetry. It was evidenced that the ability of R. leguminosarum bv. trifolii to produce EPS significantly affected bacterial attachment and biofilm formation on both abiotic and biotic surfaces. In addition, the presence of this polysaccharide influenced the zeta potential of rhizobial cells. Mutant strains having a mutation in genes involved in EPS synthesis were significantly impaired in attachment, whereas strains overproducing this polysaccharide showed higher adhesion efficiency to all of the tested materials. EPS facilitated attachment of bacterial cells to the tested surfaces most probably due to hydrophobic interactions and heterogeneity of the envelope surface. EPS produced by R. leguminosarum bv. trifolii plays a significant role in attachment and biofilm formation to both abiotic and biotic surfaces as well as bacterial surface properties.

Assessment of core and accessory genetic variation in Rhizobium leguminosarum symbiovar trifolii strains from diverse locations and host plants using PCR-based methods

The nitrogen-fixing symbiosis between Rhizobium leguminosarum and host legumes is recognized as a key part of sustainable agriculture. A culture collection containing rhizobia isolated from legumes of economic importance in the UK and worldwide, maintained at Rothamsted Research for many years, provided material for this study. We aimed to develop and validate efficient molecular diagnostics to investigate whether the host plant or geographical location had a greater influence on the genetic diversity of rhizobial isolates, and the extent to which the core bacterial genome and the accessory symbiosis genes located on plasmids were affected. To achieve this, core housekeeping genes and those involved in symbiosis interactions were sequenced and compared with genome-sequenced strains in the public domain. Results showed that some Rh.leguminosarum symbiovar trifolii strains nodulating clovers and Rh.leguminosarum sv.viciae strains nodulating peas and vicias shared identical housekeeping genes, clover nodule isolates from the same location could have divergent symbiosis genes, and others isolated on different continents could be very similar. This illustrates the likely co-migration of rhizobia and their legume hosts when crops are planted in new areas and indicates that selective pressure may arise from both local conditions and crop host genotypes. The nitrogen-fixing symbiosis between Rhizobium leguminosarum and host legumes has been recognized as a key part of sustainable agriculture for many years; this study provides new tools to study rhizobial biogeography which will be invaluable for extending the cultivation of legumes and indicating whether or not inoculation is necessary.

Genome sequence of Rhizobium leguminosarum bv trifolii strain WSM1689, the microsymbiont of the one flowered clover Trifolium uniflorum.

Rhizobium leguminosarum bv. trifolii is a soil-inhabiting bacterium that has the capacity to be an effective N2-fixing microsymbiont of Trifolium (clover) species. R. leguminosarum bv. trifolii strain WSM1689 is an aerobic, motile, Gram-negative, non-spore-forming rod that was isolated from a root nodule of Trifolium uniflorum collected on the edge of a valley 6 km from Eggares on the Greek Island of Naxos. Although WSM1689 is capable of highly effective N2-fixation with T. uniflorum, it is either unable to nodulate or unable to fix N2 with a wide range of both perennial and annual clovers originating from Europe, North America and Africa. WSM1689 therefore possesses a very narrow host range for effective N2 fixation and can thus play a valuable role in determining the geographic and phenological barriers to symbiotic performance in the genus Trifolium. Here we describe the features of R. leguminosarum bv. trifolii strain WSM1689, together with the complete genome sequence and its annotation. The 6,903,379 bp genome contains 6,709 protein-coding genes and 89 RNA-only encoding genes. This multipartite genome contains six distinct replicons; a chromosome of size 4,854,518 bp and five plasmids of size 667,306, 518,052, 341,391, 262,704 and 259,408 bp. This rhizobial genome is one of 20 sequenced as part of a DOE Joint Genome Institute 2010 Community Sequencing Program.