The genome of Rhizobium leguminosarum has recognizable core and accessory components

Background

Rhizobium leguminosarum is an α-proteobacterial N₂-fixing symbiont of legumes that has been the subject of more than a thousand publications. Genes for the symbiotic interaction with plants are well studied, but the adaptations that allow survival and growth in the soil environment are poorly understood. We have sequenced the genome of R. leguminosarum biovar viciae strain 3841.

Results

The 7.75 Mb genome comprises a circular chromosome and six circular plasmids, with 61% G+C overall. All three rRNA operons and 52 tRNA genes are on the chromosome; essential protein-encoding genes are largely chromosomal, but most functional classes occur on plasmids as well. Of the 7,263 protein-encoding genes, 2,056 had orthologs in each of three related genomes (Agrobacterium tumefaciens, Sinorhizobium meliloti, and Mesorhizobium loti), and these genes were over-represented in the chromosome and had above average G+C. Most supported the rRNA-based phylogeny, confirming A. tumefaciens to be the closest among these relatives, but 347 genes were incompatible with this phylogeny; these were scattered throughout the genome but were over-represented on the plasmids. An unexpectedly large number of genes were shared by all three rhizobia but were missing from A. tumefaciens.

Conclusion

Overall, the genome can be considered to have two main components: a 'core', which is higher in G+C, is mostly chromosomal, is shared with related organisms, and has a consistent phylogeny; and an 'accessory' component, which is sporadic in distribution, lower in G+C, and located on the plasmids and chromosomal islands. The accessory genome has a different nucleotide composition from the core despite a long history of coexistence.     

A network-based approach to classify the three domains of life

Background

Chromosomal orthologs can reveal the shared ancestral gene set and their evolutionary trends. Additionally, physico-chemical properties of encoded proteins could provide information about functional adaptation and ecological niche requirements.

Results

We analyzed 7080 genes (five groups of 1416 orthologs each) from Rhizobiales species (S. meliloti, R. etli, and M. loti, plant symbionts; A. tumefaciens, a plant pathogen; and B. melitensis, an animal pathogen). We evaluated their phylogenetic relationships and observed three main topologies. The first, with closer association of R. etli to A. tumefaciens; the second with R. etli closer to S. meliloti; and the third with A. tumefaciens and S. meliloti as the closest pair. This was not unusual, given the close relatedness of these three species. We calculated the synonymous (dS) and nonsynonymous (dN) substitution rates of these orthologs, and found that informational and metabolic functions showed relatively low dN rates; in contrast, genes from hypothetical functions and cellular processes showed high dN rates. An alternative measure of sequence variability, percentage of changes by species, was used to evaluate the most specific proportion of amino acid residues from alignments. When dN was compared with that measure a high correlation was obtained, revealing that much of evolutive information was extracted with the percentage of changes by species at the amino acid level. By analyzing the sequence variability of orthologs with a set of five properties (polarity, electrostatic charge, formation of secondary structures, molecular volume, and amino acid composition), we found that physico-chemical characteristics of proteins correlated with specific functional roles, and association of species did not follow their typical phylogeny, probably reflecting more adaptation to their life styles and niche preferences. In addition, orthologs with low dN rates had residues with more positive values of polarity, volume and electrostatic charge.

Conclusions

These findings revealed that even when orthologs perform the same function in each genomic background, their sequences reveal important evolutionary tendencies and differences related to adaptation. This article was reviewed by: Dr. Purificación López-García, Prof. Jeffrey Townsend (nominated by Dr. J. Peter Gogarten), and Ms. Olga Kamneva.     

Genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in Nepal

Background and Aims

This study was conducted to reveal the genetic diversity of common bean (Phaseolus vulgaris L.) nodulating rhizobia in various agroecological regions in Nepal.

Method

A total of 63 strains were isolated from common bean grown in the soils collected from seven bean fields in Nepal and characterized based on the partial sequences of 16S–23S internal transcribed spacer (ITS) regions, 16S rDNA, nodC, and nifH. Symbiotic properties of some representative strains with host plants were examined to elucidate their characteristics in relation to genotype and their origin.

Results

The isolated strains belonged to Rhizobium leguminosarum, Rhizobium etli, Rhizobium phaseoli, and one unknown Rhizobium lineage, all belonging to a common symbiovar (sv.) phaseoli. Nine ITS genotypes were detected mainly corresponding to a single site, including a dominant group at three sites harboring highly diverse multiple ITS sequences. Three symbiotic genotypes corresponded to a geographical region, not to the ribosomal DNA group, suggesting horizontal transfer of symbiotic genes separately in each region. Great differences in nitrogenase activity and nodule forming ability among the strains irrespective of their species and origin were observed.

Conclusions

Nepalese Himalaya harbor phylogenetically highly diverse and site-specific strains of common bean rhizobia, some of which could have high potential of symbiotic nitrogen fixation.     

Distinctive Mesorhizobium populations associated with Cicer arietinum L. in alkaline soils of Xinjiang, China

Background and aims

Rhizobia associated with chickpea in the main chickpea production zone of Xinjiang, China have never been investigated. Here, we present the first systematic investigation of these rhizobia’s genetic diversity and symbiotic interactions with their host plant.

Methods

Ninety-five isolates obtained from chickpea nodules in eight alkaline-saline (pH?8.24–8.45) sites in Xinjiang were characterized by nodulation test, symbiotic gene analysis, PCR-based restriction fragment length polymorphism (RFLP) of the 16S rRNA gene and 16S–23S rRNA intergenic spacer (IGS), BOX-PCR, phylogenies of 16S rRNA and housekeeping genes (atpD, recA and glnII), multilocus sequence analysis (MLSA) and DNA–DNA hybridization.

Results

All 95 isolates were identified within the genus of Mesorhizobium. Similarities less than 96.5% in MLSA and DNA–DNA hybridization values (<50%) between the new isolates and the defined Mesorhizobium species, and high similarities (>98%) of symbiotic genes (nodC and nifH) with those of the well studied chickpea microsymbioints Mesorhizobium ciceri and Mesorhizobium mediterraneum were found.

Conclusions

Chickpea rhizobia in alkaline-saline soils of Xinjiang, China, form a population distinct from the defined Mesorhizobium species. All these chickpea rhizobia in Xinjiang harbored symbiotic genes highly similar to the type strains of two well-studied chickpea rhizobia, M. ciceri and M. mediterraneum, evidencing the possible lateral transfer of symbiotic genes among these different rhizobial species. On the other hand, chickpea may strongly select rhizobia with a unique symbiotic gene background.     

The NifA-RpoN Regulon of Mesorhizobium loti Strain R7A and Its Symbiotic Activation by a Novel LacI/GalR-Family Regulator

Mesorhizobium loti is the microsymbiont of Lotus species, including the model legume L. japonicus. M. loti differs from other rhizobia in that it contains two copies of the key nitrogen fixation regulatory gene nifA, nifA1 and nifA2, both of which are located on the symbiosis island ICEMlSym^R7A. M. loti R7A also contains two rpoN genes, rpoN1 located on the chromosome outside of ICEMlSym^R7A and rpoN2 that is located on ICEMlSym^R7A. The aims of the current work were to establish how nifA expression was activated in M. loti and to characterise the NifA-RpoN regulon. The nifA2 and rpoN2 genes were essential for nitrogen fixation whereas nifA1 and rpoN1 were dispensable. Expression of nifA2 was activated, possibly in response to an inositol derivative, by a novel regulator of the LacI/GalR family encoded by the fixV gene located upstream of nifA2. Other than the well-characterized nif/fix genes, most NifA2-regulated genes were not required for nitrogen fixation although they were strongly expressed in nodules. The NifA-regulated nifZ and fixU genes, along with nifQ which was not NifA-regulated, were required in M. loti for a fully effective symbiosis although they are not present in some other rhizobia. The NifA-regulated gene msi158 that encodes a porin was also required for a fully effective symbiosis. Several metabolic genes that lacked NifA-regulated promoters were strongly expressed in nodules in a NifA2-dependent manner but again mutants did not have an overt symbiotic phenotype. In summary, many genes encoded on ICEMlSym^R7A were strongly expressed in nodules but not free-living rhizobia, but were not essential for symbiotic nitrogen fixation. It seems likely that some of these genes have functional homologues elsewhere in the genome and that bacteroid metabolism may be sufficiently plastic to adapt to loss of certain enzymatic functions.     

Nitrogen contributions from faba bean (Vicia faba L.) reliant on soil rhizobia or inoculation

Background and Aims

Understanding the impact of soil rhizobial populations and inoculant rhizobia in supplying sufficient nodulation is crucial to optimising N₂ fixation by legume crops. This study explored the impact of different rates of inoculant rhizobia and contrasting soil rhizobia on nodulation and N₂ fixation in faba bean (Vicia faba L.).

Methods

Faba beans were inoculated with one of seven rates of rhizobial inoculation, from no inoculant to 100 times the normal rate of inoculation, sown at two field sites, with or without soil rhizobia present, and their nodulation and N₂ fixation assessed.

Results

At the site without soil rhizobia, inoculation increased nodule number and increased N₂ fixation from 21 to 129 kg shoot N ha^?1, while N₂ fixation increased from 132 to 218 kg shoot N ha^?1 at the site with high background soil rhizobia. At the site without soil rhizobia, inoculation increased concentrations of shoot N from 14 to 24 mg g^?1, grain N from 32 to 45 mg g^?1, and grain yields by 1.0 Mg (metric tonne) ha^?1. Differences in nodulation influenced the contributions of fixed N to the system, which varied from the net removal of 20 kg N ha^?1 from the system in the absence of rhizobia, to a net maximum input of 199 kg N ha^?1 from legume shoot and root residues, after accounting for removal of N in grain harvest.

Conclusions

The impact of inoculation and soil rhizobia strongly influenced grain yield, grain N concentration and the potential contributions of legume cropping to soil N fertility. In soil with resident rhizobia, N₂ fixation was improved only with the highest inoculation rate.     

Symbiotic mutants deficient in nodule establishment identified after T-DNA transformation of Lotus japonicus

Nitrogen-fixing root nodules develop on legumes as a result of an interaction between host plants and soil bacteria collectively referred to as rhizobia. The organogenic process resulting in nodule development is triggered by the bacterial microsymbiont, but genetically controlled by the host plant genome. Using T-DNA insertion as a tool to identify novel plant genes that regulate nodule ontogeny, we have identified two putatively tagged symbiotic loci, Ljsym8 and Ljsym13, in the diploid legume Lotus japonicus. The sym8 mutants are arrested during infection by the bacteria early in the developmental process. The sym13 mutants are arrested in the final stages of infection, and ineffective nodules are formed. These two plant mutant lines were identified in progeny from 1112 primary transformants obtained after Agrobacterium tumefaciens T-DNA-mediated transformation of L. japonicus and subsequent screening for defects in the symbiosis with Mesorhizobium loti. Additional nontagged mutants arrested at different developmental stages were also identified and genetic complementation tests assigned all the mutations to 16 monogenic symbiotic loci segregating recessive mutant alleles. In the screen reported here independent symbiotic loci thus appeared with a frequency of ～1.5%, suggesting that a relatively large set of genes is required for the symbiotic interaction.     

Plasmids impact on rhizobia-legumes symbiosis in diverse environments

Rhizobia are a well-known group of soil bacteria that establish symbiotic relationship with leguminous plants, fix atmospheric nitrogen, and improve soil fertility. To fulfill multiple duties in soil, rhizobia are elaborated with a large and complex multipartite genome composed of several replicons. The genetic material is divided among various replicons, in a way to cope with, and satisfy the diverse functions of rhizobia. In addition to the main chromosome, which is carrying the essential (core) genes required for sustaining cell life, the rhizobia genomes contain several extra-chromosomal plasmids, carrying the nonessential (accessory) genes. Occasionally, some mega-plasmids, denoted as secondary chromosomes or chromids, carry some essential (core) genes. Furthermore, specific accessory gene sequences (the symbiotic chromosomal islands) are incorporated in the main chromosome of some rhizobia species in Bradyrhizobium and Mesorhizobium genera. Plasmids in rhizobia are of variable sizes. All of the plasmids in a Rhizobium cell constitute about 30–50% of the genome. Rhizobia plasmids have specific characters such as miscellaneous genes, independent replication system, self-transmissibility, and instability. The plasmids regulate several cellular metabolic functions and enable the host rhizobia to survive in diverse habitats and even under stress conditions. Symbiotic plasmids in rhizobia are receiving increased attention because of their significance in the symbiotic nitrogen fixation process. They carry the symbiotic (nod, nif and fix) genes, and some non-symbiotic genes. Symbiotic plasmids are conjugally-transferred by the aid of the non-symbiotic, self-transmissible plasmids, and hence, brings about major changes in the symbiotic interactions and host specificity of rhizobia. Besides, the rhizobia cells harbor one or more accessory, non-symbiotic plasmids, carrying genes regulating various metabolic functions, rhizosphere colonization, and nodulation competitiveness. The entire rhizobia-plasmid pool interacting in harmony and provides rhizobia with substantial abilities to fulfill their complex symbiotic and non-symbiotic functions in variable environments. The above concepts are extensively reviewed and fairly discussed.     

Selecting improved Lotus nodulating rhizobia to expedite the development of new forage species

Aims

In the past decades the increasing focus by Australian pasture development programs on the genus Lotus has seen the evaluation of many species previously untested in Australia. In field trials, nodulation failure was commonplace. This work was undertaken to select effective symbionts for Lotus to ensure further agronomic evaluation of the genus was not compromised. The symbiotic needs of Lotus ornithopodioides were a particular focus of the studies.

Methods

Glasshouse experiments were undertaken to evaluate symbiotic relationships between 15 Lotus spp and 23 strains of nodulating Mesorhizobium loti. This was followed by evaluation of elite rhizobial strains for their ability to persist and form nodules under field conditions.

Results

Complex symbiotic interactions were recorded between strains of lotus rhizobia and the different species of Lotus. Notably, the rhizobia that are currently provided commercially in Australia for the inoculation of Lotus corniculatus (strain SU343) and Lotus uliginosus (strain CC829) did not form effective symbioses with the promising species L. ornithopodioides and L. maroccanus. No strain we evaluated was compatible with all the Lotus species, however several strains with a broad host range were identified. WSM1293 and WSM1348 were the most effective strains on L. ornithopodioides and L. peregrinus.These strains were also moderately effective on L. corniculatus (79 and 52% of SU343), less effective on L. maroccanus (26 and 49% of SRDI110) but were ineffective on L. uliginosus. The latter species overall had very specific rhizobial needs. Both WSM1293 and WSM1348 produced adequate levels of nodulation when inoculated on L. ornithopodioides, over two seasons at three field sites.

Conclusions

Effective and persistent strains are now available that should allow the un-compromised evaluation of many of the contemporary Lotus species in the field. Selecting a strain for use in commercial inoculants will be more problematic, given the very large host-strain interactions for nitrogen fixation. Here, the balance of Lotus species which are adopted by farmers will have a strong bearing on which rhizobial strains are progressed to commerce.     

Boron deficiency affects rhizobia cell surface polysaccharides important for suppression of plant defense mechanisms during legume recognition and for development of nitrogen-fixing symbiosis

Background and aims

Legumes of the genus Lessertia have recently been introduced to Australia in an attempt to increase the range of forage species available in Australian farming systems capable of dealing with a changing climate. This study assessed the diversity and the nodulation ability of a collection of Lessertia root nodule bacteria isolated from different agro-climatic areas of the Eastern and Western Capes of South Africa.

Methods

The diversity and phylogeny of 43 strains was determined via the partial sequencing of the dnaK, 16srRNA and nodA genes. A glasshouse experiment was undertaken to evaluate symbiotic relationships between six Lessertia species and 17 rhizobia strains.

Results

The dnaK and 16S rRNA genes of the majority of the strains clustered with the genus Mesorhizobium. The position of the strains at the intra-genus level was incongruent between phylogenies with few exceptions. The nodA genes from Lessertia spp. formed a cluster on their own, separate from the previously known Mesorhizobium nodA sequences. Strains showed differences in their nodulation and nitrogen fixation patterns that could be correlated with nodA gene phylogeny. L. diffusa, L. herbacea and L. excisa nodulated with nearly all the strains examined while L. capitata, L. incana and L. pauciflora were more stringent.

Conclusion

Root nodule bacteria from Lessertia spp. were identified mainly as Mesorhizobium spp. Their nodA genes were unique and correlated with the nodulation and nitrogen fixation patterns of the strains. There were marked differences in promiscuity within Lessertia spp. and within strains of root nodule bacteria.     

Rhizobial counts in peat inoculants vary amongst legume inoculant groups at manufacture and with storage: implications for quality standards

Background and aims

Inoculation of legumes at sowing with rhizobia has arguably been one of the most cost-effective practices in modern agriculture. Critical aspects of inoculant quality are rhizobial counts at manufacture/registration and shelf (product) life.

Methods

In order to re-evaluate the Australian standards for peat-based inoculants, we assessed numbers of rhizobia (rhizobial counts) and presence of contaminants in 1,234 individual packets of peat–based inoculants from 13 different inoculant groups that were either freshly manufactured or had been stored at 4 °C for up to 38 months to determine (a) rates of decline of rhizobial populations, and (b) effects of presence of contaminants on rhizobial populations. We also assessed effects of inoculant age on survival of the rhizobia during and immediately after inoculation of polyethylene beads.

Results

Rhizobial populations in the peat inoculants at manufacture and decline rates varied substantially amongst the 13 inoculant groups. The most stable were Sinorhizobium, Bradyrhizobium and Mesorhizobium with Rhizobium, particularly R. leguminosarum bv. trifolii the least stable. The presence of contaminants at the 10^?6 level of dilution, i.e. >log 6.7 g^?1 peat, reduced rhizobial numbers in the stored inoculants by an average of 37 %. Survival on beads following inoculation improved 2–3 fold with increasing age of inoculant.

Conclusions

We concluded that the Australian standards for peat-based rhizobial inoculants should be reassessed to account for the large differences amongst the groups in counts at manufacture and survival rates during storage. Key recommendations are to increase expiry counts from log 8.0 to log 8.7 rhizobia g^?1 peat and to have four levels of inoculant shelf life ranging from 12 months to 3 years.     

Characterization of Strains unlike Mesorhizobium loti That Nodulate Lotus spp. in Saline Soils of Granada,Spain

Lotus species are forage legumes with potential as pastures in low-fertility and environmentally constrained soils, owing to their high persistence and yield under those conditions. The aim of this work was the characterization of phenetic and genetic diversity of salt-tolerant bacteria able to establish efficient symbiosis with Lotus spp. A total of 180 isolates able to nodulate Lotus corniculatus and Lotus tenuis from two locations in Granada, Spain, were characterized. Molecular identification of the isolates was performed by repetitive extragenic palindromic PCR (REP-PCR) and 16S rRNA, atpD, and recA gene sequence analyses, showing the presence of bacteria related to different species of the genus Mesorhizobium: Mesorhizobium tarimense/Mesorhizobium tianshanense, Mesorhizobium chacoense/Mesorhizobium albiziae, and the recently described species, Mesorhizobium alhagi. No Mesorhizobium loti-like bacteria were found, although most isolates carried nodC and nifH symbiotic genes closely related to those of M. loti, considered the type species of bacteria nodulating Lotus, and other Lotus rhizobia. A significant portion of the isolates showed both high salt tolerance and good symbiotic performance with L. corniculatus, and many behaved like salt-dependent bacteria, showing faster growth and better symbiotic performance when media were supplemented with Na or Ca salts.Legumes can establish nitrogen-fixing associations with Gram-negative soil bacteria collectively known as rhizobia. Although the symbiotic relationships among rhizobia and many legume species of agricultural importance have been intensively studied, relatively little is known about the symbiotic bacteria of certain plant genera. Lotus is a genus of legumes that includes 125 to 130 species of herbs and small shrubs, mainly distributed in the Northern Hemisphere. Several Lotus species, particularly Lotus corniculatus, Lotus uliginosus, and Lotus tenuis, are used as pasture forage worldwide and are included by phylogenetic studies in the same clade as the model legume Lotus japonicus (4). Until recently, bacteria nodulating Lotus included both intermediate-growing (mesorhizobia) and slow-growing bacteria (12, 16). The mesorhizobia can form effective symbioses with certain Lotus spp. (group I, e.g., L. corniculatus, L. tenuis, or L. japonicus) but form tumor-like structures that do not contain bacteria on L. uliginosus, Lotus subbiflorus, and Lotus angustissimus (group II Lotus spp.) (21, 24). On the other hand, slow-growing strains are usually efficient with Lotus group II species but form no nodules or form inefficient nodules in group I species (12). However, there are rare exceptions to this rule, like strain NZP2037, that can form effective symbioses with both groups of Lotus spp. (23, 25, 28). Furthermore, fast-growing Ensifer meliloti bv. lancerottense strains have been shown to be the symbionts of Lotus lancerottensis but are unable to fix nitrogen with either group I or group II Lotus spp. (19).No apparent relationship exists between the phylogenetic position of Lotus spp. and the type of rhizobia associated. For instance, L. uliginosus and L. angustissimus, which are efficiently nodulated by the bradyrhizobia, are clustered in the same clade as L. corniculatus, L. tenuis, and L. japonicus (clade B) (4), species associated with mesorhizobia. In contrast L. subbiflorus, usually associated with the same rhizobia as L. uliginosus, is clustered in a different clade.The narrow-host-range rhizobia associated with L. corniculatus and other Lotus species were initially classified as Rhizobium loti (13). Later, when the genus Mesorhizobium was created, R. loti was reclassified as Mesorhizobium loti (14), which is considered the type species. Besides the expected differences between the moderate- and the slow-growing Lotus rhizobia, large variabilities in nitrogen-fixing effectiveness (23) as well as in total DNA-DNA hybridization (3, 6) and phylogeny (5, 40) have been shown among the “meso-growing” rhizobia strains classified as M. loti, indicating that they do not form a homogeneous group. Indeed, one of the best-characterized strains of M. loti, strain MAFF303099, has been reclassified as Mesorhizobium huakuii biovar loti (35). In fact, diverse rhizobia have recently been reported to establish symbiosis with Lotus group I species. For instance, bacteria belonging to the newly described species Mesorhizobium gobiense and Mesorhizobium tarimense, were isolated from Lotus frondosus and L. tenuis in China (10). Also, rhizobia assigned to different genera (Rhizobium, Mesorhizobium, Agrobacterium, and Aminobacter) have recently been reported as symbionts of L. tenuis in the Salado River Basin in Argentina (7). While these recent reports indicate that bacteria nodulating Lotus spp. are diverse, their symbiotic genes are rather homogeneous. In fact, most isolates from Argentina and China, regardless their taxonomic assignment, had symbiotic genes closely related to M. loti (7, 10).Soil salinity is a serious and expanding threat to agricultural productivity. Improving crop productivity in saline soils requires selection of well-adapted plant genotypes and, in the case of legumes, highly efficient rhizobial partners adapted to soil conditions. As part of the Euro-South American cooperation project LOTASSA (http://www.lotassa.com/), and aiming to isolate and select for salt-tolerant bacteria able to establish efficient symbiosis with forage Lotus spp., we explored the diversity of Lotus rhizobia in two different locations of Granada province, Spain, where the presence of native Lotus spp. had previously been reported (30).     

Silencing Agrobacterium oncogenes in transgenic grapevine results in strain-specific crown gall resistance

Key message

Grapevine rootstock transformed with an Agrobacterium oncogene-silencing transgene was resistant to certain Agrobacterium strains but sensitive to others. Thus, genetic diversity of Agrobacterium oncogenes may limit engineering crown gall resistance.

Abstract

Crown gall disease of grapevine induced by Agrobacterium vitis or Agrobacterium tumefaciens causes serious economic losses in viticulture. To establish crown gall-resistant lines, somatic proembryos of Vitis berlandieri × V. rupestris cv. ‘Richter 110’ rootstock were transformed with an oncogene-silencing transgene based on iaaM and ipt oncogene sequences from octopine-type, tumor-inducing (Ti) plasmid pTiA6. Twenty-one transgenic lines were selected, and their transgenic nature was confirmed by polymerase chain reaction (PCR). These lines were inoculated with two A. tumefaciens and three A. vitis strains. Eight lines showed resistance to octopine-type A. tumefaciens A348. Resistance correlated with the expression of the silencing genes. However, oncogene silencing was mostly sequence specific because these lines did not abolish tumorigenesis by A. vitis strains or nopaline-type A. tumefaciens C58.     

Establishment and survival of the South African legume Lessertia spp. and rhizobia in Western Australian agricultural systems

Background and aims

The South African herbaceous legume species Lessertia capitata, L. diffusa, L. excisa L. incana and L. herbacea were introduced to Australia to assess plant establishment and survival, as well as the saprophytic ability of their root nodule bacteria (RNB).

Methods

Five Lessertia spp., were inoculated with selected RNB strains and were sown in five different agroclimatic areas of the Western Australian wheat-belt during 2007 and 2008. Plant population and summer survival were evaluated in situ. Soil samples and nodules from host plants were also taken from each site. The re-isolated rhizobia were RPO1-PCR fingerprinted and their partial dnaK and nodA genes were sequenced to confirm their identity.

Results

Plants achieved only poor establishment followed by weak summer survival. More than 83 % of the rhizobia re-isolated from Lessertia did not correlate with the original inoculants’ fingerprints, and were identified as Rhizobium leguminosarum. The nodA sequences of the naturalised strains were also clustered with R. leguminosarum sequences, thus eliminating the likelihood of lateral gene transference from Mesorhizobium and suggesting a competition problem with indigenous rhizobia.

Conclusion

The stressful soil conditions and high numbers of resident R. leguminosarum strains in Western Australian soils, and their ability to rapidly nodulate Lessertia spp. but not fix nitrogen are likely to preclude the adoption of Lessertia as an agricultural legume in this region.     

Identification of ferredoxin II as a major calcium binding protein in the nitrogen-fixing symbiotic bacterium Mesorhizobium loti

Background

Legumes establish with rhizobial bacteria a nitrogen-fixing symbiosis which is of the utmost importance for both plant nutrition and a sustainable agriculture. Calcium is known to act as a key intracellular messenger in the perception of symbiotic signals by both the host plant and the microbial partner. Regulation of intracellular free Ca²⁺ concentration, which is a fundamental prerequisite for any Ca²⁺-based signalling system, is accomplished by complex mechanisms including Ca²⁺ binding proteins acting as Ca²⁺ buffers. In this work we investigated the occurrence of Ca²⁺ binding proteins in Mesorhizobium loti, the specific symbiotic partner of the model legume Lotus japonicus.

Results

A soluble, low molecular weight protein was found to share several biochemical features with the eukaryotic Ca²⁺-binding proteins calsequestrin and calreticulin, such as Stains-all blue staining on SDS-PAGE, an acidic isoelectric point and a Ca²⁺-dependent shift of electrophoretic mobility. The protein was purified to homogeneity by an ammonium sulfate precipitation procedure followed by anion-exchange chromatography on DEAE-Cellulose and electroendosmotic preparative electrophoresis. The Ca²⁺ binding ability of the M. loti protein was demonstrated by ⁴⁵Ca²⁺-overlay assays. ESI-Q-TOF MS/MS analyses of the peptides generated after digestion with either trypsin or endoproteinase AspN identified the rhizobial protein as ferredoxin II and confirmed the presence of Ca²⁺ adducts.

Conclusions

The present data indicate that ferredoxin II is a major Ca²⁺ binding protein in M. loti that may participate in Ca²⁺ homeostasis and suggest an evolutionarily ancient origin for protein-based Ca²⁺ regulatory systems.

Electronic supplementary material

The online version of this article (doi:10.1186/s12866-015-0352-5) contains supplementary material, which is available to authorized users.     

Soil nitrogen balance resulting from N fixation and rhizodeposition by the symbiotic association Anthyllis vulneraria/Mesorhizobium metallidurans grown in highly polluted Zn,Pb and Cd mine tailings

Background and aims

The association of the legume Anthyllis vulneraria and the grass Festuca arvernensis, was found to be very efficient for the phytostabilisation of highly multi-metal contaminated mine tailings. Our objective was to quantify the contribution of Anthyllis inoculated with its symbiotic bacteria Mesorhizobium metallidurans to the soil N pool and to test whether a starter nitrogen fertilization may improve symbiotic nitrogen fixation and the growth of Festuca.

Methods

Plants of Festuca and of Anthyllis inoculated with M. metallidurans were grown separately during eight months in pots filled with mine contaminated soil. Estimation of the N fluxes was realized using ¹⁵?N isotopic methods.

Results

Starter N fertilization (28 kg N ha^?1) improved symbiotic N₂ fixation and the growth of both species. Belowground N balance (N rhizodeposition – soil N uptake) of the non-fertilized Anthyllis at maturity was negative (?30.6 kg N ha^?1). However, the amount of N derived from fixation, including above- and belowground parts, was 78.6 kg N ha^?1, demonstrating the ability of this symbiotic association to improve soil N content after senescence.

Conclusions

i) soil N enrichment by the N₂-fixing symbiotic association occurs after plant senescence, when decaying leaves and shoots are incorporated into the soil; ii) application of a starter fertilization is an efficient solution to improve phytostabilisation of highly contaminated sites.