Novel putative Mesorhizobium and Ensifer genomospecies together with a novel symbiovar psoraleae nodulate legumes of agronomic interest grown in Tunisia.

Forty rhizobial strains were isolated from Lotus creticus, L. pusillus and Bituminaria bituminosa endemic to Tunisia, and they belonged to the Mesorhizobium and Ensifer genera based on 16S rDNA sequence phylogeny. According to the concatenated recA and glnII sequence-based phylogeny, four Bituminaria isolates Pb5, Pb12, Pb8 and Pb17 formed a monophyletic group with Mesorhizobium chacoense ICMP14587T, whereas four other strains Pb1, Pb6, Pb13 and Pb15 formed two separate lineages within the Ensifer genus. Among the L. pusillus strains, Lpus9 and Lpus10 showed a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas six other strains could belong to previously undescribed Mesorhizobium and Ensifer species. For L. creticus strains, Lcus37, Lcus39 and Lcus44 showed 98% sequence identity with Ensifer aridi JNVU TP6, and Lcus42 shared a 96% identical nucleotide with Ensifer meliloti CCBAU83493T; whereas another four strains were divergent from all the described Ensifer and Mesorhizobium species. The analysis of the nodC gene-based phylogeny identified four symbiovar groups; Mesorhizobium sp. sv. anthyllidis (Lpus3 and Lpus11 from L. pusillus, Lcus43 from L. creticus), Ensifer medicae sv. meliloti (four strains from L. creticus and two strains from L. pusillus), E. meliloti sv. meliloti (four from L. creticus, four from L. pusillus and four from B. bituminosa). In addition, four B. bituminosa strains (Pb5, Pb8, Pb12, and Pb17) displayed a distinctive nodC sequence distant from those of other symbiovars described to date. According to their symbiotic gene sequences and host range, the B. bituminosa symbionts (Pb5, Pb8, Pb12 and Pb17) would represent a new symbiovar of M. chacoense for which sv. psoraleae is proposed.

Elucidation of quorum sensing components and their role in regulation of symbiotically important traits in Ensifer nodulating pigeon pea.

Quorum sensing (QS) in rhizobia regulates diverse processes determining the success and efficiency of association with the legume host. Despite the notable importance of QS as well as the well-known underlying variability in the genomic and metabolic components thereof, its study in rhizobia is largely restricted to few laboratory strains. In this work, QS phenomenon in the rhizobia nodulating pigeon pea- one of the most important legume crops of the global-south, is characterized. Using 16S rRNA and recombinaseA sequencing analysis, the selected QS-positive and host-beneficial isolates were identified to be taxonomically affiliated to the genus Ensifer. Their QS components, including homologues of QS genes, and the repertoire of N-acyl homoserine lactone (AHL) autoinducers were identified. Sequences of the QS homologues showed significant variabilities ranging from 10 to >20% with the known Ensifer sequences. Autoinducer profiling using LC-MS/MS revealed the production of long and short chain AHLs variably by the isolates, including 3-oxo-C12-homoserine lactone (3-O-C12-HSL) and 3-OH-C16-HSL as their first report in Rhizobiaceae. Motility and attachment- two of the most crucial traits for effective establishment on host roots were discovered to be QS dependent in in vitro analysis and the same was confirmed using expression analysis of their regulatory genes using qRT-PCR; both revealing a QS mediated repression of motility and promotion of attachment. This study highlights that Ensifer nodulating pigeon pea, although with significant variance in the anatomy of their QS components, regulate symbiotically crucial cell-processes via QS in a scheme that is conserved in multiple genera.

Illumina sequencing of 16S rRNA tag shows disparity in rhizobial and non-rhizobial diversity associated with root nodules of mung bean (Vigna radiata L.) growing in different habitats in Pakistan.

In Rhizobium-legume symbiosis, the nodule is the most frequently studied compartment, where the endophytic/symbiotic microbiota demands critical investigation for development of specific inocula. We identified the bacterial diversity within root nodules of mung bean from different growing areas of Pakistan using Illumina sequencing of 16S rRNA gene. We observed specific OTUs related to specific site where Bradyrhizobium was found to be the dominant genus comprising of 82-94% of total rhizobia in nodules with very minor fraction of sequences from other rhizobia at three sites. In contrast, Ensifer (Sinorhizobium) was single dominant genus comprising 99.9% of total rhizobial sequences at site four. Among non-rhizobial sequences, the genus Acinetobacter was abundant (7-18% of total sequences), particularly in Bradyrhizobium-dominated nodule samples. Rhizobia and non-rhizobial PGPR isolated from nodule samples include Ensifer, Bradyrhizobium, Acinetobacter, Microbacterium and Pseudomonas strains. Co-inoculation of multi-trait PGPR Acinetobacter sp. VrB1 with either of the two rhizobia in field exhibited more positive effect on nodulation and plant growth than single-strain inoculation which favors the use of Acinetobacter as an essential component for development of mung bean inoculum. Furthermore, site-specific dominance of rhizobia and non-rhizobia revealed in this study may contribute towards decision making for development and application of specific inocula in different habitats.

Mitigation of soil contaminated with diesel fuel using bioelectrokinetics.

This study investigated the effectiveness of bioelectrokinetics in rehabilitating a silty clayey sand contaminated with diesel fuel using three novel bacterial strains; Acinetobacter calcoaceticus, Sphingobacterium multivorum, and Sinorhizobium, isolated form agriculture land. Three electrokinetic bioremediation cells were used to conduct the tests and a novel electrode configuration technique was used to stabilize pH and water content in the soil specimen. Solar photovoltaic panels were used to generate sustainable energy for the process. The tests were carried out in outdoors for 55 days. Applied voltage, current passing through the electrokinetic cell, and the temperature of the soil specimen were recorded periodically during the test. The pH, water content, and diesel concentration were determined at the end of the tests. Over the test period, the voltage typically increased from zero before sunrise, remained relatively stabilized for about 4 h, and then started to decrease and dropped to zero by sunset. The temperatures in the cells were found to be 5-7 °C higher than the ambient temperature. The innovative electrode configuration succeeded in keeping the pH of soil to remain the same and thereby prevented the development of a pH gradient in the soil, an important development for survival of the bacteria. The diesel degradation in the soil after bioelectrokinetics were 20-30%, compared to 10-12% in the control test. The study was successful in developing environmentally friendly technology employing novel bacterial strains to degrade diesel fuel and utilizing solar panel to produce renewable energy for bioelectrokinetic during the winter season.

Adaptive evolution of rhizobial symbiotic compatibility mediated by co-evolved insertion sequences.

Mutualism between bacteria and eukaryotes has essential roles in the history of life, but the evolution of their compatibility is poorly understood. Here we show that different Sinorhizobium strains can form either nitrogen-fixing nodules or uninfected pseudonodules on certain cultivated soybeans, while being all effective microsymbionts of some wild soybeans. However, a few well-infected nodules can be found on a commercial soybean using inocula containing a mixed pool of Tn5 insertion mutants derived from an incompatible strain. Reverse genetics and genome sequencing of compatible mutants demonstrated that inactivation of T3SS (type three secretion system) accounted for this phenotypic change. These mutations in the T3SS gene cluster were dominated by parallel transpositions of insertion sequences (ISs) other than the introduced Tn5. This genetic and phenotypic change can also be achieved in an experimental evolution scenario on a laboratory time scale using incompatible wild-type strains as inocula. The ISs acting in the adaptive evolution of Sinorhizobium strains exhibit broader phyletic and replicon distributions than other ISs, and prefer target sequences of low GC% content, a characteristic feature of symbiosis plasmid where T3SS genes are located. These findings suggest an important role of co-evolved ISs in the adaptive evolution of rhizobial compatibility.

Phenotypic and genetic diversity of Moroccan rhizobia isolated from Vicia faba and study of genes that are likely to be involved in their osmotolerance.

Rhizobia are symbiotic nitrogen-fixing bacteria in root nodules of legumes. In Morocco, faba bean (Vicia faba L.), which is the main legume crop cultivated in the country, is often grown in marginal soils of arid and semi-arid regions. This study examines the phenotypic diversity of rhizobia nodulating V. faba isolated from different regions in Morocco for tolerance to some abiotic stresses. A total of 106 rhizobia strains isolated from nodules were identified at the species level by analysing 16S rDNA. Additionally, for selected strains recA, otsA, kup and nodA fragments were sequenced. 102 isolates are likely to belong to Rhizobium leguminosarum or R. laguerreae and 4 isolates to Ensifer meliloti. All strains tolerating salt concentrations of 428 or 342mM NaCl as well as 127 or 99mM Na2SO4 were highly resistant to alkaline conditions (pH 10) and high temperature (44°C). Three strains: RhOF4 and RhOF53 (both are salt-tolerant) and RhOF6 (salt-sensitive) were selected to compare the influence of different levels of salt stress induced by NaCl on growth and on trehalose and potassium accumulation. We find a direct correlation between the trehalose contents of the rhizobial strains and their osmotolerance.

Co-invading symbiotic mutualists of Medicago polymorpha retain high ancestral diversity and contain diverse accessory genomes.

Exotic, invasive plants and animals can wreak havoc on ecosystems by displacing natives and altering environmental conditions. However, much less is known about the identities or evolutionary dynamics of the symbiotic microbes that accompany invasive species. Most leguminous plants rely upon symbiotic rhizobium bacteria to fix nitrogen and are incapable of colonizing areas devoid of compatible rhizobia. We compare the genomes of symbiotic rhizobia in a portion of the legume's invaded range with those of the rhizobium symbionts from across the legume's native range. We show that in an area of California the legume Medicago polymorpha has invaded, its Ensifer medicae symbionts: (i) exhibit genome-wide patterns of relatedness that together with historical evidence support host-symbiont co-invasion from Europe into California, (ii) exhibit population genomic patterns consistent with the introduction of the majority of deep diversity from the native range, rather than a genetic bottleneck during colonization of California and (iii) harbor a large set of accessory genes uniquely enriched in binding functions, which could play a role in habitat invasion. Examining microbial symbiont genome dynamics during biological invasions is critical for assessing host-symbiont co-invasions whereby microbial symbiont range expansion underlies plant and animal invasions.

Characterization of Sinorhizobium sp. LM21 Prophages and Virus-Encoded DNA Methyltransferases in the Light of Comparative Genomic Analyses of the Sinorhizobial Virome.

The genus Sinorhizobium/Ensifer mostly groups nitrogen-fixing bacteria that create root or stem nodules on leguminous plants and transform atmospheric nitrogen into ammonia, which improves the productivity of the plants. Although these biotechnologically-important bacteria are commonly found in various soil environments, little is known about their phages. In this study, the genome of Sinorhizobium sp. LM21 isolated from a heavy-metal-contaminated copper mine in Poland was investigated for the presence of prophages and DNA methyltransferase-encoding genes. In addition to the previously identified temperate phage, ΦLM21, and the phage-plasmid, pLM21S1, the analysis revealed the presence of three prophage regions. Moreover, four novel phage-encoded DNA methyltransferase (MTase) genes were identified and the enzymes were characterized. It was shown that two of the identified viral MTases methylated the same target sequence (GANTC) as cell cycle-regulated methyltransferase (CcrM) of the bacterial host strain, LM21. This discovery was recognized as an example of the evolutionary convergence between enzymes of sinorhizobial viruses and their host, which may play an important role in virus cycle. In the last part of the study, thorough comparative analyses of 31 sinorhizobial (pro)phages (including active sinorhizobial phages and novel putative prophages retrieved and manually re-annotated from Sinorhizobium spp. genomes) were performed. The networking analysis revealed the presence of highly conserved proteins (e.g., holins and endolysins) and a high diversity of viral integrases. The analysis also revealed a large number of viral DNA MTases, whose genes were frequently located within the predicted replication modules of analyzed prophages, which may suggest their important regulatory role. Summarizing, complex analysis of the phage protein similarity network enabled a new insight into overall sinorhizobial virome diversity.

Wide distribution range of rhizobial symbionts associated with pantropical sea-dispersed legumes.

To understand the geographic distributions of rhizobia that associated with widely distributed wild legumes, 66 nodules obtained from 41 individuals including three sea-dispersed legumes (Vigna marina, Vigna luteola, and Canavalia rosea) distributed across the tropical and subtropical coastal regions of the world were studied. Partial sequences of 16S rRNA and nodC genes extracted from the nodules showed that only Bradyrhizobium and Sinorhizobium were associated with the pantropical legumes, and some of the symbiont strains were widely distributed over the Pacific. Horizontal gene transfer of nodulation genes were observed within the Bradyrhizobium and Sinorhizobium lineages. BLAST searches in GenBank also identified records of these strains from various legumes across the world, including crop species. However, one of the rhizobial strains was not found in GenBank, which implies the strain may have adapted to the littoral environment. Our results suggested that some rhizobia, which associate with the widespread sea-dispersed legume, distribute across a broad geographic range. By establishing symbiotic relationships with widely distributed rhizobia, the pantropical legumes may also be able to extend their range much further than other legume species.

Alfalfa microsymbionts from different ITS and nodC lineages of Ensifer meliloti and Ensifer medicae symbiovar meliloti establish efficient symbiosis with alfalfa in Spanish acid soils.

Alfalfa (Medicago sativa L.) is an important crop worldwide whose cropping in acid soils is hampered by the poor nodulation and yield commonly attributed to the sensitivity of its endosymbionts to acid pH. In this work, we isolated several acid-tolerant strains from alfalfa nodules in three acid soils in northwestern Spain. After grouping by RAPD fingerprinting, most strains were identified as Ensifer meliloti and only two strains as Ensifer medicae according to their 16S-23S intergenic spacer (ITS) sequences that allowed the differentiation of two groups within each one of these species. The two ITS groups of E. meliloti and the ITS group I of E. medicae have been previously found in Medicago nodules; however, the group II of E. medicae has been only found to date in Prosopis alba nodules. The analysis of the nodC gene showed that all strains isolated in this study belong to the symbiovar meliloti, grouping with the type strains of E. meliloti or E. medicae, but some harboured nodC gene alleles different from those found to date in alfalfa nodules. The strains of E. medicae belong to the symbiovar meliloti which should be also recognised in this species, although they harboured a nodC allele phylogenetically divergent to those from E. meliloti strains. Microcosm experiments showed that inoculation of alfalfa with selected acid-tolerant strains significantly increased yields in acid soils representing a suitable agricultural practice for alfalfa cropping in these soils.

Effect of co-inoculations of native PGPR with nitrogen fixing bacteria on seedling traits in Prosopis cineraria.

Prosopis cineraria significantly contribute to sand dune stabilization, soil fertility rejuvenation and is an integral component of agro-forestry systems in arid regions of India. Effect of different rhizobacterial seed treatments on seed germination and seedling traits in two genotypes of P. cineraria (HPY-1) and (FG-1) were tested. Observations on seed germination (%) and seedling traits viz., root length (cm), shoot length (cm), seedling weight (g) and seedling length of different treatments were recorded. Whereas, germination index (GI), seedling vigour index (SVI) and root/shoot length ratio were derived from the observed data. The scarification treatment with sulphuric acid for 10 minutes substantially enhanced germination from < 20% to 80-82% in control treatments. Treatments with co-inoculations of Bacillus licheniformis and Sinorhizobium kostiense or S. saheli supported the maximum seed germination and seedling growth and vigour. The maximum germination per cent (92.5%), seedling length (10.94 cm), seedling vigour index (10.12) and germination index (7.97) were recorded with treatment (V2T6) wherein seeds of high pod yielding genotype were co-inoculated with Bacillus licheniformis and S. kostiense. The higher positive correlations of seedling length v/s shoot length followed by SVI v/s seedling length, SVI v/s root length and seedling length v/s root length is a fair indicative of inter dependency of these characteristics. Higher R2 values of root length v/s shoot length followed by that of SVI v/s GI indicates that a regression line fits the data well and future outcomes of observed seedling traits are likely to be predicted by the model.

Arsenite oxidation by a facultative chemolithoautotrophic Sinorhizobium sp. KGO-5 isolated from arsenic-contaminated soil.

A chemolithoautotrophic arsenite-oxidizing bacterium, designated strain KGO-5, was isolated from arsenic-contaminated industrial soil. Strain KGO-5 was phylogenetically closely related with Sinorhizobium meliloti with 16S rRNA gene similarity of more than 99%, and oxidized 5 mM arsenite under autotrophic condition within 60 h with a doubling time of 3.0 h. Additions of 0.01-0.1% yeast extract enhanced the growth significantly, and the strain still oxidized arsenite efficiently with much lower doubling times of approximately 1.0 h. Arsenite-oxidizing capacities (11.2-54.1 µmol h(-1) mg dry cells(-1)) as well as arsenite oxidase (Aio) activities (1.76-10.0 mU mg protein(-1)) were found in the cells grown with arsenite, but neither could be detected in the cells grown without arsenite. Strain KGO-5 possessed putative aioA gene, which is closely related with AioA of Ensifer adhaerens. These results suggest that strain KGO-5 is a facultative chemolithoautotrophic arsenite oxidizer, and its Aio is induced by arsenic.

Nitrogen-fixing rhizobial strains isolated from common bean seeds: phylogeny, physiology, and genome analysis.

Rhizobial bacteria are commonly found in soil but also establish symbiotic relationships with legumes, inhabiting the root nodules, where they fix nitrogen. Endophytic rhizobia have also been reported in the roots and stems of legumes and other plants. We isolated several rhizobial strains from the nodules of noninoculated bean plants and looked for their provenance in the interiors of the seeds. Nine isolates were obtained, covering most known bean symbiont species, which belong to the Rhizobium and Sinorhizobium groups. The strains showed several large plasmids, except for a Sinorhizobium americanum isolate. Two strains, one Rhizobium phaseoli and one S. americanum strain, were thoroughly characterized. Optimal symbiotic performance was observed for both of these strains. The S. americanum strain showed biotin prototrophy when subcultured, as well as high pyruvate dehydrogenase (PDH) activity, both of which are key factors in maintaining optimal growth. The R. phaseoli strain was a biotin auxotroph, did not grow when subcultured, accumulated a large amount of poly-ß-hydroxybutyrate, and exhibited low PDH activity. The physiology and genomes of these strains showed features that may have resulted from their lifestyle inside the seeds: stress sensitivity, a ribulose-1,5-bisphosphate carboxylase/oxygenase (RubisCO) complex, a homocitrate synthase (usually present only in free-living diazotrophs), a hydrogenase uptake cluster, and the presence of prophages. We propose that colonization by rhizobia and their presence in Phaseolus seeds may be part of a persistence mechanism that helps to retain and disperse rhizobial strains.

Assessing genotypic diversity and symbiotic efficiency of five rhizobial legume interactions under cadmium stress for soil phytoremediation.

In the framework of soil phytoremediation using local legume plants coupled with their native root-nodulating bacteria to increase forage yields and preserve contaminated soils in arid regions of Tunisia, we investigated the diversity of bacteria from root nodules of Lathyrus sativus, Lens culinaris, Medicago marina, M. truncatula, and M. minima and the symbiotic efficiency of these five legume symbiosis under Cadmium stress. Fifty bacterial strains were characterized using physiological and biochemical features such heavy metals resistant, and PCR-RFLP of 16S rDNA. Taxonomically, the isolates nodulating L. sativus, and L. culinaris are species within the genera Rhizobium and the ones associated to Medicago sp, within the genera Sinorhizobium. The results revealed also that the cadmium tolerance of the different legumes-rhizobia interaction was as follows: M. minima < M. truncatula < M. marina < L. sativus < L. culinaris indicating that the effect of Cadmium on root nodulation and biomass production is more deleterious on M. minima-S. meliloti and M. truncatula-S. meliloti than in other symbiosis. Knowledge on genetic and functional diversity of M. marina, L. sativus and L. culinaris microsymbiotes is very useful for inoculant strain selection and can be selected to develop inoculants for soil phytoremediation.

The diversity of Rhizobia, Sinorhizobia and novel non-Rhizobial Paenibacillus nodulating wild herbaceous legumes.

The objective of the present study was to isolate and characterize nodulating bacteria associated with wild legumes. For this purpose, we recovered twenty isolates from root nodules of five wild legume species: Melilotus alles, Melilotus officinalis, Trifolium pratense, Trifolium repens and Medicago sp. Most of the isolates were morphologically analogous with only few exceptions in colony shape, appearance and incubation time. All isolates were Gram negative except T.P2-4. Random amplification of polymorphic DNA showed genetic variation among isolates. The 16S rRNA sequence analysis revealed these isolates as Rhizobium, Sinorhizobium and Paenibacillus. Each of these was also screened for nod D and nod F genes with marked variation at these loci; however, the nucleotide sequence analysis confirmed the presence of nod genes. The assignment of strains to their hosts revealed a unique symbiotic association of Paenibacillus sp. nodulating T .pratense which is being reported here for the first time.

Identification of antimony- and arsenic-oxidizing bacteria associated with antimony mine tailing.

Antimony (Sb) is a naturally occurring toxic element commonly associated with arsenic (As) in the environment and both elements have similar chemistry and toxicity. Increasing numbers of studies have focused on microbial As transformations, while microbial Sb interactions are still not well understood. To gain insight into microbial roles in the geochemical cycling of Sb and As, soils from Sb mine tailing were examined for the presence of Sb- and As-oxidizing bacteria. After aerobic enrichment culturing with As(III) (10 mM) or Sb(III) (100 µM), pure cultures of Pseudomonas- and Stenotrophomonas-related isolates with Sb(III) oxidation activities and a Sinorhizobium-related isolate capable of As(III) oxidation were obtained. The As(III)-oxidizing Sinorhizobium isolate possessed the aerobic arsenite oxidase gene (aioA), the expression of which was induced in the presence of As(III) or Sb(III). However, no Sb(III) oxidation activity was detected from the Sinorhizobium-related isolate, suggesting the involvement of different mechanisms for Sb and As oxidation. These results demonstrate that indigenous microorganisms associated with Sb mine soils are capable of Sb and As oxidation, and potentially contribute to the speciation and mobility of Sb and As in situ.

The abundance and diversity of legume-nodulating rhizobia in 28-year-old plantations of tropical, subtropical, and exotic tree species: a case study from the Forest Reserve of Bandia, Senegal.

Several fast-growing and multipurpose tree species have been widely used in West Africa to both reverse the tendency of land degradation and restore soil productivity. Although beneficial effects have been reported on soil stabilization, there still remains a lack of information about their impact on soil microorganisms. Our investigation has been carried out in exotic and native tree plantations of 28 years and aimed to survey and compare the abundance and genetic diversity of natural legume-nodulating rhizobia (LNR). The study of LNR is supported by the phylogenetic analysis which clustered the isolates into three genera: Bradyrhizobium, Mesorhizobium, and Sinorhizobium. The results showed close positive correlations between the sizes of LNR populations estimated both in the dry and rainy seasons and the presence of legume tree hosts. There were significant increases in Rhizobium spp. population densities in response to planting with Acacia spp., and high genetic diversities and richness of genotypes were fittest in these tree plantations. This suggests that enrichment of soil Rhizobium spp. populations is host specific. The results indicated also that species of genera Mesorhizobium and Sinorhizobium were lacking in plantations of non-host species. By contrast, there was a widespread distribution of Bradyrhizobium spp. strains across the tree plantations, with no evident specialization in regard to plantation type. Finally, the study provides information about the LNR communities associated with a range of old tree plantations and some aspects of their relationships to soil factors, which may facilitate the management of man-made forest systems that target ecosystem rehabilitation and preservation of soil biota.

Nonlegume Parasponia andersonii deploys a broad rhizobium host range strategy resulting in largely variable symbiotic effectiveness.

The non-legume genus Parasponia has evolved the rhizobium symbiosis independent from legumes and has done so only recently. We aim to study the promiscuity of such newly evolved symbiotic engagement and determine the symbiotic effectiveness of infecting rhizobium species. It was found that Parasponia andersonii can be nodulated by a broad range of rhizobia belonging to four different genera, and therefore, we conclude that this non-legume is highly promiscuous for rhizobial engagement. A possible drawback of this high promiscuity is that low-efficient strains can infect nodules as well. The strains identified displayed a range in nitrogen-fixation effectiveness, including a very inefficient rhizobium species, Rhizobium tropici WUR1. Because this species is able to make effective nodules on two different legume species, it suggests that the ineffectiveness of P. andersonii nodules is the result of the incompatibility between both partners. In P. andersonii nodules, rhizobia of this strain become embedded in a dense matrix but remain vital. This suggests that sanctions or genetic control against underperforming microsymbionts may not be effective in Parasponia spp. Therefore, we argue that the Parasponia-rhizobium symbiosis is a delicate balance between mutual benefits and parasitic colonization.

Sinorhizobium americanum symbiovar mediterranense is a predominant symbiont that nodulates and fixes nitrogen with common bean (Phaseolus vulgaris L.) in a Northern Tunisian field.

A total of 40 symbiotic bacterial strains isolated from root nodules of common bean grown in a soil located in the north of Tunisia were characterized by PCR-RFLP of the 16S rRNA genes. Six different ribotypes were revealed. Nine representative isolates were submitted to phylogenetic analyses of rrs, recA, atpD, dnaK, nifH and nodA genes. The strains 23C40 and 23C95 representing the most abundant ribotype were closely related to Sinorhizobium americanum CFNEI 156(T). S. americanum was isolated from Acacia spp. in Mexico, but this is the first time that this species is reported among natural populations of rhizobia nodulating common bean. These isolates nodulated and fixed nitrogen with this crop and harbored the symbiotic genes of the symbiovar mediterranense. The strains 23C2 and 23C55 were close to Rhizobium gallicum R602sp(T) but formed a well separated clade and may probably constitute a new species. The sequence similarities with R. gallicum type strain were 98.7% (rrs), 96.6% (recA), 95.8% (atpD) and 93.4% (dnaK). The remaining isolates were, respectively, affiliated to R. gallicum, E. meliloti, Rhizobium giardinii and Rhizobium radiobacter. However, some of them failed to re-nodulate their original host but promoted root growth.

Biogeography of symbiotic and other endophytic bacteria isolated from medicinal Glycyrrhiza species in China.

A total of 159 endophytic bacteria were isolated from surface-sterilized root nodules of wild perennial Glycyrrhiza legumes growing on 40 sites in central and northwestern China. Amplified fragment length polymorphism (AFLP) genomic fingerprinting and sequencing of partial 16S rRNA genes revealed that the collection mainly consisted of Mesorhizobium, Rhizobium, Sinorhizobium, Agrobacterium and Paenibacillus species. Based on symbiotic properties with the legume hosts Glycyrrhiza uralensis and Glycyrrhiza glabra, we divided the nodulating species into true and sporadic symbionts. Five distinct Mesorhizobium groups represented true symbionts of the host plants, the majority of strains inducing N2-fixing nodules. Sporadic symbionts consisted of either species with infrequent occurrence (Rhizobium galegae, Rhizobium leguminosarum) or species with weak (Sinorhizobium meliloti, Rhizobium gallicum) or no N2 fixation ability (Rhizobium giardinii, Rhizobium cellulosilyticum, Phyllobacterium sp.). Multivariate analyses revealed that the host plant species and geographic location explained only a small part (14.4%) of the total variation in bacterial AFLP patterns, with the host plant explaining slightly more (9.9%) than geography (6.9%). However, strains isolated from G. glabra were clearly separated from those from G. uralensis, and strains obtained from central China were well separated from those originating from Xinjiang in the northwest, indicating both host preference and regional endemism.