Genera Sinorhizobium Research Articles

Soil Giant Phage: Genome and Biological Characteristics of Sinorhizobium Jumbo Phage.

This paper presents the first in-depth research on the biological and genomic properties of lytic rhizobiophage AP-J-162 isolated from the soils of the mountainous region of Dagestan (North Caucasus), which belongs to the centers of origin of cultivated plants, according to Vavilov N.I. The rhizobiophage host strains are nitrogen-fixing bacteria of the genus Sinorhizobium spp., symbionts of leguminous forage grasses. The phage particles have a myovirus virion structure. The genome of rhizobiophage AP-J-162 is double-stranded DNA of 471.5 kb in length; 711 ORFs are annotated and 41 types of tRNAs are detected. The closest phylogenetic relative of phage AP-J-162 is Agrobacterium phage Atu-ph07, but no rhizobiophages are known. The replicative machinery, capsid, and baseplate proteins of phage AP-J-162 are structurally similar to those of Escherichia phage T4, but there is no similarity between their tail protein subunits. Amino acid sequence analysis shows that 339 of the ORFs encode hypothetical or functionally relevant products, while the remaining 304 ORFs are unique. Additionally, 153 ORFs are similar to those of Atu_ph07, with one-third of the ORFs encoding different enzymes. The biological properties and genomic characteristics of phage AP-J-162 distinguish it as a unique model for exploring phage-microbe interactions with nitrogen-fixing symbiotic microorganisms.

Diversity, Genomics and Symbiotic Characteristics of Sinorhizobia That Nodulate Desmanthus spp. in Northwest Argentina.

Desmanthus spp. are legumes with the ability to associate with diverse α-proteobacteria-a microsymbiont-in order to establish nitrogen-fixing root nodules. A previous investigation from our laboratory revealed that the main bacteria associated with Desmanthus paspalaceus in symbiosis in central Argentina (Province of Santa Fe) were quite diverse and belonged to the genera Rhizobium and Mesorhizobium. To achieve a more extensive view of the local microsymbionts associated with Desmanthus spp., we sampled three different sites in Jujuy and Salta, in northwest Argentina. Matrix-assisted Laser-Desorption-Ionization Time-of-Flight mass spectrometry (MALDI-TOF) typing, 16S-rDNA analysis, and genome sequencing demonstrated that the dominant root-nodule microsymbionts belonged to the genus Sinorhizobium, with some sequenced genomes related to Sinorhizobium mexicanum, Sinorhizobium chiapanecum, and Sinorhizobium psoraleae. An analysis of nodA and nodC markers indicated that, in some of the isolates, horizontal gene transfer appeared to be responsible for the lack of congruence between the phylogenies of the chromosome and of the symbiotic region. These results revealed diverse evolutionary strategies for reaching the current Desmanthus-microsymbiont diversity. What is remarkable beside their observed genetic diversity is that the tolerance profiles of these isolates to abiotic stresses (temperature, salt concentration, pH) were quite coincident with the separation of the sinorhizobia according to place of origin, suggesting possible ecoedaphic adaptations. This observation, together with the higher aerial dry-weight matter that some isolates generated in Desmanthus virgatus cv. Marc when compared to the biomass generated by the commercial strain Sinorhizobium terangae CB3126, distinguish the collected sinorhizobia as constituting valuable germplasm for evaluation in local fields to select for more efficient symbiotic pairs.

Complete Genome Sequences of the Species Type Strains Sinorhizobium garamanticum LMG 24692 and Sinorhizobium numidicum LMG 27395 and CIP 109850.

The genus Sinorhizobium comprises rhizobia that fix nitrogen in symbiosis with legumes. To support taxonomic studies of this genus and of rhizobia more broadly, we report complete genome sequences and annotations for the species type strains Sinorhizobium garamanticum LMG 24692 and Sinorhizobium numidicum LMG 27395 and CIP 109850.

"New Rhizobial Bacteria of the Genus Sinorhizobium Fredii Entering Symbiosis with Soybean Plants"

"New Rhizobial Bacteria of the Genus Sinorhizobium Fredii Entering Symbiosis with Soybean Plants"

Sinorhizobium medicae 419 vs S. meliloti 1021: differences in root nodules induced by these two strains on the Medicago truncatula host

Symbiosis between plants of family Fabaceae and bacteria genus Rhizobium is the most widespread interaction, in which atmospheric nitrogen is reduced into ammonia. This highly oxygen-sensitive process is performed by endosymbiotc forms of rhizobia called bacteroids, which occupy newly formed plant organs called root nodules. The goal in this paper was to explore the differences in the (ultra)structure and Reactive Oxygen Species (ROS) localization in two symbiotic interactions between legume model plant Medicago truncatula Gaertn. and bacteria from genus Sinorhizobium. Two bacterial strains—Sinorhizobium meliloti 1021 and Sinorhizobium medicae 419 have the ability of inducing root nodules on the Medicago truncatula, however, such nodules differ in effectiveness of biological nitrogen fixation. We demonstrated that root nodules made of S. medicae 419 [fully effective (F++)] induce a lower number of nodule per plant in comparison to S. meliloti 1021—(partially effective F±). Although the differences in the structure are scant, there are some discrepancies in the ultrastructure. In the meristem of F± nodules, there are Cajal bodies and a lower number of vacuole profiles by larger size in comparison to the F++. Nodules F++ have a thicker interzone and store less starch than F+/, while they have higher esterase activity. Localization of different ROS seems to be comparable in the two type of nodules but interestingly, there is not any Nitric Oxide (NO) in the infection thread of F± root nodules.

Inoculation of native symbiotic effective Sinorhizobium spp. enhanced soybean [Glycine max (L.) Merr.] grain yield in Ethiopia

BackgroundSoybean [Glycine max (L) Merr.] is an annual leguminous crop serving as a source of food and feed, green manure, biodiesel and fiber. It is nodulated by diverse slow growing and fast growing rhizobia belonging to the genus Bradyrhizobium and Sinorhizobium, respectively. In Ethiopia, it has been cultivated since 1950s with lower grain yield history. Yield improvement efforts have been more concentrated on agronomic studies, inoculation of exotic Bradyrhizobium japonicum including TAL379 and/or fertilizer application. The results have usually been unsatisfactory and inconsistent. This study was initiated to identify promising indigenous soybean rhizobial inoculant that can enhance yield of the crop in the country.MethodsNative soybean rhizobia, designated GMR for Glycine max rhizobia, were trapped using soybean (cv. Ethio-Yugoslavia) from soils collected across agro-ecologies of Ethiopia. They were screened for in vitro tolerance against physico-chemical stresses, plant growth promoting (PGP) traits and symbiotic performances at greenhouse and field levels. A reference B. japonicum (TAL379) was included in all experiments. A soybean plant growth promoting Achromobacter sp. was also included in field trials for co-inoculation. Quantitative data were assessed by analysis of variance (ANOVA) employing SAS computer software package version 9.3. Mean separations were undertaken using Duncan’s Multiple Range Test at p ≤ 0.05. Phenotypic variability of the test bacteria was undertaken using PAST4.03 Computer Software.ResultGMR that produced acid and grew faster with larger colonies were identified as Sinorhizobium spp. and those which produced alkali and grew slowly with smaller colonies were identified as Bradyrhizobium spp. though further genetic analysis should be performed for verification and identification of their genus and species, respectively. Two Sinorhizobium spp. (GMR120C and GMR125B) profoundly nodulated different soybean cultivars under greenhouse conditions and significantly improved grain yield (p ≤ 0.05; maximum 3.98 tons ha−1) compared to 2.41, 2.82 and 2.69 recorded as maximum grain yield (tons ha−1) for TAL379 inoculation, positive control and negative control, respectively in field trials. Higher yield was recorded when GMR125B was co-inoculated with Achromobacter sp., but when GMR120C was inoculated singly. These GMR also showed efficient utilization of numerous substrates, some PGP traits and potential adaptation to various ecological stresses.ConclusionThe two Sinorhizobium spp. (GMR120C and GMR125B) are promising soybean inoculants that can be used to enhance the productivity of the crop in the country.

Medicago-Sinorhizobium-Ralstonia Co-infection Reveals Legume Nodules as Pathogen Confined Infection Sites Developing Weak Defenses.

Legumes have the capacity to develop root nodules hosting nitrogen-fixing bacteria, called rhizobia. For the plant, the benefit of the symbiosis is important in nitrogen-deprived conditions, but it requires hosting and feeding massive numbers of rhizobia. Recent studies suggest that innate immunity is reduced or suppressed within nodules [1-10]; this likely maintains viable rhizobial populations. To evaluate the potential consequences and risks associated with an altered immuni`ty in the symbiotic organ, we developed a tripartite system with the model legume Medicago truncatula [11, 12], its nodulating symbiont of the genus Sinorhizobium (syn. Ensifer) [13, 14], and the pathogenic soil-borne bacterium Ralstonia solanacearum [15-18]. We show that nodules are frequent infection sites where pathogen multiplication is comparable to that in the root tips and independent of nodule ability to fix nitrogen. Transcriptomic analyses indicate that, despite the presence of the hosted rhizobia, nodules are able to develop weak defense reactions against pathogenic R.solanacearum. Nodule defense response displays specificity compared to that activated in roots. In agreement with nodule innate immunity, optimal R.solanacearum growth requires pathogen virulence factors. Finally, our data indicate that the high susceptibility of nodules is counterbalanced by the existence of a diffusion barrier preventing pathogen spreading from nodules to the rest of the plant.

Sinorhizobium meliloti Bacteriophage HMSP‐1‐Susan: Genome Sequence, Molecular Characterization and Implications for Symbiosis

Bacteria of the genus Sinorhizobium are plant‐growth promoting microbes that form symbiotic relationships with legumes. These bacteria are important in agriculture because of their ability to convert atmospheric nitrogen gas to ammonia. Ammonia is used to synthesize proteins that are essential for plant growth. Bacteriophages are bacterial viruses that decrease microbial populations in soils and other environments. Despite extensive research on the molecular mechanisms governing the process of nitrogen fixation in Sinorhizobium, little is known about the biotic factors that influence the persistence of these bacteria in soil and impact their ability to fix nitrogen. The presence of bacteriophages in soils has been shown to select for specific types of Sinorhizobium strains and to affect the populations nitrogen‐fixing bacteria. In spite of this observation, there are only a limited number of Sinorhizobium phages that have been characterized. The goal of this project was to carry out a molecular characterization of phage HMSP‐1, a bacteriophage isolated from an agricultural soil that infects strains of Sinorhizobium. Viral DNA was isolated and used to obtain the complete nucleotide sequence of HMSP‐1. Genome analyses revealed that HMSP‐1 is a double stranded DNA virus of 52 kb in size. The genome has a GC content of 52.5% and encodes 96 proteins. 83% percent of these proteins were classified as hypothetical proteins of unknown function. HMSP‐1 did not have significant DNA sequence homology to known bacteriophages; these data suggest HMSP‐1 is novel bacterial virus. We investigated the effects of bacteriophage HMSP‐1 on plant growth and the numbers of Sinorhizobium associated with the model legume, Medicago truncatula. No significant differences in growth or microbial numbers were found between the plants exposed to the phage and unexposed the controls. These data suggest that under the conditions tested, HMSP‐1 does not affect the bacterial populations associated with Medicago truncatula.Support or Funding InformationThe work was supported by grant 1237993 from the National Science Foundation and the Hamline University Ridgeway Fund for Undergraduate Collaborative Research.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

In Silico Phage‐Hunting in Sinorhizobium Genomes: Incorporating Original Research in Undergraduate Molecular Biology and Microbiology Courses

The study of bacterial viruses (bacteriophages) is an excellent tool to teach basic principles of molecular biology and microbiology. The availability of numerous complete bacterial genome sequences in public databases provides a unique opportunity for in‐silico‐phage hunting. This approach consists of identifying and characterizing bacteriophages integrated in bacterial chromosomes using computational analyses. The objective of this project was to develop a set of investigative laboratory exercises to allow undergraduate students to participate in a study about the prevalence of bacteriophages in bacteria of the genus Sinorhizobium. These bacteria play a critical role in agriculture due their ability to form nitrogen‐fixing symbioses with alfalfa and related legumes. The role of bacteriophages in N2 fixation and on the physiology of rhizobia remains largely unexplored. The first part of the project consisted of searching Sinorhizobium genomes for the presence of bacteriophages using PHAST, a bioinformatics tool that identifies viral sequences integrated in bacterial chromosomes. Further bioinformatics analyzes were conducted to determine the numbers and types of proteins encoded by the viruses. The students then designed several procedures to activate and excise the predicted bacteriophages out of the Sinorhizobium genomes. Exposure to UV light and Mitomycin treatments led to the successful isolation and molecular characterization of three novel bacteriophages. This set of laboratory exercises was field tested in two undergraduate molecular biology and microbiology courses. The learning objectives of the project were assessed by comparing the scores of pre and post‐tests and by evaluating the bacteriophage characterization protocols designed by the students. Post‐tests were higher than pre‐test scores at or below p=0.005. Normalized learning gains showed improvement in the student's of bioinformatics analyzes skills, basic virology concepts and the structure and function of proteins encoded by bacteriophage genomes.Support or Funding InformationThis project was supported by a grant of the National Science Foundation.

Characterization of Rhizobial Bacteria Nodulating Astragalus corrugatus and Hippocrepis areolata in Tunisian Arid Soils.

Fifty seven bacterial isolates from root nodules of two spontaneous legumes (Astragalus corrugatus and Hippocrepis areolata) growing in the arid areas of Tunisia were characterized by phenotypic features, 16S rDNA PCR-RFLP and 16S rRNA gene sequencing. Phenotypically, our results indicate that A. corrugatus and H. areolata isolates showed heterogenic responses to the different phenotypic features. All isolates were acid producers, fast growers and all of them used different compounds as sole carbon and nitrogen source. The majority of isolate grew at pHs between 6 and 9, at temperatures up to 40°C and tolerated 3% NaCl concentrations. Phylogenetically, the new isolates were affiliated to four genera Sinorhizobium, Rhizobium, Mesorhizobium and Agrobacterium. About 73% of the isolates were species within the genera Sinorhizobium and Rhizobium. The isolates which failed to nodulate their host plants of origin were associated to Agrobacterium genus (three isolates).

Assessing Genotypic Diversity and Symbiotic Efficiency of Five Rhizobial Legume Interactions Under Cadium 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.

Rhizobial Plasmids That Cause Impaired Symbiotic Nitrogen Fixation and Enhanced Host Invasion

The genetic rules that dictate legume-rhizobium compatibility have been investigated for decades, but the causes of incompatibility occurring at late stages of the nodulation process are not well understood. An evaluation of naturally diverse legume (genus Medicago) and rhizobium (genus Sinorhizobium) isolates has revealed numerous instances in which Sinorhizobium strains induce and occupy nodules that are only minimally beneficial to certain Medicago hosts. Using these ineffective strain-host pairs, we identified gain-of-compatibility (GOC) rhizobial variants. We show that GOC variants arise by loss of specific large accessory plasmids, which we call HR plasmids due to their effect on symbiotic host range. Transfer of HR plasmids to a symbiotically effective rhizobium strain can convert it to incompatibility, indicating that HR plasmids can act autonomously in diverse strain backgrounds. We provide evidence that HR plasmids may encode machinery for their horizontal transfer. On hosts in which HR plasmids impair N fixation, the plasmids also enhance competitiveness for nodule occupancy, showing that naturally occurring, transferrable accessory genes can convert beneficial rhizobia to a more exploitative lifestyle. This observation raises important questions about agricultural management, the ecological stability of mutualisms, and the genetic factors that distinguish beneficial symbionts from parasites.

Symbiotic nitrogen fixation of wild legumes in Tunisia: Soil fertility dynamics, field nodulation and nodules effectiveness

Wild legumes (herbs, shrubs or trees) play a critical role in natural ecosystems, agriculture, and agroforestry, where their ability to fix nitrogen in symbiosis makes them excellent colonizers of low-N environments, and hence an economic and environmentally friendly species. The field natural nodulation of the wild Tunisian legumes, the genetic diversity and symbiotic-efficiency of their root nodulating bacteria (RNB) and the dynamics of soil fertility under symbiotic association in arid regions of Tunisia were investigated during this study. Natural nodulation varied according to the species and site. Morphologically, nodules were spindle-shaped (Trigonella maritima, Hedysarum spinosissimum, Argyrolobium uniflorum, Genista saharae, Retama reteam, Vicia sativa). The colour of the nodules was not related to that of the roots, but depends on soil quality and colour, and on the presence of leghaemoglobin. Based on molecular techniques (16S rRNA sequencing) and phenotypic characteristics, the root-nodule bacteria isolated from the Tunisian wild legumes were classified into four genera (Rhizobium, Mesorhizobium, Sinorhizobium). No Bradyrhizobium genus was recovered in our results. Non-nodulating bacteria considered as endophytic to nodules were also reported in our results and four genera belonging to Phyllobacterium, Starkeya, Pseudomonas, and Kocuria were taxonomically defined, for Lotus sp. and H. spinosissimum. Bacteria belonging to Sinorhizobium genus were widely distributed in area under study. Wild legumes belonging to Lotus spp., Vicia spp. and Hedysarum spp. were nodulated by this genus. Mesorhizobium genus was restricted to Lotus sp., and Rhizobium genus nodulated Vicia and Lotus. Phenotypically, our results showed that the root nodulating bacteria belonging to the genus Sinorhizobium exhibited higher tolerance to salt stress and elevated temperatures. Based on symbiotic properties, our reports indicated that the wild-legume rhizobia formed effective and successful symbioses with their legume hosts. The diversity and effectiveness of the nitrogen-fixing wild legumes are of major significance to soil fertility dynamics in the arid regions. Effects on enzyme activities, microbial biomass and respiration, were evaluated in different agricultural soils and in the presence of different wild legumes such as G. saharae and R. raetam. Results showed that legumes–rhizobia symbiosis improves enzyme activities, microbial biomass and respiration of field soils and regenerate microbiological properties and the microflora activity involved in the decomposition of organic matter.

Symbiotic properties and first analyses of the genomic sequence of the fast growing model strain Sinorhizobium fredii HH103 nodulating soybean

Glycine max (soybean) plants can be nodulated by fast-growing rhizobial strains of the genus Sinorhizobium as well as by slow-growing strains clustered in the genus Bradyrhizobium. Fast-growing rhizobia strains with different soybean cultivar specificities have been isolated from Chinese soils and from other geographical regions. Most of these strains have been clustered into the species Sinorhizobium fredii. The S. fredii strain HH103 was isolated from soils of Hubei province, Central China and was first described in 1985. This strain is capable to nodulate American and Asiatic soybean cultivars and many other different legumes and is so far the best studied fast-growing soybean-nodulating strain. Additionally to the chromosome S. fredii HH103 carries five indigenous plasmids. The largest plasmid (pSfrHH103e) harbours genes for the production of diverse surface polysaccharides, such as exopolysaccharides (EPS), lipopolysaccharides (LPS), and capsular polysaccharides (KPS). The second largest plasmid (pSfrHH103d) is a typical symbiotic plasmid (pSym), carrying nodulation and nitrogen fixation genes. The present mini review focuses on symbiotic properties of S. fredii HH103, in particular on nodulation and surface polysaccharides aspects. The model strain S. fredii HH103 was chosen for genomic sequencing, which is currently in progress. First analyses of the draft genome sequence revealed an extensive synteny between the chromosomes of S. fredii HH103 and Rhizobium sp. NGR234.

Caractéristiques symbiotiques et génotypiques des Rhizobia associés à Acacia saligna (Labili.) Wendl. dans quelques pépinières en Algérie

This study on the nodulation and symbiotic nitrogen fixation by Acacia saligna in nine nurseries from North and South areas of Algeria indicates that Acacia saligna is mostly nodulated and fix nitrogen. Therefore, nodulation and nitrogen fixation potential were clearly superior for plants from northern nurseries than those from southern ones. Symbiotic and genotypic characteristics of the associated strains indicate that Acacia saligna is nodulated by strains of the genus Bradyrhizobium in northern nurseries and by strains of the genera Sinorhizobium and Rhizobium in the southern ones.

Diversity of bacteria that nodulate Prosopis juliflora in the eastern area of Morocco

A total of 274 bacterial strains were isolated from the root nodules of Prosopis juliflora, growing in two arid soils of the eastern area of Morocco. A physiological plate screening allowed the selection of 15 strains that could tolerate NaCl concentrations between 175 and 500 mM. These were compared with 15 strains chosen from among the ones which did not tolerate high salinity. The diversity of strains was first assessed by rep-PCR amplification fingerprinting using BOXA1R and ERIC primers. An analysis of the PCR-amplified 16S rDNA gene digestion profiles using five endonucleases indicated the presence of different lineages among the taxa associated with P. juliflora nodules in the soils studied. Nucleotide sequencing of the small subunit rRNA gene and BLAST analysis showed that P. juliflora could host at least six bacterial species in this region and that the identity of those associated with high salt tolerance was clearly distinct from that of the salt-sensitive ones. Among the former, the first type displayed 99% similarity with different members of the genus Sinorhizobium, the second 97% similarity with species within the genus Rhizobium, while the third ribosomal type had 100% homology to Achromobacter xylosoxidans. Within the salt-sensitive isolates the prevailing type observed showed 98% similarity with Rhizobium multihospitium and R. tropici, a second type had 98% similarity to R. giardinii, and a further case displayed 97% colinearity with the Ensifer group including E. maghrebium and E. xericitae. All of the thirty strains encompassing these types re-nodulated P. juliflora in microbiologically controlled conditions and all of them were shown to possess a copy of the nodC gene. This is the first report detecting the betaproteobacterial genus Achromobacter as nodule-forming species for legumes. The observed variability in symbiont species and the abundance of nodulation-proficient strains is in line with the observation that the plant always appears to be nodulated and efficiently fixing nitrogen in spite of a wide range of soil and environmental conditions.

Phylogenetic and symbiotic characterization of rhizobial bacteria nodulating Argyrolobium uniflorum in Tunisian arid soils

Forty-two bacterial isolates from root nodules of Argyrolobium uniflorum growing in the arid areas of Tunisia were characterized by phenotypic features, RFLP, and sequencing of PCR-amplified 16S rRNA genes. The isolates were found to be phenotypically diverse. The majority of the isolates tolerated 3% NaCl and grew at temperatures up to 40 degrees C. Phylogenetically, the new isolates were grouped in the genera Sinorhizobium (27), Rhizobium (13), and Agrobacterium (2). Except for the 2 Agrobacterium isolates, all strains induced nodulation on Argyrolobium uniflorum, but the number of nodules and nitrogen fixation efficiency varied among them. Sinorhizobium sp. strains STM 4034, STM 4036, and STM 4039, forming the most effective symbiosis, are potential candidates for inoculants in revegetalisation programs.

Multilocus sequence analysis of Ensifer and related taxa

Multilocus sequence analysis (MLSA) was performed on representatives of Ensifer (including species previously assigned to the genus Sinorhizobium) and related taxa. Neighbour-joining (NJ), maximum-parsimony (MP) and maximum-likelihood (ML) phylogenies of dnaK, gltA, glnA, recA, thrC and 16S rRNA genes were compared. The data confirm that the potential for discrimination of Ensifer species is greater using MLSA of housekeeping genes than 16S rRNA genes. In incongruence-length difference tests, the 16S rRNA gene was found to be significantly incongruent with the other genes, indicating that this gene should not be used as a single indicator of relatedness in this group. Significant congruence was detected for dnaK, glnA and thrC. Analyses of concatenated sequences of dnaK, glnA and thrC genes yielded very similar NJ, MP and ML trees, with high bootstrap support. In addition, analysis of a concatenation of all six genes essentially produced the same result, levelling out potentially conflicting phylogenetic signals. This new evidence supports the proposal to unite Ensifer and Sinorhizobium in a single genus. Support for an alternative solution preserving the two genera is less strong. In view of the opinions expressed by the Judicial Commission, the name of the genus should be Ensifer, as proposed by Young [Young, J. M. (2003). Int J Syst Evol Microbiol 53, 2107-2110]. Data obtained previously and these new data indicate that Ensifer adhaerens and 'Sinorhizobium morelense' are not heterotypic synonyms, but represent separate species. However, transfer to the genus Ensifer is not possible at present because the species name is the subject of a pending Request for an Opinion, which would affect whether a novel species in the genus Ensifer or a new combination based on a basonym would be created.

Indigenous rhizobia associated with native shrubby legumes in Taiwan

Root-nodulating bacteria were isolated and characterized from seven native shrubby legumes growing in Taiwan. Phenotypic characteristics measured included growth rates in various media, colony morphology, and tolerances to extremes of temperature, salt and pH. The isolates were very diverse phenotypically. Among the 83 isolates that were screened, the majority were fast-growing rhizobia. Twenty eight strains tolerated high concentration of salt (4.5% NaCl) and grew well between temperatures of 37 and 45 °C. The majority of the strains also tolerated extreme pH in their medium from 3.5 to 12. All strains formed nitrogen fixing nodules, and the highest activity was detected in the legume Hedysarum crinita L. PCR restriction fragment length polymorphism (PCR-RFLP) and sequencing of the small subunit ribosomal RNAs revealed that the majority of the isolates belonged to the genera Rhizobium, Bradyrhizobium and Agrobacterium. Only a single strain represented the genus Sinorhizobium. In addition, a strain related to Burkholderia from the β-class of the Proteobacteria (CC-CC-5) was found within nodules of the legume Catenaria caudatum. The study contributes to the understanding of symbiotic nitrogen fixation in selected wild legumes that are native to Taiwan and provides insights into the distribution of nodulating and nitrogen-fixing bacteria from other distinct lineages.

Polymerase chain reaction denaturing gradient gel electrophoresis analysis of the N2-fixing bacterial diversity in soil under Acacia tortilis ssp. raddiana and Balanites aegyptiaca in the dryland part of Senegal.

Denaturing gradient gel electrophoresis (DGGE) of amplified nifH gene fragments was used to study the diazotrophic community of soil samples under Acacia tortilis ssp. raddiana (legume tree) and Balanites aegyptiaca (non-legume tree), two dominant plant species growing naturally in the dryland part of Senegal. Samples were taken along transects from the stem up to 10 m distance from it, at depths of 0-0.25 m and 0.25-0.50 m. Sampling was done in the dry season (25 June 1999) and in the rainy season (28 August 1999). The community structure and diversity of the bacterial groups from the different samples was analysed further using different techniques, such as statistical analysis and diversity index evaluation of the band patterns. Diazotrophic diversity was lower under B. aegyptiaca than under A. tortilis ssp. raddiana. Multidimensional scaling (MDS) analysis and ANOSIM tests showed a significant effect of the tree on the diazotroph assemblages. SIMPER analysis showed that the major elements responsible for the dissimilarity are a member of the genus Sinorhizobium, which is characteristic of the samples taken under A. tortilis ssp. raddiana and a member of the cluster Bradyrhizobium for the samples taken under B. aegyptiaca. Forty-four major bands were partially sequenced, yielding 33 different nifH sequences, which were used in phylogenetic reconstructions. Most sequences were affiliated with the alpha- beta- and gamma-proteobacteria. Five nifH sequences were identical to those of Pseudomonas stutzeri, and one sequence showed 100% similarity to that of Azotobacter vinelandii. Four bands were affiliated with the Cyanobacteria and a single one with the Firmicutes. For both trees, there were also clear differences between the samples taken in the dry and rainy seasons. Only for the samples taken under A. tortilis ssp. raddiana was a significant difference found between the two sampling depths.