Genetic Diversity and Phylogeny of Rhizobia That Nodulate Acacia spp. in Morocco Assessed by Analysis of rRNA Genes

Forty rhizobia nodulating four Acacia species (A. gummifera, A. raddiana, A. cyanophylla, and A. horrida) were isolated from different sites in Morocco. These rhizobia were compared by analyzing both the 16S rRNA gene (rDNA) and the 16S-23S rRNA spacer by PCR with restriction fragment length polymorphism (RFLP) analysis. Analysis of the length of 16S-23S spacer showed a considerable diversity within these microsymbionts, but RFLP analysis of the amplified spacer revealed no additional heterogeneity. Three clusters were identified when 16S rDNA analysis was carried out. Two of these clusters include some isolates which nodulate, nonspecifically, the four Acacia species. These clusters, A and B, fit within the Sinorhizobium lineage and are closely related to S. meliloti and S. fredii, respectively. The third cluster appeared to belong to the Agrobacterium-Rhizobium galegae phylum and is more closely related to the Agrobacterium tumefaciens species. These relations were confirmed by sequencing a representative strain from each cluster.     

Phenotypic characterization of Astragalus glycyphyllos symbionts and their phylogeny based on the 16S rDNA sequences and RFLP of 16S rRNA gene

In this study, the nitrogen fixing Astragalus glycyphyllos symbionts were characterized by phenotypic properties, restriction fragment length polymorphism (RFLP), and sequences of 16S rDNA. The generation time of A. glycyphyllos rhizobia in yeast extract mannitol medium was in the range 4–6 h. The studied isolates exhibited a low resistance to antibiotics, a moderate tolerance to NaCl, assimilated di- and trisaccharides, and produced acid in medium containing mannitol as a sole carbon source. In the cluster analysis, based on 86 phenotypic properties of A. glycyphyllos symbionts and the reference rhizobia, examined isolates and the genus Mesorhizobium strains were placed on a single branch, clearly distinct from other lineages of rhizobial genera. By the comparative analysis of 16S rRNA gene sequences and 16S rDNA–RFLP, A. glycyphyllos nodulators were also identified as the members of the genus Mesorhizobium. On the 16S rDNA sequence phylogram, the representatives of A. glycyphyllos nodule isolates formed a robust, monophyletic cluster together with the Mesorhizobium species at 16S rDNA sequence similarity of these bacteria between 95 and 99 %. Similarly, the cluster analysis of the combined RFLP–16S rDNA patterns, obtained with seven restriction endonucleases, showed that A. glycyphyllos rhizobia are closely related to the genus Mesorhizobium bacteria. The taxonomic approaches used in this paper allowed us to classify the studied bacteria into the genus Mesorhizobium.     

Novel associations between rhizobial populations and legume species within the genera Lathyrus and O xytropis grown in the temperate region of China

Fifty rhizobial isolates of Lathyrus and Oxytropis collected from northern regions of China were studied in their genotypic characterization based upon analyses of ARDRA, 16S-23S IGS PCR-RFLP, TP-RAPD, MLEE, sequences of 16S rDNA gene and housekeeping genes of atpD, recA and glnII. The results demonstrated that most of the Lathyrus rhizobia belonged to Rhizobium and most of the Oxytropis rhizobia belonged to Sinorhizobium. A novel group of Rhizobium sp. I and S. meliloti were identified as the main microsymbionts respectively associated with Lathyrus and Oxytropis species in the collection area, which were new associations between rhizobia and the mentioned hosts. This study also provides new evidence for biogeography of rhizobia. Supported by the National Program for Basic S&T Platform Construction (Grant No. 2005DKA21201-1), the National Natural Science Foundation of China (Grant No. 30670001), and the National Basic Research Program of China (Grant No. 2006CB100206)     

Phylogenetically diverse group of native bacterial symbionts isolated from root nodules of groundnut (Arachis hypogaea L.) in South Africa

Groundnut is an economically important N_?2-fixing legume that can contribute about 100–190 kg N ha^?1 to cropping systems. In this study, groundnut-nodulating native rhizobia in South African soils were isolated from root nodules. Genetic analysis of isolates was done using restriction fragment length polymorphism (RFLP)-PCR of the intergenic spacer (IGS) region of 16S-23S rDNA. A total of 26 IGS types were detected with band sizes ranging from 471 to 1415 bp. The rhizobial isolates were grouped into five main clusters with Jaccard's similarity coefficient of 0.00–1.00, and 35 restriction types in a UPGMA dendrogram. Partial sequence analysis of the 16S rDNA, IGS of 16S rDNA-23S rDNA, atpD, gyrB, gltA, glnII and symbiotic nifH and nodC genes obtained for representative isolates of each RFLP-cluster showed that these native groundnut-nodulating rhizobia were phylogenetically diverse, thus confirming the extent of promiscuity of this legume. Concatenated gene sequence analysis showed that most isolates did not align with known type strains, and may represent new species from South Africa. This underscored the high genetic variability associated with groundnut Rhizobium and Bradyrhizobium in South African soils, and the possible presence of a reservoir of novel groundnut-nodulating Bradyrhizobium and Rhizobium in the country.     

Different species and symbiotic genotypes of field rhizobia can nodulate Phaseolus vulgaris in Tunisian soils

A collection of 160 isolates of rhizobia nodulating Phaseolus vulgaris in three geographical regions in Tunisia was characterized by restriction fragment length polymorphism analysis of polymerase chain reaction (PCR)-amplified 16S rDNA, nifH and nodC genes. Nine groups of rhizobia were delineated: Rhizobium gallicum biovar (bv.) gallicum, Rhizobium leguminosarum bv. phaseoli and bv. viciae, Rhizobium etli bv. phaseoli, Rhizobium giardinii bv. giardinii, and four groups related to species of the genus Sinorhizobium, Sinorhizobium meliloti, Sinorhizobium medicae and Sinorhizobium fredii. The most abundant rhizobial species were R. gallicum, R. etli, and R. leguminosarum encompassing 29–20% of the isolates each. Among the isolates assigned to R. leguminosarum, two-thirds were ineffective in nitrogen fixation with P. vulgaris and harbored a symbiotic gene typical of the biovar viciae. The S. fredii-like isolates did not nodulate soybean plants but formed numerous effective nodules on P. vulgaris. Comparison of nodC gene sequences showed that their symbiotic genotype was not related to that of S. fredii, but to that of the S. fredii-like reference strain GR-06, which was isolated from a bean plant grown in a Spanish soil. An additional genotype including 16% of isolates was found to be closely related to species of the genus Agrobacterium. However, when re-examined, these isolates did not nodulate their original host.     

Phenotypic and genotypic diversity of rhizobia nodulating Pterocarpus erinaceus and P. lucens in Senegal

A total of fifty root nodules isolates of fast-growing and slow growing rhizobia from Pterocarpus ennaceus and Pterocarpus lucens respectively native of sudanean and sahelian regions of Senegal were characterized. These isolates were compared to representative strains of known rhizobial species. Twenty-two new isolates were slow growers and twenty-eight were fast growers. A polyphasic approach was performed including comparative total protein sodium dodecyl sulphate polyacrylamide gel (SDS-PAGE) profile analysis; 16S rDNA and 16S-23S rDNA intergenic spacer (IGS) sequence analysis. By SDS-PAGE the slow growing isolates grouped in one major cluster containing reference strains of Bradyrhizobium sp. including strains isolated in Africa, in Brazil and in New Zealand. Most of the fast-growing rhizobia grouped in four different clusters or were separate strains related to Rhizobium and Mesorhizobium strains. The 16S rDNA and 16S-23S rDNA IGS sequences analysis showed accurately the differentiation of fast growing rhizobia among the Rhizobium and Mesorbizobium genospecies. The representative strains of slow growing rhizobia were identified as closely related to Bradyrbizobium elkanii and Bradyrhizobium japonicum. Based on 16S rDNA sequence analysis, one slow growing strain (ORS199) was phylogenetically related to Bradyrbizobium sp. (Lupinus) and Blastobacter denitrificans. This position of ORS 199 was not confirmed by IGS sequence divergence. We found no clear relation between the diversity of strains, the host plants and the ecogeographical origins.     

Genista tinctoria microsymbionts from Poland are new members of Bradyrhizobium japonicum bv. genistearum

The phylogeny and taxonomic position of slow-growing Genista tinctoria rhizobia from Poland, Ukraine and England were estimated by comparative 16S rDNA, atpD, and dnaK sequence analyses, PCR-RFLP of 16S rDNA, DNA G + C content, and DNA–DNA hybridization. Each core gene studied placed the G. tinctoria rhizobia in the genus Bradyrhizobium cluster with unequivocal bootstrap support. G. tinctoria symbionts and bradyrhizobial strains shared 96–99% similarity in 16S rDNA sequences. Their similarity for atpD and dnaK sequences was 93–99% and 89–99%, respectively. These data clearly showed that G. tinctoria rhizobia belonged to the genus Bradyrhizobium. 16S rDNA sequence analysis was in good agreement with the results of the PCR-RFLP of the 16S rRNA gene. Although the tested strains formed separate lineages to the reference bradyrhizobia their RFLP 16S rDNA patterns were quite similar. The genomic DNA G + C content of three G. tinctoria rhizobia was in the range from 60.64 to 62.83 mol%. Data for species identification were obtained from DNA–DNA hybridization experiments. G. tinctoria microsymbionts from Poland were classified within Bradyrhizobium japonicum genomospecies based on 56–82% DNA–DNA similarity.     

Diversity of endophytic bacteria associated with nodules of two indigenous legumes at different altitudes of the Qilian Mountains in China

A total of 201 endophytic root nodule-associated bacteria collected from two legumes indigenous to different Qilian Mountain altitudes (Hexi Corridor) were characterized through 16S rDNA polymerase chain reaction (PCR)-restriction fragment length polymorphism, 16S rRNA gene sequence analysis, and enterobacterial repetitive intergenic consensus-PCR clustering. The isolates phylogenetically belonged to 35 species in the Phyllobacterium, Ensifer, Rhizobium, Microvirga, Sphingomonas, Paracoccus, Mycobacterium, Paenibacillus, Cohnella, Sporosarcina, Bacillus, Staphylococcus, Brevibacterium, Xenophilus, Erwinia, Leclercia, Acinetobacter, and Pseudomonas genera. Phylogenetic nodA sequence analysis showed higher similarity to Sinorhizobium meliloti with strains related to the Rhizobium, Sinorhizobium, and Acinetobacter genera. Sequence analysis of the nifH gene revealed that the strains belonging to Xenophilus, Acinetobacter, Phyllobacterium, and Rhizobium had genes similar to those of Mesorhizobium and Sinorhizobium. The results indicated that horizontal gene transfer could have occurred between rhizobia and non-rhizobial endophytes. Canonical correspondence analysis revealed that altitude and host plant species contributed more to the bacterial endosymbiont separation than other ecological factors. This study provided valuable information on the interactions between symbiotic bacteria, non-symbiotic bacteria and their habitats, and thus provided knowledge on their genetic diversity and ecology.     

黄土高原地区大豆根瘤菌的遗传多样性和系统发育

【目的】研究黄土高原地区大豆根瘤菌的遗传多样性和系统发育。【方法】采用BOX-PCR、16S rDNAPCR-RFLP、16S-23S IGS PCR-RFLP和16S rRNA基因序列分析方法对分离自我国黄土高原地区4个省的15个地区的130株大豆根瘤菌及部分参比菌株进行了遗传多样性和系统发育分析。【结果】BOX-PCR反映的菌株多样性最丰富,形成的遗传群最多,16S rDNA PCR-RFLP方法在属、种水平上聚群较好,16S-23S IGSPCR RFLP反映的多样性介于BOX-PCR和16S rDNA PCR-RFLP之间,能够较好地反映出属、种和亲缘关系很近的菌株间的差异,3种方法聚类分析结果基本一致,可将所有供试菌株分为两大类群,中华根瘤菌属(Sinorhizobium)和慢生根瘤菌属(Bradyrhizobium)。从系统发育来看,供试的快生大豆根瘤菌为费氏中华根瘤菌(Sinorhizobium fredii),慢生大豆根瘤菌为日本慢生大豆根瘤菌(Bradyrhizobium japonicum)和辽宁慢生根瘤菌(Bradyrhizobium liaoningense)。【结论】我国黄土高原地区大豆根瘤菌具有较丰富的遗传多样性,S.fredii优势种,慢生大豆根瘤菌仅占10%,同时,分离到2株B.liaoningense。     

Genetic Diversity of <Emphasis Type="Italic">Acacia seyal</Emphasis> Del. Rhizobial Populations Indigenous to Senegalese Soils in Relation to Salinity and pH of the Sampling Sites

The occurrence and the distribution of rhizobial populations naturally associated to Acacia seyal Del. were characterized in 42 soils from Senegal. The diversity of rhizobial genotypes, as characterized by polymerase chain reaction restriction fragment length polymorphism (RFLP) analysis of 16S–23S rDNA, performed on DNA extracted from 138 nodules resulted in 15 clusters. Results indicated the widespread occurrence of compatible rhizobia associated to A. seyal in various ecogeographic areas. However, the clustering of rhizobial populations based on intergenic spacer (IGS) RFLP profiles did not reflect their geographic origin. Four genera were discriminated on the basis of 16S rRNA gene sequences of the strains representative for the IGS-RFLP profiles. The majority of rhizobia associated to A. seyal were affiliated to Mesorhizobium and Sinorhizobium 64 and 29%, respectively, of the different IGS-RFLP profiles. Our results demonstrate the coexistence inside the nodule of plant-pathogenic non-N₂-fixing Agrobacterium and Burkholderia strains, which induced the formation of ineffective nodules, with symbiotic rhizobia. Nodulation was recorded in saline soils and/or at low pH values or in alkaline soils, suggesting adaptability of natural rhizobial populations to major ecological environmental stress and their ability to establish symbiotic associations within these soil environments. These results contribute to the progressing research efforts to uncover the biodiversity of rhizobia and to improve nitrogen fixation in agroforestry systems in sub-Saharan Africa.     

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.     

Bradyrhizobium sp. nodulating the Mediterranean shrub Spanish broom (Spartium junceum L.)

AIMS: The molecular diversity of 25 strains of rhizobia, isolated in Sicily from root nodules of the Mediterranean shrubby legume Spanish broom (Spartium junceum L.), is presented in relation to the known rhizobial reference strains. METHODS AND RESULTS: Our approach to the study of the S. junceum rhizobial diversity combined the information given by the 16S and the intergenic spacer (IGS) 16S-23S rDNA polymorphic region by obtaining them in a single polymerase chain reaction (PCR) step. The PCR fragment size of the S. junceum isolates was 2400-2500 bp and that of the reference strains varied from 2400 in Bradyrhizobium strains to 2800 in Sinorhizobium strains. Inter- and intrageneric length variability was found among the reference strains. Restriction fragment length polymorphisms (RFLP) analysis allowed us to identify eight genotypes among the S. junceum rhizobia that were clustered into two groups, both related to the Bradyrhizobium lineage. Sequencing of representative strains of the two clusters confirmed these data. The 16S-IGS PCR-RFLP approach, when applied to rhizobial reference strains, allowed very close species (i.e. Rhizobium leguminosarum/R. tropici) to be separated with any of the three enzymes used; however, cluster analysis revealed inconsistencies with the 16S-based phylogenesis of rhizobia. CONCLUSIONS: Rhizobia nodulating S. junceum in the Mediterranean region belong to the Bradyrhizobium lineage. Our results confirm the resolution power of the 16S-23S rDNA in distinguishing among rhizobia genera and species, as well as the usefulness of the PCR-RFLP method applied to the entire 16S-IGS region for a rapid tracking of the known relatives of new isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: The present paper is, to our knowledge, the first report on rhizobia nodulating a Mediterranean wild woody legume.     

Phylogenetic diversity of rhizobia associated with horsegram [Macrotyloma uniflorum (Lam.) Verdc.] grown in South India based on glnII, recA and 16S-23S intergenic sequence analyses

Horsegram [Macrotyloma uniflorum (Lam.) Verdc.) is an important grain legume and fodder crop in India. Information on root nodule endosymbionts of this legume in India is limited. In the present study, 69 isolates from naturally occurring root nodules of horsegram collected from two agro-eco-climatic regions of South India was analyzed by generation rate, acid/alkali reaction on YMA medium, restriction fragment length polymorphism analysis of 16S-23S rDNA intergenic spacer region (IGS), and sequence analyses of IGS and housekeeping genes glnII and recA. Based on the rDNA IGS RFLP by means of three restriction enzymes rhizobia were grouped in five clusters (I–V). By sequence analysis of 16S-23S rDNA IGS identified genotypes of horsegram rhizobia were distributed into five divergent lineages of Bradyrhizobium genus which comprised (I) the IGS type IV rhizobia and valid species B. yuanmingense, (II) the strains of IGS type I and Bradyrhizobium sp. ORS 3257 isolated from Vigna sp., (III) the strains of the IGS type II and Bradyrhizobium sp. CIRADAc12 from Acacia sp., (IV) the IGS type V strains and Bradyrhizobium sp. genospecies IV, and (V) comprising genetically distinct IGS type III strains which probably represent an uncharacterized new genomic species. Nearly, 87% of indigenous horsegram isolates (IGS types I, II, III, and V) could not be related to any other species within the genus Bradyrhizobium. Phylogeny based on housekeeping glnII and recA genes confirmed those results found by the analysis of the IGS sequence. All the isolated rhizobia nodulated Macrotyloma sp. and Vigna spp., and only some of them formed nodules on Arachis hypogeae. The isolates within each IGS type varied in their ability to fix nitrogen. Selection for high symbiotic effective strains could reward horsegram production in poor soils of South India where this legume is largely cultivated.     

Diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in an agricultural-forestry ecosystem

To investigate the diversity of rhizobia and interactions among the host legumes and rhizobial genotypes in the same habitat, a total of 97 rhizobial strains isolated from nine legume species grown in an agricultural-forestry ecosystem were identified into seven genomic species and 12 symbiotic genotypes within the genera Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium based upon analyses of genomic DNA regions and symbiotic genes. The results evidenced that the symbiotic genotypes of rhizobia were consistent with their hosts of origin; revealed that vertical transfer was the main mechanism in rhizobia to maintain the symbiotic genes but lateral transfer of symbiotic genes might have happened between the closely related rhizobial species; suggested the existence of co-distribution and co-evolution among the legume hosts and compatible rhizobia. All of these data demonstrated that the biogeography of rhizobia was a result of interactions among the host legumes, bacterial genomic backgrounds and environments.     

Characterization of Astragalus sinicus Rhizobia by Restriction Fragment Length Polymorphism Analysis of Chromosomal and Nodulation Genes Regions

Two hundred and four isolates of rhizobia were sampled from root nodules of Astragalus sinicus grown in rice fields of six southern provinces of China. Genotypic diversity was determined by Southern hybridization using nodDBC genes as a probe, restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S-23S rDNA intergenic spacers (IGS), and plasmid profile. Our results show that rhizobia associated with A. sinicus were very diverse, and 10 genotypes were resolved within the previously identified dominant 16S rDNA type. Diversity levels varied greatly between different geographical locations. The same nod gene genotypes were harbored by distinct chromosomal types, suggesting that lateral plasmid transfer occurred during the evolution process. Received: 14 June 1999 / Accepted: 20 July 1999     

Diversity and Biogeography of Rhizobia Isolated from Root Nodules of <Emphasis Type="Italic">Glycine max</Emphasis> Grown in Hebei Province,China

A total of 215 rhizobial strains were isolated and analyzed with 16S rRNA gene, 16S–23S intergenic spacer, housekeeping genes atpD, recA, and glnII, and symbiotic genes nifH and nodC to understand the genetic diversity of soybean rhizobia in Hebei province, China. All the strains except one were symbiotic bacteria classified into nine genospecies in the genera of Bradyrhizobium and Sinorhizobium. Surveys on the distribution of these rhizobia in different regions showed that Bradyrhizobium japonicum and Bradyrhizobium elkanii strains were found only in neutral to slightly alkaline soils whereas Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense-related strains and strains of five Sinorhizobium genospecies were found in alkaline–saline soils. Correspondence and canonical correspondence analyses on the relationship of rhizobial distribution and their soil characteristics reveal that high soil pH, electrical conductivity, and potassium content favor distribution of the B. yuanmingense and the five Sinorhizobium species but inhibit B. japonicum and B. elkanii. High contents of available phosphorus and organic matters benefit Sinorhizobium fredii and B. liaoningense-related strains and inhibit the others groups mentioned above. The symbiotic gene (nifH and nodC) lineages among B. elkanii, B. japonicum, B. yuanmingense, and Sinorhizobium spp. were observed in the strains, signifying that vertical gene transfer was the main mechanism to maintain these genes in the soybean rhizobia. However, lateral transfer of symbiotic genes commonly in Sinorhizobium spp. and rarely in Bradyrhizobium spp. was also detected. These results showed the genetic diversity, the biogeography, and the soil determinant factors of soybean rhizobia in Hebei province of China.     

Diversity and geographical distribution of rhizobia associated with <Emphasis Type="Italic">Lespedeza</Emphasis> spp. in temperate and subtropical regions of China

Eighty-eight root-nodule isolates from Lespedeza spp. grown in temperate and subtropical regions of China were characterized by a polyphasic approach. Nine clusters were defined in numerical taxonomy and SDS-PAGE analysis of whole cell proteins. Based upon further characterizations of amplified 16S rDNA restriction analysis (ARDRA), PCR-based restriction fragment length polymorphism of ribosomal IGS, 16S rDNA sequence analysis and DNA-DNA hybridization, these isolates were identified as Bradyrhizobium japonicum, B. elkanii, B. yuanmingense, Mesorhizobium amorphae, M. huakuii, Sinorhizobium meliloti and three genomic species related to B. yuanmingense, Rhizobium gallicum and R. tropici. The Bradyrhizobium species and R. tropici-related rhizobia were mainly isolated from the subtropical region and the species of Mesorhizobium, S. meliloti and R. gallicum-related species were all isolated from the temperate region. Phylogenetic analyses of nifH and nodC indicated that the symbiotic genes of distinct rhizobial species associated with Lespedeza spp. might have different origins and there was no evidence for lateral gene transfer of symbiotic genes. The results obtained in the present study and in a previous report demonstrated that Lespedeza spp. are nodulated by rhizobia with diverse genomic backgrounds and these Lespedeza-nodulating rhizobia were not specific to the host species, but specific to their geographic origins. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. GenBank sequence accession numbers: The GenBank accession numbers were EF61095 through EF061114 and EF051240 for acquired 16S rDNA sequences; EF153395 through EF153402 for nifH sequences; and EF153403 through EF153410 for nodC sequences.     

Genetic diversity of rhizobia associated with Desmodium species grown in China

AIMS: Desmodia are leguminous plants used as important forage and herbal medicine in China. Little information is available about the nodule bacteria of Desmodium species. To understand the genetic diversity of rhizobia associated with Desmodium species grown in China, isolates from temperate and subtropical regions were obtained and analysed. METHODS AND RESULTS: A total of 39 rhizobial strains isolated from 9 Desmodium species grown in China were characterized by PCR-based 16S rDNA gene and 16S-23S rDNA intergenic spacer gene restriction fragment length polymorphism (RFLP) and 16S rRNA gene sequencing. The results showed high diversity among rhizobia symbiotic with Desmodium species. Most microsymbionts of Desmodium species belonged to Bradyrhizobium closely related to Bradyrhizobium elkanii, Bradyrhizobium japonicum and Bradyrhizobium yuanmingense. Several small groups or single strain were related to Rhizobium, Sinorhizobium or Mesorhizobium. CONCLUSIONS: Desmodium species formed nodules with diverse rhizobia in Chinese soils. SIGNIFICANCE AND IMPACT OF THE STUDY: These results offered the first systematic information about the microsymbionts of desmodia grown in the temperate and subtropical regions of China.     

Genetic diversity of elite rhizobial strains of subtropical and tropical legumes based on the 16S rRNA and <Emphasis Type="Italic">glnII</Emphasis> genes

Biodiversity of diazotrophic symbiotic bacteria in the tropics is a valuable but still poorly studied resource. The objective of this study was to determine if a second housekeeping gene, glnII, in addition to the 16S rRNA, can be employed to improve the knowledge about taxonomy and phylogeny of rhizobia. Twenty-three elite rhizobial strains, very effective in fixing nitrogen with twenty-one herbal and woody legumes (including species from fourteen tribes in the three subfamilies of the family Leguminosae) were selected for this study; all strains are used as commercial inoculants in Brazil. Complete sequences of the 16S rRNA and partial sequences (480 bp) of the glnII gene were obtained. The same primers and amplification conditions were successful for sequencing the glnII genes of bacteria belonging to five different rhizobial genera—Bradyrhizobium, Mesorhizobium, Methylobacterium, Rhizobium, Sinorhizobium)—positioned in distantly related branches. The analysis of the concatenated genes (16S rRNA + glnII) considerably improved information about phylogeny and taxonomy of rhizobia in comparison to the single analysis of the 16S rRNA. Nine strains might belong to new species. The complementary analysis of the glnII gene was successful with all strains and improved the phylogenetic clustering and clarified the taxonomic position of several strains. The strategy of including the analysis of glnII, in addition to the 16S rRNA, is cost- and time- effective for the characterization of large rhizobial culture collections or in surveys of many isolates.