Rhizobia with different symbiotic efficiencies nodulate Acaciella angustissima in Mexico, including Sinorhizobium chiapanecum sp. nov. which has common symbiotic genes with Sinorhizobium mexicanum

Bacteria from nodules of the legume Acaciella angustissima native to the south of Mexico were characterized genetically and their nodulation and competitiveness were evaluated. Phylogenetic studies derived from rpoB gene sequences indicated that A. angustissima is nodulated by Sinorhizobium mexicanum, Rhizobium tropici, Mesorhizobium plurifarium and Agrobacterium tumefaciens and by bacteria related to Sinorhizobium americanum, Sinorhizobium terangae, Rhizobium etli and Rhizobium gallicum . A new lineage related to S. terangae is recognized based on the sequences of gyrA, nolR, recA, rpoB and rrs genes, DNA–DNA hybridization and phenotypic characteristics. The name for this new species is Sinorhizobium chiapanecum and its type strain is ITTG S70^T. The symbiotic genes nodA and nifH were similar to those from S. mexicanum strains, which are Acaciella symbionts as well, with nodA gene sequences grouped within a cluster of nod genes from strains that nodulate plants from the Mimosoideae subfamily of the Leguminosae. Sinorhizobium isolates were the most frequently obtained from A. angustissima nodules and were among the best strains to promote plant growth in A. angustissima and to compete in interstrain nodule competition assays. Lateral transfer of symbiotic genes is not evident among the genera that nodulate A. angustissima ( Rhizobium, Sinorhizobium and Mesorhizobium ) but may occur among the sympatric and closely related sinorhizobia that nodulate Acaciella .     

Characterisation of wild legume nodulating bacteria (LNB) in the infra-arid zone of Tunisia

We report on the isolation and the characterization of nitrogen-fixing root nodule bacteria isolated from natural legumes in a region of South Tunisia corresponding to the infra-arid climatic zone. A collection of 60 new bacterial root nodule isolates were obtained from 19 legume species belonging to the genera Acacia, Anthyllis, Argyrolobium, Astragalus, Calycotome, Coronilla, Ebenus, Genista, Hedysarum, Hippocrepis, Lathyrus, Lotus, Medicago, Ononis. The isolates were characterised by (1) comparative 16S ARDRA using 7 enzymes, (2) total cell protein SDS-PAGE analysis and (3) 16S rDNA sequencing. The results show that these isolates are diverse and belong to the genera Rhizobium, Sinorhizobium, Mesorhizobium and Bradyrhizobium. Bradyrhizobium were further characterised by 16S-23S rDNA IGS sequencing. Surprisingly strains nodulating Astragalus cruciatus, Lotus creticus and Anthyllis henoniana were identified as Rhizobium galegae, a species recorded only as endosymbiont of Galega officinalis and G. orientalis in northern regions so far.     

Phylogenetic analysis of 23S rRNA gene sequences of. Agrobacterium, Rhizobium and Sinorhizobium strains

The phylogenetic relationship among twelve Agrobacterium, four Rhizobium, and two Sinorhizobium strains originating from various host plants and geographical regions was studied by analysis of the 23S rDNA sequences. The study included Agrobacterium strains belonging to biovars 1, 2 (with tumor- or hairy-root inducing and non-pathogenic strains), A. vitis, A. rubi; representative species of the Rhizobium genus: R. galegae, R. leguminosarum and R. tropici and Sinorhizobium meliloti strains. The phylogenetic analysis showed that within Agrobacterium, the biovar designation was reflected in the 23S rDNA similarity and that strains of Agrobacterium and Rhizobium are closely related to each other. The results suggest that the taxonomic definition of Agrobacterium and Rhizobium should be considered for revision and that the Agrobacterium-biovar identity is probably a reliable taxonomic trait.     

黄华属根瘤茵的16S rDNA-RFLP分析

采用16S rDNA-RFLP及序列分析方法,对分离自黄华属的披针叶黄华、喀什黄华和光叶黄华根瘤菌进行分析研究.结果表明,分离得到的33株根瘤菌在种水平上具有丰富的遗传多样性,它们分别归属于中慢生根瘤菌属(Mesorhizobium)、中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)和土壤杆菌属(Agrobacterium).其中,以CCNWGS0011和CCNWGS0010-1为代表的5株根瘤菌构成独立的分支,可能为潜在的新种.     

Colonization of Phaseolus vulgaris nodules by Agrobacterium-like strains

Non-nodulating Agrobacterium-like strains identified among root nodule isolates of common bean were labeled with gusA, a reporter gene encoding beta-glucuronidase (GUS). Bean plants were then co-inoculated with an infective Rhizobium strain and labeled transconjugants of Agrobacterium-like strains. Blue staining of nodules showed that Agrobacterium-like strains were able to colonize these symbiotic organs. Isolation and characterization by restriction fragment length polymorphism analysis of PCR-amplified 16S rRNA genes revealed a mixed population of Rhizobium and Agrobacterium-like strains in all nodules showing GUS activity. PCR amplification of the nifH gene and nodulation tests did not show any evidence of acquisition of symbiotic gene by lateral transfer from Rhizobium to Agrobacterium-like strains. Moreover, these strains were able to invade mature nodules. Based on sequencing of the 16S rRNA gene, one of these Agrobacterium-like strains showed 99.4% sequence similarity with Agrobacterium bv. 1 reference strains and 99% similarity with an Agrobacterium bv. 1 strain isolated from Acacia mollisima in Senegal. Agrobacterium tumefaciens C58 and the disarmed variant AT123 did not show any ability to colonize nodules. Co-inoculation of bean seeds with Agrobacterium and Rhizobium strains did not enhance nodulation and plant yield under controlled conditions.     

海南省快生根瘤菌的多型性

本文报告了对25株来自不同寄主植物的海南快生型根瘤菌及7株已知快生型根瘤菌的数值分类。结果表明,海南快生型根瘤菌在分类上有多样性,其中一部分属于不同的已知根瘤菌种,有13株海南快生型根瘤菌构成一个具有独立分类地位的菌群。这一独立的海南根瘤菌群来自10个属的寄主植物,并有较强的生活能力和扰逆性。     

Genetic diversity of rhizobia isolated from Astragalus adsurgens growing in different geographical regions of China

The genetic diversity among 95 isolates from Astragalus adsurgens was investigated using molecular biological methods. All of the isolates and 24 reference strains could be differentiated by AFLP, REP-, ERIC- and BOX-PCR fingerprinting analysis. By cluster analysis of the data, 31 AFLP and 38 Rep-PCR genomic groups were delineated, indicating considerable genetic diversity among the isolates. Fifty-four representative strains were further analyzed by RFLP of PCR-amplified 16S and 23S rDNA, revealing 26 rDNA genotypes among the isolates. The phylogenetic relationship of the isolates was determined by partial sequencing of 16S rRNA genes of 16 strains. The results suggest that the A. adsurgens rhizobia belong to the genera Agrobacterium, Mesorhizobium, Rhizobium and Sinorhizobium.     

攀枝花市银合欢根瘤菌遗传多样性

【目的】研究分离自四川攀枝花的银合欢根瘤菌的遗传多样性。【方法】采用联合16S rDNA RFLP和IGS RFLP的综合聚类分析(16S-IGS RFLP)、AFLP及多位点持家基因(16S rDNA,atpD,recA)序列的联合分析对供试银合欢根瘤菌进行研究。【结果】31株未知菌具有15种16S-IGS遗传图谱类型、27种AFLP类型。16S-IGS RFLP结果表明,没有未知菌与Bradyrhizobium的参比菌株聚在一起。在71.4%的相似水平上,31个未知菌按属的水平分成3个分支:S、M和R,分别分布在Sinorhizobium属(28株)、Mesorhizobium属(2株)和Rhizobium属(1株)。S分支的28个菌在84%的相似水平上,16S-IGS RFLP聚类图中构成3个群:群S1、群S2、群S3;在AFLP聚类图中构成9个AFLP群:S1–S9。多位点基因序列表明,代表菌株SCAU215、SCAU231分别与M.Plurifarium、R.huautlense亲缘关系最近。而分布于Sinorhizobium属SCAU222和SCAU228、SCAU213、SCAU216可能代表Sinorhizobium的3个新类群。【结论】攀枝花市银合欢根瘤菌遗传多样性丰富,分布于Sinorhizobium、Mesorhizobium和Rhizobium三个属,且优势类群为Sinorhizobium。     

西北地区天蓝苜蓿根瘤菌16S rDNA RFLP分析

利用RFLP和序列测定方法,对分离自西北地区的67株天蓝苜蓿根瘤菌16S rDNA进行了分析研究。结果表明:所有供试菌株分别归属于中华根瘤菌属(Sinorhizobium)、根瘤菌属(Rhizobium)和土壤杆菌属(Agrobac-terium)。以CCNWNX0042-2为代表的大部分天蓝苜蓿根瘤菌属于草木樨中华根瘤菌(Sinorhizobium meliloti),其余菌株在分群上表现出了较为明显的地域特征。     

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.     

Genetic characterization of fast-growing rhizobia able to nodulate Prosopis alba in North Spain

Prosopis is a Mimosaceae legume tree indigenous to South America and not naturalized in Europe. In this work 18 rhizobial strains nodulating Prosopis alba roots were isolated from a soil in North Spain that belong to eight different randomly amplified polymorphic DNA groups phylogenetically related to Sinorhizobium medicae, Sinorhizobium meliloti and Rhizobium giardinii according to their intergenic spacer and 16S rRNA gene sequences. The nodC genes of isolates close to S. medicae and S. meliloti were identical to those of S. medicae USDA 1,037(T) and S. meliloti LMG 6,133(T) and accordingly all these strains were able to nodulate both alfalfa and Prosopis. These nodC genes were phylogenetically divergent from those of the isolates close to R. giardinii that were identical to that of R. giardinii H152(T) and therefore all these strains formed nodules in common beans and Prosopis. The nodC genes of the strains isolated in Spain were phylogenetically divergent from that carried by Mesorhizobium chacoense Pr-5(T) and Sinorhizobium arboris LMG 1,4919(T) nodulating Prosopis in America and Africa, respectively. Therefore, Prosopis is a promiscuous host which can establish symbiosis with strains carrying very divergent nodC genes and this promiscuity may be an important advantage for this legume tree to be used in reforestation.     

The diversity of rhizobia nodulating beans in Northwest Argentina as a source of more efficient inoculant strains

The common bean (Phaseolus vulgaris L.) is cultivated widely in Central and South America and particularly in the Northwest of Argentina. In order to describe the diversity of the common bean nodulating rhizobial population from the bean producing area in Northwest Argentina (NWA), a collection of about 400 isolates of common beans recovered from nodules and soil samples from NWA were characterized by using nifH-PCR, analysis of genes coding for 16S rRNA and nodC, and REP-fingerprinting, respectively. It was found that species Rhizobium etli is predominant in common bean nodules although a high degree of diversity was found within the species. Other bean nodulating genotypes recovered from soils by using Leucaena sp. as the trapping host was found to have the 16S rDNA alleles of species such as Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium teranga, Mesorhizobium loti, and Rhizobium tropici. Some of the bean genotypes that were found to be more efficient in green house experiments were selected and assayed in two successive bean-cropping seasons in the field environment in NWA, and an increase in yields with inoculation was found. The performance of strains isolated from the region indicates potential for exploiting the diversity.     

Occurrence of choline and glycine betaine uptake and metabolism in the family rhizobiaceae and their roles in osmoprotection

The role of glycine betaine and choline in osmoprotection of various Rhizobium, Sinorhizobium, Mesorhizobium, Agrobacterium, and Bradyrhizobium reference strains which display a large variation in salt tolerance was investigated. When externally provided, both compounds enhanced the growth of Rhizobium tropici, Sinorhizobium meliloti, Sinorhizobium fredii, Rhizobium galegae, Agrobacterium tumefaciens, Mesorhizobium loti, and Mesorhizobium huakuii, demonstrating their utilization as osmoprotectants. However, both compounds were inefficient for the most salt-sensitive strains, such as Rhizobium leguminosarum (all biovars), Agrobacterium rhizogenes, Rhizobium etli, and Bradyrhizobium japonicum. Except for B. japonicum, all strains exhibit transport activity for glycine betaine and choline. When the medium osmolarity was raised, choline uptake activity was inhibited, whereas glycine betaine uptake was either increased in R. leguminosarum and S. meliloti or, more surprisingly, reduced in R. tropici, S. fredii, and M. loti. The transport of glycine betaine was increased by growing the cells in the presence of the substrate. With the exception of B. japonicum, all strains were able to use glycine betaine and choline as sole carbon and nitrogen sources. This catabolic function, reported for only a few soil bacteria, could increase competitiveness of rhizobial species in the rhizosphere. Choline dehydrogenase and betaine-aldehyde dehydrogenase activities were present in the cells of all strains with the exception of M. huakuii and B. japonicum. The main physiological role of glycine betaine in the family Rhizobiaceae seems to be as an energy source, while its contribution to osmoprotection is restricted to certain strains.     

Population genetic structure of Sinorhizobium meliloti and S. medicae isolated from nodules of Medicago spp. in Mexico

We studied the genetic structure of 176 bacterial isolates from nodules of Medicago sativa, M. lupulina and M. polymorpha in fifteen sites distributed in three localities in Mexico. The strains were characterized by multilocus enzyme electrophoresis, plasmid profiles, PCR restriction fragment length polymorphism of 16S rRNA genes and of the intergenic spacer between 16S and 23S rRNA genes, and partial sequences of glnII, recA and nodB. Most of the strains were classified as Sinorhizobium meliloti, and a high genetic diversity was recorded. Six strains were classified as Sinorhizobium medicae, with no genetic variation. Phylogenetic and population genetic analyses revealed evidence of frequent recombination and migration within species.     

Characterization of symbiotic and endophytic bacteria isolated from root nodules of herbaceous legumes grown in Qinghai–Tibet plateau and in other zones of China

Qinghai-Tibet plateau is the highest place in the world and the environment in that plateau is hard for animals and plants, with low temperature, low concentration of oxygen and high solar radiation. In this study, 61 root nodule isolates from Vicia, Oxytropis, Medicago, Melilotus and Onobrychis species grown in Qinghai-Tibet plateau and in loess plateau were comparatively characterized. Based upon the results of numerical taxonomy, ARDRA, AFLP, DNA-DNA hybridization and 16S rDNA sequencing, the isolates were classified as Rhizobium leguminosarum, Sinorhizobium meliloti, Sinorhizobium fredii, Mesorhizobium sp., Phyllobacterium sp., Stenotrophomonas sp. and two non-symbiotic groups related to Agrobacterium and Enterobacteriaceae. The strains isolated from Qinghai-Tibet plateau and from the loess plateau were mixed in these species or groups. Oxytropis spp. and Medicago archiducis-nicolai grown in Qinghai-Tibet plateau were recorded as new hosts for R. leguminosarum, as well as Oxytropis glabra and Medicago lupulina for S. fredii. In addition, strains resistant to high alkaline (pH 11) and high concentration of NaCl (3-5%, w/v) were found in each of the rhizobial species. This was the first systematic study of rhizobia isolated from Qinghai-Tibet plateau.     

Rapid and specific identification of four Agrobacterium species and biovars using multiplex PCR

On the basis of 23S rRNA gene sequences, 1 universal forward and 4 taxon (species/biovar)-specific reverse primers were designed for multiplex PCR to aid in identification and differentiation of Agrobacterium rubi, Agrobacterium vitis and Agrobacterium biovars 1 and 2. In reactions with DNA of 119 bacterial strains belonging to: Agrobacterium, Allorhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Phyllobacterium, as well as phytopathogenic bacteria representing various genera, the primers developed for identification of A. vitis, A. rubi or Agrobacterium biovar 1 amplified only DNA of strains belonging to these taxa, producing fragments of the expected sizes: 478, 1006 and 184bp, respectively. However, in the case of the primer developed for identification of Agrobacterium biovar 2, the characteristic 1066bp PCR product was obtained not only with DNA of this biovar, but also with DNA of 3 atypical biovar 1 strains and some rhizobial strains. Differentiation between Agrobacterium biovar 2 and the other strains was possible using the restriction analysis of this product with endonuclease Alw26I. The method developed is an excellent tool for rapid classification of these 4 taxa of Agrobacterium.     

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.     

Genes for utilization of deoxyfructosyl glutamine (DFG), an amadori compound, are widely dispersed in the family Rhizobiaceae

Amadori compounds form spontaneously in decomposing plant material and can be found in the rhizosphere. As such, these compounds could influence microbial populations by serving as sources of carbon, nitrogen and energy to microorganisms expressing suitable catabolic pathways. Two distinct sets of genes for utilization of deoxyfructosyl glutamine (DFG), an Amadori compound, have been identified in isolates of Agrobacterium spp. One, the soc gene set, is encoded by pAtC58, a 543 kb plasmid in A. tumefaciens strain C58. The second, mocD dissimilates DFG formed in the pathway for catabolism of mannopine (MOP) a non-Amadori, imine-type member of the mannityl opine family characteristic of certain Ti and Ri plasmids. To assess the level of dispersal of these two Amadori-utilizing systems, isolates of Agrobacterium spp. and related bacteria in the family Rhizobiaceae were examined by Southern analysis for homologs of socD and mocD. Homologs of mocD were associated only with Ti plasmid-encoded pathways for catabolism of MOP. Homologs of socD were more widely distributed, being detectable in many but not all of the isolates of Agrobacterium, Sinorhizobium, and Rhizobium spp. tested. However, this gene was never associated with the virulence elements, such as the Ti and Ri plasmids, in these strains. Regardless of genus most of the isolates containing socD homologs could utilize DFG as sole source of carbon, nitrogen and energy. Correlation studies suggested that mocD has evolved uniquely as part of the mannityl opine catabolic pathway while socD has evolved for the general utilization of Amadori compounds. Certain isolates of Agrobacterium and Rhizobium that lacked detectable homologs of socD and mocD also could utilize DFG suggesting the existence of additional, unrelated pathways for the catabolism of this Amadori compound. These results suggest that Amadori compounds constitute a source of nutrition that is important to microflora in the rhizosphere.     

Role of trehalose transport and utilization in Sinorhizobium meliloti--alfalfa interactions

Genes thuA and thuB in Sinorhizobium meliloti Rm1021 code for a major pathway for trehalose catabolism and are induced by trehalose but not by related structurally similar disaccharides like sucrose or maltose. S. meliloti strains mutated in either of these two genes were severely impaired in their ability to grow on trehalose as the sole source of carbon. ThuA and ThuB show no homology to any known enzymes in trehalose utilization. ThuA has similarity to proteins of unknown function in Mesorhizobium loti, Agrobacterium tumefaciens, and Brucella melitensis, and ThuB possesses homology to dehydrogenases containing the consensus motif AGKHVXCEKP. thuAB genes are expressed in bacteria growing on the root surface and in the infection threads but not in the symbiotic zone of the nodules. Even though thuA and thuB mutants were impaired in competitive colonization of Medicago sativa roots, these strains were more competitive than the wild-type Rml021 in infecting alfalfa roots and forming nitrogen-fixing nodules. Possible reasons for their increased competitiveness are discussed.     

Nodulation of Cyclopia spp. (Leguminosae, Papilionoideae) by Burkholderia tuberum

BACKGROUND AND AIMS: Species of the genus Burkholderia, from the Betaproteobacteria, have been isolated from legume nodules, but so far they have only been shown to form symbioses with species of Mimosa, sub-family Mimosoideae. This work investigates whether Burkholderia tuberum strains STM678 (isolated from Aspalathus carnosa) and DUS833 (from Aspalathus callosa) can nodulate species of the South African endemic papilionoid genera Cyclopia (tribe Podalyrieae) and Aspalathus (Crotalarieae) as well as the promiscuous legume Macroptilium atropurpureum (Phaseoleae). METHODS: Bacterial strains and the phylogeny of their symbiosis-related (nod) genes were examined via 16S rRNA gene sequencing. Seedlings were grown in liquid culture and inoculated with one of the two strains of B. tuberum or with Sinorhizobium strain NGR 234 (from Lablab purpureus), Mesorhizobium strain DUS835 (from Aspalathus linearis) or Methylobacterium nodulans (from Crotalaria podocarpa). Some nodules, inoculated with green fluorescence protein (GFP)-tagged strains, were examined by light and electron microscopy coupled with immunogold labelling with a Burkholderia-specific antibody. The presence of active nitrogenase was checked by immunolabelling of nitrogenase and by the acetylene reduction assay. B. tuberum STM678 was also tested on a wide range of legumes from all three sub-families. KEY RESULTS: Nodules were not formed on any of the Aspalathus spp. Only B. tuberum nodulated Cyclopia falcata, C. galioides, C. genistoides, C. intermedia and C. pubescens. It also effectively nodulated M. atropurpureum but no other species tested. GFP-expressing inoculant strains were located inside infected cells of C. genistoides, and bacteroids in both Cyclopia spp. and M. atropurpureum were immunogold-labelled with antibodies against Burkholderia and nitrogenase. Nitrogenase activity was also shown using the acetylene reduction assay. This is the first demonstration that a beta-rhizobial strain can effectively nodulate papilioinoid legumes. CONCLUSIONS: Papilionoid legumes from widely different tribes can be nodulated by beta-rhizobia, forming both indeterminate (Cyclopia) and determinate (Macroptilium) nodules.