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Bradyrhizobium guangxiense CCBAU53363 efficiently nodulates peanut but exhibits incompatible interaction with mung bean. By comparing the common nod region with those of other peanut bradyrhizobia efficiently nodulating these two hosts, distinctive characteristics with a single nodD isoform ( nodD1 ) and a truncated nolA were identified. However, the regulatory roles of NodD1 and NolA and their coordination in legume-bradyrhizobial interactions remain largely unknown in terms of explaining the contrasting symbiotic compatibility. Here, we report that nolA was important for CCBAU53363 symbiosis with peanut but restricted nodulation on mung bean, while nodD1 was dispensable for CCBAU53363 symbiosis with peanut but essential for nodulation on mung bean. Moreover, nolA exerted a cumulative contribution with nodD1 to efficient symbiosis with peanut. Additionally, mutants lacking nolA delayed nodulation on peanut, and both nolA and nodD1 were required for competitive nodule colonization. It is noteworth that most of the nodulation genes and type III secretion system (T3SS)-related genes were significantly downregulated in a strain 53Δ nodD1nolA mutant compared to wild-type strain CCBAU53363, and the downregulated nodulation genes also had a greater impact than T3SS-related genes on the symbiotic defect of 53Δ nodD1nolA on peanut, which was supported by a more severe symbiotic defect induced by 53Δ nodC than that with the 53Δ nodD1nopP , 53Δ nodD1rhcJ , and 53Δ nodD1ttsI mutants. NolA did not regulate nod gene expression but did regulate the T3SS effector gene nopP in an indirect way. Meanwhile, nolA , nodW , and some T3SS-related genes besides nopP were also demonstrated as new "repressors" that seriously impaired CCBAU53363 symbiosis with mung bean. Taken together, the roles and essentiality of nolA and nodD1 in modulating symbiotic compatibility are sophisticated and host dependent. IMPORTANCE The main findings of this study were that we clarified that the roles and essentiality of nodD1 and nolA are host dependent. Importantly, for the first time, NolA was found to positively regulate T3SS effector gene nopP to mediate incompatibility on mung bean. Additionally, NolA does not regulate nod genes, which are activated by NodD1. nolA exerts a cumulative effect with nodD1 on CCBAU53363 symbiosis with peanut. These findings shed new light on our understanding of coordinated regulation of NodD1 and NolA in peanut bradyrhizobia with different hosts.

Bradyrhizobium arachidis strain CCBAU 051107 could differentiate into swollen and nonswollen bacteroids in determinate root nodules of peanut (Arachis hypogaea) and indeterminate nodules of Sophora flavescens, respectively, with different N 2 fixation efficiencies. To reveal the mechanism of bacteroid differentiation and symbiosis efficiency in association with different hosts, morphologies, transcriptomes, and nitrogen fixation efficiencies of the root nodules induced by strain CCBAU 051107 on these two plants were compared. Our results indicated that the nitrogenase activity of peanut nodules was 3 times higher than that of S. flavescens nodules, demonstrating the effects of rhizobium-host interaction on symbiotic effectiveness. With transcriptome comparisons, genes involved in biological nitrogen fixation (BNF) and energy metabolism were upregulated, while those involved in DNA replication, bacterial chemotaxis, and flagellar assembly were significantly downregulated in both types of bacteroids compared with those in free-living cells. However, expression levels of genes involved in BNF, the tricarboxylic acid (TCA) cycle, the pentose phosphate pathway, hydrogenase synthesis, poly-β-hydroxybutyrate (PHB) degradation, and peptidoglycan biosynthesis were significantly greater in the swollen bacteroids of peanut than those in the nonswollen bacteroids of S. flavescens , while contrasting situations were found in expression of genes involved in urea degradation, PHB synthesis, and nitrogen assimilation. Especially higher expression of ureABEF and aspB genes in bacteroids of S. flavescens might imply that the BNF product and nitrogen transport pathway were different from those in peanut. Our study revealed the first differences in bacteroid differentiation and metabolism of these two hosts and will be helpful for us to explore higher-efficiency symbiosis between rhizobia and legumes. IMPORTANCE Rhizobial differentiation into bacteroids in leguminous nodules attracts scientists to investigate its different aspects. The development of bacteroids in the nodule of the important oil crop peanut was first investigated and compared to the status in the nodule of the extremely promiscuous medicinal legume Sophora flavescens by using just a single rhizobial strain of Bradyrhizobium arachidis, CCBAU 051107. This strain differentiates into swollen bacteroids in peanut nodules and nonswollen bacteroids in S. flavescens nodules. The N 2 -fixing efficiency of the peanut nodules is three times higher than that of S. flavescens . By comparing the transcriptomes of their bacteroids, we found that they have similar gene expression spectra, such as nitrogen fixation and motivity, but different spectra in terms of urease activity and peptidoglycan biosynthesis. Those altered levels of gene expression might be related to their functions and differentiation in respective nodules. Our studies provided novel insight into the rhizobial differentiation and metabolic alteration in different hosts.

Type I peanut bradyrhizobial strains can establish efficient symbiosis in contrast to symbiotic incompatibility induced by type II strains with mung bean. The notable distinction in the two kinds of key symbiosis-related regulators nolA and nodD close to the nodABCSUIJ operon region between these two types of peanut bradyrhizobia was found. Therefore, we determined whether NolA and NodD proteins regulate the symbiotic adaptations of type I strains to different hosts. We found that NodD1-NolA synergistically regulated the symbiosis between the type I strain Bradyrhizobium zhanjiangense CCBAU51778 and mung bean, and NodD1-NodD2 jointly regulated nodulation ability. In contrast, NodD1-NolA coordinately regulated nodulation ability in the CCBAU51778-peanut symbiosis. Meanwhile, NodD1 and NolA collectively contributes to competitive nodule colonization of CCBAU51778 on both hosts. The Fucosylated Nod factors and intact type 3 secretion system (T3SS), rather than extra nodD2 and full-length nolA, were critical for effective symbiosis with mung bean. Unexpectedly, T3SS-related genes were activated by NodD2 but not NodD1. Compared to NodD1 and NodD2, NolA predominantly inhibits exopolysaccharide production by promoting exoR expression. Importantly, this is the first report that NolA regulates rhizobial T3SS-related genes. The coordinated regulation and integration of different gene networks to fine-tune the expression of symbiosis-related genes and other accessory genes by NodD1-NolA might be required for CCBAU51778 to efficiently nodulate peanut. This study shed new light on our understanding of the regulatory roles of NolA and NodD proteins in symbiotic adaptation, highlighting the sophisticated gene networks dominated by NodD1-NolA., Competing Interests: Conflict of Interest The authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier GmbH. All rights reserved.)

Aims: To investigate the effects of three symbiotic Bradyrhizobium strains on peanut growth and on rhizobacterial communities in flowering and harvest stages in an organic farm, also to evaluate the role of plant development in influencing peanut rhizobacterial microbiota and correlations among the inoculants, rhizobacterial communities and plant growth., Methods and Results: Peanut seeds were inoculated with three individual Bradyrhizobium strains, plant growth performance was measured in two developmental stages and rhizobacterial communities were analysed by Illumina sequencing of rpoB gene amplicons from peanut rhizosphere. The three bradyrhizobial inoculants significantly increased the nodule numbers and aboveground fresh weight of peanut plants regardless of the different growth stages, and the pod yields were increased to some extent and significantly positively correlated with Bradyrhizobium abundances in rhizosphere. Principal coordinate analysis indicated that the rhizobacterial communities were strongly influenced by the inoculation and peanut developmental stages. The bradyrhizobia inoculation increased relative abundances of potentially beneficial bacteria in peanut rhizosphere, and also altered rhizobacterial co-occurrence association networks and important network hub taxa. Similarly, plant development also significantly influenced the structure, composition and co-occurrence association networks of rhizobacterial communities., Conclusions: Bradyrhizobial inoculants increased peanut growth and yields, they and plant development affected the assembly of peanut rhizobacterial communities., Significance and Impact of the Study: Rhizobial inoculants improved the host plant performance that might also be associated with the dynamic changes in rhizobacterial community except enhancing the biological nitrogen fixation and helps to profoundly understand the mechanism how rhizobia inoculants improve plant growth and yields., (© 2021 The Society for Applied Microbiology.)

Currently, symbiotic rhizobia (sl., rhizobium) refer to the soil bacteria in α- and β-Proteobacteria that can induce root and/or stem nodules on some legumes and a few of nonlegumes. In the nodules, rhizobia convert the inert dinitrogen gas (N 2 ) into ammonia (NH 3 ) and supply them as nitrogen nutrient to the host plant. In general, this symbiotic association presents specificity between rhizobial and leguminous species, and most of the rhizobia use lipochitooligosaccharides, so called Nod factor (NF), for cooperating with their host plant to initiate the formation of nodule primordium and to inhibit the plant immunity. Besides NF, effectors secreted by type III secretion system (T3SS), exopolysaccharides and many microbe-associated molecular patterns in the rhizobia also play important roles in nodulation and immunity response between rhizobia and legumes. However, the promiscuous hosts like Glycine max and Sophora flavescens can nodulate with various rhizobial species harbouring diverse symbiosis genes in different soils, meaning that the nodulation specificity/efficiency might be mainly determined by the host plants and regulated by the soil conditions in a certain cases. Based on previous studies on rhizobial application, we propose a '1+n-N' model to promote the function of symbiotic nitrogen fixation (SNF) in agricultural practice, where '1' refers to appreciate rhizobium; '+n' means the addition of multiple trace elements and PGPR bacteria; and '-N' implies the reduction of chemical nitrogen fertilizer. Finally, open questions in the SNF field are raised to future think deeply and researches., (© 2020 The Society for Applied Microbiology.)

Aeschynomene indica is a semiaquatic legume that forms both stem and root nodules with rhizobia. Some A. indica rhizobia (AIRs) have been reported to nodulate the host using a Nod factor-independent pathway and possess photosynthetic abilities. To investigate the diversity and community structure of AIRs in China, a total of 300 rhizobial isolates were acquired from the root and stem nodules of A. indica grown at 4 sites in Shandong Peninsula, China. Nineteen representative strains were selected according to their recA phylogeny. With further classification in comparison with reference strains, 10 Bradyrhizobium genospecies were defined based on the 16S rRNA gene phylogeny and multilocus sequence analysis (MLSA) of housekeeping genes (HKGs) recA , atpD , glnII , dnaK , gyrB , and rpoB In addition, 6 genospecies were found only in China. No nodulation gene ( nodA , nodB , nodC , or nodZ ) was detected in the AIRs isolates by PCR amplification and Southern blotting. Phylogenetic analysis of nifH and the photosynthesis-related gene pufLM revealed their common origins. All representative strains formed root nodules, but only 9 representative strains for 4 genospecies formed stem nodules on A. indica , indicating that the stem nodulation process of A. indica is limited to some strains. The nucleotide diversity and recombination events of the HKGs, as well as nifH and pufLM genes, showed that mutation contributes more than recombination in evolution. The distribution of dominant AIR genospecies was mainly affected by available nitrogen, organic carbon, total nitrogen, and pH. Our study helps to characterize the diversity and evolution of AIRs. IMPORTANCE Aeschynomene indica rhizobia (AIRs) can form both root and stem nodules via Nod factor-independent processes, which distinguishes them from other rhizobia. This study systematically uncovered the diversity and community composition of A. indica rhizobia distributed in eastern China. Our results reclassified all the A. indica rhizobia across the world and represent a useful contribution to evaluating the diversity and distribution of the symbiont. The presence of novel genospecies specifically distributed in China enriched the A. indica rhizobia resources and provided insight into the geographic distribution of rhizobia. The phylogenetic relationship between nifH and pufLM of A. indica rhizobia across the world provides insight into the evolution of their nitrogen fixation and photosynthetic abilities., (Copyright © 2019 American Society for Microbiology.)

Mutualistic symbiosis and nitrogen fixation of legume rhizobia play a key role in ecological environments. Although many different rhizobial species can form nodules with a specific legume, there is often a dominant microsymbiont, which has the highest nodule occupancy rates, and they are often known as the "most favorable rhizobia". Shifts in the most favorable rhizobia for a legume in different geographical regions or soil types are not well understood. Therefore, in order to explore the shift model, an experiment was designed using successive inoculations of rhizobia on one legume. The plants were grown in either sterile vermiculite or a sandy soil. Results showed that, depending on the environment, a legume could select its preferential rhizobial partner in order to establish symbiosis. For perennial legumes, nodulation is a continuous and sequential process. In this study, when the most favorable rhizobial strain was available to infect the plant first, it was dominant in the nodules, regardless of the existence of other rhizobial strains in the rhizosphere. Other rhizobial strains had an opportunity to establish symbiosis with the plant when the most favorable rhizobial strain was not present in the rhizosphere. Nodule occupancy rates of the most favorable rhizobial strain depended on the competitiveness of other rhizobial strains in the rhizosphere and the environmental adaptability of the favorable rhizobial strain (in this case, to mild vermiculite or hostile sandy soil). To produce high nodulation and efficient nitrogen fixation, the most favorable rhizobial strain should be selected and inoculated into the rhizosphere of legume plants under optimum environmental conditions., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

To evaluate the interactions among endophytes, plants and heavy metal/arsenic contamination, root endophytic bacteria of Prosopis laevigata (Humb and Bonpl. ex Willd) and Sphaeralcea angustifolia grown in a heavy metal(loid)-contaminated zone in San Luis Potosi, Mexico, were isolated and characterized. Greater abundance and species richness were found in Prosopis than in Sphaeralcea and in the nutrient Pb-Zn-rich hill than in the poor nutrient and As-Cu-rich mine tailing. The 25 species identified among the 60 isolates formed three groups in the correspondence analysis, relating to Prosopis/hill (11 species), Prosopis/mine tailing (4 species) and Sphaeralcea/hill (4 species), with six species ungrouped. Most of the isolates showed high or extremely high resistance to arsenic, such as ≥100 mM for As(V) and ≥20 mM for As(III), in mineral medium. These results demonstrated that the abundance and community composition of root endophytic bacteria were strongly affected by the concentration and type of the heavy metals and metalloids (arsenic), as well as the plant species.

With the increasing cultivation of medicinal legumes in agricultural fields, the rhizobia associated with these plants are facing new stresses, mainly from fertilization and irrigation. In this study, investigations on the nodulation of three cultivated medicinal legumes, Astragalus mongholicus, Astragalus membranaceus and Hedysarum polybotrys were performed. Bacterial isolates from root nodules of these legumes were subjected to genetic diversity and multilocus sequence analyses. In addition, the distribution of nodule bacteria related to soil factors and host plants was studied. A total 367 bacterial isolates were obtained and 13 genospecies were identified. The predominant microsymbionts were identified as Mesorhizobium septentrionale, Mesorhizobium temperatum, Mesorhizobium tianshanense, Mesorhizobium ciceri and Mesorhizobium muleiense. M. septentrionale was found in most root nodules especially from legumes grown in the barren soils (with low available nitrogen and low organic carbon contents), while M. temperatum was predominant in nodules where the plants were grown in the nitrogen-rich fields. A. mongholicus tended to be associated with M. septentrionale, M. temperatum and M. ciceri in different soils, while A. membranaceus and H. polybotrys tended to be associated with M. tianshanense and M. septentrionale, respectively. This study showed that soil fertility may be the main determinant for the distribution of rhizobia associated with these cultured legume plants., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Six slow-growing rhizobial strains isolated from effective nodules of Erythrophleum fordii were classified into the genus Bradyrhizobiumbased on their 16S rRNA gene sequences. The results of multilocus sequence analysis of recA, glnII and gyrB genes and 16S-23S rRNA intergenic spacer (IGS) sequence phylogeny indicated that the six strains belonged to two novel species, represented by CCBAU 53325T and CCBAU 51502T, which were consistent with the results of DNA-DNA hybridization; CCBAU 53325T had 17.65-25.59 % relatedness and CCBAU 51502T had 22.69-44.58 % relatedness with five closely related type strains, Bradyrhizobium elkanii USDA 76T, B. pachyrhizi LMG 24246T, B. lablabi CCBAU 23086T, B. jicamae LMG 24556T and B. japonicum USDA 6T. In addition, analysis of phenotypic characteristics and fatty acid profiles also distinguished the test strains from defined species of Bradyrhizobium. Two novel species, Bradyrhizobium erythrophlei sp. nov., represented by the type strain CCBAU 53325T ( = HAMBI 3614T = CGMCC 1.13002T = LMG 28425T), and Bradyrhizobium ferriligni sp. nov., represented by the type strain CCBAU 51502T ( = HAMBI 3613T = CGMCC 1.13001T), are proposed to accommodate the strains.

The nodulation of Erythrophleum fordii has been recorded recently, but its microsymbionts have never been studied. To investigate the diversity and biogeography of rhizobia associated with this leguminous evergreen tree, root nodules were collected from the southern subtropical region of China. A total of 166 bacterial isolates were obtained from the nodules and characterized. In a PCR-based restriction fragment length polymorphism (RFLP) analysis of ribosomal intergenic sequences, the isolates were classified into 22 types within the genus Bradyrhizobium. Sequence analysis of 16S rRNA, ribosomal intergenic spacer (IGS), and the housekeeping genes recA and glnII classified the isolates into four groups: the Bradyrhizobium elkanii and Bradyrhizobium pachyrhizi groups, comprising the dominant symbionts, Bradyrhizobium yuanmingense, and an unclassified group comprising the minor symbionts. The nodC and nifH phylogenetic trees defined five or six lineages among the isolates, which was largely consistent with the definition of genomic species. The phylogenetic results and evolutionary analysis demonstrated that mutation and vertical transmission of genes were the principal processes for the divergent evolution of Bradyrhizobium species associated with E. fordii, while lateral transfer and recombination of housekeeping and symbiotic genes were rare. The distribution of the dominant rhizobial populations was affected by soil pH and effective phosphorus. This is the first report to characterize E. fordii rhizobia., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

Three bacterial isolates (CCBAU 101002(T), CCBAU 101000 and CCBAU 101001) originating from root nodules of the herbaceous legume Kummerowia stipulacea grown in the campus lawn of China Agricultural University were characterized with a polyphasic taxonomic approach. Comparative 16S rRNA gene sequence analysis showed that the isolates shared 99.85-99.92% sequence similarities and had the highest similarities to the type strains of Rhizobium mesoamericanum (99.31%), R. endophyticum (98.54%), R. tibeticum (98.38%) and R. grahamii (98.23%). Sequence similarity of four concatenated housekeeping genes (atpD, glnII, recA and rpoB) between CCBAU 101002(T) and its closest neighbor (R. grahamii) was 92.05%. DNA-DNA hybridization values between strain CCBAU 101002(T) and the four type strains of the most closely related Rhizobium species were less than 28.4±0.8%. The G+C mol% of the genomic DNA for strain CCBAU 101002(T) was 58.5% (Tm). The major respiratory quinone was ubiquinone (Q-10). Summed feature 8 (18:1ω7cis/18:1ω6cis) and 16:0 were the predominant fatty acids. Strain CCBAU 101002(T) contained phosphatidylcholine and phosphatidylethanolamine as major polar lipids, and phosphatidylglycerol and cardiolipin as minor ones. No glycolipid was detected. Unlike other strains, this novel species could utilize dulcite or sodium pyruvate as sole carbon sources and it was resistant to 2% (w/v) NaCl. On the basis of the polyphasic study, a new species Rhizobium cauense sp. nov. is proposed, with CCBAU 101002(T) (=LMG 26832(T)=HAMBI 3288(T)) as the type strain., (Copyright © 2012 Elsevier GmbH. All rights reserved.)

Four bacterial strains isolated from root nodules of Phaseolus vulgaris, Mimosa pudica and Indigofera spicata plants grown in the Yunnan province of China were identified as a lineage within the genus Rhizobium according to the analysis of 16S rRNA gene sequences, sharing most similarity with Rhizobium lusitanum P1-7(T) (99.1 % sequence similarity) and Rhizobium rhizogenes IAM 13570(T) (99.0 %). These strains also formed a distinctive group from the reference strains for defined species of the genus Rhizobium in a polyphasic approach, including the phylogenetic analyses of the 16S rRNA gene and housekeeping genes (recA, atpD, glnII), DNA-DNA hybridization, BOX-PCR fingerprinting, phenotypic characterization, SDS-PAGE of whole-cell proteins, and cellular fatty acid profiles. All the data obtained in this study suggested that these strains represent a novel species of the genus Rhizobium, for which the name Rhizobium vallis sp. nov. is proposed. The DNA G+C content (mol%) of this species varied between 60.9 and 61.2 (T(m)). The type strain of R. vallis sp. nov. is CCBAU 65647(T) ( = LMG 25295(T) =HAMBI 3073(T)), which has a DNA G+C content of 60.9 mol% and forms effective nodules on Phaseolus vulgaris.

Five strains isolated from root nodules of Lablab purpureus and Arachis hypogaea grown in the Anhui and Sichuan provinces of China were classified as members of the genus Bradyrhizobium. These strains had identical 16S rRNA gene sequences which shared 99.48 %, 99.48 % and 99.22 % similarity with the most closely related strains of Bradyrhizobium jicamae PAC68(T), Bradyrhizobium pachyrhizi PAC48(T) and Bradyrhizobium elkanii USDA 76(T), respectively. A study using a polyphasic approach, including 16S rRNA gene RFLP, IGS-RFLP, BOX-PCR, comparative sequence analysis of the 16S-23S rRNA intergenic spacer (IGS) and the recA, atpD and glnII genes, DNA-DNA hybridization and phenotypic tests, showed that the five strains clustered into a coherent group that differentiated them from all recognized species of the genus Bradyrhizobium. Sequencing of nifH and nodC genes and cross-nodulation tests showed that the representative strains CCBAU 23086(T), CCBAU 23160 and CCBAU 61434, isolated from different plants, had identical nifH and nodC gene sequences and were all able to nodulate Lablab purpureus, Arachis hypogaea and Vigna unguiculata. Based upon these results, the name Bradyrhizobium lablabi sp. nov. is proposed for this novel species and strain CCBAU 23086(T) ( = LMG 25572(T) = HAMBI 3052(T)) is designated as the type strain. The DNA G+C mol% is 60.14 (T(m)).

Seven Rhizobium strains associated with various legume species grown in different geographical regions of China were defined into four genomic groups related to Rhizobium giardinii, based upon ribosomal intergenic spacer RFLP, phylogenies of 16S rRNA and housekeeping (atpD, recA and glnII) genes, and DNA relatedness. Three strains in group I were classified as R. giardinii, as they showed high gene sequence similarities (>97 %) and DNA relatedness (64.3-67.5 %) to R. giardinii H152(T). Groups II, III and IV differed from all defined Rhizobium species based upon the consensus of all analyses. As group II contained two strains that originated from two distinct populations, we propose this group as a novel species, Rhizobium herbae sp. nov., with strain CCBAU 83011(T) ( = LMG 25718(T) = HAMBI 3117(T)) as the type strain.

A total of 115 endophytic bacteria were isolated from root nodules of the wild legume Sphaerophysa salsula grown in two ecological regions of Loess Plateau in China. The genetic diversity and phylogeny of the strains were revealed by restriction fragment length polymorphism and sequencing of 16S rRNA gene and enterobacterial repetitive intergenic consensus-PCR. Their symbiotic capacity was checked by nodulation tests and analysis of nifH gene sequence. This is the first systematic study on endophytic bacteria associated with S. salsula root nodules. Fifty of the strains found were symbiotic bacteria belonging to eight putative species in the genera Mesorhizobium, Rhizobium and Sinorhizobium, harboring similar nifH genes; Mesorhizobium gobiense was the main group and 65 strains were nonsymbiotic bacteria related to 17 species in the genera Paracoccus, Sphingomonas, Inquilinus, Pseudomonas, Serratia, Mycobacterium, Nocardia, Streptomyces, Paenibacillus, Brevibacillus, Staphylococcus, Lysinibacillus and Bacillus, which were universally coexistent with symbiotic bacteria in the nodules. Differing from other similar studies, the present study is the first time that symbiotic and nonsymbiotic bacteria have been simultaneously isolated from the same root nodules, offering the possibility to accurately reveal the correlation between these two kinds of bacteria. These results provide valuable information about the interactions among the symbiotic bacteria, nonsymbiotic bacteria and their habitats., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

A total of 99 bacterial isolates that originated from root nodules of Glycine soja were characterized with restriction analyses of amplified 16S ribosomal DNA and 16S-23S rDNA intergenic spacers (ITS), and sequence analyses of 16S rRNA, rpoB, atpD, recA and nodC genes. When tested for nodulation of G. soja, 72 of the isolates were effective symbionts, and these belonged to five species: Bradyrhizobium japonicum, Bradyrhizobium elkanii, Bradyrhizobium yuanmingense, Bradyrhizobium liaoningense and Sinorhizobium fredii. All of these, except some B. yuanmingense strains, also formed effective nodules on the domesticated soybean Glycine max. The remaining 27 isolates did not nodulate either host, but were identified as Rhizobium. Phylogeny nodC in the G. soja symbionts suggested that this symbiosis gene was mainly maintained by vertical gene transfer. Different nodC sublineages and rrs-ITS clusters reflected the geographic origins of isolates in this study., (© 2011 Federation of European Microbiological Societies. Published by Blackwell Publishing Ltd. All rights reserved.)

As an introduced plant, Lablab purpureus serves as a vegetable, herbal medicine, forage and green manure in China. In order to investigate the diversity of rhizobia associated with this plant, a total of 49 rhizobial strains isolated from ten provinces of Southern China were analyzed in the present study with restriction fragment length polymorphism and/or sequence analyses of housekeeping genes (16S rRNA, IGS, atpD, glnII and recA) and symbiotic genes (nifH and nodC). The results defined the L. purpureus rhizobia as 24 IGS-types within 15 rrs-IGS clusters or genomic species belonging to Bradyrhizobium, Rhizobium, Ensifer (synonym of Sinorhizobium) and Mesorhizobium. Bradyrhizobium spp. (81.6%) were the most abundant isolates, half of which were B. elkanii. Most of these rhizobia induced nodules on L. purpureus, but symbiotic genes were only amplified from the Bradyrhizobium and Rhizobium leguminosarum strains. The nodC and nifH phylogenetic trees defined five lineages corresponding to B. yuanmingense, B. japonicum, B. elkanii, B. jicamae and R. leguminosarum. The coherence of housekeeping and symbiotic gene phylogenies demonstrated that the symbiotic genes of the Lablab rhizobia were maintained mainly through vertical transfer. However, a putative lateral transfer of symbiotic genes was found in the B. liaoningense strain. The results in the present study clearly revealed that L. purpureus was a promiscuous host that formed nodules with diverse rhizobia, mainly Bradyrhizobium species, harboring different symbiotic genes., (Copyright © 2011 Elsevier GmbH. All rights reserved.)

A group of rhizobial strains isolated from nodules of multiple legume species grown in different geographical regions of China had identical 16S rRNA genes. Phylogenetic analysis based on the 16S rRNA gene sequences showed that the novel strains formed a subclade in the genus Rhizobium together with Rhizobium galegae, Rhizobium huautlense and Rhizobium alkalisoli, with 99.8  % gene sequence similarity between the strains. The DNA-DNA relatedness values between the representative strain CCBAU 05176(T) and R. galegae ATCC 43677(T), R. huautlense S02(T) and R. alkalisoli CCBAU 01393(T) were 22.6  %, 8.9  % and 15.9  %, respectively. The novel strains were distinguished from recognized species of the genus Rhizobium by using a polyphasic approach, including PCR-based restriction fragment length polymorphism analysis (RFLP) of the 16S-23S intergenic spacer (IGS), phenotypic and physiological tests, sequence comparisons of housekeeping genes and cellular fatty acid profiles. Therefore, it is suggested that this group of strains represents a novel species for which the name Rhizobium vignae sp. nov. is proposed. The type strain is CCBAU 05176(T) (=HAMBI 3039(T)=LMG 25447(T)).

Agrobacterium sp. II CCBAU 21244 isolated from root nodules of Wisteria sinensis was verified as an endophytic bacterium by inoculation and reisolation tests. However, inoculation with a mixture of this strain and a Sinorhizobium meliloti strain could induce root nodules on W. sinensis and two other woody legumes, which do not form a symbiosis with S. meliloti alone. Rod-shaped and irregular nodules were found on the inoculated plants, in which the S. meliloti strain was detected in all of the nodules; while the Agrobacterium strain was inside of the rod-shaped nodules, or occupied only the nodule surface of the irregular globe-shaped nodules. These findings revealed novel interactions among the symbiotic bacteria, endophytic bacteria and the legume plants, although the mechanisms are still unknown.

The associations among rhizobia chromosomal background, nodulation genes, legume plants, and geographical regions are very attractive but still unclear. To address this question, we analyzed the interactions among rhizobia rDNA genotypes, nodC genotypes, legume genera, as well as geographical regions in the present study. Complex relationships were observed among them, which may be the genuine nature of their associations. The statistical analyses indicate that legume plant is the key factor shaping both rhizobia genetic and symbiotic diversity. In the most cases of our results, the nodC lineages are clearly associated with rhizobial genomic species, demonstrating that nodulation genes have co-evolved with chromosomal background, though the lateral transfer of nodulation genes occurred in some cases in a minority. Our results also support the hypothesis that the endemic rhizobial populations to a certain geographical area prefer to have a wide spectrum of hosts, which might be an important event for the success of both legumes and rhizobia in an isolated region.

Thirteen novel strains were isolated from root nodules of three different leguminous plants of the genera Albizia, Kummerowia and Dalbergia grown in China. Cells of these strains were Gram-negative, strictly aerobic, non-spore-forming, motile rods. Phylogenetic analysis of 16S rRNA gene sequences revealed that they belong to the genus Rhizobium. A representative strain, CCBAU 25010T, showed 98.8% 16S rRNA gene sequence similarity to its closest phylogenetic relative, Rhizobium sullae IS123T. The 16S-23S intergenic spacer (ITS) sequence of CCBAU 25010T shared 91.5 and 87.2% sequence similarity, respectively, with those of Rhizobium etli CFN 42T and Rhizobium leguminosarum LMG 14904T. Analysis of the sequences of the housekeeping genes atpD and recA was in agreement with the results of ITS sequence analysis. The nodC gene sequence of CCBAU 25010T was identical to that of Rhizobium tropici CFN 299. DNA-DNA hybridization values for strain CCBAU 25010T ranged from 20.7% (with Rhizobium mongolense USDA 1844T) to 34.4% (with R. leguminosarum USDA 2370T). Cell protein SDS-PAGE, BOX-PCR and several phenotypic characteristics, such as use of sole carbon sources and antibiotic resistance, could differentiate the novel strains from defined Rhizobium species. We therefore propose that the novel strains reported in this study form a novel species, Rhizobium mesosinicum sp. nov., with the type strain CCBAU 25010T (=LMG 24135T =JCM 14777T).

Adzuki bean (Vigna angularis) is an important legume crop native to China, but its rhizobia have not been well characterized. In the present study, a total of 60 rhizobial strains isolated from eight provinces of China were analyzed with amplified 16S rRNA gene RFLP, IGS-RFLP, and sequencing analyses of 16S rRNA, atpD, recA, and nodC genes. These strains were identified as genomic species within Rhizobium, Sinorhizobium, Mesorhizobium, Bradyrhizobium, and Ochrobactrum. The most abundant groups were Bradyrhizobium species and Sinorhizobium fredii. Diverse nodC genes were found in these strains, which were mainly co-evolved with the housekeeping genes, but a possible lateral transfer of nodC from Sinorhizobium to Rhizobium was found. Analyses of the genomic and symbiotic gene backgrounds showed that adzuki bean shared the same rhizobial gene pool with soybean (legume native to China) and the exotic Vigna species. All of these data demonstrated that nodule formation is the interaction of rhizobia, host plants, and environment characters.

A total of 128 bacterial test strains originated from Astragalus, Caragana, Gueldenstaedtia, Medicago, Melilotus, Oxytropis, Trifolium, and Vicia grown in Tibet were characterized phenotypically and genomically. Based upon the consensus of grouping results, they were identified as 16 putative species. Twenty-five test strains belonging to seven putative species of Agrobacterium, Bradyrhizobium, and Rhizobium might be nonsymbiotic bacteria and the remaining 103 test strains were symbiotic bacteria belonging to Mesorhizobium, Rhizobium, and Sinorhizobium meliloti. Although no novel taxon was detected in the symbiotic bacteria, several characters including the alkaliphilic psychrotolerance revealed that the Tibetan rhizobia could be ecotypes adapted to the local conditions. The results also demonstrated that frequent lateral transfer of symbiotic genes might have happened in the Tibetan rhizobia since nodC genes similar to that of S. meliloti were found in several Rhizobium test strains and all the Mesorhizobium species had very similar nodC genes despite their genomic background. All of these findings demonstrated that the Tibetan rhizobia were an important resource for further studies on rhizobial ecology and application.

Twenty-four Mesorhizobium strains were isolated from desert soils in the Xinjiang region of China and were characterized by a polyphasic approach. These strains grouped into three clusters in IGS-RFLP, SDS-PAGE analysis of whole-cell proteins and BOX-PCR analysis, corresponding to genomic species V, VI and VII as found in a previous study. The results were supported by sequencing analyses of rrs, IGS, atpD and recA genes. Genospecies VII was most related to Mesorhizobium septentrionale, while genospecies V and VI were both most closely related to Mesorhizobium tianshanense, but were distinct from each other and from M. tianshanense. The DNA-DNA hybridization value between the representative strain CCBAU 83284 (genospecies VII) and the type strain of M. septentrionale was 90.1 %. Genospecies VII was thus defined as M. septentrionale. The DNA-DNA relatedness value for representative strains of genospecies V or VI with the related reference strains of recognized species were always lower than 60 %. Low values of DNA-DNA hybridization (32.79 %) between representative strains of genospecies V (CCBAU 83330(T)) and of VI (CCBAU 83306(T)) were also observed. Based upon these results, two novel species are proposed: Mesorhizobium gobiense sp. nov. represented by genospecies V (type strain, CCBAU 83330(T)=LMG 23949(T)=HAMBI 2974(T)) and Mesorhizobium tarimense sp. nov. represented by genospecies VI (type strain, CCBAU 83306(T)=LMG 24338(T)=HAMBI 2973(T)). Strain CCBAU 83278 grouped as the most peripheral member with genospecies VI in SDS-PAGE of whole-cell proteins and BOX-PCR analysis and in the phylogenetic tree of 16S-23S rRNA intergenic spacer (IGS) sequences. The results of analyses of rrs, atpD and recA gene sequences, as well as those of DNA-DNA hybridization studies, strongly supported the suggestion that this strain belonged to a species quite different from genospecies V and VI and from any other recognized species of the genus Mesorhizobium. As only one strain has been isolated to date, strain CCBAU 83278 was not proposed as a novel species in this study. Mesorhizobium gobiense sp. nov. and Mesorhizobium tarimense sp. nov. could be differentiated from each other as well as from recognized species of the genus Mesorhizobium on the basis of phenotypic characteristics. The symbiotic loci (nodC and nifH) of the two novel species formed two phylogenetic branches related to Mesorhizobium loti and M. tianshanense. The type strains of the two novel species were able to nodulate Glycyrrhiza uralensis, Lotus corniculatus, Oxytropis glabra and Robinia pseudoacacia but not Astragalus membranaceus, Leucaena leucocephala, Phaseolus vulgaris, Pisum sativum or Medicago sativa.

A total of 111 rhizobial strains were isolated from wild legumes in Xinjiang, an isolated region of northwest China. Nine genomic species belonging to four genera of Rhizobium, Mesorhizobium, Ensifer, and Bradyrhizobium were defined among these strains based on the characterization of amplified 16S ribosomal DNA restriction analysis (ARDRA), restriction fragment length polymorphism (RFLP) analysis of 16S-23S rDNA intergenic spacers (IGS), 16S rRNA gene sequencing and multilocus sequence analysis (MLSA). Twenty-five nodC types corresponding to eight phylogenetic clades were divided by RFLP and sequence analysis of the PCR-amplified nodC gene. The acid-producing Rhizobium and Mesorhizobium species were predominant, which may be related to both the local environments and the hosts sampled. The present study also showed the limitation of using nod genes to estimate the host specificity of rhizobia.

Cowpea (Vigna unguiculata) and mung bean (Vigna radiata) are important legume crops yet their rhizobia have not been well characterized. In the present study, 62 rhizobial strains isolated from the root nodules of these plants grown in the subtropical region of China were analyzed via a polyphasic approach. The results showed that 90% of the analyzed strains belonged to or were related to Bradyrhizobium japonicum, Bradyrhizobium liaoningense, Bradyrhizobium yuanmingense and Bradyrhizobium elkanii, while the remaining represented Rhizobium leguminosarum, Rhizobium etli and Sinorhizobium fredii. Diverse nifH and nodC genes were found in these strains and their symbiotic genes were mainly coevolved with the housekeeping genes, indicating that the symbiotic genes were mainly maintained by vertical transfer in the studied rhizobial populations.

Thirty-one rhizobial strains isolated from nodules of legumes native of Xinjiang, China, were characterized. These strains were classified as belonging to the genus Rhizobium based on amplified 16S rDNA restriction analysis (ARDRA). The strains were distinguished from recognized Rhizobium species using analysis of 16S-23S rDNA intergenic spacers (IGS-RFLP), SDS-PAGE analysis of whole proteins and BOX-PCR; the test strains always formed a distinct cluster with patterns that were quite different from those of the reference rhizobial strains used. According to the phylogenetic analysis based on the 16S rRNA gene, the test strains belonged to the genus Rhizobium, with Rhizobium tropici, Rhizobium rhizogenes and Rhizobium lusitanum as the closest related species, with 99.6, 99.2 and 99.4 % sequence similarities, respectively, between the type strains of the three Rhizobium species and strain CCBAU 83401(T). Phylogenetic analyses of the representative strains using IGS and atpD, recA and glnII genes all confirmed the phylogenetic arrangements obtained using the 16S rRNA gene. The DNA-DNA relatedness values between strain CCBAU 83401(T) and strains CCBAU 83364, CCBAU 83345 and CCBAU 83523 ranged from 80.8 to 100 %, showing that they belong to the same species. The DNA-DNA relatedness between strain CCBAU 83401(T) and R. tropici IIB CIAT 899(T), R. tropici IIA CFN 299, R. rhizogenes LMG 150(T) and R. lusitanum P1-7(T) were 26.9, 27.7, 38.2 and 22.6 %, respectively, clearly indicating that strain CCBAU 83401(T) represents a novel species. Phenotypic characterization of four representative strains, CCBAU 83401(T), CCBAU 83364, CCBAU 83345 and CCBAU 83523, showed several distinctive features that differentiated them from closely related species. The 31 strains had identical nodD and nifH genes, which were very similar to those of the bean-nodulating R. lusitanum, Devosia neptuniae and R. tropici IIB. Based upon these results, the strains from this study are considered to represent a novel species, for which the name Rhizobium multihospitium sp. nov. is proposed. The DNA G+C content ranged from 65.3 to 66.0 mol% (T(m)). The type strain is CCBAU 83401(T) (=LMG 23946(T)=HAMBI 2975(T)), which nodulates Robinia pseudoacacia, but not Leucaena leucocephala, Phaseolus vulgaris, Pisum sativum or Medicago sativa.

Bacterial strain CCBAU 25048(T) was isolated from root nodules of Kummerowia stipulacea grown in Shandong province of China. Cells of the strain were Gram-negative, strictly aerobic, non-spore-forming, motile short rods. Phylogeny of 16S rRNA gene sequences revealed that the strain belonged to the genus Shinella, a member of family Rhizobiaceae. Its closest phylogenetic relatives were Shinella granuli Ch06(T) and Shinella zoogloeoides IAM 12669(T), respectively showing 98.3 and 98.9 % 16S rRNA gene sequence similarity. Strain CCBAU 25048(T) had DNA-DNA relatedness of 43.5 and 34.8 %, respectively, with S. zoogloeoides JCM 20728(T) and S. granuli JCM 13254(T). In addition, in TP-RAPD analysis, different patterns were obtained for these three strains and some rhizobial strains. The nifH, nodC and nodD sequences of CCBAU 25048(T) were identical or very similar to those of bean-nodulating Rhizobium tropici strains. Several phenotypic characteristics, including the use of citrate and d-ribose as carbon sources and growth at pH 11.0, as well as the fatty acid composition, could differentiate CCBAU 25048(T) from the two defined Shinella species. Therefore, a novel species Shinella kummerowiae sp. nov. is proposed, with strain CCBAU 25048(T) (=JCM 14778(T) =LMG 24136(T)) as the type strain.

A total of 63 bacterial strains were isolated from root nodules of Kummerowia striata and K. stipulacea grown in different geographic regions of China. These bacteria could be divided into fast-growing (FG) rhizobia and slow-growing (SG) rhizobia according to their growth rate. Genetic diversity and taxonomic relationships among these rhizobia were revealed by PCR-based 16 S rDNA RFLP and sequencing, 16 S-IGS RFLP, SDS-PAGE of whole cell soluble proteins, BOX-PCR and symbiotic gene (nifH/nodC) analyses. The symbiotic FG strains were mainly isolated from temperate regions and they were identified as four genomic species in Rhizobium and Sinorhizobium meliloti based on the consensus of grouping results. The SG strains were classified as five genomic species within Bradyrhizobium and they were mainly isolated fron the subtropic and tropical regions. The phylogenetic analyses of nifH and nodC genes showed relationships similar to that of 16 S rDNA but the symbiotic genes of Bradyrhizobium strains isolated from Kummerowia were distinct from those isolated from Arachis and soybean. These results offered evidence for rhizobial biogeography and demonstrated that the Kummerowia-nodulating ability might have evolved independently in different regions in association with distinctive genomic species of rhizobia.

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.

To analyze the diversity and relationships of rhizobia in the subtropical and tropical zones of China, we characterized 67 bacterial strains isolated from root nodules of five legume species in the genera Trifolium, Crotalaria and Mimosa . PCR-amplified 16S rDNA RFLP, numerical taxonomy, SDS-PAGE of whole cell proteins, sequencing of 16S rDNA and DNA-DNA hybridization grouped the isolates into 17 lineages belonging to Bradyrhizobium, Mesorhizobium, Rhizobium, Sinorhizobium and Burkholderia, as well as a non-symbiotic group of Agrobacterium. The Rhizobium group contained twenty strains isolated from Mimosa pudica, Crotalaria pallida and two species of Trifolium. Fifteen of them were R. leguminosarum. Twenty-one strains isolated from four species of Trifolium, Crotalaria and Mimosa were classified into five groups of Bradyrhizobium, including B. japonicum. Agrobacterium group composed of 20 isolates from Mimosa pudica, C. pallida and Trifolium fragiferum. In addition, several strains of Sinorhizobium and Mesorhizobium associated with Trifolium and Burkholderia associated with Mimosa pudica were also identified. The predominance of Bradyrhizobium in the nodules of Trifolium was a novel finding and it demonstrated that the nodule microsymbionts might be selected by both the geographic factors and the legume hosts.

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.

Conzattia multiflora is a leguminous tree present only in Mexico and Guatemala. There is no record about its symbiotic or pathogenic microbes. In this study, we found that numerous bacteria with 10(4)-10(6) individuals per gram of fresh epidermis were distributed in the tissue of this plant. All the bacteria isolated from the Conzattia epidermis were Gram-negative, facultative anaerobic rods and formed yellow or colorless colonies. They were identified as endophytes by inoculation tests. Some of the bacteria could significantly promote the growth of Conzattia seedlings. Nine different groups were defined by PCR-based RFLP, which were classified as Pantoea, Erwinia, Salmonella, Enterobacter, Citrobacter and Klebsiella by the phylogenetic analysis of 16S rRNA genes. The existence of plant-borne lineages of Salmonella indicates that the unexplored plants may harbor some unknown microbes.

Fifty-nine bacterial isolates from root nodules of the woody legumes Wisteria sinensis, Cercis racemosa and Amorpha fruticosa grown in the central and eastern regions of China were characterized with phenotypic analysis, PCR-based 16S and 23S rRNA gene RFLP, Box PCR and 16S rRNA gene sequencing. Seven main phena were defined in numerical taxonomy, which corresponded to distinct groups within the genera Agrobacterium, Bradyrhizobium, Mesorhizobium and Rhizobium in 16S and 23S rRNA gene PCR-RFLP. The phylogenetic relationships of the 16S rRNA genes supported the grouping results of PCR-RFLP. Most of the isolates from Amorpha fruticosa were classified into two groups closely related to Mesorhizobium amorphae. Seventeen of the 21 isolates from Wisteria sinensis were identified as two groups related to Rhizobium and Agrobacterium. Six out of 10 isolates from Cercis racemosa were identified as a group related to Bradyrhizobium. Our results indicated that each of the investigated legumes nodulated mainly with one or two rhizobial groups, although isolates from different plants intermingled in some small bacterial groups. In addition, correlation between geographic origin and grouping results was found in the isolates from Amorpha fruticosa. These results revealed that the symbiotic bacteria might have been selected by both the legume hosts and the geographic factors.

Eighty bacterial isolates from root nodules of the leguminous plants Phaseolus vulgaris, Campylotropis spp. and Cassia spp. grown in China were classified into five groups by phenotypic analyses, SDS-PAGE of whole-cell proteins, PCR-based 16S rRNA gene restriction-fragment-length-polymorphism and sequencing. Thirty-three isolates from the three plant genera were identified as Agrobacterium tumefaciens because they are closely related to the type strain of A. tumefaciens. Fourteen isolates from P. vulgaris grown in Yunnan and Inner Mongolia were classified as R. leguminosarum bv. phaseoli based on their close relationship with the type strain in numerical taxonomy and in 16S rDNA phylogeny. Twenty-seven isolates from Campylotropis delavayi, P. vulgaris and four species of Cassia grown in the central zones of China were classified into three groups within the genus Bradyrhizobium. One of these three groups could be defined as Bradyrhizobium japonicum. Our results demonstrated that P. vulgaris and the species of Campylotropis and Cassia could form nodules with diverse rhizobia in Chinese soils, including novel lineages associated with P. vulgaris. These results also offered information about the convergent evolution between rhizobia and legumes since the rhizobial populations associated with P. vulgaris in Chinese soils were completely different from those in Mexico, the original cite of this plant. Some rhizobial species could be found in all of the three leguminous genera.

Forty-six Mesorhizobium strains associated with the leguminous plants Leucaena leucocephala and Sesbania herbacea in an uncultivated Mexican field were characterized using a polyphasic approach. The strains were identified as Mesorhizobium plurifarium based upon the close relationships with the reference strains for this species in PCR-based restriction fragment length polymorphism analyses, sequencing of 16S rRNA genes, multilocus enzyme electrophoresis, and DNA-DNA hybridization. Although the strains isolated from both plants formed the same group in multilocus enzyme electrophoresis and cross-nodulations were observed in the laboratory, different electrophoretic types were obtained from the two plants grown in natural soils, indicating the existence of a preferable association between the plants and the rhizobia. The M. plurifarium strains from Mexico and the reference strains from Africa and Brazil formed different phenotypic clusters in a numerical taxonomy. The Mexican strains did not grow at 37 degrees C and were sensitive to salty-alkaline conditions, while the reference strains from Africa and Brazil grew at 42 degrees C and were more resistant to salty-alkaline conditions. These results demonstrate that both the plants and environmental factors affected the evolution of rhizobia and that the Mexican strains had adapted to the neutral soils and the cool climate where they were isolated.

Forty-eight rhizobial isolates from root nodules of Indigofera and Kummerowia, two genera of annual or perennial wild legumes growing in the Loess Plateau in north-western China, were characterized by a polyphasic approach. Two main groups, cluster 1 and cluster 2, were defined based upon the results of numerical taxonomy, SDS-PAGE of whole-cell proteins and DNA relatedness. All the isolates within cluster 1 were isolated from Indigofera and they were identified as Rhizobium strains by 16S rRNA gene analysis. DNA relatedness of 29.5-48.9% was obtained among the cluster 1 isolates and the reference strains for defined Rhizobium species. Cluster 2 consisted of isolates from Kummerowia stipulacea and was identified as belonging to Sinorhizobium by 16S rRNA gene analyses. DNA relatedness varied from 5.2 to 41.7% among the isolates of cluster 2 and reference strains for Sinorhizobium species. Considering the existence of distinctive features among these two groups and related species within the genera Rhizobium and Sinorhizobium, we propose two novel species, Rhizobium indigoferae sp. nov. for cluster 1, with isolate CCBAU 71714(T) (= AS 1.3046(T)) as the type strain, and Sinorhizobium kummerowiae sp. nov. for cluster 2, with isolate CCBAU 71042(T) (= AS 1.3045(T)) as the type strain.

Amorpha fruticosa was inoculated with rhizosphere soil from Iowa, USA, and 140 rhizobia isolated from root nodules were compared with Mesorhizobium amorphae originating from Chinese soils. PCR-RFLP patterns of the 16S rRNA gene from the isolates and from M. amorphaewere the same. All isolates had a symbiotic plasmid of the same size with a single nifHgene. DNA:DNA hybridization values, DNA G+C content, and induced Nod factor patterns also were similar. We concluded that the four genotypes distinguished among 53 representative American isolates were M. amorphae. Since A. fruticosa is native to the Americas and is highly specific in its nodulation requirement, M. amorphae probably was transmitted to China.

Of 42 rhizobial isolates from Medicago sativa and Melilotus spp. growing in arid saline fields in Xinjiang, China, 40 were identified as Sinorhizobium meliloti by a polyphasic approach. However, diverse groups were obtained from these isolates in numerical taxonomy and SDS-PAGE of proteins. They could grow at pH 10.5 and were tolerant to 2.5-4.0% (w/v) NaCl.

Fifty rhizobial isolates from root nodules of Mimosa affinis, a small leguminous plant native to Mexico, were identified as Rhizobium etli on the basis of the results of PCR-RFLP and RFLP analyses of small-subunit rRNA genes, multilocus enzyme electrophoresis and DNA-DNA homology. They are, however, a restricted group of lineages with low genetic diversity within the species. The isolates from M. affinis differed-from the R. etli strains that orginated from bean plants (Phaseolus vulgaris) in the size and replicator region of the symbiotic plasmid and in symbiotic-plasmid-borne traits such as nifH gene sequence and organization, melanin production and host specificity. A new biovar, bv. mimosae, is proposed within R. etli to encompass Rhizobium isolates obtained from M. affinis. The strains from common bean plants have been designated previously as R. etli bv. phaseoli. Strains of both R. etli biovars could nodulate P. vulgaris, but only those of bv. mimosae could form nitrogen-fixing nodules on Leucaena leucocephala.

Nodule isolates from 11 species of wild legumes in north-western China were characterized by numerical taxonomy, PCR-based 16S rRNA gene RFLP and sequence analyses, DNA-DNA hybridization, restriction patterns of nodDAB and nifH genes, and symbiotic properties. Based on the results of numerical taxonomy, most of the 35 new isolates were grouped into five clusters (clusters 7, 9, 12, 14 and 15). Clusters 7 and 12 were identified as Mesorhizobium amorphae and Agrobacterium tumefaciens, respectively, based on their high DNA homologies with the reference strains for these species, their 16S rRNA gene analysis and their phenotypic features. Results of 16S rDNA PCR-RFLP analysis showed that cluster 9 belonged to Rhizobium. Clusters 14 and 15 were identified as Mesorhizobium based on their moderately slow-growing, acid-producing characters and the high similarity of their 16S rDNA PCR-RFLP patterns to those of Mesorhizobium species. These two clusters were genomic species distinct from all described species based on analysis of DNA relatedness within this genus. The isolates in cluster 12 (Agrobacterium tumefaciens) failed to nodulate their original host and other selected hosts and they did not hybridize to nif or nod gene probes. The possibility of opportunistic nodulation of these isolates is discussed. Identical restriction patterns were obtained in the nif or nod gene hybridization studies from the three isolates within cluster 15, which were isolated from the same host species. The isolates from different host plants in each of clusters 9 and 14 produced different nodDAB RFLP patterns, but similar nifH RFLP patterns appeared (one band for each isolate). Different patterns were observed among different clusters from both the nod and nif gene hybridization studies. Crossnodulation was recorded among the isolates and the host plants in the same cluster and promiscuous properties were found among some of the hosts tested.

A fast-growing rhizobial group isolated from leguminous plants in Hainan Province, a tropical region of China, is proposed as a new Rhizobium species on the basis of 16S rRNA gene sequencing. DNA-DNA hybridization, and phenotypic characterization. This new species belongs to the phylogenetic branch which includes Rhizobium leguminosarum. We propose the name Rhizobium hainanense sp. nov. for this species. The strain CCBAU 57015 (166) is the type strain; it has been deposited in the culture collection of Beijing Agricultural University, People's Republic of China.