Evidence for Phosphate Starvation of Rhizobia without Terminal Differentiation in Legume Nodules.

Phosphate homeostasis is tightly modulated in all organisms, including bacteria, which harbor both high- and low-affinity transporters acting under conditions of fluctuating phosphate levels. It was thought that nitrogen-fixing rhizobia, named bacteroids, inhabiting root nodules of legumes are not phosphate limited. Here, we show that the high-affinity phosphate transporter PstSCAB, rather than the low-affinity phosphate transporter Pit, is essential for effective nitrogen fixation of Sinorhizobium fredii in soybean nodules. Symbiotic and growth defects of the pst mutant can be effectively restored by knocking out PhoB, the transcriptional repressor of pit. The pst homologs of representative rhizobia were actively transcribed in bacteroids without terminal differentiation in nodules of diverse legumes (soybean, pigeonpea, cowpea, common bean, and Sophora flavescens) but exhibited a basal expression level in terminally differentiated bacteroids (alfalfa, pea, and peanut). Rhizobium leguminosarum bv. viciae Rlv3841 undergoes characteristic nonterminal and terminal differentiations in nodules of S. flavescens and pea, respectively. The pst mutant of Rlv3841 showed impaired adaptation to the nodule environment of S. flavescens but was indistinguishable from the wild-type strain in pea nodules. Taken together, root nodule rhizobia can be either phosphate limited or nonlimited regarding the rhizobial differentiation fate, which is a host-dependent feature.

Ensifer glycinis sp. nov., a rhizobial species associated with species of the genus Glycine.

Rhizobial strains from root nodules of Astragalus mongholicus and soybean (Glycine max) were characterized phylogenetically as members of the genus Ensifer (formerly named Sinorhizobium), based on 16S rRNA gene sequence comparisons. Results based upon concatenated sequence analysis of three housekeeping genes (recA, atpD and glnII, ≤ 93.8 % similarities to known species) and average nucleotide identity (ANI) values of whole genome sequence comparisons (ranging from 89.6 % to 83.4 % to Ensifer fredii and Ensifer saheli, respectively) indicated the distinct positions of these novel strains within the genus Ensifer. Phylogeny of symbiotic genes (nodC and nifH) of three novel strains clustered them with rhizobial species Ensifer fredii and Ensifer sojae, both isolated from nodules of Glycine max. Cross-nodulation tests showed that the representative strain CCBAU 23380T could form root nodules with nitrogen fixation capability on Glycine soja, Albizia julibrissin, Vigna unguiculata and Cajanus cajan, but failed to nodulate Astragalus mongholicus, its original host legume. Strain CCBAU 23380T formed inefficient nodules on G. max, and it did not contain 18 : 0, 18 : 1ω7c 11-methyl or summed feature 1 fatty acids, which differed from other related strains. Failure to utilize malonic acid as a carbon source distinguished strain CCBAU 23380T from the type strains of related species. The genome size of CCBAU 23380T was 6.0 Mbp, comprising 5624 predicted genes with DNA G+C content of 62.4 mol%. Based on the results above, a novel species, Ensifer glycinis sp. nov., is proposed, with CCBAU 23380T (=LMG 29231T =HAMBI 3645T) as the type strain.

Brevibacterium metallicus sp. nov., an endophytic bacterium isolated from roots of Prosopis laegivata grown at the edge of a mine tailing in Mexico.

A Gram-positive, aerobic, nonmotile strain, NM2E3(T) was identified as Brevibacterium based on the 16S rRNA gene sequence analysis and had the highest similarities to Brevibacterium jeotgali SJ5-8(T) (97.3 %). This novel bacterium was isolated from root tissue of Prosopis laegivata grown at the edge of a mine tailing in San Luis Potosí, Mexico. Its cells were non-spore-forming rods, showing catalase and oxidase activities and were able to grow in LB medium added with 40 mM Cu(2+), 72 mM As(5+) and various other toxic elements. Anteiso-C15:0 (41.6 %), anteiso-C17:0 (30 %) and iso-C15:0 (9.5 %) were the major fatty acids. MK-8(H2) (88.4 %) and MK-7(H2) (11.6 %) were the major menaquinones. The DNA G + C content of the strain NM2E3(T) was 70.8 mol % (Tm). DNA-DNA hybridization showed that the strain NM2E3(T) had 39.8, 21.7 and 20.3 % relatedness with B. yomogidense JCM 17779(T), B. jeotgali JCM 18571(T) and B. salitolerans TRM 45(T), respectively. Based on the phenotypic and genotypic analyses, the strain NM2E3(T) (=CCBAU 101093(T) = HAMBI 3627(T) = LMG 8673(T)) is reported as a novel species of the genus Brevibacterium, for which the name Brevibacterium metallicus sp. nov., is proposed.

Mesorhizobium acaciae sp. nov., isolated from root nodules of Acacia melanoxylon R. Br.

Three novel strains, RITF741T, RITF1220 and RITF909, isolated from root nodules of Acacia melanoxylon in Guangdong Province of China, have been previously identified as members of the genus Mesorhizobium, displaying the same 16S rRNA gene RFLP pattern. Phylogenetic analysis of 16S rRNA gene sequences indicated that the three strains belong to the genus Mesorhizobium and had highest similarity (100.0 %) to Mesorhizobium plurifarium LMG 11892T. Phylogenetic analyses of housekeeping genes recA, atpD and glnII revealed that these strains represented a distinct evolutionary lineage within the genus Mesorhizobium. Strain RITF741T showed >73 % DNA­DNA relatedness with strains RITF1220 and RITF909, but < 60 % DNA­DNA relatedness with the closest type strains of recognized species of the genus Mesorhizobium. They differed from each other and from their closest phylogenetic neighbours by presence/absence of several fatty acids, or by large differences in the relative amounts of particular fatty acids. While showing distinctive features, they were generally able to utilize a wide range of substrates as sole carbon sources based on API 50CH and API 20NE tests. The three strains were able to form nodules with the original host Acacia melanoxylon and other woody legumes such as Acacia aneura, Albizia falcataria and Leucaena leucocephala. In conclusion, these strains represent a novel species belonging to the genus Mesorhizobium based on the data obtained in the present and previous studies, for which the name Mesorhizobium acaciae sp. nov. is proposed. The type strain is RITF741T ( = CCBAU 101090T = JCM 30534T), the DNA G+C content of which is 64.1 mol% (T m).

Bradyrhizobium erythrophlei sp. nov. and Bradyrhizobium ferriligni sp. nov., isolated from effective nodules of Erythrophleum fordii.

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.

Bradyrhizobium guangdongense sp. nov. and Bradyrhizobium guangxiense sp. nov., isolated from effective nodules of peanut.

Seven slow-growing rhizobia isolated from effective nodules of Arachis hypogaea were assigned to the genus Bradyrhizobium based on sharing 96.3-99.9 % 16S rRNA gene sequence similarity with the type strains of recognized Bradyrhizobium species. Multilocus sequence analysis of glnII, recA, gyrB and dnaK genes indicated that the seven strains belonged to two novel species represented by CCBAU 51649T and CCBAU 53363T. Strain CCBAU 51649T shared 94, 93.4, 92.3 and 94.9 % and CCBAU 53363T shared 91.4, 94.5, 94.6 and 97.7 % sequence similarity for the glnII, recA, gyrB and dnaK genes, respectively, with respect to the closest related species Bradyrhizobium manausense BR 3351T and Bradyrhizobium yuanmingense CCBAU 10071T. Summed feature 8 and C16 : 0 were the predominant fatty acid components for strains CCBAU 51649T and CCBAU 53363T. DNA-DNA hybridization and analysis of phenotypic characteristics also distinguished these strains from the closest related Bradyrhizobium species. The strains formed effective nodules on Arachis hypogaea, Lablab purpureus and Aeschynomene indica, and they had identical nodA genes to Bradyrhizobium sp. PI237 but were phylogenetically divergent from other available nodA genes at less than 66 % similarity. Based in these results, strains CCBAU 51649T ( = CGMCC 1.15034T = LMG 28620T) and CCBAU 53363T ( = CGMCC 1.15035T = LMG 28621T) are designated the type strains of two novel species, for which the names Bradyrhizobium guangdongense sp. nov. and Bradyrhizobium guangxiense sp. nov. are proposed, respectively.

Phyllobacterium sophorae sp. nov., a symbiotic bacterium isolated from root nodules of Sophora flavescens.

Two novel Gram-stain-negative strains (CCBAU 03422(T) and CCBAU 03415) isolated from root nodules of Sophora flavescens were classified phylogenetically into the genus Phyllobacterium based on the comparative analysis of 16S rRNA and atpD genes. They showed 99.8 % rRNA gene sequence similarities to Phyllobacterium brassicacearum LMG 22836(T), and strain CCBAU 03422(T) showed 91.2 and 88.6 % atpD gene sequence similarities to strains Phyllobacterium endophyticum LMG 26470(T) and Phyllobacterium brassicacearum LMG 22836(T), respectively. Strain CCBAU 03422(T) contained Q-10 as its major quinone and showed a cellular fatty acid profile, carbon source utilization and other phenotypic characteristics differing from type strains of related species. DNA-DNA relatedness (lower than 48.8 %) further confirmed the differences between the novel strains and the type strains of related species. Strain CCBAU 03422(T) could nodulate and fix nitrogen effectively on its original host plant, Sophora flavescens. Based upon the results mentioned above, a novel species named Phyllobacterium sophorae is proposed and the type strain is CCBAU 03422(T) ( = A-6-3(T) = LMG 27899(T) = HAMBI 3508(T)).

Rhizobium sophorae sp. nov. and Rhizobium sophoriradicis sp. nov., nitrogen-fixing rhizobial symbionts of the medicinal legume Sophora flavescens.

Five bacterial strains representing 45 isolates originated from root nodules of the medicinal legume Sophora flavescens were defined as two novel groups in the genus Rhizobium based on their phylogenetic relationships estimated from 16S rRNA genes and the housekeeping genes recA, glnII and atpD. These groups were distantly related to Rhizobium leguminosarum USDA 2370(T) (95.6 % similarity for group I) and Rhizobium phaseoli ATCC 14482(T) (93.4 % similarity for group II) in multilocus sequence analysis. In DNA-DNA hybridization experiments, the reference strains CCBAU 03386(T) (group I) and CCBAU 03470(T) (group II) showed levels of relatedness of 17.9-57.8 and 11.0-42.9 %, respectively, with the type strains of related species. Both strains CCBAU 03386(T) and CCBAU 03470(T) contained ubiquinone 10 (Q-10) as the major respiratory quinone and possessed 16 : 0, 18 : 0, 19 : 0 cyclo ω8c, summed feature 8 and summed feature 2 as major fatty acids, but did not contain 20 : 3 ω6,8,12c. Phenotypic features distinguishing both groups from all closely related species of the genus Rhizobium were found. Therefore, two novel species, Rhizobium sophorae sp. nov. for group I (type strain CCBAU 03386(T) = E5(T) = LMG 27901(T) = HAMBI 3615(T)) and Rhizobium sophoriradicis sp. nov. for group II (type strain CCBAU 03470(T) = C-5-1(T) = LMG 27898(T) = HAMBI 3510(T)), are proposed. Both groups were able to nodulate Phaseolus vulgaris and their hosts of origin (Sophora flavescens) effectively and their nodulation gene nodC was phylogenetically located in the symbiovar phaseoli.

Rhizobium anhuiense sp. nov., isolated from effective nodules of Vicia faba and Pisum sativum.

Four rhizobia-like strains, isolated from root nodules of Pisum sativum and Vicia faba grown in Anhui and Jiangxi Provinces of China, were grouped into the genus Rhizobium but were distinct from all recognized species of the genus Rhizobium by phylogenetic analysis of 16S rRNA and housekeeping genes. The combined sequences of the housekeeping genes atpD, recA and glnII for strain CCBAU 23252(T) showed 86.9 to 95% similarity to those of known species of the genus Rhizobium. All four strains had nodC and nifH genes and could form effective nodules with Pisum sativum and Vicia faba, and ineffective nodules with Phaseolus vulgaris, but did not nodulate Glycine max, Arachis hypogaea, Medicago sativa, Trifolium repens or Lablab purpureus in cross-nodulation tests. Fatty acid composition, DNA-DNA relatedness and a series of phenotypic tests also separated these strains from members of closely related species. Based on all the evidence, we propose a novel species, Rhizobium anhuiense sp. nov., and designate CCBAU 23252(T) ( = CGMCC 1.12621(T) = LMG 27729(T)) as the type strain. This strain was isolated from a root nodule of Vicia faba and has a DNA G+C content of 61.1 mol% (Tm).

Rhizobium pakistanensis sp. nov., isolated from groundnut (Arachis hypogaea) nodules grown in rainfed Pothwar, Pakistan.

A Gram-negative, white, non-motile, rod shaped bacterial strain BN-19(T) was isolated from a root nodule of groundnut (Arachis hypogaea) in Pakistan. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain BN-19(T) formed a subclade in the genus Rhizobium together with Rhizobium alkalisoli CCBAU 01393(T), Rhizobium vignae CCBAU 05176(T), Rhizobium huautlense SO2(T) and Rhizobium tarimense PL-41(T) with sequence similarities of 97.5, 97.3, 97.2 and 97.1 % respectively. Sequence analysis of housekeeping genes atpD, glnII and recA (with sequence similarities of ≤92 %) confirmed the unique position of BN-19(T) in the genus Rhizobium. DNA-DNA relatedness between the strain BN-19(T) and R. alkalisoli CCBAU 01393(T), R. vignae CCBAU 05176(T), R. huautlense SO2(T) and R. tarimense PL-41(T) were 20.6, 22.5, 15.9 and 20.5 % respectively, further confirming that BN-19(T) represents a novel species in the genus Rhizobium. The DNA G + C content was 60.1 mol%. The dominant fatty acids of strain BN-19(T) were C19:0 cyclo ω8c, summed feature 2 (C14:0 3OH and/or C16:1 iso I) and summed feature 8 (C18:1 ω7c). Some phenotypic features also differentiate the strain BN-19(T) from the related species. On the basis of these results, strain BN-19(T) is considered to represent a novel species in the genus Rhizobium, for which the name Rhizobium pakistanensis sp. nov. is proposed. The type strain is BN-19(T) (=LMG 27895(T) = CCBAU 101086(T)).

Genetic diversity and evolution of Bradyrhizobium populations nodulating Erythrophleum fordii, an evergreen tree indigenous to the southern subtropical region of China.

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.

Genetic divergence of bradyrhizobium strains nodulating soybeans as revealed by multilocus sequence analysis of genes inside and outside the symbiosis island.

The genus Bradyrhizobium has been considered to be a taxonomically difficult group. In this study, phylogenetics and evolutionary genetics analyses were used to investigate divergence levels among Bradyrhizobium strains nodulating soybeans in China. Eleven genospecies were identified by sequence analysis of three phylogenetic and taxonomic markers (SMc00019, thrA, and truA). This was also supported by analyses of eight genes outside the symbiosis island ("off-island" genes; SMc00019, thrA, truA, fabB, glyA, phyR, exoN, and hsfA). However, seven genes inside the symbiosis island ("island" genes; nifA, nifH, nodC, nodV, fixA, trpD, and rhcC2) showed contrasting lower levels of nucleotide diversity and recombination rates than did off-island genes. Island genes had significantly incongruent gene phylogenies compared to the species tree. Four phylogenetic clusters were observed in island genes, and the epidemic cluster IV (harbored by Bradyrhizobium japonicum, Bradyrhizobium diazoefficiens, Bradyrhizobium huanghuaihaiense, Bradyrhizobium liaoningense, Bradyrhizobium daqingense, Bradyrhizobium sp. I, Bradyrhizobium sp. III, and Bradyrhizobium sp. IV) was not found in Bradyrhizobium yuanmingense, Bradyrhizobium sp. II, or Bradyrhizobium elkanii. The gene flow level of island genes among genospecies is discussed in the context of the divergence level of off-island genes.

Rhizobium flavum sp. nov., a triazophos-degrading bacterium isolated from soil under the long-term application of triazophos.

A Gram-stain-negative, non-motile, pale yellow, rod-shaped bacterial strain, YW14(T), was isolated from soil and its taxonomic position was investigated by a polyphasic study. Strain YW14(T) did not form nodules on three different legumes, and the nodD and nifH genes were not detected by PCR. Strain YW14(T) contained Q-10 as the predominant ubiquinone. The major cellular fatty acid was C(18 : 1)ω7c. Phylogenetic analyses based on 16S rRNA gene sequences and seven housekeeping gene sequences (recA, atpD, glnII, gyrB, rpoB, dnaK and thrC) showed that strain YW14(T) belonged to the genus Rhizobium. Strain YW14(T) showed 16S rRNA gene sequence similarity of 93.4-97.3% to the type strains of recognized species of the genus Rhizobium. DNA-DNA relatedness between strain YW14(T) and the type strains of Rhizobium sullae IS123(T) and Rhizobium yanglingense CCBAU 71623(T) was 19.6-25.7%, indicating that strain YW14(T) was distinct from them genetically. Strain YW14(T) could also be differentiated from these phylogenetically related species of the genus Rhizobium by various phenotypic properties. On the basis of phenotypic properties, phylogenetic distinctiveness and genetic data, strain YW14(T) is considered to represent a novel species of the genus Rhizobium, for which the name Rhizobium flavum sp. nov. is proposed. The type strain is YW14(T) ( = KACC 17222(T) = CCTCC AB2013042(T)).

Bradyrhizobium ganzhouense sp. nov., an effective symbiotic bacterium isolated from Acacia melanoxylon R. Br. nodules.

Three slow-growing rhizobial strains, designated RITF806(T), RITF807 and RITF211, isolated from root nodules of Acacia melanoxylon grown in Ganzhou city, Jiangxi Province, China, had been previously defined, based on amplified 16S rRNA gene restriction analysis, as a novel group within the genus Bradyrhizobium. To clarify their taxonomic position, these strains were further analysed and compared with reference strains of related bacteria using a polyphasic approach. According to 16S rRNA gene sequence analysis, the isolates formed a group that was closely related to 'Bradyrhizobium rifense' CTAW71, with a similarity value of 99.9%. In phylogenetic analyses of the housekeeping and symbiotic gene sequences, the three strains formed a distinct lineage within the genus Bradyrhizobium, which was consistent with the results of DNA-DNA hybridization. In analyses of cellular fatty acids and phenotypic features, some differences were found between the novel group and related species of the genus Bradyrhizobium, indicating that these three strains constituted a novel group distinct from any recognized species of the genus Bradyrhizobium. Based on the data obtained in this study, we conclude that our strains represent a novel species of the genus Bradyrhizobium, for which the name Bradyrhizobium ganzhouense sp. nov. is proposed, with RITF806(T) ( = CCBAU 101088(T) = JCM 19881(T)) as the type strain. The DNA G+C content of strain RITF806(T) is 64.6 mol% (T(m)).

Bradyrhizobium daqingense sp. nov., isolated from soybean nodules.

Thirteen slow-growing rhizobial strains isolated from root nodules of soybean (Glycine max L.) grown in Daqing city in China were classified in the genus Bradyrhizobium based on 16S rRNA gene sequence analysis. Multilocus sequence analysis of IGS, atpD, glnII and recA genes revealed that the isolates represented a novel clade in this genus. DNA-DNA relatedness lower than 42.5 % between the representative strain CCBAU 15774(T) and the type strains of the closely related species Bradyrhizobium liaoningense USDA 3622(T), Bradyrhizobium yuanmingense CCBAU 10071(T) and Bradyrhizobium betae LMG 21987(T), further confirmed that this group represented a novel species. CCBAU 15774(T) shared seven cellular fatty acids with the three above-mentioned species, but the fatty acids 15 : 0 iso and summed feature 5 (18 : 2ω6,9c and/or 18 : 0 anteiso) were unique for this strain. The respiratory quinone in CCBAU 15774(T) was ubiquinone-10 and the cellular polar lipids were phosphatidylethanolamine, phosphatidylglycerol, phosphatidylcholine, cardiolipin and unknown aminolipid, polar lipid and phospholipid. In addition, some phenotypic features could be used to differentiate the novel group from the related species. On basis of these results, we propose the name Bradyrhizobium daqingense sp. nov., with CCBAU 15774(T) ( = LMG 26137(T) = HAMBI 3184(T) = CGMCC 1.10947(T)) as the type strain. The DNA G+C content of the type strain is 61.2 mol% (T(m)).

Mesorhizobium qingshengii sp. nov., isolated from effective nodules of Astragalus sinicus.

In a study on the diversity of rhizobia isolated from root nodules of Astragalus sinicus, five strains showed identical 16S rRNA gene sequences. They were related most closely to the type strains of Mesorhizobium loti, Mesorhizobium shangrilense, Mesorhizobium ciceri and Mesorhizobium australicum, with sequence similarities of 99.6-99.8%. A polyphasic approach, including 16S-23S intergenic spacer (IGS) RFLP, comparative sequence analysis of 16S rRNA, atpD, glnII and recA genes, DNA-DNA hybridization and phenotypic tests, clustered the five isolates into a coherent group distinct from all recognized Mesorhizobium species. Except for strain CCBAU 33446, from which no symbiotic gene was detected, the four remaining strains shared identical nifH and nodC gene sequences and nodulated with Astragalus sinicus. In addition, these five strains showed similar but different fingerprints in IGS-RFLP and BOX-repeat-based PCR, indicating that they were not clones of the same strain. They were also distinguished from recognized Mesorhizobium species by several phenotypic features and fatty acid profiles. Based upon all the results, we suggest that the five strains represent a novel species for which the name Mesorhizobium qingshengii sp. nov. is proposed. The type strain is CCBAU 33460(T) (=CGMCC 1.12097(T)=LMG 26793(T)=HAMBI 3277(T)). The DNA G+C content of the type strain is 59.52 mol% (Tm).

Bacteroid Development, Transcriptome, and Symbiotic Nitrogen-Fixing Comparison of Bradyrhizobium arachidis in Nodules of Peanut (Arachis hypogaea) and Medicinal Legume Sophora flavescens.

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 N2 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-ß-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 N2-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.

Scrutiny of NolA and NodD1 Regulatory Roles in Symbiotic Compatibility Unveils New Insights into Bradyrhizobium guangxiense CCBAU53363 Interacting with Peanut (Arachis hypogaea) and Mung Bean (Vigna radiata).

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 53ΔnodD1nolA 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 53ΔnodD1nolA on peanut, which was supported by a more severe symbiotic defect induced by 53ΔnodC than that with the 53ΔnodD1nopP, 53ΔnodD1rhcJ, and 53ΔnodD1ttsI 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.

Bacillus endoradicis sp. nov., an endophytic bacterium isolated from soybean root.

A gram-positive, aerobic, motile rod, designated strain CCBAU 05776(T), was isolated from the inner tissues of a healthy soybean (Glycine max L.) root collected from an agricultural field in the countryside of Shijiazhuang city, Hebei Province, China. Phylogenetic analysis of the 16S rRNA gene indicated that this strain was most closely related to Bacillus muralis LMG 20238(T) and Bacillus simplex NBRC 15720(T) with similarity of 96.5 % and 96.3 %, respectively, lower than the suggested threshold (97.0 %) for separating bacterial species. In phenotypic characterization, the novel strain differed from the two most related species in that it did not hydrolyse casein or starch but could grow on MacConkey agar. It grew between 15 and 45 °C and tolerated up to 7 % NaCl (w/v). Strain CCBAU 05776(T) grew in media with pH 5.5 to 10 (optimal growth at pH 7.0-8.0). The predominant cellular fatty acids were iso-C(15 : 0) (40.81 %) and C(16 : 1)ω7c alcohol (10.61 %). The predominant isoprenoid quinone was menaquinone 7 (MK-7). The cell-wall peptidoglycan contained meso-diaminopimelic acid. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol. The DNA G+C was 40.8 mol% (T(m)). DNA-DNA relatedness of the novel isolate with B. muralis and B. simplex was 42.4 % and 32.7 %, respectively. Based upon the consensus of phylogenetic and phenotypic analyses, strain CCBAU 05776(T) represents a novel species within the genus Bacillus, for which the name Bacillus endoradicis sp. nov. is proposed. The type strain is CCBAU 05776(T) ( = LMG 25492(T)  = HAMBI 3097(T)).

Bradyrhizobium huanghuaihaiense sp. nov., an effective symbiotic bacterium isolated from soybean (Glycine max L.) nodules.

In a survey of the biodiversity and biogeography of rhizobia associated with soybean (Glycine max L.) in different sites of the Northern (Huang-Huai-Hai) Plain of China, ten strains were defined as representing a novel genomic species in the genus of Bradyrhizobium. They were distinguished from defined species in restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene and the 16S-23S rRNA gene intergenic spacer (IGS). In BOX-PCR, these strains presented two patterns that shared 94% similarity, demonstrating that they were a homogenous group with limited diversity. In phylogenetic analyses of the 16S rRNA gene, IGS and housekeeping gene sequences, four representative strains formed a distant lineage within the genus Bradyrhizobium, which was consistent with the results of DNA-DNA hybridization. The strains of this novel group formed effective nodules with G. max, Glycine soja and Vigna unguiculata in cross-nodulation tests and harboured symbiotic genes (nodC and nifH) identical to those of reference strains of Bradyrhizobium japonicum, Bradyrhizobium liaoningense and 'Bradyrhizobium daqingense' originating from soybean, implying that the novel group may have obtained these symbiotic genes by lateral gene transfer. In analyses of cellular fatty acids and phenotypic features, some differences were found between the novel group and related Bradyrhizobium species, demonstrating that the novel group is distinct phenotypically from other Bradyrhizobium species. Based upon the data obtained, these strains are proposed to represent a novel species, Bradyrhizobium huanghuaihaiense sp. nov., with CCBAU 23303(T) ( = LMG 26136(T)  = CGMCC 1.10948(T)  = HAMBI 3180(T)) as the type strain. The DNA G+C content of strain CCBAU 23303(T) is 61.5 mol% (T(m)).