Chang Fu Tian

Comparative genomics of rhizobia nodulating soybean suggests extensive recruitment of lineage-specific genes in adaptations

The rhizobium–legume symbiosis has been widely studied as the model of mutualistic evolution and the essential component of sustainable agriculture. Extensive genetic and recent genomic studies have led to the hypothesis that many distinct strategies, regardless of rhizobial phylogeny, contributed to the varied rhizobium–legume symbiosis. We sequenced 26 genomes of Sinorhizobium and Bradyrhizobium nodulating soybean to test this hypothesis. The Bradyrhizobium core genome is disproportionally enriched in lipid and secondary metabolism, whereas several gene clusters known to be involved in osmoprotection and adaptation to alkaline pH are specific to the Sinorhizobium core genome. These features are consistent with biogeographic patterns of these bacteria. Surprisingly, no genes are specifically shared by these soybean microsymbionts compared with other legume microsymbionts. On the other hand, phyletic patterns of 561 known symbiosis genes of rhizobia reflected the species phylogeny of these soybean microsymbionts and other rhizobia. Similar analyses with 887 known functional genes or the whole pan genome of rhizobia revealed that only the phyletic distribution of functional genes was consistent with the species tree of rhizobia. Further evolutionary genetics revealed that recombination dominated the evolution of core genome. Taken together, our results suggested that faithfully vertical genes were rare compared with those with history of recombination including lateral gene transfer, although rhizobial adaptations to symbiotic interactions and other environmental conditions extensively recruited lineage-specific shell genes under direct or indirect control through the speciation process.

Biodiversity and Biogeography of Rhizobia Associated with Soybean Plants Grown in the North China Plain

ABSTRACT As the putative center of origin for soybean and the second largest region of soybean production in China, the North China Plain covers temperate and subtropical regions with diverse soil characteristics. However, the soybean rhizobia in this plain have not been sufficiently studied. To investigate the biodiversity and biogeography of soybean rhizobia in this plain, a total of 309 isolates of symbiotic bacteria from the soybean nodules collected from 16 sampling sites were studied by molecular characterization. These isolates were classified into 10 genospecies belonging to the genera Sinorhizobium and Bradyrhizobium, including four novel groups, with S. fredii (68.28%) as the dominant group. The phylogeny of symbiotic genes nodC and nifH defined four lineages among the isolates associated with Sinorhizobium fredii, Bradyrhizobium elkanii, B. japonicum, and B. yuanmingense, demonstrating the different origins of symbiotic genes and their coevolution with the chromosome. The possible lateral transfer of symbiotic genes was detected in several cases. The association between soil factors (available N, P, and K and pH) and the distribution of genospecies suggest clear biogeographic patterns: Sinorhizobium spp. were superdominant in sampling sites with alkaline-saline soils, while Bradyrhizobium spp. were more abundant in neutral soils. This study clarified the biodiversity and biogeography of soybean rhizobia in the North China Plain.

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

Qinghai–Tibet plateau is the highest place in the world and the environment in that plateau is hard for animals and plants, with low temperature, low concentration of oxygen and high solar radiation. In this study, 61 root nodule isolates from Vicia, Oxytropis, Medicago, Melilotus and Onobrychis species grown in Qinghai–Tibet plateau and in loess plateau were comparatively characterized. Based upon the results of numerical taxonomy, ARDRA, AFLP, DNA–DNA hybridization and 16S rDNA sequencing, the isolates were classified as Rhizobium leguminosarum, Sinorhizobium meliloti, Sinorhizobium fredii, Mesorhizobium sp., Phyllobacterium sp., Stenotrophomonas sp. and two non-symbiotic groups related to Agrobacterium and Enterobacteriaceae. The strains isolated from Qinghai–Tibet plateau and from the loess plateau were mixed in these species or groups. Oxytropis spp. and Medicagoarchiducis-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

Rhizobium fabae sp. nov., a bacterium that nodulates Vicia faba

Six strains were isolated from root nodules of Vicia faba grown in Nanchang, Yifeng, Taihu, Huaibei, Bengbu and Lujiang, in the middle and lower reaches of the Yangtze River. According to phylogenetic analyses of 16S rRNA gene, atpD and recA sequences, these strains belong to the genus Rhizobium, with Rhizobium etli and Rhizobium leguminosarum as the closest related species. CCBAU 33202(T), a representative of these novel isolates, showed sequence similarity to its closest relatives R. etli CFN 42(T) and R. leguminosarum USDA 2370(T) of 99.5 and 99.1 % for the 16S rRNA gene, 91.9 and 91.9 % for atpD and 90.3 and 93.2 % for recA. The strains from this study could also be differentiated from R. etli CFN 42(T) and R. leguminosarum USDA 2370(T) by 16S-IGS RFLP and SDS-PAGE of whole-cell proteins, fatty acid profiles and several phenotypic characteristics. DNA-DNA hybridization yielded relatedness of 19 and 14-43 %, respectively, with R. etli CFN 42(T) and strains representing different biovars of R. leguminosarum. All data obtained in this study showed that these V. faba isolates belong to a novel species, for which the name Rhizobium fabae sp. nov. is proposed. The type strain, CCBAU 33202(T) (=LMG 23997(T) =JCM 14381(T)), was isolated from Nanchang.

Population mixing of Rhizobium leguminosarum bv. viciae nodulating Vicia faba: the role of recombination and lateral gene transfer.

The level and mechanisms of population mixing among faba bean (Vicia faba) rhizobia of different geographic origins (three ecoregions of China and several Western countries) were analysed by sequencing three chromosomal housekeeping loci (atpD, recA and glnII) and one nodulation gene (nodD). Eight distinct sublineages of Rhizobium leguminosarum bv. viciae (Rlv) were identified by concatenated sequences of chromosomal loci. structure analysis revealed admixture patterns of Rlv populations of different geographic origins. Recombination, particularly among these chromosomal loci, was revealed to be an important microevolutionary force in shaping the observed genetic diversity and the phylogeny of Rlv. The phylogeny of nodD is largely independent of that of the chromosomal loci, reflecting multiple gene transfers between sublineages and possibly selection imposed by different faba bean gene pools. The dominant nodulation genotype of faba bean rhizobia in the spring growing region of China is identical to the prevalent type of Europe, while the winter growing region of China has another related, but distinct, dominant nodulation genotype. Although several geographically specific sublineages of Rlv were observed, recombination and lateral gene transfer have driven the process of population mixing among different ecoregions of China or between China and countries to the west.

Rhizobium multihospitium sp. nov., isolated from multiple legume species native of Xinjiang, China

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.

Diversity and Biogeography of Rhizobia Isolated from Root Nodules of Glycine max Grown in Hebei Province, China

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

Genetic diversity of rhizobia associated with Vicia faba in three ecological regions of China

Great genetic diversity was revealed among 75 rhizobal isolates associated with Vicia faba grown in Chinese fields with AFLP, ARDRA, 16S rDNA sequencing, DNA–DNA hybridization, BOX-PCR and RFLP of PCR-amplified nodD and nodC. Most of the isolates were Rhizobium leguminosarum, and six isolates belonged to an unnamed Rhizobium species. In the homogeneity analysis, the isolates were grouped into three clusters corresponding to (1) autumn sowing (subtropical) region where the winter ecotype of V. faba was cultivated, (2) spring sowing (temperate) region where the spring ecotype was grown, and (3) Yunnan province where the intermediate ecotype was sown either in spring or in autumn. Nonrandom associations were found among the nod genotypes, genomic types and ecological regions, indicating an epidemic symbiotic gene transfer pattern among different genomic backgrounds within an ecological region and a relatively limited transfer pattern between different regions. Conclusively, the present results suggested an endemic population structure of V. faba rhizobia in Chinese fields and demonstrated a novel rhizobium associated with faba bean.

Bradyrhizobium elkanii, Bradyrhizobium yuanmingense and Bradyrhizobium japonicum are the main rhizobia associated with Vigna unguiculata and Vigna radiata in the subtropical region of China

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.

Genetic diversity of nodulating and non‐nodulating rhizobia associated with wild soybean (Glycine soja Sieb. & Zucc.) in different ecoregions of China

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.