Comparative Genomics And Phylogeny Of Rhizobia Isolated From Phaseolus Vulgaris

Phaseolus vulgaris(common bean)is an important economic crop in the world,which could establish symbiotic nitrogen fixation relationships with rhizobia.Its microsymbionts belonging to three genera Rhizobium,Sinorhizobium,and Bradyrhizobium have been isolated in its introduced country-China.The related symbiotic gene in these bacteria is similar among strains within each genus,but different among distinct genera.Little information is available about the genetic differentiation,structure,and evolution of symbiotic plasmids among bean-nodulating Rhizobium species,or the emergence of symbiotic ability to associate with bean plants in Bradyrhizobium and Sinorhizobium strains.Furthermore,the biogeographic patterns have been revealed on Rhizobium microsymbionts,but genetic differences associated with biogeographic patterns in these microsymbionts remain poorly understood.In this study,we sequenced genomes of 29 representative bean microsymbionts(21 Rhizobium,four Sinorhizobium,and four Bradyrhizobium)and compared them with closely related reference strains to estimate the origins of symbiotic genes among bean microsymbionts.We performed phylogenetic analyses of 69 Rhizobium strains mainly isolated from root nodules of common bean and clover(Trifolium spp.)to investigate the diversity of bean rhizobia at the species level.We performed comparative genome sequence analyses on nodulation genes of these 29 representative bean microsymbionts with related rhizobia.We also compared the genomes assignments of strains isolated from either acidic or neutral/alkaline soils in different Clusters of Orthologous Genes categories to determine whether genetic differences were associated with biogeographic patterns.And a potential novel-species was characterized using polyphasic taxonomy approach.The results are as follows:1.Based on genome average nucleotide identity,digital DNA:DNA hybridization,and phylogenetic analysis of 1,458 single-copy core genes,we classfied 69 Rhizobium strains into 28 species(clusters),consistent with species definition based on multilocus sequence analysis of three concatenated housekeeping genes(atpD,glnII,and recA).Bean rhizobia were found in 16 defined species and nine potentially novel species,suggesting the 35strains previously described as R.etli,R.phaseoli,R.vallis,R.gallicum,R.leguminosarum and Rhizobium spp.should be renamed according to genome data.In addition,phylogenetic patterns of two symbiotic genes(nodC and nifH)were highly host-specific and inconsistent with genomic phylogeny.Multiple symbiovars(sv.)within the same species is a common feature in genus Rhizobium;R.anhuiense contained three symbiovars(sv.phaseoli,sv.trifolii and sv.viciae);sv.phaseoli and sv.mimosae,sv.phaseoli and sv.trifolii,sv.phaseoli and sv.viciae,sv.trifolii and sv.viciae were suggestive of R.sophoriradicis/R.etli/Rhizobium sp.III,R.hidalgonense/R.acidisoli,R.leguminosarum/Rhizobium sp.IX,R.laguerreae,respectively.Thus,genomic comparison revealed great diversity in bean rhizobia at the species level,and clarified the exact taxonomic position of some previously misnamed strains.2.The 19 native bean Rhizobium microsymbionts were assigned to four defined species and six putative novel species.Comparative genomics among Rhizobium strains demonstrated horizontal gene transfer exclusively at the symbiotic region,leading to expanded diversity of bean-nodulating Rhizobium strains.The symbiotic genes of R.phaseoli,R.sophorriradicis,and R.esperanzae strains originated from Mexican bean-nodulating strains possibly introduced alongside bean seeds.Among R.anhuiense and the six putative novel species identified only in China,horizontal transfer of symbiotic genes suggested symbiosis with other indigenous legumes and loss of originally symbiotic regions or non-symbionts before the introduction of common bean.Genome data for Sinorhizobium and Bradyrhizobium strains indicated symbiotic compatibility between microsymbionts of common bean and other hosts such as soybean.3.Strains from acidic soils(group I)had an average of 6,222 proteins per genome assigned to 20 COG functional categories,with 3,997 core genes(4.24 Mb)shared by all seven genomes.Strains from neutral/alkaline soils(group II)had an average of 6,015proteins per genome,with 3,955(4.17 Mb)core genes shared by all seven genomes.The genes specific to strains isolated from either acidic or neutral/alkaline soils were enriched in different Clusters of Orthologous Genes categories.Across both full genomes,group I contained significantly more genes associated with cell motility than did group II.Significant differences between the two groups were identified in 10 of the 20 functional categories associated with the core genomes.The core genes specific to group I included a T4SS,whereas those specific to group II included a T3SS.4.J15~T was isolated from root nodules of Phaseolus vulgaris L.in Chutan,Jiangxi Province,China.Based on the the phylogenetic analysis of 16S rRNA gene,concatenated housekeeping genes,and core genes with closest neighbors,genomic,chemotaxonomic,and phenotypic characteristics,we propose J15~T as a novel member in Rhizobium and named as Rhizobium phaseona.The strain J15~T has the advantages of a wide pH growth range(pH5-11)and a large number of available carbon sources.This study revealed the homology and acquisition of related symbiotic genes of common bean microsymbionts in three genera at the genomic level.Our results provide insights into symbiotic strategies during rhizobial evolution with the introduction of new host legume.And these insights could be helpful in guidance for the selection and application of rhizobia inoculants in different bean ecological production areas.