Diversity And Evolution History Of Rhizobia Nodulated With Coastal Salt-tolerant Plant Aeschynomene Indica

Coastal saline-alkali land has a high potential for agricultural development,but only a few salt-tolerant plants can grow due to the limitations of water and salt conditions.The symbiosis systems between coastal salt-tolerant legume and rhizobia was applied to improve saline-alkali soil and increase the soil fertility.In this thesis,we studied the diversity of legume Aeschynomene indica rhizobia in coastal saline-alkali areas,and analyzed its evolutionary history by means of comparative genome analysis,which is of great significance in expanding the A.indica rhizobia resources of rhizobia,deepening the understanding of the genetic development history of rhizobia,and improving coastal saline-alkali areas.A.indica is a kind of semiaquatic legume that forms both stem and root nodules with rhizobia.To investigate the diversity and community structure of Aeschynomene indica rhizobia 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 gene sequences.With further classification in comparison with reference strains,10 Bradyrhizobium genospecies were defined based on the multilocus sequence analysis.In addition,6 genospecies were first reported.No nodulation gene(nodA,nodB,nodC or nodZ)was detected in the Aeschynomene indica rhizobia by PCR amplification.The phylogenetic trees of nitrogen fixation gene nifH and photosynthetic gene pufLM were highly consistent with the phylogenetic tree of housekeeping genes.All representative strains formed root nodules,but only 8 representative strains in 4 genospecies formed stem nodules on A.indica.The results of recombination events and mutant events analysis of the housekeeping genes,as well as nifH and pufLM genes,showed that mutation contributes more than recombination in evolution history.The Neutral test results showed that the A.indica rhizobia were in an expanding state,and there were still undiscovered A.indica rhizobium species.The symbiosis system and the A.indica rhizobia have a certain tolerance ability to saline-alkali.The A.indica rhizobial isolated from the sample with a high degree of salinization could tolerate a salt concentration of 3%-4%,and the symbiosis system could grow in soil with 0.9%salt content.Four strains of Bradyrhizobium sp.? with high diversity and abundance were identified by polyphasic classification.With B.oligotrophicum S58T and B denitrificans LMG8443T as the reference strains,the physiological and biochemical indexes of the tested strains were determined,and the whole genome ANI values were calculated,and the species tree was constructed.The optimal growth temperature of this species is 38?.The optimal salt concentration was 0%-0.5%.The optimal pH was 7.The bacteria were rods with a length of about 3-4 um.With urease and catalase activity,it is resistant to neomycin,trimethoprim,lincomycin,ofloxacin and erythromycin.The unique carbon source and fatty acid component results,genome-wide ANI value and species tree showed that this species belonged to the genus Bradyrhizobium,but were significant different from the type strains in this genus Bradyrhizobium.The name Bradyrhizobium asechynomene sp.nov.was proposed and strain 83002T was chosen as the type strain.The genome size of 83002T is 7.52 Mbp,and the DNA G+C content is 65.42%A.indica rhizobia has a special evolutionary history,forming three branches in the phylogenetic trees of 16S rRNA gene,housekeeping genes,nitrogen fixation gene and photosynthetic gene.In this study,a comparative analysis was conducted on the genomes of A.indica rhizobia and the reference genome of genus Bradyrizobium.It was found that A.indica rhizobia had three different origin ways.The first way was early differentiation of Bradyrhizobium strain.Strains of this branch has a long evolutionary history and high diversity.The second way was non-symbiotic strain in Bradyrhizobium differentiated into A.indica rhizobia.Strains of this branch species had low diversity but photosynthesis-related genes.The last way was modification of symbiotic strain in Bradyrhizobium,which has resulted in the deletion of many genomic fragments in evolutionary history,but lacked photosynthesis-related genes.The A.indica rhizobia formed by the latter two methods has low diversity and high consistency with the genomic components of the reference strains in genus Bradyrhizobium,which were recently differentiated A.indica rhizobia groups.The comparison and analysis of the genome of A.indica rhizobia and the reference strain genomes of genus Bradyrhizobia indicated that the genes Nod gene and type III secretory system effectors were generally absent in the genome of A.indica rhizobia,but the nitrogen fixation genes and photosynthetic genes were conservative.The specific genes in the genome of A.indica rhizobia were mostly related to pathogen infection,and these genes may be related to the Nod factor-independent nodulation pattern of A.indica rhizobia.