Rhizobium Diversity And Nodulation And Nitrogen Fixation Mechanism In Trifolium Ambiguum

Trifolium ambiguum(Caucasian clover)is a perennial rooting and tillering forage legume with desirable characteristics,such as strong adaptability and high cold resistance.However,poor nodulation and nitrogen fixation capacities are some of the main factors limiting its popularization and cultivation.Therefore,the present study used Caucasian clover as the test material and analyzed bacterial diversity in the root nodules to identify bacterial strains that could facilitate nodulation and nitrogen fixation,in addition to exploring the associated mechanisms.The aim of the present study was to provide a nodulation and nitrogen fixation for Caucasian clover popularization and cultivation.In this study,we isolated and purified bacterial strains from the root nodules of Caucasian clover,and determined phenotypic diversity and genetic diversity of these strains.The phenotypic diversity of bacterial strains was studied from application of the sole carbon source,acid and alkaline resistance,antibiotic resistance,salt tolerance and physiological and biochemical reactions.The genetic diversity of the bacterial strains was identified by BOX-PCR and 16 S r RNA sequencing.The identified rhizobia were inoculated into Caucasian clover and the nutritional,rhizosphere and physiological indices of Caucasian clover were measured.Using transcriptomics and metabolomics,we measured the changes of gene level and metabolite level of Caucasian clover root system after inoculation with rhizobia.We sampled and sequenced Caucasian clover root systems at 3 d of rhizobia inoculation,6 d of rhizobia inoculation,and 9 d of rhizobia inoculation,and set up 5comparison groups,G＿3 vs CK＿G＿3,G＿6 vs CK＿G＿6,G＿9 vs CK＿G＿9,CK＿G＿6 vs CK＿G＿3 and CK＿G＿9 vs CK＿G＿6,and the differentially expressed genes and differential metabolites in the comparative groups were analyzed.The main results obtained are as follows.(1)Fifteen endophytic bacterial strains were isolated from Caucasian clover root nodules.According to the phenotypic diversity analysis results,bacterial strains identified had high carbon source utilization capacity,with the exception of gentamicin-resistant bacteria,which exhibited good tolerance to antibiotics at low concentrations,with strong alkaline tolerance,weak acid tolerance,and salt tolerance.The 15 bacterial strains and one reference strain could be divided into six groups at 87% similarity level.Genetic diversity analysis of the 15 isolated bacterial strains and one reference strain revealed that six strains(G-1,QG-1,QG-2,QG-3,QG-5,and QG-9)were Rhizobium,three strains(G-7,G-4039,and G-50)were Bacillus,and six strains(G-2,G-4,G-6,G-8,G-48,and G-49)were Pseudomonas.(2)The inoculation of Caucasian clover with the obtained rhizobia has different growth promoting effects.Based on a comprehensive analysis of membership function,the order of Rhizobium inoculation effects was QG-5 > QG-1 > QG-9 > G-1 > QG-2 > QG-3 > G-47.QG-5 strain had a significant effect on nodule area,markedly increased plant root index.QG-1 strain markedly increased plant aboveground and belowground biomass,and significantly increased root index.QG-3 strain had a significant effect on plant height,significantly increased leaf chlorophyll content.Inoculation of rhizobia had minor effects on plant nutritional indexes of plants.(3)A total of 97,430 genes were identified in five comparative combinations based on analysis of Caucasian clover inoculated with rhizobia,using transcriptomics technologies,and 96,410 annotated genes were obtained from various databases.The results of differential gene expression and functional analysis indicated that genes leading to nodule nitrogen fixation in Caucasian clover were mainly concentrated in phenylpropanoid biosynthesis,flavonoid biosynthesis and nitrogen metabolism pathways.(4)The expression of chalcone synthase(CHS),protein kinase R(PKR),and cytochrome P450(CYP)75B subfamily(CYP75B)enzymes in the flavonoid biosynthetic pathway changed following inoculation with Rhizobium for three days,and the expression of nitrite transporter(Nrt)and carbonic anhydrase(CA)enzymes changed during nitrogen metabolism.The expression of CHS,caffeoyl coenzyme A 3-O-methyltransferase(CCo AOMT),PKR,and CYP73 A enzymes associated with the flavonoid biosynthetic pathway changed following inoculation with Rhizobium for six days,and the expression of nitrate reductase(NR)and CA genes changed during nitrogen metabolism.The gene expression levels of hydroxycinnamoyltransferase(HCT),peptidoglycan glycosyltransferase(PGT),chalcone isomerase(CHI),flavanone-3-hydroxylase(F3H),flavonol synthase(FLS),leucoanthocyanidin reductase(LAR),and CCo AOMT enzymes associated with the flavonoid biosynthetic pathway changed significantly following inoculation with Rhizobium for nine days.The gene expression levels of Nrt,NR,and nitrite reductase enzymes changed significantly during nitrogen metabolism.(5)A total of 828 metabolites were identified in five comparative combinations using metabonomics technologies.The content of phenylpropanoids and flavonoids was highly variable.The results of differential expression and functional analysis of the metabolites showed that the main metabolites leading to nodule formation and nitrogen fixation were the synthesis of secondary metabolites and the biosynthesis of flavonoids.(6)After inoculation with Rhizobium for three days,amino acid contents(tryptophan,glutamine,and histidine)increased significantly,whereas phenylpropanoid contents(cinnamaldehyde,sinapic acid,sinapyl alcohol,and coniferol)decreased significantly.In addition,flavonoid contents(Eriodictyol,poncirin,diosmetin,and methylkaempferol)decreased significantly.Tryptophan content increased significantly,whereas phenylpropanoid(coniferol and coumarin)and flavonoid(poncirin)contents decreased significantly following inoculation with Rhizobium for six days.Amino acid(aspartic acid and arginine),phenylpropanoid(coumarin),and flavonoid(methylkaempferol,isorhamnetin,kaempferol,quercetin,and myricetin)contents increased significantly following inoculation with Rhizobium for nine days.(7)According to results of transcriptomics and metabolomics analyses,the regulation of NRT and CA changed following inoculation with Rhizobium for three days,resulting in increases in glutamine content,expression of phenylalanine ammonia lyase,CHS,and CYP75 B,decreases in coniferaldehyde,sinapic acid,sinapyl alcohol,and eriodictyol contents,and an increase in phlorizin content.The regulation of bgl X,CYP73 A,and caffeic acid 3-O-methyltransferase(COMT)changed following inoculation with Rhizobium for six days,resulting in decreases in coumarin and coniferaldehyde contents.The regulation of cynamyl alcohol dehydrogenase,CHI,F3 H,COMT,ferulate-5-hydroxylase,and FLS changed following inoculation with Rhizobium for nine days,resulting in increases in coumarin,kaempferol,phlorizin,quercetin,and myricetin contents,and a decrease in sinapyl alcohol content.