| Research on genetic diversity and symbiotic specificity of rhizobia is helping to enable the classification,identification and accurate utilization of rhizobia resources.Rhizobial strains from identical species have different physiological and biochemical characteristics and may exhibit varying symbiotic ability with different alfalfa cultivars.Therefore,rhizobia can be distinguished into different biotypes according to their phenotypic differences and their symbiotic efficiency on alfalfa cultivars.In this study,alfalfa cultivars Medicago sativa cvs.Gannong No.3,Gannong No.9,Longzhong,Qingshui and WL168 HQ,planted in forage test stations of Gansu Agricultural University in Huishi Town,Huining County,Baiyin City,Huangyang Town,Liangzhou District,Wuwei City and Anning District,Lanzhou City,were chosen as materials.Rhizobial strains from root nodule,epidermis,stele,stem,leaf,flower,seed,rhizosphere soil,and field soil were isolated.They were used to conduct physiological and biochemical characteristics assay,restriction fragment length polymorphism(RFLP)analysis,16 S r RNA gene sequencing,multilocus sequence typing(MLST),nod and nif gene sequencing and symbiotic efficiency analysis.By studying the phenotypic and genetic diversity as well as the symbiotic specificity,rhizobia were classified into different biotypes,and the symbiotic specificity system with a highly efficient matching of rhizobia and alfalfa cultivars was analyzed by transcriptomics.The purpose of this study was to explore the underlying molecular mechanism of specific symbiosis between rhizobial biotypes and alfalfa cultivars and to provide a theoretical basis for enriching the resources of specific rhizobia,strengthening the efficient symbiotic breeding between alfalfa and rhizobia,and cultivating high-quality symbiotic alfalfa cultivars.Results showed that:(1)Of the 53 rhizobia strains isolated,43 were endophytic rhizobia,accounting for 81.1% of the total strains,and 10 were non-endophytic rhizobia,accounting for 18.9% of the total strains.They showed abundant phenotypic diversity,and endophytic rhizobia were more diverse than non-endophytic ones.They had a wide utilization spectrum of carbon and nitrogen sources,strong resistance to antibiotics and dyes,and were resistant to low concentrations of Na Cl,weak acid,weak base,and high temperature.The Ensifer and Rhizobium strains grouped into six and eight clusters at 77% coefficient,and 25 and 20 clusters at 100% coefficient,respectively.Phenotypic diversity of Ensifer and Rhizobium strains from different cultivation areas,alfalfa cultivars,and tissue parts differed.Those isolated from Anning District,Lanzhou City(with Shannon-Weiner diversity indexes reaching 1.099 and 1.733),WL168HQ(1.099 and 1.733)and nodule(0.757 and 1.332)were the most diverse,respectively.(2)The 53 rhizobial strains were genetically abundant and contained 22 kinds of 16 S r RNA-RFLP types.The genetic diversity of rhizobia from different cultivation areas,alfalfa cultivars,and tissue parts varied obviously.Strains isolated from Liangzhou District,Wuwei City(with a diversity index reaching 1.864),Gannong No.3(1.268)and rhizosphere soil(1.386)gave the highest diversity index,respectively.Based on 16 S r RNA and MLST analysis,53 rhizobial strains were phylogenetically related to Ensifer meliloti LMG 6133 T,Rhizobium radiobacter ATCC 19358 T and Rhizobium rosettiformans W3 T.The sequence similarities between rhizobial strains and the type strains were more than 97%,and strains were identified as E.meliloti(32),R.radiobacter(20)and R.rosettiformans(1).The nod C and nif H genes could not be amplified from Rhizobium strains,whereas those of E.meliloti strains originated from the same ancestor,with sequence similarities ranging from 97% to 100%;the diversity of nif H gene was higher than that of nod C gene.There was no direct relationship between phenotypic and genetic diversity.(3)The symbiotic efficiency between rhizobial strains and different alfalfa cultivars differed.In terms of individual inoculation efficiency,strain QL2 and Gannong No.3,strain G3L3 and Gannong No.9,strain LP3 and Qingshui exhibited strong symbiotic matching and compatibility.The inoculation efficiencies of different strains on Longzhong and WL168 HQ varied.For the overall symbiotic efficiencies,after inoculation of 32 E.meliloti strains,the dispersion degree of symbiotic parameters of Gannong No.3,Gannong No.9 and Qingshui was lower than that of Longzhong and WL168 HQ.This suggested that the symbiotic efficiency of E.meliloti strains on the former three alfalfa cultivars was similar but on the latter two was discrepant.Strains' symbiotic efficiency differed primarily according to alfalfa cultivars and,to a lesser extent,to the tested strains,indicating the difference in mutual recognition ability and sensitivity between different alfalfa cultivars and rhizobial strains.(4)The shoot dry weight,which contributed the most to the symbiotic efficiency in the principal component analysis(PCA),represented the symbiotic efficiency of rhizobia and alfalfa cultivars.The symbiotic efficiency was then marked effective(E),inhibitive(I)or noneffective(O)when the shoot dry weight of plants inoculated with rhizobia was significantly higher than(P<0.05),lower than(P<0.05)or not significantly different from(P>0.05)that of the uninoculated controls,respectively.Twelve symbiotic combinations were obtained by combining the marked symbiotic efficiency of each rhizobium together with the alfalfa cultivar order of M.sativa cvs.Gannong No.3,Gannong No.9,Longzhong,Qingshui and WL168 HQ.And the 32 E.meliloti strains were divided into six symbiotic patterns based on the number of E,O,and I components in the symbiotic combinations.Strains subjected to symbiotic combinations EOOOO,OEOOO,OOEOO and OOOEO displayed strong effective symbiotic-specificity with Gannong No.3,Gannong No.9,Longzhong,and Qingshui,respectively.The symbiotic combinations EEOOO(Gannong No.3 and Gannong No.9)and OEOEO(Gannong No.9 and Qingshui)represented strains which showed effective symbiosis with two alfalfa cultivars.Strains of OIOEO presented not only effective symbiosis with Qingshui but also inhibitive symbiosis with Gannong No.9.Those belonging to OOOOI,OIOOO and OOOIO displayed strong inhibitive symbiotic-specificity with WL168 HQ,Gannong No.9 and Qingshui,respectively.The symbiosis between strains of OOOII and two alfalfa cultivars,Qingshui and WL168 HQ,was also inhibitive.No symbiotic efficiency was observed between the strains of OOOOO and the five alfalfa cultivars.(5)Thirty-two E.meliloti strains were classified into 28 biotypes according to 25 kinds of phenotypes and six kinds of symbiotic patterns.Effective specificity was observed for Gannong No.3(biotypes ?(QL2)and ?(WLG1)),Gannong No.9(biotype ???(LL11)),Qingshui(biotype ?(LL1)),and Longzhong(biotypes ?(LP3)and ?(G3L3)),whereas inhibitive specificity was observed for Gannong No.9(biotypes ?(G3L4),?(G3L7),??(G3L10 and G3L13),???(G3L12),?(G9L5)and ??(LL10)),Qingshui(biotype ?(G3L9)),and WL168HQ(biotypes ??(LL8),??(QL5),???(QL4)and ???(G3L5)).Effective non-specificity was observed for Gannong No.9 and Qingshui(biotypes ?(WLP2))as well as Gannong No.3 and Gannong No.9(biotype ???(LL2)),while inhibitive non-specificity was observed for Qingshui and WL168HQ(biotype ??(G9L7)).Effective and inhibitive specificity for biotype ??(G9L3 and G9L8)was observed for Qingshui and Gannong No.9,respectively.Biotypes ?(G3L2),?(G3T2),??(G9L6,LL5,LL6),??(LL7),??(G3L6),??(G3L8)and ???(G9L4)showed symbiotic efficiency with none of the five alfalfa cultivars.No effective symbiosis was observed on WL168 HQ after inoculation of the 32 strains,indicating that introduced alfalfa cultivars could not match well with local rhizobium resources.(6)To investigate the molecular basis of symbiotic-specificity biotype classifications based on phenotype and symbiotic efficiency,four specific biotype strains(?,?,?,and ?)and two non-specific biotype strains(?,and ???)as well as alfalfa cultivars Gannong No.3,Gannong No.9,Longzhong and Qingshui were selected for root transcriptome analysis.The discrepancy of inoculation efficiencies of different biotype strains on the same alfalfa cultivar is the main basis for rhizobial biotype classification,and different alfalfa cultivars have diverse molecular mechanisms.G9-LL2 vs G9-WLP2(Gannong No.9 inoculated with two non-specific biotype strains ???(LL2)and ?(WLP2),the same below)shared no differentially expressed genes(DEGs)with G3-LL2 vs G3-QL2(non-specific vs specific),Q-LL1 vs Q-WLP2(specific vs non-specific),and L-G3L3 vs L-LP3(specific vs specific),and none of the GO(Gene ontology)terms were significantly enriched by the DEGs in G9-LL2 vs G9-WLP2 as well.This indicated that biotype classification mechanism based on Gannong No.9 may dramatically differ from that on other three alfalfa cultivars.G3-LL2 vs G3-QL2,Q-LL1 vs Q-WLP2,and L-G3L3 vs L-LP3 shared 68 DEGs,which were involved in 23 KEGG(Kyoto encyclopedia of genes and genomes)and five unknown pathways.The stronger the specificity,the higher the number and expression amount of gene as well as metabolic pathways involved at the transcriptional level.Weighted gene coexpression network analysis(WGCNA)determined three modules that were predominantly associated with both shoot dry weight and root dry weight,which confirmed the reliability of symbiotic efficiency measured according to shoot dry weight.Of the 68 shared DEGs,45 were overlapped with the representative genes involved in WGCNA modules,indicating that these genes were putatively associated with biotype classification.They were involved in plant secondary metabolite synthesis,carbohydrate and amino acid metabolism,and hormone signal transduction.They mainly encode signal factors of nod gene inducers such as flavonoids from alfalfa to rhizobia,and nod factors synthesized by rhizobia,and they play important roles in symbiotic specificity between rhizobial biotypes and alfalfa cultivars,plant innate immunity,and rhizobial suppression of plant immune defence.This provides a molecular basis for the classification of symbiotic-specificity rhizobial biotypes on M.sativa cvs.Gannong No.3,Longzhong and Qingshui. |
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