| In addition to rhizobium,it is a common objective phenomenon that there are nonrhizobium in legume nodules.Compared with endophytes in other plant tissues,there is a lack of systematic research on non-rhizobium in root nodules,and the significance of their existence and their potential ecological functions are not clear,especially the interaction mechanism between them and rhizobium needs to be revealed.In view of this,this study took the root nodules of Sophora davidii(Franch.)Skeels,a native wild legume nitrogenfixing shrub in six different counties of northern Shaanxi,as the research object.Traditional culture methods and high-throughput sequencing techniques were used to conduct a comprehensive and systematic study on the genetic diversity of microorganisms in root nodules of Sophora davidii(Franch.)Skeels,to clarify their phylogenetic status,and to explore the community composition,distribution and species abundance of endophytes in root nodules.Then,the horizontal gene transfer between non-rhizobium and rhizobium was discussed by means of multi-site sequence analysis.The nodulation ability of the strains was verified by plant re-inoculation experiment,and the growth promoting characteristics of the isolated strains were studied by pot experiment of wheat.The results of this study not only have important scientific significance for further exploring the role of such microbial resources in the natural ecosystem and enriching the pool of endophytic resources,but also provide a theoretical basis for their application in ecological restoration in arid areas of northern Shaanxi.The main results are as follows:1.The results of high-throughput sequencing of Sophora davidii root nodule and its rhizosphere soil 16 S r DNA V3+V4 region were analyzed.The results showed that among the root nodule samples of 6 different counties in northern Shaanxi,the most dominant bacteria were Mesorhizobium(60.15%～83.74%),and the dominant non-rhizobium was Denitratisoma(4.95%)in Baota County nodule(BTN).Lactobacillus in Fuxian nodule(FXN)and Zichang County nodule(ZCN)(4.61% and 4.48%).At the same time,in the rhizosphere soil samples,the dominant genera in Huangling county rhizosphere soil(HLS)and Yanchang county rhizosphere soil(YCS)were Sphingomonas(3.04% and 3.15%),Fuxian rhizosphere soil(FXS)was Mesorhizobium(4.85%),Baota rhizosphere soil(BTS)and Zichang county rhizosphere soil(ZCS)were RB41(3.84% and 4.74%).The rhizosphere soil(ZDS)in Zhidan County is Arthrobacter(4.73%),which indicates that there are differences in endophytic bacteria and rhizosphere soil bacterial communities in different habitats.Based on the correlation analysis of physicochemical properties of rhizosphere soil and endophytic bacteria in different sampling sites,it was found that available phosphorus was the main factor affecting the diversity and composition of bacterial community in the rhizosphere of Sophora davidii.This showed that there was a correlation between the distribution of endophytic bacteria and the physicochemical properties of the soil(p < 0.05).2.High-throughput sequencing of ITS1-1F region in root nodules and rhizosphere soil of Sophora davidii.The results showed that in root nodule samples,the dominant bacteria of HLN,YCN,ZDN and ZCN were Dactylonectria(15.77%～24.29%),while those of FXN were Titaea(22.97%),Cyphellophora(14.59%)and Ceratobasidium(13.78%).The dominant genus in BTN is Fusarium(6.65%).In the rhizosphere soil samples,the dominant genus was Fusarium(11.09%～28.94%),and the other dominant genus in FXS was Tuber(15.08%).In the control soil,the dominant genera of HLCK and FXCK were Cladosporium,accounting for 17.21% and 24.99%,respectively.The dominant genus of BTCK is Fusarium(26.87%.)The dominant genera of ZDCK are undetermined genera(15.15%)in Verrucariaceae family and Ceratobasidium(10.57%).This shows that there are some differences in the composition of endophytic fungal communities in root nodules of Sophora davidii in different habitats,and there are also differences in fungal communities in different niches of Sophora davidii growing in the same habitat.Based on the correlation analysis of physicochemical properties of rhizosphere soil and endophytic fungi in different sampling sites,it was found that available phosphorus and nitrate nitrogen were the main factors affecting the diversity and composition of rhizosphere fungi.3.The nif H gene in the root nodules of Sophora davidii was sequenced and the results showed that the dominant nitrogen-fixing bacteria in the nodule samples was Mesorhizobium.The analysis of α-diversity and β-diversity showed that there were some differences among endophytic nitrogen-fixing bacteria communities in root nodules of Sophora davidii.Based on the correlation analysis of environmental factors between the physicochemical properties of rhizosphere soil and endophytic nitrogen-fixing bacteria in different sampling sites,it was found that available potassium was the main factor affecting the distribution of endophytic nitrogen-fixing bacteria in root nodules at different sampling sites.4.320 strains of endophytic bacteria were screened from root nodules of Sophora davidii in 6 different counties of northern Shaanxi by preparing root nodule extract in buffer containing antioxidants and cultured in different culture medium.belonging to 4 phylums,7 classes,17 orders,35 families and 55 genera.16 S r DNA phylogenetic analysis showed that the dominant bacteria in Sophora davidii root nodules included Pseudomonas(18.44%),Bacillus(17.81%)and Mesorhizobium(11.56%).Multi-site sequence analysis of housekeeping genes(atp D,dna K and rec A)and symbiotic genes(nod A and nif H)was carried out to detect horizontal gene transfer between non-symbiotic endophytes and rhizobia.The results showed that the phylogeny of housekeeping genes of some strains was consistent with that of 16 S r DNA sequences,but there were some differences.Phylogenetic analysis of symbiotic genes(nod A and nif H)showed that both nod A and nif H genes of the tested strains were located in Mesorhizobium.The re-inoculation experiment showed that the Mesorhizobium could symbiosis with the host plant Sophora davidii to form root nodules.Although HL-2 did not amplify the related nodulation genes,it had obvious nodulation phenomenon.5.The growth-promoting characteristics of 192 strains of Sophora davidii root nodule endophytic bacteria in Baota area were studied.The results showed that 115 strains of bacteria had the ability of nitrogen fixation,20 strains had good ability of dissolving phosphorus,78 strains had the ability of secreting IAA,and 18 strains could secrete iron carrier.Four strains of endophytic baacteria with good growth promoting effect were selected,and the agronomic characters of wheat planted in pots were measured by single bacteria inoculation and multi-bacteria mixed inoculation.the results showed that the plant height and fresh weight of wheat in treatment group HIJ increased by 49.65% and 140%respectively,the root length of wheat in treatment group HK increased by 45.84%,and the chlorophyll content of wheat seedlings increased by 25.48% in treatment group IK.Therefore,these three groups of compound bacterial agents can be selected as materials for further research,which lays a good foundation for the development of microbial fertilizer. |
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