| Rhizobia are symbiotic nitrogen fixation microorganisms participating in nitrogen cycling and agricultural production,Rhizobial inoculation can promote the growth and development of leguminous crops,increase crop yield,reduce fertilizer application,reduce agricultural production costs and promote sustainable agricultural development.Based on the biogeographical distribution of soybean rhizobia at various experimental sites(Wudalianchi,Tongliao,Feicheng,Linyi,Jining and Sanya)in China soybean growing ecological zone,the efficient rhizobia were screened from these sites,and the fermentation conditions of the efficient strains were optimized and the shelf life of rhizobia was explored.According to the relationship between soil physico-chemical properties and the distribution of rhizobia structure community,effective strains were screened,the compound rhizobia formula was developed and the rhizobia inoculation was conducted in the above experiment sites.Total 483 rhizobial strains(belonging to 44 BOX genotypes)obtained from greenhouse experiments were classified into six known species,including Sinorhizobium fredii,Bradyrhizobium elkanii,B.japonicum,B.huanghuaihaiense,B.yuanmingense and B.diazoefficiens,and two unknown Bradyrhizobium sp.I and Bradyrhizobium sp.? using rpoB gene comparison.Another total 921 rhizobia strains(belonging to 75 BOX genotypes)obtained from field experiments were identified as 7 populations(S.fredii,B.elkanii,Sinorhizobium sp.,B.japonicum,B.huanghuaihaiense,B.daqingense and B.diazoefficiens)using rpoB gene comparison.The dominant rhizobia in these experiment sites were B.elkanii or S.fredii.A species isolated from Jining's experimental site was identified as a novel species in the Sinorhizobium using polyphasic taxonomy and whole genomic comparison.It was named as Sinorhizobium shofinae sp.nov.with the type strain of CCBAU 251167T.The effects of soil physical and chemical properties,soybean varieties,geographical factors and climatic factors on the structure distribution of soybean rhizobial community were explored.Results showed that B.elkanii distributed in acid soils while S.fredii existed in alkali soils.Available iron was detected as the first determinant for distribution of the two rhizobial species,and the soybean varieties(Xudou 18,Heihe 43 and Nansheng 270)did not greatly affect the rhizobial compatibility.Geographic latitude and mean rain precipitation in June were the main geographic and climate factors affecting the rhizobial distribution.Based on the Bray-Curtis distance analysis,the distribution of soil bacteria(16 rDNA level)in soybean rhizosphere soil had a significant positive correlation with soil organic matter content,available iron content,available boron content and water-soluble calcium content,electrolyte,pH,geographic latitude and annual effective rainfall.However,the geographical longitude,geographical height,soybean cultivars and soybean growth period(sowing,flowering and maturity)with bacterial community structure did not reach to significant level.Based on the UniFrac distance analysis,it was found that there was a significant positive correlation between soil bacterial(OTU level)community structure and geographical factors and soil physico-chemical properties,but was not closely related to the soybean varieties and growth stages of soybean.The response surface methodology was used to optimize the fermentation conditions of B.elkanii L18-31 and S.fredii J18-3.The maximum viable count of B.elkanii L18-31 and S.fredii J18-3 reached 8.5×109 CFU/mL and 5.1×109 CFU/mL,respectively.The effective viable count of rhizobia supplied with 20%trehalose reached to the highest after being stored for 90 days at 20?25?,which were 3.4×109 CFU/mL and 6.0 ×108CFU/mL for B.elkanii L18-31 and S.fredii J18-3,respectively.The inoculation of 22 rhizobial strains in six experimental sites showed that the number of nodules and fresh weight of nodule were higher than those in the non-inoculation treatments.Inoculation with rhizobia could increase the soybean yields ranging from 4.96%to 31.67%and the protein content ranging from 0.16%to 7.80%.In summary,we studied the biogeographic distribution of soybean rhizobia,screened high-efficiency rhizobia,optimized their fermentation conditions,explored the shelf life of microbial agents,and inoculated field trials,in order to provide theoretical and technic guidances for the promotion and application of soybean rhizobia in the future. |
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