Discovery And Application Of Effective Rhizobium Strains For Peanut On Acid Soils

Peanut(Arachis hypogaea L.)is an important oil and economic crop in the world.Most of the peanuts are cultivated in acidic soils in South China.Since low pH value,and N(Nitrogen)and P(Phosphorus)deficiencies on acidic soils severely limit yield and BNF(Biological Nitrogen Fixation)capacity in peanut,it is of great importance to isolate acidic soils adaptive rhizobium stains to improve BNF and yield of peanut,as well as to remediate acidic soils.In this study,root nodules of 17 peanuts were collected from three typical acidic soil sites in Fujian Province: Anxi,Fuan and Youxi.The nodule endophyte were isolated and purified from fresh peanut nodules,which were collected from the field by plate streaking combined with microscopic examination.The mainly results were as follows:(1)The 16 S V4-V5 fragment was used to identify the isolates and 189 culturable endophytic bacteria which were isolated from peanut nodules.There are 32 genera of bacteria,including Rhizobium,Bradyrhizobium,Bacillus,Rafsonia,Microbacterium,Bacillus,Staphylococcus Aeromonas and so on.Through 16 S V4-V5 fragments alignment in NCBI,32 of them belonged to peanut rhizobium.Through phylogenetic tree analysis,27 Bradyrhizobium strains were clustered with Bradyrhizobium SEMIA6396,Bradyrhizobium japonicun MN-139,and 5 Rhizobium strains were clustered with Rhizobium G2312 and Rhizobium 5502 G.(2)The structural genes nifH encoding nitrogenase ferritin components and nodA encoding nod genes were used to amplify and identify 32 strains of Peanut rhizobium by PCR.Among them,10 strains including the structural genes nifH and nodA.16 S rDNA was used to identify full length of these Rhizobium sequences and phylogenetic tree analysis was carried out.By 16 S rDNA sequencing,8 strains belonged toBradyrhizobium and 2 strains belonged to Rhizobium.Phylogenetic tree analysis showed that 8 strains belonging to Bradyrhizobium were clustered with Bradyrhizobium SEMIA6396 and Bradyrhizobium japonicun MN-139 respectively.2strains of Rhizobium were clustered with Rhizobium miluonense strain 5502 G.(3)The peanut Minhua 8,Minhua 6 and Quanhua 3 and 19 strains of the 32 Rhizobium were identified in hydroponics.It was further confirmed that 10 strains were Rhizobium sp.,for which could form functional nodules with peanuts in hydroponics.The results showed that all the 10 Rhizobium strains with the structure gene nifH and nodA of nitrogenase ferritin could form effective nodules,which confirmed that they were Peanut rhizobium with nodulation and nitrogen fixation.(4)The peanut Minhua 8,Quanhua 3 and Jihua 26 and Rhizobium(4 strains:AXLQ1-6,FAMB12-6,YZMB13-3,YZMO14-4)with effective BNF(Biological Nitrogen Fixation)capacity were selected for field trials on acidic soils.The results showed that all the 4 strains successfully formed functional nodules with peanuts in the field.On contrary,none of nodules could be formed in peanuts without inoculation(CK).Besides,Rhizobia inoculation significantly increased the plant height,branch number and SPAD value of peanut leaves.It also improved N nutrition,P nutrition,K nutrition,and increased biomass and yield of peanut.By comparison with CK,biomass,yield and N content of peanuts inoculated with rhizobium strains were significantly increased.In conclusion,this study isolated and identified several high-effective rhizobia for peanut,which were widely compatible to different hosts and could effectively fix nitrogen,promote the growth and of peanut on acidic soils,so as to exhibit prospective application potentials.