Phylogenetic Study Of Rhizobia Nodulating With Acacia Melanoxylon R. Br. And Identification Of Novel Species

Acacia melanoxylon R.Br.,a tall tree,belongs to Acicia of the subfamily Mimosaceae,witch is native to southeast in Australia.In the 1900s,Acacia melanoxylon was introduced into China,and it now has become one of the important fast-growing species of tropical and subtropical regions of China,because Acacia melanoxylon has many good qualities,such as excellent furniture material;cold,drought and infertile resistant;fixing nitrogen nodulating with rhizobia.However,contamination-free microbial fertilizer has attracted many attention in recently years because ecosystem has been polluted seriously by overusing of chemical fertilizer.In order to ameliorate polluted ecosystem and improve the growing rate of Acacia melanoxylon,this issue studies the phylogeny situation of rhizobia nodulating with Acacia melanoxylon,expecting to discover and select diverse rhizobial germplasm resources providing theoretical basis for microbial fertilizer and agent.On the 16S rDNA PCR-RFLP of previous research,33 strains related to known but unnamed novel species have been selected.This study carried out phylogenetic analysis and identification of new species by phylogenetic analysis of 16S rDNA,housekeeping genes(recA,atpD,glnII),MLSA,symbiotic genes(nifH,nodC),assay of fatty acid components,DNA-DNA hybridization,DNA(G+C)mol%determination,physiology and biochemistry experiment and nodule experiment on these strains.The results of phynogenetic analysis based on the 16S rDNA and housekeeping genes were mostly in consonance with the 16S rDNA PCR-RFLP.These tested strains were divided into 12 genotypes,three of which belonged to Mesorhizobium and the others were Bradyrhizobium.Symbiotic genes of rhizobia nodulating with Acacia melanoxylon were grouped into two big branches,and the similarities among all tested strains in the same branch were close to100%.Moreover,in the nifH tree,the relationship between the tested strains and the reference strains varied significantly according to the genera.However,in the nodC tree,the relationship had smaller correlation with the genera;for example,all the tested strains belonged to the genus Bradyrhizobium and Mesorhizobium had the closest similarity with the reference strain Mesorhizobium gobiense CCBAU 83330~T.This study confirmed 5 novel rhizobial species:Bradyrhizobium sp.nov.?(RITF1106),Bradyrhizobium sp.nov.?(RITF1313),Bradyrhizobium sp.nov.?(RITF1631,RITF1633),Mesorhizobium aciciae sp.nov.(the type strain RITF741~T=CCBAU 101090~T=JCM 30534~T)and Mesorhizobium zengchengense sp.nov.(the type strain RITF1218~T=CCBAU101091~T=JCM 30535~T).In addition,genotype 3 had been identified as Bradyrhizobium rifense.Noduling experiment found that tested strains could nodulate with the original host,Leucaena leucocephala,Albizia falcataria and Acacia aneura four woody legumes of different genera,but had different effect on them.In the case of biomass,the amount of increasing in Acacia melanoxylon was 2.7%~110.3%,and strain RITF522 has the best promotion.Strain RITF741 to Leucaena leucocephala and strain RITF908 to Albizia falcataria increased significantly,with increasing 66.4%and 34.5%,respectively.While,the effect on biomass to Acacia aneura was quite different,ranging from-19.4%to 117.9%.In conclusion,this study indicates that rhizobia nodulating with Acacia melanoxylon has abundant species diversity and symbiotic diversity.And it again proves that species distribution is related to locality.