Diversity And Phylogeny Of Rhizobia Isolated From Exotic Plant Coronilla Varia

Coronilla varia, native to the Mediterranean region, was introduced to China fromEurope and America in the1960s-70s. Because of the developed root system, strongresistance, high nutritional value as well as its beautiful color, it has been widely used forerosion control, soil rehabilitation, pasturage, roadside planting, ornamental plants and greenmanure throughout the Northwest, North and Eastern China and other many places. However,the variety of rhizobia from root nodules of Coronilla varia hasn’t been studied systematically.It is not clear that which and how the soil factors affect its variety. Also, it is urgent thatCoronilla varia, as an exotic plant, how find the matched rhizobia to soon form the symbiosisin the introduced places.To deplore the problem above,90symbiotic rhizobial isolates from root nodules ofCoronilla varia, collected in6sites in the central part of Shaanxi, were characterized on thebasis of phenotypic and genetic analysis. They included123physiological and biochemicalindex,16S rRNA, nodC, nifH PCR-RFLP and the sequences analysis of16S rRNA, recA,nodC, nifH. Then, the rhizobial variety, their symbiotic evolution and the related soil factorswere studied. The main results were followed.1.84of90isolates could form nodules on the roots of Coronilla varia by nodulationtests on their original host plant. The most16, the least1, averaged six nodules were observedon each inoculated plant. Most of nodules located on the lateral roots, whereas, few were ontaproots or both taproots and lateral roots. The majority of nodules shaped in spherical andshort rod, with the size of about0.4mm, in white or pink color.2. The123physiological and biochemical index of90isolates from root nodules ofCoronilla varia showed they had the good ability of using the various carbon and nitrogensources, and the excellent resistance to the stresses.17.8%strains could grow on3.0%NaClmedium. Most of the isolates had fair tolerance to phosphorus mine mycin, penicillin andampicillin. All isolates could grow well on0.1%to0.2%deoxysodium cholate and NaNO2medium. They had fair tolerance to most dye. All isolates could grow on pH5.0-12.0medium. 12.2%and32.2%isolates could grow at10℃and40℃, respectively.3.16S rRNA PCR-RFLP and sequences analysis of16S rRNA, recA gene showed theconsistent phylogenetic relationships for all isolates except for the representative strainCCNWSX661.90isolates belonged to3genura, Mesorhizobium, Rhizobium andAgrobacterium and6species, M. sp., M. alhagi, M. amorphae, M. caraganae, R.yanglingense and A. tumefaciens. It showed the high species diversity of rhizobia from rootndules of Coronilla varia.78isolates, accounting for86.7%, had4genotypes, all belongingto Mesorhizobium. It indicated Coronilla varia mainly formed the symbiosis with rhizobiafrom Mesorhizobium.4. nodC PCR-RFLP and sequences analysis showed there were5genotypes of nodCgene of rhizobia from root nodules of Coronilla varia. They belonged to Mesorhizobium andRhizobium, but Mesorhizobium was the dorminant, including4genotypes and accounting for92.86%isolates. It indicated nodC gene of Mesorhizobium were probably from the nativehabitat through the lateral transfer and long-term adaptation, finally evolved towards M.alhagi.5. nifH PCR-RFLP and sequences analysis showed there were4genotypes of nifH geneof rhizobia from root nodules of Coronilla varia, belonging to Mesorhizobium,Agrobacterium and Rhizobium. The phylogenetic trees based on16S rRNA, nodC and nifHdisplayed obvious differences, implied that possible lateral transfer of the symbiotic genesoccurred between different species or genera.6. It was down that the correlation analysis of16S rRNA of rhizobia from root nodulesof Coronilla varia in6sampling sites and the soil physicochemical characteristics, includingcontent of the organic matter, total and available nitrogen, phosphorus, potassium and pH. Theresults showed that pH had significantly positive correlation with the diversity of16S rRNA(P＜0.05). Within a certain range, the diversity of16S rRNA gene tended to be higher as thepH of soil increased. It implied soil pH had stronger effect on variety of rhizobia fromCoronilla varia in Shannxi.