| Nitrogen fixed by the Rhizobium-Legume symbiosis accounts for approximately 65% of the total biologically fixed nitrogen, and it’s the system which has the most biologically fixed nitrogen and highest efficiency. Therefore in order to know more about this symbiosis, it is of vital importance to carry out a survey of the strains of rhizobium and to launch researches into the genetic diversity and mechanism of nodulation. The fertile soils and mild climate of Chengdu plain provide plants such as Soybeans, Peas, Vicia fabas with favorable condition for their growth. What the effect will the long term crop rotation and relay intercropping of multiple legumes have on the distribution of the rhizobium in the soil?In this study, root nodules of Peas and the soil samples were collected from Chengdu plain, and rhizobiums were captured by planting Vicia faba, Clover, Soybeans in soils mentioned above. The strains analyzed by BOXAIR-PCR,16S rDNA PCR-RFLP,16S rDNA sequencing, housekeeping genes and symbiotic genes sequenceing. The results were as the follow:1. Rhizobial stains form symbiotic nodulation with Peas, Vicia fabas, Clovers, Soybeans are widely distributed in the soils in Chengdu plain.2. Through the BOXAIR-PCR fingerprint, the 105 rhizobial strains isolated from root nodules of Peas were divided into 6 types, and genotype II was the dominant one, and consisted of 54 strains. The strains were divided into 11 types based on 16S rDNA PCR-RFLP analysis. On the basis of above analysis,38 representative srains were selected to do 16S rDNA sequencing analysis, which had yielded results that the representative strains belong to 3 different biological types of R. leguminosarum according to phylogenetic tree.3. The results of 16S rDNA sequencing analysis and housekeeping genes sequencing showed that these strains forming root nodules with Peas, Vicia fabas, Clovers and Soybeans belonged to R. leguminosarum, S. meliloti, S. fredii, B. betae, B. japonicum, B. liaoningense and B. elkanii based on the phylogenetic analysis. Furthermore, the results proved that there were different rhizobium populations existed in the soils.4. The result of the comparison among the symbiotic genes(nodC and nifH) and 16S rDNA, housekeeping genes analysis indicated that the horizontal transfer of symbiotic genes are prevalent between the rhizobiums, for instance, according to the 16S rDNA analysis and housekeeping gene analysis of strains which isolated from root nodules of Clovers, all tested strains were phylogenetically in the genus of S. meliloti, while the result of symbiotic genes sequence analysis indicated that all tested strains were phylogenetically closest to S. kummerowiae. This result showed that the symbiotic genes of S. kummerowiae had transferred to S. meliloti in the long term process of evolution. The same results were appeared in the strains isolated from nodules of Soybeans and Vicia fabas.5. There was some correlation between the genotypes of rhizobium and the environmental factors such as the soil physical and chemical properties. The results of the BOXAIR-PCR of the tested strains isolated from root nodules of Peas showed that there existed certain regularity between the distribution of rhizobium based on different genotypes and the physical and chemical properties of soil.In conclusion, the soil in Chengdu plain exited diverse rhizobium populations forming nodules with various legumes, and the geographic distribution of rhizobium based on different genotypes was affected by environmental factors. Furthermore, the horizontal transfers of symbiotic genes were studied. The outcomes of this study enriched the knowledge of the rhizobium populations in the soil of Chengdu plain, and laid the foundation for the selective breeding of the highly efficient rhizobacteria in the future. |
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