Biological Characterization Of The Chickpea Rhizobia From Xinjiang

Chickpea is a kind of legumes whose seeds can both be used for medicine and food, also can form nodules with rhizobia and fix nitrogen. While rhizobia associated with chickpea grown in China had never been surveyed until now. Therefore it is meaningful to explore the biological characterization of chickpea rhizobia from the cities of Mulei and Qitai in Xinjiang-the main planting area in China. And it will be a necessary supplementary for research of chickpea rhizobia all over the world.In this study, the phylogeny and diversity of chickpea rhizobia from Xinjiang were analyzed. The nodules were collected and total of95chickpea rhizobial strains were obtained from Mulei and Qitai in Xinjiang. These strains were analyzed by RFLP of genes of16S rRNA and1GS, the phylogenetic analysis of16S rRNA, three housekeeping genes (atpD, recA and glnⅡ), and symbiotic genes (nodC and nifH), and molecular evolution analysis. It showed that,1) all of the chickpea rhizobia in Xinjiang belonged to the genus of Mesorhizobium which were different from the known species from Mesorhizobium genus and were proposed as a distinctive genomic species;2) there was strong host-specificity of the symbiotic genes among the tested chickpea rhizobia which were above98.6%of nodC and above97.9%of nifH compared with the well studied chickpea rhizob’ia-Mesorhizobium mediterraneum and Mesorhizobium ciceri;3) the soil samples were all with the alkaline characteristic and pH values ranging from8.24to8.45, while the strains from Mesorhizobium can produce acids through the metabolism which may be used for neutralizing and adapting to the alkaline soils;4) The genetic differentiation index Kst*between the chickpea rhizobial group from China and the known species of M. mediterraneum, M. ciceri or M. temperatum was0.44227,0.48364or0.31383respectively, and the gene flow index Nm was0.03,0.02or0.04respectively while Kst*was1.00000and Nm was0.00between each couple of M. mediterraneum, M. ciceri and M. temperatum. It suggested that the tested species were significantly and geneticly differentiated from each other and that the distinct chickpea rhizobial group from China may result from the long history of geogrophical seperation and the selection of symbiotic genes from their host chickpea.Based on the research above, the distinctive genomic species of chickpea rhizobia from Xinjiang was further identified by polyphasic taxonomy including the multi locus sequence analysis (MLSA), the analysis of whole cell protein profiles, fatty acids profiles, polar lipids profiles, DNA-DNA hybridization, numerical taxonomy and cross nodulation. It confirmed the chickpea rhizobial group from Xinjiang as a novel species which was named as Mesorhizobium muleiense sp. nov..In order to find the evolution principle and ecological adaptation of rhizobia in natural conditions, four years after the original research, the chickpea nodules and soil samples from the original sites were collected again. Then housekeeping gene (recA), ecological adaptation and competitive nodulation tests were analyzed. It showed that recA sequences of the resampling chickpea rhizobia still kept well of the diversity and stability of recA gene types when compared with representative strains from M, muleiense four years ago; while recA sequences from the chickpea rhizobia trapped from soils in the condition of greenhouse were lack of the recA gene type related with CCBAU83939. However, no matter resampling or trapping, the chickpea rhizobia isolated both belonged to the single species of M. muleiense. It suggested that M. muleiense was the relatively stable species associated with chickpea in Xinjiang.Furthermore M. muleiense, isolated four years ago. M. mediterraneum and M. ciceri were tested for their soil adaptation and competitive nodulation. No matter in sterilized vermiculate or sterilized soil samples, M. ciceri showed higher nodule occupancy rates and M. mediterraneum followed behind. While when it comes to the unsterilized soil samples, M. muleiense both in inoculation and uninoculation treatments was the more competitive species and with higher nodule occupancy rates than the other two species.