Molecular Ecologic Aspects Of Bacteria In Soybean Root Nodules Collected In Chengdu Plain, Sichuan

In this study, the soybean nodule samples and soybean rhizospheric soil were collected from16sites in Chengdu Plain, the quantity of soil bacteria was counted by pour plate method, and the indigenous soybean rhizobia was determined by MPN method, and the physical and chemical characteristics of the soybean rhizospheric soil was done. DNA from root nodule was isolated and the genetic diversity of rhizobia in the soybean root nodules and phylogeny was analyzed by BOXAIR-PCR,16S rDNA PCR-RFLP,16S rDNA sequencing techniques; Furthermore, the diversity of unculturable bacteria, nifH gene of the oybean root nodules was analyzed by PCR-DGGE using nodule DNA as template, and the dominant representative DGGE bands were coloned and sequenced. The results are as follows.1. The number of indigenous rhizobia of soybean rhizospheric soil in Chengdu Plain was from3.6×10to16.3×10cfu/g·DW soil, and the number of bacteria was from3.7×106to18.8×106g-DW soil. The number of indigenous rhizobia was correlated positively with soil total N content, and soil organic matter content. The number of indigenous rhizobia was different in the two kinds of soil.2. Analysis of the genetic diversity of soybean rhizobia in Chengdu Plain soil was done, the results of BOXAIR-PCR and16S rDNA PCR-RFLP analysis divided45samples into11groups at76%and96%level of similarity, respectively; both of the results were in good agreement.Phylogeny based on16S rDNA sequence of representative nodules DNA showed that the rhizobia in the soybean root nodules formed2phylogentic branches, i.e. Sinorhizobium fredii, Bradyrhizobium japonicum and B. liaoningense. The dominant species were genus of Bradyrhizobium, and contained37nodule samples, the rhizobium in other8nodule samples belong to genus of Sinorhizobium, most of the strains of the same area together in the same group3. PCR-DGGE analysis of unculturable bacteria in nodules showed that, the bacteria in the root nodules collected from the Chengdu Plain in different locations was abundant, and the Shannon diversity index ranged from1.3662to1.9386among the different nodules. The dominant and representative bands in the DGGE profiles were clonded and sequenced, and the phylogenetic tree was constructed, the results showed that the bacterial communities in soybean nodules were mainly unculturable. Interestingly, one strong band picked from each nodule DGGE samples and sequenced, and blasted from GenBank, the result showed that the sequnce of clone of this band was high similar to genus Agrobacterium tumefaciense, and it located the same phylogenic branch, which suggested that these bands represented Agrobacterium strains existed in the soybean nodules, which helps to explain why the Agrobacterium strains were always founde and isolated from the root nodules of leguminous plants.NifH gene PCR-DGGE analysis showed that the types of nifH gene in the soybean root nodules were similar, but some were relatively abundant; the Shannon Shannon diversity index ranged from0.6538to1.7529. The diversity of nifH gene types was higher in the north part than that in central and south part of Chengdu Plain. The predominant DGGE bands were cloned and sequenced, and phylogenetic tree was constructed, the results showed that the nifH gene types in soybean nodules mainly belonged to Bradyrhizobium, but DGGE band NA8-2belonged to the uncultured nitrogen-fixing bacteria nifH gene, DGEE band NA12-1was close to uncultured sinorhizobium nifH gene, and formed a separate branch, respectively.