Screening And Analysis For Efficient Co-inoculation Of Soytean Rhizobia And Plant Growth-promoting Rhizobacteria

Plant growth promoting rhizobacteria (PGPR) are a class of bacteria survived in the range of plant rhizosphere, which can secret plant hormone or inhibit pathogenic bacteria, Many studies showed that PGPR can promote nodulation, including increasing the number of nodules, promoting root development and so on. This paper reported that screening PGPR from soybean rhizosphere soil and the collection strains, selecting five combinations of PGPR and soybean rhizobium which improved nodulation and nitrogen fixation in vermiculite and soil pot experiment and studying the mechanism of co-inoculation.Through phosphate solubilization and IAA production experiment, twenty four solubilizing phosphate strains and thirteen strains of growth hormone secretion were screened from seventy-three strains. Nine strains both have capacity of dissolving phosphorus and producing growth hormone.The fourteen strains were selected to co-inoculate with Sinorhizobium fredii 4644 and Bradyrhizobium japonicum 4219 on soybean in vermiculite pot experiment. This experiment showed five high efficient combinations of PGPR and soybean rhizobia were acquired, which were Sinorhizobium fredii 4644 with Bacillus subtilis 1.15, strain P13 and strain P7 and Bradyrhizobium japonicum 4219 with Bacillus megaterium 92075 and strain 1-3. The co-inoculation promoted soybean nodulation and growth and improved efficiently in the number of nodule, nodule dry weight, shoot dry weight and plant nitrogen content. The results of soil pot experiment of Fenyang's, Yan'an and Jining's soils were basically agreed with the results of vermiculite pot experiment. However, for the five combinations of co-inoculation, the increase of the number of nodule, nodule dry weight, shoot dry weight and plant nitrogen content were varied in different soils.Phosphorus quatitive determination showed the five PGPR strains dissolved inorganic phosphorus efficiently. The solubilizing ability of Bacillus subtilis 1.15 and strain P13 was the highest, which reached to 255.58mg/L and 266.81mg/L respectively. The total P content of soybean increased 10.0% to 62.5% in soil pot experiment through co-inodulation.The composition of five PGPR strains' fermentation of by HPLC showed these strains had ability to produce plant hormones including IAA and organic acids. Except strain 1-3, the other strains didn't produce zeatin.Strain P7, P13 and 1-3 were isolated from soybean rhizosphere soil. 16SrDNA sequencing analysis showed strain P7, strain P13 and strain 1-3 had 99% homology with Phyllobacterium ifriqiyense, Sinorhizobium meliloti and Bacillus firmus respectively. They were related to Phyllobacterium sp., Sinorhizobium sp. and Bacillus sp..The molecular mechanism of improving nodulation and nitrogen fixation of rhizobium through PGPR was studied using proteomics research. Strain P7 improved fast growing strain 4644's ability of nitrogen-fixing and nodulation on soybean, but Psendomonas fluorescens 10040 didn't. Strain 4644 was treated with daidzein from Zhonghuang13 and extracellular materials of P7. Then twenty two up-regulated proteins and four down proteins were found by two-dimensional electrophoresis and mass spectrometry. Those proteins included metabolism proteins and hypothetical proteins. Strain 4644 was treated with daidzein from Zhonghuang13 and extracellular materials of strain 10040. Sixty up-regulated proteins and 14 down-regulated proteins were found, which involved in metabolism, DNA replication and transcription. One of the proteins isβlactamases. The remaining proteins showed differences that didn't match retrieving information in the database.