Studies On Selective Breeding And Effects Of Nitrogen-fixing Bacillus Strain

In this thesis, eleven strains of nitrogen-fixing bacillus with high nitrogenase activity were selected from 105 samples of rhizosphere soil. At the same time, the further studies for them were conducted. The results obtained are as follows:The selection and identification of nitrogen-fixing bacillus: A total 347 isolates were selectively obtained, based on their growth on nitrogen-free medium and their resistance to 100℃for 10-15 min. These isolates were tested for their nitrogenase activity using acetylene reduction assay (ARA). The result suggested that 170 isolates showed nitrogenase activity. The nitrogenase activities of 12 isolates were higher than the reference strain (Azotobocter chroococcum). These 12 isolates were Y1, C2, B3, D4, E5, Y6, S7, S8, S9, N10, Y11 and Y12. They were identified based on configuration, physiological and biochemical characterization, 16S rDNA sequence. Isolates Y12 was identified as Microbacterium sp. Isolates Y1, S9 and N10 were identified as Paenibacillus sp. Isolates C2, B3, D4, E5, Y6, S7, S8 and Y11 were identified as Bacillus sp.The effect of inoculation on the pakchoi: The pakchoi was inoculated with 11 bacillus strains of high nitrogenase activity respectively. The results indicated that the treatments with nitrogen-fixing bacillus had positive effect on pakchoi's seedling growth and led to the significant increase in fresh, dry plant weights of pakchoi. The plant fresh weight was increased by 38.2%～69.8% and the plant dry weight was increased by 25.9%～69%. Meanwhile, the total nitrogen content of pakchoi seeding was also increased considerablely, reaching significant level for 10 strains of them respectively. In addition, the experiment showed that both B3 and S9 strains had slight influence on total nitrogen, available phosphorus and quick-acting potassium contents of the rhizosphere soil.The impact of bacterial diversity in rhizosphere soil: With PCR-DGGE method, it was detected the fluctuation of bacterial community diversity in pakchoi rhizosphere soil, which was influenced by nitrogen-fixing bacillus. The results showed that the bacterial community diversity increased as the treatment with nitrogen-fixing bacillus, and the different treatments had different impact of the diversity.The applicability to diverse environmental factors: Effects of diverse environmental factors on nitrogenase activity (ARA) of both B3 and S9 strains in nitrogen-free culture were investigated in our experiments. The results implied that these two strains could easily adapt to different cultural conditions: it could propagate quickly and fix nitrogen at a temperature range of 15℃to 40℃and at a pH range of 4 to 9. Moreover, low NH₄⁺ concentration had little effect on its nitrogenase activity. When the NH₄⁺ concentration was about 0.5mmol/L and 1mmol/L respectively, their highest nitrogenase activities could be detected.