Isolation And Identification Of Nitrogen-fixing Bacillus And Colonization Of Maize, Rice And Wheat By Nitrogen-fixing Bacillus Megaterium C4

Bacillus megaterium C4, a nitrogen-fixing bacterium, was marked with gfp gene. Maize , rice and wheat seedlings were inoculated with the GFP-labeled B. megaterium C4 and then grown in gnotobiotic condition. The maize roots, stems, leaves, rice roots and wheat roots were optically sectioned and observed under confocal laser scanning microscope. Observations of the maize root sections at 1,3,5,7, 9 and 11 days after inoculation showed significantly the infection process that the bacterial cells infected the maize roots from the root surfaces to the steles. One day after inoculation, the bacteria colonized mainly the surfaces of primary and lateral roots, and the junctions between primary root and lateral root. Three days after inoculation, the bacterial cells colonized cortexes. Five days after inoculation, the bacteria had progressed towards inner cortex. Seven days after inoculation, the bacteria reached vascular stele and penetrated into xylem and pith. The bacteria were mainly located in the intercellular spaces, although a few bacterial cells also were present within the xylem vessels, root hair cells, epidermis, cortical parenchyma and pith cells. A probable site of entry into maize roots for the bacteria was cracks formed at the lateral root junctions. Microscopic observations of the sections of maize stems and leaves at 30 and 40 days after inoculation revealed that the bacteria were present in stems and leaves at 30 and 40 days after inoculation, indicating that the bacteria migrated slowly from roots to stems and leaves.Colonization studies of rice roots and wheat roots at 1, 3 , 5 , 7 days after inoculation revealed clearly the infection process on rice roots and wheat roots was similar to that on maize roots, and the bacterial cells were found to colonize epidermis, cortex and vascular tissues of rice root and wheat root. It also indicated that the root tip in the zone of elongation and the junctions between the primary and the lateral roots were the two sites for the bacteria entry into rice root.The population of the bacterial cells colonized in the maize roots, stems and leaves were estimated by counting colony-forming units on selective medium. The population in roots reached 10^3.2CFU/g FW at 3 days after inoculation, and gradually increased to 10^7.1 CFU/g FW at 9 days after inoculation. At 30 days after inoculation, the bacterial population reached 10^8.2 CFU/g FW in leaves and 10^4.3 CFU/g FW in stems.Soil samples of different plant rhizosphere were collected from fifteen provinces. They were made into suspensions and seethed for 15 min, then plated on nitrogen-free medium in order to isolate nitrogen- fixing bacillus. Colony PCR was used to amplify nifH gene segment and nitrogenase activity was determined by acetylene reduction method. 116 strains of nitrogen-fixing bacillus which both have nifH gene segment and nitrogenase activity were acquired, and nitrogenase activities of 7 strains exceeded that of type strain P .azotopxans DSM1735.