Isolation And Biological Characteristic Investigation On Endophytic Diazotroph Of Crop

In this study,154 nitrogen-fixing bacteriaes screened from wheat,maize and rice were studied on their nitrogenase activity, ACC deaminase,16S rDNA gene amplification, nifH gene amplification, solubilizing phosphate activity and fungil antagonism.The endophytic diazotrophs from the crops were isolated using two different methods,surface sterilization and water scrubbing.A total of 154 isolates were obtained by cultivating on CCM medium and nitrogen-free selective medium. The population size of of endophytes had a significant difference between species and cultivars of crops,range from 1.50×104 to 9.0×107CFU/g fresh weight.The result showed that the density of endophytes were much higer by using water scrubbing than using surface sterilization.These isolates were tested for their nitrogenase activity using acetylene reduction assay (ARA). The result suggested that all of the 154 isolates had nitrogenase and nitrogenase activity.and the enzymatic activity were from 0.210 to 236.385nmol/mg*h.3 isolates(SD1-1-1, SD2-2-6,SD2-2-8) had a higher nitrogenase activity than the reference strain(Azotobocter chroococcum).The whole 16S rDNA of 94 nitrogen-fixing bacteriaes were obaited. The diversity results showed that these 94 nitrogenfixing bacterias belonged to 28 genus with the dominant population from Pseudomonas,Bacillus and Agrobacterium.The nifH gene were detected from 2 isolates, SD1-1-land SD2-2-6.The 16 ACC deaminase positive strains were chosen by preliminary and second screening from 154 isolates and mainly were isolated from wheat and rice.The ACC deaminase activity of SD1-1-2 was higer than the test of positive strains, reached 12.016μmol·mg-1·h-1.Out of the 154 isolates,8 isolates had solubilizing phosphate activity by the way of dissolved phosphorus circle.4 isolates with a strong inhibitory activity against Gibberella zeae and the 8 isolates against Sclerolinia sclerotiorum were screened from the 154 strains using slab confrontation.The physiological and biochemical characterization of 11 endophytic bacteriaes were tested.