An Environmentally Friendly Engineered Azotobacter Strain That Replaces a Substantial Amount of Urea Fertilizer while Sustaining the Same Wheat Yield

In our endeavor to improve the nitrogen fixation efficiency of a soil diazotroph that would be unaffected by synthetic nitrogenous fertilizers, we have deleted a part of the negative regulatory gene nifL and constitutively expressed the positive regulatory gene nifA in the chromosome of Azotobacter chroococcum CBD15, a strain isolated from the local field soil. No antibiotic resistance gene or other foreign gene was present in the chromosome of the engineered strain. Wheat seeds inoculated with this engineered strain, which we have named Azotobacter chroococcum HKD15, were tested for 3 years in pots and 1 year in the field. The yield of wheat was enhanced by 60% due to inoculation of seeds by A. chroococcum HKD15 in the absence of any urea application. Ammonium only marginally affected acetylene reduction by the engineered Azotobacter strain. When urea was also applied, the same wheat yield could be sustained by using seeds inoculated with A. chroococcum HKD15 and using 85 kg less urea ( 40 kg less nitrogen) than the usual 257 kg urea ( 120 kg nitrogen) per hectare. Wheat plants arising from the seeds inoculated with the engineered Azotobacter strain exhibited far superior overall performance, had much higher dry weight and nitrogen content, and assimilated molecular 15N much better. A nitrogen balance experiment also revealed much higher total nitrogen content. Indole-3acetic acid (IAA) production by the wild type and that by the engineered strain were about the same. Inoculation of the wheat seeds with A. chroococcum HKD15 did not adversely affect the microbial population in the field rhizosphere