An environment friendly engineered Azotobacter can replace substantial amount of urea fertilizer and yet sustain same wheat yield.

In our endeavor to improve upon 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 there in the chromosome of the engineered strain. Wheat seeds inoculated with this engineered strain, which we have named Azotobacter chroococcum HKD15, were tested for three years in pots and one year in the field. 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 ∼85kg less urea (∼40kg 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 15 N much better. A nitrogen balance experiment also revealed much higher total nitrogen content. IAA production by the wild type and the engineered strain was about the same. Inoculation of the wheat seeds with A. chroococcum HKD15 did not adversely affect the microbial population in the field rhizosphere soil.IMPORTANCE Application of synthetic nitrogenous fertilizers is a standard agricultural practice to augment crop yield. Plants, however, utilize only a fraction of the applied fertilizers, while the unutilized fertilizers cause grave environmental problems. Wild type soil diazotrophic microrganisms cannot replace synthetic nitrogenous fertilizers, as these reduce atmospheric nitrogen very inefficiently and almost none at all in presence of added nitrogenous fertilizers. If the nitrogen fixing ability of soil diazotrophs could be improved upon and sustained even in the presence of synthetic nitrogenous fertilizers, then a mixture of the bacterial and reduced quantity of chemical nitrogenous fertilizers could be employed to obtain the same grain yield, but at much reduced environmental cost. The engineered Azotobacter that we have reported here has considerably enhanced nitrogen fixation and excretion abilities and can replace ∼85 kg of urea per hectare, but sustain same wheat yield, if the seeds are inoculated with it before sowing.