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 and constitutively expressed the positive regulatory gene in the chromosome of 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 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 HKD15 in the absence of any urea application. Ammonium only marginally affected acetylene reduction by the engineered strain. When urea was also applied, the same wheat yield could be sustained by using seeds inoculated with 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 strain exhibited far superior overall performance, had much higher dry weight and nitrogen content, and assimilated molecular N much better. A nitrogen balance experiment also revealed much higher total nitrogen content. Indole-3-acetic acid (IAA) production by the wild type and that by the engineered strain were about the same. Inoculation of the wheat seeds with HKD15 did not adversely affect the microbial population in the field rhizosphere soil. 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 microorganisms cannot replace synthetic nitrogenous fertilizers, as these reduce atmospheric nitrogen very inefficiently and almost none at all in the presence of added nitrogenous fertilizers. If the nitrogen-fixing ability of soil diazotrophs could be improved and sustained even in the presence of synthetic nitrogenous fertilizers, then a mixture of the bacteria and a reduced quantity of chemical nitrogenous fertilizers could be employed to obtain the same grain yield but at a much-reduced environmental cost. The engineered strain that we have reported here has considerably enhanced nitrogen fixation and excretion abilities and can replace ∼85 kg of urea per hectare but sustain the same wheat yield, if the seeds are inoculated with it before sowing.