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-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 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 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 Azotobacter 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.

Light promoted brown staining of protoplasm by Ag+ is ideal to test wheat pollen viability rapidly.

Pollen viability is crucial for wheat breeding programs. The unique potential of the protoplasm of live cells to turn brown due to the synthesis of silver nanoparticles (AgNPs) through rapid photoreduction of Ag+, was exploited for testing wheat pollen viability. Ag+-viability test medium (consisting of 0.5 mM AgNO3 and 300 mM KNO3) incubated with wheat pollen turned brown within 2 min under intense light (~600 µmol photon flux density m-2s-1), but not in dark. The brown medium displayed AgNPs-specific surface plasmon resonance band in its absorption spectrum. Light microscopic studies showed the presence of uniformly stained brown protoplasm in viable pollen incubated with Ag+-viability medium in the presence of light. Investigations with transmission electron microscope coupled with energy dispersive X-ray established the presence of distinct 5-35 nm NPs composed of Ag. Powder X-ray diffraction analysis revealed that AgNPs were crystalline and biphasic composed of Ag0 and Ag2O. Conversely, non-viable pollen and heat-killed pollen did not turn brown on incubation with Ag+-medium in light. We believe that the viable wheat pollen turn brown rapidly by bio-transforming Ag+ to AgNPs through photoreduction. Our findings furnish a novel simplest and rapid method for testing wheat pollen viability.

Reducing strength prevailing at root surface of plants promotes reduction of Ag+ and generation of Ag(0)/Ag2O nanoparticles exogenously in aqueous phase.

Potential of root system of plants from wide range of families to effectively reduce membrane impermeable ferricyanide to ferrocyanide and blue coloured 2,6-dichlorophenol indophenol (DCPIP) to colourless DCPIPH2 both under non-sterile and sterile conditions, revealed prevalence of immense reducing strength at root surface. As generation of silver nanoparticles (NPs) from Ag+ involves reduction, present investigations were carried to evaluate if reducing strength prevailing at surface of root system can be exploited for reduction of Ag+ and exogenous generation of silver-NPs. Root system of intact plants of 16 species from 11 diverse families of angiosperms turned clear colorless AgNO3 solutions, turbid brown. Absorption spectra of these turbid brown solutions showed silver-NPs specific surface plasmon resonance peak. Transmission electron microscope coupled with energy dispersive X-ray confirmed the presence of distinct NPs in the range of 5-50 nm containing Ag. Selected area electron diffraction and powder X-ray diffraction patterns of the silver NPs showed Bragg reflections, characteristic of crystalline face-centered cubic structure of Ag(0) and cubic structure of Ag2O. Root system of intact plants raised under sterile conditions also generated Ag(0)/Ag2O-NPs under strict sterile conditions in a manner similar to that recorded under non-sterile conditions. This revealed the inbuilt potential of root system to generate Ag(0)/Ag2O-NPs independent of any microorganism. Roots of intact plants reduced triphenyltetrazolium to triphenylformazon and impermeable ferricyanide to ferrocyanide, suggesting involvement of plasma membrane bound dehydrogenases in reduction of Ag+ and formation of Ag(0)/Ag2O-NPs. Root enzyme extract reduced triphenyltetrazolium to triphenylformazon and Ag+ to Ag(0) in presence of NADH, clearly establishing potential of dehydrogenases to reduce Ag+ to Ag(0), which generate Ag(0)/Ag2O-NPs. Findings presented in this manuscript put forth a novel, simple, economically viable and green protocol for synthesis of silver-NPs under ambient conditions in aqueous phase, using root system of intact plants.