Chlorpyrifos inhibits nitrogen fixation in rice-vegetated soil containing Pseudomonas stutzeri A1501

Chlorpyrifos, a common organophosphorus pesticide, is widely used for agricultural pest control and can inhibit nitrogen-fixing bacteria biomass in paddy. In this study, the additions of chlorpyrifos (1 and 8 mg kg^-1) to soil, with or without Pseudomonas stutzeri A1501, resulted in a significant decrease in nitrogen fixation, despite insignificant effects on the abundances of P. stutzeri A1501 and bacteria in soil. Toxic effect of chlorpyrifos on P. stutzeri A1501 nitrogenase activity in medium was also observed, accompanied by a significant reduction in the expression of nitrogen-fixing related genes (nifA and nifH). Furthermore, rhizosphere colonization and biofilm formation by P. stutzeri A1501 were repressed by chlorpyrifos, leading to decreased nitrogenase activity in the rhizosphere. Biofilm formation in medium was inhibited by bacterial hyperkinesis and reduction of extracellular polymeric substance, including exopolysaccharides and proteins. Together, these findings showed that chlorpyrifos-induced production of reactive oxygen species (ROS) which was directly responsible for reduced nitrogenase activity in the medium, soil, and rhizosphere by inhibiting the expressions of nitrogen-fixing related genes. Furthermore, the inhibition of biofilm formation by chlorpyrifos or ROS likely aggravated the reduction in rhizospherere nitrogenase activity. These findings provide potentially valuable insights into the toxicity of chlorpyrifos on nitrogen-fixing bacteria and its mechanisms. Furthermore, for sustainable rice production, it is necessary to evaluate whether other pesticides affect nitrogen fixation and select pesticides that do not inhibit nitrogen fixation.