Abstract

The rhizobium–legume symbiosis plays a vital role in the global nitrogen cycle. Although microplastics have been shown to affect this symbiotic system, the accumulation and impacts of nanoplastics (NPs) in rhizobia and their root nodules remain poorly understood, particularly regarding the interactive effects of NPs of different sizes on symbiotic nitrogen fixation. This study demonstrated that polystyrene (PS) NPs exhibited a significant size difference effect on rhizobia and their symbiotic nitrogen-fixing association with soybean ( Glycine max ). We found that both rhizobia and soybean nodules efficiently internalized PS NPs, with differently sized NPs showing mutual enhancement during the cellular uptake of rhizobia. 100 mg/kg of 20 nm PS NPs severely disrupted the symbiotic nitrogen fixation, reducing nitrogenase activity by 51.3% in single exposures and 28.6% in combined exposure to 200 nm PS NPs. This observed disruption caused by 20 nm PS NPs was associated with suppressed nodule formation (26.0% reduction in number, 50.4% decrease in fresh biomass), diminished leghemoglobin content (64.9% reduction), impaired nutrient acquisition (26.5% decrease in nodule Mo content), reduced rhizobia infection efficiency, impaired plant growth, and modified expression of nodulation- and nitrogen-fixation-related genes. These findings revealed that small-sized PS NPs posed a substantial threat to the rhizobium–legume symbiosis, underscoring the ecological risks of NP pollution in agricultural systems.