Long term crumb rubber leachate reduces symbiotic nitrogen fixation and nitrogen metabolism in Medicago sativa L.

Tire wear particles (TWPs) generated by vehicle tire abrasion mainly enter soil ecosystems via deposition and runoff pathways. However, the toxicity of TWPs to terrestrial plants, particularly legumes, remain largely unexplored. Long-term leachate was obtained by immersing crumb rubber (CR) with varying particle sizes (0.5–1 mm, 1–2 mm, and 2–4 mm) in water from 2004 to 2022. Alfalfa ( Medicago sativa L.), a typically leguminous forage, was used as a model plant, and the effects of long-term CR leachates on symbiotic nitrogen fixation in an alfalfa–rhizobium system and the underlying mechanisms were investigated through transcriptome analysis. The results demonstrate that long-term CR leachate significantly decreased root growth, inhibited photosynthesis, and induced oxidative stress. Especially, root biomass, root length, root vitality, and total chlorophyll content decreased by 24.5%, 28.7%, 37.0%, and 64.3%, respectively, under 0.5–1 mm CR leachate ( p < 0.05). Furthermore, qualitative live/dead staining revealed that cell death in root tips increased with decreasing CR size. Compared with the control plants, the alfalfa stressed with the CR leachates had fewer root nodules, and decreased nitrogen fixation parameters such as the leghemoglobin (Lb) content, nitrate reductase activity, and ammonia content. Transcriptomic analysis revealed that leachate stress triggered downregulation of key nitrogen assimilation genes (e.g., nitrate/nitrite transporter, nitrate/nitrite reductase, formamidase, and glutamine synthase). The integrated data suggest that CR leachate impairs nodulation, induces oxidative stress, and disrupts the transcriptional regulation of nitrogen assimilation, ultimately suppressing symbiotic nitrogen fixation. However, the specific chemical characterization of the leachate and qRT-PCR validation of nitrogen metabolism genes were not performed, which represents limitations of the study for mechanistic attribution.