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The extract of crofton weed (Eupatorium adenophorum) inhibits seed germination and weed growth;however, the physiological mechanisms underlying the effect of crofton weed extract on the modulation of seedling growth and root system development remain largely unclear. In this study, we investigated the effects of the leaf extract of crofton weed (LECW) on primary root (PR) growth in maize seedlings. Treatment with LECW markedly inhibited seed germination and seedling growth in a dose-dependent manner. Physiological analysis indicated that the LECW induced reactive oxygen species (ROS) accu-mulation in root tips, thereby leading to cell swelling and deformation both in the root cap and elon-gation zone of root tips, finally leading to cell death in root border cells (RBCs) and PR growth inhibition. The LECW also inhibited pectin methyl esterase (PME) activity, thereby decreasing the RBC number. Taken together, our results indicated that the LECW inhibited PR growth by inducing ROS accumulation and subsequent cell death in RBCs. The present study provides a better understanding of how the LECW modifies root system development and provides insight for evaluating the toxicity of crofton weed ex-tracts in plants.