Objectives The secretion of phenolic phytochemicals is a significant factor that contributes to difficulties in tobacco crop rotation. Investigating the mechanism through which these phytochemicals affect tobacco offers a theoretical foundation for addressing challenges in tobacco crop rotation.

Methods Using Yunyan 87 as the test crop and benzoic acid as the exogenous allelochemical, germination tests were conducted using the Petri dish method. Six enzoic acid concentrations were applied: 0, 0.1, 10, 100, 500, and 1000 mg/L, with tobacco seed germination traits measured. Soilless cultivation experiments were conducted with seedlings grown under identical benzoic acid concentrations. After 21 days of growth, samples were collected to investigate seedling development, leaf photosynthetic efficiency, and enzyme activity. Transcriptome sequencing was employed to investigate the major enrichment pathways of differentially expressed genes (DEGs) in flue-cured tobacco seedlings under varying benzoic acid treatments.

Results Low concentrations of benzoic acid (0.1 mg/L) slightly promoted seed germination, whereas concentrations ≥10 mg/L significantly inhibited seed germination and impeded radicle elongation. Low concentrations of benzoic acid (0.1−10 mg/L) significantly increased chlorophyll content in seedlings, enhancing photosynthetic capacity, whereas high concentrations (100−1000 mg/L) severely inhibited growth. Regardless of the concentration, benzoic acid significantly suppressed root development in seedlings, indicating heightened root sensitivity to benzoic acid. As benzoic acid concentration increased, leaf malondialdehyde (MDA) content progressively decreased. Superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) activities initially rose before declining, peaking at 100 mg/L. Root vitality, however, exhibited a continuous decline with increasing concentration. GO functional enrichment analysis revealed that under high benzoic acid concentrations, DEGs in leaf tissues predominantly enriched into entries such as photosynthesis and tetrapyrrole metabolism, whereas root DEGs significantly enriched into functional entries including cell wall biogenesis and modification. KEGG metabolic pathway analysis further revealed that in leaves, plant hormone signaling and MAPK signalling pathways were up-regulated, while photosynthesis-antenna proteins and carbon metabolism pathways were significantly down-regulated with increasing benzoic acid concentration. In roots, phenylpropanoid biosynthesis, hormone signalling, and flavonoid biosynthesis pathways were up-regulated, whilst kinesin, plant-pathogen interaction, and pentose and glucuronic acid conversion pathways were down-regulated.

Conclusion The accumulation of benzoic acid significantly inhibits the germination of flue-cured tobacco seeds and the growth of seedlings, with the most pronounced effect observed on root development. The mechanism likely involves benzoic acid severely impeding photosynthetic energy capture and energy synthesis by markedly suppressing the leaf photosynthesis-antenna proteins pathway and carbon metabolism processes. Concurrently, within the root system, it activates the synthesis of defensive secondary metabolites such as phenylpropanoids and flavonoids while inhibiting pathways related to actin, pentose conversion, and pathogen defence, leading to metabolic dysfunction. Ultimately, the coupling of leaf energy depletion with root defence resource exhaustion forms an interactive system of “photosynthetic inhibition-defence imbalance”, systematically impeding normal plant development.