Objective The objective of this study is to elucidate the effects and to analyze the function of melatonin in enhancing resistance against East Asian Passiflora Virus (EAPV) in Nicotiana benthamiana.

Methods N. benthamiana plant were treated with various concentrations of melatonin(0, 50, 100, 200, 400 μmol/L), applied both before and after EAPV inoculation, healthy plants treated with melatonin-free solution and not inoculated with virus were used as control (CK). An optimal concentration was screened for by monitoring external symptoms, molecular virus detection, and changes in salicylic acid (SA) and jasmonic acid (JA) levels. Subsequently, the protective (anti-infection) and inhibitory (anti-replication) effects of melatonin were quantitatively assessed by measuring the host OD₄₀₅ values.

Results EAPV infection significantly reduced plant height. Untreated infected plants (T0) showed a height of 6.4 (±0.20) cm, 52.2% of CK. Foliar application of melatonin (200 μmol/L, T3) most effectively alleviated stunting. When inoculated at day 1, day 4, and day 7 post-treatment, infected plant heights were 1.66, 1.27, and 1.06 times higher than T0, respectively.Despite failing to protect plants from infection (protection rate < 0), 200 μmol/L melatonin exhibited a substantial inhibitory effect on EAPV, suppressing viral accumulation by 26.9% on average.JA levels increased significantly from 10.38 ng/g (day 0) to 123.37 ng/g (day 7), while SA levels remained unchanged.

Conclusion After inoculating EAPV, the plant significantly dwarfed. The application of exogenous melatonin could significantly alleviate the dwarfism of the native tobacco, with the optimal concentration being 200 μmol/L. After spraying melatonin, the JA level in the native tobacco significantly increased, while the SA level did not change significantly, suggesting that exogenous melatonin mitigates EAPV-induced stunting by activating JA-mediated induced systemic resistance (ISR), rather than directly preventing viral entry.