Background: Tobacco mosaic virus (TMV) is a harmful plant pathogen that causes a decline in the quality and yield of many economic crops. Natural products are important potential sources of biopesticides for the prevention and treatment of TMV. This study focuses on the discovery of anti-TMV active compounds from Aspergillus versicolor and investigates their activities against TMV. Results: In this study, four isocoumarins 7-methoxy-3-(2-oxopropy)-5-hydroxymethyl-isocoumarin (1), 7-methyl-3-(2-oxopropy)-5-hydroxymethyl-isocoumarin (2), oryzaein A (4) and oryzaein B (5), two indole alkaloids aspergilline F (6) and aspergilline G (7), and one indole alkaloid and isocoumarin hybrid aspergillactone A (3) were isolated from Nicotiana tabacum-derived A. versicolor YNCA0363. Among them, compounds 1–3 are new isolates, compound 3 represents the first example of indole alkaloid and isocoumarin connected by C(12)-N(1′) bond. The inactivation efficacies for compounds 1, 2 and 3 were 58.9, 43.8 and 52.6% at the concentration of 50 μg/mL, respectively, which were significantly higher than that of positive control, ningnanmycin. The protective effects of these three compounds ranged from 48.6 to 62.3%, which were significantly higher than that of positive control. At the same time, the content of TMV-CP was also significantly lower than that of positive control, and compound 1 was the lowest. The curative efficacy for compound 1 was also much better than that of positive control. Transmission electron microscopy (TEM) showed that compound 1 could directly destroy viral particles into small fragments. The results of molecular docking showed that the binding ability of compounds 1, 3, 2 to TMV-CP protein decreased in turn, which was consistent with the results of activities assays. Conclusion: Compounds 1–3 from A. versicolor showed potent antiviral activities against TMV including inactivation, protective and curative effects. Compound 1 can directly destroy the virus particles to achieve the effect of anti-TMV. In addition, compounds 1–3 can bind to TMV-CP protein in molecular docking experiments. The above experimental results show that TMV-CP was an important target for active indole alkaloid and isocoumarin derivatives to fracture TMV particle. The results provided evidence that indole alkaloid and isocoumarin derivatives from A. versicolor have the potential to control TMV. Graphical

Three new isochromenes, (5-methoxy-7-prenyl-1H-isochromen-3-yl)methanol (1), 3-(3-(hydroxymethyl)-5-methoxy-1H-isochromen-7-yl)propan-1-ol (2), and (5-methoxy-7-methyl-1H-isochromen-3-yl)methanol (3), along with three known analogues (4–6) were isolated from the fermentation products of a Nicotiana tabacum-derived endophytic fungus Aspergillus versicolor. Their structures were elucidated by spectroscopic methods, including extensive 1 D and 2 D NMR techniques. Compounds 1–3 and 6 were evaluated for their anti-tobacco mosaic virus (anti-TMV) activities. The results showed that compound 2 exhibited high anti-TMV activity with inhibition rate of 46.4%, and this rate is higher than that of positive control. Compounds 1, 3, and 6 also showed potential anti-TMV activity with inhibition rates of 28.6, 30.5, and 26.2%, respectively. The IC₅₀ of compounds 1–3 and 6 were also tested, and showed IC₅₀ values of 49.3, 22.4, 42.2, and 54.1 µM, respectively. Graphical Abstract