Screening And Mechanism Of Secondary Metabolites Inhibiting Tobacco Mosaic Virus From Dendrobium Findlayanum

Plant virus disease is the second largest crop disease after fungal disease.It is also known as "plant cancer" due to its serious harm and difficult control.Among them,Tobacco mosaic virus(TMV)can infect more than 400 kinds of plants belonging to 36 families including Solanaceae,Cruciferae,Compositae,etc.,and seriously endanger the crop production.With the concept of green prevention and the "two zero growth" goals of chemical fertilizers and pesticides application,the development of biological pesticides has attracted more study interests.In recent years,plant-derived antiviral substances stand out due to their advantages of novel structure and good activity,and become one of the hot spots in the screening of antiviral agents.Dendrobium findlayanum belongs to the Dendrobium genus(Orchidaceae)and has a long history of medicinal use.Previous studies have shown that the genus contains various types of chemical constituents with diverse pharmacological activities,however,there is no report on the anti-plant virus at present.In this study,we evaluated the anti-virus activity of secondary metabolites in Dendrobium candidum on three host plants including Nicotiana glutinosa L.,N.tabacum var.Samsun NN and Nicotiana benthamiana,which were infected TMV.The mechanism of their anti-virus activity against TMV was also investigated.The main research results are as follows:(1)A total of 8 compounds were isolated from the petroleum ether and ethyl acetate extracts of Dendrobium findlayanum by multiple chromatography.Based on spectrum data and relevant literature,their chemical structures were identified as2-hydroxy-4,6-dimethoxy-trans-cinnamic acid(1),dendrofindline B(2),findlayline A(3),2,7-dihydroxy-4-methoxy-9-fluorenone(4),the novel ester n-triacontyl coumarae(5),β-sitosterol-3-O-β-D-galactopyranoside(6),daucosterol(7)andβ-sitosterol(8),wherein findlayline A and dendrofindline B are alkaloid compounds.(2)The anti-TMV activity of the above compounds was determined by half leaf blight method.The results showed that findlayline A and dendrofindline B exhibit the best anti-TMV activity with inhibition rates of 14.63% and 38.59%,respectively.(3)The research on the anti-TMV mechanism showed that findlayline A and dendrofindline B could protect the morphological integrity of TMV-infected tobacco protoplast cells.Treated by compounds,the activities of alanine ammonia lyase(PAL),peroxidase(POD)and superoxide dismutase(SOD)in infected plants were significantly higher than in control group.Moreover,the increased activities of POD and PAL,and decreased activity of SOD demonstrated that findlayline A and dendrofindline B can assist cells to remove harmful substances,thereby reducing the activity of SOD.Western blot proves that findlayline A and dendrofindline B can significantly inhibit the expression of TMV virus coat protein.Further real-time fluorescence quantitative PCR detection shows that the virus content in the leaves treated by compounds tend to be higher than that of the control group.In order to verify this conjecture,molecular docking simulations were used to predict the potential interaction between small molecules and TMV virus coat protein.It found that findlayline A and dendrofindline B could bind to the active site of the viral coat protein via polarity interactions,thereby inhibiting the activity of coat protein of Tobacco mosaic virus.