Study On Anti-TMV Activity Of Coreopsis Drummondii And Parthenium Hysterophorus

The antiviral activity of ethanol extracts from 108 plant species belonging to 56 families against Tobacco mosaic virus (TMV) was determined, using local lesion and leaf discs assay. The results showed that about 2/3 plant extracts exhibited certain antiviral activity of inhibiting virus infection, and 48．1% extracts showed more than 50% inhibiting rate at concentration of 10 mg/mL. However, only a few of plant extracts (6 out of 108) showed higher inhibition than other plants on TMV replication. The extracts with over 50% inhibiting rate were then partitioned in turn by use of chloroform and n-butanol, most components with antiviral activity were in the n-butanol phase.By bioassay-guided, the antiviral active constituents from Coreopsis drummondii and Parthenium hysterophorus were isolated and purified. Five constituents (A₁, A₂, A₃, A₄ and A₅) were isolated from Coreopsis drummondii and tow constituents (B₁ and B₂) were from Parthenium hysterophorus. A₁ was first obtained from Coreopsis drummondii. These constituents were identified as 1-phenyl-1,3,5-triheptalkyne (A₁) , (7E,8Z)-1,2,4-Trihydroxy-1-(3-hydroxy-1-butenyl)-2,6,6-trimethycyclohexene (A₂) ,β-stigmasterol (A₃) , stigmasterol (A₄) ,β-sitosterol-3-O-β-D-glucopyranoside (A₅) , Parthenolide (B₁) and Vomifoliol (B₂) , respectively. The results of bioassay indicated that tow constituents (A₁ and B₁) could not only inhibit TMV infection, with 81．3% and 72．1% inhibition at a concentration of 0．2 mg/mL, but also exhibit significant activity against TMV replication, with 84．3% and 72．3% inhibition while they were used at 0．2 mg/mL, respectively.The antiviral mechanism of tow constituents (A₁ and B₁) was studied. A serial experiment, i.e. electron microscope observing, dialyzing and ELISA, was performed to investigate the effect of A₁ and B₁ on TMV particles. The results indicated that A₁ and B₁ could not destroy TMV particles and its antigenic determinant of coat protein. While they could significantly affect the polymerization of coat protein in vitro and showed strong activity reducing TMV-RNA infectivity. The results of real-time PCR showed that A₁ and B₁ could strongly inhibit the expression of RdRp and CP gene. For A₁, the expression levels of RdRp and CP gene were respectively 25．7% and 20．2% of the contrasts at 0．2 mg/mL. As for B₁．the expression levels of RdRp and CP gene were respectively 37．3% and 23．1% of the contrasts at 0．2 mg/mL.The effect of extracts on TMV infection or replication was as the evaluating index. Three extracts with high antiviral effect on TMV infection or replication were selected to mixed by orthogonal design(L₉(3³) ).The evaluating results showed that the most optimum ratio was 1．0mg/mL Brucea javanica, 0．8 mg/mL Coreopsis drummondii, 1．2 mg/mL Epimedium grandiflorum, three extracts mixed according to that ratio showed significantly inhibition on TMV infection, inhibiting rate was 99．6%; The evaluating results about TMV replication showed that the most optimum ratio was 1．0 mg/mL Brucea javanica, 0．5 mg/mL Coreopsis drummondii, 0．8 mg/mL Epimedium grandiflorum, three extracts mixed according to that ratio showed significant inhibition on TMV replication, inhibiting rate was 89．6%. Furthermore, the process of ME of mixed extracts was studied, the composition of 10% ME of mixed extracts was as follows: mixed extracts (10%), DBS-Ca (5%), Nongru 603 (15%), methanol (25%), water (45%).Mixed extracts were sprayed on the Tobaccos before and after inoculating TMV, concentration of TMV is below the contrast in evidence; The results in pot showed that the therapeutic effect with spraying 100 times 10% ME of mixed extracts was 45．6%; On the other hand, The activity of enzymes relating to defendant and contention of chlorophyll in Nicotiana tabacum leaves was determined, after spraying mixed extracts, it showed that the activities of PPO, POD, PAL and SOD, and contentions of chlorophyll a ,chlorophyll b, as well as the gross of chlorophyll were elevated on different degree, after spraying mixed extracts, it indicated that mixed extracts could boost up resistance of tobaccos for TMV at a certain extent.