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Antiviral activity of Eupatorium adenophorum leaf extract against tobacco mosaic virus
Crop Protection 60 (2014) 28e33
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Antiviral activity of Eupatorium adenophorum leaf extract against tobacco mosaic virus Yi Jin a,1, Lingyu Hou a,1, Miaozhi Zhang a, Zhaofeng Tian b, Aocheng Cao c, Xiangming Xie a, * a
College of Biological Sciences and Biotechnology, Beijing Forestry University, 35 QingHua East Road, Beijing 100083, China Institute of Plant & Environment Protection, Beijing Academy of Agriculture & Forestry Sciences, Beijing 100097, China c Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2013 Received in revised form 15 February 2014 Accepted 23 February 2014
Eupatorium adenophorum Spreng is a major invasive plant in China causing great economic losses. Considerable effort has been expended searching for new and innovative methods for integrative management of this species. In this study, an E. adenophorum extract was tested for antiviral activity against tobacco mosaic virus (TMV) using the local lesion assay method, and also characterized. The E. adenophorum leaf extract was able to strongly inhibit TMV infection, with electron microscopic observations indicating that the virus particles had been disaggregated. The active components in the extract are thermostable, stable at acidic pH, and largely not dialyzable. Tobacco seedlings treated with the extract exhibited significantly elevated superoxide dismutase, peroxidase, and polyphenol oxidase activities. Our resultsdthe first reported observations of antiviral activity from leaf extracts of E. adenophorum d suggest that this species is a promising source of antiviral substances for practical use. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Eupatorium adenophorum Spreng Tobacco mosaic virus Antiviral activity Leaf extract
1. Introduction Plants possess many constitutive and inducible mechanisms for protecting themselves against infection by viruses, bacteria, and fungi (Bell, 1981). Many higher plants contain inhibitory substances against virus infection, with some of the most potent of these having been isolated and characterized (Ragetli and Weintraub, 1962; Wyatt and Shepherd, 1969; Irvin, 1975; Straub et al., 1986). Although studies on inhibitors have primarily focused on their distribution and modes of action in plants (Kammen et al., 1961; Smookler, 1971; Grasso and Shepherd, 1978; Nelson and Wheeler, 1978; Sako, 1980; Stirpe et al., 1981), their use as sources of antiviral substances for practical application has also drawn attention. Eupatorium adenophorum Spreng, a member of the family Asteraceae and a native of Mexico and Costa Rica in Central America, is a worldwide noxious invasive weed (Lu et al., 2007). E. adenophorum is a perennial semi-shrubby herbaceous plant with a woody stem basis in older plants. The seeds of E. adenophorum weigh 4.8 2.5 105 g in China. Each mature plant produces 10,000e100,000 seeds per year, which can be transported by wind
* Corresponding author. Tel.: þ86 (0)10 62336016. E-mail address: [email protected] (X. Xie). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.cropro.2014.02.008 0261-2194/Ó 2014 Elsevier Ltd. All rights reserved.
over long distances due to their low weight (Sang et al., 2010). E. adenophorum can now be found in the city of Chongqing, and Yunnan, Sichuan, Guizhou, Guangxi, Taiwan, and Hubei provinces (Lu and Ma, 2004). Although much attention has been paid to management of E. adenophorum, with several control methodsd manual, chemical, and biologicaldhaving been attempted, there has been no significant progress towards its eradication (Gu et al., 2008). E. adenophorum is strongly stress-resistant and contains many biologically active substances. Various types of compounds have been isolated from E. adenophorum, including terpenoids, sterols, flavonoids, phenylpropanoids, coumarins, alkaloids, caffeic acid, polysaccharides, essential oils and so on, and different biological activities (such as antiviral, antibacterial and antifeedant activities) of these constituents have also been reported (Kurade et al., 2010; Wei et al., 2010, 2011; Shi et al., 2012; Jin et al., 2013). Because of the wide distribution and abundance in areas of China prone to disasters, it may be possible to exploit its active substances as biological pesticides to prevent major plant viral diseases. Although such usage would be beneficial for protecting the environment and would bring significant social, ecological, and economic profits, only a few studies assessing the utility of E. adenophorum as a biological pesticide have been reported (Sharma and Mehta, 2009; Zou et al., 2009; Shi et al., 2012). In this study, we investigated the effects of leaf extract of E. adenophorum against Tobacco mosaic
Y. Jin et al. / Crop Protection 60 (2014) 28e33
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TMV was maintained on its systemic host, Nicotiana tabacum cv. Samsun NN. Systemically infected leaves were used as inoculum sources in all experiments. Fresh infected N. tabacum leaves (1 g) were crushed together with 15 mL phosphate-buffered saline (PBS, pH 7.0; 0.01 M) in a sterilized mortar and pestle. The pulp was strained through two layers of muslin cloth, and the filtrate was centrifuged at 3000 rpm for 15 min. The resulting clear supernatant was used for inoculation purposes. The virus inoculum plaqueforming unit (PFU) count was 1.62 103 lesions/mL.
clay pots, 10e15 cm in diameter and filled with sterilized soil. Plants of the same height, age, and vigor were selected for experiments. For the antiviral assay, the tobacco leaves were sprayed with 5 mL of different E. adenophorum leaf extract dilutions, while the control group was treated with distilled water or tobacco leaf extract. Before or after distilled water or extract treatments, the leaves were inoculated with 0.2 mL TMV inoculums. The TMV inoculation was performed using a standard mechanical rubbing method based on Cao et al. (2006). The inoculated plants were maintained at 20e28 C under cool-white fluorescent lamps (Daye night fluctuation). Lesions on the inoculated leaves were photographed 3 d after infection. Each treatment consisted of three replicates, with at least five tobacco seedlings per replicate. Percent inhibition was calculated using the formula (C T) 100 / C, where C is the number of lesions on control leaves and T is the number of lesions on treated leaves. Lesion data was statistically analyzed, and differences in percent inhibition were evaluated at a p 0.05 significance level using Duncan’s multiple range test.
2.2. Extract preparation
2.5. Thermal stability
Leaves were collected from E. adenophorum plants in Yunnan Province, China. An extract was prepared from fresh leaves using a previously described protocol (Chakravarty et al., 2004; Chakravarty and Yasmin, 2005, 2008; Chakravarty et al., 2009). The leaves were sliced and dried in the shade; the dried materials were then pulverized in a mixer and filtered through a 40-mesh screen. A 100 g portion of the powdered leaf tissue was extracted by ultrasonic treatment with 1000 mL ethanol for 30 min. After collecting the supernatant, the precipitate was resuspended in another 1000 mL of ethanol and subjected to a second 30-min extraction. The resulting supernatant was combined with the one obtained from the first extraction. The final supernatant was evaporated to dryness on a rotary evaporator. The final quantity of extract obtained per 100 g leaves was about 7 g. This leaf extract was diluted with water to give a final concentration of 0.1 g/mL and filtered.
Test tubes containing partially clarified E. adenophorum leaf extract were heated at 40, 50, 60, 70, and 80 C for 10 min in a temperature-controlled water bath. Tube contents were allowed to cool, and then centrifuged at 5000 rpm for 15 min. The clear supernatant was tested for antiviral activity as described above.
virus (TMV) and found that it could induce resistance against the virus. Because of the low cost of E. adenophorum leaf extract production, this species has potential as a pesticide in the control of TMV disease in tobacco seedlings. 2. Materials and methods 2.1. Virus and virus inoculum preparation
2.3. Virus purification and electron microscopic observation Wild-type TMV from infected N. tabacum was isolated and purified as described by Gooding and Hebert (1967). Virions were further purified by centrifugation at 22,000 rpm for 2 h in a 10e40% sucrose density gradient at 4 C. The white band corresponding to the virus layer was extracted, and then pelleted by centrifugation in a solution of water at 30,000 rpm in a Beckman TI80 rotor for 2 h at 4 C. The pellet was resuspended in water and analyzed for virus concentration using a Unico UV-2102C spectrophotometer. Virus concentrations for TMV were determined by measuring absorbance at 260 nm, corrected for light scattering at 325 nm, using an extinction coefficient of 3.01 cm2/mg. Purified virus particles were mixed with an equal volume of E. adenophorum extract (0.05 g/mL) for 30, 60, or 90 min at room temperature. The mixture was placed on carbon-coated grids and negatively stained with 0.01 mL of 2% phosphotungstic acid for 1 min at room temperature before being allowed to dry. The samples were examined with a Hitachi electron microscope. An untreated virus sample served as a control. 2.4. Antiviral activity assay N. tabacum cv. Samsun NN plants were used as hypersensitive hosts for TMV. Seeds of the test hosts were sown in composted soil in the greenhouse, with the resulting seedlings transplanted into
2.6. Effect of pH The pH of E. adenophorum extract was adjusted to 4e11 by adding HCl or NaOH, and maintained for 1 h at each pH. The solutions were then brought to pH 7 with NaOH or HCl and rubbed on nine leaves of N. tabacum cv. Samsun NN plants. Samples were tested for antiviral activity as described above. 2.7. Dialysis assay E. adenophorum leaf extract was placed in a dialysis bag and dialyzed against three times its volume of distilled water at 4 C for 48 h. The molecular weight cut off of the dialysis bags was 14.4 KDa (Realtimes, USA). Undialyzed sample diluted to 0.05 g/mL and maintained for 48 h at 4 C served as a control. The dialyzate and bag contents were tested for antiviral activity as described above. 2.8. Determination of superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) in N. tabacum cv. Samsun NN Tobacco seedlings were sprayed with 5 mL of extract (0.05 g/mL) or distilled water 3 d and 1 d before TMV infection. Each day after infection, 1 g of leaf material was crushed with 4 mL of 50 mM phosphate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 2% (w/v) polyvinylpolypyrrolidone (PVP) in a sterilized mortar and pestle. The homogenate was centrifuged at 12,000 g for 25 min at 4 C. SOD activity was determined using the method of Beauchamp and Fridovich (1971), which measures inhibition of the photochemical reduction of nitroblue tetrazolium spectrophotometrically at 560 nm. POD activity was determined using guaiacol as a hydrogen donor by measuring change in optical density at 460 nm as reported previously (Ippolito et al., 2000). PPO activity was assayed using catechol as a substrate by measuring absorbance change at 398 nm, as described by Liu et al. (2007). POD and PPO activities were expressed in terms of units, where one unit represents the absorbance increase per mass of protein per min. Each
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Y. Jin et al. / Crop Protection 60 (2014) 28e33
treatment consisted of three replicates, with five tobacco seedlings per replicate.
Table 2 Effect of temperature on the inhibitory activity of Eupatorium adenophorum extract. Temperatureb
2.9. Statistical analysis
Number of lesions on 6 half leavesc Treatment
40 C
The experiments were arranged in a completely randomized block design with three replicates. Enzyme activity was calculated based on non-linear regression analysis of the raw data using GraphPad PRISMÒ software. Comparisons with p-values 0.05 were considered significantly different. In tables, values followed by the same letters within a column were not significantly different based on Duncan’s multiple range tests at p 0.05. All values reported were means SD of three replicates.
Inhibition (%)
Control 566 34
79.69a
729 53
78.6a
555 32
76.94a
927 67
74.3a
1032 84
72.87a
606 51
29.29a
98 13
50 C
156 21
60 C
128 14
70 C
238 27
80 C
280 36 Control 119 19
3. Results
Test plants were treated with 5 mL of leaf extract (0.05 g/mL) before infection. b Each test sample was placed in a glass tube, heated for 10 min, and then cooled. c Total number of local lesions on six half-leaves of Nicotiana tabacum cv. Samsun N.
3.1. Antiviral activity assay Inhibition of TMV infection by an E. adenophorum leaf extract was examined in N. tabacum cv. Samsun NN seedlings. Seedlings treated with the E. adenophorum leaf extract 24 h before virus challenge displayed significant inhibitory activity to TMV infection compared to the control group and the tobacco leaf extract treated group (Table 1). As shown in Table 1, the inhibition rate was 83.0% when a 1:1 dilution was used, and 66.6% with a 1:2 dilution; when the extract was diluted 1:5, the inhibition rate dropped to 40.4%. When the extract was applied 24 h after TMV infection, however, the inhibitory effect of the extract declined and showed only slight antiviral activity. At a dilution of 1:1, the inhibition rate was only 49.3%. It appears that the E. adenophorum extract had a relatively good preventive effect on TMV infection, but an inadequate curative effect. Data analysis also indicated that inhibitory rates at different dilutions differed significantly (Table 1). The antiviral activity of the extract was obviously dose-dependent. 3.2. Thermal stability As shown in Table 2, the inhibitory activity of the E. adenophorum extract was fairly thermostable between 40 and 80 C. After heating aliquots of the extract for 10 min at various temperatures ranging from 40 to 80 C, the inhibition rate remained above 70%. Even heating to 80 C for 10 min did not reduce its antiviral activity.
Table 3 Effect of pH on the inhibitory activity of Eupatorium adenophorum extract. pHd
Number of lesions on 9 half leavese Treatment
4 5 6 7 8 9 10 11
324 639 695 213 980 1160 1575 1460
Inhibition (%)
Control
18 26 24 11 42 46 32 43
608 1356 1157 786 1363 1575 1894 1323
29 42 42 31 76 87 78 27
46.71b 53.16b 66.81a 72.63a 28.10c 26.35c 20.21c 19.94c
Test plants were treated with 5 mL of leaf extract (0.05 g/mL) before infection. d Each test sample was adjusted to pH 4e11, incubated for 1 h, and then brought to pH 7. e Total number of local lesions on nine half-leaves of Nicotiana tabacum cv. Samsun NN.
relatively active at acidic pH (pH 4e6), but was not stable at basic pH: inhibition at pH 8e11 was less than half of that observed at acidic pH.
3.3. Effect of pH
3.4. Effect of dialysis
As indicated by the results given in Table 3, inhibitory activity was influenced by pH. Optimum activity was observed at pH 7, providing an inhibition rate of 72.6%. The crude extract was
After dialysis against distilled water at 4 C for 48 h, inhibitory activity was retained within the bag, providing 71.2% inhibition. By comparison, viral infection was 75.4% inhibited in a control group
Table 1 Effect of Eupatorium adenophorum leaf extract on TMV infection when applied before or after virus inoculation. Dilutiond
Applied before or after virus inoculation
Number of lesionse Treatment 1f
1:1 1:2 1:5
Before After Before After Before After
614 483 816 621 1007 711
35 28 56 39 103 68
Test plants were treated with 5 mL of leaf extract 24 h before or after TMV infection. d Eupatorium adenophorum leaf extract was diluted 1:1 to 1:5 with distilled water. e Total number of local lesions on nine half-leaves of Nicotiana tabacum cv. Samsun NN. f This treatment was treated by Eupatorium adenophorum extract. g Nicotiana tabacum cv. Samsun NN extract treated.
Inhibition (%) Treatment 2g 1453 935 1241 975 1354 980
89 52 94 76 84 75
Control 1704 979 1412 1014 1512 1014
275 69 194 112 147 89
Treatment 1
Treatment 2
82.97a 49.29a 66.58b 38.76b 40.4c 29.88c
14.73a 4.49a 12.11b 3.85b 10.45c 3.35c
Y. Jin et al. / Crop Protection 60 (2014) 28e33
treated with 0.05 g/mL extract but not subjected to dialysis treatment. This inhibition rate did not differ significantly from that observed after dialysis treatment. Virus particles appeared to be destroyed by the E. adenophorum extract and their infection capability was not restored by dialysis. The inhibitory rate was consequently nearly identical to that of the control. These results also demonstrate that the active component in the extract was nondialyzable and apparently macromolecular. 3.5. Electron microscopic observations Samples containing E. adenophorum extract and TMV solution were examined under an electron microscope as described in Materials and Methods. As shown in Fig. 1, differences in particle morphology or size distribution were found after treatment with the extract for different durations. Compared with the control (Fig. 1A), TMV particles were more-or-less destroyed and obviously shortened (Fig. 1B and C), and then became disaggregated (about 70 damaged particles/100 particles) (Fig. 1D). The observed effect was also dependent upon mixing time. 3.6. Determination of superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) in N. tabacum cv. Samsun NN Tobacco seedlings were sprayed with 5 mL of extract (0.05 g/mL) or distilled water, and re-sprayed 48 h later. After an additional 24 h, leaves were inoculated with TMV. Extractions for enzyme testing were performed the following day. Activity of SOD, which catalyzes the dismutation of O2 to H2O2 and O2 (Bowler et al., 1992), was shown in Fig. 2A for water-treated (WT), E. adenophorum extract-treated (EAET), and untreated virus free plants control (Control) groups. The highest total SOD activity in tobacco leaves was observed at day 4 post-infection. At that time
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point, SOD levels were 2.1-fold higher in WT and EAET groups than in the Control group. SOD levels were always lower in the Control group than in the other groups. SOD activity in the EAET group was higher than that of WT at days 2 and 3 post-infection. Extract treatment and TMV inoculation also increased POD activity in tobacco seedlings. On day 5, POD levels in the EAET group were 2.2and 5.2-fold higher than in WT and Control leaves, respectively (Fig. 2B). PPO activity was maintained at a relatively higher level in the EAET group than in the WT and Control groups; on day 3, PPO levels were 2.5- and 8.3-fold higher in this group than in WT and Control, respectively (Fig. 2C). These results suggest E. adenophorum leaf extract can induce a host resistance reaction against viral infection.
4. Discussion In this study, we investigated the antiviral activities of E. adenophorum leaf extract. We first observed that the leaf extract could inactivate TMV particles. Further experiments demonstrated that the extract is effective for prevention, and also has a slight curative effect. The putative inhibitor is thermostable, stable at acidic pH, and largely not dialyzable. The antiviral mechanism appears to involve direct inhibition of viral particles and promotion of a response in the host plant. Although the active compound has not yet been isolated and determined, E. adenophorum holds promise as a source of antiviral substances for practical use. In addition, the use of E. adenophorum leaf extract represents an alternative strategy in the development of novel plant-based biological pesticides and comprehensive management of invasive weeds. Plant viral diseases are some of the most serious biological diseases in agricultural production, with affected areas and degree of virus-caused damage gradually increasing. Although chemical controls have been used previously (Wang et al., 2008), they have
Fig. 1. Electron microscopy of morphological changes in TMV particles. (A) Normal TMV particles; (B) TMV after treatment with E. adenophorum extract for 30 min; (C) TMV after treatment with E. adenophorum extract for 60 min; (D) TMV after treatment with E. adenophorum extract for 90 min. The concentration of the E. adenophorum extract was 0.05 g/mL, and that of purified TMV was 0.5 mg/mL. The sample was observed under 12000 magnification using a Hitachi electron microscope.
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Y. Jin et al. / Crop Protection 60 (2014) 28e33
Fig. 2. Effect of E. adenophorum extract on SOD, POD, and PPO activity. Tobacco seedlings were sprayed with 5 mL extract (0.05 g/mL) or distilled water 3 d and 1 d before TMV infection. Activities of (A) SOD, (B) POD, and (C) PPO in E. adenophorum extract-treated (EAET), distilled water-treated (WT), and Control groups were examined 24 h after infection. Results were the mean SD values obtained from three independent experiments.
negative ecological impacts. Much attention is currently focused on research and development of environmentally-friendly biological pesticides because of their simple extraction protocols and obvious effects. Because E. adenophorum was widely distributed in China as an invasive plant, considerable effort has being made to find new and innovative methods for its integrative management. The chemical composition and comprehensive application of E. adenophorum have been extensively studied, with its use as a biofuel, biological medicine, bio-fertilizer, and dyestuff (Sun et al., 2004). Various plants, including: Phytolacca Americana L., Pelargonium hortorum Bailey, Chenopodium album Linn, Chenopodium amaranticolor Coste et Reyn, Capsicum frutescens L., Azadirachta indica A. Juss., Vitis vinifera L., and Rosa banksiae Ait., possess antiviral factors (Blaszczak et al., 1959; Sangar and Dhingra, 1982; Zarling et al., 1991; Vivanco et al., 1999). Plant-derived antiviral compounds are active against plant, animal and human viruses (Vivanco et al., 1999). We previously reported that the polysaccharide from E. adenophorum leaf extract was a prophylactic and immune enhancement agent against H5N1 influenza virus infection in mouse model (Jin et al., 2013). In this study, the E. adenophorum leaf extract showed significant inhibitory activity against TMV infection when plants were treated prior to viral challenge. Antioxidant enzyme system responses are some of the most important environmental acclimatization mechanisms in plants (Lu et al., 2008). The role of these enzyme systems in development of plant tolerance to extreme environments has been clearly demonstrated (Dhindsa and Matowe, 1981; Schoner and Heinrich Krause, 1990; Hernandez et al., 2001). To mitigate and repair damage initiated by active oxygen species, plants have evolved cellular adaptive responses such as upregulation of oxidative stress protectors and accumulation of protective solutes (Horling et al., 2003). Antioxidant defense enzymes such as superoxide dismutase (SOD), polyphenol oxidase (PPO), and guaiacol peroxidase (POD) minimize superoxide and hydrogen peroxide concentrations. POD is generally considered to be potentially important in host resistance mechanisms because it can catalyze the last step of lignin biosynthesis (Hammerschmidt et al., 1982) and can oxidize phenolic compounds to quinones (Campos-Vargas and Saltveit,
2002). In addition, high concentrations of phenolic compounds around an infected area can restrict or weaken pathogen growth (Reimers and Leach, 1991). In addition to possessing their own antimicrobial activities, phenolic compounds can be oxidized by PPO and POD, resulting in substances such as quinones that are even more toxic to pathogens (Wang et al., 2009). In this study, SOD, POD, and PPO activities were elevated by E. adenophorum leaf extract treatment. Based on observed morphological changes to extract-treated TMV particles, the antiviral effect of E. adenophorum leaf extract is most likely due to direct destruction of viral particles and induction of host response against TMV. In conclusion, we have demonstrated in this study that E. adenophorum contains a potent inhibitor against TMV infection. Although the isolation of the active antiviral constituents still requires lots of further studies, E. adenophorum may eventually be exploited as a pesticide source, providing a foundation for comprehensive use of this invasive weed and for its ecologically sustainable development. Acknowledgments We thank Professor Chenggui Han, College of Agricultural Biotechnology, Chinese Agricultural University, China, for his kind help in the purification of virus. This work was supported by the Fundamental Research Funds for the Central University (No. TD2012-03), the Public Welfare Project from Ministry of Agriculture of the People’s Republic of China (Grant No. 201103027) and the Beneficial Professional Project Grant (Ministry of Agriculture, China, Grant No. 200803021-030). References Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276e287. Bell, A.A., 1981. Biochemical mechanisms of disease resistance. Annu. Rev. Plant Physiol. 32, 21e81. Blaszczak, W., Ross, A., Larson, R., 1959. The inhibitory activity of plant juices on the infectivity of potato virus X. Phytopathology 49, 784e791. Bowler, C., Montagu, M.v., Inze, D., 1992. Superoxide dismutase and stress tolerance. Annu. Rev. Plant Biol. 43, 83e116.
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Lu, P., Sang, W.G., Ma, K.P., 2008. Differential responses of the activities of antioxidant enzymes to thermal stresses between two invasive Eupatorium species in China. J. Integr. Plant Biol. 50, 393e401. Lu, Z., Ma, K., 2004. The influence of topographical factors on the invasion of the alien species, Eupatorium adenophorum. Acta Phytoecol. Sin. 28, 761. Nelson, M., Wheeler, R., 1978. Biological and serological characterization and separation of potyviruses that infect peppers. Phytopathology 68, 979e984. Ragetli, H., Weintraub, M., 1962. Purification and characteristics of a virus inhibitor from Dianthus caryophyllus L.: II. Characterization and mode of action. Virology 18, 241e248. Reimers, P., Leach, J., 1991. Race-specific resistance to Xanthomonas oryzae pv. oryzae conferred by bacterial blight resistance gene Xa-10 in rice (Oryza sativa) involves accumulation of a lignin-like substance in host tissues. Physiol. Mol. Plant Pathol. 38, 39e55. Sako, N., 1980. Loss of aphid transmissibility of turnip mosaic virus. Phytopathology 70, 647. Sangar, R., Dhingra, M., 1982. Potato virus inhibitor from neem leaf extract. J. Indian Potato Assoc. 9, 143e149. Sang, W., Zhu, L., Axmacher, J.C., 2010. Invasion pattern of Eupatorium adenophorum Spreng in southern China. Biol. Invasions 12, 1721e1730. Schoner, S., Heinrich Krause, G., 1990. Protective systems against active oxygen species in spinach: response to cold acclimation in excess light. Planta 180, 383e389. Sharma, S., Mehta, P.K., 2009. Bioefficacy of plant extracts against cabbage aphid. Res. Crops 10, 98e100. Shi, W., Luo, S.H., Li, S.H., 2012. Defensive Sesquiterpenoids from leaves of Eupatorium adenophorum. Chin. J. Chem. 30, 1331e1334. Smookler, M., 1971. Properties of inhibitors of plant virus infection occurring in the leaves of species in the Chenopodiales. Ann. Appl. Biol. 69, 157e168. Stirpe, F., Williams, D.G., Onyon, L.J., Legg, R.F., Stevens, W.A., 1981. Dianthins, ribosome-damaging proteins with anti-viral properties from Dianthus caryophyllus L. (carnation). Biochem. J. 195, 399. Straub, P., Adam, G., Mundry, K., 1986. Isolation and characterization of a virus inhibitor from spinach (Spinacia oleracea L.). J. Phytopathol. 115, 357e367. Sun, X., Lu, Z., Sang, W., 2004. Review on studies of Eupatorium adenophoruman important invasive species in China. J. For. Res. 15, 319e322. Vivanco, J.M., Querci, M., Salazar, L.F., 1999. Antiviral and antiviroid activity of MAPcontaining extracts from Mirabilis jalapa roots. Plant Dis. 83, 1116e1121. Wang, F., Feng, G., Chen, K., 2009. Defense responses of harvested tomato fruit to burdock fructooligosaccharide, a novel potential elicitor. Postharvest Biol. Technol. 52, 110e116. Wang, Z., Peng, W., Cai, J., He, J., Cai, Q., 2008. Extracting mycostatic materials of two plants and mycostatic test. Chin. Agric. Sci. Bull. 2, 72e78. Wei, Y., Gao, Y., Zhang, K., Ito, Y., 2010. Isolation of caffeic acid from eupatorium adenophorum Spreng by high-speed countercurrent chromatography and synthesis of caffeic acid-intercalated layered double hydroxide. J. Liq. Chromatogr. Relat. Technol. 33, 837e845. Wei, Y., Zhang, K., Zhang, G.L., Ito, Y., 2011. Isolation of five bioactive components from Eupatorium adenophorum Spreng using stepwise elution by high-speed countercurrent chromatography. J. Liq. Chromatogr. Relat. Technol. 34, 2505e2515. Wyatt, S., Shepherd, R., 1969. Isolation and characterization of a virus inhibitor from Phytolacca americana. Phytopathology 59, 1787. Zarling, J.M., Moran, P.A., Haffar, O., Diegel, M., Myers, D.E., Kuelbeck, V., Ledbetter, J.A., Uckun, F.M., 1991. Inhibition of HIV-1 replication in seropositive patients’ CD4þ T-cells by pokeweed antiviral protein-monoclonal antibody conjugates. Int. J. Immunopharmacol. 13 (Suppl. 1), 63e68. Zou, F.C., Duan, G., Xie, Y.J., Zhou, Y., Dong, G.D., Lin, R.Q., Zhu, X.Q., 2009. Molluscicidal activity of the plant Eupatorium adenophorum against Oncomelania hupensis, the intermediate host snail of Schistosoma japonicum. Ann. Trop. Med. Parasitol. 103, 549e553.
Contents lists available at ScienceDirect
Crop Protection journal homepage: www.elsevier.com/locate/cropro
Antiviral activity of Eupatorium adenophorum leaf extract against tobacco mosaic virus Yi Jin a,1, Lingyu Hou a,1, Miaozhi Zhang a, Zhaofeng Tian b, Aocheng Cao c, Xiangming Xie a, * a
College of Biological Sciences and Biotechnology, Beijing Forestry University, 35 QingHua East Road, Beijing 100083, China Institute of Plant & Environment Protection, Beijing Academy of Agriculture & Forestry Sciences, Beijing 100097, China c Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 27 June 2013 Received in revised form 15 February 2014 Accepted 23 February 2014
Eupatorium adenophorum Spreng is a major invasive plant in China causing great economic losses. Considerable effort has been expended searching for new and innovative methods for integrative management of this species. In this study, an E. adenophorum extract was tested for antiviral activity against tobacco mosaic virus (TMV) using the local lesion assay method, and also characterized. The E. adenophorum leaf extract was able to strongly inhibit TMV infection, with electron microscopic observations indicating that the virus particles had been disaggregated. The active components in the extract are thermostable, stable at acidic pH, and largely not dialyzable. Tobacco seedlings treated with the extract exhibited significantly elevated superoxide dismutase, peroxidase, and polyphenol oxidase activities. Our resultsdthe first reported observations of antiviral activity from leaf extracts of E. adenophorum d suggest that this species is a promising source of antiviral substances for practical use. Ó 2014 Elsevier Ltd. All rights reserved.
Keywords: Eupatorium adenophorum Spreng Tobacco mosaic virus Antiviral activity Leaf extract
1. Introduction Plants possess many constitutive and inducible mechanisms for protecting themselves against infection by viruses, bacteria, and fungi (Bell, 1981). Many higher plants contain inhibitory substances against virus infection, with some of the most potent of these having been isolated and characterized (Ragetli and Weintraub, 1962; Wyatt and Shepherd, 1969; Irvin, 1975; Straub et al., 1986). Although studies on inhibitors have primarily focused on their distribution and modes of action in plants (Kammen et al., 1961; Smookler, 1971; Grasso and Shepherd, 1978; Nelson and Wheeler, 1978; Sako, 1980; Stirpe et al., 1981), their use as sources of antiviral substances for practical application has also drawn attention. Eupatorium adenophorum Spreng, a member of the family Asteraceae and a native of Mexico and Costa Rica in Central America, is a worldwide noxious invasive weed (Lu et al., 2007). E. adenophorum is a perennial semi-shrubby herbaceous plant with a woody stem basis in older plants. The seeds of E. adenophorum weigh 4.8 2.5 105 g in China. Each mature plant produces 10,000e100,000 seeds per year, which can be transported by wind
* Corresponding author. Tel.: þ86 (0)10 62336016. E-mail address: [email protected] (X. Xie). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.cropro.2014.02.008 0261-2194/Ó 2014 Elsevier Ltd. All rights reserved.
over long distances due to their low weight (Sang et al., 2010). E. adenophorum can now be found in the city of Chongqing, and Yunnan, Sichuan, Guizhou, Guangxi, Taiwan, and Hubei provinces (Lu and Ma, 2004). Although much attention has been paid to management of E. adenophorum, with several control methodsd manual, chemical, and biologicaldhaving been attempted, there has been no significant progress towards its eradication (Gu et al., 2008). E. adenophorum is strongly stress-resistant and contains many biologically active substances. Various types of compounds have been isolated from E. adenophorum, including terpenoids, sterols, flavonoids, phenylpropanoids, coumarins, alkaloids, caffeic acid, polysaccharides, essential oils and so on, and different biological activities (such as antiviral, antibacterial and antifeedant activities) of these constituents have also been reported (Kurade et al., 2010; Wei et al., 2010, 2011; Shi et al., 2012; Jin et al., 2013). Because of the wide distribution and abundance in areas of China prone to disasters, it may be possible to exploit its active substances as biological pesticides to prevent major plant viral diseases. Although such usage would be beneficial for protecting the environment and would bring significant social, ecological, and economic profits, only a few studies assessing the utility of E. adenophorum as a biological pesticide have been reported (Sharma and Mehta, 2009; Zou et al., 2009; Shi et al., 2012). In this study, we investigated the effects of leaf extract of E. adenophorum against Tobacco mosaic
Y. Jin et al. / Crop Protection 60 (2014) 28e33
29
TMV was maintained on its systemic host, Nicotiana tabacum cv. Samsun NN. Systemically infected leaves were used as inoculum sources in all experiments. Fresh infected N. tabacum leaves (1 g) were crushed together with 15 mL phosphate-buffered saline (PBS, pH 7.0; 0.01 M) in a sterilized mortar and pestle. The pulp was strained through two layers of muslin cloth, and the filtrate was centrifuged at 3000 rpm for 15 min. The resulting clear supernatant was used for inoculation purposes. The virus inoculum plaqueforming unit (PFU) count was 1.62 103 lesions/mL.
clay pots, 10e15 cm in diameter and filled with sterilized soil. Plants of the same height, age, and vigor were selected for experiments. For the antiviral assay, the tobacco leaves were sprayed with 5 mL of different E. adenophorum leaf extract dilutions, while the control group was treated with distilled water or tobacco leaf extract. Before or after distilled water or extract treatments, the leaves were inoculated with 0.2 mL TMV inoculums. The TMV inoculation was performed using a standard mechanical rubbing method based on Cao et al. (2006). The inoculated plants were maintained at 20e28 C under cool-white fluorescent lamps (Daye night fluctuation). Lesions on the inoculated leaves were photographed 3 d after infection. Each treatment consisted of three replicates, with at least five tobacco seedlings per replicate. Percent inhibition was calculated using the formula (C T) 100 / C, where C is the number of lesions on control leaves and T is the number of lesions on treated leaves. Lesion data was statistically analyzed, and differences in percent inhibition were evaluated at a p 0.05 significance level using Duncan’s multiple range test.
2.2. Extract preparation
2.5. Thermal stability
Leaves were collected from E. adenophorum plants in Yunnan Province, China. An extract was prepared from fresh leaves using a previously described protocol (Chakravarty et al., 2004; Chakravarty and Yasmin, 2005, 2008; Chakravarty et al., 2009). The leaves were sliced and dried in the shade; the dried materials were then pulverized in a mixer and filtered through a 40-mesh screen. A 100 g portion of the powdered leaf tissue was extracted by ultrasonic treatment with 1000 mL ethanol for 30 min. After collecting the supernatant, the precipitate was resuspended in another 1000 mL of ethanol and subjected to a second 30-min extraction. The resulting supernatant was combined with the one obtained from the first extraction. The final supernatant was evaporated to dryness on a rotary evaporator. The final quantity of extract obtained per 100 g leaves was about 7 g. This leaf extract was diluted with water to give a final concentration of 0.1 g/mL and filtered.
Test tubes containing partially clarified E. adenophorum leaf extract were heated at 40, 50, 60, 70, and 80 C for 10 min in a temperature-controlled water bath. Tube contents were allowed to cool, and then centrifuged at 5000 rpm for 15 min. The clear supernatant was tested for antiviral activity as described above.
virus (TMV) and found that it could induce resistance against the virus. Because of the low cost of E. adenophorum leaf extract production, this species has potential as a pesticide in the control of TMV disease in tobacco seedlings. 2. Materials and methods 2.1. Virus and virus inoculum preparation
2.3. Virus purification and electron microscopic observation Wild-type TMV from infected N. tabacum was isolated and purified as described by Gooding and Hebert (1967). Virions were further purified by centrifugation at 22,000 rpm for 2 h in a 10e40% sucrose density gradient at 4 C. The white band corresponding to the virus layer was extracted, and then pelleted by centrifugation in a solution of water at 30,000 rpm in a Beckman TI80 rotor for 2 h at 4 C. The pellet was resuspended in water and analyzed for virus concentration using a Unico UV-2102C spectrophotometer. Virus concentrations for TMV were determined by measuring absorbance at 260 nm, corrected for light scattering at 325 nm, using an extinction coefficient of 3.01 cm2/mg. Purified virus particles were mixed with an equal volume of E. adenophorum extract (0.05 g/mL) for 30, 60, or 90 min at room temperature. The mixture was placed on carbon-coated grids and negatively stained with 0.01 mL of 2% phosphotungstic acid for 1 min at room temperature before being allowed to dry. The samples were examined with a Hitachi electron microscope. An untreated virus sample served as a control. 2.4. Antiviral activity assay N. tabacum cv. Samsun NN plants were used as hypersensitive hosts for TMV. Seeds of the test hosts were sown in composted soil in the greenhouse, with the resulting seedlings transplanted into
2.6. Effect of pH The pH of E. adenophorum extract was adjusted to 4e11 by adding HCl or NaOH, and maintained for 1 h at each pH. The solutions were then brought to pH 7 with NaOH or HCl and rubbed on nine leaves of N. tabacum cv. Samsun NN plants. Samples were tested for antiviral activity as described above. 2.7. Dialysis assay E. adenophorum leaf extract was placed in a dialysis bag and dialyzed against three times its volume of distilled water at 4 C for 48 h. The molecular weight cut off of the dialysis bags was 14.4 KDa (Realtimes, USA). Undialyzed sample diluted to 0.05 g/mL and maintained for 48 h at 4 C served as a control. The dialyzate and bag contents were tested for antiviral activity as described above. 2.8. Determination of superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) in N. tabacum cv. Samsun NN Tobacco seedlings were sprayed with 5 mL of extract (0.05 g/mL) or distilled water 3 d and 1 d before TMV infection. Each day after infection, 1 g of leaf material was crushed with 4 mL of 50 mM phosphate buffer (pH 7.0) containing 1 mM ethylenediaminetetraacetic acid (EDTA) and 2% (w/v) polyvinylpolypyrrolidone (PVP) in a sterilized mortar and pestle. The homogenate was centrifuged at 12,000 g for 25 min at 4 C. SOD activity was determined using the method of Beauchamp and Fridovich (1971), which measures inhibition of the photochemical reduction of nitroblue tetrazolium spectrophotometrically at 560 nm. POD activity was determined using guaiacol as a hydrogen donor by measuring change in optical density at 460 nm as reported previously (Ippolito et al., 2000). PPO activity was assayed using catechol as a substrate by measuring absorbance change at 398 nm, as described by Liu et al. (2007). POD and PPO activities were expressed in terms of units, where one unit represents the absorbance increase per mass of protein per min. Each
30
Y. Jin et al. / Crop Protection 60 (2014) 28e33
treatment consisted of three replicates, with five tobacco seedlings per replicate.
Table 2 Effect of temperature on the inhibitory activity of Eupatorium adenophorum extract. Temperatureb
2.9. Statistical analysis
Number of lesions on 6 half leavesc Treatment
40 C
The experiments were arranged in a completely randomized block design with three replicates. Enzyme activity was calculated based on non-linear regression analysis of the raw data using GraphPad PRISMÒ software. Comparisons with p-values 0.05 were considered significantly different. In tables, values followed by the same letters within a column were not significantly different based on Duncan’s multiple range tests at p 0.05. All values reported were means SD of three replicates.
Inhibition (%)
Control 566 34
79.69a
729 53
78.6a
555 32
76.94a
927 67
74.3a
1032 84
72.87a
606 51
29.29a
98 13
50 C
156 21
60 C
128 14
70 C
238 27
80 C
280 36 Control 119 19
3. Results
Test plants were treated with 5 mL of leaf extract (0.05 g/mL) before infection. b Each test sample was placed in a glass tube, heated for 10 min, and then cooled. c Total number of local lesions on six half-leaves of Nicotiana tabacum cv. Samsun N.
3.1. Antiviral activity assay Inhibition of TMV infection by an E. adenophorum leaf extract was examined in N. tabacum cv. Samsun NN seedlings. Seedlings treated with the E. adenophorum leaf extract 24 h before virus challenge displayed significant inhibitory activity to TMV infection compared to the control group and the tobacco leaf extract treated group (Table 1). As shown in Table 1, the inhibition rate was 83.0% when a 1:1 dilution was used, and 66.6% with a 1:2 dilution; when the extract was diluted 1:5, the inhibition rate dropped to 40.4%. When the extract was applied 24 h after TMV infection, however, the inhibitory effect of the extract declined and showed only slight antiviral activity. At a dilution of 1:1, the inhibition rate was only 49.3%. It appears that the E. adenophorum extract had a relatively good preventive effect on TMV infection, but an inadequate curative effect. Data analysis also indicated that inhibitory rates at different dilutions differed significantly (Table 1). The antiviral activity of the extract was obviously dose-dependent. 3.2. Thermal stability As shown in Table 2, the inhibitory activity of the E. adenophorum extract was fairly thermostable between 40 and 80 C. After heating aliquots of the extract for 10 min at various temperatures ranging from 40 to 80 C, the inhibition rate remained above 70%. Even heating to 80 C for 10 min did not reduce its antiviral activity.
Table 3 Effect of pH on the inhibitory activity of Eupatorium adenophorum extract. pHd
Number of lesions on 9 half leavese Treatment
4 5 6 7 8 9 10 11
324 639 695 213 980 1160 1575 1460
Inhibition (%)
Control
18 26 24 11 42 46 32 43
608 1356 1157 786 1363 1575 1894 1323
29 42 42 31 76 87 78 27
46.71b 53.16b 66.81a 72.63a 28.10c 26.35c 20.21c 19.94c
Test plants were treated with 5 mL of leaf extract (0.05 g/mL) before infection. d Each test sample was adjusted to pH 4e11, incubated for 1 h, and then brought to pH 7. e Total number of local lesions on nine half-leaves of Nicotiana tabacum cv. Samsun NN.
relatively active at acidic pH (pH 4e6), but was not stable at basic pH: inhibition at pH 8e11 was less than half of that observed at acidic pH.
3.3. Effect of pH
3.4. Effect of dialysis
As indicated by the results given in Table 3, inhibitory activity was influenced by pH. Optimum activity was observed at pH 7, providing an inhibition rate of 72.6%. The crude extract was
After dialysis against distilled water at 4 C for 48 h, inhibitory activity was retained within the bag, providing 71.2% inhibition. By comparison, viral infection was 75.4% inhibited in a control group
Table 1 Effect of Eupatorium adenophorum leaf extract on TMV infection when applied before or after virus inoculation. Dilutiond
Applied before or after virus inoculation
Number of lesionse Treatment 1f
1:1 1:2 1:5
Before After Before After Before After
614 483 816 621 1007 711
35 28 56 39 103 68
Test plants were treated with 5 mL of leaf extract 24 h before or after TMV infection. d Eupatorium adenophorum leaf extract was diluted 1:1 to 1:5 with distilled water. e Total number of local lesions on nine half-leaves of Nicotiana tabacum cv. Samsun NN. f This treatment was treated by Eupatorium adenophorum extract. g Nicotiana tabacum cv. Samsun NN extract treated.
Inhibition (%) Treatment 2g 1453 935 1241 975 1354 980
89 52 94 76 84 75
Control 1704 979 1412 1014 1512 1014
275 69 194 112 147 89
Treatment 1
Treatment 2
82.97a 49.29a 66.58b 38.76b 40.4c 29.88c
14.73a 4.49a 12.11b 3.85b 10.45c 3.35c
Y. Jin et al. / Crop Protection 60 (2014) 28e33
treated with 0.05 g/mL extract but not subjected to dialysis treatment. This inhibition rate did not differ significantly from that observed after dialysis treatment. Virus particles appeared to be destroyed by the E. adenophorum extract and their infection capability was not restored by dialysis. The inhibitory rate was consequently nearly identical to that of the control. These results also demonstrate that the active component in the extract was nondialyzable and apparently macromolecular. 3.5. Electron microscopic observations Samples containing E. adenophorum extract and TMV solution were examined under an electron microscope as described in Materials and Methods. As shown in Fig. 1, differences in particle morphology or size distribution were found after treatment with the extract for different durations. Compared with the control (Fig. 1A), TMV particles were more-or-less destroyed and obviously shortened (Fig. 1B and C), and then became disaggregated (about 70 damaged particles/100 particles) (Fig. 1D). The observed effect was also dependent upon mixing time. 3.6. Determination of superoxide dismutase (SOD), peroxidase (POD), and polyphenol oxidase (PPO) in N. tabacum cv. Samsun NN Tobacco seedlings were sprayed with 5 mL of extract (0.05 g/mL) or distilled water, and re-sprayed 48 h later. After an additional 24 h, leaves were inoculated with TMV. Extractions for enzyme testing were performed the following day. Activity of SOD, which catalyzes the dismutation of O2 to H2O2 and O2 (Bowler et al., 1992), was shown in Fig. 2A for water-treated (WT), E. adenophorum extract-treated (EAET), and untreated virus free plants control (Control) groups. The highest total SOD activity in tobacco leaves was observed at day 4 post-infection. At that time
31
point, SOD levels were 2.1-fold higher in WT and EAET groups than in the Control group. SOD levels were always lower in the Control group than in the other groups. SOD activity in the EAET group was higher than that of WT at days 2 and 3 post-infection. Extract treatment and TMV inoculation also increased POD activity in tobacco seedlings. On day 5, POD levels in the EAET group were 2.2and 5.2-fold higher than in WT and Control leaves, respectively (Fig. 2B). PPO activity was maintained at a relatively higher level in the EAET group than in the WT and Control groups; on day 3, PPO levels were 2.5- and 8.3-fold higher in this group than in WT and Control, respectively (Fig. 2C). These results suggest E. adenophorum leaf extract can induce a host resistance reaction against viral infection.
4. Discussion In this study, we investigated the antiviral activities of E. adenophorum leaf extract. We first observed that the leaf extract could inactivate TMV particles. Further experiments demonstrated that the extract is effective for prevention, and also has a slight curative effect. The putative inhibitor is thermostable, stable at acidic pH, and largely not dialyzable. The antiviral mechanism appears to involve direct inhibition of viral particles and promotion of a response in the host plant. Although the active compound has not yet been isolated and determined, E. adenophorum holds promise as a source of antiviral substances for practical use. In addition, the use of E. adenophorum leaf extract represents an alternative strategy in the development of novel plant-based biological pesticides and comprehensive management of invasive weeds. Plant viral diseases are some of the most serious biological diseases in agricultural production, with affected areas and degree of virus-caused damage gradually increasing. Although chemical controls have been used previously (Wang et al., 2008), they have
Fig. 1. Electron microscopy of morphological changes in TMV particles. (A) Normal TMV particles; (B) TMV after treatment with E. adenophorum extract for 30 min; (C) TMV after treatment with E. adenophorum extract for 60 min; (D) TMV after treatment with E. adenophorum extract for 90 min. The concentration of the E. adenophorum extract was 0.05 g/mL, and that of purified TMV was 0.5 mg/mL. The sample was observed under 12000 magnification using a Hitachi electron microscope.
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Y. Jin et al. / Crop Protection 60 (2014) 28e33
Fig. 2. Effect of E. adenophorum extract on SOD, POD, and PPO activity. Tobacco seedlings were sprayed with 5 mL extract (0.05 g/mL) or distilled water 3 d and 1 d before TMV infection. Activities of (A) SOD, (B) POD, and (C) PPO in E. adenophorum extract-treated (EAET), distilled water-treated (WT), and Control groups were examined 24 h after infection. Results were the mean SD values obtained from three independent experiments.
negative ecological impacts. Much attention is currently focused on research and development of environmentally-friendly biological pesticides because of their simple extraction protocols and obvious effects. Because E. adenophorum was widely distributed in China as an invasive plant, considerable effort has being made to find new and innovative methods for its integrative management. The chemical composition and comprehensive application of E. adenophorum have been extensively studied, with its use as a biofuel, biological medicine, bio-fertilizer, and dyestuff (Sun et al., 2004). Various plants, including: Phytolacca Americana L., Pelargonium hortorum Bailey, Chenopodium album Linn, Chenopodium amaranticolor Coste et Reyn, Capsicum frutescens L., Azadirachta indica A. Juss., Vitis vinifera L., and Rosa banksiae Ait., possess antiviral factors (Blaszczak et al., 1959; Sangar and Dhingra, 1982; Zarling et al., 1991; Vivanco et al., 1999). Plant-derived antiviral compounds are active against plant, animal and human viruses (Vivanco et al., 1999). We previously reported that the polysaccharide from E. adenophorum leaf extract was a prophylactic and immune enhancement agent against H5N1 influenza virus infection in mouse model (Jin et al., 2013). In this study, the E. adenophorum leaf extract showed significant inhibitory activity against TMV infection when plants were treated prior to viral challenge. Antioxidant enzyme system responses are some of the most important environmental acclimatization mechanisms in plants (Lu et al., 2008). The role of these enzyme systems in development of plant tolerance to extreme environments has been clearly demonstrated (Dhindsa and Matowe, 1981; Schoner and Heinrich Krause, 1990; Hernandez et al., 2001). To mitigate and repair damage initiated by active oxygen species, plants have evolved cellular adaptive responses such as upregulation of oxidative stress protectors and accumulation of protective solutes (Horling et al., 2003). Antioxidant defense enzymes such as superoxide dismutase (SOD), polyphenol oxidase (PPO), and guaiacol peroxidase (POD) minimize superoxide and hydrogen peroxide concentrations. POD is generally considered to be potentially important in host resistance mechanisms because it can catalyze the last step of lignin biosynthesis (Hammerschmidt et al., 1982) and can oxidize phenolic compounds to quinones (Campos-Vargas and Saltveit,
2002). In addition, high concentrations of phenolic compounds around an infected area can restrict or weaken pathogen growth (Reimers and Leach, 1991). In addition to possessing their own antimicrobial activities, phenolic compounds can be oxidized by PPO and POD, resulting in substances such as quinones that are even more toxic to pathogens (Wang et al., 2009). In this study, SOD, POD, and PPO activities were elevated by E. adenophorum leaf extract treatment. Based on observed morphological changes to extract-treated TMV particles, the antiviral effect of E. adenophorum leaf extract is most likely due to direct destruction of viral particles and induction of host response against TMV. In conclusion, we have demonstrated in this study that E. adenophorum contains a potent inhibitor against TMV infection. Although the isolation of the active antiviral constituents still requires lots of further studies, E. adenophorum may eventually be exploited as a pesticide source, providing a foundation for comprehensive use of this invasive weed and for its ecologically sustainable development. Acknowledgments We thank Professor Chenggui Han, College of Agricultural Biotechnology, Chinese Agricultural University, China, for his kind help in the purification of virus. This work was supported by the Fundamental Research Funds for the Central University (No. TD2012-03), the Public Welfare Project from Ministry of Agriculture of the People’s Republic of China (Grant No. 201103027) and the Beneficial Professional Project Grant (Ministry of Agriculture, China, Grant No. 200803021-030). References Beauchamp, C., Fridovich, I., 1971. Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal. Biochem. 44, 276e287. Bell, A.A., 1981. Biochemical mechanisms of disease resistance. Annu. Rev. Plant Physiol. 32, 21e81. Blaszczak, W., Ross, A., Larson, R., 1959. The inhibitory activity of plant juices on the infectivity of potato virus X. Phytopathology 49, 784e791. Bowler, C., Montagu, M.v., Inze, D., 1992. Superoxide dismutase and stress tolerance. Annu. Rev. Plant Biol. 43, 83e116.
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