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Herbicidal potential of pseudoguaninolide sesquiterpenes on wild oat, Avena fatua L.
Biochemical Systematics and Ecology 44 (2012) 333–337
Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Herbicidal potential of pseudoguaninolide sesquiterpenes on wild oat, Avena fatua L. Mona M.G. Saad a, Samir A.M. Abdelgaleil a, *, Toshihiko Suganuma b a b
Department of Chemistry of Pesticides, Faculty of Agriculture, 21545-El-Shatby, Alexandria University, Alexandria, Egypt Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2011 Accepted 2 June 2012 Available online 4 July 2012
The inhibitory effects of five pseudoguaninolide sesquiterpenesdneoambrosin, damcinic acid, damsin, ambrosin and hymenindisolated from the aerial parts of Ambrosia maritima were evaluated on seed germination and seedling growth of wild oat, Avena fatua L. The tested sesquiterpenes caused pronounced reduction in seed germination, particularly at the higher concentrations of 1 and 2 mM. Damsin was the most potent compounds with seed germination percentage of 16.7% at 2 mM. The results of seedling growth test showed that the sesquiterpenes are potent inhibitors of root growth and ambrosin and damsin were the most potent compounds with EC50 values of 0.22 and 0.24 mM, respectively. Similarly, strong inhibition of shoot growth was observed with ambrosin (EC50 ¼ 0.30) being the most active compound. Nevertheless, the inhibition of root growth by all compounds was greater than that of shoot growth. Interestingly, the five tested sesquiterpenes showed greater inhibitory effect on germination, root and shoot growth than a reference herbicide, imazamethabenz. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Pseudoguaninolide sesquiterpenes Ambrosia maritima Avena fatua Herbicidal activity
1. Introduction Controlling weeds is very important in agriculture because weeds are widely distributed in the world and the yield losses caused by 1800 kinds of weeds are about 9.7% of total crop production every year (Li et al., 2003). Chemical herbicides have been used effectively to control various weeds but they can cause herbicide-resistant weeds, herbicide residues as well as reduce water and soil quality and affect non-target organisms (Heap et al., 1993; Batish et al., 2002; Vyvyan, 2002). These side effects are shifting the attention to alternative weed control technologies based on natural products (Dayan et al., 1999; Rimando and Duke, 2006; Copping and Duke, 2007). Plant compounds with a wide diversity of skeletal types have allelopathic and herbicidal properties. (Dakshini and Einhellig, 1995; Cespedes et al., 2000; Lotina-Hennsen et al., 1998; Chon et al., 2005; Amoo et al., 2008). Sesquiterpenes are colorless, bitter, relatively stable, lypophilic constituents, which are biogenetically derived from trans,trans farnesyl pyrophosphate (Geissman, 1973) following an initial cyclization and subsequent oxidative modifications. They are mainly found in several genera of Asteraceae, but also in Umbelliferae, Acanthaceae, Amaranthaceae, Apiaceae and Magnoliaceae. In addition to anti-inflammatory, antimicrobial, antiprotozoal, and antitumoral properties of sesquiterpenes (Picman, 1986; Galindo et al., 1999; Neerman, 2003), many of these compounds have shown to possess strong phytotoxic
* Corresponding author. Fax: þ20 3 592 0067. E-mail address: [email protected] (S.A.M. Abdelgaleil). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.06.004
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activity against several weeds (Pandey, 1996; Duke et al., 1987; Batish et al., 1997, 2002; Macias et al., 1998; Wedge et al., 2000; Abdelgaleil et al., 2009). Ambrosia maritima L. (Asteraceae) is a widely distributed weed in southern parts of Egypt, Sudan, Senegal, and neighboring countries (Ghazanfar, 1994). Some sesquiterpenes isolated from the plant, such as damsin, ambrosin and tribromoambrosin were previously found to have toxic effect against the fresh water snails Biomphalaria alexandrina and Bulinus truncatus (Shoeb and El-Emam, 1976). Recently, the insecticidal, molluscicidal and antifungal activities of some sesquiterpenes isolated from A. maritima were described (Abdelgaleil, 2010; Abdelgaleil et al., 2011). No studies are available in the literature on allelopathic and/or herbicidal activity of sesquiterpenes isolated from A. maritima except our preliminary study on the effect of sesquiterpenes on barnyard grass, Echinochloa crusgalli L. (Abdelgaleil, 2010) In this study, we described the effect of five sesquiterpenes neoambrosin (1), damcinic acid (2), damsin (3), ambrosin (4) and hymenin (5) isolated from the aerial parts of A. maritima on seed germination, root and shoot growth of wild oat, Avena fatua L. 2. Materials and methods 2.1. Tested seeds Wild oat (Avena fatua) field biotype seeds were collected from Alexandria Desert Research Station Farm, Alexandria, Egypt in June 2009. All undersized or damaged seeds were discarded, and the assayed seeds of uniform size were selected. Germination of the seeds was tested before experiments and was 60%. 2.2. Test sesquiterpenes Five pseudoguaninolide sesquiterpenes, neoambrosin (1), damcinic acid (2), damsin (3), ambrosin (4) and hymenin (5) were isolated from chloroform extract of Ambrosia maritima as previously described by Abdelgaleil (2010). The chemical structure (Fig. 1) of these sesquiterpenes (1–5) was confirmed on the basis of their spectroscopic data of ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR) and mass spectroscopy (MS) (Nagaya et al., 1994; Iskander et al., 1988). 2.3. Seed germination and seedling growth tests Phytotoxic effects of the tested sesquiterpenes were evaluated on wild oat (Avena fatua) germination and subsequent seedling growth. The tested sesquiterpenes and a reference herbicide, imazamethabenz, were dissolved in dimethyl sulfoxide (DMSO) followed by dilution with distilled water to obtain a stock solution of 2 mM. The concentration of DMSO in this solution was 0.5% v/v. Then a series of concentrations (0.125, 0.25, 0.5 and 1 mM) were prepared by dilution with distilled water. An aqueous solution of DMSO (0.5% v/v) was used as control treatment. Three replicates, each of 20 seeds, were prepared for each treatment using glass Petri dishes (9 cm) lined with Whatman No. 2 filter paper. Six milliliters of each test solution were added to each Petri dish. Afterward, Petri dishes were placed in the bottom of 0.1 mm thick polyethylene bags (15 30 cm) that were expanded to contain air and then closed at the top with rubber bands to prevent the loss of moisture. The Petri dishes were kept on a germination cabinet at 20 1 C with 12 h photoperiod, 3.3 mmol m2 s1. After 10 days of sowing, germination and root and shoot lengths were determined. The growth inhibition percentages of root and shoot lengths were calculated from the following equation: I(%) ¼ [1 T/C] 100; T is the length of treatment (cm) and C is the length of control (cm). The concentrations causing 50% inhibition (EC50s) of root and shoot growth were calculated from a probit analysis (Finney, 1971). 2.4. Statistical analysis Germination percentages, root lengths and shoot lengths were subjected to one-way analysis of variance followed by Student–Newman–Keuls test (Cohort software Inc. 1985) to determine significant differences among mean values at the probability level of 0.05. 3. Results and discussion 3.1. Effects on wild oat seed germination Table 1 showed the germination percentages of wild oat seeds treated with neoambrosin (1), damcinic acid (2), damsin (3), ambrosin (4) and hymenin (5) and a reference herbicide imazamethabenz. In general, the tested sesquiterpenes caused significant reduction of seed germination at concentrations of 0.25, 0.5, 1 and 2 mM compared to control, except damcinic acid (2). The five sesquiterpenes showed greater reduction in seed germination than imazamethabenz at the highest concentration 2 mM. At this concentration, damsin (3) was the most potent compounds with seed germination percentage of 16.7%, followed by neoambrosin (1) and ambrosin (4), while hymenin (5) was the less effective. Neoambrosin (1), damsin (3) and ambrosin (4) were more potent at inhibiting germination of wild oat than the sesquiterpenes lactones, costunolide, parthenolide and 1,10-epoxyparthenolide isolated from southern magnolia, Magnolia grandiflora L. (Abdelgaleil et al., 2009).
M.M.G. Saad et al. / Biochemical Systematics and Ecology 44 (2012) 333–337
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Fig. 1. Chemical structure of sesquiterpenes (1–5).
Similarly, the seed germination reduction observed in this study was broadly comparable with that of a sesquiterpene lactone parthenin tested against the same weed (Batish et al., 2002). Other sesquiterpenes and sesquiterpene lactones have been shown to inhibit seed germination. For examples, artemisinin and its sesquiterpene analogs reduced the seed germination of lettuce, rye, and Arabidopsis (Dayan et al., 1999). Sesquiterpene lactones tagitinin A and B isolated from Mexican sunflower (Tithonia diversifolia (Hemsley) A. Gray) also inhibited seed germination of radish, cucumber, and onion (Baruah et al., 1994). Sesquiterpene lactones derivatives of glaucolide B isolated from Vernonia fruticulosa inhibited seed germination of the dicotyledons Physalis ixocarpa and Trifolium alexandrinum and of the monocotyledons Lolium multiflorum and Amaranthus hypochondriacus (Barbosa et al., 2004). 3.2. Effect of sesquiterpenes on root growth The results showed that the tested compounds exhibited significant reduction of root growth at all of the tested concentrations compared to control, except for damsinic (2) and hymenin (5) at the lowest concentration (0.125 mM; Table 2). Table 1 Effect of sesquiterpenes on wild oat germination 10 d after sowing.a Conc (mM)
0 0.125 0.25 0.5 1 2 a b
Germination (%) Neoambrosin (1)
Damsinic acid (2)
Damsin (3)
Ambrosin (4)
Hymenin (5)
Imazamethabenz
50.0 2.89ab 46.7 1.67ab 40.0 2.89bc 36.7 1.67c 40.0 0.0bc 23.3 1.67d
50.0 2.89a 41.7 1.67a 40.0 2.89a 38.3 4.21ab 38.3 4.21ab 28.3 1.67b
50.0 2.89a 46.7 4.21a 35.0 2.89b 30.0 0.0b 26.7 1.67b 16.7 4.21c
50.0 2.89a 50.0 0.0a 36.7 4.21b 28.3 1.67c 26.7 1.67c 26.7 1.67c
50.0 2.89a 38.3 1.67b 36.7 1.67b 33.3 4.21b 30 .0 0.0b 30.0 2.89b
50.0 2.89a 35.0 2.89b 38.3 4.21b 40.0 0.0b 30.0 0.0b 31.7 1.67b
Data are expressed as means SE from experiments with three replicates of 20 seeds each. Means within a column sharing the same letter are not significantly different at the 0.05 probability level.
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Table 2 Effect of sesquiterpenes on wild oat root growth.a Conc (mM)
Neoambrosin (1)
0 0.125 0.25 0.5 1 2 EC50d (mM) a b c d
Root length (cm)
I (%)b
9.6 0.36ac 8.5 0.21b 5.7 0.37c 5.2 0.35c 3.7 0.14d 0.5 0.12 0.48
0.0 11.5 40.6 45.6 61.5 94.8
Damsinic acid (2)
Damsin (3)
Root length (cm)
I (%)
Root length (cm)
I (%)
Ambrosin (4) Root length (cm)
I (%)
Hymenin (5) Root length (cm)
I (%)
Imazamethabenz Root length (cm)
I (%)
9.6 0.36a 9.2 0.06a 6.6 0.10b 4.8 0.09c 3.4 0.03d 2.0 0.34e 0.60
0.0 4.2 31.3 50.0 64.6 79.2
9.6 0.36a 6.7 0.22b 4.3 0.26c 3.3 0.17d 0.73 0.09e 0.17 0.07e 0.24
0.0 30.2 55.2 65.6 92.4 98.2
9.6 0.36a 7.4 0.31b 4.1 0.27c 1.4 0.24d 0.73 0.07de 0.10 0.0e 0.22
0.0 22.9 57.3 85.4 92.4 99.0
9.6 0.36a 8.8 0.32ab 8.1 0.38b 6.0 0.27c 2.6 0.25d 0.47 0.47e 0.57
0.0 8.3 15.6 37.5 72.9 95.6
9.6 0.36a 7.8 0.14b 6.7 0.31c 6.5 0.09c 3.7 0.37d 2.8 0.20e 0.74
0.0 18.8 30.2 32.3 61.5 70.8
Data are expressed as means SE from experiments with three replicates of 20 seeds each. I ¼ inhibition. Means within a column sharing the same letter are not significantly different at the 0.05 probability level. EC50 ¼ concentration of compound causing 50% root growth inhibition.
Ambrosin (4) and damsin (3) were the most potent compounds with EC50 values of 0.22 and 0.24 mM, respectively. In contrast, hymenin (5) and damsinic (2) were the less effective. The five tested sesquiterpenes showed greater inhibitory effect of root growth than a reference herbicide, imazamethabenz. The results of this study are consistent with previous studies in which sesquiterpene lactones costunolide and parthenolide strongly reduced the root growth of wheat, lettuce radish, onion and wild oat (Abdelgaleil and Hashinaga, 2007; Abdelgaleil et al., 2009). 3.3. Effect of sesquiterpenes on shoot growth The tested sesquiterpenes exhibited significant reduction of shoot growth particularly at the highest concentrations 0.5, 1 and 2 mM (Table 3). Ambrosin (4) (EC50 ¼ 0.30 mM) was the most active compound, while damsinic (2) (EC50 ¼ 1.09 mM) was the less effective one. Based on the EC50 values, all of the tested compounds had grater shoot growth reduction than a reference herbicide, imazamethabenz. The inhibitory effects of some sesquiterpenes on the root and shoot growth have been described (Baruah et al., 1994; Dayan et al., 1999; Batish et al., 2002; Vyvyan, 2002). The results showed that the inhibitory effects of the tested compounds on root growth were greater than on shoot growth. These results are consistent with those reported elsewhere for other sesquiterpenes and some plant extracts (Chung and Miller, 1995; Turk and Tawaha, 2002; Abdelgaleil et al., 2009). This finding might be expected, because it is likely that roots are the first to absorb the allelochemicals compounds from the environment (Turk and Tawaha, 2002). It was also observed that the inhibitory effect of tested sesquiterpenes on root and shoot growth was greater than that on germination. Similarly, Leather and Einhellig (1985) demonstrated that bioassays determining seedling growth are usually more sensitive than those measuring germination. Moreover, ambrosin (4) with C2–C3 double bond was more active than neoambrosin (1) with C1–C2 double bond. The results indicated that beside the presence of a-methylene- g-lactone, which is essential for potent bioactivity of sesquitrpene lactones, the other function groups and their position on the skeleton may also enhance or reduce the activity of sesquitrpene lactones. Regarding the mechanism of action of sesquiterpenes, it has been reported that sesquiterpene lactones react with the –SH group of amino acids, proteins, and enzymes (Picman, 1986). Parthenolide has been shown to inhibit acetolactate synthase, which is a target enzyme for several classes of commercial herbicides (Abdelgaleil et al., 2009). Other sesquiterpenes have been shown to inhibit cell division and reduce chlorophyll content (Dayan et al., 1999), damage the cell membrane and reduce
Table 3 Effect of sesquiterpenes on wild oat shoot growth.a Conc (mM)
Neoambrosin (1) Shoot length (cm)
0 0.125 0.25 0.5 1 2 EC50d (mM)
11.2 0.35ac 10.9 0.46a 10.0 0.5ab 8.9 0.46b 6.3 0.41c 1.7 0.29d 0.96
a b c d
Damsinic acid (2)
Damsin (3)
I (%)
Shoot length (cm)
I (%)
Shoot length (cm)
I (%)
Shoot length (cm)
I (%)
Shoot length (cm)
I (%)
Shoot length (cm)
I (%)
0.0 2.7 10.7 20.5 43.8 84.8
11.2 0.35a 10.8 0.58a 10.5 0.85a 7.9 0.43b 6.2 0.26b 3.3 0.75c 1.09
0.0 3.6 6.3 29.5 44.6 70.5
11.2 0.35a 11.0 0.35a 10.6 0.49a 8.7 0.57b 3.8 0.24c 1.8 0.25d 0.83
0.0 1.8 5.4 22.3 66.1 83.9
11.2 0.35a 9.0 0.32b 5.4 0.18c 2.9 0.15d 2.7 0.18d 1.5 0.14e 0.30
0.0 19.6 51.8 74.1 75.9 86.6
11.2 0.35a 10.0 0.53a 8.4 0.17b 6.8 0.55c 4.4 0.61d 2.2 0.21e 0.67
0.0 10.7 25.0 39.3 60.7 80.4
11.2 0.35a 9.7 0.57b 8.6 0.45b 8.4 0.33b 5.8 0.24c 4.7 0.24c 1.33
0.0 13.4 23.2 25.0 48.2 58.0
b
Ambrosin (4)
Hymenin (5)
Data are expressed as means SE from experiments with three replicates of 20 seeds each. I ¼ inhibition. Means within a column sharing the same letter are not significantly different at the 0.05 probability level. EC50 ¼ concentration of compound causing 50% shoot growth inhibition.
Imazamethabenz
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dehydrogenase, protease, and peroxidase activities (Batish et al., 2002), affect protein content and respiration (Pandey, 1996). In conclusion, the present study indicates that the sesquiterpene lactones ambrosin (4) and damsin (3) had potent herbicidal activity on seedling growth of wild oat. However, further studies on field efficiency and toxicological effects of these sesquiterpenes are highly recommended. References Abdelgaleil, S.A.M., Abdel-Razeek, N., Soliman, S.A., 2009. Herbicidal activity of three sesquiterpene lactones on wild oat (Avena fatua) and their possible mode of action. Weed Science 57, 6–9. Abdelgaleil, S.A.M., 2010. Assessment of mosquitocidal, herbicidal and molluscicidal potentials of extracts and phytochemicals isolated from three Egyptian plants. Alexandria Journal of Agricultural Research 55, 59–73. Abdelgaleil, S.A.M., Badawy, M.E.I., Suganuma, T., Kitahara, K., 2011. Antifungal and biochemical effects of pseudoguaianolide sesquiterpenes isolated from Ambrosia maritima L. African Journal of Microbiology Research 5, 3385–3392. Abdelgaleil, S.A.M., Hashinaga, F., 2007. Allelopathic potential of two sesquiterpene lactones from Magnolia grandiflora L. Biochemical Systematics and Ecology 35, 737–742. Amoo, S.O., Ojo, A.U., Van Staden, J., 2008. Allelopathic potential of Tetrapleura tetraptera leaf extracts on early seedling growth of five agricultural crops. South African Journal of Botany 74, 149–152. Barbosa, L.C.A., Costa, A.V., Pilo-Veloso, D., Lopes, J.L.C., Hernandez-Terrones, M.G., Lotina-Hennsen, B.K., 2004. Phytogrowth-inhibitory lactones derivatives of glaucolide B. Zeitschrift für Naturforschung 59c, 803–810. Baruah, N.C., Sarma, J.C., Barua, N.C., Sarma, S., Sharma, R.P., 1994. Germination and growth inhibitory sesquiterpene lactones and a flavone from Tithonia diversifolia. Phytochemistry 36, 29–36. Batish, D.R., Kohli, R.K., Singh, H.P., Xaxena, D.B., 1997. Studies on herbicidal activity of parthenin, a constituent of Parthenium hysterophorus towards billgoat weed (Ageratum conyzoides). Current Science 73, 369–371. Batish, D.R., Singh, H.P., Kohli, R.K., Xaxena, D.B., Kaur, S., 2002. Allelopathic effects of parthenin against two weedy species, Avena fatua and Bidens pilosa. Environmental and Experimental Botany 47, 149–155. Cespedes, C., Achine, L., Alarcon, J., Becerra, J., Lotina Hennsen, B., 2000. Photosynthetic inhibitory activity of dihydro-b-agarofuran sesquiterpenes from Maytenus disticha and Maytenus boaria (Celastraceae). Zeitschrift für Naturforschung 55c, 631–637. Chon, S.-U., Jang, H.-G., Kim, D.-K., Kim, Y.-M., Boo, H.-O., Kim, Y.-J., 2005. Allelopathic potential in lettuce (Lactuca sativa L.) plants. Scientia Horticulturae 106, 309–317. Chung, I.M., Miller, D.A., 1995. Natural herbicide potential of alfalfa residues on selected weed species. Agronomy Journal 87, 920–925. Copping, L., Duke, S.O., 2007. Natural products that have been used commercially as crop protection agents. Pest Management Science 63, 524–554. Dakshini, K.M.M.E., Einhellig, F.A., 1995. Allelopathy: Organisms, Processes and Applications. ACS, Washington, DC, USA. Dayan, F.E., Hern_andez, A., Allen, S.T., Moraes, R.T., Vroman, J.A., Avery, M.A., Duke, S.O., 1999. Comparative phytotoxicity of artemisininand several sesquiterpene analogues. Phytochemistry 50, 607–614. Duke, S.O., Vaughn, K.C., Croom, E.M.J., Elsohly, H.N., 1987. Artemisinin, a constituent of annual wormwood (Artemisia annua), is a selective phytotoxin. Weed Science 35, 499–505. Finney, D.J., 1971. Probit Analysis, third ed. Cambridge University Press, London, 318 p. Galindo, J.C.G., Hernandez, A., Dayan, F.E., Tellez, M.R., Macıas, F.A., Paul, R.N., Duke, S.O., 1999. Dehydrozaluzanin C, a natural sesquiterpenolide, causes rapid plasma membrane leakage. Phytochemistry 52, 805–813. Geissman, T.A., 1973. Terpanoids: Biogenesis and Distribution. In: Runeckles, V. (Ed.), Recent Advances in Phytochemistry, Vol. 6, pp. 65–94. Ghazanfar, S., 1994. CRC Handbook of Arabian Medicinal Plants. CRC Press, Boca Raton, p. 265. Heap, I.M., Murray, B.G., Loeppky, H.A., Morreson, I.N., 1993. Resistance to aryloxypropionate and cyclohexanedione herbicides in wild oat (Avena fatua). Weed Science 41, 232–238. Iskander, G.M., Modawi, B.M., Ahmed, H.E., 1988. Crystal and molecular structure of ambrosin and damsin, sesquiterpene lactone isolates of Ambrosia maritima L. Journal für Praktische Chemie 330, 182–190. Leather, G.R., Einhellig, F.A., 1985. Mechanisms of allelopathic action in bioassay. In: Thompson, A.C. (Ed.), The Chemistry of Allelopathy. ACS, Washington, pp. 197–205. Li, Y., Sun, Z., Zhuang, X., Xu, L., Chen, S., Li, M., 2003. Research progress on microbial herbicides. Crop Protection 22, 247–252. Lotina-Hennsen, B., Mata, R., Calderon, J.S., Cespedes- Acunea, C.L., Jimenez Estrada, M., 1998. Secondary metabolites isolated from Mexican plants: target and mechanism of action on photosynthesis. In: Pandalai, S.G. (Ed.), Secondary Metabolites from Mexican Plants: Chemistry and Biological Properties. Research Signpost, Trivandrum, India, pp. 59–68. Macias, F.A., Varela, R.M., Torres, A., Oliva, R.M., Molinillo, J.M.G., 1998. Bioactive norsesquiterpenes from Helianthus annuus with potential allelopathic activity. Phytochemistry 48, 631–636. Nagaya, H., Nagae, T., Usami, A., Itokawa, H., Takeya, A., Omar, A.A., 1994. Cytotoxic chemical constituents from Egyptian medicinal plant, Ambrosia maritima L. Natural Medicines 48, 223–226. Neerman, M.F., 2003. Sesquiterpene lactones: a diverse class of compounds found in essential oils possessing antibacterial and antifungal properties. International Journal of Aromatherapy 13, 114–120. Pandey, D.K., 1996. 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Contents lists available at SciVerse ScienceDirect
Biochemical Systematics and Ecology journal homepage: www.elsevier.com/locate/biochemsyseco
Herbicidal potential of pseudoguaninolide sesquiterpenes on wild oat, Avena fatua L. Mona M.G. Saad a, Samir A.M. Abdelgaleil a, *, Toshihiko Suganuma b a b
Department of Chemistry of Pesticides, Faculty of Agriculture, 21545-El-Shatby, Alexandria University, Alexandria, Egypt Department of Biochemistry and Applied Biosciences, Faculty of Agriculture, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 29 November 2011 Accepted 2 June 2012 Available online 4 July 2012
The inhibitory effects of five pseudoguaninolide sesquiterpenesdneoambrosin, damcinic acid, damsin, ambrosin and hymenindisolated from the aerial parts of Ambrosia maritima were evaluated on seed germination and seedling growth of wild oat, Avena fatua L. The tested sesquiterpenes caused pronounced reduction in seed germination, particularly at the higher concentrations of 1 and 2 mM. Damsin was the most potent compounds with seed germination percentage of 16.7% at 2 mM. The results of seedling growth test showed that the sesquiterpenes are potent inhibitors of root growth and ambrosin and damsin were the most potent compounds with EC50 values of 0.22 and 0.24 mM, respectively. Similarly, strong inhibition of shoot growth was observed with ambrosin (EC50 ¼ 0.30) being the most active compound. Nevertheless, the inhibition of root growth by all compounds was greater than that of shoot growth. Interestingly, the five tested sesquiterpenes showed greater inhibitory effect on germination, root and shoot growth than a reference herbicide, imazamethabenz. Ó 2012 Elsevier Ltd. All rights reserved.
Keywords: Pseudoguaninolide sesquiterpenes Ambrosia maritima Avena fatua Herbicidal activity
1. Introduction Controlling weeds is very important in agriculture because weeds are widely distributed in the world and the yield losses caused by 1800 kinds of weeds are about 9.7% of total crop production every year (Li et al., 2003). Chemical herbicides have been used effectively to control various weeds but they can cause herbicide-resistant weeds, herbicide residues as well as reduce water and soil quality and affect non-target organisms (Heap et al., 1993; Batish et al., 2002; Vyvyan, 2002). These side effects are shifting the attention to alternative weed control technologies based on natural products (Dayan et al., 1999; Rimando and Duke, 2006; Copping and Duke, 2007). Plant compounds with a wide diversity of skeletal types have allelopathic and herbicidal properties. (Dakshini and Einhellig, 1995; Cespedes et al., 2000; Lotina-Hennsen et al., 1998; Chon et al., 2005; Amoo et al., 2008). Sesquiterpenes are colorless, bitter, relatively stable, lypophilic constituents, which are biogenetically derived from trans,trans farnesyl pyrophosphate (Geissman, 1973) following an initial cyclization and subsequent oxidative modifications. They are mainly found in several genera of Asteraceae, but also in Umbelliferae, Acanthaceae, Amaranthaceae, Apiaceae and Magnoliaceae. In addition to anti-inflammatory, antimicrobial, antiprotozoal, and antitumoral properties of sesquiterpenes (Picman, 1986; Galindo et al., 1999; Neerman, 2003), many of these compounds have shown to possess strong phytotoxic
* Corresponding author. Fax: þ20 3 592 0067. E-mail address: [email protected] (S.A.M. Abdelgaleil). 0305-1978/$ – see front matter Ó 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.bse.2012.06.004
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activity against several weeds (Pandey, 1996; Duke et al., 1987; Batish et al., 1997, 2002; Macias et al., 1998; Wedge et al., 2000; Abdelgaleil et al., 2009). Ambrosia maritima L. (Asteraceae) is a widely distributed weed in southern parts of Egypt, Sudan, Senegal, and neighboring countries (Ghazanfar, 1994). Some sesquiterpenes isolated from the plant, such as damsin, ambrosin and tribromoambrosin were previously found to have toxic effect against the fresh water snails Biomphalaria alexandrina and Bulinus truncatus (Shoeb and El-Emam, 1976). Recently, the insecticidal, molluscicidal and antifungal activities of some sesquiterpenes isolated from A. maritima were described (Abdelgaleil, 2010; Abdelgaleil et al., 2011). No studies are available in the literature on allelopathic and/or herbicidal activity of sesquiterpenes isolated from A. maritima except our preliminary study on the effect of sesquiterpenes on barnyard grass, Echinochloa crusgalli L. (Abdelgaleil, 2010) In this study, we described the effect of five sesquiterpenes neoambrosin (1), damcinic acid (2), damsin (3), ambrosin (4) and hymenin (5) isolated from the aerial parts of A. maritima on seed germination, root and shoot growth of wild oat, Avena fatua L. 2. Materials and methods 2.1. Tested seeds Wild oat (Avena fatua) field biotype seeds were collected from Alexandria Desert Research Station Farm, Alexandria, Egypt in June 2009. All undersized or damaged seeds were discarded, and the assayed seeds of uniform size were selected. Germination of the seeds was tested before experiments and was 60%. 2.2. Test sesquiterpenes Five pseudoguaninolide sesquiterpenes, neoambrosin (1), damcinic acid (2), damsin (3), ambrosin (4) and hymenin (5) were isolated from chloroform extract of Ambrosia maritima as previously described by Abdelgaleil (2010). The chemical structure (Fig. 1) of these sesquiterpenes (1–5) was confirmed on the basis of their spectroscopic data of ultraviolet (UV), infrared (IR), nuclear magnetic resonance (NMR) and mass spectroscopy (MS) (Nagaya et al., 1994; Iskander et al., 1988). 2.3. Seed germination and seedling growth tests Phytotoxic effects of the tested sesquiterpenes were evaluated on wild oat (Avena fatua) germination and subsequent seedling growth. The tested sesquiterpenes and a reference herbicide, imazamethabenz, were dissolved in dimethyl sulfoxide (DMSO) followed by dilution with distilled water to obtain a stock solution of 2 mM. The concentration of DMSO in this solution was 0.5% v/v. Then a series of concentrations (0.125, 0.25, 0.5 and 1 mM) were prepared by dilution with distilled water. An aqueous solution of DMSO (0.5% v/v) was used as control treatment. Three replicates, each of 20 seeds, were prepared for each treatment using glass Petri dishes (9 cm) lined with Whatman No. 2 filter paper. Six milliliters of each test solution were added to each Petri dish. Afterward, Petri dishes were placed in the bottom of 0.1 mm thick polyethylene bags (15 30 cm) that were expanded to contain air and then closed at the top with rubber bands to prevent the loss of moisture. The Petri dishes were kept on a germination cabinet at 20 1 C with 12 h photoperiod, 3.3 mmol m2 s1. After 10 days of sowing, germination and root and shoot lengths were determined. The growth inhibition percentages of root and shoot lengths were calculated from the following equation: I(%) ¼ [1 T/C] 100; T is the length of treatment (cm) and C is the length of control (cm). The concentrations causing 50% inhibition (EC50s) of root and shoot growth were calculated from a probit analysis (Finney, 1971). 2.4. Statistical analysis Germination percentages, root lengths and shoot lengths were subjected to one-way analysis of variance followed by Student–Newman–Keuls test (Cohort software Inc. 1985) to determine significant differences among mean values at the probability level of 0.05. 3. Results and discussion 3.1. Effects on wild oat seed germination Table 1 showed the germination percentages of wild oat seeds treated with neoambrosin (1), damcinic acid (2), damsin (3), ambrosin (4) and hymenin (5) and a reference herbicide imazamethabenz. In general, the tested sesquiterpenes caused significant reduction of seed germination at concentrations of 0.25, 0.5, 1 and 2 mM compared to control, except damcinic acid (2). The five sesquiterpenes showed greater reduction in seed germination than imazamethabenz at the highest concentration 2 mM. At this concentration, damsin (3) was the most potent compounds with seed germination percentage of 16.7%, followed by neoambrosin (1) and ambrosin (4), while hymenin (5) was the less effective. Neoambrosin (1), damsin (3) and ambrosin (4) were more potent at inhibiting germination of wild oat than the sesquiterpenes lactones, costunolide, parthenolide and 1,10-epoxyparthenolide isolated from southern magnolia, Magnolia grandiflora L. (Abdelgaleil et al., 2009).
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Fig. 1. Chemical structure of sesquiterpenes (1–5).
Similarly, the seed germination reduction observed in this study was broadly comparable with that of a sesquiterpene lactone parthenin tested against the same weed (Batish et al., 2002). Other sesquiterpenes and sesquiterpene lactones have been shown to inhibit seed germination. For examples, artemisinin and its sesquiterpene analogs reduced the seed germination of lettuce, rye, and Arabidopsis (Dayan et al., 1999). Sesquiterpene lactones tagitinin A and B isolated from Mexican sunflower (Tithonia diversifolia (Hemsley) A. Gray) also inhibited seed germination of radish, cucumber, and onion (Baruah et al., 1994). Sesquiterpene lactones derivatives of glaucolide B isolated from Vernonia fruticulosa inhibited seed germination of the dicotyledons Physalis ixocarpa and Trifolium alexandrinum and of the monocotyledons Lolium multiflorum and Amaranthus hypochondriacus (Barbosa et al., 2004). 3.2. Effect of sesquiterpenes on root growth The results showed that the tested compounds exhibited significant reduction of root growth at all of the tested concentrations compared to control, except for damsinic (2) and hymenin (5) at the lowest concentration (0.125 mM; Table 2). Table 1 Effect of sesquiterpenes on wild oat germination 10 d after sowing.a Conc (mM)
0 0.125 0.25 0.5 1 2 a b
Germination (%) Neoambrosin (1)
Damsinic acid (2)
Damsin (3)
Ambrosin (4)
Hymenin (5)
Imazamethabenz
50.0 2.89ab 46.7 1.67ab 40.0 2.89bc 36.7 1.67c 40.0 0.0bc 23.3 1.67d
50.0 2.89a 41.7 1.67a 40.0 2.89a 38.3 4.21ab 38.3 4.21ab 28.3 1.67b
50.0 2.89a 46.7 4.21a 35.0 2.89b 30.0 0.0b 26.7 1.67b 16.7 4.21c
50.0 2.89a 50.0 0.0a 36.7 4.21b 28.3 1.67c 26.7 1.67c 26.7 1.67c
50.0 2.89a 38.3 1.67b 36.7 1.67b 33.3 4.21b 30 .0 0.0b 30.0 2.89b
50.0 2.89a 35.0 2.89b 38.3 4.21b 40.0 0.0b 30.0 0.0b 31.7 1.67b
Data are expressed as means SE from experiments with three replicates of 20 seeds each. Means within a column sharing the same letter are not significantly different at the 0.05 probability level.
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Table 2 Effect of sesquiterpenes on wild oat root growth.a Conc (mM)
Neoambrosin (1)
0 0.125 0.25 0.5 1 2 EC50d (mM) a b c d
Root length (cm)
I (%)b
9.6 0.36ac 8.5 0.21b 5.7 0.37c 5.2 0.35c 3.7 0.14d 0.5 0.12 0.48
0.0 11.5 40.6 45.6 61.5 94.8
Damsinic acid (2)
Damsin (3)
Root length (cm)
I (%)
Root length (cm)
I (%)
Ambrosin (4) Root length (cm)
I (%)
Hymenin (5) Root length (cm)
I (%)
Imazamethabenz Root length (cm)
I (%)
9.6 0.36a 9.2 0.06a 6.6 0.10b 4.8 0.09c 3.4 0.03d 2.0 0.34e 0.60
0.0 4.2 31.3 50.0 64.6 79.2
9.6 0.36a 6.7 0.22b 4.3 0.26c 3.3 0.17d 0.73 0.09e 0.17 0.07e 0.24
0.0 30.2 55.2 65.6 92.4 98.2
9.6 0.36a 7.4 0.31b 4.1 0.27c 1.4 0.24d 0.73 0.07de 0.10 0.0e 0.22
0.0 22.9 57.3 85.4 92.4 99.0
9.6 0.36a 8.8 0.32ab 8.1 0.38b 6.0 0.27c 2.6 0.25d 0.47 0.47e 0.57
0.0 8.3 15.6 37.5 72.9 95.6
9.6 0.36a 7.8 0.14b 6.7 0.31c 6.5 0.09c 3.7 0.37d 2.8 0.20e 0.74
0.0 18.8 30.2 32.3 61.5 70.8
Data are expressed as means SE from experiments with three replicates of 20 seeds each. I ¼ inhibition. Means within a column sharing the same letter are not significantly different at the 0.05 probability level. EC50 ¼ concentration of compound causing 50% root growth inhibition.
Ambrosin (4) and damsin (3) were the most potent compounds with EC50 values of 0.22 and 0.24 mM, respectively. In contrast, hymenin (5) and damsinic (2) were the less effective. The five tested sesquiterpenes showed greater inhibitory effect of root growth than a reference herbicide, imazamethabenz. The results of this study are consistent with previous studies in which sesquiterpene lactones costunolide and parthenolide strongly reduced the root growth of wheat, lettuce radish, onion and wild oat (Abdelgaleil and Hashinaga, 2007; Abdelgaleil et al., 2009). 3.3. Effect of sesquiterpenes on shoot growth The tested sesquiterpenes exhibited significant reduction of shoot growth particularly at the highest concentrations 0.5, 1 and 2 mM (Table 3). Ambrosin (4) (EC50 ¼ 0.30 mM) was the most active compound, while damsinic (2) (EC50 ¼ 1.09 mM) was the less effective one. Based on the EC50 values, all of the tested compounds had grater shoot growth reduction than a reference herbicide, imazamethabenz. The inhibitory effects of some sesquiterpenes on the root and shoot growth have been described (Baruah et al., 1994; Dayan et al., 1999; Batish et al., 2002; Vyvyan, 2002). The results showed that the inhibitory effects of the tested compounds on root growth were greater than on shoot growth. These results are consistent with those reported elsewhere for other sesquiterpenes and some plant extracts (Chung and Miller, 1995; Turk and Tawaha, 2002; Abdelgaleil et al., 2009). This finding might be expected, because it is likely that roots are the first to absorb the allelochemicals compounds from the environment (Turk and Tawaha, 2002). It was also observed that the inhibitory effect of tested sesquiterpenes on root and shoot growth was greater than that on germination. Similarly, Leather and Einhellig (1985) demonstrated that bioassays determining seedling growth are usually more sensitive than those measuring germination. Moreover, ambrosin (4) with C2–C3 double bond was more active than neoambrosin (1) with C1–C2 double bond. The results indicated that beside the presence of a-methylene- g-lactone, which is essential for potent bioactivity of sesquitrpene lactones, the other function groups and their position on the skeleton may also enhance or reduce the activity of sesquitrpene lactones. Regarding the mechanism of action of sesquiterpenes, it has been reported that sesquiterpene lactones react with the –SH group of amino acids, proteins, and enzymes (Picman, 1986). Parthenolide has been shown to inhibit acetolactate synthase, which is a target enzyme for several classes of commercial herbicides (Abdelgaleil et al., 2009). Other sesquiterpenes have been shown to inhibit cell division and reduce chlorophyll content (Dayan et al., 1999), damage the cell membrane and reduce
Table 3 Effect of sesquiterpenes on wild oat shoot growth.a Conc (mM)
Neoambrosin (1) Shoot length (cm)
0 0.125 0.25 0.5 1 2 EC50d (mM)
11.2 0.35ac 10.9 0.46a 10.0 0.5ab 8.9 0.46b 6.3 0.41c 1.7 0.29d 0.96
a b c d
Damsinic acid (2)
Damsin (3)
I (%)
Shoot length (cm)
I (%)
Shoot length (cm)
I (%)
Shoot length (cm)
I (%)
Shoot length (cm)
I (%)
Shoot length (cm)
I (%)
0.0 2.7 10.7 20.5 43.8 84.8
11.2 0.35a 10.8 0.58a 10.5 0.85a 7.9 0.43b 6.2 0.26b 3.3 0.75c 1.09
0.0 3.6 6.3 29.5 44.6 70.5
11.2 0.35a 11.0 0.35a 10.6 0.49a 8.7 0.57b 3.8 0.24c 1.8 0.25d 0.83
0.0 1.8 5.4 22.3 66.1 83.9
11.2 0.35a 9.0 0.32b 5.4 0.18c 2.9 0.15d 2.7 0.18d 1.5 0.14e 0.30
0.0 19.6 51.8 74.1 75.9 86.6
11.2 0.35a 10.0 0.53a 8.4 0.17b 6.8 0.55c 4.4 0.61d 2.2 0.21e 0.67
0.0 10.7 25.0 39.3 60.7 80.4
11.2 0.35a 9.7 0.57b 8.6 0.45b 8.4 0.33b 5.8 0.24c 4.7 0.24c 1.33
0.0 13.4 23.2 25.0 48.2 58.0
b
Ambrosin (4)
Hymenin (5)
Data are expressed as means SE from experiments with three replicates of 20 seeds each. I ¼ inhibition. Means within a column sharing the same letter are not significantly different at the 0.05 probability level. EC50 ¼ concentration of compound causing 50% shoot growth inhibition.
Imazamethabenz
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dehydrogenase, protease, and peroxidase activities (Batish et al., 2002), affect protein content and respiration (Pandey, 1996). In conclusion, the present study indicates that the sesquiterpene lactones ambrosin (4) and damsin (3) had potent herbicidal activity on seedling growth of wild oat. However, further studies on field efficiency and toxicological effects of these sesquiterpenes are highly recommended. References Abdelgaleil, S.A.M., Abdel-Razeek, N., Soliman, S.A., 2009. Herbicidal activity of three sesquiterpene lactones on wild oat (Avena fatua) and their possible mode of action. Weed Science 57, 6–9. Abdelgaleil, S.A.M., 2010. Assessment of mosquitocidal, herbicidal and molluscicidal potentials of extracts and phytochemicals isolated from three Egyptian plants. Alexandria Journal of Agricultural Research 55, 59–73. Abdelgaleil, S.A.M., Badawy, M.E.I., Suganuma, T., Kitahara, K., 2011. 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