J Arid Land (2013) 5(3): 324–330 doi: 10.1007/s40333-013-0170-2 jal.xjegi.com; www.springer.com/40333

∗Corresponding author: Chi ZHANG (E-mail: zc@ms.xjb.ac.cn) Received 2012-11-07; revised 2013-01-18; accepted 2013-01-28 © Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Science Press and Springer-Verlag Berlin Heidelberg 2013

Chemical composition and phytotoxic activity of the volatile oil of invasive Xanthium italicum Moretti from Xinjiang, China

Hua SHAO1, YuanMing ZHANG1, Peng NAN2, XiaoLi HUANG1, Chi ZHANG3∗

1 Key Laboratory of Biogeography and Bioresource in Arid Land, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China;

2 Ministry of Education Key Laboratory for Biodiversity Science and Ecological Engineering, School of Life Sciences, Fudan University, Shanghai 200433, China;

3 State Key Laboratory of Desert and Oasis Ecology, Xinjiang Institute of Ecology and Geography, Chinese Academy of Sciences, Urumqi 830011, China

Abstract: Aerial parts of Xanthium italicum in an air tight container greatly inhibited root elongation of radish, implying that this invasive plant could release biologically active volatile organic compounds (VOCs) into the envi-ronment to affect other plants’ growth. This phenomenon was further studied by evaluating the phytotoxic effects of X. italicum essential oil against two dicot plants, amaranth (Amaranthus mangostanus L.) and lettuce (Lectuca sa-tiva L.), and two monocot plants, wheat (Triticum aestivum Linn) and ryegrass (Lolium multiforum), and analyzing the chemical composition of the oil. Among the 4 test species, amaranth was the most sensitive plant, 0.5µl/mL essential oil application resulted in a 50% reduction on root elongation, and 2.5 µl/mL essential oil almost com-pletely inhibited its seedling growth. Wheat was the least sensitive species, whose root growth was reduced to 36% of control by 5 µl/mL essential oil. The essential oil exerted moderate inhibitory effect on both lettuce and ryegrass. Compared to a commercial herbicide–Harness, X. italicum oil exhibited stronger phytotoxicity on amaranth, lettuce and wheat, but weaker activity on ryegrass. The chemical composition of the essential oil isolated by hydrodistilla-tion from the aerial parts of X. italicum Moretti was analyzed by GC/MS. Thirty two compounds were identified, representing 94.89% of total oil, which was found to be rich in monoterpene hydrocarbons (60.71%). The main constituents of the oil were limonene (51.61%), germacrene B (6.98%), δ-cadinol (5.94%), β-pinene (5.23%), α-caryophyllene (5.1%) and bornyl acetate (3.15%). Bioassay revealed the dominant constituent–limonene, was unlikely the responsible phytotoxic compound due to its low biological activity; rather, there might be other oil con-stituent(s) that either act alone, or work together, and possibly assisted by synergistic effect, to display the phyto-toxic activity. Our results suggested that X. italicum might produce allelopathic VOCs to facilitate its invasion suc-cess. This is the first report on the phytotoxic activity and the chemical composition of the essential oil of X. italicum Moretti from China.

Keywords: allelopathy; phytotoxicity; essential oil; volatile organic compounds; Xanthium italicum

Citation: Hua SHAO, YuanMing ZHANG, Peng NAN, XiaoLi HUANG, Chi ZHANG. 2013. Chemical composition and phytotoxic activity of the volatile oil of invasive Xanthium italicum Moretti from Xinjiang, China. Journal of Arid Land, 5(3): 324–330.

China is one of the many countries seriously affected by biological invasion. Among the various exotic spe-cies, Italian cocklebur (Xanthium italicum Moretti), a

herbaceous annual weed belonging to the genus Xan-thium (family Asteraceae) that is represented by 25 species distributing throughout Eurasia and America,

Hua SHAO et al.: Chemical composition and phytotoxic activity of the volatile oil of invasive Xanthium italicum Moretti from … 325

was first spotted in Beijing, the capital city of China (Liu et al., 2002; Kovács et al., 2009). In the past two decades, the exotic plant has quickly expanded to six provincial regions, which are not only geographically far away but also characterized by distinctive climates, indicating that X. italicum is capable of adapting to different environmental conditions (Li et al., 2010; Wang et al., 2010). For example, among the six pro-vincial regions, Xinjiang Uygur autonomous region, where our study site was located, is a typical arid re-gion in far northwestern China on the border with Mongolia and Kazakhstan, where the world’s second largest desert, Taklamakan Desert, is located (Zhao, 1995); and it is noteworthy to mention that arid eco-systems are relatively simple in structure and function compared with other terrestrial ecosystems, which makes them more susceptible to invasions (Kitayama and Mueller-Dombois, 1995; Goodall and Perry, 2009). So far, the distribution of X. italicum is some-what limited in China; however, ecological modeling revealed that X. italicum has the potential to spread further to most regions in China, except for several areas with extreme environmental conditions (Wang et al., 2010).

Invasive exotic plants can negatively affect native plant community by either displacing resident species or inhibiting the establishment of new individuals (Yurkonis et al., 2005). X. italicum is a very competi-tive weed; once established, X. italicum is often found to form dense monocultures as long as light, moisture and nutrition are sufficient, which consequently re-sults in adverse impacts on native plant communities. Young Xanthium plants are poisonous to animals, which may contribute in part to its rapid spread (Witte, 1990). Xanthium plants are found to be very difficult to control either chemically or manually, therefore they were listed on the “People’s Republic of China imported plant quarantine pests list” in 2007 (Ministry of Agriculture of the People’s Republic of China, 2007).

Although biological invasion has caused great damage worldwide, the mechanisms underlying this phenomenon are still unclear (Levine et al., 2003). A number of hypotheses have been proposed in the past decades in an effort to explain why some species can

become extremely invasive in invaded regions but not in their native range; among them, the AARS (“al-lelopathic advantage against resident species”) hy-pothesis posits that allelopathy may contribute to the invasion success of some exotic species because they are more allelopathic, or better biochemically de-fended, than source populations (Callaway and Ridenour, 2004; Inderjit et al., 2006). Allelopathy re-fers to any direct and indirect harmful or beneficial effect by one plant on another through the production of chemical compounds that release into the nearby environment (Rice, 1984). Many studies suggest that allelopathy may facilitate the invasiveness of some exotic plants (Callaway and Aschehoug, 2000; Shao et al., 2005; Shanab et al., 2010). Most of the responsible allelochemicals are identified as secondary metabo-lites, including volatile organic compounds (VOCs; Barney et al., 2009; Inderjit et al., 2011). These VOCs can either function directly in their volatile gaseous state, or accumulate in soil matrix to affect neighbor-ing plants’ growth over time (Barney et al., 2009; Inderjit et al., 2011). Our preliminary study showed that aerial parts of X. italicum in an air tight container could inhibit root elongation of radish by 79%, im-plying that this plant can release biologically active VOCs into the environment to affect other plants’ growth (data not shown). There was a recent report on the chemical composition of X. italicum oil from Cor-sica (Andreani et al., 2012); however, no study on the chemical composition and the phytotoxitiy of X. itali-cum oil from Chinese origin has ever been conducted. In the current study, we evaluated the phytotoxic ac-tivity of X. italicum oil and analyzed its chemical composition, which could help us understand the pos-sible involvement of X. italicum VOCs as potential allelochemicals in its invasion success.

1 Materials and methods

1.1 Materials

Aerial plant parts of X. italicum plants, including leaves, stems, flowers and young fruits, were collected from a dense monoculture growing along the roadside in Urumqi city, Xinjiang Uygur autonomous region in China in September 2011 (43°54′40.2″N, 87°17′7.6″E).

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The plants were harvested at their early fruiting stage and fresh plant materials were used directly for ex-tracting essential oil. (R)-(+)-Limonene (analytical standard) was purchased from Sigma-Aldrich Co. (St. Louis, USA).

1.2 Isolation of the essential oil

Fresh plant material (200 g) was hydrodistillated for 3 h using a Clevenger type apparatus to isolate the essen-tial oil. The oil was dried over anhydrous sodium sul-fate and was kept in a sealed vial at 4ºC until required. The isolation procedure was repeated until sufficient oil was obtained for the gas chromatography/mass spectrometry (GC-MS) analysis and phytotoxic as-says.

1.3 Gas chromatography-mass spectrometry ana-lysis

The oil was analyzed by GC-MS using a Perkin-Ele-mer Autosystem XL-Turbemass system with a PE-5MS capillary column (60 m×0.25 mm; 0.25 μm film thickness). The carrier gas was helium, with a flow rate of 1 ml/min. The oven temperature was held at 60ºC for 5 min then programmed at rate of 2ºC/min to 270ºC and then held at this temperature for 3 min. Mass spectra were taken at 70 ev. Mass range was from m/z 35–350 amu. Injector port temperature is 280ºC, detector 280ºC, injected volume 0.1 μL, split ratio 1:50. Relative amounts of individual components were calculated based on GC peak areas without flame ionization detector (FID) response factor correction. Identification of the constituents of the essential oil was made by comparison of their mass spectra and retention indices (RI, calculated by linear interpolation relative to retention times of a standard mixture of C8-C28 n-alkanes) with the National Institute of Stan-dards and Technology (NIST) and a home-made li-brary and those given in the literature (Andreani et al., 2012; Esmaeili et al., 2012).

1.4 Phytotoxic effect of the essential oil

The phytotoxic effect of essential oil as well as the major constituent (i.e. limonene, 51.61% of the oil) were evaluated by conducting bioassays against two dicot plants, amaranth (Amaranthus mangostanus L.) and lettuce (Lectuca sativa L.), along with two

monocot plants, wheat (Triticum aestivum Linn) and ryegrass (Lolium multiforum). A commercial herbicide, Harness (Monsanto Co., St. Louis, USA), was used as a reference. Seeds of 4 test species were surface ster-ilized with 0.5% HgCl2 before use. Essential oil and limonene were diluted in 0.5% acetone in distilled H2O to give 0.5, 2.5, 5.0 µl/mL; with acetone as the initial solvent (previous study showed that acetone at such concentration did not significantly affect seedling growth of 4 test plants). Harness was diluted with dis-tilled H2O. The volume of 3 mL of 0.5% acetone in distilled H2O (control) or diluted solutions was added to each petri dish (9-cm in diameter), followed by ad-dition of 10 test seeds. Petri dishes were sealed with parafilm to prevent water loss and essential oil vola-tilization and stored in the dark at 25°C. Seedlings were measured after 4 days of cultivation for amaranth, lettuce and wheat, and 5 days for ryegrass. Three rep-licates were made for all phytotoxic bioassays (in total 30 seedlings were measured).

1.5 Statistical analyses

The significance of phytotoxic effects of essential oil, limonene and Harness on seedling growth of test spe-cies was first examined by ANOVA (P<0.05) and then analyzed using Fisher’s LSD test at P<0.05 level. All of the statistical analyses were performed using SPSS 13 software package.

2 Results and discussion

2.1 Composition of the essential oil

Essential oil of X. italicum obtained by hydrodistilla-tion of fresh aerial plant parts had a bright yellow color with an herbal odor. The yield was 0.05% (v/w, volume/fresh weight). In total 33 oil compounds were identified, accounting for 94.89% of the total oil, while 5.1% of the oil remained unidentified (Table 1). Monoterpene hydrocarbons displayed the highest con-tribution (60.71%), whereas oxygenated monoterpenes represented only 7.02% of the total oil. In comparison with monoterpenes, sesquiterpenes were relatively weak (23.93%), with 17% of sesquiterpene hydrocar-bons and only 6.93% oxygenated sesquiterpenes. Among the 33 oil constituents, the most abundant

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Table 1 Essential oil composition of X. italicum Moretti

Compounds Rotation time (min)

Percentage (%)

α-Pinene 6.32 1.08

Camphene 6.90 0.84

β-Pinene 7.80 5.60

β-Myrcene 8.34 0.41

Limonene 10.72 51.61

γ-Terpinene 11.74 0.13

Nonanal 14.34 0.39

2,6-Dimethyl-1,3,5,7-octatetraene, E,E- 16.53 1.04

Pinocarvone 17.81 0.57

Thujol 18.59 1.86

3,5-Nonadien-7-yn-2-ol 19.04 0.42

Myrtenal 19.89 0.11

Trans-3(10)-Caren-2-ol 20.04 0.24

β-Cyclocitral 21.55 0.92

Bornyl acetate 25.98 3.15

3-Tridecene 26.53 0.21

α-Clovene 32.68 0.79

Eugenol methyl ether 33.58 0.17

α-Gurjunene 33.81 0.06

Caryophyllene 34.60 0.56

γ-Elemene 35.46 1.57

α-Caryophyllene 37.13 5.10

Acetic acid, undec-2-enyl ester 38.90 1.00

Cadina-1,4-diene 39.24 0.29

2- Tridecanone 39.77 0.40 Germacrene D 41.14 1.65 Germacrene B 43.67 6.98 Caryophyllene oxide 46.40 0.22 α-trans-Bergamotenol 48.70 0.18 τ-Cadinol 49.21 0.59

δ-Cadinol 51.73 5.94

Phytol 73.31 0.81

compounds were limonene (51.61%), germacrene B (6.98%), δ-cadinol (5.94%), β-pinene (5.23%), α-caryophyllene (5.1%) and bornyl acetate (3.15%). The limonene percent in the test essential oil was in agreement with the reported composition of X. cava-nillesii (43.6%), X. pennsylvanicum (65%), X. pun-gens (40.2%), X. strumarium L. (24.7%) essential oil (Taher et al., 1985; Ammar et al., 1992; Esmaeili et al., 2012), and X. italicum (35.3%) from Corsica (Andre-ani et al., 2012).

2.2 Phytotoxic effect of the essential oil, limonene and Harness

Among the 4 test species, amaranth was the most sen-sitive plant to essential oil treatment, whereas wheat was the least sensitive species (Fig. 1). The applica-tion of 0.5 µl/mL essential oil resulted in a 50% re-duction in root elongation of amaranth seedlings, and 2.5 µl/mL essential oil almost completely inhibited its seedling growth. In comparison, root growth of wheat seedlings was still 36% of control even when essential oil concentration reached 5.0 µl/mL. Essential oil ex-erted relatively moderate inhibitory effect on lettuce and ryegrass, whose root elongation was inhibited by 56%, 71%, 90%, and 44%, 58%, 82%, by 0.5, 2.5 and 5.0 µl/mL essential oil, respectively. Shoot growth of all test species responded to essential oil treatment in a similar way compared to root growth (data not shown).

Fig. 1 Phytotoxic effect of X. italicum oil on root growth of amaranth, lettuce, ryegrass and wheat. Means with different letters indicate significant differences at P<0.05 level ac-cording to Fisher’s LSD test.

As the only dominant component of the essential oil, limonene exhibited very weak plant growth effect on 4 receiver species (Fig. 2). Root growth of amaranth was unaffected at tested concentrations; for lettuce, the root growth was promoted by 0.5 and 2.5 µl/mL essential oil and not significantly influenced by 5.0 µl/mL oil; in comparison, root growth of ryegrass and wheat was significantly suppressed at 5.0 and 2.5 µl/mL, respectively. On the other hand, the positive control, Harness, exhibited slightly weaker inhibitory activity on amaranth, lettuce and wheat, but much stronger activity on ryegrass (Fig. 3). At the highest

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Fig. 2 Phytotoxic effect of limonene on root growth of amaranth, lettuce, ryegrass and wheat. Means with different letters indicate significant differences at P<0.05 level ac-cording to Fisher’s LSD test.

Fig. 3 Phytotoxic effect of Harness on root growth of ama-ranth, lettuce, ryegrass and wheat. Means with different let-ters indicate significant differences at P<0.05 level according to Fisher’s LSD test.

concentration (5.0 µl/mL), root length of amaranth, lettuce, ryegrass and wheat was reduced by 44%, 70%, 100% and 43%, respectively, with ryegrass being the most sensitive species.

It is speculated that allelopathy might play a role in the invasion of X. italicum. This plant contains pheno-lic acids, a group of chemicals reported to be the re-sponsible allelochemicals of some plants (David et al., 2005); besides, aqueous and organic extracts of X. italicum were reported to be phytotoxic (Casini, 2004;

Shao et al., 2012). In a separate study, we have con-firmed the presence of three xanthanolides, i.e. xan-thinin, xanthatin and xanthinosin, with moderate to strong phytotoxicity, in soils infested by X. italicum, suggesting these xanthanolides might contribute, at least in part, to the allelopathic property of this plant (unpublished data). Results from the present study demonstrated that X. italicum oil had phytotoxic effect on seedling growth of amaranth, lettuce, ryegrass and wheat. Essential oils have been reported to possess a variety of biological activities, such as antibacterial, antifungal, antioxidant, insecticidal and herbicidal activities (Scherer et al., 2010; Krifa et al., 2011; Zu-zarte et al., 2012). Besides numerous studies on the phytotoxic activity of essential oil from various plant species, field experiments also demonstrated the al-lelopathic activity of essential oil. Barney et al. (2009) found that volatile monoterpenes released from an invasive perennial in North America, Artemisia vul-garis, exhibited little direct inhibitory activity in their volatile gaseous state; rather, they concentrate in the soil and adversely affect growth of vegetation in the vicinity, which consequently fosters its invasiveness in the invaded region. In another study, leaf litter of in-vasive Ageratina adenophora was found to be able to emit biologically active volatile compounds to affect other plants’ growth, which was considered to facili-tate its invasion success (Inderjit et al., 2011). VOCs produced by A. adenophora as invaders differed quan-titatively but not qualitatively than those produced in its native habitat, suggesting that this plant may be experiencing selection on oil composition in its in-vaded ranges. Our results revealed that X. italiucm oil of Chinese origin differred from that of Corsica origin both qualitatively and quantitatively, suggesting that biogeographic differences might be factors contribut-ing to its invasion (Andreani et al., 2012).

Due to the fact that X. italicum oil consists of doz-ens of compounds, it is difficult to identify which one(s) is responsible phytotoxins. Monoterpenes (67.73% of the oil) dominated in X. italicum oil. Pre-viously, monoterpenes have been reported to possess plant growth regulatory activity; they were found to stimulate plant germination at low concentration and inhibited it at high concentration (Asplund, 2001; Martino et al., 2010). Limonene, a monoterpene con-

Hua SHAO et al.: Chemical composition and phytotoxic activity of the volatile oil of invasive Xanthium italicum Moretti from … 329

stitutes 51.61% of the oil, was previously reported to have allelopathic activity (Ibrahim, 2001; however, it is unlikely the responsible compound in X. italicum oil, due to its low biological activity. Even at its highest concentration (5.0 µl/mL), limonene only inhibited root growth of ryegrass and wheat by 24% and 22%, respectively, indicating that there might be other chemicals contributing to the phytotoxicity of X. italicum oil. These compounds, either act alone, or work together or possibly facilitated by synergistic effect, to display the phytotoxic activity.

Finally, it is noteworthy to mention that the release of volatile phytoxoxins into the soil matrix may result in complicated ecological consequences other than their toxicity alone, which may subsequently alter the soil properties. It was reported that phytotoxins passed into the soil had important impacts on soil nutrient dynamics, such as organic matter dynamics and nutri-ent cycling (Kuiters, 1990; White, 1994); further, phytoxoxins may also alter the soil microbial commu-nity structure and function, which could presumably create unfavorable growth conditions for other plants (Kourtev et al., 2002; Ehrenfeld, 2003). Therefore, VOCs released by X. italicum, possibly along with other allelochemicals, might function directly and in-directly to influence neighboring plants’ growth, and foster its dominance in the invaded regions.

3 Conclusions

The essential oil of X. italicum exhibited strong phy-totoxic activity on two monocot plants and two dicot plants. GC-MS revealed that monoterpenes were the dominant constituents, which have been reported to possess plant growth regulatory activity. Limonene was the main component of the oil (51.61%) but unlikely the responsible toxic compound. Our results suggested that VOCs emitted by X. italicum might function as active allelochemicals, and foster its inva-sion success in China.

Acknowledgements

This study is financially supported by the International Sci-ence and Technology Cooperation Program of China (2010 DFA 92720-06), the One Hundred Person Project of the Chinese Academy of Sciences granted to Chi ZHANG, and

the West Light Foundation of the Chinese Academy of Sci-ences granted to Hua SHAO (LHXZ201202).

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