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Keywords:

  • activated carbon;
  • allelopathy;
  • biological invasions;
  • goldenrod;
  • novel weapons hypothesis;
  • root exudates;
  • soil biota feedbacks

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
  • 1
    One mechanism explaining the success of invasive weeds may be the production and release of allelopathic compounds by the invader that, due to a lack of co-evolutionary history, have harmful effects on plant neighbours in the introduced range.
  • 2
    We partially tested this hypothesis by growing seven competing native European plant species either with the introduced Solidago canadensis s.l., one of the most successful invasive plants in Europe or on soil pre-cultivated with S. canadensis. We added activated carbon to the soil to neutralize organic chemical compounds with putative allelopathic effects. Furthermore, we added unsterilized soil inocula from the introduced (Switzerland) or native (USA) range to the soil to test potential confounding effects of soil microbes on invasion success. Untreated sterilized soil served as control.
  • 3
    Five out of the seven native species were more competitive against the invasive species in soils with activated carbon than without, supporting the allelopathy hypothesis. However, competitive outcomes were also influenced by the two sources of soil inoculum and by interactive effects of soil inoculum and Solidago origin suggesting that soil microbes alter allelopathic effects.
  • 4
    Achillea millefolium, the species least affected by the presence of S. canadensis and with no response to the activated carbon treatment is the only species used in this experiment reported to grow within Solidago stands in Europe, whereas the other European species tested tend to grow at the periphery of invasive Solidago stands.
  • 5
    Chemical analysis by LC-MS of Solidago root extracts revealed four main secondary chemical compounds with potential allelopathic effects. Root exudates of Solidago showed a significant inhibitory effect on growth of the model plant species Arabidopsis thaliana. The magnitude of inhibition increased with increasing concentration of the extract.
  • 6
    Synthesis. Levels of the four compounds were lower in Solidago populations from the invasive range than in populations of the same ploidy level from the native range. This suggests lower investment of invasive plants into these secondary compounds, possibly because of a higher susceptibility of plant competitors in the invasive range to these substances.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Solidago canadensis s.l. L. (Canada goldenrod) is an exceptionally successful worldwide invader of North American origin (Semple & Cook 2006) that has to date conquered Europe, large parts of Asia, Australia and New Zealand (Weber 2003; Lu et al. 2007). However, the traits enabling the species to successfully establish in natural ecosystems around the world and dominate the new ecosystems remain unclear. Even though S. canadensis s.l. in the invasive range only possesses a fraction of the insect herbivores that are found within the native range, a controlled biogeographical comparison did not find any evidence for a competitive advantage due to enemy release or due to the introduction of pre-adapted especially vigorous genotypes (van Kleunen & Schmid 2003): In North America, 314 insect herbivores have been reported to feed on the species (reviewed in Jobin et al. 1996), whereas a survey in Switzerland revealed a total of 55 generalist species upon Solidago of which only 18 species were detected to actually feed on the plant (but this feeding did not lead to any noticeable reduction in plant performance; Jobin et al. 1996). Despite this reduction in herbivore pressure, no indication of a rapid evolutionary shift in allocation from defence to growth due to enemy release has been observed until now: populations of S. canadensis from the introduced range in Europe have smaller inflorescences, tend to have fewer vegetative offspring and do not grow as tall as plants from the native North American range (van Kleunen & Schmid 2003) where stem density is a key mechanism to limit colonization dynamics of competing plant species (Crutsinger et al. 2007). Hence, other mechanisms are likely to be involved in the invasion success of this species. One alternative explanation for the establishment and spread of invasive plants in undisturbed communities is the release of novel (not experienced before in the invaded ecosystem) phytochemicals by the invader, such as allelopathic compounds that have phytotoxic or at least fitness-reducing effects on plant neighbours that have not been co-evolved (Rabotnov 1982; Callaway & Aschehoug 2000; Callaway & Ridenour 2004; Prati & Bossdorf 2004; Cappuccino & Arnason 2006; Schenk 2006). These non-coevolved competitors may be more susceptible to novel phytochemicals than adapted competitors in the native range (novel weapons hypothesis; Callaway & Aschehoug 2000). Allelopathy is the phytotoxicity of a compound or a group of compounds released from plant parts by leaching, root exudation, volatilization or residue decomposition to susceptible plants (Inderjit et al. 2006). These compounds are diverse in chemical structure. They vary in concentration and localization in plant tissues between plant species and they differ in concentration depending on (seasonal) changes in both biotic and abiotic environmental conditions (Inderjit & Duke 2003). There is accumulating evidence for negative effects of allelopathic plants on the fitness of other competing plants, for example, the reduction of seed germination and seedling growth (see Tarayre et al. 1995; Hierro & Callaway 2003). However, it is difficult to separate direct allelopathic effects from indirect negative effects of invasive non-indigenous plants mediated by soil micro-organisms (Mangla et al. 2008). Indirect negative effects due to other organisms may be quite common and lead to net changes in ecosystem properties (Wardle et al. 1998). Affected organisms include soil pathogens as well as symbiotic and saprophytic soil micro-organisms (Wardle et al. 1998; Mangla et al. 2008). Hence, invasiveness may be driven by the combined effects of the novel phytochemistry of the invader (Callaway & Ridenour 2004; Cappuccino & Arnason 2006; Reinhart & Callaway 2006) and the alteration of soil microbial communities (Kourtev et al. 2002). Dominant invasive plant species may often show strong positive feedback loops with soil biota, such as receiving more benefits from biotic interactions with mycorrhizal fungi than their native plant competitors (Klironomos 2002) or through having escaped their native microbial pathogens and viruses (Mitchell & Power 2003; Mitchell et al. 2006). Hence the success of invasive species may also be ascribed to the influence of soil micro-organisms. Therefore, examining allelopathic effects in the context of native and non-native soils may provide a greater generality to the overall effect.

To investigate the influence of allelopathy and soil microbes on invasion success, we compared the germination and early growth of European competitor species growing either with invasive European or with native American populations of S. canadensis s.l. on sterile soils or on (sterile) soils where an unsterilized Swiss and American soil inoculum were added. All treatments were planted with and without the addition of finely ground activated carbon to the substrate to reduce potential interference by allelopathic chemicals in the soils (see Callaway & Aschehoug 2000). An enhanced performance of competitor species if activated carbon is added to the soil is regarded as an indication of the presence of allelopathic substances in the soil (see Prati & Bossdorf 2004; Vivanco et al. 2004). As it is not clear if invasive plants must be ‘pre-adapted’ or if they evolve these traits after invasion (Maron 2006), we chose a biogeographical approach (Hierro et al. 2005), comparing a range of native North American populations of S. canadensis s.l. differing in ploidy level with populations from the invasive European range. Specifically, we tested whether plant competitor performance depended on (i) the presence of activated carbon in the soil, (ii) the origin of the Solidago plants (native vs. invasive), (iii) the soil inoculum, or (iv) interactive effects of these factors.

Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

study species

Solidago canadensis s.l. L. was introduced into Europe in the 17th century (Weber 2001). It is a perennial herb forming large clonal colonies that tend to reduce the abundance of native vegetation (Weber 1998). Flowers are self-sterile (Schmid & Dolt 1994) and seeds are wind-dispersed (Weber 1997). Solidago canadensis is one of the most aggressive invaders in Europe (Zwoelfer 1976; Kowarik & Starfinger 2002) and has also been introduced to parts of Asia and Australia (Weber 2003; Dong et al. 2006). The taxon is highly variable and includes diploid, tetraploid and hexaploid plants in the native range (Semple & Cook 2006; Halverson et al. 2008), but only diploid cytotypes are found in Europe (van Kleunen & Schmid 2003).

experimental setup i

Five native American populations of S. canadensis s.l. from different sites on the east coast of the United States and eight invasive European populations of S. canadensis from different sites in Austria, Czech Republic, France, Germany and Switzerland (Table 1) were chosen and two maternal genotypes were used per population and block. Ploidy level was either known from an earlier study using plant material from the same populations (van Kleunen & Schmid 2003) or determined by flow cytometry (Partec, Münster, Germany: see Schlaepfer et al. in press for a description of the ploidy determination). In October 2004, seedlings were individually planted in 1-L-pots (11 × 11 × 12 cm) containing 1 : 1 = sterile sand: commercial potting compost (Tref Substrates Coervorden B.V. The Netherlands), which was autoclaved twice for 20 min at 121 °C within a time period of 12 h (see Bartelt-Ryser et al. 2005). One gram of ‘Osmocote,’ a slow release fertilizer, was added to each pot to minimize possible confounding effects of activated carbon on nitrogen availability (Lau et al. 2008). In a fully crossed design, 2% (20 mL, 8 g) of pure, finely ground activated carbon, particle size < 0.8 mm (puriss. p.a., Fluka), was added to half of the pots. The carbon treatment was crossed with a soil treatment: to one-third of the pots 50 mL of unsterilized soil from a Swiss Solidago site, or 50 mL of unsterilized soil from an American Solidago site, or 50 mL of sterilized soil, was added. The total N content (determined with a CHN-analyzer; LECO-932; St. Joseph, MI) of the Swiss soil inoculum was similar to the sterilized potting compost (0.62% and 0.79%, respectively) and an order of magnitude higher than the US soil inoculum (0.027%). However, these differences in total N content should not have had an effect on plant growth, as pots were additionally fertilized (see above) and the soil inoculum comprised 5% of the total soil only (see also Bartelt-Ryser et al. 2005). In total, we had 249 pots distributed in two blocks (due to seedling mortality, there were often less than four plants left per population and treatment). We randomized the positions of the pots in each block and the pots were placed in 14.2× 16.2 m slug exclosure cages in an experimental garden at the University of Zurich, Switzerland (47°33′ N, 8°37′ E, Altitude 534 m a.s.l.).

Table 1.  Names, latitude, and longitude of the cities nearest the seeds’ harvest location; ploidy level of the parent population is also indicated
SiteLatitudeLongitudePloidy
  • *

    Populations used in experiment II.

  • Populations used in experiment I.

North America
 Petersburg (North Dakota)*48°00′ N97°59′ W4
 Huntington (Vermont)*44°19′ N72°59′ W2
 Saukville (Wisconsin)*43°26′ N87°58′ W2/6
 Claremont (New Hampshire)*†43°22′ N72°20′ W6
 Haverhill (Massachusetts)*42°46′ N71°04′ W6
 Ann Harbor A (Michigan)*42°27′ N83°45′ W2
 Ann Harbor B (Michigan)*42°27′ N83°45′ W2
 Ann Harbor C (Michigan)*42°27′ N83°45′ W6
 Ithaca (New York)*†42°26′ N76°30′ W6
 Boston A (Massachusetts)*†42°17′ N71°25′ W2
 Boston B (Massachusetts)42°17′ N71°25′ W2
 Boston C (Massachusetts)42°17′ N71°25′ W4
 Lone tree (Iowa)*41°30′ N91°26′ W6
Europe
 Friedrichsthal (Germany)*†52°47′ N13°17′ E2
 Göttingen (Germany)*†51°31′ N09°55′ E2
 Katowice (Poland)*50°15′ N19°01′ E2
 Prague (Czech Republic)*†50°05′ N14°25′ E2
 Hegenheim (France)*†47°33′ N07°31′ E2
 Arlesheim (Switzerland)47°28′ N07°37′ E2
 Sihlbrugg (Switzerland)*†47°14′ N08°33′ E2
 Landeck (Austria)*†47°08′ N10°34′ E2
 Fribourg (Switzerland)*†46°48′ N07°08′ E2
 Chiasso (Switzerland)*45°50′ N09°01′ E2

After 10 weeks, pots were shifted to an indoor plant-growing chamber (20–24 °C; 16 h light, 8 h dark) where all Solidago plants were measured and then trimmed to the same height (20 cm). Afterwards, 10 seeds each of the three European competitor species, Arrhenatherum elatius (L.)Presl., Trifolium pratense L., and Lythrum salicaria L., were sown around the S. canadensis plants in each pot. As a control, all species were also sown in additional pots in monocultures. These competitor species have been recorded to grow within or at the periphery of S. canadensis stands in Europe (Voser-Huber 1983; Gruber & Eichberger 2006). Seeds were acquired from a commercial distributor of local ecotypes (UFA Seed Company, Switzerland). All pots were regularly watered. The germination rate was assessed after 5 weeks; after 3 months, above-ground biomass of A. elatius and T. pratense was harvested and dried for 48 h at 80 °C before being weighed. At the time of harvest, height and phenological state of Solidago plants were measured and these data were included as a covariate in the statistical analyses to correct for direct effects of competition for light depending on size of Solidago plants. Seeds of L. salicaria did not germinate during this period, hence seeds of Lythrum were re-sown and pots were transferred to the experimental garden to trigger germination of this species. Four weeks after sowing, the germination rate of Lythrum was assessed; after 8 weeks, above-ground biomass was harvested and dried for 48 h at 80 °C.

experimental setup ii

Solidago canadensis plants were pre-cultivated from April 2000 to July 2006 in 5-L pots in the experimental garden of the Institute of Environmental Sciences, Zürich, Switzerland. In July 2006, we took 250 mL soil inoculum from each of the pots representing 11 native American and nine invasive European populations of S. canadensis (see Table 1). Two maternal genotypes were used per population and treatment. These ‘pre-cultivated’ soil samples were transferred to 1-L pots (11 × 11 × 12 cm) and mixed with peat soil and sand (1 : 1) and 1 g of ‘Osmocote,’ a slow release fertilizer. To half of the 80 pots, 2% (8 g) activated Carbon (puriss. p.a.; Fluka) was added. Five native competitor species, which have been recorded to grow either at the edge of S. canadensis stands in Europe (A. elatius, T. repens L., Stachys officinalis (L.) Trevis., Dactylis glomerata L.) or are commonly found growing within Solidago stands (Achillea millefolium L.; Voser-Huber 1983; J. Joshi, pers. obs.) were selected. Ten seeds per species were sown into each pot. Pots were randomly grouped within trays with the restriction that all pots in a tray contained pots with or without activated carbon and afterwards were placed in snail-proof exclosure cages at the experimental garden of the University of Zurich. After germination, pots were randomized among trays every 7 days. One set of controls of individual competitor species were sown either as monocultures or mixtures in pots with activated carbon but without the Solidago-soil inoculum. The other set of controls contained no activated carbon and no Solidago-soil inoculum. Carbon addition in control pots without any Solidago soil inoculum did not influence germination (P > 0.8, mean germination rate without carbon 51% vs. 53% if carbon was added) nor did the diversity of the control pots (monocultures vs. mixture) influence the germination rate (P > 0.8). The plants were watered daily with automatic watering sprinklers. However, the automatic watering sprinkler ceased to work during a long hot weekend in summer causing premature death of most plants. Therefore, only germination data are presented for this experiment.

phytotoxic bioassay

Arabidopsis thaliana Columbia-0 seeds purchased from Lehle Seeds (Roundrock, TX) were surface-sterilized with 100% commercial bleach for 1 min, rinsed three times in sterile distilled water and germinated on solid Murashige and Skoog medium (MS medium; Murashige & Skoog 1962) in a 25 °C incubator with a 16 h/8 h day/night schedule. Seven-day-old plants were transferred into 1 mL of liquid MS medium supplemented with 3% sucrose in 24-well plates (VWR Scientific). A crude extract of Solidago root exudates was obtained from an aeroponic culture: seeds of S. canadensis acquired from a commercial distributor of native North American ecotypes (Western Native Seed, Coaldale, CO) were germinated in sand. After roots were long enough to put in an aeroponic system (2–3 week old seedlings), they were transferred to the system (EZ Clone 120, Green Coast hydroponic) in the greenhouse and the plants were grown in 1/2 strength Hoagland's solution for 8 weeks. Exudates (around 70 L) were collected and filtered through Whatman paper no. 1 prior to the concentration process. Oasis HLB (6 g) solid phase extraction cartridges (Part # 186000118; Waters Corporation Milford, MA) were chosen to concentrate the exudates. The column was conditioned with 100 mL of methanol followed by 100 mL of distilled water before the addition of the exudates. Once the exudates were eluted, the column was washed with 500 mL of distilled water and the recovery of the metabolites was completed by eluting the column with methanol. The methanol extract was collected and dried under vacuum.

After 24 h, the Arabidopsis seedlings were spiked with five concentrations of the crude extract (0, 25, 50, 250 and 500 µg mL−1), with four replicates per treatment. The crude extract of the root exudates was dissolved in methanol, and applied to the 24-well plates containing the seedlings. MeOH (50 µL) was added to the control wells. After spiking, well plates were sealed with parafilm and incubated on rotary shakers for 7 days. After 7 days the fresh weight of each plant was recorded. The effects of the extracts were analysed using an anova and Dunnetts two-tailed comparison (XLSTAT Pro 2006; Addinsoft, Paris, France) to determine if the effects were significantly different to control plants.

chemical analysis

Medium extraction: To examine the compounds present in different rhizome samples from S. canadensis, 40 randomized (two samples/population), neutrally numbered (sample identity was only known to a researcher not involved in chemical analysis), dried and powdered samples of this species were extracted two times over 24 h with dichloromethane at room temperature. The dried extract was dissolved in 1 mL of absolute dimethyl sulfoxide (Fisher Scientific) and analyzed by LC-ESI-MS.

The HPLC system was equipped with a P680 pump, an ASI-100 autosampler and a PDA-100 photodiode array detector (Dionex). Samples were chromatographed (20 µL) on an analytical Dionex Acclaim 120 C18 column (5 µm, 4.6 × 150 mm) using gradient elution. The mobile phase consisted of 0.1% (v/v) acetic acid in water (A)-methanol (B). The solvent regimen used comprised 3 min at 10% B, a linear gradient to 90% B over 40 min, held at 90% B for 8 min, with a flow rate of 0.7 mL min−1. The mass of the peaks was determined by a Thermo Finnigan Surveyor MSQ mass spectral detector. Ionization for MS analysis was performed in positive and negative ion mode using electrospray ionization with a nitrogen flow at 80 psi, a cone voltage of 70 V, needle voltage of 3 kV, and cone temperature of 600 °C. Mass data were collected over the range of the gradient program at the rate of one scan per 2 s. UV detection was recorded from 200 to 800 nm. All the solvents used were HPLC grade. Relative abundance of the compounds was given based on the peak heights present in the chromatogram with measurements in milli absorption units (mAU). As the identity of the compounds is yet unknown, we could not use size standards. Therefore, the measurements were grouped into five concentration classes (0; 1–100 mAU; 101–500 mAU; 501–1000 mAU; 1001–6000 mAU).

statistical analysis

Statistical analyses were conducted using R (version 2.3.1). The data were analyzed using general linear models to calculate summary analyses of variance tables. Significance tests were based on F-tests (anova). For concentration-level data, we took the median value of each concentration level (see Appendix S1 in Supplementary Material) and transformed the data to the power of 1/4 to meet analysis of variance assumptions of homoscedasticity and normality. The sequential analysis included main effects of treatments in this sequence: addition of activated carbon, soil sterilization, addition of unsterilized soil inocula from Solidago sites, ploidy level, native vs. invasive populations (fixed factor) as well as interactions of ploidy level and native vs. invasive origin with treatment factors. Ploidy level was fitted before continent of origin (native-vs.-invasive) to test and adjust for differences between hexaploid, tetraploid and diploid plants (van Kleunen & Schmid 2003). The effects of blocks (Experiment 1), tray and population identity were considered random factors. Tray number was included in experiment 2 in the analysis of seedling number. The effects of ploidy level and of native vs. invasive populations were tested for significance with F-ratios using the pooled variation among both invasive and native populations (term population) as denominator (Joshi & Vrieling 2005). Pots where no competitor species germinated were excluded from the biomass analyses.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

effects of solidago ploidy level and origin as well as carbon addition on performance of competitors of the invasive range

Of the seven competitor species tested, only one species, L. salicaria, showed a trend to be differentially affected by Solidago plants of different ploidy levels having a 49% lower germination rate when grown in pots with hexaploid compared to diploid Solidago plants (F1,11 = 4.59; P = 0.055). Carbon addition in Lythrum pots, however, did not have differential effects depending on ploidy level (P > 0.5). Overall, four of the seven competitor species tested germinated better if activated carbon was added to the soil (Fig. 1): L. salicaria germinated consistently better in pots with Solidago plants if activated carbon was added (Experiment 1; Fig. 1). The germination of D. glomerata, S. officinalis and to a lesser extent T. repens, was lower even in pots that contained only a soil inoculum conditioned with S. canadensis without carbon addition (Experiment 2, Fig. 1).

image

Figure 1. The effect of carbon addition on germination success of plant competitors grown in soils conditioned by Solidago canadensis s.l.. Vertical bars denote ± 1 SE.

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The positive effect of carbon addition did in some species depend on the origin of the Solidago plants: In A. elatius, the germination was positively affected by the addition of activated carbon only in pots with invasive European S. canadensis plants (Fig. 2; carbon × native vs. invasive: F1,10 = 5.60; P < 0.05). Three months after sowing, the biomass of Arrhenatherum was more than twofold higher in pots where activated carbon was added than in pots without (0.77 g dry mass ± 0.10 vs. 0.32 g ± 0.05; F1,145 = 9.56; P < 0.01). In contrast to Arrhenatherum, the germination of Dactylis was lowest if grown in pots where a soil inoculum of native American Solidago populations was added; additionally the carbon effect was biggest in this treatment (carbon × native vs. invasive: F1,17 = 4.71; P < 0.05; carbon addition to pots with a soil inoculum of native Solidagos: 3.27 seedlings ± 0.38 [RIGHTWARDS ARROW] 5.32 ± 0.37 compared with soil of invasive European Solidagos: 4.67 ± 0.43 [RIGHTWARDS ARROW] 4.72 ± 0.38). Trifolium pratense did not react to any addition of activated carbon to the soil. However, the species tended to grow better in competition with native American Solidago plants than with invasive European Solidagos (0.12 g dry mass ± 0.044 vs. 0.07 g ± 0.017; F1,11 = 4.26; P = 0.06). A similar increase in biomass, though only marginally significant, was observed when T. repens, was grown in competition with native American Solidagos versus invasive European plants (0.21 g dry mass ± 0.012 vs. 0.18 g ± 0.014; F1,17 = 3.07; P < 0.1).

image

Figure 2. In Arrhenatherum elatius, the germination was only positively affected by the addition of activated carbon in pots with invasive European S. canadensis plants (carbon × native-vs.-invasive: F1,10 = 5.60; P < 0.05). Vertical bars denote ± 1 SE.

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interactive effects of carbon addition and soil microbial origin on performance of competitors of the invasive range

The species used in experiment 1 where the carbon treatment was added to three different soil types (sterilized soil, pots with Swiss (invasive) soil inoculum, pots with (native) US soil inoculum) revealed complex patterns as overall allelopathic effects were altered by soil types: the biomass of L. salicaria, a species native to Europe that is found in the same habitats as Solidago and is currently invasive in North America, was positively influenced by carbon addition everywhere except when grown with native American plants on American soil (carbon × soil × native vs. invasive: F4,14 = 7.23; P < 0.01). If carbon was added, Lythrum grew best in Swiss soil together with invasive European Solidagos. In contrast, Arrhenatherum grew best on US soil (Fig. 3) and observed positive effects of carbon addition on biomass differed among the soil types tested (carbon × soil: F2,145 = 9.05; P < 0.01): the addition of carbon mainly had a beneficial effect on growth of Arrhenatherum in native US soils (Fig. 3), whereas the effect was less distinct in sterilized soil and in Swiss soil (Fig. 3).

image

Figure 3. Arrhenatherum elatius grew best on US soil and the positive carbon effect differed among the soil types tested (carbon × soil: F2,145 = 9.05; P < 0.01). Vertical bars denote ± 1 SE.

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Two species were not influenced by the addition of activated carbon to the soil and hence by putative allelopathic compounds released by Solidago plants: in T. pratense, carbon addition had no effect on germination and growth, but germination did depend on soil type, this being highest on sterilized soil (F1,163 = 24.7; P < 0.001), a result which indicates the probable existence of specific pathogens in non-sterile soil. The germination and growth of A. millefolium, a dicot herb, which grows amidst Solidago stands in Switzerland (Voser-Huber, 1983) and is also a part of the native American flora, was not at all influenced by activated carbon in the soil (all P < 0.5; Fig. 1).

effects of root exudates of solidago canadensis on performance of arabidopsis thaliana

The crude extract of the root exudates showed a significant inhibitory effect on growth of Arabidopsis (Fig 4; P < 0.05). The magnitude of inhibition was directly proportional to the concentration of the extract added. The growth of Arabidopsis seedlings decreased when the Solidago exudates were applied in doses between 25 and 250 µg mL−1, whereas complete mortality occurred at 500 µg mL−1 (Fig. 4).

image

Figure 4. Phytotoxic activity of root exudates of Solidago canadensis s.l..The crude extract of Solidago root exudates showed a significant inhibitory effect on growth of Arabidopsis thaliana grown for 7 days in media containing different concentrations of root exudates (P < 0.05). MeOH (50 µL) was added to control wells. Vertical bars denote ± 1 SE. n = 4. *P < 0.05.

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chemical analysis of the root extracts of solidago canadensis

A double-blind chemical analysis by LC-MS of Solidago root extracts revealed four main chemical compounds that had different molecular masses and were consistently present in the 40 different Solidago samples analysed (see Appendix S1). In the following, the four chemical substances are named after their molecular mass (MS). Polyacetylenes and diterpenes have already been isolated from roots of S. canadensis (Hegnauer 1977). However, the molecular masses of the compounds isolated in this study are higher than any of the previously detected compounds present in the roots. The UV spectrum suggests that the compound with mass 517 is a polyacetylene derivative maybe with sugar residues incorporated which could increase the water solubility of the compounds and thus increase the molecular mass while other compounds isolated in this study may be diterpene lactone derivatives.

The secondary chemical compound that occurred at lowest concentrations (MS 517; Appendix S1) did not significantly differ between native and invasive (diploid) populations or among different ploidy levels in the native range (all P > 0.2); but the three other compounds showed different concentrations among ploidy levels (MS 363: Fdev.2,19 = 14.18; P < 0.001; MS 349: Fdev.2,19 = 8.27; P < 0.01; MS 335: Fdev.2,19 = 4.11; P < 0.05) and in the case of MS 349 and MS 363 (Appendix S1) between diploid native vs. invasive populations (MS 349: Fdev.1,17 = 15.2; P < 0.001; MS 363: Fdev.1,17 = 16.6; P < 0.001). However, none of the four compounds had a higher concentration in invasive populations compared with plants from the native range. MS 349 and 363 were present in the highest concentrations in the native American hexaploid and tetraploid populations, whereas in MS 335 and MS 517 the concentration was highest in native American diploid plants (Appendix S1). In invasive, diploid populations, the concentration of all four compounds was on average about half the concentration (albeit with variation among populations) of that observed in diploid native American plants. This suggests lower investment by invasive plants into secondary compounds and raises the question of a higher susceptibility of plant competitors in the invasive than in the native range to these putatively allelopathic substances. Of the five European competitor species that were positively influenced by the addition of activated carbon to the soil, two (S. officinalis, T. repens) were either not significantly influenced by Solidago origin (non-significant interaction of carbon addition × native vs. invasive) or actually showed a higher carbon effect if grown with invasive Solidagos (Arrhenatherum, Lythrum – effect only in Swiss soil, see above), despite the lower concentration of secondary compounds in invasive Solidago rhizomes. Only for Dactylis was the allelopathic effect of Solidago plants from the native range greater than the effect of Solidago plants from the invaded range (see results above).

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Five out of the seven European grassland species tested exhibited a reduction in performance when grown in substrate without activated carbon and conditioned by S. canadensis s.l., a plant species that forms dominant stands and is hence likely to have a greater influence on the biochemistry of the soil than less abundant species (Wardle et al. 1998). Root extracts from Solidago rhizomes did have harmful dosage-dependent effect on Arabidopsis germination. These results corroborate findings of in vitro studies, in which native American S. canadensis roots extracted in water were shown to have phytotoxic effects on lettuce and radish (Butcko & Jensen 2002), and invasive Chinese S. canadensis plants extracted in ethanol were shown to be phytotoxic to native Chinese plant competitors (Yang et al. 2007). In the latter study, which focused primarily on grasses, reduced germination and growth were observed when extracts were added to Petri dishes (Yang et al. 2007). In addition, S. canadensis may also harm mycorrhizal interactions of plant competitors by the release of allelochemicals (Zhang et al. 2007). Fischer et al. (1978) discovered in a series of experiments allelopathic effects of water extracts of leaves and decaying plant material of S. canadensis on maple seedlings, but observed that the deleterious effects of goldenrod were overcome by large additions of soluble phosphorous fertilizer. Allelopathic effects have also been detected in vitro for other species within the Asteraceae (e.g. Menelaou et al. 1993; Tongma et al. 1998; Azania et al. 2003).

We have not yet identified the active compounds in our study, but the UV spectrum suggests that one of the compounds we detected is a polyacetylene derivative while the other three main compounds isolated in this study may be diterpene lactone derivatives. A range of putative allelopathic substances have been described in S. canadensis s.l. roots, including seven clerodane-type diterpenes related to kolavenic acid and five matricaria ester-type polyacetylenes (Werner et al. 1980, Lu et al. 1993). Some of theses natural products have been reported to have phytotoxic, antifungal, and anti-ant properties (Lu et al. 1993). In particular, a cis-Dehydromatricaria ester (DME), a polyacetylene compound, which is released into the soil and also present in roots, showed inhibitory effects on the growth of other plants in vitro (Kobayashi et al. 2004). This polyacetylene may be considered an allelopathic compound with ecological relevance. However, the ecological role of allelochemicals is difficult to assess as external biotic factors such as herbivory can stimulate the production of allelochemicals (Thelen et al. 2005), and the degradation of allelopathic compounds in the soil may depend on soil type (Kobayashi et al. 2004), on abiotic soil conditions such as humidity (Blair et al. 2006), on soil nutrient content (Fischer et al. 1978) and on the specific composition of the microbial soil community (Weidenhamer & Romeo 2004).

Our findings support other studies that have found allelopathic effects of S. canadensis s.l. on competing plant species in vitro. Root exudates of Solidago demonstrated clear inhibitory effects on Arabidopsis growth dependent on root exudate concentration. Under more realistic conditions in soil, the addition of activated carbon had a beneficial effect on the majority of species tested (Figs 1, 2). However, germination and growth of the surviving plants did not only depend on the addition of activated carbon, but also on (i) plant-species identity, (ii) the soil type, (iii) the origin of the Solidago plants (native vs. invasive); and (iv) on interacting effects of these factors. Hence, allelopathic effects can be altered by soil microbes and the competing plant species tested might have been differentially affected by the soil microbial community, for example, by specific soil pathogens. This might explain why T. pratense, a plant species known to suffer from root-rot pathogens that can accumulate in the soil (Taylor & Quesenberry 1996) germinated best on sterilized soil, or why A. elatius, a common plant species in Europe, grew better on US soils if activated carbon was added to the soil. These complex biochemical interactions within the soil compartment play an important, yet poorly understood, role in plant invasions (e.g. Reinhart et al. 2005; Levine et al. 2006).

The lower concentration of the four main compounds found in diploid invasive populations compared to diploid, tetraploid and hexaploid plants from the native range might support the novel weapons hypothesis (Callaway & Aschehoug 2000: old weapons in the native, co-evolved range need to be stronger than in a biogeographically new range with new and naïve plant competitors) as the majority of the European competitor species tested also reacted negatively to the lower concentrations of the compounds produced by the invasive European Solidago. However, we did not perform experiments with native American prairie species that might have evolved tolerance to the rhizosphere biochemistry of the co-occurring S. canadensis s.l. (Callaway & Ridenour 2004). Stronger effects caused by root exudates of non-indigenous invasive species against their new and naïve neighbours have been observed in Alliaria petiolata (Prati & Bossdorf 2004) and in Vaccinium myrtillis (Mallik & Pellissier 2000). In addition, in the closely related Solidago gigantea, a lower concentration of diterpenes in invasive European compared with native American plants was recently reported (Johnson et al. 2007) partially supporting the EICA hypothesis of lower investment in defensive secondary compounds, which in this case, did not translate into higher allocation to growth and reproduction.

In conclusion, the invasion success of S. canadensis in Europe may in part be attributable to the release of allelopathic compounds and the subsequent effects this has on some of the competing flora in the new range. However, to what extent such allelopathic effects are influenced by the soil microbial flora and the reason why some species such as A. millefolium are able to persist in Solidago stands, seemingly without influence from secondary metabolites excreted by Solidago, needs to be further investigated.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

This work was supported by a grant from Swiss National Science Foundation to J. J. (no. 3100AO-104006) and by a grant from DOD-SERDP (SI-1388 to J.M.V.). Authors thank Rene Husi for CHN analysis and Katja Schächle, Theres Zwimpfer and Alex Schwendener for their help and support in conducting the experiments, the Experimental Station of the Institute of Plant Sciences of ETH, Eschikon for use of their flowcytometer, and Daniel Schläpfer for help with the same. Comments by Ray Callaway, the handling editor, and by Bernhard Schmid, Alexander Fergus and two anonymous referees greatly improved the manuscript.

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  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information
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Supporting Information

  1. Top of page
  2. Summary
  3. Introduction
  4. Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  9. Supporting Information

Appendix S1 Molecular mass of LC-MS peaks present in the root extracts of 40 different Solidago canadensis samples.

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