Activated carbon may have undesired side effects for testing allelopathy in

invasive plants
Karoline Weißhuhna,, Daniel Pratib,1
a
UFZ-Helmholtz Centre for Environmental Research, Department Community Ecology, Theodor-Lieser-Straße 4, D-06120
Halle, Germany
b
Institute for Biochemistry & Biology, University of Potsdam, Maulbeerallee 1, D-14467 Potsdam, Germany

Received 28 January 2008; accepted 23 October 2008

Abstract
Activated carbon has become a widely used tool to investigate root-mediated allelopathy of plants,
especially in plant invasion biology, because it adsorbs and thereby neutralizes root exudates.
Allelopathy has been a controversially debated phenomenon for years, which revived in plant
invasion biology as one possible reason for the success of invasive plants. Noxious plant exudates
may harm other plants and provide an advantage to the allelopathic plant. However, root exudates are
not always toxic, but may stimulate the microbial community and change nutrient availability in the
rhizosphere. In a greenhouse experiment, we investigated the interacting effects of activated carbon,
arbuscular mycorrhiza and plant competition between the invasive Senecio inaequidens and the native
Artemisia vulgaris. Furthermore, we tested whether activated carbon showed any undesired effects by
directly affecting mycorrhiza or soil chemistry. Contrary to the expectation, S. inaequidens was a
weak competitor and we could not support the idea that allelopathy was involved in the competition.
Activated carbon led to a considerable increase in the aboveground biomass production and reduced
the infection with arbuscular mycorrhiza of both plant species. We expected that arbuscular
mycorrhiza promotes plant growth by increasing nutrient availability, but we found the contrary when
activated carbon was added. Chemical analyses of the substrate showed, that adding activated carbon
resulted in a strong increase in plant available phosphate and in a decrease of the Corganic/Ntotal
ratio, both of which suggest stimulated microbial activity. Thus, activated carbon not only reduced
potential allelopathic effects, but substantially changed the chemistry of the substrate. These results
show that activated carbon should be handled with great care in ecological experiments on allelopathy
because of possible confounding effects on the soil community.

r 2009 Gesellschaft fu¨r O¨ kologie. Published by Elsevier GmbH. All rights reserved.

Introduction
Allelopathy – the negative effect of one plant on another via toxic substances – has been a
controversially debated phenomenon for years (Harper, 1977; Inderjit & Mallik, 2002; Williamson,
1990). Because allelopathy is difficult to distinguish from resource competition and because plants
generally show a high ability to adapt to toxic compounds (Harper, 1977), the importance of
allelopathy in natural communities has often been questioned. Recently, allelopathy revived in plant
invasion biology as a possible mechanism for the success of invasive plants (Callaway & Ridenour,
2004; Cappuccino & Arnason, 2006). The main argument is that non-indigenous plants may possess
biochemicals that are novel to the invaded community. Species in this community may not be adapted
to these chemicals and therefore particularly susceptible to them (Callaway & Aschehoug, 2000).
Moreover, allelopathy may indirectly disrupt co-evolutionary associations of plants with mutualistic
mycorrhizal fungi, as shown in Alliaria petiolata, a European plant invasive in North America
(Callaway et al., 2008; Stinson et al., 2006). However, this indirect allelopathic effect via mycorrhizal
fungi was demonstrated only for one species and it still remains unclear how general these
allelopathic effects are in natural systems.
For studying the role of allelopathic compounds in the rhizosphere in the context of invasive plants,
activated carbon (AC) has been a widely used approach (e.g. Callaway & Aschehoug, 2000;
Semchenko, Hutchings, & John, 2007). Activated carbon is expected to adsorb allelopathic plant
exudates in the soil with only little effects on soil nutrients (Callaway & Aschehoug, 2000). By its
very large surface-to-volume ratio, AC has long been known as an adsorbent of organic compounds in
soils (Zackrisson, Nilsson, & Wardle, 1996), and has been used in greenhouse and field experiments
(e.g. Callaway, Ridenour, Labowski, Weir, & Vivanco, 2005; Kulmatiski & Beard, 2006; Prati &
Bossdorf, 2004).
In natural communities, plants interact in various ways with symbionts such as arbuscular mycorrhizal
fungi (AMF) or pathogens and these interactions often involve the release of organic compounds to
the soil (Bais, Park, Weir, Callaway, & Vivanco, 2004). Plants release a diverse array of secondary
compounds that influence microorganisms and these can exhibit positive or negative feedbacks on
plant growth (Bever, 2002). Some secondary compounds may simultaneously affect different soil
organisms (Bais, Vepachedu, Gilroy, Callaway, & Vivanco, 2003). Inderjit and Callaway (2003)
reviewed the use of AC and recommend further experiments to test the possible effects of AC on soil.
Thus, given the complexity of the interactions of plants with microorganisms, it is possible that
disrupting plant–soil communication may have unwanted side effects. However, only few studies
directly addressed the potential confounding effects of AC on soil chemistry and resulting effects on
plant growth (see Lau et al., 2008).
In this study, we tested the suitability of AC to investigate allelopathic effects in a multifactorial
greenhouse experiment. We analysed the effect of AC on plant competition that would show direct
allelopathic effects, mycorrhizal colonisation that would show indirect allelopathic effects and soil
chemistry. We used the potentially allelopathic invasive South African ragwort Senecio inaequidens
and the native Artemisia vulgaris, a plant generally associated with AMF. Our main objectives were
to test (i) whether S. inaequidens exhibits allelopathic effects on a native plant, either directly or
indirectly via disrupting the association with AMF, and (ii) whether activated carbon in addition to
neutralizing putative allelopathic effects exhibits undesired side effects on soil chemistry.

Material and methods

Study species
Senecio inaequidens DC. (Asteraceae), native to South Africa, was introduced to Europe at the end of
the 19th century and is currently one of the most rapidly spreading alien plant in Europe (Heger &
Bo¨hmer, 2005). In Central Europe it mainly invades disturbed habitats such as roadsides and
railways (Ernst, 1998), whereas in the Mediterranean it is considered as a serious weed in pastures
and vineyards (Lopez-Garcia & Maillet, 2005). S. inaequidens contains pyrrolizidine alkaloids
(Bicchi, Damato, & Cappelletti, 1985) which are toxic to livestock and may cause allelopathic effects.
Roots of S. inaequidens sampled in the field were not associated with AMF (Weißhuhn, unpublished
data). For the experiment, seeds of S. inaequidens were collected from ten populations from all parts
of its distributional range. As a target species we used A. vulgaris L. (Asteraceae), a native European
perennial herb that co-occurs with invasive populations of S. inaequidens, exhibits a similar growth
form and is generally associated with AMF (Harley & Harley, 1987). Seeds from A. vulgaris were
bought from Rieger-Hofmann GmbH, Blaufelden-Raboldshausen, Germany.

Experimental design
The experiment consisted of two factorially combined soil treatments: with or without activated
carbon and with or without arbuscular mycorrhizal fungi (see details below). To test for the effect of
competitor identity two individuals of S. inaequidens or A. vulgaris were planted in a replacement
series either as monocultures or mixtures. For each soil treatment, 50 replicates were set up with two
S. inaequidens, 50 replicates with one S. inaequidens and one A. vulgaris, and 10 replicates with two
A. vulgaris. We did not replicate the last combination as often because we focused on different
populations of S. inaequidens rather than on A. vulgaris. This resulted in a total of 440 pots with 880
plants. Seven to eight pots with the same soil treatment were placed together in trays of 45.5 27.5 cm,
which were randomly distributed across 20 blocks over two benches in a greenhouse.
The substrate used in the experiment contained 80% washed silica sand (0.3–0.8 mm, Schlingmeier
Quarzsand GmbH, Schwu¨lper, Germany) and 20% Chernozem soil (collected in Bad Lauchsta¨dt,
Germany, 51123 2 0 N, 11151 0 E). The substrate was heated for 48 h at 200 1C to exclude AMF.
Studies showed that this temperature reduced fungal spores by 99.6% while leaving C and P contents
unchanged (Guerrero, MataixSolera, Go´mez, Garcı´a-Orenes, & Jorda´n, 2005). Half of the substrate
was then mixed with finely ground pure AC (Merck, Darmstadt, Germany) at a concentration of 20
mL/L. Pots of 1 L volume were filled with a layer of 2 cm slate for drainage and the substrate. Half of
these pots received 3 g of a natural mycorrhizal inoculum consisting of washed and cut roots of A.
vulgaris, expected to inoculate the substrate with AMF (Grime, Mackey, Hillier, & Read, 1987). The
roots were collected from natural populations of A. vulgaris around Halle and Bad Lauchsta¨dt
(Germany). To establish natural microbial communities we added 10 mL soil solution to all pots. For
non-AMF pots we filtered the solution through 20–25 mm Whatman filter paper to exclude AMF-
spores. In May 2006 seeds of S. inaequidens, orA. vulgaris, or both species were added to the pots and
thinned out to two individuals per pot after germination. The plants grew at a 7–15 1 C/15–20 1C
day/night cycle for seven weeks, and afterwards at 15 1C/25 1C day/night with additional light
provided by 400 W lamps.

Measurements
In June 2006, we measured the initial plant height of 4-week-old seedlings. In August 2006, after 14
weeks when the majority of plants flowered, all plants were harvested to assess aboveground biomass
production, mycorrhization status and soil chemistry. Plants were clipped aboveground, dried for 48 h
at 80 1C and weighed. To analyse arbuscular mycorrhizal colonisation, roots of five individuals per
species, competitor identity, and soil treatment (N ¼ 80) were washed and stored in formaldehyde
acetic acid (6.0% formaldehyde, 2.3% glacial acetic acid, 45.8% ethanol, 45.9% H2O (v/v)), stained
with ink (Vierheilig et al., 2001) and analysed by the line intersect method (Brundrett, Bougher, Dell,
Grove, & Malajczuk, 1996) using a light microscope at 400-fold magnification. For each root sample,
the occurrence of arbuscules, vesicles and intraradical mycelium was counted along 300 root
segments. For the soil chemistry analysis, two samples of 200 g of the substrate with and without AC
were taken before seeds were added and three samples in each of the four soil treatments were taken
at the end of the experiment. Each sample was composed of pooled and thoroughly mixed soil from
all flower pots from one randomly chosen tray which received a particular treatment. These samples
were dried at 60 1C and sieved (2 mm) for all further investigations. Plant available P was extracted in
double lactate and detected by inductively coupled plasma–atomic emission spectrometry using a
Spectro Ciros CCD analyser (SPECTRO Analytical Instruments GmbH, Kleve, Germany). Total N
and C were determined in finely grounded samples by Vario EL III l (Elementar Analysesysteme
GmbH, Hanau, Germany) and the Corganic/Ntotal ratio was calculated after subtracting the amount of
carbon added as AC. To determine pH, 2 10 g dry soil of each sample was extracted in 25 mL
demineralized water, shaken and measured after 24 h by pH-meter (Knick Elektronische Messgera¨te,
Berlin, Germany). The substrate was tested for inorganic C by dissolving the samples in 10% HCl,
but no inorganic C could be detected.

Data analysis

Individual biomass production was analysed using analysis of covariance (ANCOVA) for a split-plot
design with the effects of the four soil treatments as a plot-level treatment and competitor identity and
its interaction with soil treatments as within-plot level (Table 1). The effect of blocks was tested, but
because it was not significant it was excluded from the analyses presented here. To achieve
ndependence of data points, one plant individual per pot was chosen at random whereas the identity of
the second plant (S. inaequidens or A. vulgaris) indicated inter- versus intraspecific competition.
Initial plant height of the focal individual and the biomass of the second individual in the pot were
used as co-variables. The soil treatments were the plotlevel treatments (the trays in our case) tested
against the variation among trays, whereas the competitor identity and its interactions were the within-
plot treatment tested against the residual. Analysis of variance (ANOVA) was used to test for
differences in mycorrhizal colonization and soil parameters followed by a posthoc Student’s t-test. All
analyses were carried out with the software JMP5.1.2 (SAS Institute Incorporated, Cary, NC 27513,
USA).

Results

Aboveground biomass
Generally, S. inaequidens and A. vulgaris reached similar final aboveground biomass in our
experiment (Fig. 1). Plant height at the beginning of the experiment was positively correlated with
final biomass (Table 1, line 1). The biomass of the competitor reduced thebiomass of the focal plant,
indicating that the two plants growing in the same flower pot competed with each other (Table 1, line
2). After correcting for these effects and pooling the data over soil treatments, S. inaequidens grew by
45% better together with a conspecific than with A. vulgaris. On the other hand, A. vulgaris grew by
20% better with S. inaequidens than with a -conspecific (Table 1, line 7). Taken together, these results
showed that, contrary to our expectation, A. vulgaris was competitively superior over S. inaequidens.
On average, adding AC increased biomass by 42% for S. inaequidens and 75% for A. vulgaris (Table
1, line 4). This stimulating effect of AC also occurred when plants grew together with a conspecific
which indicates a fertilizing effect of AC rather than a neutralization of an allelopathic effect.
Inoculating the soil with AMF decreased average biomass by 8.3% for S. inaequidens

Fig. 1. Plant biomass production (least square means7SE) of (A) Senecio inaequidens and (B)
Artemisia vulgaris in intra- and interspecific competition, cultivated with (hatched bars) or without
arbuscular mycorrhiza (open bars) and with (grey bars) or without activated carbon (white bars).
Different letters show significant differences based on a posthoc Student’s t-test.

and 11% for A. vulgaris (Table 1, line 3). However, the effects of AMF and AC strongly interacted
with each other (Table 1, line 5, Fig. 1). AMF did not profoundly affect biomass of either species in
soil without AC. In contrast, adding AC to the soil strongly increased biomass production in the
absence of AMF which was especially true for A. vulgaris. In addition, for S. inaequidens this
interacting effect depended on the identity of the competitor as indicated by a significant three-way
interaction (Table 1, line 10). Here we found that the combined treatment of AC and AMF increased
biomass only when S. inaequidens grew with a conspecific, but not when it grew with A. vulgaris.
Taken together these results do not support the idea that mycorrhizal fungi were suppressed by root
exudates and that this effect was ameliorated by adding activated carbon.
Mycorrhizal colonisation
In the treatments without AMF we could not detect any mycorrhizal structures in the roots of either
species and we therefore omitted all interactions with this treatment from the model (Table 2). A.
vulgaris showed twice as many mycorrhizal structures compared with S. inaequidens (Table 3).
Adding AC decreased mycorrhizal structures in both species to a similar degree (Table 2) and
decreased the ratio of arbuscules to vesicles more strongly in A. vulgaris than in S. inaequidens
although this was not significant. This again is contrary to our expectation of an indirect, allelopathic
effect acting on mycorrhizal fungi.

Substrate
Substrate analysis further supports the notion of a fertilizing effect of activated carbon. The major
influence of AC was a strong increase of plant available phosphorus by 94% compared with the
substrate at the start of the experiment and by 54% compared with the treatment without AC (Table
4). Values of pH also differed between the particular treatments (Table 4) but were in the neutral
range in all treatments. Total nitrogen content differed little between the treatments and slightly
increased N values in the mycorrhizal treatment could be due to contamination of the substrate with
fungal hyphae. Total carbon values showed opposite trends during the experiment: while there was a
small but significant increase of Ctotal in the substrate without AC, Ctotal decreased during the
experiment when AC was added. The added carbon was subtracted to calculate the ratio of organic
carbon to nitrogen, this ratio decreased by 77% in the treatment with AC.

Discussion
Several invasive plant species proved to be superior competitors via allelopathic effects (e.g. Bais et
al., 2003; Callaway & Aschehoug, 2000; Callaway et al., 2008; Stinson et al., 2006), but results from
our greenhouse experiment do not support this idea for the invasive S. inaequidens, at least in
comparison with A. vulgaris. Neither could we find evidence for direct allelopathic effects of S.
inaequidens on its competitor (Fig. 1) nor for indirect effects on AMF associated with its competitor
(Table 2). Presently, the occurrence of S. inaequidens in Central Europe is restricted to disturbed
habitats and early successional stages, for which competition and mycorrhizal fungi are generally
regarded as relatively unimportant (Grime, 1979; Read, 1991). Although we could not confirm that
allelopathy was involved in the case of S. inaequidens, it is still possible that this species turns into an
aggressive invader in natural communities. But because competitive relationships are often species-
specific, further studies with other potential competitor species and from other plant communities are
required before this species can be considered as unproblematic in terms of invasion.
The most important effect of our study was a strong increase of plant biomass in both species by the
addition of AC (Fig. 1). In addition, AC modified the substrate chemistry, negatively affected the
degree of mycorrhization, and changed the competitive interaction between S. inaequidens and A.
vulgaris. Allelopathy could explain this pattern only under the very unlikely assumption that both
species exhibit allelopathic effects, including auto-allelopathy, which would be the intoxication of a
plant by its own products. A much more plausible explanation is that AC directly affected the
substrate chemistry, modified the cost–benefit relationship from mycorrhizal fungi and thereby caused
increased vigour in both species.
The substrate chemistry differed strongly between the treatments with and without AC (Table 4). In
particular, plant available phosphorus increased with AC and this may have caused the better growth
of plants in our experiment. Wardle, Zackrisson, and Nilsson (1998) showed that AC could foster the
soil microbial community and consequently stimulate the decomposition of organic matter. Such an
increase in microbial activity may have occurred in our experiment too, but we lack data on
composition and activity of microbes in the substrate used. In addition, AC can change the
relationship between compounds in the soil, e.g. by reducing phenolic compounds (Bicchi et al., 1985;
Zackrisson, Nilsson, & Wardle, 1996). Root exudates do not always have an allelopathic influence on
plant competitors, but plants also manipulate their rhizosphere by promoting mutualistic symbiosis,
nutrient availability and defence against pathogens (Bais et al., 2004). Therefore, addition of AC
could be responsible for suppressing these effects in addition to changing the substrate directly. Taken
together, our data show a substantial shift in phosphorus availability resulting in different growing
conditions for the plants.
Nutrient availability is one of the functions in the plant–mycorrhiza relationship which directly affects
the benefit plants receive from mycorrhizal associations. But host plants profit from mycorrhizal
associations only when the benefit of receiving nutrients exceeds the costs of supplying the fungi.
Increasing the availability of a limiting soil resource can disturb the mutualism and even turn it into
parasitism (Johnson, Rowland, Corkidi, Egerton-Warburton, & Allen, 2003). Arbuscules are the
structures for nutrient exchange whereas vesicles serve for storage. Therefore, the arbuscule/ vesicle
ratio may be a useful index for arbuscular mycorrhiza activity (Johnson, 1993). In our experiment,
the arbuscule/vesicle ratio for A. vulgaris was lower under the influence of AC, suggesting that AC
turned the symbiosis less profitable for the host plant (Table 3). Thus, we explain the negative
influence of AMF in the treatment with AC by a shift from a mutualistic into a parasitic interaction, as
previously shown by Johnson (1993). Furthermore, if allelopathic effects had interrupted the
symbiosis with AMF, AC should have increased mycorrhizal colonisation, contrary to our
data.
In conclusion, our results demonstrate that adding AC could change plant–plant and plant–soil
interactions in several undesired ways. Consequently, AC does not only reduce allelopathy as
advocated in previous experiments. Activated carbon may also directly influence nutrient availability
and may interact with mutualists and possibly pathogens. Thus, future experiments with AC for the
study of allelopathy should be handled with great care and should involve analyses of possible
changes in the soil.

Acknowledgements
We thank Verena Schmidt, Antje Thondorf, Ines Volkmann and the UFZ crew from the experimental
station for help with the experiment, Verena Blanke for discussing mycorrhizal structures and
Franc¸ois Buscot, Carsten Renker, Harald Auge and Stefan Hempel for helpful discussion and
support.

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