Biol Invasions DOI 10.1007/s10530-009-9608-z
ORIGINAL PAPER
Soil microbial communities alter allelopathic competition between Alliaria petiolata and a native species Richard Lankau
Received: 17 June 2009 / Accepted: 6 October 2009 Ó Springer Science+Business Media B.V. 2009
Abstract Allelopathy has been increasingly invoked as a mechanism facilitating exotic plant invasions. However, studies even on the same target species often yield varying results concerning the strength and importance of allelopathic inhibition, suggesting that the process may depend on the specific environmental context. Here I studied how the allelopathic inhibition of sycamore (Platanus occidentalis) seedlings by garlic mustard (Alliaria petiolata) depended on the presence of a soil microbial community. Using three analytical approaches to quantifying allelopathy, I consistently found allelopathic inhibition only in sterilized soils, suggesting that certain microbial taxa inhibit the process, possibly by degrading the allelochemicals. Determining the environmental contexts that reduce or eliminate allelopathic inhibition could lead to a greater understanding of the spatial variation in invasion success and potentially lead to new avenues for management. Keywords Glucosinolates Platanus occidentalis Size asymmetry Allelopathy
R. Lankau (&) Illinois Natural History Survey, Institute of Natural Resource Sustainability, University of Illinois at Urbana-Champaign, 1816 S. Oak Street, Champaign, IL 61820, USA e-mail:
[email protected];
[email protected]
Introduction Invasive plants have transformed many ecosystems by out competing native species. Many mechanisms have been proposed to explain the competitive superiority of invasive species, including a release from natural enemies, a greater ability to exploit anthropogenic disturbance and the presence of empty niches unfilled by any native species (Catford et al. 2009). Allelopathy, in which one plant species produces toxic secondary compounds which directly or indirectly reduce the germination, growth, or survival of competing species, has been documented in several invasive plant species (Hierro and Callaway 2003). The Novel Weapons Hypothesis suggests that invasive species may be likely to display allelopathic activities in their introduced range where their competitors have no evolutionary history with the invader and so likely have not evolved specific counter-measures to the invader’s allelochemicals (Callaway and Ridenour 2004). Although allelopathy has been implicated in a number of plant invasions (Hierro and Callaway 2003), studies may find contradictory results even when investigating the same invasive species (Bais et al. 2003; Blair et al. 2006). These differences can result from methodological differences (Blair et al. 2009), but can also stem from context dependency in the behavior of allelochemicals. For instance, the toxicity of the putative allelochemical in Centaurea diffusa differs based on which metal it chelates
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(Tharayil et al. 2009). This context dependency suggests that allelopathy may not be a constant trait of a species, but the outcome of specific plantenvironment interactions. Thus, research on allelopathic inhibition by plants requires investigating the phenomenon in multiple environmental contexts. Little is known about the exact mechanisms by which allelochemicals affect neighboring plants. Since soils are such complex environments, considerable changes may occur to allelochemicals between the time they are exuded by a source species and their coming in contact with the competitor. Soil microbes, especially, could influence allelopathy in several ways. First, mutualistic microbes may be a primary target for allelochemicals, indirectly resulting in reduced growth of competitor plants. For instance, most plant species rely on associations with mycorrhizal fungi for increased nutrient uptake. Plants in the Brassicaceae family do not form mycorrhizal connections, and thus could potentially gain a competitive advantage by releasing allelochemicals toxic to mycorrhizal fungi (Lankau and Strauss 2007; Stinson et al. 2006). Microbial species may enhance the effectiveness of allelochemicals by transforming them into more toxic by-products (Inderjit 2005). Alternatively, microbial species may reduce the effectiveness of allelochemicals by breaking down the compounds into less toxic forms, or consuming them altogether (Inderjit 2005). Alliaria petiolata (garlic mustard) is an aggressive invader of forest understories in eastern North America. Like most invasive species, A. petiolata benefits from disturbance, but unlike many invaders it is also able to invade relatively intact forest understories (Nuzzo 1999; Rodgers et al. 2008). A. petiolata’s competitive success likely stems from multiple sources, including reduced herbivory (especially from white tailed deer, (Knight et al. 2009), an unusual phenology that allows it to resume active growth earlier than most native understory species (Myers and Anderson 2003; Rodgers et al. 2008), and through the production of toxic compounds that reduce the growth and germination of both native plants and mycorrhizal fungi (Callaway et al. 2008; Roberts and Anderson 2001; Stinson et al. 2006; Vaughn and Berhow 1999). Several studies have investigated the allelopathic potential of A. petiolata on both native plants and mutualistic soil microbes, and these studies highlight
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the variety of indirect methods employed to study allelopathy. Studies have tested the effect of extracts of A. petiolata tissues on the growth or germination of plant seeds or mycorrhizal fungal spores (Callaway et al. 2008; Roberts and Anderson 2001; Vaughn and Berhow 1999). Others have focused on the indirect allelopathic effects of this species mediated by effects on soil microbial communities by using A. petiolata to cultivate soil communities, then growing native plants in the resulting soils (Callaway et al. 2008; Wolfe et al. 2008). Two studies have used activated carbon to absorb any allelopathic compounds of A. petiolata. Prati and Bossdorf (2004) found that adding activated carbon to soils in which A. petiolata plants grew led to higher seed germination of a North American native species as compared to soils with A. petiolata but no carbon. Cipollini et al. (2008) found that incorporation of activated carbon into field soils led to greater growth of a native annual in the presence of A. petiolata. Despite the many studies finding evidence for allelopathic inhibition by A. petiolata, several others have found weak or no evidence for the phenomenon. McCarthy and Hanson (1998) found no evidence for allelopathic inhibition using aqueous tissue extracts, and Burke (2008) found relatively weak differences in arbuscular mycorrhizal fungal communities under A. petiolata stands. This variation in results could stem from methodological differences, but could also suggest that the allelopathic affects of A. petiolata are context-dependent, varying in strength based on environmental conditions or among different A. petiolata populations. I performed a factorial experiment to investigate how the presence of an intact, native soil community would influence the competitive and allelopathic effects of A. petiolata on a native species, Platanus occidentalis (sycamore). I grew each plant species alone or in combination in soils that included either a living or sterilized inoculum of forest soil with or without the addition of activated carbon (in all combinations). Activated carbon is frequently used to test for allelopathy since it absorbs small organic molecules, although its use entails certain caveats (Lau et al. 2008). Since allelopathy is a difficult phenomenon to study directly, I used three different indirect approaches to quantifying the strength of allelopathic inhibition. If the allelochemicals of A. petiolata primarily affect mutualistic microbes
Soil microbial communities alter allelopathic competition
rather than plants directly, or if microbial species enhance the toxicity of the allelochemicals, then one would predict stronger evidence of allelopathy in soils with an intact microbiota. On the other hand, if microbial communities interfere with the effectiveness of the allelochemicals then one would predict stronger allelopathic effects in the sterilized soil.
Methods Plant and soil sources I collected Platanus occidentalis seeds from several trees growing in Urbana, Illinois in fall of 2008, and Alliaria petiolata seeds from several populations in Wisconsin, Michigan, and Illinois to provide a wide range in phenotypic traits. Populations likely differ in their production of allelochemicals, among other traits (Lankau et al. 2009). Unfortunately, I was not able to replicate within populations in this experiment, and thus do not evaluate population differences. However, I did ensure that all populations were equally represented in all treatments, when possible, and did not use multiple individuals from the same maternal family within a treatment to insure that population differences could not lead to biases across experimental treatments. I collected field soil from the Trelease Woods *10 km east of Urbana, IL. This site has been invaded with A. petiolata in the past, but has been actively managed for the last decade to reduce or eliminate the population. No A. petiolata individuals were present within *50 m2 of the site where soil was collected. Experimental treatments I factorially crossed three competition treatments (one P. occidentalis individual, one A. petiolata, or one of each in a pot) with two soil community treatments (a fresh or sterilized field soil inoculum) and the presence or absence of activated carbon. I germinated seeds in sterilized soil and then transplanted the seedlings into pots after one week of growth. I filled conical pots with 333 ml of a sterilized background soil (a 3:1 mix of potting soil and sand) and 167 ml of the live or sterilized field inoculum. I sterilized soils by autoclaving for two 1 h periods, with 1 h in between. For the activated carbon
treatments, I added 20 ml of finely ground activated carbon powder (Marineland Black DiamondÒ Premium Activated Carbon, United Pet Group, Inc. Cincinnati, OH) to each liter of soil prior to filling the pots, following methods in previous studies (Lau et al. 2008). Sample sizes were unbalanced with respect to competition treatments (6 replicates of the A. petiolata alone treatments, 10 replicates of the P. occidentalis alone treatments, and 15 replicates of the A. petiolata ? P. occidentalis treatments) in order to allow greater power to correlate phenotypic traits of A. petiolata with A. petiolata and P. occidentalis biomass in the combined treatments. I completely randomized pots on a single greenhouse bench. This experiment attempted to determine if A. petiolata and P. occidentalis interacted competitively, defined broadly as a mutually negative interaction between two species, which could result from scramble competition for resources or interference mechanisms like allelopathy. To do that I used a limited response surface design, in that the density of each species is varied independently of the other species (either 1 or 0 individuals per pot), allowing the total density to vary. This contrasts with another common design, the replacement series, in which the total density is held constant but the frequency of the two species varies. Replacement series can provide information on the relative strength of intra- vs. interspecific competition, but cannot determine the absolute effect of one species on another because the design confounds the density and frequency of each species (Inouye 2001). My design allows me to calculate the absolute effect of A. petiolata on P. occidentalis (and vice versa), but provides no information on the strength of intraspecific competition. However, the main goal of this study was to elucidate the dominate mechanisms underlying the competitive effect of the invasive on the native species, which required the ability to calculate and compare absolute competitive effects in different soil treatments, and not to compare the relative strengths of inter- vs. intraspecific competition. Measurements After 6 months of growth, I measured above and below ground biomass by collecting all shoot material and carefully washing root material free from
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soil. In mixed species pots, I took care to separate P. occidentalis and A. petiolata roots with minimum tissue loss, but some error was inevitable. I weighed plant material after drying for 48 h at 60°C. I took a small sample of fresh root tissue from each A. petiolata plant prior to drying in order to quantify the concentration of several putative allelochemicals. Root tissue was placed immediately in 95% methanol, and then later weighed for fresh mass. I ground samples by bead beating, shook them for 1 h, and then passed them through an anion exchange column. I collected the flow through to analyze flavanoids (including the putative allelochemical alliarinoside). Glucosinolates were retained on the column due to their negative charge, and were separated from the beads by the addition of sulfatase enzyme. The resulting desulphoglucosinolates were collected for analysis. I analyzed both fractions on a Shimadzu HPLC system. I identified alliarinoside with LC-mass spectroscopy on several samples and then via retention time and UV spectra. Glucosinolates were identified by comparison of retention time and UV spectra with standards (for complete methods see Kliebenstein et al. 2001). Analysis I evaluated evidence for allelopathy in three ways. First, I tested whether the presence of activated carbon affected P. occidentalis or A. petiolata fitness differentially when competing versus when individuals grew alone, by testing for a significant competition by activated carbon treatment interaction in an ANOVA. A three way interaction of competition by activated carbon by soil community treatments would indicate that the allelopathic affect was influenced by an intact soil community. I used this same ANOVA model to determine whether concentrations of putative allelochemicals differed among the soil and competition treatments. Because differences in glucosinolate concentrations between A. petiolata individuals may affect the strength of allelopathy (see below), I performed an additional analysis where I used only the ‘‘high’’ or ‘‘low’’ glucosinolate individuals (defined as the top or bottom 50% of individuals in a given treatment combination). I then used the same ANOVA model with these subsets to
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see if the evidence for allelopathy (i.e. the competition*activated carbon interaction) differed among them. Secondly, I used just those pots containing both plant species to test whether competition was size symmetric or asymmetric, and whether this depended on the soil community or activated carbon treatments. Size symmetric competition occurs when the biomass of one species is significantly negatively correlated with the biomass of a competitor species. Such a pattern is indicative of exploitative resource competition in which resource uptake is primarily determined by surface area available for resource acquisition (Schenk 2006). Size asymmetric competition, on the other hand, occurs when there is no significant relationship between the biomass of a target plant and its neighbor. This pattern implies some form of resource preemption, in which one competitor is able to monopolize a resource (Schenk 2006). For instance, one species could preempt light by overtopping its competitors (since any additional increases in plant size once a plant is taller than its neighbor would have little additional impact on the neighbor’s light levels), or preempt soil nutrients by producing allelopathic compounds that interfere with its competitor’s ability to acquire resources. A size asymmetric pattern can be more confidently attributed to allelopathy if it only occurs in the absence of activated carbon. Finally, since I directly measured several putative allelochemicals, I tested whether P. occidentalis responded to quantitative variation in allelochemical concentrations, and whether this response varied across treatments. If P. occidentalis biomass declined with increasing concentrations of an allelochemical, this would imply that that chemical had a direct or indirect allelopathic activity, especially if this pattern disappears with the addition of activated carbon. If this pattern differs with the presence or absence of soil microbes, this would again imply that the microbial community influences the allelopathic activity of A. petiolata. These last two approaches were analyzed with ANCOVA, using the soil microbial and activated carbon treatments as continuous variables, and either A. petiolata biomass or allelochemical concentrations as covariates, with all interactions.
Soil microbial communities alter allelopathic competition
Results
In addition to affecting biomass, competition and soil communities also induced changes in glucosinolate concentrations in A. petiolata roots. When A. petiolata plants were grown alone the glucosinolate concentrations, the sum of allyl- and benzylglucosinolates, in their roots tended to be higher in sterilized versus live soils, although this difference was not significant. However, when competing with P. occidentalis glucosinolate concentrations were significantly higher in live soils (Table 1; Fig. 1c). These patterns did not change when controlling for differences among the A. petiolata seed source populations by including population as a random effect in the model. Finally, the effect of activated carbon on P. occidentalis biomass differed somewhat between subsets using only high or only low glucosinolate A. petiolata competitors. For the low glucosinolate subset, the activated carbon by competition treatment interaction was far from significant (F1,63 = 0.046, P = 0.83), and contrasts showed that this did not differ between live or sterile soils (F1,63 \ 0.31, P [ 0.56 for both). In this subset, P. occidentalis biomass was consistently lower in the presence of activated carbon, regardless of whether it was grown alone or in competition, or in live or sterilized soil. However, in the high glucosinolate subset, there was a marginally significant activated carbon*competition interaction (F1,59 = 2.71, P = 0.10). Contrast showed that this interaction was significant in sterile soils (F1,59 = 4.53, P = 0.04) but not in live soils (F1,59 = 0.044, P = 0.83). In this subset activated carbon
Effects of activated carbon and soil communities on plant biomass and allelochemical production The presence of an intact soil microbial community interacted strongly with the competition from A. petiolata to determine P. occidentalis biomass (Table 1; Fig. 1a). In sterilized soils, P. occidentalis biomass was high when grown alone, but declined greatly when grown with an A. petiolata individual. However, in live soils, P. occidentalis biomass was relatively low when grown alone, and was not significantly decreased by the addition of an A. petiolata competitor. This led to a highly significant soil treatment by competition interaction (Table 1). P. occidentalis biomass showed a marginally significant trend to decrease when activated carbon was added to the soil; this effect did not depend on the presence or absence of an A. petiolata competitor or a live soil community (Table 1). Alliaria petiolata biomass was also significantly decreased by the presence of a live soil community and the addition of a P. occidentalis competitor, but these factors did not interact (Table 1). Thus, the relative decline in biomass due to a live soil community was roughly similar between A. petiolata plants grown singly and between A. petiolata plants grown with P. occidentalis competitors (Fig. 1b). Activated carbon had no significant effect on A. petiolata biomass (Table 1).
Table 1 ANOVA results of P. occidentalis and A. petiolata biomass, as well as glucosinolate concentration in A. petiolata root tissue, responses to experimental treatments Source
Soil treatment
P. occidentalis biomass
A. petiolata biomass
Glucosinolate concentration
F
P
F
P
F
