2012 NORTHEASTERN NATURALIST 19(2):297–312 The Allelopathic Potentials of the Non-native Invasive Plant Microstegium vimineum and the Native Ageratina altissima: Two Dominant Species of the Eastern Forest Herb Layer Brian F. Corbett1,2 and Janet A. Morrison*,1 Abstract - Allelopathy is one explanation for non-native plant invasion, but native plants also can be allelopathic. We tested the allelopathic potentials of the non-native, invasive grass Microstegium vimineum (Japanese Stilt-grass) and the native herb Ageratina altissima (White Snakeroot), which both can dominate the herb layer in central New Jersey forests. Aqueous extracts from roots and shoots of both species negatively affected the speed of germination and the percent germination of Lettuce and Radish seeds in Petri dishes, and White Snakeroot shoot extract had the strongest effect. In a factorial experiment in pots of forest soil that combined extract treatments with activated carbon addition (to manipulate allelochemicals) and soil sterilization (to investigate indirect allelopathic effects via the soil microflora), Lettuce and Radish seedling establishment was reduced by extracts, especially from the native White Snakeroot. However, growth of surviving seedlings was unaffected by the extracts or their interactions with carbon or soil sterilization. These results show that a native species had stronger allelopathic potential than an aggressive, non-native invader from the same forest and that allelopathy was effective on the earliest developmental stages of the target plant species. In addition, activated carbon and sterilization interacted to directly influence plant growth. Growth was greater in sterilized than unsterilized soils, but only when carbon was added, suggesting caution in using these techniques in allelopathy studies. Introduction Expanding urbanization throughout much of the northeastern region of the United States has relegated an increasing amount of forest land to relatively small fragments surrounded by residential and commercial development. A common, striking feature of these fragmented forests is the presence of large populations of non-native, invasive plant species. Any attempt to understand the ecology of forests in the region must therefore include consideration of invasive plants as key community members. An important example is one of the subjects of our study, Microstegium vimineum (Trin.) A. Camus (Japanese Stilt-grass or Nepalese Browntop; Poaceae), a non-native grass that is spreading rapidly in the region’s forests. Many possible causes and mechanisms of non-native plant invasion have been proposed and studied. In the past decade, allelopathy has attracted considerable attention because of the “novel weapons” hypothesis. It posits that native plants have no opportunity to evolve defenses against an invader’s allelochemistry and therefore may be particularly vulnerable, thereby allowing invasion (Callaway 1Department of Biology, The College of New Jersey, PO Box 7718, Ewing, NJ 08628. 2Current address - Thomas Jefferson University Hospital for Neuroscience, 900 Walnut Street, Suite 444, Philadelphia, PA 19107. *Corresponding author - morrisja@tcnj.edu. 298 Northeastern Naturalist Vol. 19, No. 2 and Aschehoug 2000, Callaway and Ridenour 2004). This attention to allelopathy in non-native, invasive species should not lead us to ignore the possibility that allelopathy may also be important for the ecological competiveness of native species. Indeed, we still have little understanding of the overall relevance of allelopathy for natural communities in general (Inderjit et al. 2005). To determine whether allelopathy is more important for non-native competiveness than for native competitiveness and therefore an important general mechanism of nonnative invasion, it ultimately will be necessary to compare allelopathy among non-native, invasive species and native, resident species (e.g., Kim and Lee 2010), preferably in situ. A first step toward this ambitious goal is to compare species for their allelopathic potential using screening assays against test species, which was the aim of our study. Results of screening assays give some indication of the relative strength of allelopathy in each species and can indicate which species should be subjected to further field studies, where the ecological importance of allelopathy can be investigated (e.g., Butcko and Jensen 2002, Jefferson and Pennachio 2003, McCarthy and Hanson 1998, Rashid et al. 2010). In our study, we compared the allelopathic potential of Japanese Stilt-grass to that of Ageratina altissima (L.) King & H. Rob. var. altissima (White Snakeroot; Asteraceae), a native herb layer species in eastern North American deciduous forests. Native species abundance is typically low where Japanese Stilt-grass has invaded, but we commonly observe abundant White Snakeroot growing intermingled with Japanese Stilt-grass stands in the fragmented forests of central New Jersey. Within Washington Crossing State Park (Titusville, NJ), for example, these two species are the dominant members of an otherwise species-poor herb layer community. The co-dominant pattern of Japanese Stilt-grass and White Snakeroot suggests that they tolerate each other’s chemical properties, while the generally depauperate community suggests the possibility of broad allelopathic effects against other plant species from Japanese Stilt-grass and/or White Snakeroot. In our assay, we made initial tests of both species’ allelopathic potential against two commercially bred species commonly used in allelopathy assays, Lactuca sativa L. (Lettuce) and Raphanus sativus L. (Radish). Since neither assay species belongs to the natural communities in which Japanese Stilt-grass and White Snakeroot co-occur, our results must be interpreted as a first look at the potential that these two species have for using allelopathy as a competitive strategy. We focused on allelopathic effects in the early stages of the assay plants’ growth (seed germination, seedling establishment, seedling growth), reasoning that allelopathy is bound to be most effective as a competitive strategy when deployed early, to prevent neighboring plants from becoming large enough for significant resource competition. Study Species and Field Site Japanese Stilt-grass is a species of growing concern as a strong competitor threatening native biodiversity in eastern forests (Leicht et al. 2005). It is a C4 annual grass (Horton and Neufeld 1998, Winter et al. 1982) that was introduced 2012 B.F. Corbett and J.A. Morrison 299 into eastern North America from eastern Asia some time prior to 1919 (Fairbrothers and Gray 1972). Currently, it ranges from Massachusetts to Missouri, south to Florida and Texas (USDA 2011). Its seeds germinate in early spring, with flowering and fruiting in late summer and fall. Growth is rapid in midsummer; by summer’s end, the forest floor can be completely obscured by nearly monotypic stands, and it is negatively associated with native species richness (Oswalt et al. 2007, Vidra et al. 2006). Although Japanese Stilt-grass is considered shade-tolerant, it performs well under a range of light levels (Cheplick 2005, Claridge and Franklin 2002, Horton and Neufeld 1998, Morrison et. al 2007). Studies show that Japanese Stilt-grass can alter forest soil chemistry (Ehrenfeld et. al 1997) and that it has allelopathic potential, as assayed against Radish seed germination (Pisula and Meiners 2010). White Snakeroot is a native perennial herb that also is common in the eastern deciduous forest herb layer. Its range extends from Maine and Québec to Florida, west to Texas and North Dakota (USDA 2011). Its phenology and habitat are similar to Japanese Stilt-grass; it flowers and fruits in late summer to fall, and it grows in shady to partially shady habitats (Clewell and Wooten 1971). It is known to be toxic to herbivores (Beier et al. 1987), and its allelopathic toxicity toward plants has been demonstrated in a laboratory assay against Lettuce and Radish radicles in a study from Korea, where White Snakeroot is considered a non-native, invasive species (Park et al. 2011). The site we observed and collected from at Washington Crossing State Park (Titusville, NJ) consists of closed hardwood canopy with a very depauperate understory. There are few shrubs or juvenile trees, but there is high cover in the herb layer (87%, based on cover estimates in 1-m2 plots arrayed across a 50-m x 50-m area, n = 80, SE = 2.05), which is almost entirely composed of Japanese Stilt-grass and White Snakeroot (B.F. Corbett and J.A. Morrison, pers. observ.). The canopy trees with the highest importance values (IV) in this site are Acer rubrum L. (Red Maple), IV = 136.28; Fraxinus pennsylvanica Marsh. (Green Ash), IV = 72.70; Quercus rubra L. (Red Oak), IV = 37.64; Robinia pseudoacacia L. (Black Locust), IV = 26.59; Acer saccharum Marsh. (Sugar Maple), IV = 14.03; Prunus serotina Ehrh. (Wild Black Cherry), IV = 7.32; and Juniperus virginiana L. (Eastern Red Cedar), IV = 5.44. We excavated entire plants (roots and shoots) of both Japanese Stilt-grass and White Snakeroot from this site in early October, when in central New Jersey the forest canopy is still intact and almost entirely green, White Snakeroot is in flower with fully green foliage, and Japanese Stiltgrass is in fruit and fully green or beginning to senesce (B.F. Corbett and J.A. Morrison, pers. observ.). We collected approximately 100 fully green individuals of each species for our experiments. Methods Seed germination We separated shoots (stems plus foliage) and roots of both White Snakeroot and Japanese Stilt-grass, cleaned the roots of all forest soil, and dried the material for three days at 60 ºC. We made aqueous extracts of all four tissue types from 300 Northeastern Naturalist Vol. 19, No. 2 1 g dry weight/10 mL diH2O by chopping the tissue in the water for 90 seconds in a blender, stirring for 16 hours, and filtering with cheesecloth. Plain deionized water was the control. This extract concentration is within the range of values used in studies of allelopathy with a variety of plant species (Bruckner et al. 2003, Butcko and Jensen 2002, Eppard at al. 2005, Machado 2007, Morgan and Overholt 2005, Park et al. 2011, Pisula and Meiners 2010). We tested the extracts against seed germination of Lettuce and Radish, which we chose for their fast germination times, lack of stratification period, and use in previous experiments on allelopathic potential (e.g., Aliotta et al. 1994, Butcko and Jensen 2002, Epperd et al. 2005, Gannon et al. 2006, Jefferson and Pinnachio 2003, McCarthy and Hanson 1998, Park et al. 2011, Rashid et al. 2010). We placed 25 Lettuce or Radish seeds 1 cm apart in Petri dishes lined with Whatman no. 1 filter paper, added 2 mL of extract solution to each dish, and sealed it with parafilm. For both Lettuce and Radish, we made 12 replicates of each extract treatment (Japanese Stilt-grass roots, Japanese Stilt-grass shoots, White Snakeroot roots, White Snakeroot shoots, water control), and arranged the 60 plates into four randomized blocks (different shelves) in an incubator for germination. We kept them slightly cooler than room temperature (20 ºC) to promote germination but avoid fungal growth and in the dark to avoid any growth effects from phototropism. On each day for seven days, we counted the number of germinated seedlings. Because of their tiny mass, we pooled all seedlings from one extract treatment at the end of Day 7, let them air-dry for one day, and then measured the combined mass from each treatment. We calculated an average mass per seedling for each extract by dividing this combined mass by the number of germinated seedlings on Day 7. We analyzed percent germination over time as a repeated measures analysis of variance with main effects of EXTRACT and BLOCK and the repeated effect of DAY. We also analyzed final percent germination (Day 7) with factorial analysis of variance (ANOVA), with main effects of EXTRACT and BLOCK; we arcsinetransformed the Radish data to meet the normality assumption for ANOVA. All ANOVAs were performed using PROC GLM in SAS v. 9.2. We present the data for average mass of germinated seedlings in each treatment, but did not analyze it statistically since the data were not replicated, due to pooling of seedlings. Seedling establishment and growth in soil We collected soil from a forested site at The College of New Jersey, Ewing, NJ. In order to experimentally manipulate the soil microflora, we autoclaved half of the soil for one hour at 122 ºC and 17 psi (Trevors 1996). To manipulate the level of allelopathy we added 11.7 g/kg soil of activated carbon (Darco G-60, -100 mesh powder, Aldrich Chemical Co.), which has been shown to adsorb some classes of allelochemicals (Cookson 1978, Virender et al. 2009, Zackrisson at el. 1996) to half of both the sterilized and non-sterilized soil. We based the C : soil ratio on previous allelopathy studies that used activated carbon (Callaway and Aschehoug 2000, Cheremisinoff and Ellerbush 1978, Nilsson 1994, Rich 2004). In order to demonstrate allelopathy, growth of an assay plant would need to be 2012 B.F. Corbett and J.A. Morrison 301 significantly lower in plants exposed to the allelopathic agent compared to a control, and this effect would need to be eliminated upon addition of carbon to the soil. If this difference were present in pots with unsterilized soil but not with sterilized soil, then we would conclude that that the allelochemicals are operating on plant growth indirectly, via the soil microflora. We added each soil type to 120 small pots (35-mm film canisters with drainage holes, 3 cm diameter × 5 cm height), for a total of 480 pots, and planted half with Lettuce seeds and half with Radish seeds. We randomly assigned 60 pots to each soil type × assay seed combination, and assigned each pot to an extract treatment: Japanese Stilt-grass, White Snakeroot, or water control. Extract treatments used whole-plant aqueous extracts of Japanese Stilt-grass and White Snakeroot (combining shoots and roots), made with the protocol described above. We arranged the 20 replicates of each soil type × assay seed × extract combination into 8 randomized blocks under 8 banks of 6 fluorescent lights, positioned 110 cm above the pots. We added 2 mL of the appropriate extract or water to each pot on the first day of the experiment and twice more, one week and two weeks later (we made fresh extracts each week). During the first week, we grew the seedlings under clear, plastic domes in flats, in order to aid germination and retain moisture, but we removed the domes for the remaining two weeks. After three weeks, we noted seedling establishment in each pot and measured the dry mass of each seedling. The lack of a seedling in a pot after three weeks could have been due to either a failure of seed germination or early seedling mortality; our measure of “establishment” did not discriminate. We analyzed establishment with a G-test for heterogeneity (using PROC FREQ in SAS v. 9.2), and used factorial ANOVA for the dry mass data (log-10 transformed), with main effects EXTRACT TYPE, CARBON, SOIL STERILIZATION, BLOCK, and their interactions (using PROC GLM in SAS v. 9.2). Results Seed germination All extracts delayed seed germination of both Lettuce and Radish relative to plain water controls (Fig. 1), as shown by the significant DAY x EXTRACT effect in the repeated measures analyses of variance (Table 1). For example, by Day 2, 97% of Lettuce and Radish had germinated in the water treatment, while mean germination in the extract treatments was just 7–40% for Lettuce and 15–64% for Radish. The cumulative effect of the extracts after seven days of exposure was dependent on the extract species and type of tissue used in the extract. Lettuce germination was lowest with Japanese Stilt-grass root and White Snakeroot shoot extracts, at only 35%, while White Snakeroot shoot extract nearly halved the germination rate of Radish relative to water and the other extracts (Fig. 1). Mean dry mass of germinated seedlings of both species was lower when exposed to all extracts (but note that this result is for illustration purposes only, since we could not test it for significance). Lettuce was particularly sensitive; its dry mass was nearly an order of magnitude lower in most extract treatments than in the water control (Fig. 2). 302 Northeastern Naturalist Vol. 19, No. 2 Figure 1. Percent germination (means ± SE, n = 12) of Lettuce and Radish seeds exposed over seven days to water or aqueous extracts from shoots or roots of Japanese Stilt-grass (non-native, invasive) or White Snakeroot (native). Day 7 values with different letters are significantly different (based on post-hoc Fisher’s LSD tests; ANOVAs on Day 7 data, EXTRACT effect: Lettuce, F = 106.64, df = 4,40, P < 0.0001; Radish, F = 57.49, df = 4,40, P < 0.0001). Figure 2. Mean mass per seedling (total g of seedlings/number of seedlings) of germinated seedlings exposed to water or aqueous extracts from shoots or roots of Japanese Stilt-grass (non-native, invasive) or White Snakeroot (native). 2012 B.F. Corbett and J.A. Morrison 303 Seedling establishment For both Lettuce and Radish, the percent seedling establishment was not significantly different among blocks nor among carbon treatment, so we pooled the data across blocks and carbon treatment. It is reasonable to ignore the carbon treatment for these data in any case, since seed and early seedling mortality would likely occur from direct application of the extracts to the soil surface where the seeds were located, and would not be mediated by added carbon in the soil. Seedling establishment for both assay species was significantly variable among the three treatments overall, but only when the soil was sterilized (Fig. 3; G-tests for heterogeneity of ALIVE [yes/no] x EXTRACT [water/Japanese Stilt-grass/ White Snakeroot], df = 2 for each test: Lettuce, unsterilized soil, G = 0.61, not significant [ns]; Lettuce, sterilized soil, G = 7.31, P = 0.026; Radish, unsterilized soil, G = 1.83, ns; Radish, sterilized soil, G = 10.9, P = 0.006). The significance in the sterilized soils was largely due to the difference between the water control and the White Snakeroot extract (G-tests of heterogeneity of ALIVE [yes/no] x EXTRACT [water/White Snakeroot], df = 1 for each test: Lettuce, G = 7.31, P = 0.007; Radish, G = 10.12, P = 0.0015). All other pairwise G tests were not significant, although the water/Japanese Stilt-grass test for Radish was nearly so (G = 3.23, P = 0.07). Table 1. Repeated measures analysis of variance of percent seed germination over seven days, for Lettuce and Radish, when treated with plain water or aqueous root or shoot extracts from Japanese Stilt-grass (non-native, invasive) or White Snakeroot (native). ns = not significant. Adjusted P Source of variation df MS F P (G-G) Lettuce Between-subjects effects Extract 4 81713.90 103.81 <0.0001 Block 3 557.72 0.001 ns Error (= E x B) 12 787.18 Within-subjects effects Day 6 14469.06 297.11 <0.0001 <0.0001 Day x Extract 24 1144.29 23.5 <0.0001 <0.0001 Day x Block 18 55.38 1.14 ns ns Error (= E x B x D) 72 48.70 Greenhouse-Geiser ε = 0.518 Radish Between-subjects effects Extract 4 41243.80 128.79 <0.0001 Block 3 92.22 0.29 ns Error (= E x B) 12 320.25 Within-subjects effects Day 6 26197.70 773.48 <0.0001 <0.0001 Day x Extract 24 2029.29 59.91 <0.0001 <0.0001 Day x Block 18 41.50 1.23 ns ns Error (= E x B x D) 72 33.87 Greenhouse-Geiser ε = 0.516 304 Northeastern Naturalist Vol. 19, No. 2 Growth Dry mass was not significantly different for assay plants grown with water or an extract, nor was there any significant variation due to the interactions of the extract treatments with the carbon or sterilization treatments (Table 2, Fig. 4). However, the carbon and sterilization treatments themselves interacted to directly affect growth (Table 4). Dry mass of both Lettuce and Radish were greater in sterilized soil, but only when carbon was added (Fig. 5). Discussion Comparison of Japanese Stilt-grass and White Snakeroot Allelopathy has been hypothesized as an important mechanism of invasion by non-native species, but allelopathy also may be a common competitive strategy in native species. We found that the allelopathic potential of an invasive, non-native species against sensitive assay species was not greater than that of a co-occurring native species; rather, it appeared somewhat weaker. While extracts of both the invasive Japanese Stilt-grass and the native White Snakeroot decreased germination speed, percentage of seed germination, and size of germinated seedlings of assay species, the most striking germination effect was by the shoot extract of White Snakeroot, which decreased percent germination of Radish seeds by nearly 50%. Similarly, the seedling establishment phase (in sterilized soils) was negatively affected by the extracts, most strongly from White Snakeroot. Both species Figure 3. Percent seedling establishment (out of 40 seeds per group) of Lettuce and Radish in pots under lights, in unsterilized or sterilized forest soils, watered with water or whole-plant aqueous extracts of Japanese Stilt-grass (non-native invasive) or White Snakeroot (native). Figure 4 (opposite page). Mean dry mass (± 95% CL) of Lettuce or Radish plants grown in pots under lights, in unsterilized or sterilized forest soils, watered with water or wholeplant aqueous extracts of Japanese Stilt-grass (non-native invasive) or White Snakeroot (native), and with or without activated carbon added to the soil. Sample sizes from left to right: A) 11, 13, 12, 12, 10, 11; B) 10, 14, 8, 12, 8, 6; C) 6, 8, 14, 7, 9, 9; D) 13, 12, 11, 6, 5, 6. Data were back-transformed from log10. 2012 B.F. Corbett and J.A. Morrison 305 Table 2. Analysis of variance of dry mass of Lettuce or Radish seedlings, grown with plain water or aqueous extracts of Japanese Stilt-grass (non-native invasive) or White Snakeroot (native), with soil sterilized or not, and activated carbon added or not. Data were log10-transformed. The data were pooled across blocks, since all terms that included the block effect in the complete model were not significant (ns). Source of variation df MS F Lettuce Extract (E) 2 0.0074 0.10, ns Carbon (C) 1 0.0361 0.47, ns Soil sterilization (S) 1 0.0933 1.23, ns E x C 2 0.0391 0.51, ns E x S 2 0.0674 0.89, ns C x S 1 0.4210 5.54, P = 0.02 E x C x S 2 0.0858 1.13, ns Error 115 0.0738 Radish Extract (E) 2 0.0197 0.46, ns Carbon (C) 1 0.0112 0.26, ns Soil sterilization (S) 1 0.7142 16.72, P < 0.0001 E x C 2 0.0002 0.00, ns E x S 2 0.0159 0.37, ns C x S 1 0.2400 5.62, P = 0.02 E x C x S 2 0.0885 2.07, ns Error 94 306 Northeastern Naturalist Vol. 19, No. 2 have at least the potential for allelopathy to be a mechanism for their competitive success, but the native White Snakeroot may have more potential than the nonnative, invasive species. The next step would be to determine how the allelopathic potentials of Japanese Stilt-grass and White Snakeroot play out in natural communities, with a biogeographical approach (Inderjit et al. 2008b) testing allelopathy against each other and against forest species native to North America. Their effects against naturally evolved species may be quite different from those on commercially bred Lettuce and Radish. We may expect allelopathy by Japanese Stilt-grass to be generally effective against native forest species, which have no long ecological history with this invader from another continent, and so have had little time to evolve defenses against its allelochemistry (“novel weapons” hypothesis). However, the prevalence of White Snakeroot within stands of Japanese Stiltgrass suggests that not all native species are vulnerable. The idea of a novel allelochemical weapon should also apply in the opposite direction. Japanese Stilt-grass should experience the allelochemistry of White Snakeroot (and any other allelopathic natives) as novel, yet Japanese Stilt-grass thrives among White Snakeroot. Our ability to understand the role that allelopathy plays in structuring invaded communities clearly will require comparisons of allelopathy among a wide range of co-occurring native and non-native invasive species, such as Kim and Lee (2010) have begun to do. Allelopathic effects on different life-history stages This study demonstrated allelopathic effects from Japanese Stilt-grass and White Snakeroot extracts on seed germination and seedling establishment, but not on plant biomass (i.e., growth). We observed this effect in the seed germination experiment, in which both Lettuce and Radish seeds exposed to the plant extracts had slower germination rates and reduced total germination than when exposed to water, and in the soil experiment, in which seedling Figure 5. Mean dry mass (± 95% CL) of Lettuce or Radish plants grown in pots under lights, in unsterilized or sterilized forest soils, with or without activated carbon added to the soil. Data were pooled across the non-significant extract treatments. Sample sizes from left to right: A) 33, 26, 36, 32; B) 29, 29, 25, 24. Data were back-transformed from log10. 2012 B.F. Corbett and J.A. Morrison 307 establishment of both assay species was much greater with water than with the plant extracts (in sterilized soil). The inhibitory effects of allelopathy may be viewed as a dynamic process with respect to the life-history stage of its target (Keeley et al. 1985, McPherson and Muller 1969). Allelopathic effectiveness at the earliest life-history stages, as shown here, may be a particularly effective strategy to use against potential competitors, if it prevents them from becoming large enough to compete against the allelopathic plant species for space or resources. Furthermore, a given dosage of allelochemicals on a small, germinating seed or on seedlings, which generally are susceptible to environmental stress, would likely have a stronger effect than that same dosage on a larger, growing plant, making allelopathy targeted to seed germination and seedlings very efficient. These results illustrate the importance of examining potential allelopathic effects on plants at different life-history stages. The use of seed germination as the sole measure of allelopathy has been questioned (Inderjit and Dakshini 1995), but, similarly, studies that focus only on allelopathic effects on mature plant growth and/or reproduction may miss the most crucial and dramatic effects of allelopathic species—those on seeds and seedlings. Allelopathic effects of roots vs. shoots Tissue-specific allelopathic potential has been shown in various species, including, for example, Eichhornia crassipes (Mart.) Solms (Common Water Hyacinth; Chen et al. 2005), Pueraria lobata (Willd.) Ohwi (Kudzu; Rashid et al. 2010), Alliaria petiolata (M. Bieb.) Cavara & Grande (Garlic Mustard; Mc- Carthy and Hanson 1998), Chenopodiaceae species (Jefferson and Pinnachio 2003), and Asteraceae species (Butcko and Jensen 2002). In our experiment, we noted differences in allelopathic potential between root and shoot tissue of both species, but in contrasting ways. First, Japanese Stilt-grass root extract was more toxic to Lettuce germination than was shoot extract (there was no difference for Radish germination). Allelopathy takes place in the soil, so it makes sense for a species to preferentially allocate its toxic secondary chemicals to roots, which produce exudates that are in constant contact with the soil. In our experiment, we used extracts derived from ground plant material, which includes chemicals that could be released as exudates or upon physical disruption by decomposers (although it also should be noted that atypical chemicals could have been present due to the stress of the collection and drying process). Second, the shoot extract from White Snakeroot was much more toxic to both assay species’ seed germination than was the root extract. White Snakeroot is known to be avoided by deer, sheep, cattle, a variety of insects, and other grazing herbivores that feed on aboveground shoot tissue (Sharma et al. 1998). It produces tremetone (Beier et al. 1993), a toxic chemical that is secreted in leaf tissue (Curtis and Lersten 1986). Leaves of White Snakeroot come in contact with the forest soil upon leaf abscission and decomposition, so if leaf chemicals that caused the toxic effects we observed on Lettuce and Radish seeds (perhaps tremetone) persist in the soil, then White Snakeroot shoot tissue could have a strong inhibitory influence on forest plant species. Persistence of allelochemicals has been demonstrated for 308 Northeastern Naturalist Vol. 19, No. 2 other species, e.g., Garlic Mustard (Cipollini and Gruner 2007) and Centaurea maculosa Lam. (Spotted Knapweed; Perry et al. 2007). The marked difference in the shoot vs. root allocation of allelopathic potential between the two species we studied suggests that the threat of aboveground herbivory may be more important for the longer-lived perennial, White Snakeroot, than for the annual Japanese Stilt-grass, which, as a grass, is likely to be more tolerant of grazing. The role of soil microflora, and experimental considerations An important aspect of our experimental design was to manipulate the soil microflora, since phytochemicals can affect microorganisms (e.g., Boufalis and Pellessier 1994, Kong et al. 2004, Souto et al. 2000), and microorganisms have various, complex effects on plants (Pena and Reyes 2007, Saravanan et al. 2008, Shefferson et al. 2008, Van Der Heijden et al. 2008, Winder 1997). Any study of allelopathy should therefore attempt to detect whether allelochemicals affect plants directly, or indirectly via effects on microorganisms (e.g., Callaway et al. 2008). We found that sterilization of the soil strongly influenced the allelopathic effect against seedling establishment by both Japanese Stilt-grass and White Snakeroot extracts. There was no significant difference in establishment among water and extract treatments in unsterilized soil, but in sterilized soil seedling establishment was lower in the extract treatments. Similar to Winder’s (2007) study, this result suggests that the soil microflora protected the seeds from the toxic effects of the plant extracts, perhaps attributed to degradation of allelochemicals by certain species of bacteria (Inderjit et al. 2008a). However, Figure 3 shows that in the control pots watered with plain water, sterilization tended to increase survival, suggesting that, in the absence of allelochemicals, the soil microflora inhibited seed germination and/or survival (similar to Nikitina et al. 2004). It also is possible that autoclaving caused these effects by altering the physical property of the soil in a manner that interacted with the extracts (Trevors 1996). We observed complex effects from soil sterilization in the biomass data as well, combined with direct effects of activated carbon (i.e., not due to its effect on the extract treatments, which were all nonsignificant). Growth decreased when carbon was added to the unsterilized soil relative to the no-carbon treatment, but growth increased when carbon was added to sterilized soils relative to the no-carbon treatment. The overall result was that plants grown in sterilized soil were double the size, on average, of plants grown in unsterilized soil, but only when carbon was added (Fig. 5). This shows that added carbon can have unintended effects in allelopathy experiments. Instead of acting to increase growth in unsterilized soil by neutralizing allelochemicals, as expected, carbon had a negative effect on growth. Instead of having no effect in sterilized soils as expected, carbon increased growth. Although widely used in allelopathy experiments, activated carbon is not a neutral substance. It can both enhance soil fertility (Lau et al. 2008) and suppress the action of beneficial soil microorganisms (Weißhuhna and Pratib 2009), which can cause decreased growth (Wurst et al. 2010). Also, as an adsorber of small organic molecules like allelochemicals (Bais et al. 2003), activated carbon probably would adsorb toxins that may be 2012 B.F. Corbett and J.A. Morrison 309 lysed from microbial cells during autoclaving. Therefore, caution is required when utilizing activated carbon and soil sterilization in allelopathy studies. They can interact in unpredictable ways and have a direct effect on target plants, regardless of allelochemicals. Finally, extracts made from disrupted tissue include a wide range of phytochemicals, including those that have specific allelopathic potential, but many others as well (Inderjit and Dakshini 1995). In addition, the quantity and quality of allelochemicals in the extract may or may not correspond to the level of allelochemicals released by living plants and their decomposing tissues and their persistence under field conditions. 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