Soil sterilization alters interactions between the native grass Bouteloua gracilis and invasive Bromus tectorum

Soil sterilization alters interactions between the native grass Bouteloua gracilis and invasive Bromus tectorum

Journal of Arid Environments 111 (2014) 91e97 Contents lists available at ScienceDirect Journal of Arid Environments journal homepage: www.elsevier...

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Journal of Arid Environments 111 (2014) 91e97

Contents lists available at ScienceDirect

Journal of Arid Environments journal homepage:

Soil sterilization alters interactions between the native grass Bouteloua gracilis and invasive Bromus tectorum Taraneh M. Emam a, *, Erin K. Espeland b, Matthew J. Rinella c a

Department of Plant Sciences, University of California, Davis, Mail Stop 1, One Shields Ave, Davis, CA 95616, USA Pest Management Research Unit, US Department of Agriculture, Agricultural Research Service, 1500 N Central Avenue, Sidney, MT 59270, USA c US Department of Agriculture, Agricultural Research Service, 243 Fort Keogh Road, Miles City, MT 59301, USA b

a r t i c l e i n f o

a b s t r a c t

Article history: Received 24 February 2014 Received in revised form 7 August 2014 Accepted 25 August 2014 Available online 16 September 2014

The invasive grass Bromus tectorum negatively impacts grass and shrublands throughout the western U.S., particularly in arid and semiarid regions. We asked whether soil microbes associated with a native grass (Bouteloua gracilis) affect growth of Bromus and competition between Bromus and Bouteloua. We also examined whether plant responses varied between soils from 15 sites in the Northern Great Plains. Bromus and Bouteloua were grown in media with sterilized or unsterilized soil, alone and together. Soil sterilization reduced biomass of Bouteloua and Bromus grown alone by an estimated 50% and 48%, respectively. Additionally, results provided evidence that sterilization increased the effect of competition on Bromus, and may have reduced the effect of competition on Bouteloua. Bouteloua likely had a stronger negative effect on Bromus in sterilized soils because sterilization reduced Bromus biomass by a greater absolute amount. Response to sterilization varied appreciably by site for Bromus, but not Bouteloua. Our results support the hypothesis that invasive species such as Bromus often have positive responses to soil biota in the invaded range. Soil microbes are one factor that may be important in determining dynamics of plant invasions, and plant responses to new sites and competition with natives. © 2014 Elsevier Ltd. All rights reserved.

Keywords: Competition Great Plains Plantesoil feedback Soil microbes

1. Introduction The non-native grass Bromus tectorum L. (hereafter referred to as Bromus) currently occupies over 22 million hectares in the western United States (Duncan et al., 2009), and during the first decades of invasion from 1889 to 1929, it spread at one of the fastest documented rates of invasion for plants (Pysek and Hulme, 2005). Bromus has greatly altered ecosystem processes in the arid western U.S. by increasing fire frequency (Balch et al., 2013) and outcompeting native plants (Mack, 1981). Much research effort has been expended to determine the traits of Bromus that lead to its dominance in arid and semiarid lands. Explanations include prolific seed production, a phenology which enables early access to spring moisture, and being adapted to large grazers (Harris, 1967; Hulbert, 1955; Mack, 1989). Relationships with soil biota may be another determinant of Bromus invasion dynamics. Past research has demonstrated effects of Bromus invasion on resident soil biota and nutrient cycling (e.g., Hawkes et al., 2006; Schaeffer et al., 2012). However, the effects of resident soil biota on Bromus remain * Corresponding author. Tel.: þ1 530 752 1701; fax: þ1 530 752 4361. E-mail addresses: [email protected] (T.M. Emam), [email protected] gov (E.K. Espeland), [email protected] (M.J. Rinella). 0140-1963/© 2014 Elsevier Ltd. All rights reserved.

unclear, as past studies have shown positive, negative, or no detected effects of soil biota on Bromus growth (Al-Qarawi, 2002; Rowe et al., 2009; Wilson and Hartnett, 1998). Factors such as variation in abiotic conditions across sites may influence the net effect of soil biota on Bromus, and interspecific competition may alter responses to soil biota (e.g., Callaway et al., 2004). Examining the role of soil biota in Bromus invasion may assist in identifying new methods for preventing or controlling Bromus (e.g., Meyer and Nelson, 2006; Rowe et al., 2009). In addition, interactions between plants and soil biota influence multiple processes including succession, plant community diversity, and productivity (Bever et al., 2010; Inderjit and van der Putten, 2010), but further study is needed to understand how planteplant interactions and geographic variation shape these interactions. 1.1. Plantesoil feedbacks influence invasion Plantesoil feedbacks e plant effects on soil biotic and/or abiotic factors which affect subsequent plant growth e can influence invasion processes (Inderjit and van der Putten, 2010; Klironomos, 2002). Most native species exhibit a negative conspecific feedback: a species' fitness is often lower in soil previously occupied by conspecifics than in sterilized soil or soil previously occupied by


T.M. Emam et al. / Journal of Arid Environments 111 (2014) 91e97

heterospecifics (Brinkman et al., 2010; Kulmatiski et al., 2008). These negative plantesoil feedbacks are thought to be due to the accumulation of specialist pathogens, parasites, and herbivores (Reynolds et al., 2003). Negative feedbacks are particularly prevalent among grasses: Kulmatiski et al. (2008) suggest that characteristics of grasses adapted to competing for water in semiarid lands may lead to greater exposure to soil enemies. The prevalence of negative conspecific feedbacks may promote coexistence of competing plant species and thereby contribute to maintenance of biodiversity (Bever, 2003). However, among invasive plant species, many studies have found positive (or failed to detect negative) conspecific feedbacks in the invaded range (Inderjit and van der Putten, 2010). Positive feedbacks with soil biota occur when the benefits of soil mutualists outweigh the negative effects of natural enemies, which are thought to lead to increased interspecific competition and competitive exclusion of native plant species by invasive species (Bever, 2003). Several studies have found that while native plant species tend to generate feedbacks that benefit community diversity and coexistence of multiple species (i.e., negative conspecific effects but positive effects on others), invasive species tend to generate feedbacks that promote their own growth; this phenomenon has been documented in arid lands as well as through meta-analysis of many systems (Kulmatiski et al., 2008; Perkins and Nowak, 2013). Using the context of feedbacks between plants and soil biota, we attempted to examine how interactions between an invasive species (Bromus) and a native species (Bouteloua gracilis) responded to the soil community across a range of sites. 1.2. Study aims We conducted a greenhouse study comparing the responses of Bromus and a native grass (Bouteloua gracilis H.B.K. Lag. ex Steud, hereafter referred to as Bouteloua) to growth media inoculated with soil gathered beneath Bouteloua in the field. We chose Bouteloua gracilis as a study species because it is a key late-seral species of Great Plains mixed and shortgrass prairie, and was historically the dominant species in terms of frequency and biomass (Costello, 1944). Moreover, Bouteloua is common throughout western North America and occurs throughout much of the invasive range of Bromus. Bromus interacting with Bouteloua-associated soil microbes therefore represents a realistic invasion scenario in the Great Plains. We expected Bouteloua and Bromus to differ appreciably in their response to soil microbes. Bromus is a non-native cool-season annual grass, while Bouteloua is a native, warmseason perennial C4 grass. C4 grasses are known to respond more positively to mycorrhizal fungi (Hoeksema et al., 2010), and differences in photosynthetic pathways between these two species may affect species compositions of associated soil bacteria (Porazinska and Bardgett, 2003). We collected soil samples from beneath patches of Bouteloua at 15 sites in the semiarid Northern Great Plains to serve as a source of microbial inoculum. Bromus and Bouteloua were grown alone and together, with soil sterilized by autoclaving and unsterilized, in a factorial experiment to examine whether soil sterilization affected planteplant interactions, and whether there was substantial variation in the responses of Bromus and Bouteloua among sites of soil collection. We tested the following hypotheses: (1) Unsterilized soil from beneath Bouteloua plants will have a negative effect on Bouteloua biomass and a positive effect on Bromus biomass, because native species tend to generate negative conspecific feedbacks and positive heterospecific feedbacks (Perkins and Nowak, 2013).

(2) If Bouteloua biomass is reduced in the unsterilized soil, Bromus presence will have a greater negative effect on Bouteloua when the two species are grown together in unsterilized soil as opposed to sterilized soil. Testing these two hypotheses provides information regarding how Bromus might invade established communities of the Great Plains or those undergoing restoration: if Bromus has an advantage in unsterilized soils, this would suggest that soil microbes may contribute to competitive exclusion of Bouteloua by Bromus. Because we studied multiple sites, we were also able to examine variation in response to soil sterilization. 2. Materials and methods 2.1. Collection of soil samples and seeds In summer 2012, soil samples were collected from 15 sites in eastern Montana or northeastern Wyoming, U.S. (Appendix Fig. A.1). Sites were located within 6 sampling areas: 1) Caballo, 2) Eagle Butte, and 3) Spring Creek mines, 4) North and 5) South regions of the Thunder Basin National Grassland, and 6) Fort Keogh Livestock and Range Research Laboratory. Climate, landscape, soil, and site history are described in Table 1. Landscape and soil data for mined sites were collected by the U.S. Department of Agriculture as part of a study of these sites. At Thunder Basin and Fort Keogh, soil pH and texture were analyzed by A&L Western Laboratories, Inc. (Modesto, CA), and site slope and aspect were assessed in November 2013. Sites with a mining history had been strip-mined for coal. Reclamation activities at mines included soil replacement, mulching and fertilizing at some sites, and seeding with Bouteloua and a variety of other species. Vegetative cover at sites was predominantly made up of native perennial grasses, Agropyron cristatum (L.) Gaertn. and annual Bromus species. Total cover was relatively low across sites, averaging between approximately 5% and 30%. Though cover was low and soil was sampled from directly beneath Bouteloua, it is possible that any neighboring plant species present may have influenced the soil community as well. Table 1 Characteristics of the 15 sites used in this study. Sampling area names are followed by total annual precipitation (Precip) in mm and mean annual temperature (MAT) in  C averaged from 1983 to 2012. Aspect of “Und” indicates undulating terrain where aspect could not be determined. Soil texture: C ¼ clay, F ¼ fine, L ¼ loam, S ¼ sand. Sites with no mining history have a reclamation year of “N/A”. Missing data are indicated by “e”. Climate data were obtained from PRISM Climate Group (available online at Sampling area

Site# Slope Aspect (%)

Soil Soil pH Reclamation texture year

Caballo Mine Precip: 384 MAT: 7.4

1 2 3 4 1 2 3 4 1 2

1 1 2 2 3 2 2 3 4 7



7.4 7.4 7.6 7.4 7.2 8.0 7.4 7.6 7.4 7.3

1990 1990 1998 1998 1992 1999 1999 1992 N/A N/A







1 2

0 3

Neutral C NNE SL

6.1 6.0


1 2

e e

e e

e e


Eagle Butte Mine Precip: 378 MAT: 7.5 Fort Keogh Precip: 348 MAT: 7.7 Spring Creek Mine Precip: 378 MAT: 7.0 Thunder Basin North Precip: 384 MAT: 7.4 Thunder Basin South Precip: 325 MAT: 8.1

e e

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Soil and Bouteloua root fragment samples were collected from beneath 4 to 11 Bouteloua patches per site using an 11-cm tall conical bulb planter (base and top diameter 6 and 7.5 cm, respectively) to collect one soil core per Bouteloua patch. Samples were then pooled by site, yielding between approximately 1.5 and 4 L per site (the number of cores taken, and consequently soil volume, was limited by the number of Bouteloua patches available at each site). The pooled samples were oven-dried for 4 days at 60  C. Oven drying is likely to have excluded soil organisms aside from microbes (e.g., nematodes, arthropods, etc), and may have altered the microbial community to favor heat-tolerant species. However, this is the optimal method for preserving mycorrhizal propagules (Habte and Byappanhalli, 1998); allowing soils to retain moisture during storage would have also resulted in an altered community due to the creation of anaerobic conditions and activity by decomposers (Harris et al., 1993). Soils were coarsely ground to ~4 mm to homogenize them and allow mixing with artificial growth media, and stored at ambient temperature in a laboratory. Utilizing field-collected soils as opposed to greenhouseconditioned soils (i.e., growing a certain plant species to culture its associated soil microbes in the greenhouse prior to the start of a feedback experiment) enabled us to examine among-site variation. Seeds from individual Bromus plants were collected from populations in eastern Montana. Seeds of Bouteloua, Bad River variety, were purchased from Granite Seed Company (Lehi, UT). 2.2. Experimental design To create a “sterilized soil” treatment, half of the soil collected from each site was sterilized by autoclaving 3 times at 121  C for 1 h with 36e48 h between each autoclaving in order to remove soil biota (modification of Meiman et al., 2006). Autoclaving can affect a number of soil properties, and soil characteristics are affected by all currently available sterilization methods (Perkins et al., 2013). As described below, we diluted the soils with artificial media and applied fertilizer in an attempt to diminish the effects of autoclaving that were unrelated to soil biota. The effect of autoclaving on soil nutrient availability is typically much smaller than effect of applying fertilizer (e.g., He and Cui (2009) detected a change of 5 mg/kg NH4 with autoclaving, while we applied a fertilizer containing 200 mg/L N in this study). Sterilized and unsterilized soil was mixed with PROFILE Field and Fairway® Natural, a ceramic particle soil replacement (PROFILE® Products LLC, Buffalo Grove, IL) at a rate of 12% soil to 88% PROFILE® (v:v), and this growth media was added to pots. Although using artificial media led to less realistic growing conditions, mixtures comprising between 1 and 15% field soil are widely used for studying effects of soil microbes on plants while reducing the effects of abiotic soil characteristics (Brinkman et al., 2010). PROFILE® was used to make up the remainder of the growth media because it has higher water and nutrient retention than sand (McCoy and Stehouwer, 1999). The growth media was placed in 6.4-cm wide by 25-cm deep conical Deepots™ (Stuewe and Sons, Corvallis, OR). Approximately 1 cm of PROFILE® was added on top of the media to reduce transmission of soil microbes across pots. We collected one sample of growth media (PROFILE® and soil mixture) from sterilized and unsterilized treatments for 8 of the sites to determine whether soil sterilization had significant effects on soil chemical properties after dilution with PROFILE®. Samples were analyzed for plant-available P, K, Mg, Ca, Na, S, pH, and cation exchange capacity (CEC) at A&L Western Laboratories, Inc. (Modesto, CA). Growth media samples submitted for analysis did not include the fertilizer that was later added. Pots were arranged within 5 blocks in the greenhouse (i.e., 5 replicates at start of experiment; however, this changed due to mortality e see Table A1). Unsterilized and sterilized treatments


were separated by approximately 15 cm to reduce crosscontamination during watering. Bromus individuals, which tend to have a very high germination rate, were planted as seeds. Because Bouteloua germination can be low, seeds were pregerminated in the lab on wet paper towels in ambient natural and artificial light and then the 1e2 day old germinants placed in the pots. Bouteloua germinants that died were replaced by new germinants for up to a week following the initial planting. Within soil sterilization treatments in each block, we applied two different competition treatments to each species for each of the 15 sites: 1 pot was planted with a single Bromus individual, 1 pot with a single Bouteloua individual, and 2 pots containing an individual of each species. Pots containing one live individual of each species after 3 weeks of growth were defined as “with competition” or grown together; pots with only one species present at this point were defined as “without competition” or grown alone. We use the terms “competition” and “competitive effect” throughout this text; however, we did not implement a replacement series to determine the effects of inter-versus intra-specific competition and intend only to describe the effects of Bouteloua and Bromus grown together versus alone. Plants were grown in a greenhouse from November 8th 2012 to January 14th 2013 (approximately 9 weeks) at 32  C (day) and 16  C (night), with 13 h of supplemental daytime lighting and daily watering. Pots were fertilized with a P-free nutrient solution prior to planting in order to prevent excessive nutrient deficiency. The solution contained 0.2 g/L N, 0.04 g/L K, 0.02 g/L S, and 0.002 g/L Fe. On Dec. 6th 2012, a more dilute solution containing 0.08 g/L N, 0.01 g/L K, 0.007 g/L S, and 0.0007 g/L Fe was added. Any other species that germinated from the existing seed bank were removed upon detection. Aboveground biomass was harvested from pots by block from Jan. 7th 2013 to Jan. 14th 2013. Biomass was oven-dried to constant weight at 60  C then weighed. 2.3. Statistical analysis Because some plants failed to establish, some combinations of site, soil sterilization, and competition treatments did not occur in all blocks. When Bromus and Bouteloua were planted together and one of the two plants failed to establish within three weeks of the experiment start date, the surviving plant served as a subsample of the corresponding “grown alone” treatment in the analysis. This type of establishment failure led to 0e3 subsamples per treatment combination within blocks. Subsamples were averaged within the five blocks. Replication of treatment combinations is described in the Appendix, Table A1. According to initial analyses, the effects of soil sterilization, site  soil sterilization, competition treatment  soil sterilization, and site  competition treatment  soil sterilization on mortality of Bromus and Bouteloua were not substantial; therefore, we do not present mortality results here. Our two response variables were natural-log transformed Bromus and Bouteloua aboveground biomass per plant. We fit the same linear model to both response variables. Initial models indicated no substantial difference between plant response to soils from non-mined versus mined sites, and this term was not included in subsequent models. Additionally, including a term for sampling area did not substantially improve the model when site was also included; therefore, sampling area was excluded from the final model. The final model had terms indicating block, site, competition treatment (plants grown alone or with an individual of the other species), and whether or not soil was sterilized. Site  competition treatment, site  soil sterilization, competition treatment  soil sterilization, and site  competition treatment  soil sterilization interactions were also included in the model. Site, and all


T.M. Emam et al. / Journal of Arid Environments 111 (2014) 91e97

interactions involving site, were modeled as random effects due to the high number of levels for this factor (Gelman and Hill, 2007). Block was also modeled as random, and all other parameters were modeled as fixed. We wrote a FORTRAN program to fit the models (Intel Corporation, 2013). Inferences were based on backtransformed parameter point estimates and 95% confidence intervals, and P values calculated from uncertainty estimates on the regression parameters using methods outlined by Gelman and Hill (2007). Effects of site and sterilization treatment on properties of the growth media were analyzed using the GLM procedure in SAS 9 (SAS Institute, Inc.) using untransformed response variables for soil P, K, Mg, Ca, Na, S, pH, and CEC. 3. Results When grown alone, soil sterilization decreased aboveground biomass of Bromus by an estimated 48% (P ¼ 0.01, confidence interval 30e61%, Fig. 1a). Similarly, Bouteloua grown alone had 50% less biomass when soil was sterilized (P ¼ 0.01, confidence interval 10e71%, Fig. 1b). In unsterilized soil, Bromus biomass was not substantially different when grown with or without a Bouteloua individual (P ¼ 0.22). However, soil sterilization increased the effect of competition on Bromus (P ¼ 0.04, Fig. 1a). Conversely, in unsterilized soils competition reduced Bouteloua biomass by 61% (P ¼ 0.01, confidence interval 25e79%), but there was some evidence that sterilization lessened the effect of competition on Bouteloua (P ¼ 0.13, Fig. 1b): Bouteloua biomass decreased less in response to competition when soil was sterilized than when soil was unsterilized.

Fig. 1. Aboveground biomass of Bromus (a) and Bouteloua (b) in unsterilized and sterilized soils, when grown alone or with a competitor of the other species (bars shown with standard errors).

The effect of soil sterilization on Bromus biomass varied appreciably by site (Fig. 2a): Bromus had less biomass in response to sterilization of soil from most, but not all, sites (Fig. 2b). Compared to the mean across sites, sterilization reduced Bromus biomass more drastically at one Eagle Butte mine site and one Fort Keogh site, and less drastically at one Eagle Butte mine site and two Thunder Basin National Grassland sites (Fig. 2a). Interestingly, siteto-site variation in Bromus response to sterilization appeared to be similar in magnitude within and among sampling areas. The site of soil collection appeared to affect Bromus biomass both when soil was sterilized and unsterilized (Fig 2b). Bouteloua responses to soil sterilization did not differ substantially by site, and the effect of competition in sterilized or unsterilized soils did not vary substantially by site for either species. See Appendix Table A2 for detailed results. Some properties of the growth media (soil mixed with PROFILE®) were altered by soil sterilization (Table 2). Sterilization increased CEC by 11% (P ¼ 0.03, confidence interval 2e21%), while sterilization decreased pH by 5% (P ¼ 0.004, confidence interval 2e7%). Site of soil origin affected CEC, S, Ca, Mg, and pH (all confidence intervals not overlapping zero and P < 0.05, results not shown). Point estimates of site effects tended to be higher than sterilization effects; for example, point estimates of the effect of site were 46% and 23% on CEC and pH in sites with the largest effects, compared to estimates of 11% and 5% for sterilization. 4. Discussion 4.1. Soil sterilization decreased Bromus tectorum and Bouteloua gracilis biomass when grown alone We found that overall, soil sterilization decreased aboveground biomass of Bromus grown alone. This result is consistent with our hypothesis that Bromus would benefit from unsterilized soil due to soil microbes associated with a native plant, similar to invasive plants in other studies (Inderjit and van der Putten, 2010). However, our results show that Bouteloua biomass also decreased when soil was sterilized, which runs counter to our hypothesis that Bouteloua would have a positive response to soil sterilization. This could be because late-seral or climax species such as Bouteloua may promote their own dominance through positive feedback mechanisms (Connell and Slatyer, 1977), despite the accumulation of natural enemies. Meta-analysis has shown that overall, native species tend to have negative plantesoil feedbacks, but later successional species such as Bouteloua may have less negative plantesoil feedbacks (Kulmatiski et al., 2008). Relationships between plants and soil microbes may explain our results. One key way that soil microbes affect plants is by influencing quantities and availabilities of soil nutrients. For example, soil bacteria perform the processes of N mineralization and immobilization, which heavily affect soil N availability (Myrold and Bottomley, 2008). Past research has found that Bromus increases rates of microbial N immobilization and N cycling relative to native grassland plants (Schaeffer et al., 2012). AM fungi can also increase plant biomass by providing nutrients, particularly P (Smith and Read, 2008). Biomass of Bromus has been shown to increase (AlQarawi, 2002), or remain similar (Wilson and Hartnett, 1998), in response to AM fungi. C4 grasses such as Bouteloua tend to have more positive responses to AM fungi than C3 grasses (Hoeksema et al., 2010), and Bouteloua has been shown to have high levels of AM colonization and substantial increases in carbon fixation rates with AM symbiosis (Allen et al., 1984). In addition, soil microbes can have positive non-nutritive effects on plants. For example, AM fungi have been shown to reduce effects of soil pathogens (Wehner et al., 2010), and plant growth promoting rhizobacteria can increase plant

T.M. Emam et al. / Journal of Arid Environments 111 (2014) 91e97


Fig. 2. a) Point estimates (dots) and 95% confidence intervals (lines) estimating site-to-site differences in the effects of soil sterilization on Bromus grown alone. Confidence intervals that do not overlap zero (dashed line) indicate sites that differed from the mean of all sites in terms of the effect of soil sterilization on Bromus biomass. b) Aboveground biomass (solid bars shown with standard errors) of Bromus grown alone, with unsterilized or sterilized soil from each site. Asterisks indicate biomass was substantially different between paired bars, i.e., 95% confidence intervals of the relative effect of sterilization (not shown) did not overlap zero.

biomass by producing hormones such as auxin, which stimulates root growth (Lugtenberg and Kamilova, 2009). Another important consideration is that plant responses to soil biota are not uniform over the life of plants. Past research has shown that feedback responses of native grasses tend to become more negative over time as deleterious soil microbes accumulate, while this may not be true for non-native congeners (Hawkes et al., 2012). While our results suggest positive effects of soil biota on both a native and a nonnative grass, our experiment was conducted over a 2-month period and the response of Bouteloua to soil microbes may have become negative over a longer time period. In addition, our soil preparation methods may have favored heat-tolerant microbes, particularly fungi, and we did not attempt to identify which taxa or functional groups of soil biota may have affected Bromus and Bouteloua or the mechanism(s) underlying the positive responses to unsterilized soils. An alternative explanation is that plants may have responded to soil changes imposed by autoclaving (Perkins et al., 2013) despite steps taken to minimize these effects. Soil sterilization had effects on soil CEC and pH. Increased CEC enables the soil to retain more

nutrients, but may have somewhat reduced plant-available nutrients. However, levels of the nutrients we assessed did not change substantially with sterilization. Decreased pH may have affected nutrient availability as well; however, the effect of sterilization on pH was relatively small, particularly in comparison to the level of variation across sites. 4.2. Soil sterilization altered competition between Bouteloua gracilis and Bromus tectorum Both species had greater biomass in unsterilized soil, but in absolute terms Bromus size increased to a greater degree than Bouteloua. This likely conferred greater competitive ability to Bromus in unsterilized soil, lessening the effect of competition on Bromus and increasing the effect of competition on Bouteloua when soil was not sterilized. The influence of soil sterilization on competition may reflect one or more of several possible mechanisms. One possibility is that Bromus size increased to a greater degree than Bouteloua in response to soil microbes, conferring greater competitive ability to Bromus in unsterilized soil. If soil

Table 2 Growth medium properties in sterilized and unsterilized treatments. CEC ¼ Cation exchange capacity; averages are given ±standard error. Treatment


P ppm

K ppm

Mg ppm

Ca ppm

Na ppm

CEC meq/100 g

Unsterilized Sterilized

6.3 ± 0.2 6.0 ± 0.2

25 ± 2 30 ± 2

446 ± 20 391 ± 10

305 ± 21 304 ± 28

1847 ± 216 2000 ± 267

22 ± 1 20.5 ± 0.8

14.5 ± 0.9 16 ± 1


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microbes increased nutrient availability, then these results may reflect the demonstrated ability of Bromus to better exploit soil nutrients compared to native grasses such as Bouteloua (Lowe et al., 2003; Vasquez et al., 2008). Past research has shown that nonnative species are often able to rapidly use added or fluctuating resources, while native species are better able to compete with non-native species in nutrient-poor soils (Davis et al., 2000). In addition, changes in soil pH and CEC imposed by autoclaving may have also affected nutrient dynamics. Competition between Bromus and Bouteloua could have shifted in response to these abiotic effects as well as in response to microbes. We noted that Bouteloua had a lower survival rate than Bromus (62% compared to 93% across all treatments); had Bouteloua survival been higher, the effect of competition on Bromus may have been stronger overall. However, Bouteloua mortality was not affected by soil sterilization or interactions between soil sterilization and other treatments (results not shown), so this does not explain differences between competition in sterilized and unsterilized soils. If other non-nutritive mechanisms of increasing biomass discussed in the previous section (e.g., growth stimulation) had a stronger positive effect on Bromus than Bouteloua, this could also explain how microbes increased Bromus size and thus competitive ability. In addition, because Bromus can alter microbial community composition as a dynamic process during growth (Schaeffer et al., 2012), Bromus may have changed microbial communities in unsterilized soils in our experiment, thereby reducing Bouteloua growth indirectly. 4.3. Response of Bromus tectorum to soil sterilization varies appreciably by site Bromus biomass varied by site of soil origin, as did the response of Bromus to soil sterilization (Fig. 2). Site-to-site variation in the effect of soil sterilization on Bromus (i.e., the difference in biomass between sterilized and unsterilized soils) may reflect differences in taxa (or abundances) of certain soil mutualists or pathogens that had stronger effects on Bromus than Bouteloua. Abiotic conditions such as climate and soil pH, and biotic factors such as plant taxa, are known to shape soil microbial communities (Fierer and Jackson, 2006; Kivlin et al., 2011). It is uncertain which environmental factors were most important in determining Bromus response to soil sterilization across sites in our study. However, regardless of the source of the variability, our results indicate that Bromus is more sensitive to it than Bouteloua in terms of aboveground biomass response. Variation in Bromus biomass among sites when soils were sterilized likely reflects differences in soil properties that were not fully compensated for by diluting the soil with PROFILE® and applying fertilizer. Site of soil origin affected chemical properties of the growth media such as CEC, S, Ca, Mg, and pH. The effects of site on soil properties tended to be larger than the effect of soil sterilization. 5. Conclusions Our results support prior work showing that invasive species such as Bromus often have a positive (or non-negative) response to soil biota in their invasive range (Inderjit and van der Putten, 2010; Perkins and Nowak, 2013). Conversely, our hypothesis that the native grass Bouteloua would respond negatively to conspecific soil microbes was not supported e this species also responded positively to unsterilized soil. The increase in biomass of both species in unsterilized soils could be due to microbial-mediated increases in nutrient availability, non-nutritive effects of soil microbes such as protection from pathogens or stimulation of plant growth, effects of autoclaving on some abiotic soil properties, or a combination of factors.

Soil sterilization affected competition between the invasive grass Bromus tectorum and the native grass Bouteloua gracilis. When soil was sterilized, Bromus biomass was reduced by competition from Bouteloua, but in unsterilized soil Bromus was not substantially affected by Bouteloua. This implies that soil microbes may facilitate Bromus growth and competition in some instances, and may contribute in part to the invasiveness of this species. In addition, our findings show that Bromus responded differently to sterilization of soil from different sites, while there was no appreciable effect of site on the response of Bouteloua. The microbial community composition may have varied across these sites in a way that strongly affected Bromus, but not Bouteloua, possibly due to a longer history of Bouteloua presence at these sites. Bromus growth may be facilitated by the microbial community at some sites, and Bouteloua may not be able to compete as effectively with Bromus at these sites as a result. However, because we did not examine the composition of the soil microbial community, we cannot say for certain that microbes alone were driving this effect. Bromus biomass differed by site of soil origin in sterilized soils as well, likely due to differences among sites in soil properties such as pH, CEC, Ca, Mg, and S. Plant biomass responses to soil biota are likely to be influenced by many factors, such as environmental conditions and abiotic soil characteristics which shape the microbial community. Responses to soil sterilization in one context (e.g., using soil from one site or one greenhouse conditioning environment) may not accurately represent responses under other conditions, and this should be taken into account when studying relationships between plants and soil biota. In addition, soil sterilization and site of soil origin may affect abiotic soil properties even after diluting soil in other growth media; in our study variation in soil properties was greater across sites than between sterilized and unsterilized soil treatments. While soil microbial communities are clearly important for plant growth and competitive outcomes, other soil characteristics may play roles as well. Determining the importance of microbial communities on invasion dynamics requires an approach that makes distinctions between the effect of experimental methods and the effect of soil biota themselves. Our data suggest soil microbes play a role in interactions between Bromus and Bouteloua; however, the soil microbial community is just one of many factors that can influence plant growth and invasiveness. Acknowledgments This material is based upon work supported by the National Science Foundation Graduate Research Fellowship Program under Grant No. 1148897. We thank Valerie Eviner, Lora Perkins, Kevin Rice, Kate Scow, and four anonymous reviewers for comments on previous versions of this work; and Lian Rother, James Mizoguchi, Kao Saelee, Maureen O'Mara, Darcy Hammond, Ming-Yu Stephens, Annalisa Bryant, Bruce Moffat, Bob Haynes, and Brian Kozar for research assistance. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture or the National Science Foundation. Appendix A. Supplementary data Supplementary data related to this article can be found at http:// References Allen, M., Allen, E., Stahl, P., 1984. Differential niche response of Bouteloua gracilis and Pascopyrum smithii to VA mycorrhizae. Bull. Torrey Bot. Club 111, 361e365.

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