Root herbivores, pathogenic fungi, and competition between Centaureamaculosa and Festuca idahoensis

Wendy L. Ridenour and Ragan M. Callaway*Division of Biological Sciences, University of Montana, Missoula, Montana 59812, USA; *Author forcorrespondence (e-mail: callaway@selway.umt.edu; phone: 406–243-5077; fax: 406–243-4184)

Received 9 October 2001; accepted in revised form 21 June 2002

Key words: Agapeta zoegana, Allelopathy, Biological control, Centaurea maculosa, Compensatory growth, Com-petition, Festuca idahoensis, Indirect interactions, Interference, Sclerotinia sclerotiorum

Abstract

We used a common garden experiment to evaluate the isolated and combined effects of a biocontrol agent, theinsect (Agapeta zoegana, Lepidoptera), and a native North American fungal pathogen (Sclerotinia sclerotiorum)on competition between the noxious weed Centaurea maculosa and the native Festuca idahoensis. In 0.5-m2

plots with 24 plants per plot, competition between Centaurea and Festuca was highly asymmetrical, with Cen-taurea strongly reducing the final biomass and reproduction of Festuca, and Festuca having no effect, or possi-bly a positive effect, on Centaurea. The direct effects of the biological control agents differed entirely. AllCentaurea individuals died in plots receiving Sclerotinia, but Agapeta did not significantly reduce the growth ofCentaurea, and apparently stimulated a compensatory reproductive response in the weed. Individual Centaureaplants that had been damaged by Agapeta produced more flowerheads, and the number of Centaurea plants withAgapeta root damage in a plot was positively correlated with total Centaurea biomass. These differences in thedirect effects of the consumers were reflected in their indirect effects. In plots where Sclerotinia killed Centaurea(strong direct effects) Festuca growth and reproduction was equal to that in Festuca plots without Centaurea andthe reproductive output of Festuca increased substantially (strong indirect effects). However, in the absence ofSclerotinia, the application of Agapeta did not significantly decrease Centaurea biomass (weak direct effects)and actually stimulated small, but significant decreases in Festuca reproduction and trends towards lower Fes-tuca biomass (weak and opposite indirect effects – Agapeta does not eat Festuca). If the direct effects of bio-control agents on Centaurea are weak, as suggested by our results, natives are unlikely to be released from thecompetitive effects of Centaurea, and natives may suffer from Centaurea’s compensatory response to herbivory.

Introduction

A large body of research has demonstrated that her-bivory can alter interactions among plants (Louda etal. 1990; Clay 1990; Crawley 1992). Plants experi-encing herbivory are typically at a competitive disad-vantage, an observation which provides the theoreti-cal rationale for importing large numbers of exoticinsect herbivores to control exotic, highly competitiveinvasive plants. However, there have been relativelyfew controlled experimental studies of the effects ofconsumers on competition between exotic and nativeplants in general (Clay 1990; Clay et al. 1993; Calla-

way et al. 1999), and most studies of the effects ofbiological control agents on the growth of their targetspecies or inter-plant interactions have focused on asingle biocontrol species. In natural and biocontrol-weed systems, however, the additive effects of mul-tiple consumers can be much greater than their iso-lated effects (Charudattan 1986; Berlow 1999), andthe assumption that multiple biocontrol agents can dowhat one agent cannot is widespread (Harris 1984;Goeden and Andrés 1999). However, the effects ofmultiple species cannot always be predicted frompaired experiments (Adler and Morris 1994; Kareiva1994; Wootton 1994). Van der Putten et al. (2001)

161Plant Ecology 169: 161–170, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

have emphasized the importance of linking mul-titrophic interactions of plants, herbivores, and patho-gens in order to understand community process.

Intermountain grassland of western Montana is agood system in which to investigate the direct and in-direct effects of biocontrols. A diverse native floraexists, but intermountain grassland has been exten-sively invaded by exotic perennials, including Cen-taurea maculosa Lam. which was introduced intoNorth America from Eurasia (Müller-Schärer andSchroeder 1993; Sheley et al. 1998). Centaurea mac-ulosa is among the most widespread and destructivegrassland invaders in the Western United States andCanada (Tyser and Key 1989; Griffith and Lucey1991; Sheley and Jacobs 1997). The negative effectsof Centaurea species on native plants are well docu-mented (Muir and Majak 1983; Rice et al. 1992; Les-ica and Shelley 1996). Near our experimental site C.maculosa appears to have reduced the diversity ofnative grassland species by more than 90% (Ridenourand Callaway 2001). Centaurea maculosa and theclosely related C. diffusa may suppress natives viaallelopathic effects (Muir and Majak 1983; Callawayand Aschehoug 2000; Ridenour and Callaway 2001),and competition for resources (Callaway and Asche-houg 2000). Despite more than 50 years of chemicalcontrol efforts and biological control introductionsstarting in 1970 (the seed attacking Urophora affınis)C. maculosa had spread onto > 2.5 million hectaresin the Northwest by the mid-1980’s (Chicoine et al.1985) and the number of counties in the region re-porting C. maculosa increased from 39 in 1974 to 133in 1994 (Rice 1994).

At least 12 insect species have now been intro-duced to North America from Eurasia in an effort toincrease “cumulative stress” (i.e. Harris (1984)) andreduce the competitive effects of C. maculosa onNorth American natives (Sheley et al. 1998). Addi-tionally, a fungus species native to the intermountainprairie, Sclerotinia sclerotiorum, has been shown todamage C. maculosa and benefit native Pseudoroeg-neria spicata grasses (Jacobs et al. 1996). Therefore,the intermountain prairie has native competitors, na-tive pathogens that damage Centaurea, and intro-duced insect herbivores that may interact with Cen-taurea in complex ways. Studies of complexinteractions among C. maculosa, competitors, andconsumers in its native range in Europe have pro-vided valuable insight into the natural ecology of thisspecies (Müller 1989; Müller-Schärer 1991; Müllerand Steinger 1990; Steinger and Müller-Schärer 1992;

Weiner et al. 1997). However, these studies providelittle evidence that C. maculosa is strongly limited byherbivory in its natural range. Quantitative studies ofinteractions in invaded North American communitiesare crucial to formulating realistic evaluations andexpectations for controlling Centaurea species in in-termountain prairie.

Here we report on the results from a common gar-den experiment (see also Callaway et al. (1999)) de-signed to study: 1) the direct effects of C. maculosaon F. idahoensis, 2) the isolated and combined indi-rect effects of Sclerotinia and Agapeta on F. idahoe-nsis, and 3) the isolated and combined direct effectsof two consumers (the fungus Sclerotinia sclerotio-rum and Agapeta zoegana, Lepidoptera) and one na-tive competitor (Festuca idahoensis) on C. maculosa.

Methods

We conducted the common garden experiment at TheUniversity of Montana Diettert Experimental Gar-dens. These gardens occupy land once covered by in-termountain grassland, and are near natural inter-mountain grasslands that have been heavily invadedby C. maculosa. Centaurea maculosa and F. idahoe-nsis plants were started from seed in March 1994 in agreenhouse and transplanted into the garden plots inMay 1994. The experimental design consisted of sixtreatments, each of which was replicated ten times fora total of 60 0.25-m2 plots that were randomly locatedwith respect to each other in the experimental area(see Callaway et al. (1999)). Treatments were: 1)Centaurea alone, no consumers, 2) Festuca alone, noconsumers 3) Centaurea with Festuca, no consumers4) Centaurea with Festuca and Agapeta, 5) Centau-rea with Festuca and Sclerotinia, and 6) Centaureawith Festuca, Agapeta, and Sclerotinia. Each plot was0.50 m from any other plot, and within each plot, in-dividual plants were located 10 cm from all neigh-bors. Twenty-four individuals were grown in eachplot. In “control” (1 & 2) plots, all 24 individualswere either C. maculosa or F. idahoensis. In “treat-ment” (3,4,5, & 6) plots, 12 Centaurea and 12 Fes-tuca were planted such that individual plants alter-nated by species in rows and columns of a grid. Thisdesign permitted two scales at which we could mea-sure treatment effects; that of the individual plant, andthat of the combined 12 target plants in the entire0.25-m2 plot (reported as plot biomass). In otherwords, because the two “control” monoculture plots

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had 24 conspecific individuals we only weighed thecombined biomass of the 12 individuals that occurredin the same locations within the plots as their 12 con-specifics in the plots with Centaurea and Festucaplanted together. This standardized all measuredplants so that they had the same number of neighbors.Biomass in the 0.25 m2 plots was converted to g/m2.

To investigate the effects of treatments on soil wa-ter availability, PVC monitoring tubes were installed30-cm deep in the center of each of the 60 gardenplots. Soil moisture was measured using FrequencyDomain Reflectometry (Troxler, Sentry 200-AP) onceper week at 15-cm and 30-cm depths from April tolate October 1994 and April to late October 1995.

Two biocontrol treatments were applied, both sin-gly and in combination. These biocontrols were thelepidopteran root-mining herbivore Agapeta zoeganaand the fungal pathogen Sclerotinia sclerotiorum. Ag-apeta was introduced into the United States from Eur-asia, where both Agapeta and C. maculosa are native,for the biocontrol of Centaurea species. Agapeta ishighly host specific to several closely related speciesof Centaurea. Adult insects deposit their eggs at theroot-shoot interface of Centaurea plants, and the lar-vae mine Centaurea taproots. We acquired Agapetafrom the Montana State University Agricultural Ex-perimental Station in Corvallis, Montana. In August1994, twenty plots with mixtures of Centaurea andFestuca were treated with Agapeta to determinewhether or not root herbivory on Centaurea wouldshift the balance of competition in favor of Festuca.After a period of plant establishment and growth,fine-mesh cages were placed over all plots and threeadult Agapeta were introduced into each of the tenAgapeta treatment plots and the ten Agapeta-Sclero-tinia plots. Cages were placed over the other 40 plotsthat were not treated with Agapeta to control for theeffects of caging. After ten days, the time allowed forAgapeta egg-laying, cages were removed from allplots.

Sclerotinia is a soil-borne fungus native to theNorthern Rocky Mountains and was acquired fromDavid Sands, Montana State University. In August,two days after applying Agapeta, ten of the plots thatwere planted with mixtures of Centaurea and Festucaand treated with Agapeta were also infected withSclerotinia by applying 2 cm3 of Sclerotinia-infectedgrain to the base of each Centaurea stem, and tenother plots were treated with Sclerotinia without Ag-apeta. Several hours before applying Sclerotinia,plots were watered manually to promote establish-

ment, and plots were watered for several days after-wards.

In September of 1995, volumetric measurements(maximum height, basal diameter, and crown diam-eter) and number of green leaves (fall green-up) of thefour Festuca plants nearest the center in each plotwere collected. At the scale of individuals, we non-destructively measured the growth of the four interiorF. idahoensis and C. maculosa individuals, thus con-trolling for the number of interspecific neighbors,edge effect, and intraspecific density. At the whole-plot scale, all plots were harvested at the end of thegrowing season in September 1995, and abovegroundbiomass was dried at 60 °C and weighed. Althoughwe did not measure belowground biomass because ofdifficulty inherent to collecting the fine fragile rootsof Centaurea, we pulled out the taproots of Centau-rea, measured their diameters and searched them forsigns of Agapeta damage. We also counted the num-ber of total Festuca florets on the 12 target individu-als per plot (see first paragraph of methods) and Cen-taurea flowerheads per individual and total on the 12target individuals per plot to quantify reproductiveoutput.

Results

The total aboveground biomass of the 12 target indi-viduals in the Festuca control plots was more thantwo times greater than conspecifics in the Centaurea× Festuca treatment plots (One-way ANOVA, Ftreat-

ment = 4.32, df = 5,59, P = 0.003, post-ANOVA TukeyHSD, P < 0.01), (Figure 1). Festuca floret productionwas 10–20 times greater in Festuca control plots thanin Centaurea × Festuca treatment plots (One-wayANOVA, Ftreatment = 8.34, df = 5,59, P < 0.001 post-ANOVA Tukey HSD, P < 0.001; Figure 2). The in-terfering effect of Centaurea on Festuca, however,did not appear to be caused by decreased soil water(see Callaway et al. (1999)). Soil moisture content at15 and 30 cm depths did not vary significantly be-tween treatments throughout the summer in eitheryear (Repeated-measures ANOVA, Ftreatment × time at15 cm = 0.93, df = 5,59, P = 0.606; Ftreatment × timeat 30 cm = 1.04, df = 5,59, P = 0.412). Likewise, theinterfering effect of Centaurea on Festuca was notassociated with reduced levels of soil nutrients. Avail-able soil nitrate, ammonium and phosphorous did notvary significantly between treatments during the firstyear of the study (One-way ANOVA, NO3: Ftreatment

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= 0.511, df = 5,59, P = 0.767; NH4: Ftreatment = 0.446,df = 5,58, P = 0.814; P: Ftreatment = 1.20, df = 5,59, P= 0.321). In contrast to the strong negative effect ofCentaurea on Festuca, Centaurea plants grown withFestuca competitors were almost 4 times larger thanCentaurea’s grown with conspecific competitors (Fig-ure 3).

The direct effects of the two biocontrol agents dif-fered markedly (Figure 3). All of the Centaurea plantsin both Sclerotinia treatments (Centaurea with Fes-tuca with Sclerotinia and Centaurea with Festucawith Agapeta and Sclerotinia) died within two weeksof application. However, Centaurea biomass in Cen-taurea x Festuca with Agapeta treatments was notless than when the biocontrol larvae were absent (Fig-ure 3). Examination of the taproots of the 12 focalCentaurea plants in the ten plots with Centaurea, Fes-tuca, and Agapeta found that a mean of 4.0 ± 0.7 (1s.e.) Centaurea individuals (30%) had signs of rootdamage.

Elimination of Centaurea by Sclerotinia hadstrong positive effects on the growth of Festuca (Fig-ures 1 and 2). By October 1994, only two months af-ter application of the fungal pathogen, individual Fes-tuca plants in the Sclerotinia treatment plots hadsimilar basal diameters and heights as those in theFestuca controls (data not shown). By October 1995,aboveground Festuca biomass in the Sclerotinia treat-

ment plots was 50–100% greater, and floret produc-tion 100% greater, than that of the total biomass ofthe comparable 12 focal individuals in the Festucacontrol plots where conspecific competition was moreintense (Figures 1 and 2). Festuca plots were not

Figure 1. Above-ground biomass of Festuca idahoensis in a com-mon garden experiment either in a matrix of conspecifics, a matrixof Centaurea maculosa, and for the interspecific matrix with thebiocontrols Agapeta zoegana and Sclerotinia sclerotiorum appliedalone or in combination. For the conspecific matrix we only in-cluded the biomass of the 12 individuals that were in the same lo-cations occupied by F. idahoensis in the interspecific matrices. Er-ror bars represent one standard error, and letters designate meansthat were significantly different in post-ANOVA Tukey HSD tests,P < 0.05).

Figure 2. Floret production of Festuca idahoensis in a commongarden experiment either in a matrix of conspecifics, a matrix ofCentaurea maculosa, and for the interspecific matrix with the bio-controls Agapeta zoegana and Sclerotinia sclerotiorum appliedalone or in combination. For the conspecific matrix we only in-cluded the biomass of the 12 individuals that were in the same lo-cations occupied by F. idahoensis in the interspecific matrices. Er-ror bars represent one standard error, and letters designate meansthat were significantly different in post-ANOVA Tukey HSD tests,P < 0.05).

Figure 3. Above-ground biomass of Centaurea maculosa in acommon garden experiment either in a matrix of conspecifics, amatrix of Festuca idahoensis, and for the interspecific matrix withthe biocontrols Agapeta zoegana and Sclerotinia sclerotiorum ap-plied alone or in combination. For the conspecific matrices we onlyincluded the biomass of the 12 individuals that were in the samelocations occupied by C. maculosa in the interspecific matrices.Error bars represent one standard error, and letters designate meansthat were significantly different in post-ANOVA Tukey HSD tests,P < 0.05).

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more productive when Agapeta were applied to Cen-taurea neighbors, and may have actually been hurt bythe response of Centaurea to mild root herbivory. Thetotal number of Festuca florets on the 12 target plantsdecreased significantly in plots where Agapeta wasapplied to Centaurea (post-ANOVA Tukey HSD, P =0.038, Figure 2). A similar trend, although not signif-icant, was also seen for Festuca biomass (Figure 1).The negative response of Festuca to herbivory onCentaurea may have been due to a compensatory re-sponse by Centaurea. Root diameters of individualCentaurea plants with Agapeta damage were largerthan those of plants without root damage, and moreimportantly, flower production per unit of root diam-eter on damaged individuals was almost twice that ofundamaged individuals (Figure 4). For the ten plotswith Centaurea, Festuca and Agapeta, Centaurea bio-mass was positively correlated with the number ofCentaurea plants that had been damaged by Agapeta(Figure 5). Neither Festuca biomass nor floret num-ber was significantly correlated with the number ofCentaurea plants with Agapeta damage (rbiomass =−0.11, P = 0.32; rfloret = −0.22, P = 0.14, data notshown).

Discussion

The fungal pathogen Sclerotinia eliminated Centau-rea maculosa from experimental plots and substan-tially increased the biomass and reproduction of Fes-tuca idahoensis. In contrast to the effect ofSclerotinia, root herbivory by Agapeta during the rel-atively short time period of our experiment did nothave strong negative direct effects on Centaurea, anddid not reduce the aboveground biomass of C. macu-losa. In fact, for a similar root diameter, individualswith Agapeta damage had greater flowerhead produc-tion, suggesting reproductive over-compensation(Paige and Whitham 1987) and not simply Agapetachoosing larger plants. Furthermore, plots with highernumbers of Centaurea plants with Agapeta damagealso had higher total aboveground biomass. These in-creases in Centaurea flower production and plot bio-mass with Agapeta damage suggest either a compen-satory response or a shift in allocation with herbivory-induced stress. However, because of the experimentaldesign we cannot eliminate the possibility that thedifference was due to the indirect effects of Festuca.The root diameters of damaged plants were also sig-nificantly larger than those of undamaged plants;however, this may have been due to the preference ofAgapeta for larger host plants (see Story et al.(2000)).

Figure 4. The relationship between root diameter and flower num-ber for individual Centaurea maculosa plants in the ten plots withCentaurea, Festuca, and Agapeta. The upper regression line repre-sents individuals with signs of root damage and the lower regres-sion line represents individuals with no sign of root damage. Thelarge symbols represent the means and 95% confidence limits forboth root diameter (X axis) and flower number (Y axis). For indi-viduals with no root damage, r2 = 0.17, P = 0.006; for individualswith root damage, r2 = 0.11, P = 0.003.

Figure 5. Biomass of the 12 focal Centaurea maculosa individu-als as a function of the percent of the total number of Centaurea(24) plants damaged by Agapeta zoegana. P < 0.05.

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A number of other studies support our finding thatC. maculosa has a remarkable capacity to tolerateherbivory and defoliation, and under some conditions,may overcompensate (i.e. grow larger or reproducemore after herbivory [Paige and Whitham (1987) andBelsky et al. (1993), Trumble et al. (1993)]). Müller(1989) found that C. maculosa plants of German andCanadian origin increased fine root growth when in-fected by Agapeta and did not decrease in fecundity.Steinger and Müller-Schärer (1992) found that thebiomass of Centaurea maculosa seedlings grown inpots were not affected by Agapeta feeding, and attrib-uted the lack of effect to compensatory root growth.In other experiments, however, the root feeding wee-vil, Cyphocleonus achates reduced whole-plant bio-mass. In field experiments in Switzerland, Müller-Schärer (1991) found that low levels of Agapetaherbivory increased survival, shoot number, and fe-cundity of Centaurea maculosa, but the effects of her-bivory were highly complex and were negative underother conditions. Callaway et al. (1999) reported (inaddition to other analyses of some of the same fieldexperiments reported here) that Centaurea plants ex-periencing leaf herbivory from Trichoplusia ni (cab-bage looper) were stronger competitors against Fes-tuca idahoensis. Kennett et al. (1992) found thatdefoliation of potted C. maculosa (up to 4 times in� 6 months and up to intensities of 75% of theleaves) had no effect on the final biomass of the de-foliated plants. However, leaf defoliation decreasedC. maculosa carbohydrate concentrations and poolssubstantially (Lacey et al. 1994).

In our experimental conditions the effects of Aga-peta on Centaurea were weak. However, repeated in-fections over a longer period of time, higher infectionlevels on individual plants, or more stressful abioticconditions might result in completely different out-comes. Stronger doses of Agapeta, either through theeventual buildup of populations or local adaptation,would be expected to have stronger effects. However,the proportion of C. maculosa plants infested withAgapeta (30%) was within the range of proportionsfound in invasive populations in North America andnatural populations in Europe. The infestation level inour experiment was also comparable to that at anAgapeta release site after 6–7 years (31.8%; Story etal. (2000)), and higher than that reported in somenatural populations of C. maculosa in eastern Europe(15–36%; Müller et al. (1988)). Virtually all ecologi-cal interactions are conditional, or context-specific,and conditional effects of biological controls should

be expected. However, we should not exclude thepossibility that the effects of Agapeta may be evenweaker in some conditions.

Our contrasting effects of Sclerotinia and Agapetaare confounded by different application intensities.We were able to apply substantial doses of Sclerotiniato Centaurea, but as noted only 30% of the Centau-rea plants in Agapeta treatments were infected.Therefore a strict quantitative comparison is not jus-tified, but 100% mortality for Sclerotinia compared to0% mortality versus no significant effect of Agapetaon Centaurea growth does not require a strict quan-titative comparison. Although little is known aboutthe natural distribution of Sclerotinia in the field, ourapplications were undoubtedly far higher than Cen-taurea would experience in nature. Correspondingly,the efficacy of Sclerotinia as a biological controlagent has been highly limited by the spread of thefungus and difficulties inherent to delivering a lethaldose.

Story et al. (2000) compared C. maculosa biomassand reproduction at one field site where Agapeta hadbeen released to one site where they had not been re-leased. They found that Centaurea plants at releasesites were smaller and had fewer flowers, but therewere no data collected prior to Agapeta release. Fur-thermore, Agapeta were not excluded from the con-trol site and by the final sampling date the percentagesof Centaurea plants infested by Agapeta did not dif-fer between the sites. Interestingly, individual Cen-taurea plants that were infested with Agapeta had40% more flowerheads and 112% more abovegroundbiomass, a pattern that was attributed to the prefer-ence of Agapeta for larger plants. Our experiments(Figure 4), however, indicate that even when the size(root diameter) of Centaurea is accounted for, dam-age by Agapeta increases flower production substan-tially. Such compensatory growth may explain theslightly higher competitive effects of C. maculosa onF. idahoensis in the Agapeta treatment (also see Cal-laway et al. (1999)).

The slightly higher competitive effects of C. mac-ulosa on F. idahoensis in the Agapeta treatment weresignificant, but small in magnitude. Therefore, as abiological process the higher competitive effects afterherbivory appear to be of little consequence relativeto the competitive strength of C. maculosa in general.However, understanding the mechanisms behind sucha counterintuitive response may provide new insightinto complex interactions among plants and herbi-vores. Slightly higher competitive effects may have

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been due to compensatory resource uptake, or an in-duction of greater production of defensive secondarymetabolites that also functioned as allelopathic chem-icals. Centaurea maculosa contains the chemical cni-cin, a sesquiterpene lactone that has been implicatedin both anti-herbivore and allelopathic interactions(Kelsey and Locken 1987; Landau et al. 1994), but toour knowledge there is no evidence that cnicin de-fenses are inducible. Others have found dual anti-her-bivore/allelopathic roles in inducible plant metabo-lites, which increase under stress (Lovett and Holt(1995) and Tang et al. (1995), Siemens et al., in re-view). Root observation chamber experiments suggestthat C. maculosa may be allelopathic (Ridenour andCallaway 2001). In these experiments Festuca rootsgrew more slowly near Centaurea roots than whennear conspecific roots, and activated carbon, whichabsorbs organic compounds, reduced the negative ef-fects of Centaurea roots. This indicates that Centau-rea may be allelopathically interfering with its neigh-bors. Other studies indicate that C. maculosa and C.diffusa outcompete North American natives for re-sources (Callaway and Aschehoug 2000), but in ourexperiments the interfering effect of C. maculosa onF. idahoensis was not clearly manifest through deple-tion of soil resources.

A third hypothesis for Centaurea’s compensatoryresponse to herbivory and slightly higher competitiveeffects on Festuca involves mycorrhizae. Centaureamaculosa and other Centaurea species may benefitfrom a form of mycorrhizae-mediated parasitismthrough common mycorrhizal networks (Grime et al.1987; Marler et al. 1999; Carey and Callaway (1999,1999)) or a shift in the relative abundance of mutual-istic and pathogenic fungi in the presence of Festucaso that Centaurea is favored (Callaway et al. (inpress)). Carey and Callaway (1999) found that thestable carbon isotope concentration of C. maculosashoot tissue was significantly more similar to that ofF. idahoensis in the presence of mycorrhizae thanwithout mycorrhizae, indicating that carbon wastransferred from the Festuca to the Centaurea. In con-trast, experiments conducted by Zabinski et al. (un-published) suggest that AM-mediated neighbor ef-fects are the result of mycorrhizal networks thatincrease species’ access to phosphorus. In general, therole of carbon transfer among plants via AM fungiremains controversial (Robinson and Fitter 1999).

In contrast to the weak competitive effect of theNorth American native Festuca idahoensis, Müller-Schärer (1991) found that competition from the Eu-

ropean native, Festuca pratensis, was the single mostimportant factor influencing the success of Centaureamaculosa in field experiments, and the most impor-tant factor influencing the effect of Agapeta herbivoryon Centaurea. He also found that “in the absence ofgrass competition, Agapeta herbivory showed no sig-nificant impact on plant height, biomass, and fecun-dity”. The finding that Festuca pratensis, commonlyassociated with Centaurea in Europe, is a much stron-ger competitor with Centaurea than with F. idahoen-sis, may explain some of the contradictory results be-tween our study and Müller-Schärer’s and suggeststhat escape from natural consumers (the theoreticalbasis for biocontrols) is not the only mechanism driv-ing the success of this weed in North America. Fur-ther support for the role of plant communities them-selves in determining the success of invaders waspublished by Callaway and Aschehoug (2000) whofound that Eurasian grass species had much strongercompetitive effects on Centaurea diffusa, a close rela-tive of C. maculosa, than closely related North Amer-ican grass species. These differences appeared to bedue to different responses to allelopathic root exu-dates from C. diffusa. Their results indicated that thesuccess of some invasive exotics might not be dueonly to escaping consumers, but also to novel com-petitive mechanisms not previously experienced byindigenous species. In a review of studies of the ef-fects of natural enemies and competitors, Sheppard(1996) reported that the dominant factor in 10 of 12studies in natural grasslands was competition. How-ever, unlike we observed for C. maculosa, most ef-fects of competitors and natural enemies were “mul-tiplicative”; each factor had an impact but without aninteraction.

Classical biological control can be defined as theintroduction of enemies of invasive, exotic plants inorder to control their spread (Harris 1991). Many bi-ological control efforts involve the release of one bi-ological control agent (usually an insect herbivore,Wurtz (1995) and Julien (1992)), and some of thesehave been successful (Huffaker et al. 1961; Cullen etal. 1973; Kok and Surles 1975). Our study clearlydemonstrates the efficacy of a single fungal pathogen.However, species abundances are often regulated byfactors other than consumers. Even though our exper-iments found exceptionally strong negative direct andpositive indirect effects of Sclerotinia on a nativegrass, Agapeta had weak to positive (compensatoryresponse) direct effects and negative indirect effects.These results are not desired of a biocontrol and em-

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phasize the importance of achieving strong negativeeffects of biocontrols on target species in order toachieve strong indirect positive effects of biocontrolson native plants. In the case of Centaurea maculosa,other researchers have shown that large reductions inthe weed’s abundance are required to shift the balanceof competition in favor of native grasses (Sheley andJacobs 1997), which corroborates our finding of weakbiocontrol effects. Considered together, the compen-satory responses of C. maculosa to biocontrol her-bivory and defoliation shown in this study and in anumber of others, and the ability of C. maculosa tooutcompete native grasses even after large reductionsin its abundance, suggest that criteria applied prior tointroducing biological control species for this weedshould include convincing experimental evidence thatthe biocontrols will have strong direct effects on theirhosts.

Acknowledgements

We thank Jim Story for providing Agapeta and DavidSands for providing Sclerotinia. Many thanks are dueto Steve Baker and Jim Plummer for their valuableassistance with soil nutrient analysis and contributionof laboratory space; Jennifer Costich, Michael Wojdy-lak, Paul Ridenour, Todd Wojtowicz, Erik Aschehougand Raven Stevens for their assistance during thecourse of the research; and Dr Colin Henderson forhelpful discussions, especially regarding statisticalanalysis. This study was funded by The University ofMontana Grant Program, a grant to Ragan Callawayand Cathy Zabinski from the National Science Foun-dation, DEB-9726829, and a grant to Ragan Callawayfrom the Andrew W. Mellon Foundation.

References

Adler F.R. and Morris W.F. 1994. A general test for interactionmodification. Ecology 75: 1552–1559.

Belsky A.J., Carlson W.P., Jensen C.L. and Fox G.A. 1993. Over-compensation by plants - herbivore optimization or red herring?Evolutionary Ecology 7: 109–121.

Berlow E.L. 1999. Strong effects of weak interactions in ecologi-cal communities. Nature 398: 330–334.

Binkley D. and Vitousek P. 1991. Soil nutrient availability. In:Pearcy R.W., Ehleringer J.R., Mooney H.A. and Rundel P.W.(eds), Plant Physiological Ecology. Chapman and Hall, Lon-don, pp. 75–96.

Callaway R.M. and Aschehoug E.T. 2000. Invasive plants versustheir new and old neighbors: a mechanism for exotic invasion.Science 290: 521–523.

Callaway R.M., DeLuca T. and Belliveau W.M. 1999. Herbivoresused for biological control may increase the competitive abilityof the noxious weed Centaurea maculosa. Ecology 80: 1196–1201.

Callaway R.M., Newingham B., Zabinski C.A. and Mahall B.E.Compensatory growth and competitive ability of and invasiveweed are enhanced by soil fungi and native neighbors. EcologyLetters (in press).

Carey E.V. and Callaway R.M. 1999. Stable isotopes provide evi-dence for carbon transfer from the invasive weed Centaureamaculosa to a native bunchgrass. Annual Meeting of the Eco-logical Society of America., Spokane, Washington, USA.

Charudattan R. 1986. Integrated control of water hyacinth (Eich-hornia crassipes) with a pathogen, insects, and herbicides.Weed Science 34: 26–30.

Chicoine T.K., Fay P.K. and Nielson G.A. 1985. Predicting weedmigration from soil and climate maps. Weed Science 34: 57–61.

Clay K. 1990. The impact of parasitic and mutualistic fungi oncompetitive interactions among plants. In: Grace J.B. and Til-man D. (eds), Perspectives on Plant Competition. AcademicPress Inc, San Diego, USA, pp. 391–412.

Clay K., Marks S. and Cheplick G.P. 1993. Effects of insect her-bivory and fungal endophyte infection on competitive interac-tions among grasses. Ecology 74: 1767–1777.

Crawley M.J. 1992. Natural Enemies: the Population Biology ofPredators, Parasites, and Diseases. Blackwell Science, Oxford,UK.

Cullen J.M., Kable P.F. and Catt M. 1973. Epidemic spread of arust imported for biological control. Nature 244: 462–464.

Goeden R.D. and Andrés L.A. 1999. Biological control of weedsin terrestrial and aquatic environments. In: Bellows T.S. andFisher T.W. (eds), Handbook of Biological Control. AcademicPress, New York, pp. 871–890.

Griffith D. and Lucey J.R. 1991. Economic evaluation of spottedknapweed (Centaurea maculosa) control using picloran. Jour-nal of Range Management 44: 43–47.

Grime J.P., Mackey J.M.L., Hillier S.H. and Read D.J. 1987. Flo-ristic diversity in a model system using experimental micro-cosms. Nature 328: 420–422.

Harris P. 1984. Current approaches to the biological control ofweeds. In: Kelleher J.S. and Hulme M.A. (eds), BiologicalControl Programmes Against Insects and Weeds in Canada1969–1980. Commonwealth Agricultural Bureau, FarnhamRoyal, England.

Harris P. 1991. Invitation paper (C.P. Alexander Fund): classicalbiocontrol of weeds: its definition, selection of effective agents,and administrative, political problems. Canadian Entomologist123: 827–849.

Huffaker C.B., Ricker D. and Kennett C. 1961. Biological controlof puncture vine with imported weevils. California Agriculture15: 11–12.

Jacobs J.S., Sheley R.L. and Maxwell B.D. 1996. Effect of Sclero-tina sclerotiorum on the interference between bluebunch wheat-grass (Agropyron spicatum) and spotted knapweed (Centaureamaculosa). Weed Technology 10: 13–21.

168

Julien M.H. 1992. Biological control of weeds: a world catalogueof agents. Commonwealth Agricultural Bureau International,Wallingford, Oxon, UK.

Kareiva P. 1994. Higher order interactions as a foil to reductionistecology. Ecology 75: 1527–1528.

Kelsey R.G. and Locken R.J. 1987. Phytotoxic properties of cni-cin, a sesquiterpene lactone from Centaurea maculosa (spottedknapweed). Journal Chemical Ecology 13: 19–33.

Kennett G.A., Lacey J.R., Butt C.A., Olson-Rutz K.M. and Hafer-kamp M.R. 1992. Effects of defoliation, shading and competi-tion on spotted knapweed and bluebunch wheatgrass. Journalof Range Management 45: 363–369.

Kok L.T. and Surles W.W. 1975. Successful biological control ofmusk thistle by an introduced weevil. Environmental Entomol-ogy 4: 1025–1027.

Lacey J.R., Olson-Rutz K.M., Haferkamp M.R. and Kennet G.A.1994. Effects of defoliation and competition on total nonstruc-tural carbohydrates of spotted knapweed. Journal of RangeManagement 47: 481–484.

Landau I., Müller-Schärer H. and Ward P.I. 1994. Influence of cni-cin, a sesquiterpene lactone of Centaurea maculosa (Asterace-ae) on specialist and generalist insect herbivores. JournalChemical Ecology 20: 929–942.

Lesica P. and Shelley J.S. 1996. Competitive effects of Centaureamaculosa on the population dynamics of Arabis fecundis. Bul-letin Torrey Botanical Club 123: 111–121.

Lovett J.V. and Holt A.C.H. 1995. Allelopathy and self-defense inbarley. In: Derjit K.M.M., Dakshini and Einhellig F.A. (eds),Allelopathy. American Chemical Society, Washington, DC,USA, pp. 170–183.

Louda S.M., Keeler K.H. and Holt R.D. 1990. Herbivore influenceson plant performance and competitive interactions. In: GraceJ.B. and Tilman D. (eds), Perspectives on Plant Competition.Academic Press Inc, San Diego, pp. 413–444.

Marler M.J., Zabinski C.A., and Callaway R.M. 1999. Mycorrhizaeindirectly enhance competitive effects of an invasive forb on anative bunchgrass. Ecology 80: 1180–1186.

Miller T.E. 1994. Direct and indirect species interactions in an earlyold-field plant community. The American Naturalist 143: 1007–1025.

Muir A.D. and Majak W. 1983. Allelopathic potential of diffuseknapweed (Centaurea diffusa) extracts. Canadian Journal ofPlant Science 63: 989–996.

Müller H. 1989. Growth pattern of diploid and tetraploid spottedknapweed, Centaurea maculosa Lam. (Compositae) and effectsof the root mining moth Agapeta zoegana (L.) (Lepidoptera-:Cochylidae). Weed Research 29: 103–111.

Müller H., Schroeder D. and Gassmann A. 1988. Agapeta zoegana(L.) (Lepidoptera:Cochylidae), a suitable prospect for biologi-cal control of spotted and diffuse knapweed, Centaurea macu-losa Monnet de la Marck and Centaurea diffusa Monnet de laMarck (Compositae) in North America. Canadian Entomologist120: 109–124.

Müller-Schärer H. 1991. The impact of root herbivory as a func-tion of plant density and competition: survival, growth and fe-cundity of Centaurea maculosa in field plots. Journal AppliedEcology 28: 759–776.

Müller-Schärer H. and Schroeder D. 1993. The biological controlof Centaurea spp. in North America: do insects solve the prob-lem? Pesticide Science 37: 343–353.

Müller H. and Steinger 1990. Separate and joint effects of rootherbivores, plant competition, and nitrogen shortage on re-source allocation and components of reproduction in Centau-rea maculosa (Compositae). In: Szentesi A. (ed.), Proceedingsof the 7th International Symposium on Insect-Plant relation-ships. Hungarian academy of Sciences., Budapest, pp. 215–224.

Paige K.N. and Whitham T.G. 1987. Overcompensation in responseto mammalian herbivory: the advantage of being eaten. Ameri-can Naturalist 129: 407–416.

Rice P.M., Bedunah D.J. and Carlson C.E. 1992. Plant communitydiversity after herbicide control of spotted knapweed. US For-est Service Res. Paper. USDA.

Rice P.M. 1994. Invaders Database. University of Montana, Mis-soula, Montana, USA.

Ridenour W.M. and Callaway R.M. 2001. The relative importanceof allelopathy in interference: the effects of an invasive weedon a native bunchgrass. Oecologia 126: 444–450.

Robinson D. and Fitter A. 1999. The magnitude and control of car-bon transfer between plants linked by a common mycorrhizalnetwork. Journal Experimental Botany 50: 9–13.

Sheley R.L. and Jacobs J.S. 1997. “Acceptable” levels of spottedknapweed (Centaurea maculosa control. Weed Technology 11:363–368.

Sheley R.L., Jacobs J.S. and Carpinelli M.F. 1998. Distribution,biology, and management of diffuse knapweed (Centaurea dif-fusa) and spotted knapweed (Centaurea maculosa). Weed Tech-nology 12: 353–362.

Sheppard A.W. 1996. The interaction between natural enemies andinterspecific plant competition in the control of invasive pas-ture weeds. In: Moran V.C. and Hoffman J.H. (eds), Proceed-ings of the IX Inernational Symposium on Biological Controlof Weeds., Stellenbosch, South Africa, pp. 19–26.

Siemens D., Garner S., Mitchell-Olds T. and Callaway R.M. 2002.The cost of defense in the context of competition. Brassicarapa may grow and defend. Ecology 83: 505–517.

Steinger T. and Müller-Schärer H. 1992. Physiological and growthresponses of Centaurea maculosa (Asteraceae) to root her-bivory under varying levels of interspecific competition andsoil nitrogen availability. Oecologia 91: 141–149.

Story J.M., Good W.R., White L.J. and Smith L. 2000. Effects ofthe interaction of the biocontrol agent Agapeta zoegana (L.)(Lepidoptera:Cochylidae) and grass competition on spottedknapweed. Biological Control 17: 182–190.

Tang C., Cai W., Kohl K. and Nishimoto R.K. 1995. Plant stressand allelopathy. In: Derjit K.M.M., Dakshini and Einhellig F.A.(eds), Allelopathy. American Chemical Society, Washington,DC, USA, pp. 142–157.

Trumble J.T., Kolodny-Hirsch D.M. and Ting I.P. 1993. Plant com-pensation for herbivory. Annual Review Entomology 38: 93–119.

Tyser R.W. and Key C.H. 1989. Spotted knapweed in natural areafescue grasslands: an ecological assessment. Northwest Science62: 151–160.

Van der Putten W.H., Vet L.E.M., Harvey J.A. and Wäckers F.L.2001. Linking above- and belowground multitrophic interac-tions of plants, herbivores, pathogens, and their antagonists.Trends in Ecology and Evolution 16: 547–554.

169

Weiner J., Martinez S., Müller-Schärer H., Stoll P. and Schmid B.1997. How important are environmental maternal effects inplants? A study with Centaurea maculosa. Journal of Ecology85: 133–142.

Wootton J.T. 1994. Putting the pieces together: testing the indepen-dence of interactions among organisms. Ecology 75:1544–1551.

Wurtz T.L. 1995. Domestic geese: biological weed control in anagricultural setting. Ecological Applications 5: 570–578.

Zabinski C.A., Quinn L. and Callaway R.M. Phosphorus uptake,not carbon transfer, explains arbuscular mycorrhizal enhance-ment of Centaurea maculosa in the presence of native grass-land species. Functional Ecology (unpublished).

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