BELOWGROUND COMPETITION AND RESPONSE TO DEFOLIATION OF CENTAUREA MACULOSA AND TWO NATIVE GRASSES. Karla Anne Sartor

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1 BELOWGROUND COMPETITION AND RESPONSE TO DEFOLIATION OF CENTAUREA MACULOSA AND TWO NATIVE GRASSES by Krl Anne Srtor A thesis submitted in prtil fulfillment of the requirements for the degree of Mster of Science in Lnd Resources nd Environmentl Sciences MONTANA STATE UNIVERSITY Bozemn, Montn My 2005

2 COPYRIGHT by Krl Anne Srtor 2005 All Rights Reserved

3 ii APPROVAL of thesis submitted by Krl Anne Srtor This thesis hs been red by ech member of the thesis committee nd hs been found to be stisfctory regrding content, English usge, formt, cittions, bibliogrphic style nd consistency, nd is redy for submission to the college of grdute studies. Ctherine A. Zbinski Approved for the Deprtment of Lnd Resources nd Environmentl Sciences Jon M. Writh Approved for the College of Grdute Studies Bruce R. McLeod

4 iii STATEMENT OF PERMISSION TO USE In presenting this thesis in prtil fulfillment of the requirements for mster s degree t Montn Stte University- Bozemn, I gree tht the Librry shll mke it vilble to borrowers under rules of the Librry. If I hve indicted my intention to copyright this thesis by including copyright notice pge, copying is llowble only for scholrly purposes, consistent with fir use s prescribed in the U.S. Copyright Lw. Requests for permission for extended quottion from or reproduction of this thesis in whole or in prts my be grnted only by the copyright holder. Krl Srtor My 2005

5 iv ACKNOWLEDGEMENTS I would like to thnk my dvisor Cthy Zbinski for her encourgement nd support. I m lso grteful for the dvice on design, nlysis nd writing from Bret Olson nd Bruce Mxwell. Members of the Zbinski lb including Trcy McCreery, Krin Neff, Chris Seftick, Brd Wguespck, Lorn McIntyre, Leslie Eddington, Mri Erwin, Brin Eckenrod, Delis Rogers, Jeff Reynolds, Srh Henz, Adm Novotny, Ashley Bembenek, Anne Rockhold, Kim Buerly, nd Ann McCuley were instrumentl in hrvesting nd processing the mny hundreds of plnt smples. I would lso like to thnk Rosie Wllnder for her solutions to mny problems, Dve Bumbuer for excellent mngement of the plnt growth center nd Rebecc Bunn for sttisticl dvice. Finlly, I would not be where I m without the technicl dvice nd support from Brin Lrsen.

6 v TABLE OF CONTENTS 1. INTRODUCTION... 1 BACKGROUND... 1 RESEARCH OBJECTIVES BELOWGROUND COMPETITION BETWEEN CENTAUREA MACULOSA AND TWO NATIVE GRASSES... 5 INTRODUCTION... 5 METHODS... 7 Experimentl Setup... 7 Plnt Anlysis... 8 Mycorrhizl Anlysis... 9 Sttisticl Anlysis RESULTS Biomss Root Mss Rtio AM Coloniztion Extrrdicl Hyphe Plnt Nitrogen Plnt Phosphorous DISCUSSION ARBUSCULAR MYCORRHIZAE AND NEIGHBOR EFFECTS ON C. MACULOSA AND NATIVE GRASS RESPONSE TO HERBIVORY INTRODUCTION METHODS Experimentl Setup Plnt Anlysis Mycorrhizl Anlysis Sttisticl Anlysis RESULTS Biomss RMR AM Coloniztion Extrrdicl Hyphe Plnt Nitrogen DISCUSSION AM influence on competition AM nd Herbivory Clipping effect on AM fungi... 58

7 vi Nutrients Summry CONCLUSIONS ARBUSCULAR MYCORRHIZAL FUNCTION PLANT COMPETITION AM AND CLIPPING INTERACTIONS IMPLICATIONS FOR MANAGEMENT OF CENTAUREA MACULOSA REFERENCES CITED... 68

8 vii LIST OF TABLES Tble Pge 1. Results of ANOVA for totl biomss Results of ANOVA for RMR Results of ANOVA for AM coloniztion % in AM tretment pots Results of ANOVA for extrrdicl hyphl density Results of ANOVA for C. mculos extrrdicl hyphl length density when plnts were grown lone Results of ANOVA for tissue percent N Results of ANOVA for N content (g plnt -1 ) Results of ANOVA for tissue percent P Results of ANOVA for P content (g plnt -1 ) Results of ANOVA for totl biomss (g) Results of ANOVA for C. mculos totl biomss (g) with ll neighbors Results of ANOVA for RMR Results of ANOVA for C. mculos RMR Results of ANOVA for AM Coloniztion (%) of F. idhoensis nd P. spict roots Results of ANOVA for AM Coloniztion (%) of C. mculos roots Results of ANOVA for ERH density of F. idhoensis nd P. spict roots Results of ANOVA for ERH density of C. mculos roots Results of ANOVA for N concentrtion Results of ANOVA for C. mculos N concentrtion with ll neighbors (g) Results of ANOVA for N content Results of ANOVA for C. mculos N content with ll neighbors (g)

9 viii LIST OF TABLES - CONTINUED Tble Pge 22. Results of ANOVA for P concentrtion Results of ANOVA for C. mculos P concentrtion with ll neighbors (g) Results of ANOVA for P content Results of ANOVA for C. mculos P content with ll neighbors (g) Plnt P content for ech species by clipping tretment... 56

10 ix LIST OF FIGURES Figure Pge 1. Totl Biomss (g) of ech species by () AM nd (b) neighbor fctors Neighbor nd brrier effects on totl biomss for ech species AM effects nd brrier effects on RMR for ech species Neighbor effects on root mss rtio (RMR) of ech species AM coloniztion (%) by () brrier nd (b) neighbor fctors Difference in extrrdicl hyphe length density for AM tretments vs. men of non- AM tretments (bckground level) N concentrtion (%) with m nd brrier fctors Nitrogen concentrtion for ech species by neighbor tretment N content (g plnt -1 ) of ech species by AM nd brrier tretments () Percent P by AM tretment Pseudoroegneri spict percent P by AM, brrier nd neighbor interctions Nitrogen content of ech species grown lone with nd without brrier for AM nd non-am plnts Root length (cm) of ech species by AM tretment ) Totl biomss (g) for ech species by AM tretment Clipping tretment effects on totl biomss (g) for ech species Clipping nd AM tretment effects on totl biomss (g) for ech species growing with C. mculos Clipping nd neighbor tretment effects on C. mculos totl biomss (g) under AM nd non-am conditions ) AM effect on RMR for ech species grown with C. mculos neighbors. b) Neighbor nd AM effects on RMR of C. mculos

11 x LIST OF FIGURES - CONTINUED Figure Pge 19. Clipping tretment effects on RMR for ech species Clipping nd AM effects on RMR of ech species ) Clipping tretment effects on AM coloniztion of ech species. b) Totl hyphl length for C. mculos with ech neighbor species ) AM effect on N concentrtion of ech species. b) Clipping effect on N concentrtion of ech species Clipping effects on N content of ech species ) Clipping effects on P concentrtion. b) Neighbor nd AM effects on C. mculos P content... 54

12 xi ABSTRACT Invsion of ntive rngelnds in the western United Sttes hs serious ecologicl nd economic effects. Understnding the mechnisms behind invsion of Centure mculos (spotted knpweed) is necessry to effectively mnge this species. Arbusculr mycorrhize (AM), which re type of plnt fungl symbiosis, re ubiquitous in grsslnds. My reserch explores the role of AM for incresing the competitive bility of C. mculos. A greenhouse experiment tested the effects of AM fungi nd neighbor species on growth of C. mculos, Festuc idhoensis (Idho fescue) nd Pseudoroegneri spict (bluebunch whetgrss). A mesh brrier permeble to AM hyphe llowed comprison of species interctions by either roots nd/or AM hyphe in pots without brrier or by AM hyphe lone in pots with brrier. Centure mculos plnts hd high AM coloniztion levels within roots nd ERH (extrrdicl hyphe), nd my hve incresed AM coloniztion of neighboring plnts. I found no evidence, however, tht ERH ffected competition, s C. mculos neighbors hd the gretest effect on ntive grss neighbors there ws the potentil for root contct. Additionlly, plnts grown with AM fungi were lwys smller thn non-mycorrhizl plnts. In the second experiment, I investigted growth response fter herbivory (simulted by clipping), with different neighboring species nd AM fungi. Centure mculos, F. idhoensis nd P. spict were grown in the greenhouse with C. mculos neighbor, with or without AM fungi, nd with one of three clipping tretments (no clipping, focl plnt clipped or neighbor plnt clipped to remove 75% of boveground biomss). Compenstory growth ws dependent on AM fungi nd neighbor species. Centure mculos compensted for herbivory only when grown with conspecific nd with AM fungi, or with F. idhoensis neighbor nd no AM fungi. Clipping decresed AM coloniztion in F. idhoensis only, nd coloniztion lso decresed in F. idhoensis when C. mculos neighbors were clipped. This reserch suggests tht AM fungl effects vry between species in the grsslnd system, nd is importnt for determining plnt species response to herbivory. I lso find tht high levels of herbivory my reduce C. mculos biomss enough to be method for weed control, but neighbor species is importnt to determining plnt response to herbivory.

13 1 1. INTRODUCTION Bckground Invsive plnt species hve serious impcts on mny ecosystems. The exotic plnt Centure mculos hs invded over four million hectres of ntive rngelnds nd grsslnds in western North Americ (Boggs & Story 1987). Centure mculos is perennil tp-rooted forb of the Astercee fmily. This species hs very high seed production nd it cn overwinter s rosette fter fll germintion (Wtson & Renney 1974). It ws first introduced from its ntive rnge of estern Europe to western North Americ in the erly 1900s (Wtson & Renney 1974), likely originting from contminted lflf. Centure mculos invdes disturbed nd undisturbed lnds. Dense stnds of C. mculos commonly occur nd reduce lnd vlue becuse this species is not desirble forge for domestic livestock. Trditionl methods of noxious weed control, such s herbicide ppliction, hve hd only limited success. Additionlly, herbicide ppliction cn be reltively expensive nd some weeds my develop tolernce to commonly used herbicides (Bucom & Muricio 2004). Alterntive methods to control C. mculos spred include using sheep or got grzing, species tht re known to consume both ntive nd exotic plnts (Olson & Wllnder 2001). To use these tools effectively, however, greter understnding of the fctors controlling plnt response to herbivory is necessry. An rbusculr mycorrhiz (AM) is symbiosis present in most grsslnd plnt species (Smith & Red 1997). Soil fungi colonize the roots of plnts nd receive 10-20% of plnt s photosynthte (Jkobsen et l. 2002) in exchnge for incresed nutrient

14 2 cquisition for the plnt. Mycorrhizl hyphe externl to roots re very smll in dimeter, nd re ble to extend into soil pore spces inccessible to plnt roots. The symbiosis is obligte for the fungl prtner - they cnnot grow or be cultured without the presence of plnt species - while coloniztion my hve beneficil or prsitic effect on the plnt, depending on environmentl fctors (Johnson et l. 1997). Complex interctions cn occur between plnt species medited by AM fungi. There is evidence tht AM fungi cn lter competitive interctions mong plnts through differentil effects on the host plnts or use of common network of AM hyphe (Grime et l. 1987; Hrtnett et l. 1993; Hrtnett & Wilson 2002; Simrd et l. 1997). Centure mculos is mycorrhizl in the field (Mrler et l. 1999b) nd AM fungi re importnt to interctions between this species nd ntive rngelnd species (Crey et l. 2004; Mrler et l. 1999; Zbinski et l. 2002). The mechnisms behind AM-induced increse in competitive bility, however, re not cler. Centure mculos my prsitize crbon from neighboring plnts (Mrler et l. 1999) through common mycorrhizl networks, but it ws lter shown tht no crbon is trnsferred between the species (Zbinski et l. 2002). Insted, C. mculos likely hs more efficient use of the common network of hyphe, with greter nutrient cquisition nd more hyphe tht extend beyond the roots (Wlling & Zbinski 2004). Plnt response to herbivory is controlled by mny fctors, including plnt growth stge t the time of defolition (Turner et l. 1993), the severity of defolition (Crwley 1997), nd nutrient nd wter vilbility (Chpin & McNughton 1989). Since AM fungi ffect nutrient cquisition nd vilbility, it cn be involved with plnt regrowth

15 3 fter herbivory (Gehring & Whithm 1994). To predict the outcome of grzing s weed control method, understnding the mechnisms ffecting compenstory growth is necessry. Compenstory growth, defined here is growth of plnts fter clipping which my result in compenstion for herbivory. Compenstion cn be defined s prtil, equl or greter yield thn unclipped plnts (Briske & Richrds 1994). Others hve used the definition in terms of fruits nd seeds (Mschinski & Whithm 1989) or fitness (Struss & Agrwl 1999) in reltion to unffected plnts, but in this study I will be using the term to describe biomss s n indictor of compenstion. In ddition, I re interested in the effect of herbivory on AM function, becuse AM is key component of grsslnd ecosystems. Previous literture hs been mixed on whether herbivory hs positive or negtive effects on AM coloniztion. Incresed coloniztion following herbivory my result from incresed nutrient demnd by the plnt when plnts increse growth rte to replce lost tissue. Alterntively, AM coloniztion my decrese in plnts following defolition since photosynthetic tissue is lower nd stored crbon is llocted to regrowth insted of to AM fungl symbionts. The im of my first experiment (Chpter 2) ws to investigte the role of AM extrrdicl hyphe (ERH) in the interctions between plnts. In the second experiment (Chpter 3) I ttempted to test conceptul model in which the fctors of neighboring species nd nutrient vilbility regulte the bility of plnts to compenste for herbivory. Both of these fctors re in turn ffected by AM fungi nd cn ffect ech other.

16 4 Reserch Objectives My reserch investigted mechnisms involved in AM fungl influence on competition between C. mculos nd ntive grsses, nd the role of AM fungi in the competition between these plnts nd their response to herbivory. The objectives of the experiment discussed in Chpter 2 were: 1) to mesure the influence of rbusculr mycorrhize nd root-root interctions on competition between C. mculos nd two ntive grsses; nd 2) to ssess the importnce of AM hyphe in plnt competition when root competition is eliminted. The objectives of the experiment included in Chpter 3 were to determine: 1) whether AM influences biomss of C. mculos nd ntive grsses when grown together; 2) whether clipping lters the effect of AM fungi on host plnt competitive bility; 3) whether AM fungi enhnces compenstory growth in C. mculos or the ntive grsses when grown with neighbor; nd 4) how herbivory simulted by clipping ffects AM coloniztion, ERH density nd effectiveness in incresing plnt nutrient uptke nd subsequent compenstory growth.

17 5 2. BELOWGROUND COMPETITION BETWEEN CENTAUREA MACULOSA AND TWO NATIVE GRASSES Introduction Centure mculos is n invsive tp-rooted forb introduced to the western United Sttes in the erly 1900s. This species invdes disturbed res nd intct grsslnds (Tyser & Key 1988), nd hs invded over four million hectres of ntive rngelnds nd grsslnds in the western United Sttes. Its estblishment my pose serious ecologicl impcts (Wtson & Renney 1974) including reduced plnt diversity (Tyser & Key 1988), nd economic impcts due to reduction in preferred forge plnts (Wtson & Renney 1974). The invsive bility of C. mculos hs been ttributed to lrge seed production, rpid erly seson growth, incresed bility to tke up nutrients (LeJeune & Sestedt 2001), more plstic response to N supply (Blicker et l. 2002), lck of nturl enemies (Kene & Crwley 2002), nd llelopthic chemicls, such s (-)-ctechin which my lso ffect the estblishment nd growth of neighboring plnts (Bis et l. 2003). My reserch investigtes the reltive importnce of rbusculr mycorrhizl (AM) fungi nd root interctions of C. mculos in competition with the ntive grsses Pseudoroegneri spict nd Festuc idhoensis. Arbusculr mycorrhize (AM) re n ncient symbiosis (Bidrtondo et l. 2002) present in over 80% of lnd plnts (Smith & Red 1997). The soil fungl symbiont ids plnts in cquiring nutrients, while the plnt symbiont dontes crbon to the fungus. Arbusculr mycorrhize cn increse plnt N nd P (Frey & Schüepp 1993; Smith & Red 1997), which re the most limiting nutrients to plnt growth (Aerts & Chpin 2000).

18 6 There is lso some evidence tht AM fungi provide defense ginst fungl pthogens (Newshm et l. 1995) nd improve wter reltions (Allen & Allen 1986). One fctor influencing C. mculos s bility to invde ntive grsslnds my be its bility to compete with ntive plnts through incresed nutrient cquisition. An incresed ccess to limiting resources or bility to grow t lower levels of resource supply often determines the outcome of plnt competition (Csper & Jckson 1997; Tilmn 1997). A species which benefits more thn others from the AM symbiosis my be t competitive dvntge. Arbusculr mycorrhizl fungi my influence competition between species in the tll grss pririe (Hrtnett et l. 1993; Hetrick et l. 1994) nd in the greenhouse (Allen & Allen 1984; Fitter 1977; Mrler et l. 1999; Moor & Zobel 1998). Competition my be ffected by AM fungi when plnt species differ in the benefit they receive from mycorrhize (Allen & Allen 1990; Bever et l. 2002; Grime et l. 1987), lthough tht response depends on which species of AM fungi re present (Hrt et l. 2003; Klironomos 2003). The most importnt functionl component of AM fungi is extrrdicl hyphe (ERH), the fungl mycelium externl to plnt roots which enble the plnt to cpture nutrients beyond the root s nutrient depletion zone (Fitter 1997). Extrrdicl hyphe cn rech insoluble nd immobile nutrients such s P tht would otherwise be unvilble for plnt uptke (Mrschner 1995). Centure mculos produces more extrrdicl hyphe thn F. idhoensis (Wlling & Zbinski 2004), which could confer competitive dvntge to C. mculos if it cn ccess more limiting nutrients thn ntive grsses.

19 7 Mrler et l. (1999) found tht the negtive effect of C. mculos on the biomss of F. idhoensis neighbor ws enhnced by the presence of AM fungi. The objectives of this experiment were: 1) to mesure the influence of rbusculr mycorrhize nd root-root interctions on competition between C. mculos nd two ntive grsses; nd 2) to ssess the importnce of AM hyphe in plnt competition when root competition is eliminted. Methods Experimentl Setup This experiment ws rndomized complete fctoril design, including nine species combintions, two brrier tretments, two AM tretments, nd nine replictes of ll tretments. Festuc idhoensis, P. spict nd C. mculos were grown in inter- nd intr-specific combintions nd lone. Ech species ws seeded directly into 7500 ml pots nd lter rndomly thinned to one seedling. Festuc idhoensis ws seeded nine dys prior to seeding P. spict or C. mculos becuse of its longer germintion nd estblishment time. Seedlings strted t the time of direct seeding were trnsplnted into pots without estblishment. All species combintions were grown either with no brrier or in pots with 28 µm mesh brrier (Nitex nylon, Sefr Americ, Depew, NY, USA) bisecting the pot. The brrier excluded roots but not AM hyphe, nd hs miniml effect on wter nd solute movement (Zimmerley, unpublished dt). In pots with two plnts, the brrier excluded neighbors roots but not hyphe, nd in pots with single plnt, the brriers creted comprtment tht only hyphe could ccess.

20 8 Plnts in ll tretments were grown under either AM or non-am conditions. Unpsteurized field soil served s mycorrhizl inoculum for AM tretment plnts, while for non-am tretments the entire soil mix ws erted stem psteurized t 80 C for 90 minutes. To reintroduce non-am microbes, 800 ml of field soil ws mixed with 4000 ml of DI wter nd vcuum-filtered to pss through n 11µm filter (Whtmn No. 1) three times. Ten ml of microbil wsh ws dded to ech side of ll pots in the non-am tretment. Plnts were grown for 16 weeks under greenhouse conditions mintined t 21 ºC dytime nd 16 ºC nighttime temperture with supplementl lighting (GE Multi- Vpor MVR1000/C/U) s necessry for 16-hour dy length. Plnts were grown in 4:1 snd:soil mix, with 30 grit silic snd nd 1:1 mix of psteurized lom top soil nd field soil. Field soil ws collected djcent to the Red Bluff Reserch Rnch ner Norris, Montn (45.60, ) in n F. idhoensis, P. spict dominted grsslnd with C. mculos present, nd sieved through 1.5cm screen. The soil mixture hd initil NO 3 nd P levels of 4.9mg/kg nd 3.3mg/kg, respectively. I dded 50ml of 1/2 strength Hoglnd s solution to ech side of the pot two times during the experiment s plnts showed signs of deficiency. Plnt Anlysis Root nd shoot tissue ws seprted, clened nd dried until weight did not chnge, nd biomss ws recorded. Three replictes of plnts were rndomly chosen from ech tretment for ech species to be nlyzed for tissue nutrient nlysis. Root nd shoot tissues were combined nd ground to pss through 0.5µm screen (UDY mill

21 9 Model # ; Fort Collins, CO, USA). Kjeldhl digestion for N nd nitric cid/hydrogen peroxide digestion for P nlysis were performed, nd then nlyzed using Inductively Coupled Plsm Spectrometry (MDS Hrris, Lincoln, NE, USA). Mycorrhizl Anlysis To determine coloniztion levels in mycorrhizl nd non-mycorrhizl tretments, fine root smples of five rndomly selected replictes of plnts from AM tretments nd two replictes of non-am tretments were clered for 48 hours in 2.5% KOH, rinsed with distilled wter, cidified for 12 hours with 3% HCL, nd stined for 12 hours with 0.05% Trypn blue solution (Phillips & Hymn 1970). Mycorrhizl structures including internl hyphe, vesicles nd rbuscules were quntified using the mgnified line intersect method (McGonigle et l. 1990). Extrrdicl hyphe were quntified from two 2.5 cm dimeter soil cores extrcted from ech pot. For pots with two plnts, cores were 3 cm from the bse of ech plnt. Pots with only one plnt were cored 3 cm from the plnt nd gin in the center of the hyphl comprtment. Extrrdicl hyphe were extrcted from these smples using modified method of Miller et l. (1995). Soil cores were mixed well, nd two replicte 5g smples were mixed with 15 ml of 3.75% sodium hexmetphosphte nd 100ml wter, stirred for 5 minutes, nd llowed to sit for 30 minutes to dissocite soil prticles. An dditionl smple of ech soil ws dried to obtin dry weight equivlents. A 10ml liquot ws diluted with 100ml wter, nd 20ml ws removed nd filtered through 20µm mesh filter. The filter ws plced in centrifuge tube nd stined with 5 ml 0.5% Trypn blue for 5 minutes. The contents were filtered through 0.45µm membrne filter.

22 10 AM hyphe were distinguished from non-am hyphe by chrcteristics such s dimeter, the presence of ngulr projections nd generl lck of septe (Mosse 1959; Nicolson 1959; Sylvi 1992). The length of AM hyphe on ech filter ws estimted using grid line intersect method (Tennnt 1975) under 200x mgnifiction nd used to clculte length of hyphe g -1 soil. Sttisticl Anlysis Vribles including root, shoot nd totl biomss, root mss rtio (clculted s the rtio of root biomss:totl biomss), AM percent coloniztion, ERH density, root length, nd N nd P concentrtion nd content were nlyzed by ANOVA using R (R Development Core Tem 2004). Three wy ANOVA s for the fctors of AM (+/-), brrier (+/-), nd neighbor species (C. mculos, F. idhoensis, P. spict or no neighbor), nd ll interctions were nlyzed for ech vrible individully for ech plnt species. For ERH mesurements, contrsts were used to identify ERH density greter thn the men bckground density for non-am pots. Power trnsformtions were identified using the Box-Cox procedure nd homogeneity of vrince ws verified with the modified Levene s test. Tukey s multiple comprison procedure ws used to seprte mens (α = 0.05). Results Biomss Centure mculos plnts hd biomss of 5.9g ± 0.2g, while P. spict nd F. idhoensis were 4.9g ± 0.2g nd 0.9g ± 0.1g respectively. The AM tretment hd

23 11 significnt effect on biomss (Tble 1) plnts were 46%, 50% nd 39% smller grown under AM thn non-am conditions for C. mculos, F. idhoensis nd P. spict plnts, respectively (Figure 1). Tble 1. Results of ANOVA for totl biomss. C. mculos nd F. idhoensis were log trnsformed nd P. spict ws squre root trnsformed. C. mculos F. idhoensis P. spict Residul df: Fctor df F p F p F p AM Neighbor Brrier AM X Neighbor AM X Brrier Neighbor X Brrier AM X Brrier X Neighbor Totl biomss of ll species ws ffected by neighbor (Tble 1). Centure mculos biomss ws smllest with C. mculos nd P. spict neighbors nd lrgest with either F. idhoensis or no neighbor (Figure 1b). For F. idhoensis, totl biomss ws smllest with C. mculos neighbor nd lrgest with either F. idhoensis neighbor or when growing lone. Festuc idhoensis nd P. spict neighbors ffected F. idhoensis biomss similrly (Figure 1b). Pseudoroegneri spict ws lrgest growing lone, nd smllest growing with C. mculos neighbor, nd ws lso smller growing with P. spict neighbor thn F. idhoensis neighbor (Figure 1b).

24 12 Totl Biomss (g) ) AM b) non-am 8 * * * b b C. mculos neighbor F. idhoensis neighbor P. spict neighbor d no neighbor bc b c c b C. mculos F. idhoensis P. spict Species C. mculos F. idhoensis P. spict Species Figure 1. Totl biomss (g) of ech species by () AM nd (b) neighbor tretments. Error brs represent one stndrd error of the men. (*) Indictes significnt difference between mens of AM vs. non-am tretments (α<0.05). Letters represent significnt differences between tretments within ech species (α<0.05). Biomss of F. idhoensis nd P. spict vried by brrier nd neighbor fctors (Tble 1). Both F. idhoensis nd P. spict plnts were lrger when brrier seprted them from C. mculos neighbor (Figure 2). For F. idhoensis, the brrier eliminted competition from ll other neighboring species, in tht plnts seprted from their neighbor by brrier were the sme size s plnts grown with no neighbor. Competition occurred with the brrier present for inter- nd intrspecific competition of C. mculos nd P. spict. Both species were lrger growing lone thn grown with C. mculos or P. spict neighbor, even with brrier present (Figure 2).

25 13 Totl Biomss (g) C. mculos bc c b Brrier No Brrier b c c F. idhoensis c Totl Biomss (g) bc bc bc bc b c Totl Biomss (g) P. spict b bc bcd b b cd d C. mculos F. idhoensis P. spict no neighbor Neighbor Figure 2. Neighbor nd brrier effects on totl biomss for ech species. Note difference in y-xis scles. Error brs represent one stndrd error of the men. Letters represent significnt differences between tretments within ech species (α<0.05).

26 14 Root Mss Rtio Root mss rtios were 0.62, 0.46, nd 0.54, for C. mculos, F. idhoensis nd P. spict, respectively. Root mss rtios were lower in the AM tretment for C. mculos nd F. idhoensis (Tble 2; Figure 3). The brrier tretment only ffected C. mculos (Tble 2), with higher RMR in pots with brrier (Figure 3). Tble 2. Results of ANOVA for RMR. No trnsformtions were performed. C. mculos F. idhoensis P. spict Residul df: Fctor df F p F p F p AM Neighbor Brrier AM X Neighbor AM X Brrier Neighbor X Brrier AM X Brrier X Neighbor Neighbor species ffected RMR for both C. mculos nd F. idhoensis (Tble 2). Both species hd higher RMR when grown with the other species. Centure mculos hd higher RMR grown with F. idhoensis neighbor nd F. idhoensis hd higher RMR grown with C. mculos neighbor thn grown with conspecific (Figure 4). The effect ws only pprent for F. idhoensis without the brrier (Tble 2, dt not shown).

27 ) * AM b) non-am 0.6 * Brrier No Brrier * RMR 0.4 * C. mculos F. idhoensis P. spict 0.0 C. mculos F. idhoensis P. spict Species Species Figure 3. AM effects nd brrier effects on RMR for ech species grown with ll neighbors. Error brs represent one stndrd error of the men. (*) Indictes significnt difference between mens of AM vs. non-am tretments (α<0.05). RMR b b b C. mculos neighbor F. idhoensis neighbor P. spict neighbor no neighbor b b C. mculos F. idhoensis P. spict Species Figure 4. Neighbor effects on root mss rtio (RMR) of ech species. Error brs represent one stndrd error of the men. Letters represent significnt differences between tretments within ech species (α<0.05).

28 16 AM Coloniztion Percent of root intersections colonized by AM fungi ws highest for C. mculos (18.3 ± 1.9%) nd lowest for F. idhoensis nd P. spict (3.3 ± 0.7% nd 7.0 ± 1.0%). Only one intersection in one plnt in the non-am tretment hd trits similr to AM hyphe (men <0.010% coloniztion), nd no vesicles or rbuscules were present in the non-am tretment roots. Tble 3. Results of ANOVA for AM coloniztion % in AM tretment pots. C. mculos nd P. spict were not trnsformed nd F. idhoensis ws log trnsformed plus one. C. mculos F. idhoensis P. spict Residul df: Fctor df F p F p F p Neighbor Brrier Neighbor X Brrier AM Coloniztion (%) ) * b) 25 C. mculos neighbor Brrier F. idhoensis neighbor No Brrier P. spict neighbor 20 no neighbor 15 b b b 10 b 5 5 C. mculos F. idhoensis P. spict Species C. mculos F. idhoensis P. spict Species Figure 5. AM coloniztion (%) by () brrier nd (b) neighbor tretments. Error brs represent one stndrd error of the men. (*) Indictes significnt difference between mens of AM vs. non-am tretments (α<0.05). Letters represent significnt differences between tretments within ech species (α<0.05).

29 17 The brrier tretment ffected coloniztion levels for only C. mculos (Tble 3). In pots with brrier, C. mculos hd 23% of intersections colonized, while pots without brrier hd 15% coloniztion (Figure 5). Neighbor identity ffected percent root length colonized in F. idhoensis (Tble 3). Men coloniztion levels in F. idhoensis were 10.3% grown with C. mculos neighbor nd only % with ech grss species neighbor nd growing lone (Figure 5b). Neighbor species influenced P. spict (p=0.078), nd coloniztion levels were higher with C. mculos neighbors thn with F. idhoensis neighbors (Figure 5b). Neighbor identity did not ffect C. mculos AM coloniztion (Tble 3). Extrrdicl Hyphe A bckground ERH density of 1.88 ± 0.6m g -1 soil ws present in non-am pots, likely due to non-vible hyphe in psteurized soil inoculum dded to the non-am soil medium. Extrrdicl hyphl density exceeded the bckground level only in C. mculos pots nd F. idhoensis pots with C. mculos neighbor (Figure 6), determined by contrsts. Hyphl density (m g -1 soil) in soil extrcted from the C. mculos side of the pot ws not ffected by neighbor species or brrier tretment (Tble 4). For the F. idhoensis side of the pot, neighbor species ffected hyphl density (Tble 4). Hyphl density exceeded bckground levels only in pots with C. mculos neighbor. Pseudoroegneri spict pots did not hve ERH density greter thn non-am pots for ny tretment combintion (Figure 6). Extrrdicl hyphl density did not vry by neighbor nd brrier tretments. Extrrdicl hyphl density did not vry by nd interction of neighbor nd brrier tretments.

30 18 ERH Length Density (m g -1 plnt) * * * * C. mculos neighbor F. idhoensis neighbor P. spict neighbor no neighbor -0.4 C. mculos F. idhoensis P. spict Species Figure 6. Difference in extrrdicl hyphe length density for AM tretments vs. men of non-am tretments (bckground level). Error brs represent one stndrd error of the men. (*) represents significnt hyphl length density bove bckground levels (α<0.05). For C. mculos plnts with no neighbor, smples tken on the opposite side of the brrier (hyphl comprtment) did not differ in ERH density from smples on the plnt side (Tble 5), indicting tht plnts were ble to ccess resources in the hyphl comprtment through ERH. Extrrdicl hyphl density in the hyphl comprtment did not differ from tht in the distnt side of pot (Tble 5). Pots with grss species grown lone did not hve significnt hyphl density bove the bckground density, so the side of pot nlysis ws not performed. Tble 4. Results of ANOVA for extrrdicl hyphl length density. C. mculos F. idhoensis P. spict Residul df: Fctor df F p F p F p Intercept Neighbor Brrier Neighbor X Brrier

31 Tble 5. Results of ANOVA for C. mculos extrrdicl hyphl length density when plnts were grown lone. Fctor df F p Brrier Side of pot Brrier X Side of pot Residul 8 19 Plnt Nitrogen Plnt tissue percent N ws 1.41 ± 0.04% in F. idhoensis, 0.92 ± 0.06% in P. spict nd 1.06 ± 0.05% in C. mculos. The AM tretment ffected nitrogen concentrtion for C. mculos nd mrginlly for F. idhoensis (Tble 6). Nitrogen concentrtion in C. mculos ws higher under AM conditions, nd the effect ws strongest without brrier (Figure 7). Festuc idhoensis lso hd trend towrd higher N concentrtion under AM conditions, but ws not ffected by the brrier (Figure 7). Tble 6. Results of ANOVA for tissue percent N. No trnsformtions were performed. C. mculos F. idhoensis P. spict Residul df: Fctor df F p F p F p AM Neighbor Brrier AM X Neighbor AM X Brrier Neighbor X Brrier AM X Neighbor X Brrier Neighbor species ffected N concentrtion in C. mculos nd F. idhoensis (Tble 6). For C. mculos, N concentrtion ws highest with P. spict neighbor while F. idhoensis tissue N concentrtion ws highest with conspecific, or growing

32 lone, nd lowest with C. mculos neighbor (Figure 8). Pseudoroegneri spict tissue N did not vry between tretments. 20 C. mculos F. idhoensis P. spict N Concentrtion (%) b Brrier No Brrier AM non-am AM non-am AM non-am Figure 7. N concentrtion (%) with m nd brrier tretments. Error brs represent one stndrd error of the men. Letters represent significnt differences between tretments within ech species (α<0.05). N Concentrtion (%N) 1.8 C. mculos neighbor c F. idhoensis neighbor bc 1.6 P. spict neighbor no neighbor 1.4 b b 1.2 b b C. mculos F. idhoensis P. spict Species Figure 8. Nitrogen concentrtion for ech species by neighbor tretment. Error brs represent one stndrd error of the men. Letters represent significnt differences between tretments within ech species (α<0.05).

33 21 Nitrogen content ws 57 ± 3.6mg plnt -1 in C. mculos nd 17 ± 1.9mg nd 42 ± 2.6mg plnt -1 for F. idhoensis nd P. spict respectively. For ll species, N content ws 43, 37, nd 31% lower under AM conditions for C. mculos, F. idhoensis nd P. spict respectively, consistent with the lower biomss of AM plnts (Figure 1). The brrier tretment ffected N content for C. mculos nd F. idhoensis (Tble 7). Nitrogen content ws higher in C. mculos without brrier, nd the effect ws greter under non-am conditions (Figure 9). For F. idhoensis the effect of brrier ws influenced by neighbor tretment s seen for totl biomss (Figure 2). Brrier nd AM tretments intercted to ffect N content in P. spict (Tble 7). When grown with brrier, P. spict tended to hve lower N content under AM conditions nd higher N content under non-am conditions compred with the no brrier tretment (Figure 9). Tble 7. Results of ANOVA for N content (g plnt -1 ). C. mculos nd F. idhoensis were log trnsformed, nd P. spict ws squre root trnsformed. C. mculos F. idhoensis P. spict Residul df: Fctor df F p F p F p AM Neighbor Brrier AM X Neighbor AM X Brrier Neighbor X Brrier AM X Brrier X Neighbor

34 22 C. mculos F. idhoensis P. spict 0.10 c N Content (g plnt -1 ) b b Brrier No Brrier b b b b b 0.00 AM non-am AM non-am AM non-am Figure 9. N content (g plnt -1 ) of ech species by AM nd brrier tretments. Error brs represent one stndrd error of the men. Letters represent significnt differences between tretments within ech species (α<0.05). Plnt Phosphorous Phosphorous concentrtions were 0.22 ± 0.01%, 0.23± 0.01% nd 0.16 ± 0.004% for C. mculos, F. idhoensis, nd P. spict respectively. The AM tretment ffected P concentrtion only for C. mculos (Tble 8). Tble 8. Results of ANOVA for tissue percent P. No trnsformtions were performed. Dt were not vilble for F. idhoensis three-wy interction due to smll plnt size nd insufficient plnt mteril for P nlysis for the AM, no brrier, nd C. mculos neighbor tretment. C. mculos F. idhoensis P. spict Residul df: Fctor df F p F p F p AM Neighbor Brrier AM X Neighbor AM X Brrier Neighbor X Brrier AM X Neighbor X Brrier NA NA

35 23 Plnts hd higher P concentrtion in the AM tretment (Figure 10). Neighbor nd brrier min effects did not influence P concentrtion of ny species (Tble 8). A mrginl three-wy interction ffected P. spict plnt P concentrtion (Tble 8). Phosphorous concentrtion ws higher when pired with C. mculos neighbor thn with P. spict neighbor under AM conditions, nd the effect ws most pronounced without brrier present (Figure 11). No interctions between fctors influenced P concentrtions in C. mculos or F. idhoensis * 0.25 AM non-am P Concentrtion (%) C. mculos F. idhoensis P. spict Species Figure 10. Percent P by AM tretment. Error brs represent one stndrd error of the men. (*) Indictes significnt difference between mens of AM vs. non-am tretments (α<0.05).

36 24 AM non-am P Concentrtion (%) C. mculos neighbor F. idhoensis neighbor P. spict neighbor no neighbor b b b b b b b Brrier No Brrier Brrier No Brrier Figure 11. Pseudoroegneri spict percent P by AM, brrier nd neighbor interctions. Error brs represent one stndrd error of the men. Letters represent significnt differences between tretments within ech species (α<0.05). Phosphorous content ws 13 ± 0.68mg plnt -1 in C. mculos nd 2.8 ± 0.30mg nd 7.5 ± 0.44mg plnt -1 in F. idhoensis nd P. spict plnts, respectively. All species P content ws ffected AM nd neighbor fctors, in wy tht prllels ech species biomss response (Tble 9, Figure 1 & b). Phosphorous content ws lower with the AM tretment nd C. mculos or P. spict neighbors. Phosphorous content for C. mculos nd P. spict ws ffected by n interction of brrier nd AM fctors (Tble 9). For C. mculos, P content ws higher without brrier under non-am conditions nd the interction for P. spict ws similr to tht for N content (Figure 9). Neighbor nd brrier tretments intercted similrly to ffect P content s they ffected totl biomss (Figure 2).

37 25 Tble 9. Results of ANOVA for P content (g plnt -1 ). Centure mculos nd F. idhoensis were log trnsformed nd P. spict ws not trnsformed. Dt were not vilble for F. idhoensis three-wy interction due to smll plnt size nd insufficient plnt mteril for nlysis of the tretment of AM, no brrier, nd C. mculos neighbor. C. mculos F. idhoensis P. spict Residul df: Fctor df F p F p F p AM Neighbor Brrier AM X Neighbor AM X Brrier Neighbor X Brrier AM X Brrier X Neighbor NA NA Discussion Arbusculr mycorrhizl fungi improve plnt growth nd nutrition for most species of grsslnd plnts (Smith & Red 1997). In this study, I found tht AM fungi were not beneficil to plnt growth, s plnts in the AM tretment hd significntly less biomss thn non-am plnts. Negtive effects of AM fungi on plnt growth cn occur if the cost of crbon given to the fungl symbiont is greter thn the benefit of cquisition of nutrients (Koide & Elliott 1989). It is difficult to interpret, however, how much AM cused growth suppression for the grss species in this study, due to low levels of coloniztion (3% nd 7% coloniztion). The non-am tretment ws produced with psteurized soil medium to kill AM propgules, with microbil wsh of smller, non-am fungi nd bcteri dded. The AM tretment soil my hve hd more pthogenic fungi thn the non-am tretment soil, resulting in depressed growth in the AM tretment. Another possibility is tht the stem

38 26 steriliztion incresed nitrogen vilbility in the non-am pots, which in turn led to greter biomss for these plnts (Koide & Li 1989), difference tht cn be especilly importnt in very low nitrogen conditions such s ours (Thompson 1990). I predicted tht for plnts grown singly, biomss would be lower in pots with brrier, with the cvet tht AM plnts would not be ffected by brrier becuse they hve ccess through ERH to nutrients from the opposite side of the pot. In generl the brrier did not limit plnt growth for plnts grown singly. Centure mculos nd F. idhoensis plnts grown lone were the sme size with nd without brrier present. Festuc idhoensis plnts were so smll, however, (0.9g) tht they were probbly not limited by resources in hlf the pot volume. For C. mculos, the brrier likely did not ffect biomss becuse dissolved nutrients such s nitrogen compounds could hve crossed the brrier through mss flow nd diffusion. Nitrogen content dt supports this interprettion since it usully did not differ between plnts with or without brrier, for both AM nd non-am plnts (Figure 12). The lck of response of C. mculos biomss to the brrier could lso reflect tht it hd significnt mounts of ERH. Arbusculr mycorrhizl plnts could hve ccessed immobile nutrients cross the brrier, however P content ws not s high in AM plnts s it ws in non-am plnts, regrdless of brrier tretment. Either AM hyphe were not involved in ccess of nutrients cross the brrier, or nother effect of the AM tretment, such s pthogens, msked the effect of AM hyphe. Alterntively, some P could be ccessed cross the brrier through roots. In studies on AM hyphl trnsport of P,

39 27 experiments using similr mesh comprtments hve found tht roots cn deplete soil P up to one centimeter into the hyphl comprtment (Jkobsen et l. 1992) nd the brrier utilized in this study hd lrge surfce re. AM non-am Brrier No Brrier N Content (g plnt -1 ) * C.mculos F. idhoensis P. spict 0.00 C.mculos F. idhoensis P. spict Species Species Figure 12. Nitrogen content of ech species grown lone with nd without brrier for AM nd non-am plnts. Error brs represent one stndrd error of the men. (*) Indictes significnt difference between mens of AM vs. non-am tretments (α<0.05). The brrier did not ffect P. spict biomss grown lone under non-am conditions s ws seen for C. mculos nd F. idhoensis, but with AM, both totl biomss nd shoot biomss were reduced in pots with the brrier. This result ws lso observed by Moor nd Zobel (1998). Root mss rtio generlly decreses with incresed nutrient vilbility (Fitter 1997), nd P. spict hd ccess to more nutrients with more spce when grown without the brrier, s reflected in higher N content (Figure 12). Under non-am conditions, where biomss ws not ffected by brrier, P. spict plnts were much lrger thn AM plnts nd potentilly the incresed trnspirtion of

40 28 lrger plnt llowed for incresed mss flow of dissolved nutrients cross the brrier. Agin, nitrogen content dt indictes tht P. spict plnts hd s much nitrogen uptke with the brrier s without the brrier (Figure 12) indicting tht plnts were probbly ble to ccess nitrogen through mss flow cross the brrier. I hypothesized tht the brrier tretment would llow belowground competitive interctions in mycorrhizl plnts vi ERH, but competition (reduction in biomss when grown with neighbor) occurred with the brrier present for ll species with nd without AM fungi. The effect of vrying neighbor species ws much stronger. For C. mculos focl plnts, C. mculos nd P. spict neighbors reduced C. mculos biomss s compred with growing lone. A F. idhoensis neighbor lso reduced C. mculos biomss, but only under non-am conditions. These effects could be due to shoot competition, or n inbility to ccess dissolved nutrients, s occurred with plnts growing lone, becuse they re used by the neighbor plnt. Festuc idhoensis biomss ws lower with P. spict neighbor cross the brrier, for similr resons discussed for C. mculos. With C. mculos neighbor, biomss ws reduced only under AM conditions, which could be the result of dditionl nutrient depletion by C. mculos AM hyphe. The trend ws similr for non-am plnts, however, nd with lrge vrinces in F. idhoensis biomss sttisticl differences were difficult to detect. Conspecifics did not ffect F. idhoensis biomss, likely becuse the two F. idhoensis plnts were not lrge enough tht resources becme limiting. All neighbors reduced P. spict biomss with brrier present, regrdless of AM tretment.

41 29 This is probbly becuse of competition for nutrients s seen for C. mculos, or through AM hyphe when grown with C. mculos neighbor. A greter reduction in the biomss of grsses grown with neighbor ws observed when the brrier ws removed nd root contct could occur. When pired with C. mculos, the grss species were significntly smller without the brrier present, indicting tht either root contct is importnt to C. mculos s competitive effect on the ntive grsses, or tht C. mculos simply grew fster nd ws ble to occupy spce more quickly thn the grsses (McConnughy & Bzzz 1991). Additionlly, C. mculos llocted more biomss to its root system thn the grsses, which could explin why C. mculos ws more successful competitor with direct root competition, s plnts with higher RMR re often more competitive (Aerts et l. 1991). Allelopthy could be involved in the more negtive effect of C. mculos on ntive grsses without the brrier (Bis et l. 2003), however, similr reduction in F. idhoensis biomss ws observed with P. spict neighbor under AM conditions. While llelopthy could be occurring with C. mculos neighbor, there is no documenttion of the llelopthic chemicl (-)-ctechin production in P. spict. Also, wter soluble llelopthic chemicls should be ble to cross the mesh brrier, but C. mculos hd lrge effect on grss biomss only without the brrier. In contrst to the effect on grss species, the effect of neighbor on C. mculos biomss ws independent of brrier presence. Biomss ws lower with neighbor with nd without brrier present.

42 30 Totl pot biomss (summing the responses of both plnts) ws greter for C. mculos nd P. spict grown together when brrier ws present, while the totl pot biomss of either two C. mculos plnts or two P. spict plnts ws not ffected by brrier presence. This is counter to the findings of Flik (2003) tht plnts produce more biomss in the presence of competitor thn in competition with itself. These results suggest tht the potentil for root contct increses competitive effects between these two species, result consistent with either llelopthic effects or plnt detection of non-self plnt which results in incresed interference competition. This experiment hs documented numerous potentil trits tht contribute to C. mculos s competitive bility, including incresed nutrient concentrtion with AM fungi present, incresed AM coloniztion nd high ERH density. Centure mculos ws the only plnt to hve higher concentrtions of P with AM fungi. While this could be prtly due to higher nutrient concentrtions in smller (AM) plnts, the grss species were lso smller under AM conditions, but did not hve higher nutrient concentrtions. With higher AM coloniztion levels thn the other two species (18% vs. 3 nd 7% coloniztion), C. mculos my hve reched threshold level of coloniztion, t which the benefits of coloniztion outweighed the costs, t lest in terms of nutrient cquisition (Gnge & Ayres 1999) Another explntion for incresed nutrient concentrtion in C. mculos in the AM tretment is its high ERH density. It ws the only species to hve significnt density of ERH, side from F. idhoensis when it ws grown with C. mculos neighbor, nd lso the only species to hve greter P concentrtion in the AM tretment.

43 31 Without significnt ERH density, it is not surprising tht AM fungi did not hve positive effect on grsses. Phosphorous uptke hs not successfully been correlted with AM coloniztion levels, but there is evidence tht ERH density is more importnt in predicting P uptke by AM fungi thn AM coloniztion (Jkobsen et l. 2001), so high ERH density cn be extremely dvntgeous to plnts. The brrier influence on nutrient uptke differed for N nd P in C. mculos plnts. Concentrtion of P in C. mculos ws higher in the AM tretment thn the non- AM tretment for both brrier tretments, wheres for N concentrtion, it ws only higher with AM fungi without the brrier present. This suggests tht lthough AM hyphe ccess N (Frey & Schüepp 1993), hyphe did not significntly id plnts in ccessing N unvilble to roots, while P cquisition through the brrier vi ERH ws more efficient. Our experimentl conditions were such tht N ws likely more limiting to plnt growth thn P becuse N:P rtios of plnt tissue rnged from 4.5 in C. mculos to 6.5 in F. idhoensis (dt not shown), which is well below the optiml level of 16 (Koerselmn & Meulemn 1996). If N is the most limiting nutrient, then those plnts with the gretest N content re the most competitive plnts, nd dditionl ccess to P did not gretly id C. mculos in terms of biomss. In our experiment, non-am C. mculos plnts grown without brrier hd the gretest N content (Figure 12). Although I predicted tht AM coloniztion would id the most competitive plnt, it ppers tht n extensive root system ws more importnt to ccessing nutrients. Centure mculos incresed its root length nd therefore bsorptive surfce re in the non-am tretment more thn the ntive grsses were ble to (Figure 13).