Research Article Determining the Impact of the AM-Mycorrhizosphere on Dwarf Sunflower Zn Uptake and Soil-Zn Bioavailability

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1 Journl of Botny Volume 21, Article ID 26854, 11 pges doi:1.1155/21/26854 Reserch Article Determining the Impct of the AM- on Dwrf Sunflower Zn Uptke nd Soil-Zn Biovilility Ptrick Audet 1, 2 nd Christine Chrest 1 1 Deprtment of Biology, Ottw-Crleton Institute of Biology, University of Ottw, 3 Mrie Curie Priv., Ottw, ON, Cnd K1N 6N5 2 Centre for Mined Lnd Rehilittion, Sustinle Minerls Institute, University of Queenslnd, St. Luci Cmpus, Brisne, QLD, 472, Austrli Correspondence should e ddressed to Ptrick Audet, pude86@uottw.c Received 16 June 21; Revised 13 August 21; Accepted 15 Octoer 21 Acdemic Editor: Andre Polle Copyright 21 P. Audet nd C. Chrest. This is n open ccess rticle distriuted under the Cretive Commons Attriution License, which permits unrestricted use, distriution, nd reproduction in ny medium, provided the originl work is properly cited. An in vivo comprtmentl pot greenhouse experiment involving dwrf sunflower nd n rusculr mycorrhizl (AM) fungus ws designed to ssess the contriution of non-am roots (rhizosphere), AM roots nd extrrdicl hyphe (mycorrhizosphere), or strictly extrrdicl hyphe (hyphosphere) on plnt growth, plnt metl uptke, nd soil prmeters using the micronutrient zinc (Zn) s typicl metl contminnt. We oserved tht, t high soil-zn concentrtions, the mycorrhizosphere tretments hd lower Zn concentrtions (especilly in shoots nd flowers) nd lower incidence of lef chlorosis thn the rhizosphere tretments. These phytoprotective effects re elieved to e relted to AM-induced iosorption processes tht reduce soil metl iovilility to dely the onset of plnt metl toxicity. We lso oserved tht the presence of extrrdicl hyphe cuses slight lklinistion of the proximl soil environment wheres roots tended to cidify it, this hving significnt consequences towrd metl iovilility. Altogether, the AM symiosis is considered to e key component of ecosystem function involved in uffering plnt growth conditions due to the processes of metl iosorption nd hyphl lklinistion which could contriute in enhncing the soil s resiliency. 1. Introduction The rusculr mycorrhizl (AM) symiosis mutully eneficil ssocition etween the roots of most herceous plnts nd Glomeromycotn fungi [1] is primrily recognized for incresing the minerl sttus of plnts vi the mycorrhizosphere (i.e., comined surfce re of AM roots nd extrrdicl hyphe). It hs lso een suggested tht the mycorrhizosphere plys key role in the regultion of soil metl iovilility through iosorption processes, then contriuting to the llevition of plnt metl toxicity nd nutrient imlnces [2 6]. Recently, we proposed conceptul model derived from met-nlyticl findings depicting the impct of the AM symiosis on plnt metl uptke in reltion to soil conditions rnging from low (trce)tohigh(toxic)metlexposurelevels[7 9], model we further investigted using n in vitro crrot root-orgn culture system [1]. From these studies, we demonstrted tht the AM fungi ply dul role in metl cquisition: first y incresing nutrient uptke t low metl exposure levels, nd then lessening it t high levels through metl iosorption. Notly,inour in vitro study [1], we suggested tht the enhnced uptke nd metl iosorption processes occur independently in shping plnt metl uptke nd, lso, likely hold n importnt role in enhncing soil resiliency despite such metl toxicity conditions. To expnd on these notions, we present here n in vivo comprtmentl pot growth system designed to ssess the contriution of non-am roots (rhizosphere), AM roots nd extrrdicl hyphe (mycorrhizosphere), or strictly extrrdicl hyphe (hyphosphere) on plnt growth nd Zn uptke, soil Zn iovilility, nd soil-ph. The comprtmentl pot system ws selected to isolte ech of these sphere environments nd susequently determine their impct on vrious plnt

2 2 Journl of Botny physiologicl nd edphic fctors in reltion to incresing soil metl exposure levels. In this regrd, the dwrf sunflower cultivr Helinthus nnuus L. vr. Pcino nd the AM fungus Glomus intrrdices Schenck & Smith (isolte DAOM-18162) were selected s suitle experimentl orgnisms for the study nd the essentil micronutrient Zn s typicl metl contminnt which is known to rech toxic levels in the environment due to extensive fertilizer ppliction, metl glvniztion, nd ruer vulcniztion [11 13]. 2. Mterils nd Methods 2.1. Experimentl Design. Dwrf sunflower plnts were grown from seeds (McKenzie Seeds, Brndon, MB, Cnd) for 1 weeks in two-comprtment pot systems contining previously utoclved, low minerl soil mixture (snd:potting soil, 1 : 1 v/v) which ws then inoculted (or not) with fungl propgules of G. intrrdices (Myke Pro Endo, Premier Tech, Rivière-du-Loup, QC, Cnd). The fungl inoculum ws integrted s 3 cm thick sustrte lyer contining 15 propgules g 1 dry sustrte (2 g inoculum sustrte dose hving totl propgules per pot) in the AM tretments (mycorrhizosphere nd hyphosphere plnts), wheres the non-am tretments (rhizosphere plnts) received n equivlent volume of the sme sustrte without ny propgules. Ech experimentl pot system (Figure 1()) ws composed of centrl comprtment (7 L cpcity) contining soil in which seeds were sown nd surrounding peripherl comprtment (7 L cpcity) contining the pre-sterilized soil treted with one of the four soil-zn concentrtions (, 5, 2, 4 mg Zn kg 1 dry soil), s determined from previous study [14]. The two comprtments were seprted y either 5 μm or 2 μm pore-size nylon filter g (Industril Filter Mnufcturing Ltd., Penetnguishene, ON, Cnd) which permitted either the prolifertion of non-am roots (Figure 1() rhizosphere 2 μm), oth AM roots nd hyphe (Figure 1(c) mycorrhizosphere 2 μm), or strictly extrrdicl hyphe (Figure 1(d) hyphosphere 5 μm) into the peripherl comprtment. In this regrd, the 5 μm filter ws deemed lrge enough to llow fungl hyphe to proliferte into the peripherl comprtment, ut smll enough to restrict roots, wheres the 2 μm filter ws permele to oth. The soil-zn tretments dded to the peripherl comprtment were chieved y weighing pproprite volumes of the pre-utoclved soil, dding Zn from ZnSO 4 7H2O stock solution, homogenizing the mixture with n industril mixer, nd ir-drying the soil. This soil pretretment method ws used to reduce the likelihood of metl diffusion from the peripherl comprtment into the centrl comprtment, compred to other point-source experimentl mendment strtegies [15]. The fctoril design of the study (1 plnt sp. 3 sphere tretments (non-am rhizosphere, AM mycorrhizosphere, or AM hyphosphere) 4 soil-zn concentrtions (, 5, 2, or 4 mg kg 1 DS) 4 reps) provided totl of 48 plnts, 1 plnt per pot. Four replictes of unseeded PC CC PC () Figure 1: Schemtiztion of the comprtmentl pot system () showing the prolifertion of hyphe cross the dividing filter. The inoculum (or control) sustrte lyer is shown. Seeds were sown in the centrl comprtment (CC) while the soil-zn tretments were incorported in the surrounding peripherl comprtment (PC) representing either the rhizosphere (), mycorrhizosphere (c), or hyphosphere environments (d). pots were lso prepred for ech of the soil-zn tretments to compre the pre- nd postexperimentl soil-zn iovilility (NNO 3 extrctle) s well s the ph of the re soil. The greenhouse conditions were mintined t 25 C/23 C (dy/night) with 16 hour photoperiod under hlogen lighting, n verge dytime light intensity of μmol m 2 s 1, nd 65% reltive humidity using n Argus greenhouse control system (Argus Control Systems Ltd., White Rock, BC, Cnd). Over the course of the experimentl period, ll plnts s well s the unseeded pots were wtered dily voiding ny leching nd fertilized iweekly (1 ml per week) from weeks 4 to 1 using low zinc Long-Ashton nutrient solution, ph 4.4 [16]: 2. mm K 2 SO 4, 4. mm CCl 2 nhydride, 1.5 mm MgSO 4 7H 2 O, 1.5 mm NH 2 PO 4 H 2 O, 5. μm NH 4 NO 3,.1mM MnSO 4 4H 2 O, 1. μm CuSO 4 5H 2 O, 1. μm ZnSO 4 7H 2 O,.5 mm H 3 BO 3, NCl.9mM,.5 μmn 2 MoO 4 2H 2 O, nd.1 mm EDTA-Fe Plnt Physiologicl nd Chemicl Anlyses. After the 1-week growth period, plnts were hrvested, the roots clen-rinsed with tp wter, nd the length of shoots nd numer of helthy versus chlorotic (e.g., spotted) leves recorded. Plnts were then prtitioned s flowers, shoots (e.g., leves nd stems), nd roots, oven-dried t 7 C for 72 h, nd weighed seprtely. To determine plnt-zn concentrtions, 1 mg of dried flower, shoot, or root smples were ground nd plced in cid-wshed Teflon oms (Nlgene, Rochester, NY, USA). Ech smple ws dissolved in 2 ml of 16 M HNO 3, heted for 1h in () (c) (d)

3 Journl of Botny 3 Tle 1: Pre-experimentl soil chrcteristics. Prmeter Unit Vlue (SE) P ppm 2 (.71) K ppm 9.2 (.86) Mg ppm 214 (5.42) Orgnic Mtter % 3.8 (.25) N ppm 58.3 (7.87) C ppm 1646 (25) CEC K meq/1 g.2 () CEC Mg meq/1 g 1.8 (.5) CEC C meq/1 g 8.2 (1.26) CEC N meq/1 g.3 (.3) CEC Totl meq/1 g 1.6 (1.36) Bse Sturtion K % 2.3 (.21) Bse Sturtion Mg % 17.6 (1.5) Bse Sturtion C % 78.1 (1.67) Bse Sturtion N % 2 (.5) NHCO 3 Extrctle; NH 4 Acette Extrctle; milli-equivlent per 1 g n 8 C wter-th, nd diluted with 2 ml pure grde H 2 O (EMD Chemicl Inc., Drmstdt, Germny). Five smples of pple leves (Stndrd Reference Mteril #1515) from the Ntionl Institute of Stndrds nd Technology (NIST, Githersurg, MD, USA) nd five lnks consisting solely of HNO 3 nd H 2 O were lso prepred to ensure the qulity nd ccurcy of the metl nlyses. All plnt smples were nlyzed using Inductively Coupled Plsm Opticl Emission Spectrometry (ICP OES 73-ES, Vrin Inc., Plo Alto, CA, USA). The instrumentl limit of detection clculted from the procedurl lnks nd reference mteril ws.36 μgg Soil Chemicl Anlyses. The pre-experimentl (e.g., prior to Zn tretment) soil chrcteristics (Tle 1) were determined from five 1 g soil smples dissolved in NHCO 3 or NH 4 cette solutions nd nlyzed using Atomic Asorption Spectrometry (Accutest Lortories, Ottw, ON, Cnd). Further to these nlyses, soil smples for ll tretments were lso collected fter the experimentl period from oth the centrl nd peripherl comprtments t hrvest, nd the unseeded pots smpled prior to nd fter the experimentl growth period. The soil-ph of these smples ws nlyzed directly from 5 g of soil suspended in pure grde H 2 O with soil : solution rtio of 1 : 2.5 (m/v) [17]. To determine soil-zn concentrtions, 5 g soil from ech tretment were suspended in.1 M NNO 3 with soil : solution rtio of 1 : 2.5 (m/v), filtered cross n 11 μm grde #1 cellulose filter pper (Whtmn Inc., Pisctwy, NJ, USA), nd nlyzed with ICP OES [18]. Five smples of Bufflo River sediment (Stndrd Reference Mteril #874) from NIST long with five lnks consisting solely of.1 M NNO 3 were lso nlyzed to ensure the qulity nd ccurcy of the metl nlysis. As with the plnt tissue nlysis, the instrumentl limit of detection clculted from the procedurl lnks nd reference mteril ws.36 μgg Root Coloniztion Assy. At hrvest, 2 g of fresh root smples from ech replicte were crefully excised from the root-zone surrounding the tp-root nd stined with n niline lue.2% dye solution (6.78 mm niline lue 5 ml glycerol 45 ml H 2 Od 5 ml 1% HCl) [19]. Fifty 1-2 cm long root segments per replicte were mounted on slides nd exmined t 1x nd 4x mgnifiction using compound microscope (CX41, Olympus Inc., Mrkhm, ON, Cnd). Smples of non-am roots were lso oserved to ensure their non-mycorrhizl sttus. Mycorrhizl coloniztion ws estimted y determining the % frequency of fungl structures nd % length of root coloniztion s evidenced y the presence of hyphe, vesicles nd ruscules [19, 2]. The equtions for the % frequency (1)nd%length of root coloniztion (2) redefineds No. Segments AM 1%, No. Segments Totl (1) Root Length AM 1%, Root Length Totl (2) Together, these respective indictors provide insight into the reltive distriution nd intensity of the AM root coloniztion [21] Sttisticl Anlyses. One- nd two-wy Anlyses of Vrince (ANOVA) with Bonferonni nd Schefféstudentized rnge tests were performed for men comprisons of plnt metl uptke, plnt growth, nd root coloniztion dt wheres univrite regression models were used to clculte the slopes of soil-ph nd soil-zn dt [22]. Anlyses of Co-Vrince (ANCOVA) were performed on the soil-ph nd soil-zn dt to compre the slopes nd intercepts of the regression equtions etween the tretments. The Kolmogorov-Schmirnoff nd Levene s tests were used to verify the normlity of distriution nd the homogeneity of residul vrince. The dt were Log-trnsformed s required to meet the ssumptions of ech prmetric nlysis. All of the Fisher sttistics (F), coefficients of determintion (r 2 ), degrees of freedom (df ), nd P-vlue estimtes were clculted using S-Plus 8. sttisticl softwre (Insightful Corp., Settle, WA, USA). 3. Results Significnt differences were oserved in flower, shoot, nd root Zn concentrtions etween the hyphosphere, mycorrhizosphere, nd rhizosphere tretments with incresing soil- Zn ddition (Figures 2(), 2(), nd2(c)). Although the plnt Zn concentrtions did not vry mong the tretments t the low soil-zn levels, the rhizosphere tretments hd 2% to 4% higher Zn concentrtions in their tissues thn the mycorrhizosphere tretments, nd 55% to 75% higher concentrtions thn the hyphosphere tretments t oth the 2 nd 4 soil-zn concentrtions. As for plnt growth

4 4 Journl of Botny Flower Zn concentrtion (mg kg 1 DM) cd d d d d d c d d Soil-[Zn] concentrtion (mg kg 1 DS) Shoot Zn concentrtion (mg kg 1 DM) c cd d e e e e e e Soil-[Zn] concentrtion (mg kg 1 DS) () Flowers () Shoots Root Zn concentrtion (mg kg 1 DM) c d d e e e e e e e Soil-[Zn] concentrtion (mg kg 1 DS) (c) Roots Figure 2: Flower (), shoot (), nd root Zn concentrtions (c). Mens (n = 4) nd stndrd errors for the rhizosphere (lck), mycorrhizosphere (grey), nd hyphosphere (white) tretments re shown. Shred letters designte tretments tht re not significntly different ccording to ANOVA coupled with Bonferonni nd Scheffé men comprison tests (P <.5). (Tle 2), the rhizosphere tretments generlly hd greter flower, shoot, nd root dry msses nd longer shoots thn mycorrhizosphere nd especilly hyphosphere tretments; however, these plnt growth prmeters did not show ny prticulr trends in reltion to soil-zn mendment. Still, s symptom of metl toxicity, rhizosphere tretments hd incresingly higher percentges (up to 45.6%) of chlorotic leves compred to mycorrhizosphere (up to 36.%) nd hyphosphere tretments (up to 7.6%). Moreover, roots collected in the peripherl comprtment of rhizosphere nd mycorrhizosphere tretments hd similr dry msses; menwhile, it ws noted tht no roots were found in this comprtment mong the hyphosphere tretments. All roots from the mycorrhizosphere nd hyphosphere tretments were shown to e well colonized s evidenced y the presence of hyphe, vesicles, nd ruscules (Tle 3). More specificlly, higher % frequency of hyphe thn vesicles nd ruscules ws oserved mong ll the tretments, ut slight decline in % hyphl-root length colonized in reltion with incresing soil-zn levels. In ddition, rhizosphere tretments were confirmed to e nonmycorrhizl. The soil-zn concentrtions were mesured in the peripherl (Figure 3()) nd centrl comprtments (Figure 3()) nd regression models clculted for ech tretment (Tle 4). The pre-experimentl soil-zn concentrtion in the peripherl comprtment indicted liner increse in reltion with incresing soil-zn mendment reching up to 44.1 mg kg 1 dry soil. The postexperimentl soils indicted polynomil (e.g., hyperolic) profiles in the descending order of unseeded re-soil (272.5 mg kg 1 dry soil), hyphosphere (263.4), mycorrhizosphere (247.2), nd rhizosphere tretments (199.2). By contrst, there were no significnt trends (e.g., no slope) for soil-zn in the centrl comprtment with vlues rnging etween.1 nd.7 mg kg 1

5 Journl of Botny 5 NNO3 extrct. soil-[zn] (mg kg 1 DS) NNO3 Extrct. Soil-[Zn] (mg kg 1 DS) Soil-[Zn] (mg kg 1 DS) () c c c 5 Soil-[Zn] (mg kg 1 DS) Pre-expt Un-seeded () Figure 3: Soil-Zn concentrtions mesured in the peripherl () nd centrl comprtments (). Mens (n = 4) nd stndrd errors for the pre-experimentl unseeded (lck dimond), post-experimentl unseeded (lck squre), hyphosphere (white squre), mycorrhizosphere (white tringle), nd rhizosphere (white circle) tretments re shown. Shred letters designte regression equtions hving slopes tht re not significntly different ccording to ANCOVA (P <.5). dry soil. The soil-ph ws lso mesured in the peripherl (Figure 4()) nd centrl comprtments (Figure 4()) nd regression models clculted for ech tretment (Tle 5). Unlike the soil-zn concentrtions, the pre-experimentl soilph in the peripherl comprtment indicted liner decline from 6.3 to 5.53 with incresing soil-zn levels. Menwhile, the postexperimentl soils lso indicted significnt decreses in ph, ut profiles hving polynomil trends in the descending order of unseeded re-soil (from 5.8 to 5.28), hyphosphere (from 5.69 to 5.26), mycorrhizosphere (from 5.59 to 5.23), nd rhizosphere tretments (from 5.52 to 5.17). Although there were no significnt trends (e.g., no slope) for soil-ph in the centrl comprtment, the rnge vlues were grdully lower in the order of pre-experimentl unseeded re-soil ( ), postexperimentl unseeded re-soil ( ), hyphosphere ( ), mycorrhizosphere ( ), nd then rhizosphere tretments ( ). 4. Discussion Consistent with our conceptul model regrding the impct of AM symiosis on plnt growth nd metl uptke [8, 1], the Zn concentrtions of plnts from the mycorrhizosphere tretments were generlly lower thn rhizosphere tretments t the highest soil-zn levels which resulted in AM plnts hving lower incidence of metl toxicity (e.g., lef chlorosis). These findings re in line with other studies tht detected lower Cd, Zn, nd P uptke nd n improved growth sttus in vrious AM (Glomus sp.) versus non-am plnts [23 26]. We ttriute such enhnced metl-stress tolernce with AM-induced iosorption processes, including hyphl metl-inding nd metl-lignd precipittion [27 29], tht reduce metl iovilility in the mycorrhizosphere to decrese plnt metl uptke [3, 5, 6]. Correspondingly, further studies hve shown tht metls cn e tken up nd sequestered in fungl tissues insted of eing trnsferred to

6 6 Journl of Botny Soil-pH Soil-[Zn] (mg kg 1 DS) c Soil-pH 5.8 () c c c 5 Soil-[Zn] (mg kg 1 DS) Pre-expt Un-seeded () Figure 4: Soil-pH mesured in the peripherl () ndcentrl comprtments (). Mens (n = 4) nd stndrd errors for the pre-experimentl unseeded (lck dimond), post-experimentl unseeded (lck squre), hyphosphere (white squre), mycorrhizosphere (white tringle), nd rhizosphere (white circle) tretments re shown. Shred letters designte regression equtions hving slopes tht re not significntly different ccording to ANCOVA (P <.5). roots [4, 3, 31], therey reducing cellulr oxidtive stress nd delying the onset of plnt metl toxicity [32]. Forthese resons, mycorrhizl iosorption nd hyphl sequestrtion re considered to e importnt stress voidnce strtegies tht protect host plnts in complement to their intrinsic detoxifiction mechnisms, such s metllotheinin nd phytocheltin metolisms [33]. In ddition to compring the metl uptke profiles of AM versus non-am plnts, our two-comprtment pot system ws specificlly designed to determine the reltive contriutions of extrrdicl hyphe to host plnt Zn uptke. Accordingly, in spite of hving significntly lower Zn concentrtion levels thn the rhizosphere nd mycorrhizosphere tretments, plnts of the hyphosphere tretments still hd incresingly higher Zn concentrtions even t the highest soil-zn tretments. In greement with our in vitro study [1], this finding indictes tht hyphl metl uptke nd its trnsfer to roots cn contriute in incresing plnt metl sttus despite the iosorption effect which typiclly reduces it, s in the cse of other metls such sni,cr,cs,ndp[34 37]. For this reson, we elieve tht the enhnced uptke nd iosorption processes occur independently: the first incresing the sorptive cpcity of roots nd the second regulting the iovilility of metls in soils. Thus, it is their comined effects tht shpe the host plnt s metl uptke profile. Contrry to our conceptul model, we did not detect ny significnt differences in plnt Zn uptke nor ny mjor symptoms of nutrient deficiency mong plnts of the - control nd 5 soil-zn levels. In this cse, these soil-zn tretments likely did not impose ny perceptile nutrient deficiency stress or growth limittions which cn otherwise led plnts to invest more in AM symiosis to supplement their nutritionl sttus [38, 39]. This cn e ttriuted to the pre-experimentl soil-zn levels nd fertiliztion regimes used in our study which likely provided sufficient Zn for n optiml plnt growth mong these tretments. As such, further experimentl investigtion my e needed to verify this fcet of our proposed model which proposed tht the AM symiosis would enhnce plnt metl uptke to circumvent deficiency conditions. Still, we did oserve the generl trends of slightly lower dry msses, fewer leves, nd shorter shoots mong the mycorrhizosphere nd especilly the hyphosphere

7 Journl of Botny 7 Tle 2: Plnt growth prmeters. Soil-Zn (mg kg 1 DS) Sphere tretments Flowers (.9) (.43) (.4) (.51) (.19) (.33) Dry mss (g) Leves Shoot Roots- Roots- % height Shoots Totl no. CC PC Chlorotic (cm).77 (.3) (.) 14.5 (1.19) (.21) (.7) (.25) (.12) (.4) (2.21) (.51) c (1.84) (.78) c (2.24) (.) d (2.9) (.14) (.11) (.12) (.32) (.2) (.34).89 (.6) (.) 14.3 (.85) (.7) (.3) (1.14) (.4) (.6) (2.3) (2.) (1.7) (1.88) (1.88) (4.18) (2.26) (.12) (.28) (.33) (.17) (.17) (.9).68 (.9) (.) 13.8 (.62) (.17) (.2) (.64) (.4) (.4) (2.12) (2.) (1.98) (6.6) (2.56) (11.6) (2.38) (.8) (.16) (.29) (.15) (.31) (.26) F-vlues nd levels of significnce.61 (.11) (.) 13.5 (1.83) (.6) (.5) (1.3) (.1) (.2) (.96) (4.63) (1.4) (1.7) (1.53) (1.9) (2.67) Soil-Zn (Zn).63 ns 1.31 ns ns 2.5 ns ns Sphere tretments (S) ns ns ZnxS.73 ns 1.27 ns.85 ns.95 ns.7 ns ns Block 1.28 ns 2.4 ns.85 ns.11 ns.58 ns.35 ns 1.41 ns Mens (n = 4) nd SE (inside prentheses) re shown. Shred letters within ech column designte tretments tht re not significntly different ccording to Bonferonni nd Scheffémencomprisontests. Centrl Comprtment; Peripherl Comprtment. ns nonsignificnt; P <.5; P <.1; P <.1. tretments compred to the rhizosphere tretments, which my hve resulted from the metolic cost of mintining the symiotic ssocition through the trnsfer of plnt crohydrtes to the AM fungus [4]. Corresponding with the considerle levels of AM root coloniztion reported here (e.g., s evidenced y the high frequency nd distriution of fungl hyphe, ruscules, nd vesicles), the reduction in AM thn non-am plnt iomss could e ssocited with the host plnts cron lloction in developing the mycorrhizospheric infrstructure. Alterntively, this tendency could lso e ttriuted, in prt, to the design of our comprtmentl pot systems, especilly mong the hyphosphere tretments. In this cse, the 5 μm filter gs themselves my hve limited the rootle volume of the hyphosphere plnts to then reduce their overll growth [41]. Nevertheless, the similr levels of AM root coloniztion nd undnce of ll fungl structures mong oth the mycorrhizosphere nd hyphosphere tretments would suggest tht the symiotic investment ws similr etween these tretments, nd tht the effect of our experimentl design on plnt growth nd metl uptke ws negligile. As for the soil conditions, the ddition of Zn in the form of ZnSO 4 cused liner decrese of the pre-experimentl soil-ph which ws likely prompted y the proportionl increse of SO 2 4 in the soil solution [24]. Furthermore, the dily wtering nd fertiliztion regimes lso ffected the soil conditions such tht the soil-ph nd soil-zn levels ll showed polynomil rther thn liner profiles. Over time, the influx

8 8 Journl of Botny Tle 3: AM root coloniztion. Soil-Zn (mg kg 1 DS) 5 2 Sphere tretments % Frequency Fungl structures Hyphe Vesicles Aruscules % Root length % Frequency % Root Length % Frequency % Root length 28.5 (1.7) 5.5 (1.9) 21. (3.1) 36.5 (8.8) 21.5 (2.9) 26. (1.8) 29. (6.2) 57.5 (4.8) 25.5 (7.3) 29. (3.3) 17.5 (3.2) 3. (2.) 34. (8.6) 56.3 (2.8) 22. (2.6) 27.5 (6.3) 27.5 (3.3) 32.5 (2.5) 26.5 (5.6) 3. (4.1) 23.5 (3.) 3. (4.1) 13.8 (4.4) 25. (2.9) 22. (5.2) 29.5 (1.) 19.5 (5.6) 29.5 (5.) 2.5 (5.1) 28.5 (1.5) 32.5 (3.8) 21.5 (14.5) 9.5 (3.4) 21.5 (6.5) 18.5 (3.) 16.3 (2.4) (7.7) 35. (6.5) 22.5 (4.6) 35. (6.5) 19.5 (3.9) 25. (6.5) 33.5 (5.2) 3.5 (1.5) 17.5 (4.9) 2.5 (3.9) 24.5 (5.7) 31.3 (3.1) F-vlues nd levels of significnce Soil-Zn (Zn).94 ns.28 ns 2.13 ns.64 ns.17 ns 1.55 ns Sphere Tretments (S).4 ns.55 ns.67 ns 3.31 ns 1.62 ns.98 ns ZnxS.27 ns ns.86 ns 1.78 ns 3.47 Block.17 ns ns.83 ns.13 ns Mens (n = 4) nd SE (inside prentheses) re shown. Shred letters within ech column designte tretments tht re not significntly different ccording to Bonferonni nd Scheffémencomprisontests. ns nonsignificnt; P <.5. Tle 4: Regression models for soil-zn s function of soil-zn tretment. Prmeter Comprtment Anlysis Tretment f (x) F r 2 df P Soil-Zn Peripherl Centrl Pre-Experimentl Unseeded 1.15x ,14 <1 3 Post-Experimentl Unseeded (4.7 e 4)x x 1.46 (4.7 e 4)x x 3.76 (7.5 e 4)x x x x ,14 < ,13 < ,13 < ,13 <1 3 Pre-Experimentl Unseeded (1. e 7)x ,14.75 Unseeded (1. e 7)x ,14.27 Post-Experimentl (1. e 7)x ,14.25 (1. e 7)x ,14.24 (1. e 7)x ,14.96 Polynomil equtions [f(x)], Fisher vlues [F], coefficients of determintion [r 2 ], degrees of freedom [df ], nd estimtes of P-vlue re shown. of SO 2 4 nd H + /H 3 O + ions in the soil solution ssocited with the ddition of Zn nd wter lters the soil s redox equilirium nd impcts the soluility of metl nutrients [42 45]: process referred to s metl geing [46]. Such inputs nd metl geing processes cn detrimentlly influence the soil s metl-inding cpcity, resulting in n incresed rte of metl leching [45, 47, 48]. For these resons, it is noteworthy tht the presence of roots nd (or) extrrdicl hyphe further ffected soil conditions in wy tht the soil-ph ws incresingly more cidic nd the soil-zn iovilility ws grdully lower in the order of hyphosphere, mycorrhizosphere, nd rhizosphere tretments. These tendencies re primrily ttriutle to the different rtes of Zn uptke etween the tretments nd the susequently different soil-zn depletion zones. Notwithstnding, the exudtion of orgnic cheltors y roots nd (or) extrrdicl hyphe my lso hve plyed prt in shping the edphic conditions, s in the cse of

9 Journl of Botny 9 Tle 5: Regression models for soil-ph s function of soil-zn tretment. Prmeter Comprtment Anlysis Tretment f (x) F r 2 df P Soil-pH Peripherl Centrl Pre-Experimentl Unseeded.2x ,14 <1 3 Post-Experimentl Unseeded (3.8 e 6)x 2 (2.8 e 6)x (3.7 e 6)x 2 (2.6 e 6)x (2.8 e 6)x 2.21x (1.9 e 6)x 2 (1.6 e 6)x ,13 < ,13 < ,13 < ,13 <1 3 Pre-Experimentl Unseeded (1. e 4)x ,14.21 Unseeded (1. e 4)x ,14.25 Post-Experimentl (3.7 e 5)x ,14.59 (3.2 e 5)x ,14.64 (2.8 e 5)x ,14.71 Polynomil equtions [f(x)], Fisher vlues [F], coefficients of determintion [r 2 ], degrees of freedom [df ], nd estimtes of P-vlue re shown. phosphorus nd nitrogen cquisition y mycorrhize [49 53]. From these studies, it hs een suggested tht the extrrdicl hyphe cn induce the moderte lklinistion of their growth sustrte in reltion to P nd N uptke wheres roots tend to cidify it. This could e relevnt to our proposed conceptul model of AM-plnt metl uptke since the process of hyphl lklistion could fvour metl iosorption to contriute in reducing metl iovilility, unlike root cidifiction tht my fcilitte leching nd then increse metl soluility. Accordingly, we consider it intriguing for future experimentl investigtions to exmine these mechnisms more closely (e.g., metl iosorption nd hyphl lklinistion) within the context of plnt metl uptke in order to further elucidte the impct of the extrrdicl hyphe on soil metl iovilility. Altogether, when tking into ccount the impct of the AM symiosis in stilizing the soil mtrix nd enhncing its wter nd nutrient retention cpcity [54 56], the mycorrhizosphere should ply key role in enhncing the soil s resiliency in reltion to metl stress conditions rnging from trce to toxicity exposure levels. In conclusion nd in line with our hypotheticl model, we demonstrted tht the AM symiosis plys n importnt prt in shping host-plnt Zn uptke, prticulrly y reducing plnt tissue Zn concentrtions t high exposure levels nd therey delying the onset of Zn toxicity in shoots nd flowers compred to non-am plnts. Accordingly, we lso suggested tht the mycorrhizosphere plys n eqully importnt role in shping the soil environment vi iosorption processes nd other AM-induced chemicl chnges which contriute in enhncing the soil s resiliency. As such, we consider the AM symiosis to e n essentil component of extrinsic stress tolernce nd key fctor of ecosystem function. Menwhile, we lso consider our model depicting the impct of the AM symiosis on plnt nd soil systems to e relevnt tool in environmentl remedition prctices y conceptully determining the fte of metl contminnts in reltion with the ctivities of plnts nd soil microorgnisms. Arevitions AM: Arusculr mycorrhiz ICP OES: Inductively coupled plsm opticl emission spectrometry Zn: Zinc. Acknowledgments Specil thnks re given y the uthors to Dr. Andre Polle (Editor) nd the nonymous evlutors for their insightful review of this mnuscript. The uthors lso thnk Dr. N. DeSilv (Deprtment of Erth Sciences, University of Ottw) for ICP OES nlyses. This study ws mde possile y Cnd Grdute Scholrship to P. Audet nd Discovery Grnt to C. Chrest from the Nturl Science nd Engineering Reserch Council of Cnd. References [1] A. Schüßler, D. Schwrzott, nd C. Wlker, A new fungl phylum, the Glomeromycot: phylogeny nd evolution, Mycologicl Reserch, vol. 15, no. 12, pp , 21. [2] P. Christie, X. Li, nd B. Chen, Arusculr mycorrhiz cn depress trnsloction of zinc to shoots of host plnts in soils modertely polluted with zinc, Plnt nd Soil, vol. 261, no. 1-2, pp , 24. [3] P. Jeffries, S. Gininzzi, S. Perotto, K. Turnu, nd J.-M. Bre, The contriution of rusculr mycorrhizl fungi in sustinle mintennce of plnt helth nd soil fertility, Biology nd Fertility of Soils, vol. 37, no. 1, pp. 1 16, 23. [4] E. J. Joner, R. Briones, nd C. Leyvl, Metl-inding cpcity of rusculr mycorrhizl mycelium, Plnt nd Soil, vol. 226, no. 2, pp , 2. [5] C. Leyvl, K. Turnu, nd K. Hselwndter, Effect of hevy metl pollution on mycorrhizl coloniztion nd function: physiologicl, ecologicl nd pplied spects, Mycorrhiz,vol. 7, no. 3, pp , [6] A. A. Mehrg, The mechnistic sis of interctions etween mycorrhizl ssocitions nd toxic metl ctions, Mycologicl Reserch, vol. 17, no. 11, pp , 23.

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