Research Article Identification of Constraining Experimental-Design Factors in Mycorrhizal Pot-Growth Studies

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1 Journl of Botny Volume, Artile ID 7113, pges doi:.1155//7113 Reserh Artile Identifition of Constrining Experimentl-Design Ftors in Myorrhizl Pot-Growth Studies Ptrik Audet nd Christine Chrest Deprtment of Biology, Ottw Crleton Institute of Biology, University of Ottw, 3 Mrie Curie, Ottw, ON, Cnd K1N N5 Correspondene should e ddressed to Ptrik Audet, pude@uottw. Reeived 1 Jnury ; Revised 1 Mrh ; Aepted 7 April Ademi Editor: Zed Rengel Copyright P. Audet nd C. Chrest. This is n open ess rtile distriuted under the Cretive Commons Attriution Liense, whih permits unrestrited use, distriution, nd reprodution in ny medium, provided the originl work is properly ited. In the ojetive of testing the design of pot-growth experiments, we onduted two greenhouse studies of dwrf sunflower ultivr nd n rusulr myorrhizl (AM) fungus to determine how pot size nd inoulum distriution ffet plnt growth nd AM symiosis. As predited, lrge-potted plnts developed greter overll iomss nd root oloniztion thn smllpotted ones whih we ttriuted to the lrger rootle volume. Furthermore, plnts grown in nd of high density inoulum sustrte showed higher prevlene of fungl vesiles (sites of lipid storge) inditing more dvned level of root oloniztion ompred to those grown in dispersed inoulum sustrte; this likely eing due to the higher frequeny of intertion etween roots nd fungl propgules. In seond experiment, lrge-potted AM plnts showed greter tolerne to wter defiit thn non-am ontrol plnts; however, this myorrhizl effet ws not deteted mong smll-potted plnts. We onlude tht reful onsidertion should e mde towrd design prmeters to limit result ises nd ultimtely filitte omprison of findings etween studies. 1. Introdution Studies involving pot-grown plnts represent n underpinning of omprtive plnt physiology. However, design prmeters suh s the size of the experimentl miroosm (e.g., pot size) my onfound the interprettion of results regrdless of the intended experimentl onditioning [1 ]. Speifi to the study of myorrhiz mutulisti ssoition etween plnt roots nd soil fungi we elieve tht the pot size nd fungl inoulum distriution my indvertently ffet plnt growth nd symioti intertion. It n e hypothesized tht pot size influenes the dynmis of resoure llotion nd myorrhizl symiosis y potentilly restriting the prolifertion of roots nd extrrdil hyphe [5]. Menwhile, the distriution of fungl inoulum my lso influene the frequeny of intertion etween roots nd fungl propgules whih then ffets the level of root oloniztion nd symioti tivity []. To test these notions, we onduted ftoril greenhouse study of dwrf sunflower nd n rusulr myorrhizl (AM) fungus to disern how the pot size nd fungl inoulum distriution ffet plnt growth nd root oloniztion. Given tht the AM symiosis is widely reognized for enefiting plnts under vrious environmentlly stressful onditions [7 11], we then onduted follow-up experiment inorporting wter defiit tretment to investigte the effets of pot size on the dynmis of symioti ssoition nd stress tolerne. By identifying key design ftors tht influene plnt growth nd myorrhizl ssoition, it is our gol to improve upon the design of pot-growth experiments to filitte more effetive omprison of findings etween studies.. Mterils nd Methods.1. Experimentl Design. In the first pot experiment, dwrf sunflower (Helinthus nnuus L. vr. Pino) plnts were grown from seeds (MKenzie Seeds, Brndon, MB, Cnd) for weeks in previously utolved, low minerl soil mixture (snd:potting soil, 1:1 v/v). Plnts were grown in either smll ( L) or lrge-sized (7.5 L) pots ontining the sme se soil mixture ut inorporting

2 Journl of Botny Pot size Lrge Smll ontrol or AM-HDL inoulum sustrtes for weeks under the sme onditions s desried ove, ut lso inorporting wter defiit regime ording to Ry nd Sinlir [3]. This regime onsisted of monitoring the wter ontent of ll soils vi time-domin refletometry [1], nd speifilly reduing the reltive soil moisture from 9% to % for the drought-treted plnts over the ourse of weeks to. All plnts reeived iweekly fertiliztion ( ml per week) s desried ove. The ftoril design of the study (1 plnt sp. soil tretments [Control; AM-HDL] pot sizes [Smll; Lrge] irrigtion tretments [Well-Wtered; Droughted] 5 reps) provided totl of plnts (1 plnt per pot). Non-AM AM-DI AM-HDL Inoulum distriution Figure 1: Shemtiztion of the pot size nd inoulum distriution tretments. one of three fungl inoulum tretments (noninoulted [Control], dispersed inoulum [AM-DI], or high density lyered inoulum [AM-HDL]) whih resulted in totl soil volume equivlent to the respetive pot size pity (Figure 1). To test the effets of fungl inoulum distriution, the AM-soil tretments were hieved y inorporting fungl propgules (e.g., spores nd hyphe) of Glomus intrrdies Shenk & Smith (isolte DAOM-11) from ommeril inoulum hving reported density of 15 propgules g 1 dry sustrte (Myke Pro Endo, Premier Teh, Premier Teh, Rivière-du-Loup, QC, Cnd). The AM- DI tretment onsisted of the inoulum sustrte eing evenly mixed into the soil using n industril mixer, wheres the AM-HDL tretment onsisted of the sme inoulum integrted s 3 m lyer of sustrte. Due to the respetive pot-size volumes, it ws estimted tht smll pots ontined 75 totl propgules (5 g inoulum sustrte dose) while lrge pots ontined 75 3 propgules ( g inoulum sustrte dose). The ftoril design of the study (1 plnt sp. 3 soil tretments [Control; AM-DI; AM-HDL] pot sizes [Smll; Lrge] 5reps)providedtotlof 3 plnts (1 plnt per pot), eh distriuted in ftoril lok design. The greenhouse onditions were mintined t 5 C/3 C (dy/night) with 1-hour photoperiod, n verge light intensity of 3. µmol m s 1, nd 5% reltive humidity. Over the ourse of the experimentl period, ll plnts were wtered dily s required voiding ny lehing nd fertilized iweekly ( ml per week) from weeks to using low phosphorus Long-Ashton nutrient solution (K SO. mm, CCl nhydride. mm, MgSO 7H O 1.5 mm, NH PO H O1.5mM,NH NO 3 5. µm, MnSO H O.1mM,CuSO 5H O1.µM, ZnSO 7H O1.µM, H 3 BO 3.5 mm, NCl.9 mm, N MoO H O.5µM, nd EDTA-Fe.1 mm). In the seond experiment, plnts were grown in similr smll nd lrge-sized pots ontining either the non-am.. Hrvest nd Determintion of Root Coloniztion. Plnts were hrvested weeks fter seeding with roots eing thoroughly lened with tp wter. Plnt orgns were then prtitioned s roots, shoots, or flowers, oven-dried t 7 C for 7 h, nd weighed seprtely. At the time of hrvest, fresh root smples ( g) from eh replite were refully exised from the pil zone ner the tproot nd stined with niline lue.% dye solution (.7 mm niline lue; 5 ml glyerol; 5 ml H Od; 5 ml 1% HCl) ording to Dlpé [13]. One hundred, 1- m long, root segments per replite were rndomly seleted, mounted on slides, nd exmined t x nd x mgnifition using ompound mirosope (CX1, Olympus In., Centre-Vlley, PA, USA). Smples of non-am roots were lso prepred to ensure their nonmyorrhizl sttus. Myorrhizl oloniztion ws estimted y determining the % frequeny of oloniztion [13], % length of root oloniztion [1], nd reltive density (# mm 1 root length) of fungl strutures (e.g., intrrdil vesiles nd rusules). The equtions for the % frequeny (1), % length of root oloniztion (), nd reltive density (3)redefineds: #Segments AMstrutures #Segments totl %, (1) RootLength AMstruture RootLength totl %, () #AMstrutures RootLength olonized. (3).3. Sttistil Anlyses. One- nd multi-wy nlyses of vrine (ANOVA) with Bonferonni nd Sheffé studentized rnge tests were performed for men omprison nlyses [15]. The Kolmogorov- Shmirnoff nd Levene s tests were used, respetively, to verify the normlity of distriution nd homogeneity of residul vrine. The dt were Logtrnsformed s required to meet the ssumptions of prmetri nlyses. All of the Fisher sttistis (F), degrees of freedom (df), nd P-vlue estimtes were lulted using S-Plus. sttistil softwre (Insightful Corp., Settle, WA, USA).

3 Journl of Botny 3 1 Flower DM (g) Shoot DM (g) Smll Lrge Smll Lrge () () Root DM (g) Smll Lrge Non-AM AM-DI AM-HDL () Figure : Flower (), shoot (), nd root () dry msses from the pot size nd inoulum distriution experiment. Mens (n = 5) nd SE for the non-am (empty rs), AM-DI (grey rs), nd AM-HDL tretments (solid rs) re shown. Shred letters indite tretments tht re not signifintly different ording to Bonferonni nd Sheffé men omprison tests. Tle 1: Root oloniztion summry for the pot size nd inoulum distriution experiment. Frequeny (%) Root Length Colonized (%) Density (mm 1 ) Pot size Inoulum Hyphe Vesiles Arusules Hyphe Vesiles Arusules Vesiles Arusules Smll AM-DI 1. (3.1) 7. (1.) 1. (3.1).5 (3.9).7 (3.1) 5. (.1) 1.5 (.5) 7. (.) AM-HDL. (.) 1. (.3). (.5) 7. (3.1) 35. (5.) 59.1 (5.9) 3. (.). (.3) Lrge AM-DI. (.1). (.7) 39. (3.5) 75.3 (.5) 1. (.) 5.9 (.5). (.1) 7. (.7) AM-HDL 3. (.) 37 (3.) 3.5 (3.5).1 (5.1) 5.1 (.1) 53. (.).9 (.) 3. (.3) F-vlues nd levels of signifine Pot size (P) ns 5.. ns.3 ns 1.5 ns Inoulum (In).5 ns.3. ns ns PxIn.1 ns..3 ns. ns 1.1 ns. ns. ns 9. Blok 3. ns 3. ns 1. ns. ns.7 ns.1 ns.3 ns.3 ns Mens (n = 5) nd SE (inside prentheses) re shown, Shred letters within eh olumn designte tretments tht re not signifintly different ording to Bonferonni nd Sheffé men omprison tests, ns nonsignifint, P <.5, P <.1, P <.1.

4 Journl of Botny Flower DM (g) Shoot DM (g) 1 1 Smll Lrge Smll Lrge () () Root DM (g) Smll Lrge Non-AM drought Non-AM well-wtered () AM drought AM well-wtered Figure 3: Flower (), shoot (), nd root () dry msses from the pot size nd wter defiit experiment. Mens (n = 5) nd SE for the non- AM drought (empty rs), non-am well-wtered (light-grey rs), AM drought (drk-grey rs), nd AM well-wtered tretments (solid rs) re shown. Shred letters indite tretments tht re not signifintly different ording to Bonferonni nd Sheffé men omprison tests. 3. Results In the first experiment, the root, shoot, nd flower dry msses were over two-fold greter for the lrge versus smll-potted plnts (Figure ); these tendenies lso eing oserved for the fresh msses (dt not shown). More speifilly, the flower dry mss of lrge-potted plnts (Figure ()) ws grdully smller when ompring the Control (7.1 g), AM- DI (.5 g), nd AM-HDL (. g) tretments; wheres smllpotted plnts (rnging etween.9 nd 1.3 g) showed no suh signifint differenes. Although smll-potted plnt roots showed some inidene of eing pot-ound, none of these plnts demonstrted ny visul signs of nutrient defiieny (dt not shown). As for the root oloniztion (Tle 1), ll AM plnt tretments were shown to e well olonized s evidened y the presene of hyphe, vesiles, nd rusules, lthough the % frequeny nd length of root oloniztion were generlly greter mong lrge versus smll-potted plnts. In ddition, the fungl inoulum distriution lso influened root oloniztion s greter length of oloniztion, nd density of vesiles were oserved for the AM-HDL tretments; yet, higher density of rusules for the AM-DI tretments. In the seond experiment, the root, shoot, nd flower dry msses were gin greter for the lrge versus smllpotted plnts (Figure 3). The well-wtered plnts generlly produed greter iomss thn the drought-treted ones (oth lrge nd smll pots) whih showed symptoms of wilting nd hlorosis (dt not shown). Notly, the AMdroughted plnts grown in lrge pots showed flower (3. g) nd shoot (9. g) dry msses similr to those of well-wtered plnts (. g flower, 9.9 g shoot), wheres non-am droughted plnts suffered redution in dry mss. The AM-drought smll-potted plnts showed no suh growth dvntge over non-am drought plnts. As in the first experiment, ll AM plnt tretments were well olonized nd lrge-potted plnts

5 Journl of Botny 5 Tle : Root oloniztion summry for the pot size nd wter defiit experiment. Frequeny (%) Root Length Colonized (%) Density (mm 1 ) Pot size Irrigtion Hyphe Vesiles Arusules Hyphe Vesiles Arusules Vesiles Arusules Smll Drought 33. (.7). (7.1) 5. (.) 5. (5.). (5.). (1.9). (.) 3. (.1) Well-Wtered. (3.) 37. (.1) 5. (.) 7. (5.1) 3. (3.) 35.7 (9.) 3. (.3) 3. (.) Lrge Drought. (9.7) 3. (9.5) 31. (.3) 71. (.1). (5.7) 39.5 (.).7 (.) 3.5 (.1) Well-Wtered 5. (.) 3. (.) 3. (7.1) 5. (1.1) 3. (1.3) 5.1 (.) 3. (.) 3.7 (.1) F-vlues nd levels of signifine Pot size (P) 1.9 ns. ns.9 ns.7 1. ns 1.. ns 1.1 ns Irrigtion (Ir). ns. ns.5 ns.7 ns. ns 1. ns. ns.9 ns PxIr. ns 1. ns. ns ns Blok. ns. ns. ns.7 ns.7 ns.. ns 1. ns Mens (n = 5) nd SE (inside prentheses) re shown, Shred letters within eh olumn designte tretments tht re not signifintly different ording to Bonferonni nd Sheffé men omprison tests, ns nonsignifint, P <.5, P <.1, P <.1. gin generlly tended to hve higher % frequeny nd length of root oloniztion thn the smll-potted plnts (Tle ). Menwhile, the overll AM root oloniztion of well-wtered plnts ws greter thn drought-treted ones.. Disussion We report tht pot size is key experimentl ftor tht, when limiting, n signifintly redue plnt growth nd AM root oloniztion. We ssoite this finding with the ft tht the pot is n rtifiil miroosm hving smller rootle volume thn nturl environments []. This rootle volume stritly determines the totl nutrient supply in the rhizosphere nd then limits the plnts reltive growth potentil (e.g., % of mximum growth) without neessrily expressing ny notiele symptoms of nutrient defiieny, s reported with mize, potto, soyen, nd young sprue [ ]. By restriting the prolifertion of roots nd extrrdil hyphe, the rootle volume lso influenes the investment in AM symiosis sine the primry enefit of myorrhizl ssoition (e.g., n enhned resoure quisition pity) is redued, then resulting in lower overll root oloniztion mong smll-potted plnts [1]. Aordingly, when the pot size is less onstrining, our results demonstrte tht plnt growth nd AM oloniztion re oth signifintly inresed. Besides pot size, we determined tht the dispersl of the fungl inoulum further ffeted AM root oloniztion suh tht plnts grown in the high density lyered (AM-HDL) sustrte showed higher frequeny nd density of vesiles thn those grown in the dispersed inoulum (AM-DI) sustrte. By ontrst, those grown in the AM-DI sustrte showed higher density of rusules. The prevlene of vesiles (sites of lipid storge) over rusules (sites of resoure exhnge) indites tht the AM fungus hd rehed more dvned stge of its life yle [1]. As predited, this tendeny my e ttriuted to the ft tht roots of AM-HDL plnts likely hd erlier oloniztion enounters with fungl propgules ompred to roots of AM-DI plnts whih hd reltively more diffuse propgule distriution. For this reson, we suspet tht the higher density inoulum distriution ould e more effetive in promoting root oloniztion thn dispersed one despite hving similr totl fungl propgules per pot. Furthermore, we lso suspet tht suh differenes in myorrhizl investment ould ount for the grdully lower flower dry msses produed y non-am ontrol, AM-DI, nd AM-HDL plnts, prtiulrly owing to the differentil resoure llotion ssoited with mintining the symiosis [17]. From the seond experiment, we demonstrted tht the pot size effet my onfound the dynmis of symioti ssoition nd plnt stress tolerne. Besides hving greter overll iomss nd root oloniztion for the lrge versus smll-potted plnts (s ove), the AM-droughted plnts produed similr shoot nd flower iomss ompred to the well-wtered plnts wheres non-am plnts suffered growth redution. Unlike smll potted-plnts whih showed no suh differenes, lrge potted AM-plnts espeilly enefited from the myorrhizosphere s enhned resoure quisition pility whih provided them with signifint growth dvntge over non-am plnts when sujeted to wter-defiit onditions [, 11, 17]. In ddition, it is possile tht the myorrhizosphere lso provided indiret enefits to host plnts y enhning soil stility [1, 19] nd moisture retention [, 1] therey uffering the soil environment. In this regrd, more thorough investigtion into the extent of the myorrhizosphere would e enefiil to speifilly verify these perspetives. In onlusion, this study hs shown tht pot size nd fungl inoulum distriution re key experimentl ftors tht ffet plnt growth nd AM root oloniztion y respetively influening () the rootle volume nd () the likelihood of intertion etween roots nd fungl propgules. When limiting, these ftors my influene the interprettion of results regrdless of the intended experimentl onditioning, suh s drought resistne. By tking these ftors into

6 Journl of Botny onsidertion, it is possile to redue result ises nd filitte the omprison of findings etween studies. Arevitions AM: Arusulr myorrhizl AM-DI: Arusulr myorrhizl dispersed inoulum AM-HDL: Ausulr myorrhizl high density lyered inoulums. Aknowledgments Speil thnks re given y the uthors to Dr. Zed Rengels (Editor-in-Chief) nd the two nonymous evlutors for their insightful review of this mnusript. This study ws mde possile y Cnd Grdute Sholrship to P.A. nd Disovery Grnt to C.C. from the Nturl Siene nd Engineering Reserh Counil of Cnd. Referenes [1] E. Bååth nd D. S. Hymn, Effet of soil volume nd plnt density on myorrhizl infetion nd growth response, Plnt nd Soil, vol. 77, no. -3, pp , 19. [] G. R. Blli, M. R. Hdi, P. Yvri, H. Bidrm, A. G. Nderi, nd A. Eslmi, Effet of pot size, plnting dte nd genotype on minituer prodution of Mrfon potto ultivr, Afrin Journl of Biotehnology, vol. 7, no. 9, pp ,. [3] J.D.RyndT.R.Sinlir, Theeffetofpotsizeongrowth nd trnspirtion of mize nd soyen during wter defiit stress, JournlofExperimentlBotny, vol. 9, no. 35, pp , 199. [] J. Townend nd A. L. Dikinson, A omprison of rooting environments in ontiners of different sizes, Plnt nd Soil, vol. 175, no. 1, pp , [5] R. T. Koide, Density-dependent response to myorrhizl infetion in Autilon theophrsti Medi, Oeologi, vol. 5, no. 3, pp , [] L. K. Aott nd A. D. Roson, Ftors influening the ourrene of vesiulr-rusulr myorrhizs, Agriulture, Eosystems nd Environment, vol. 35, no. -3, pp , [7] P. Audet nd C. C. Chrest, Dynmis of rusulr myorrhizl symiosis in hevy metl phytoremedition: metnlytil nd oneptul perspetives, Environmentl Pollution, vol. 17, no. 3, pp. 9 1, 7. [] P. Audet nd C. C. Chrest, Allotion plstiity nd plntmetl prtitioning: met-nlytil perspetives in phytoremedition, Environmentl Pollution, vol. 15, no., pp. 9 9,. [9] P. Audet nd C. C. Chrest, Contriution of AM symiosis to in vitro root metl uptke: from tre to toxi metl onditions, Botny, vol. 7, no., pp , 9. [] K. S. Surmnin nd C. Chrest, Arusulr myorrhize nd nitrogen ssimiltion in mize fter drought nd reovery, Physiologi Plntrum, vol., no., pp. 5 9, 199. [11] K. S. Surmnin nd C. Chrest, Aquisition of N y externl hyphe of n rusulr myorrhizl fungus nd its impt on physiologil responses in mize under droughtstressed nd well-wtered onditions, Myorrhiz, vol. 9, no., pp. 9 75, [1] F. N. Dlton nd M. T. vn Genuhten, The time-domin refletometry method for mesuring soil wter ontent nd slinity, Geoderm, vol. 3, no. 1, pp. 37 5, 19. [13] Y. Dlpé, Vesiulr rusulr myorrhize, in Soil Smpling nd Methods of Anlysis, M. R. Crter, Ed., pp. 7 31, CRC Press, Bo Rton, Fl, USA, 3rd edition, [1] M. Giovnnetti nd B. Mosse, An evlution of tehniques for mesuring vesiulr-rusulr myorrhizl infetion in roots, New Phytologist, vol., pp. 9 5, 19. [15] J. H. Zr, Biosttistil Anlysis, Prentie Hll, Upper Sddle River, NJ, USA, 3rd edition, [1] Y. Dlpé, F. A. de Souz, nd S. Delerk, Life yle of Glomus speies in monoxeni ulture, in In vitro Culture of Myorrhizs, S.Delerk,D.G.Strullu,ndF.A.Fortin,Eds., pp. 9 71, Springer, Berlin, Germny, 5. [17] D. D. Douds, P. E. Pfeffer, nd Y. Shhr-Hill, Cron prtitioning, ost, nd metolism of rusulr myorrhizs, in Arusulr Myorrhizs: Physiology nd Funtion, Y. Kpulnik nd D. D. Douds, Eds., pp. 7 13, Kluwer Ademi, Dordreht, The Netherlnds,. [1] B. N. Berden nd L. Petersen, Influene of rusulr myorrhizl fungi on soil struture nd ggregte stility of vertisol, Plnt nd Soil, vol. 1, no. 1-, pp ,. [19] R. M. Miller nd J. D. Jstrow, Hierrhy of ggregtion, Soil Biology nd Biohemistry, vol., no. 5, pp , 199. [] R. M. Augé,A.J.W.Stodol,J.E.Tims,ndA.M.Sxton, Moisture retention properties of myorrhizl soil, Plnt nd Soil, vol. 3, no. 1, pp. 7 97, 1. [1] J. S. Piotrowski, T. Denih, J. N. Klironomos, J. M. Grhm, nd M. C. Rillig, The effets of rusulr myorrhizs on soil ggregtion depend on the intertion etween plnt nd fungl speies, New Phytologist, vol. 1, no., pp ,.

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