Arbuscular mycorrhizas modify tomato responses to soil zinc and phosphorus addition

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1 Biol Fertil Soils (2012) 48: DOI /s x ORIGINAL PAPER Arbusculr mycorrhizs modify tomto responses to soil zinc nd phosphorus ddition Stephnie J. Wtts-Willims & Timothy R. Cvgnro Received: 6 July 2011 /Revised: 7 September 2011 /Accepted: 8 September 2011 /Published online: 11 October 2011 # Springer-Verlg 2011 Abstrct Arbusculr mycorrhizs (AM) ply n importnt role in plnt P nd Zn nutrition; however, reltively few studies hve directly investigted the interctive effects of these nutrients on plnts. Therefore, we undertook glsshouse experiment to study the effects of Zn nd P on AM formtion nd functioning. A mycorrhiz defective tomto mutnt (rmc) nd its mycorrhizl wild-type progenitor (76R) were used in this experiment. Plnts were grown in soil mended with five Zn concentrtions, rnging from deficient to toxic, nd two levels of P ddition. The ddition of Zn nd P to the soil over rnge of concentrtions hd profound effects on plnt growth nd nutrition nd mycorrhizl coloniztion. Mycorrhizl benefits were the gretest when plnts were grown under low soil P nd Zn. Furthermore, the effect of soil Zn supply on plnt growth, nutrition, nd AM coloniztion ws strongly influenced by the concentrtion of P in the soil. Thus, studies of AM nd Zn (or other nutrients of interest) should tke into ccount the impct of soil P concentrtion on the role of AM in plnt Zn cquisition, under both deficient nd toxic soil Zn concentrtions. Keywords Arbusculr mycorrhizs (AM). Zinc. Phosphorus. Mycorrhiz defective tomto mutnt (rmc). Solnum lycopersicum (Tomto) S. J. Wtts-Willims : T. R. Cvgnro (*) School of Biologicl Sciences, Monsh University, Melbourne, VIC 3800, Austrli e-mil: timothy.cvgnro@monsh.edu T. R. Cvgnro Austrlin Centre for Biodiversity, Monsh University, Melbourne, VIC 3800, Austrli Introduction Most (pproximtely 80%) terrestril plnt species form rbusculr mycorrhizs (AM) with specilized group of soil fungi (Smith nd Red 2008). While it hs been well estblished tht the formtion of AM cn significntly enhnce plnt cquisition of P (Smith nd Red 2008), AM lso ply n importnt, lbeit less well understood, role in the cquisition Zn, N, Cu, K, nd other nutrients (Frey nd Schuepp 1993; Pio nd Liu 2011; Johnsen et l. 1993; Mrschner nd Dell 1994; Cvgnro 2008; Bürkert nd Robson 1994). These improvements in plnt nutrient cquisition cn result in improvements in plnt growth (Johnson et l. 1997) nd competitive bility (Fcelli nd Fcelli 2002) nd my lso be importnt in improving the nutritive vlue of crops (Crdoso nd Kuyper 2006; Burns et l. 2010). An estimted 30% of the world s popultion experiences indequte dietry Zn intke (Brown nd Wuehler 2000). This is not unexpected given tht pproximtely one third of the world s soils re considered Zn deficient (Hcislihoglu nd Kochin 2003), nd much of the world s popultion relies on plnt-bsed diet. Consequently, incresing the density of Zn (nd other micronutrients) in crop plnts is of high priority (Burns et l. 2010; White nd Brodley 2009). Since the formtion of AM under deficient soil Zn concentrtion cn result in n increse in plnt Zn cquisition of 25 50% (Cvgnro et l. 2006; Mrschner nd Dell 1994), AM my hve n importnt role to ply in improving the nutrition of crop plnts (Cvgnro 2008; Menge 1983; Covey et l. 1981). Despite this, our understnding of how AM improve plnt Zn cquisition is limited. While AM cn improve plnt Zn cquisition in Zndeficient soils, they cn lso protect plnts ginst excessive Zn uptke when in Zn-contminted soils (Li

2 286 Biol Fertil Soils (2012) 48: nd Christie 2001; Chen et l. 2003; Christie et l. 2004). Tht is, plnts colonized by rbusculr mycorrhizl fungi (AMF) hve been found to hve lower tissue Zn concentrtions when grown under toxic soil Zn concentrtions, when compred to their non-mycorrhizl counterprts (Dueck et l. 1986; Weissenhorn nd Leyvl 1995; Liu et l. 2000). This response is prticulrly interesting when considered in light of the fct tht in Zn-deficient soils, AM cn enhnce plnt Zn cquisition (see bove) (Chen et l. 2003). Despite this, there hve been few studies tht directly compre the response of mycorrhizl nd nonmycorrhizl plnts under soil Zn concentrtions rnging from deficient through to toxic (Li nd Christie 2001; Zhu et l. 2001; Chen et l. 2003); such studies will be importnt in dvncing our understnding of this intriguing dul role of AMF in plnt Zn cquisition. Nutrient elements undergo complex interctions both in the soil environment nd in plnt (Epstein nd Bloom 2005). For exmple, the P sttus of plnts cn hve significnt impct upon plnt Zn nutrition (Mrschner 1995). In the cse of mycorrhizl plnts, AM-induced improvements in plnt P nutrition my in turn impct upon plnt Zn nutrition nd vice vers (Zhu et l. 2001; Chenetl.2003). For exmple, incresed P uptke in mycorrhizl plnts my result in incresed plnt growth nd, thence, Zn deficiency, due to tissue dilution effect (Robson nd Pitmn 1983; Mrschner 1995; Zhu et l. 2001); however, AM effects on plnt Zn cquisition must lso be tken into considertion. While there is some evidence tht P nd Zn uptke in mycorrhizl plnts is correlted (Jns et l. 2003), this is not lwys the cse (Mehrvrn et l. 2000). Despite the fct tht AM cn improve plnt cquisition of both P nd Zn, few studies hve directly ssessed the role of AM in improving plnt Zn nutrition under different soil P conditions in the one experiment (Bi et l. 2003; Liuet l. 2000). In erlier lbortory nd field-bsed studies, we hve used mycorrhiz defective tomto (Solnum lycopersicum L.) mutnt (nmed rmc) nd its mycorrhizl wildtype progenitor (nmed 76R) to study the role of AM in plnt Zn cquisition. Using this genotypic pproch for the study of AM functioning, AM hve been found to increse plnt Zn uptke under low soil Zn concentrtions (Cvgnro et l. 2006; Cvgnro nd Mrtin 2011) nd protect ginst excessive Zn uptke under toxic soil Zn concentrtions (Cvgnro et l. 2010). Here we present the results of glsshouse bsed study in which we used these tomto genotypes to ssess the interctive effects of soil P supply (low nd high soil P concentrtions) nd soil Zn supply (deficient through toxic soil Zn concentrtions) on the formtion nd functioning of AM. Mterils nd methods Soil, fungi, nd plnts Plstic, free-drining pots were filled with 1 kg of 80:20 (W/W) snd/field soil mixture. The field soil ws collected from Wllenjoe Swmp Stte Gme Prk situted ner Rochester in northern Victori, Austrli. Vegettion t the site is dominted by Euclyptus cmldulensis (Mrtin 2007). Soil t the site is lom with ph of 5.7±0.11, totl C content of 1.5±0.1%, totl N content of 0.1± 0.0%, nd hs low concentrtions of plnt vilble P nd DPTA extrctble Zn (see below) nd high AMF inoculum potentil (see Cvgnro nd Mrtin 2011 for dditionl physicochemicl properties). Furthermore, soil from this site hs been found to hve high AMF inoculum potentil (Cvgnro nd Mrtin 2011; Mrtin 2007). The snd ws corse-wshed river snd. This soil snd mixture, which is referred to s soil herefter, ws used s in erlier studies s it further reduces soil nutrient concentrtions nd permits redy nd complete extrction of fine roots t the time of hrvest (Cvgnro et l. 2010). To estblish low nd high soil P ddition tretments, CHPO 4 ws dded to the soil t rte of 20 nd 75 mg CHPO 4 kg 1 soil, respectively. These P ddition tretments, which re referred to s low nd high herefter, resulted in soil (plnt vilble Olsen) P concentrtions of 8 nd 15 mg P kg 1 soil, respectively; these soil P concentrtions support good levels of plnt growth, while mintining high levels of AM coloniztion of roots in this soil (T.R. Cvgnro, unpublished). To estblish the soil Zn ddition tretments, Zn (s ZnSO 4 7H 2 O) ws dded to the P mended soil (high nd low P ddition tretments) t rtes of 0, 25, 50, 75, nd 100 mg Zn kg 1 soil (Cvgnro et l. 2010; Chenet l. 2003) nd mixed so tht it ws evenly distributed throughout the soil. These Zn dditions resulted in soil DPTA extrctble Zn concentrtions of 1, 23, 48, 74, nd 98 mg Zn kg 1 ndrereferredtoszn1,zn2,zn3,zn 4,ndZn5,herefter. Two tomto genotypes were used in this experiment reduced mycorrhizl coloniztion (rmc, herefter) tomto (S. lycopersicum L.) mutnt nd its wild-type progenitor S. lycopersicum cv. 76R (76R, herefter) (see Brker et l. 1998). In erlier studies, it hs been shown tht these genotypes re mtched when grown under wide rnge of experimentl conditions (Cvgnro nd Mrtin 2011; Cvgnro et l. 2005; Poulsen et l. 2005), including the bsence of AMF (Cvgnro et l. 2004) indicting tht the muttion ffecting the formtion of AM by the rmc genotype hs no pleiotropic effects on other plnt processes. Seeds of both tomto genotypes were surfce-sterilized (Cvgnro et l. 2010) nd directly sown into pots contining soil mended with Zn nd P. Ech tretment

3 Biol Fertil Soils (2012) 48: ws replicted five times, giving totl of 100 pots. The plnts were then trnsferred to glsshouse on the Monsh University Clyton cmpus. Conditions in the temperturecontrolled glsshouse during the experimentl period (October November 2010) were s follows: Light levels during dylight hours (14 h dy length) verged 215± 27 μmol m 2 s 1, nd the temperture ws 22.4±0.24 C dy nd 17.2±0.95 C night. All plnts were wtered with reverse osmosis (RO) wter for the first 4 weeks. Plnts were then wtered with 1/10 strength modified Long- Ashton solution (P nd Zn omitted) (Cvgnro et l. 2001) until 7 weeks fter plnting, fter which they were irrigted with hlf strength modified Long-Ashton solution (P nd Zn omitted) to correct visul symptoms of N deficiency. The plnts were rrnged in the glsshouse s rndomized complete block design, with the position of plnts in the glsshouse rotted on weekly bsis. Hrvesting nd nlysis There ws one destructive hrvest 9 weeks fter plnting, s follows. Plnts were wshed free from their pots nd ny ttched soil with RO wter. All of the shoots nd weighed sub-smple of the roots were oven-dried nd shoot dry weights (SDW) nd root dry weights (RDW) determined. Tissue P nd Zn concentrtions were determined on plnt mteril (ground to fine powder) by rdil view inductively coupled plsm opticl emission spectrometry (by Wite Anlyticl Services, ws, lst ccessed 20 My 2011). A second weighed subsmple of roots ws used for ssessment of mycorrhizl coloniztion, using gridline intersect method (Giovnnetti nd Mosse 1980), fter the roots were clered in KOH (10%, W/V) nd stined with Trypn blue (omitting phenol from ll regents) (Phillips nd Hymn 1970). Clcultions nd dt nlysis Mycorrhizl growth responses (MGR) were clculted using the individul totl plnt dry weights of 76R plnts nd men totl plnt dry weight vlues of rmc plnts t ech tretment (Eq. 1). %MGR ¼ dry weight ð76rþ men dry weight ðrmcþ men dry weight ðrmcþ 100 ð1þ In ddition to investigtion of ptterns of P nd Zn uptke by mycorrhizl nd non-mycorrhizl plnts on tissue concentrtion bsis (see below), whole plnt tissue contents were used to determine the mycorrhizl P uptke response (MPR) nd mycorrhizl Zn uptke response (MZnR) (following Cvgnro et l. 2003). The mycorrhizl P response ws clculted using the individul P content of 76R plnts nd men P content of rmc plnts (Eq. 2). %MPR ¼ Pcontentð76RÞ men P content ðrmcþ men P content ðrmcþ 100 ð2þ The mycorrhizl Zn response ws clculted using the individul Zn content of 76R plnts nd men Zn content of rmc plnts (Eq. 3). %MZnR ¼ Zn content ð76rþ men Zn content ðrmcþ men Zn content ðrmcþ 100 ð3þ All coloniztion, biomss, nd tissue nutrient concentrtion dt were nlyzed by multi-fctor ANOVA fctors in the nlysis were Genotype, Zn ddition tretment, nd P ddition tretment. Where significnt differences were found, pirwise comprisons were mde using Tukey s honestly significntly different (HSD; Zr 1999). In the interests of clrity, the results from ll combintions of the three experimentl tretments re presented in the figures. Where the three-wy interction term in the model ws significnt, results of pirwise comprisons re presented in the figures. Where the three-wy interction term ws not significnt, mens for ech of the significnt lower-order interctions (i.e., two-wy interctions or min effects) re presented in the tbles. Mycorrhizl P response nd mycorrhizl Zn response dt were nlyzed by two-wy ANOVA, with Zn ddition tretment nd P ddition tretment s fctors in the nlysis. Where significnt differences were found, pirwise comprisons were mde using Tukey s HSD.All dt were nlyzed using JMP sttisticl softwre (version 9.0.0). Results Mycorrhizl coloniztion Roots of the rmc genotype were essentilly non-mycorrhizl (<1%; dt not shown). In contrst, the roots of 76R plnts were well colonized (Fig. 1). When considered seprtely from the rmc tretments, mycorrhizl coloniztion of 76R roots ws significntly lower in the high P ddition tretment, irrespective of Zn ddition tretment (Tble 1; Fig. 1). Similrly, coloniztion of 76R roots decresed significntly with Zn ddition, irrespective of P ddition tretment.

4 288 Biol Fertil Soils (2012) 48: Mycorrhizl coloniztion (%) Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 b b c Root dry weights For RDW there ws significnt (Tble 1) three-wy interction between the Genotype, Zn ddition tretment nd P ddition tretment (Fig. 2). The RDW of both genotypes ws higher in the high P ddition tretment nd decresed with incresing Zn supply. Furthermore, the RDW of 76R plnts ws significntly greter thn those of rmc plnts in the low P tretment t the lowest level of soil Zn ddition (Zn 1 tretment). 0 Fig. 1 Mycorrhizl coloniztion t hrvest of 76R plnts grown under low (white brs) nd high (gry brs) levels of soil P ddition nd rnge of soil Zn ddition tretments (Zn 1 5). Vlues re men±se, n=5. Mens followed by the sme letter were not significntly different t the P<0.05 level (Tukey s HSD),see Tble 1 for detils of ANOVA results Plnt biomss Shoot dry weights The SDW of both genotypes incresed with P supply nd decresed with incresing Zn supply (Fig. 2). Anlysis of the SDW dt reveled significnt two-wy interctions (Tble 1) between the Genotype nd Zn ddition tretment nd between the P ddition tretment nd Zn ddition tretment. When the interction term between the Genotype nd Zn ddition tretment ws considered (Tble 2), there ws significnt decrese in SDW with incresing soil Zn ddition nd greter SDW in the 76R genotype, thn the rmc genotype, in the low Zn ddition tretments. Similrly, when the interction between P ddition tretment nd Zn ddition tretment ws considered (Tble 2), there ws lrge increse in SDW with P ddition nd lrge decrese in SDW with incresing Zn supply, especilly in the low P ddition tretment, where ten times decrese in SDW ws observed between the Zn 1 nd Zn 5 Zn ddition tretments. Plnt phosphorus Shoot nd root P concentrtions There ws significnt three-wy interction between Genotype, P ddition tretment, nd Zn ddition tretment for shoot P concentrtions (Tble 1; Fig. 3). Specificlly, in the low P tretment, the shoot P concentrtion of the 76R genotype ws significntly higher thn tht of the rmc genotype, in the low to intermedite Zn supply tretments (Zn 1 Zn 4) (Fig. 3). Conversely, t high P, there were no differences in shoot P between ny of the tretments, nd P concentrtions were within the rnge of those of 76R plnts in the low P ddition tretment. In terms of root P concentrtions, similr pttern s tht for shoot P concentrtions ws observed; however, the interction between the three tretments ws not significnt (Tble 1). There ws, however, significnt two-wy interction between the Genotype nd P ddition tretments (Tble 3), with the 76R plnts in the low P tretment hving the highest root P concentrtions nd rmc plnts in the low P tretment hving the lowest root P concentrtions. The nlysis of root P dt lso reveled significnt two-wy interction between the Genotype nd Zn ddition tretments (Tble 3), with the root P concentrtion of 76R plnts t ll levels of Zn supply being generlly higher thn those of rmc plnts. Furthermore, for root P concentrtions, there ws lso significnt two-wy interction between the P ddition Tble 1 ANOVA summry tble for ll response vribles Fctors in the nlysis were G, P, nd Zn. Both min effects nd interctions terms re indicted G genotype, P P ddition tretment, Zn Zn ddition tretment *P<0.05; **P<0.01; ***P< G P Zn P Zn G Zn G P G P Zn % Coloniztion *** *** ns % MGR * *** ** % MPR *** *** ** % MZnR ** ** ** Shoot dry weight ** *** *** ** * ns ns Root dry weight * *** *** ** * ns * Shoot P concentrtion *** *** *** ** *** *** ** Root P concentrtion *** ns ** ** ** *** ns Shoot Zn concentrtion ns *** *** *** ns ns ns Root Zn concentrtion ns *** *** *** ns ns ns

5 Biol Fertil Soils (2012) 48: Low P High P 1.5 Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Zn1 Zn 2 Zn 3 Zn 4 Zn Biomss (g) ef gh fg gh gh gh h gh h h b bc bcd b bc bc bcd def cde 1 Fig. 2 Shoot (bove x-xis) nd root (below x-xis) dry weights of 76R (gry brs) nd rmc (white brs) plnts grown under low nd high levels of soil P ddition nd rnge of soil Zn ddition tretments (Zn 1 5). Vlues re men ± SE, n=5. Mens followed by the sme letter were not significntly different t the P<0.05 level (Tukey s HSD), see Tble 1 for detils of ANOVA results nd Tble 2 for mens for ech of the significnt lower-order interctions (i.e., two-wy interctions or min effects) tretment nd the Zn ddition tretments (Tble 3); however, no cler ptterns long the Zn nd P ddition grdients were seen. Plnt zinc Shoot nd root Zn concentrtions The concentrtion of Zn in plnts incresed to very high concentrtions with incresing soil Zn supply, especilly in the Low P ddition tretment (Fig. 3b). There ws, however, little difference between the two genotypes. To this end, there ws significnt (Tble 1) two-wy interction between the P ddition tretment nd Zn ddition tretments for both shoot nd root Zn concentrtions (Tble 4). Mycorrhizl P nd Zn responses Whole plnt P nd Zn contents Whole plnt P contents were used to clculte MPRs (Tble 5). At low P supply, MPR ws positive cross ll Zn supply tretments, Tble 2 SDW (grms) of mycorrhizl (76R) nd non-mycorrhizl (rmc) tomto genotypes grown under low nd high levels of soil P ddition nd rnge of soil Zn ddition tretments (Zn 1 5) Genotype 76R rmc Zn ddition tretment Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Men b 0.60bc 0.51cd 0.27e 0.70bc 0.62bc 0.56c 0.50cd 0.33de ± SE P ddition tretment Low P High P Zn ddition tretment Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Men 0.36d 0.30de 0.12ef 0.06f 0.03f b 1.05b 0.94b 0.57c ± SE Only results where there ws significnt tretment effect reveled in the ANOVA (Tble 1) re presented here. Dt from ll tretment combintions re given in Fig. 2. Vlues re men ± SE, n=5. Mens followed by the sme letter were not significntly different t the P<0.05 level (Tukey s HSD); full detils of sttisticl nlysis given in text

6 290 Biol Fertil Soils (2012) 48: Low P High P P concentrtion (mg kg -1 ) Zn 1 e Zn 2 Zn 3 Zn 4 Zn 5 Zn 1 Zn 2 b bc bc bc cd bcd e e e de e Zn 3 Zn 4 Zn 5 bc bc bcd bcd bcd bcd b 7000 Zn 1 Low P Zn 2 Zn 3 Zn 4 Zn 5 Zn 1 High P Zn 2 Zn 3 Zn 4 Zn Zn concentrtion (mg kg -1 ) Fig. 3 Shoot (bove x-xis) nd root (below x-xis) tissue P concentrtions nd b tissue Zn concentrtions of 76R (gry brs) nd rmc (white brs) plnts grown under low nd high levels of soil P ddition nd rnge of soil Zn ddition tretments (Zn 1 5). Vlues re men ± SE, n=5. Mens followed by the sme letter were not significntly different t the P<0.05 level (Tukey s HSD), see Tble 1 for detils of ANOVA results. See lso Tbles 3 nd 4 for mens for ech of the significnt lower-order interctions (i.e., two-wy interctions or min effects) for plnt P nd Zn concentrtions, respectively lthough decresed significntly (Tble 1) with incresingznsupply.incontrst,mprwsclosetozerointhe high P ddition tretment t ll levels of soil Zn supply. Whole plnt Zn contents were used to clculte MZnRs (Tble 5): In the low P ddition tretment, the MZnR ws positive where the soil Zn concentrtion ws low. In contrst, the MZnR ws close to zero in ll other experimentl tretments.

7 Biol Fertil Soils (2012) 48: Tble 3 Root P concentrtions (milligrms per kilogrm) of mycorrhizl (76R) nd non-mycorrhizl (rmc) tomto genotypes grown under low nd high levels of soil P ddition nd rnge of soil Zn ddition tretments (Zn 1 5) Genotype 76R rmc P ddition tretment Low P High P Low P High P Men 1,475 1,179b 771c 1,140b ± SE Genotype 76R rmc Zn ddition tretment Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Men 1,297b 1,287b 1,505 1,348b 1,198bc 905d 927d 945d 1,043cd 956d ± SE P ddition tretment Low P High P Zn ddition tretment Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Men 1,074c 1,114bc 1,286 1,169bc 972c 1,128bc 1,100bc 1,164bc 1,222b 1,182b ± SE Only results where there ws significnt tretment effect reveled in the ANOVA (Tble 1) re presented here. Dt from ll tretment combintions re given in Fig. 3. Vlues re men ± SE, n=5. Mens followed by the sme letter were not significntly different t the P<0.05 level (Tukey s HSD); full detils of sttisticl nlysis given in text Discussion Mycorrhizl coloniztion Wheres roots of the 76R tomto genotype were well colonized by AMF, those of the rmc genotype were not, s in erlier studies (Brker et l. 1998). Levels of mycorrhizl coloniztion of the 76R genotype were significntly reduced with incresing P nd Zn supply to the soil. The reduction in coloniztion with incresing soil P supply is consistent with erlier studies (Boln et l. 1984; Hsbullh nd McNeill 2011; Bon et l. 1992; Smith nd Red 2008). For Zn, the reduction in coloniztion with incresing Zn ddition to the soil is consistent with some previous studies (Gildon nd Tinker 1983;Bietl.2003;Chenetl.2004), but not others, where both positive (Zhu et l. 2001; Lee nd George 2005) nd neutrl effects hve been reported (Diz et l. 1996; Orts et l. 2002; Cvgnro et l. 2010). The resons for these differences between studies remin to be determined. The reduction in coloniztion with incresing soil Zn supply observed here my be due to differences in the mgnitude of the effect of Zn on the growth rte of roots nd AMF. Tht is, the effect of Zn on fungl growth rtes my be greter thn tht of roots. The reduction in coloniztion my lso be due to reduction in the effective inoculum potentil of the soil due to negtive effects of incresing soil Zn supply on rtes of spore germintion nd hyphl growth. Given tht the soil used in this study is nturlly low in Zn, it is likely tht the indigenous AMF my hve low level of resistnce to toxic soil Zn conditions, unlike AMF from Zn-contminted sites where there hs been selection of AMF resistnt to high Zn concentrtions (Weissenhorn et l. 1993; Shetty et l. 1994b; Diz et l. 1996; Pwlowsk et l. 1996). Finlly, irrespective of the mechnisms involved, the impct of Zn on levels of mycorrhizl coloniztion of roots ppered to operte independently of soil P supply. Tble 4 Shoot nd root Zn concentrtions (milligrms per kilogrm) of mycorrhizl (76R) nd non-mycorrhizl (rmc) tomto genotypes grown under low nd high levels of soil P ddition nd rnge of soil Zn ddition tretments (Zn 1 5) P ddition tretment Low P High P Zn ddition tretment Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Shoots Roots Men ±SE Men ±SE 85f 776e 1,869d 3,690b 5,300 38f 412ef 869e 1,424d 2,430c p 1,978n 4,680l 6,970k 7,990j 45p 1,018o 2,167n 3,340m 4,160l Only results where there ws significnt tretment effect reveled in the ANOVA (Tble 1) re presented here. Dt from ll tretment combintions re given in Fig. 3b. Vlues re men ± SE, n=5. Mens followed by the sme letter were not significntly different t the P<0.05 level (Tukey s HSD); full detils of sttisticl nlysis given in text. Vlid comprisons cnnot be mde between prmeters, s indicted by different sets of letters

8 292 Biol Fertil Soils (2012) 48: Tble 5 MGR, MPR, nd MZnR vlues of tomtoes grown under low nd high levels of soil P ddition nd rnge of soil Zn ddition tretments (Zn 1 5) P ddition tretment Low P High P Zn ddition tretment Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 Zn 1 Zn 2 Zn 3 Zn 4 Zn 5 % MGR Men b 10.9b 17.3b 14.6b 7.1b 24.7b 9.4b 7.8b 19.5b ± SE % MPR Men 452.7j 208.1k 144.8kl 43.7kl 17.1kl 9.7kl 16.2kl 22.5kl 1.7kl 17.7l ± SE % MZnR Men 45.8xy 59.4x 4.9yz 23.7z 7.7z 14.7z 2.9yz 6.3yz 2.3yz 17.4z ± SE Only results where there ws significnt tretment effect reveled in the ANOVA (Tble 1) re presented here. Vlues re men ± SE, n=5. Mens followed by the sme letter were not significntly different t the P<0.05 level (Tukey s HSD); full detils of sttisticl nlysis given in text. Vlid comprisons cnnot be mde between prmeters, s indicted by different sets of letters MGR mycorrhizl growth response, MPR mycorrhizl P response, MZnR mycorrhizl Zn response Plnt biomss The growth of the tomto plnts, irrespective of the formtion of AM, ws significntly reduced with incresing soil Zn concentrtion, s in erlier studies (Godbold et l. 1983; Runo et l. 1988). This reduction in growth ws clerly ssocited with the ccumultion of Zn in plnt tissues to very high (nd toxic) concentrtions (Påhlsson 1989; Rout nd Ds 2003). In ddition to improving plnt growth generlly, the ddition of P to the soil lso decresed the negtive impct of soil Zn ddition on plnt growth; this my be due to dilution of Zn in the plnt tissues (due to incresed plnt growth) (Nielsen nd Jensen 1983). While the supply of more P to the soil my fford plnts with some protection ginst Zn toxicity, most sites tht re contminted with hevy metls re often depleted of vilble minerl nutrients, especilly P (Shetty et l. 1994, b). Thus, the supply of P my be useful strtegy in the protection of plnts ginst toxic soil Zn concentrtions. The formtion of AM resulted in significntly incresed plnt growth (reflected in biomss nd % MGR dt) under low Zn nd low P soil concentrtions. At ll other levels of P nd Zn supply, there ws no pprent benefit or cost to plnts, in terms of biomss, of forming AM. This lck of difference in biomss between mycorrhizl nd nonmycorrhizl plnts is consistent with some (Cvgnro et l. 2010; Cvgnro nd Mrtin 2011), but not other studies (Mrschner nd Timonen 2004), using these genotypes, under wide rnge of soil conditions. Plnt phosphorus nd zinc nutrition While generlly low, the concentrtion of P in the plnts ws strongly influenced by the ddition of P nd Zn to the soil nd the formtion of AM. Where soil P ws low, the concentrtion of P in the tissues of mycorrhizl plnts ws higher thn tht of the non-mycorrhizl plnts. Interestingly, s the concentrtion of Zn in the soil incresed nd plnt biomss decresed, the difference between mycorrhizl nd non-mycorrhizl plnts ws reduced. This response my be relted to prllel reltive decrese in mycorrhizl coloniztion of roots. In contrst, tissue P concentrtions did not differ between mycorrhizl nd non-mycorrhizl plnts in the high P ddition tretment nd were not ffected by soil Zn supply. When whole plnt P contents were considered, presented s mycorrhizl P responses (% MPR) here, the formtion of AM provided the gretest benefit in terms of P uptke when soil P nd Zn concentrtions were low. Agin, this is consistent with erlier studies showing tht AM ply n importnt role in plnt P nd Zn nutrition under low soil P nd Zn concentrtions (Smith nd Red 2008). Tissue Zn concentrtions in this study rnged from deficient in the low soil Zn ddition tretments, through to extremely toxic in the moderte to high soil Zn ddition tretments (Reuter nd Robinson 1997). The reduction in tissue Zn in the high soil P ddition tretment cn be explined by the lrge increse in plnt growth with soil P supply, which in turn cn led to dilution of Zn in the plnt tissues (Robson nd Pitmn 1983; Zhu et l. 2001). Interestingly, when plnts in the low Zn ddition tretment were considered seprtely from ll other Zn ddition tretments, the concentrtion of Zn in the roots nd shoots ws significntly lower in the high P ddition tretment (dt not shown). This is consistent with reports of soclled P-induced Zn deficiency (Mrschner 1995). Even where tissue Zn concentrtions were toxic, the ddition of P to the soil resulted in lower tissue Zn concentrtions. Increses in plnt P concentrtions ssocited with the formtion of AM did not, however, result in reduction in tissue Zn concentrtions. Where the supply of Zn to the soil

9 Biol Fertil Soils (2012) 48: ws high, we did not find evidence of incresed P uptke by mycorrhizl plnts lleviting Zn toxicity (Chen et l. 2003; Christie et l. 2004). When whole plnt Zn contents were considered, presented s mycorrhizl Zn responses (% MZnR) here, the formtion of AM ws beneficil where soil P nd Zn (Zn 1 nd Zn 2 tretments) concentrtions were low. In contrst, there ws no evidence of AM hving (sttisticlly significnt) protective role when soil Zn concentrtions were high, s in erlier studies (Cvgnro et l. 2010; Chen et l. 2003). Although it is importnt to note tht % MZnR ws negtive or close to zero in the highest Zn ddition tretments. Conclusions Both formtion nd functioning of AM were strongly influenced by the ddition of P nd Zn to the soil. Importntly, this study highlights the interctive effects of P nd Zn supply on AM. While little evidence of protective role of AM in soils high in Zn ws found, this my be in prt relted to the fct tht the AMF used in this study were from soil with low endogenous Zn concentrtion. Tken together the results presented here point to the importnce of AM in plnt P nd Zn nutrition (under some conditions); more detiled moleculr nd physiologicl studies will be importnt in exploring the role of AM in these responses. Acknowledgments The uthors wish to thnk Ms. Lees Hughes for her excellent technicl ssistnce nd members of the Cv-Lb for vluble discussions. We lso grtefully cknowledge Dr. Susn Brker nd Prof. Slly Smith for continued ccess to the rmc nd 76R genotypes of tomto. This reserch ws in prt funded by the Monsh University, School of Biologicl Sciences. TRC lso wishes to cknowledge the Monsh Reserch Accelertor progrm for finncil support. References Bon JB, Smith SE, Alston AM, Wheeler RD (1992) Phosphorus efficiency of three cerels s relted to indigenous mycorrhizl infection. Aust J Agric Res 43: Brker SJ, Stummer B, Go L, Dispin I, O Connor PJ, Smith SE (1998) A mutnt in Lycopersicon esculentum Mill. with highly reduced VA mycorrhizl coloniztion: isoltion nd preliminry chrcteristion. Plnt J 15: Bi YL, Li XL, Christie P (2003) Influence of erly stges of rbusculr mycorrhiz on uptke of zinc nd phosphorus by red clover from low-phosphorus soil mended with zinc nd phosphorus. Chemosphere 50: Boln NS, Robson AD, Brrow NJ (1984) Incresing phosphorus supply cn increse the infection of plnt roots by vesiculr rbusculr mycorrhizl fungi. Soil Biol Biochem 16: Brown KH, Wuehler SE (2000) Zinc nd humn helth: results of recent trils nd implictions for progrm interventions nd reserch. Interntionl Development Reserch Center, Ottw Bürkert B, Robson A (1994) 65 Zn uptke in subterrnen clover (Trifolium subterrneum L.) by 3 vesiculr rbusculr mycorrhizl fungi in root-free sndy soil. Soil Biol Biochem 26: Burns A, Gledow R, Cliff J, Zcris A, Cvgnro T (2010) Cssv: the drought, wr nd fmine crop in chnging world. Sustinbility 2: Crdoso IM, Kuyper TW (2006) Mycorrhizs nd tropicl soil fertility. Agric Ecosyst Environ 116:72 84 Cvgnro TR (2008) The role of rbusculr mycorrhizs in improving plnt zinc nutrition under low soil zinc concentrtions: review. Plnt Soil 304: Cvgnro TR, Mrtin AW (2011) Arbusculr mycorrhizs in southestern Austrlin processing tomto frm soils. Plnt Soil 340: Cvgnro TR, Smith FA, Lorimer MF, Hskrd KA, Ayling SM, Smith SE (2001) Quntittive development of Pris-type rbusculr mycorrhizs formed between Asphodelus fistulosus nd Glomus corontum. New Phytol 149: Cvgnro TR, Smith FA, Ayling SM, Smith SE (2003) Growth nd phosphorus nutrition of Pris-type rbusculr mycorrhizl symbiosis. New Phytol 157: Cvgnro TR, Smith FA, Hy G, Crne-Cvgnro VL, Smith SE (2004) Inoculum type does not ffect overll resistnce of n rbusculr mycorrhiz-defective tomto mutnt to colonistion but inocultion does chnge competitive interctions with wildtype tomto. New Phytol 161: Cvgnro TR, Smith FA, Smith SE, Jkobsen I (2005) Functionl diversity in rbusculr mycorrhizs: exploittion of soil ptches with different phosphte enrichment differs mong fungl species. Plnt Cell nd Environ 28: Cvgnro TR, Jckson LE, Six J, Ferris H, Goyl S, Asmi D, Scow KM (2006) Arbusculr mycorrhizs, microbil communities, nutrient vilbility, nd soil ggregtes in orgnic tomto production. Plnt Soil 282: Cvgnro TR, Dickson S, Smith FA (2010) Arbusculr mycorrhizs modify plnt responses to soil zinc ddition. Plnt Soil 329: Chen BD, Li XL, To HQ, Christie P, Wong MH (2003) The role of rbusculr mycorrhiz in zinc uptke by red clover growing in clcreous soil spiked with vrious quntities of zinc. Chemosphere 50: Chen B, Shen H, Li X, Feng G, Christie P (2004) Effects of EDTA ppliction nd rbusculr mycorrhizl coloniztion on growth nd zinc uptke by mize (Ze mys L.) in soil experimentlly contminted with zinc. Plnt Soil 261: Christie P, Li X, Chen B (2004) Arbusculr mycorrhiz cn depress trnsloction of zinc to shoots of host plnts in soils modertely polluted with zinc. Plnt Soil 261: Covey RP, Koch BL, Lrsen HJ (1981) Influence of vesiculr rbusculr mycorrhize on the growth of pple nd corn in low-phosphorous soil. Phytopthology 71: TDiz G, Azcon-Aguilr C, Honrubi M (1996) Influence of rbusculr mycorrhize on hevy metl (Zn nd Pb) uptke nd growth of Lygeum sprtum nd Anthyllis cytisoides. Plnt Soil 180: Dueck TA, Visser P, Ernst WHO, Scht H (1986) Vesiculr rbusculr mycorrhizs decrese zinc toxicity to grsses in zinc polluted soil. Soil Biol Biochem 18: Epstein E, Bloom AJ (2005) Minerl nutrition of plnts: principles nd perspectives, 2nd edn. Sinuer Assocites, Sunderlnd Fcelli E, Fcelli JM (2002) Soil phosphorus heterogeneity nd mycorrhizl symbiosis regulte plnt intr-specific competition nd size distribution. Oecologi 133:54 61 Frey B, Schuepp H (1993) Acquisition of nitrogen by externl hyphe of rbusculr mycorrhizl fungi ssocited with Ze mys L. New Phytol 124:

10 294 Biol Fertil Soils (2012) 48: Gildon A, Tinker PB (1983) Interctions of vesiculr rbusculr mycorrhizl infection nd hevy metls in plnts. I. The effects of hevy metls on the development of vesiculr rbusculr mycorrhizs. New Phytol 95: Giovnnetti M, Mosse B (1980) An evlution of techniques for mesuring vesiculr rbusculr mycorrhizl infection in roots. New Phytol 84: Godbold DL, Horst JW, Mrschner H, Collins JC, Thurmn DA (1983) Root growth nd zinc uptke by two ecotypes of Deschmpsi cespitos s ffected by high zinc concentrtions. Z Pflnzenphysiol 112: Hcislihoglu G, Kochin LV (2003) How do some plnts tolerte low levels of soil zinc? Mechnisms of zinc efficiency in crop plnts. New Phytol 159: Hsbullh MP, McNeill A (2011) Legume residue influence rbusculr mycorrhizl colonistion nd P uptke by whet. Biol Fert Soils 47: doi: /s Jns J, Mozfr A, Frossrd E (2003) Long-distnce trnsport of P nd Zn through the hyphe of n rbusculr mycorrhizl fungus in symbiosis with mize. Agronomie 23: Johnsen A, Jkobsen I, Jensen ES (1993) Externl hyphe of vesiculr rbusculr mycorrhizl fungi ssocited with Trifolium subterrneum L. 3. Hyphl trnsport of 32 Pnd 15 N. New Phytol 124:61 68 Johnson NC, Grhm JH, Smith FA (1997) Functioning of mycorrhizl ssocitions long the mutulism prsitism continuum. New Phytol 135: Lee YJ, George E (2005) Contribution of mycorrhizl hyphe to the uptke of metl ctions by cucumber plnts t two levels of phosphorus supply. Plnt Soil 278: Li X, Christie P (2001) Chnges in soil solution Zn nd ph nd uptke of Zn by rbusculr mycorrhizl red clover in Zn-contminted soil. Chemosphere 42: Liu A, Hmel C, Hmilton RI, M BL (2000) Acquisition of Cu, Zn, Mn nd Fe by mycorrhizl mize (Ze mys L.) grown in soil t different P nd micronutrient levels. Mycorrhiz 9: Mrschner H (1995) Minerl nutrition of higher plnts, 2nd edn. Acdemic, Sn Diego Mrschner H, Dell B (1994) Nutrient uptke in mycorrhizl symbiosis. Plnt Soil 159: Mrschner P, Timonen S (2004) Interctions between plnt species nd mycorrhizl coloniztion on the bcteril community composition in the rhizosphere. Appl Soil Ecol 28:23 36 Mrtin AW (2007) The role of rbusculr mycorrhizl fungi in sustinble tomto production. Disserttion, The University of Adelide, Adelide Mehrvrn H, Mozfr A, Frossrd E (2000) Uptke nd prtitioning of 32 P nd 65 Zn by white clover s ffected by eleven isoltes of mycorrhizl fungi. J Plnt Nutr 23: Menge JA (1983) Utiliztion of vesiculr rbusculr mycorrhizl fungi in griculture. Cn J Bot 61: Nielsen JD, Jensen A (1983) Influence of vesiculr rbusculr mycorrhizl fungi on growth nd uptke of vrious nutrients s well s uptke rtio of fertilizer P for lucerne (Medicgo stiv). Plnt Soil 70: Orts I, Ortkci D, Ky Z, Cinr A, Onelge N (2002) Mycorrhizl dependency of sour ornge in reltion to phosphorus nd zinc nutrition. J Plnt Nutr 25: Påhlsson A-MB (1989) Toxicity of hevy metls (Zn, Cu, Cd, Pb) to vsculr plnts. Wter Air Soil Pollut 47: doi: / bf Pwlowsk TE, Blszkowski J, Ruhling A (1996) The mycorrhizl sttus of plnts colonizing clmine spoil mound in southern Polnd. Mycorrhiz 6: Phillips JM, Hymn DS (1970) Improved procedures for clering nd stining prsitic nd vesiculr rbusculr mycorrhizl fungi for rpid ssessment of infection. Trns Br Mycol Soc 55: Pio H-C, Liu C-Q (2011) Vritions in nitrogen, zinc, nd sugr concentrtions in Chinese fir seedlings grown on shrublnd nd plowed soils in response to rbusculr mycorrhizemedited process. Biol Fert Soils 47: doi: / s Poulsen KH, Ngy R, Go L-L, Smith SE, Bucher M, Smith FA, Jkobsen I (2005) Physiologicl nd moleculr evidence for Pi uptke vi the symbiotic pthwy in reduced mycorrhizl coloniztion mutnt in tomto ssocited with comptible fungus. New Phytol 168: Reuter DJ, Robinson JB (1997) Plnt nlysis: n interprettion mnul, 2nd edn. CSIRO, Melbourne Robson AD, Pitmn MG (1983) Interctions between nutrients in higher plnts. Encyclopedi plnt physiology new series, vol 15A. Springer, Berlin Rout GR, Ds P (2003) Effect of metl toxicity on plnt growth nd metbolism: I. Zinc. Agronomie 23:3 11 Runo A, Brceló J, Poschenrieder C (1988) Growth nd biomss prtitioning of zinc toxic bush bens. J Plnt Nutr 11: Shetty KG, Bnks MK, Hetrick BAD, Schwb AP (1994) Biologicl chrcteriztion of southest Knss mining site. Wter Air Soil Pollut 78: Shetty KG, Hetrick BAD, Figge DAH, Schwb AP (1994b) Effects of mycorrhize nd other soil microbes on revegettion of hevy metl contminted mine spoil. Environ Pollut 86: Smith SE, Red DJ (2008) Mycorrhizl symbiosis, 3rd edn. Acdemic, Cmbridge Weissenhorn I, Leyvl C (1995) Root coloniztion of mize by Cdsensitive nd Cd-tolernt Glomus mossee nd cdmium uptke in snd culture. Plnt Soil 175: Weissenhorn I, Leyvl C, Berthelin J (1993) Cd-tolernt rbusculr mycorrhizl (AM) fungi from hevy metl polluted soil. Plnt Soil 157: White PJ, Brodley MR (2009) Biofortifiction of crops with seven minerl elements often lcking in humn diets iron, zinc, copper, clcium, mgnesium, selenium nd iodine. New Phytol 182:49 84 Zr JH (1999) Biosttisticl nlysis, 4th edn. Prentice Hll, Upper Sddle River Zhu Y, Christie P, Lidlw AS (2001) Uptke of Zn by rbusculr mycorrhizl white clover from Zn-contminted soil. Chemosphere 42: