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Plant Physiology and Biochemistry 60 (2012) 53e58

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Plant Physiology and Biochemistry

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Research article

The effects of root endophyte and arbuscular mycorrhizal fungi on growthand cadmium accumulation in wheat under cadmium toxicity

Saleh Shahabivand a, Hassan Zare Maivan a,*, Ebrahim Mohammadi Goltapeh b, Mozafar Sharifi a,Ali Asghar Aliloo c

aDepartment of Plant Sciences, Faculty of Biological Sciences, Tarbiat Modares University, Jalal Al Ahmad Highway, Tehran 14115 175, IranbDepartment of Plant Pathology, Faculty of Agriculture, Tarbiat Modares University, Tehran, IrancDepartment of Agronomy and Plant Breeding, Faculty of Agriculture, University of Maragheh, Maragheh, Iran

a r t i c l e i n f o

Article history:Received 10 June 2012Accepted 16 July 2012Available online 2 August 2012

Keywords:CadmiumGlomus mosseaePiriformospora indicaTriticum aestivum

* Corresponding author. Tel.: þ98 21 82883484; faxE-mail addresses: shahabi70@yahoo.com (S. Shaha

(H.Z. Maivan).

0981-9428/$ e see front matter � 2012 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.07.018

a b s t r a c t

The effects of Piriformospora indica and Glomus mosseae on some growth and physiological parameters,and cadmium (Cd) amounts in Triticum aestivum cv. Sardari39 plants under the toxic levels of Cd wereinvestigated. The experiment was carried out including four treatments (P. indica inoculation, G. mosseaeinoculation, co-inoculation of G. mosseae and P. indica, and non-inoculation), each having four Cdconcentrations (0, 0.3, 0.6 and 0.9 mM Cd). In non-inoculated plants, by increasing Cd concentration inthe soil, growth parameters, chlorophyll content, performance index (PI) and Fv/Fm were reduced,whereas root and shoot Cd accumulation were increased. The colonized plants with P. indica andG. mosseae (in solitary or together) had higher growth, chlorophyll content and PI compared to control. Inthe presence of P. indica, the Cd concentration was increased in roots, whereas it was decreased in shootsunder higher soil Cd concentrations. The presence of G. mosseae decreased root Cd concentration underlower Cd concentration in the soil. Results from this study indicate that P. indica inoculation yieldedgreater readings of growth parameters, chlorophyll content, PI, root colonization and also reduced shootCd content compared to G. mosseae inoculation.

� 2012 Elsevier Masson SAS. All rights reserved.

1. Introduction

Wheat is one of the oldest and most extensively cultivatedcrops. According to FAO reports, in 2007world production of wheatwas 607 million tons, making it the third most-produced cerealafter maize and rice. Since the past century, many regions world-wide suffer from heavy metals contamination (such as cadmium)due to anthropogenic activities [1]. Cadmium (Cd) is a non-essential heavy metal for plants and human beings that maycause toxic effects on crop production [2]. Mainly areas with highindustrial and agricultural activities have to cope with increasedlevel of Cd in the soil [3]. In plants, Cd causes visible symptoms likereduction in root and shoot growth, chlorosis, leaf roll and necrosis,and even death at higher concentrations [4]. At cellular level, Cdinduces oxidative stress as evidenced by enhanced lipid perox-idation, hydrogen peroxide (H2O2) generation and ion leakage [5,6].Plants have evolved tolerance mechanisms to excessive metalconcentrations such as metal efflux, reduced metal uptake by root

: þ98 21 82884717.bivand), Zare897@yahoo.com

son SAS. All rights reserved.

immobilization or mycorrhizal action, intracellular chelation bymetal complexes with phytochelatins and compartmentalization inthe vacuoles [7].

Arbuscular mycorrhizal fungi (AMF), such as Glomus mosseae,are important soil microorganisms forming symbiotic associationswith most of the vascular plant species. The impact of AMF in metalstress alleviation in plants growing inmetal-contaminated soils hasbeen recognized [8]. Improved nutritional status and reduced oraltered metal uptake are among the most related advantages ofmycorrhizal association to host plants under metal stress [9,10].AMF act as a metal sink, reducing local concentrations in soils andcreating a more suitable environment for plants growing in metal-contaminated soils [8]. The AM association can alter plant metaluptake [9,10], with increasing or reducing metal concentrations inplant tissues. As a consequence of physiological changes, AM plantshave an improved performance under metal stress conditions [11].Nevertheless, the overall mechanisms by which AM fungi alleviatemetal toxicity in hosts are still not completely elucidated, consid-ering controversial results depending on the specific plant/fungal/metal species interactions [12].

Piriformospora indica, a rootendophytic fungus (discovered in theIndian Thar desert in 1997) of Sebacinaceae family (Basidiomycota),

S. Shahabivand et al. / Plant Physiology and Biochemistry 60 (2012) 53e5854

colonizes the roots of awide variety of plant species, promotes theirgrowth and confers tolerance against abiotic and biotic stresses[13,14,15]. In contrast to AMF, it can be easily cultivated in axenicculture where it produces chlamydospores [16]. It is suggested thatendophytesmayprotect their hosts byenhancing stress tolerance tooxidative stress. Several studies demonstrated increased productionof antioxidant enzymes and compounds in endophyte-infectedplants [15,17,18]. This antioxidant capacity may also contribute tothe enhancement of stress tolerance in their hosts [19]. Also, Walleret al. [20] showed that P. indica reprograms barley to salt stresstolerance, resistance to diseases and higher yield.

There is scarce or null information about the effects of P. indicaon physiological changes of various plants under heavy metalstress. Therefore, this study was carried out to investigate theeffects of P. indica and G. mosseae on growth, physiology and shootand root Cd accumulation in Triticum aestivum cv. Sardari39 underdifferent Cd contamination levels.

2. Results

2.1. Root colonization

In P. indica-inoculated and G. mosseae-inoculated plants, rootcolonization was significantly decreased at 0.6 and 0.9 mM Cdcompared to 0 mM Cd (Table 1). However, the sensitivity ofG. mosseae was more than P. indica to Cd treatment at 0.6 and0.9 mM Cd. In co-inoculated plants, root colonization was signifi-cantly decreased with excess of Cd in the soil from 0 to 0.3 and from0.6 to 0.9 mM Cd (Table 1). Co-inoculated plants had less rootcolonization than P. indica-inoculated ones under 0.3, 0.6 and0.9 mM Cd.

2.2. Growth parameters

In non-inoculated plants, the Cd concentrations of 0.6 and0.9 mM in the soil, significantly reduced shoot length, whereasconcentration of 0.3 mM had no significant influence on shootlength compared to 0 mM Cd (Table 1). By increasing Cd concen-tration in the soil, shoot and root dry weights were decreased,except for shoot dry weight at 0.6 mM Cd, in non-inoculated plants.This reduction was not significant on shoot dry weight, but wassignificant on root dry weight under 0.6 and 0.9 mM Cd comparedto 0 mM Cd. The highest concentrations of Cd produced the lowest

Table 1The effects of G. mosseae and P. indica on root colonization, shoot length, shoot dry weig

Cd treatment (mM) Fungal treatment RC (%)

0 C 00.0 � 0.0 h0 Pi 61.7 � 1.7 a0 Gm 45.0 � 1.1 f0 Pi þ Gm 62.0 � 1.5 a

0.3 C 00.0 � 0.0 h0.3 Pi 61.3 � 1.8 ab0.3 Gm 43.3 � 2.4 f0.3 Pi þ Gm 53.3 � 0.7 de

0.6 C 00.0 � 0.0 h0.6 Pi 58.0 � 1.1 bc0.6 Gm 25.0 � 1.1 g0.6 Pi þ Gm 54.7 � 0.7 cd

0.9 C 00.0 � 0.0 h0.9 Pi 55.3 � 1.4 cd0.9 Gm 22.7 � 0.7 g0.9 Pi þ Gm 50.0 � 1.1 e

C: control (non-inoculation); Pi: P. indica; Gm: G. mosseae; RC: root colonization; SL: shootThe same letter within each column indicates no significant difference among treatmen

shoot length and shoot and root dry weights (Table 1). The presenceof P. indica significantly increased shoot length under 0.3, 0.6 and0.9 mM Cd, shoot dry weight under 0, 0.3, 0.6 and 0.9 mM Cd androot dry weight under 0.3 mM Cd compared to non-colonizedwheats (Table 1). Inoculation of G. mosseae significantly increasedshoot length at 0.3 mM Cd, shoot dry weight at 0 and 0.3 mM Cdand root dry weight at 0.3 mM Cd in comparison with non-inoculated plants (Table 1). Co-inoculated wheats had highershoot length under 0.3 and 0.6 mM Cd, shoot dry weight under 0,0.3 and 0.6 mM Cd and root dry weight under 0.3, 0.6 and 0.9 mMCd compared to non-colonized plants (control).

2.3. Chlorophyll content, PI and Fv/Fm

In non-inoculated plants, chlorophyll content, PI and Fv/Fmwerereduced by increasing Cd concentration in the soil (Table 2). Thisreduction was significant on chlorophyll content from 0 to 0.3 mMCd, on PI from 0 to 0.6 mM Cd and on Fv/Fm from 0.3 to 0.9 mM Cd.Inoculation of P. indica significantly increased chlorophyll contentunder 0 and 0.9 mM Cd, PI under 0, 0.3, 0.6 and 0.9 mM Cd and Fv/Fm under 0.9 mM Cd compared to non-inoculated wheats (Table 2).Also, the presence of G. mosseae significantly increased chlorophyllcontent at 0 and 0.3 mM Cd, and PI at 0.3 and 0.9 mM Cd. Inocu-lation of P. indica þ G. mosseae together significantly increasedchlorophyll content under 0, 0.3 and 0.9 mM Cd, PI under 0.3, 0.6and 0.9 mM Cd in comparison to non-colonized plants (Table 2). Fv/Fm ratio did not affect by G. mosseae inoculation (in solitary ortogether with P. indica).

2.4. Shoot and root Cd accumulation

A significant increase in Cd concentration of both shoot and rootwas observed by increasing soil Cd concentration compared tocontrol treatment (Table 2). The highest soil Cd concentrationcaused higher accumulation of Cd in both root and shoot. Therewasa significant difference in the Cd concentrations in shoot and rootunder P. indica inoculation (Table 2). The presence of P. indica (insolitary or together with G. mosseae) increased Cd concentration inroot but decreased Cd content in shoot with the excess of Cdconcentrations in the soil (Table 2). For roots, this increase wassignificant under 0.6 and 0.9 mM Cd. However, under 0 and 0.3 mMCd, there was not significant reduction, but at higher soil Cdconcentrations (0.6 and 0.9 mM Cd), reduction was significant in

ht and root dry weight in wheat under increasing Cd in the soil.

SL (cm) SDW (g/plant) RDW (g/plant)

46.0 � 1.43 bcd 0.40 � 0.06 def 0.10 � 0.01 cd46.7 � 2.59 bc 0.68 � 0.05 a 0.10 � 0.01 cd46.2 � 1.69 bcd 0.59 � 0.03 ab 0.10 � 0.01 cd46.7 � 2.62 bc 0.66 � 0.07 a 0.10 � 0.01 cd

41.0 � 2.26 de 0.29 � 0.02 ef 0.07 � 0.02 def49.9 � 2.76 ab 0.55 � 0.08 abc 0.15 � 0.02 ab52.4 � 1.64 a 0.53 � 0.06 abcd 0.16 � 0.01 a49.4 � 1.25 ab 0.50 � 0.02 bcd 0.12 � 0.02 bc

39.4 � 0.24 e 0.31 � 0.02 ef 0.04 � 0.01 fgh46.4 � 1.52 bcd 0.54 � 0.03 abcd 0.08 � 0.01 def40.9 � 0.72 de 0.32 � 0.01 ef 0.06 � 0.01 efgh47.7 � 1.80 abc 0.55 � 0.08 abc 0.09 � 0.01 cde

38.2 � 1.05 e 0.27 � 0.02 f 0.02 � 0.01 h46.1 � 1.28 bcd 0.44 � 0.04 cde 0.04 � 0.01 fgh41.0 � 0.79 de 0.34 � 0.02 ef 0.03 � 0.01 gh43.1 � 1.42 cde 0.40 � 0.05 def 0.07 � 0.02 defg

length; SDW: shoot dry weight; RDW: root dry weight. Values are mean� SE; n¼ 4.ts (P < 0.05) using Duncan’s Multiple Range Test.

Table 2The effects of G. mosseae and P. indica on total chlorophyll content, PI, Fv/Fm, shoot Cd and root Cd in wheat under increasing Cd in the soil.

Cd treatment (mM) Fungal treatment Total Chl. content PI Fv/Fm Shoot Cd (mg/kg DW) Root Cd (mg/kg DW)

0 C 24.27 � 1.5 c 5.96 � 0.4 bcd 0.83 � 0.004 a 0 � 0 f 42 � 3.0 i0 Pi 34.27 � 3.8 a 7.59 � 0.5 a 0.82 � 0.003 a 0 � 0 f 51 � 1.5 i0 Gm 28.77 � 2.2 b 6.70 � 0.2 b 0.83 � 0.000 a 0 � 0 f 33 � 2.5 i0 Pi þ Gm 30.71 � 2.6 ab 6.67 � 0.4 b 0.82 � 0.003 a 0 � 0 f 50 � 1.6 i

0.3 C 07.40 � 0.3 fg 4.61 � 0.2 ef 0.82 � 0.005 a 81 � 3.5 e 4770 � 140 g0.3 Pi 10.25 � 1.0 def 6.17 � 0.7 bc 0.82 � 0.003 a 70 � 2.4 e 5078 � 125 g0.3 Gm 14.25 � 1.6 d 5.71 � 0.1 cd 0.83 � 0.000 a 74 � 1.8 e 3720 � 95 h0.3 Pi þ Gm 12.72 � 0.4 de 5.65 � 0.2 cd 0.82 � 0.003 a 77 � 3.8 e 4986 � 114 g

0.6 C 3.42 � 0.2 gh 2.95 � 0.1 gh 0.81 � 0.005 ab 181 � 6.3 b 7506 � 220 ef0.6 Pi 8.20 � 1.8 efg 5.21 � 0.2 de 0.82 � 0.005 a 154 � 5.8 d 8110 � 170 d0.6 Gm 3.58 � 0.2 gh 3.31 � 0.1 gh 0.80 � 0.016 b 171 � 5.5 bc 7148 � 148 f0.6 Pi þ Gm 7.97 � 1.2 efg 4.73 � 0.4 e 0.82 � 0.006 a 166 � 8.1 c 7868 � 162 de

0.9 C 0.98 � 0.1 h 2.59 � 0.2 h 0.80 � 0.009 b 205 � 2.9 a 9522 � 148 c0.9 Pi 8.05 � 1.0 efg 4.44 � 0.1 ef 0.82 � 0.006 a 171 � 4.1 bc 10,764 � 177 a0.9 Gm 2.45 � 0.3 h 4.56 � 0.3 ef 0.81 � 0.007 ab 199 � 4.0 a 9496 � 161 c0.9 Pi þ Gm 8.42 � 1.0 efg 3.70 � 0.2 fg 0.81 � 0.009 ab 179 � 3.5 b 9960 � 93 b

C: control (non-inoculation); Pi: P. indica; Gm: G. mosseae. Values are mean � SE, n ¼ 4. The same letter within each column indicates no significant difference amongtreatments (P < 0.05) using Duncan’s Multiple Range Test.

S. Shahabivand et al. / Plant Physiology and Biochemistry 60 (2012) 53e58 55

shoot Cd content (Table 2). G. mosseae inoculation reduced root Cdat all Cd levels in the soil, but this reduction was only significantunder 0.3 mM Cd (Table 2).

3. Discussion

The data from our study showed that increasing Cd concentra-tion in the soil caused a reduction on root colonization and thesensitivity of G. mosseae was more than that of P. indica underhigher Cd concentrations in the soil. Pawlowska and Charvat [21]showed that spores of Glomus etunicatum and Glomus intraradicesdiffered noticeably in their sensitivities to the Cd, Pb, and Znexposures. G. mosseaewas more sensitive than P. indica at differentlevels of Cd in the soil. Pawlowska and Charvat [21] showed that inG. etunicatum, the extent of presymbiotic hyphal extension wasgenerally constant but presymbiotic hyphal extension inG. intraradices increased with Cd and Pb concentrations, even atconcentrations that partially inhibited spore germination. Thisincrease in metal sensitivity in spores from the metal-rich soil mayindicate that the cellular system responsible for metal buffering issaturated with metal ions and subsequent exposure to elevatedmetal concentrations results in toxicity [21]. Substantial differencesin the spore germination, hyphal density, symbiotic extra-radicalhyphae expansion may be present not only among differentgenera of AM fungi [22] but also between other fungi such as rootendophytic fungus. The root colonization in co-inoculated plantswas less than P. indica-inoculated wheats under Cd exposure. AMFsymbiosis is known to change physiological and biochemicalproperties of the host and these changes may alter the compositionof root exudates which play a role in the modification of themicrobial population in the mycorrhizosphere [23]. It is likely thatCd treatment affects on the composition of root exudates byG. mosseae.

The toxic effects of Cd have widely been studied in differentplant species and Cd is known to reduce or inhibit plant growth. Inthe present work, with increasing Cd in the soil, shoot length andshoot and root dry weights were decreased, in agreement with theprevious reports by Lopez-Millan et al. [24] in tomato and by Wuand Zhang [25] in barley. P. indica treatment was strongly associ-ated with greater growth parameters than that of control. Similarresults were obtained by other researchers [15,20]. P. indica mayincrease host fitness and competitive abilities by increasing growthrate through evolving biochemical pathways to produce plant

growth hormones such as indole-3-acetic acid and cytokinins[26,27] or enhance the absorption of nutritional elements by thehost [28]. We also observed the positive effects of G. mosseae on thegrowth of wheats. Similar results have been reported for othermycorrhizal plant species under metal stress conditions [12,29].The positive effect likely attributed to the improvement of Pnutrition, the uptake of water by hyphae and the increase of rootlength density [30].

The results from Table 2 showed that the Cd concentration inroots was more than that of soil Cd, indicating that the Cdabsorption mechanism for roots is an active process in wheat. It issuggested that themechanisms of Cd absorption in roots and xylemloading are related to an energy-dependent active process [31,32].Based on Table 2, root accumulated more Cd than shoot. It could becalculated that only 3e4% of the total Cd accumulated in rootsreached the shoot cells [33]. Accumulation of large amounts of Cd inthe roots may limit the accumulation of Cd in above-groundportions of the plant [34]. In the presence of P. indica, the Cdconcentration was increased in root (about 10%), whereas it wasdecreased in shoot (about 10%). These results showed that chela-tion of Cd inside the fungus or adsorption of Cd to chitin in thefungal cell wall caused accumulation of Cd in root and preventedthe Cd translocation from root to shoot. Similarly, in AMF, there isevidence suggesting that fungal hyphae components may provideadditional detoxification mechanisms by storing toxic compounds[8]. It was shown that endophytes possessing suitable degradationpathways or metal sequestration or chelation systems are able toincrease host plant tolerance to presence of heavy metal, therebyassisting their hosts to survive in contaminated soil [35]. We havenot found reports of increased or reduced Cd concentrations inplant tissues as a response to excessive soil Cd concentrationsunder P. indica symbiosis. Inoculation of G. mosseae diminished Cdaccumulation in roots. AMF can bind heavymetals beyond the plantrhizosphere by releasing an insoluble glycoprotein commonlyknown as glomalin [36]. The reduction in root Cd content underG. mosseae inoculationmight be related to the adsorptive capabilityfor metals of the relatively large fungal biomass (especially extra-radical hyphal cell wall) associated with the host plant roots,which may physically minimize or exclude the entry of metals intohost plant.

The chlorophyll content and PI were decreased by increasing Cdconcentration in the soil (Table 2). A significant reduction in chlo-rophyll content under metal toxicity has been attributed to

S. Shahabivand et al. / Plant Physiology and Biochemistry 60 (2012) 53e5856

a number of effects including inhibition of chlorophyll biosynthesis,chlorophyll degradation, hastened senescence and disorganizationof chloroplasts, and oxidative stress [33]. The performance index(PI) combines three independent functional steps of photosyn-thesis, such as the density of reaction centers in the chlorophyllbed, excitation energy trapping and conversion of excitation energyto electron transport into a single multi-parametric expressionunder stress conditions [37]. According to Goncalves et al. [38], thelow value of PI under the stress condition caused by high freeenergy availability in the system suggests that adaptation to fullsunlight of wheat plants is thermodynamically unfavorable due tothe difficulty of using the excess energy in the photosyntheticprocess. In this study, inoculation of P. indica and G. mosseae (insolitary or together) increased chlorophyll content and PI incomparison with non-inoculated plants. These results indicate thepositive influence of these plantemicrobe interactions on photo-synthetic apparatus of wheat plants.

However, Fv/Fm ratio did not change in plants grown with 0.3and 0.6 mM Cd, but it significantly decreased at 0.9 mM Cd whencompared to 0 mM Cd values (Table 2). Fv/Fm is a parameter thatexpresses the maximum efficiency of PSII controlled by theprimary photochemistry of PSII (charge separation, recombinationand stabilization), non-radiative loss of excited states in light-harvesting antennae and by excited states quenched by oxidizedplastoquinone (PQ) molecules from the PQ pool [39]. According toLopez-Millan [24] the lack of major effects of Cd (at 0.3 and0.6 mM) on leaf electron transport rates support that Cd-treatedwheat plants were not affected by photo-inhibitory processes.Similar observations have been made before. For example, treat-ment of cucumber with Cd, Cu and Pb decreased the plant growthmarkedly but only a mild effect of metals was observed on Fv/Fm[40]. Shi and Cai [41] have also shown that Fv/Fm in peanut did notchange with increasing Cd concentration up to 200 mg Cd/kg soil.It is shown that Cd damages photosynthetic electron transportchain rather than PSII [42]. On the other hand, numerous studieshave indicated that heavy metals significantly alter Fv/Fm [43].Therefore, it is possible that the effects of heavy metals such as Cdon Fv/Fm depend on the plant species and even varieties, leaf ageand metal content in leaf tissue. Inoculation of P. indica signifi-cantly increased Fv/Fm under 0.9 mM Cd compared to non-inoculated wheats. Sun et al. [15] reported that Fv/Fm valuesdecreased in the control after exposure to drought, while nosignificant decrease was observed for P. indica-colonized plants.These results demonstrate that P. indica-inoculated plants sufferless from stress than the control. As the crosstalk between differentdefense signaling pathways has been suggested, response of theplant dual-inoculated with AMF and P. indica shall be furtherclarified.

In conclusion, the consistent differences were found betweeninoculated and non-inoculated wheats in response to theincreasing soil Cd. We show here that P. indica-colonized plants aremore resistant to Cd stress rather than that of G. mosseae-colonizedwheats, which include a reduction in Cd content of shoot andenhanced growth. Because P. indica, unlike G. mosseae, can easily bepropagated on a large scale in the absence of a host plant, wesuggest the consideration of this fungus as a biofertilizer forsustainable agriculture.

4. Materials and methods

4.1. Plant materials

Wheat seeds (T. aestivum cv. Sardari39) were obtained from theDryland Agricultural Research Institute,Maragheh, Iran. Seedsweresurface sterilized for 20 min in 1% NaClO, then rinsed with distilled

water five times and germinated on wet filter paper in Petri dishesat 25 �C for 48 h.

4.2. Soil preparation

The experiment soil was collected from the surface horizon ofMaragheh University Campus farm. It contained 65% sand, 23% silt,12% clay, 1.2% organic matter, 0.05% total N, 7 mg/kg available P,35mg/kg available K,1.8mg/kg total Cd, having pH of 7.3 and 1.3 ds/m EC. The soil samples were air-dried, sieved to pass 2 mm andwere steam sterilized (100 �C for 1 h, three consecutive days) byautoclaving to eliminate native AM fungal propagules as well asother microorganisms. After sterilization, four Cd concentrations(0, 0.3 mM or 33.7 mg Cd/kg soil, 0.6 mM or 67.4 mg Cd/kg soil and0.9 mM or 101.1 mg Cd/kg soil) were added to the soil (as CdCl2).The samples then were incubated at 20 �C for one month allowingmetal to distribute into various fractions and equilibrating with soilsolid phase.

4.3. Fungal materials

P. indica was cultured in Petri dishes on a modified Kaefermedium [44]. The plates were placed in a temperature-controlledgrowth chamber at 25 �C for 2 weeks. The liquid culture was keptin shaker incubator at 100 rpm for 15 d at room temperature in thedark. The amount of 50 ml liquid culture was added to pots thatwere treated with P. indica. G. mosseae inoculum consisted ofspores, soil, hyphae and infected maize root fragments (suppliedfrom the Department of Plant Pathology, School of Agriculture,Tarbiat Modares University, Tehran, Iran). The inoculated dosagewas 50 g of inoculum per pot containing approximately 20 spores/gsoil.

4.4. Planting and growth conditions

The experiment was carried out under growth chamber condi-tions and consisted of a completely randomized 4 � 4 factorialdesign. Pots were filled with 5 kg of sterilized sandy soil thatcontained four added Cd (as CdCl2) concentrations (0, 0.3, 0.6 and0.9 mM Cd) with four replicates. The fungal treatments were: (1)inoculation of P. indica (50 ml of liquid culture), (2) inoculation of G.mosseae (50 g of inoculated soil), (3) co-inoculation of G. mosseaeand P. indica together (50 ml of liquid culture þ 50 g of inoculatedsoil) and (4) non-inoculation (control). Non-P. indica and non-G. mosseae treatments received the same weights of autoclavedP. indica and G. mosseae inoculums. P. indica and G. mosseae inoc-ulums were placed 2 cm below wheat seeds at sowing time. Theexperimental pots were placed in a growth chamber underconditions of 14 h of light, 10 h darkness, 28/20 �C day/nighttemperature, relative humidity of 50e65% and photosyntheticphoton flux density of 200 mmol m�2 s�1. Watering was done at72 h intervals throughout the growth period using deionized waterto near field capacity. Plants were harvested after 45 d for growthand chemical analysis. Roots and shoots of the harvested wheatsamples were rinsed with tap water to remove soil particles andthen carefully washed with deionized water. The samples weredried by filter paper for measurement of shoot length then weredried in an oven at 70 �C for 48 h (to measure the dry weights andCd contents of roots and shoots).

4.5. Root colonization

A small fraction of the root systemwere carefully washed in tapwater, cut into 1 cm root pieces and were cleared with a 10% (w/v)KOH solution, stained with 0.05% (v/v) Trypan blue in lactic acid

S. Shahabivand et al. / Plant Physiology and Biochemistry 60 (2012) 53e58 57

[45]. The percentage of fungal root length infection, either byhyphae, arbuscules or vesicles of G. mosseae microscopically wasassessed by the grid-line intersect technique [46]. In P. indica-inoculated roots, the distribution of chlamydospores within theroot was taken as an index of colonization [14].

4.6. Total chlorophyll content and chlorophyll a fluorescenceparameters

Total chlorophyll content and chlorophyll fluorescence param-eters (Fv/Fm, PI) were measured by the CL-01 chlorophyll contentmeter (Hansatech, UK) and chlorophyll fluorometer (Hansatech,Instruments LTD, UK), respectively.

4.7. Cd determination

The dried plant samples of finely ground (0.1 g) were digestedwith a mixture (7:1, v/v) of HNO3 and HClO4 [47]. Cd concentrationsin digested solutions were determined using an atomic absorptionspectrophotometer (Shimadzu, Japan).

4.8. Statistical analysis

The analysis of variance (ANOVA) was performed on all exper-imental data using GenStat 12 software. The differences betweenmeanswere determined using Duncan’sMultiple Range Test at 0.05probability level.

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