Rhizophagus clarus and phosphate alter the physiological responses of Crotalaria juncea cultivated...

Applied Soil Ecology 91 (2015) 37–47

Rhizophagus clarus and phosphate alter the physiological responses ofCrotalaria juncea cultivated in soil with a high Cu level

Paulo Ademar Avelar Ferreira a,*, Carlos Alberto Ceretta a, Hilda Hildebrand Soriani a,Tadeu Luiz Tiecher a, Cláudio Roberto Fonsêca Sousa Soares b, Liana Veronica Rossato a,Fernando Teixeira Nicoloso a, Gustavo Brunetto a, Juçara Terezinha Paranhos a,Pablo Cornejo c

aDepartment of Soil Science, Federal University of Santa Maria, CEP 97105-900, Rio Grande do Sul, BrazilbCentre for Biological Sciences, Department of Microbiology, Immunology and Parasitology, Federal University of Santa Catarina, Florianopolis, SC, BrazilcDepartamento de Ciencias Químicas y Recursos Naturales, Universidad de La Frontera, Casilla 54-D, Temuco, Chile

A R T I C L E I N F O

Article history:Received 4 November 2014Received in revised form 3 February 2015Accepted 17 February 2015Available online xxx

Keywords:Arbuscular mycorrhizal fungiChlorophyllFluorescenceReactive oxygen speciesHeavy metal toleranceCu-based fungicide

A B S T R A C T

Symbioses with arbuscular mycorrhizal fungi (AMF) may increase plant tolerance to heavy metals, inaddition to the improvement in phosphorus (P) uptake by plants. This study evaluated the effects of theinteraction between an increase in the soil phosphorus level and the colonization by Rhizophagus claruson several biochemical and physiological parameters of Crotalaria juncea plants cultivated in a soil with ahigh copper (Cu) level. Plant growth and photosynthetic pigment fluorescence parameters, as well as thesuperoxide dismutase (SOD), catalase (CAT), ascorbate peroxidase (APX), and peroxidase (POD) enzymeactivities in C. juncea plants, were analyzed. The experiment was conducted in a greenhouse, and a3 � 2 factorial design was employed, including the natural P level, 40, and 100 mg P kg�1, with andwithout R. clarus inoculation, with three replicates in a soil with a high Cu level (65 mg kg�1). The resultsdemonstrate that the combination of the addition of P together with AMF inoculation improved the plantnutritional status, with consequent increases in the levels of P, K, Mg, Fe, Zn, and chlorophyll afluorescence parameters. Furthermore, mycorrhization and phosphorus addition increased the activitiesof the SOD and CAT enzymes, which are responsible for the removal of reactive oxygen species whenplants are exposed to high Cu levels. This synergistic effect between P application and inoculation withR. clarus could be of technological interest for achieving increased growth of C. juncea cultivated in soilswith high Cu levels via the promotion of effective mechanisms for reducing Cu phytotoxicity.

ã 2015 Published by Elsevier B.V.

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1. Introduction

Southern Brazil is a key wine-grape producing region, and theapplication of copper (Cu)-based fungicides is a common practicefor the control of various plant pathogens. The continued use ofCu-based fungicide has added large amounts of Cu to the soil,which increases the potential toxicity to plants and the level ofenvironmental contamination (Miotto et al., 2014). The

* Corresponding author. Tel.: +55 55 99733656; fax: +55 32208256.E-mail addresses: ferreira.aap@gmail.com (P. Ademar Avelar Ferreira),

carlosceretta@ufsm.br (C.A. Ceretta), hildasoriani@gmail.com(H. Hildebrand Soriani), tadeu.t@hotmail.com (T. Luiz Tiecher),crfsoares@gmail.com (C.R. Fonsêca Sousa Soares), liana.rossato@gmail.com(L.V. Rossato), ftnicoloso@yahoo.com (F.T. Nicoloso), brunetto.gustavo@gmail.com(G. Brunetto), jtparanhos@gmail.com (J.T. Paranhos), pablo.cornejo@ufro.cl(P. Cornejo).

http://dx.doi.org/10.1016/j.apsoil.2015.02.0080929-1393/ã 2015 Published by Elsevier B.V.

establishment of numerous Brazilians vineyard on sandy soilswith low organic matter levels, which are characterized by a lowersorption capacity for Cu and thus increased potential forenvironmental contamination, has aggravated this situation. Forexample, the pseudo-total level of Cu (USEPA Method 3050B) in the0–20 cm layer of a vineyard was determined to be 62.5 mg kg�1,while the natural copper content of these soils was 3.2 mg kg�1

(Miotto et al., 2014). The Brazilian environmental agency hasdetermined that a soil copper content greater than 60 mg kg�1

signals the need to perform preventive practices in order to ensurethe maintenance of soil functionality or to implement correctivemeasures aimed at restoring soil quality and promoting soilsustainability in a manner consistent with intended uses (Conama420, 2009). The presence of a high Cu level in the soil surface layerreduces the development of naturally occurring herbaceous orintroduced plants that are normally grown in the interrows ofvineyards.

Table 1Soil chemical properties after soil supplementation with P and Cu, used as growthsubstrate for C. juncea plants.

Soil chemical characterization 0 mg kg�1

P40 mg kg�1

P100 mg kg�1 Pa

pH (H2O) 5.9 5.6 5.5SOC (g kg�1) soil solution 0.65 0.67 0.63TOC (g kg�1) 8.0 8.2 8.0Cu disponível por EDTA (mg kg�1) 45.6 45.5 42.5Cu (mg L�1) soil solution 10.5 7.3 4.8Available P by Mehlich-1 (mg kg�1) 5.6 34.1 85.3P (mg L�1) soil solution 5.2 7.4 10.1Available K by Mehlich-1(mg kg�1)

190.5 183.5 170.5

Exchangeable Ca (mg kg�1) 458.4 458.4 552.8Exchangeable Mg (mg kg�1) 90.7 94.1 94.4

a The P supplementation was done on a soil with a natural level of 5.6 mg P kg�1.

38 P. Ademar Avelar Ferreira et al. / Applied Soil Ecology 91 (2015) 37–47

Cu, a plant micronutrient that acts as structural components ofproteins, fulfills catalytic functions in various enzymes (Pilon et al.,2006) involved in the processes of photosynthesis and respiration(Kabata-Pendias and Pendias, 2011). However, an excess of Cu inthe soil may lead to increased Cu levels in plant tissues (withconsequent toxicity) because Cu affects photosynthesis by alteringthe plant water content and osmotic potential, resulting in anutritional imbalance and a consequent reduction in growth(Schimdt et al., 1997; Yruela, 2005; Abdel Latef, 2011). Further-more, Cu has a high affinity for sulfhydryl groups and binds to andinactivates proteins (Ruttkay-Nedecky et al., 2013). Cu can alsogenerate reactive oxygen species (ROS) that cause lipid peroxida-tion and that may affect membrane permeability and causedamage at the genetic level (Andrade et al., 2010). The ROSgenerated by an excess of Cu may also damage the photosyntheticapparatus, primarily that of photosystem II (PSII) (Mateos-Naranjoet al., 2013), thus affecting the photosynthetic rate.

Several strategies for decreasing the solubility and bioavailabil-ity of soil Cu have been proposed to minimize the negative impactsof high Cu levels on plant tissues. Moreover, a number of studieshave shown that supplementation with phosphate may contributeto the remediation of soils contaminated with toxic elements(Kede et al., 2008; Soares and Siqueira, 2008) by promoting theformation of insoluble mineral species, thus immobilizing con-taminants (Ayati and Madsen 2001; Cao et al., 2003). Furthermore,studies indicate that an adequate supply of P also enables anincrease in the retention of toxic elements in roots via theformation of insoluble metal compounds, thus restricting thetransport of those elements to the shoots (Van Steveninck et al.,1994; Brown et al., 1995). However, the use of certain beneficialmicroorganisms associated with plant roots could also be ofinterest for their potential to improve the positive effects of Paddition on plant growth and development.

In this context, associations among plants and arbuscularmycorrhizal fungi (AMF) are of great importance because AMFimprove P uptake and assist in plant growth and because AMF mayalso contribute a reduction in the availability of heavy metals as aresult of their immobilization in fungal structures (Gonzalez-Chavezet al., 2002; Andrade et al., 2010; Cornejo et al., 2013) or glomalin(Gonzalez-Chavez et al., 2004; Cornejo et al., 2008; Aguilera et al.,2011; Abdel Latef, 2013; Gil-Cardeza et al., 2014). This latter effectresults in a decrease in the transferof potentially toxic elements fromroots to shoots (Joner et al., 2000; Christie et al., 2004). AMF may alsoact to reduce heavy metal uptake by promoting the precipitation orchelation of elements in the rhizosphere (Kaldorf et al.,1999) and bypromoting morphological, physiological and molecular changes inhost plants (Andrade et al., 2010).

As a result of the biochemical and physiological changes causedby AMF, plants increase the activities of anti-oxidant enzymes,including peroxidases and superoxide dismutases (Lambais et al.,2003; Arfaoui et al., 2007), which assist in the removal (ordetoxification) of the formed ROS, thus increasing plant toleranceto heavy metal stress (Andrade et al., 2010; Meier et al., 2011).However, little is known of the effects of AMF on the activity ofantioxidant enzymes and on the chlorophyll a fluorescenceparameters of plants grown in soils with high Cu levels. Basedon the above-described background knowledge, we have hypoth-esized that the joint use of P supplementation and AMF inoculationcould represent an interesting biotechnological tool for theimprovement of the establishment and growth of C. junceacultivated in soils with high accumulations of Cu, such as thosedesignated for wine-grape production in Southern Brazil.

Therefore, this study has aimed to evaluate the interactionbetween additions of P and colonization by the fungus Rhizophagusclarus on the biochemical and physiological characteristics ofC. juncea cultivated in a soil with a high Cu level.

2. Materials and methods

2.1. Experimental design

This study was conducted in a greenhouse of the Department ofSoil Science of the Federal University of Santa Maria (UniversidadeFederal de Santa Maria – UFSM). A 3 � 2 factorial design wasemployed in a completely randomized scheme with 3 replicates.The treatments consisted of three P levels: (i) natural level(5.6 mg kg�1), and the natural level plus the supply of (ii) 40 or (iii)100 mg P kg�1. These levels of P addition were determinedaccording to previous studies examining phosphorous and heavymetal interactions (Soares and Siqueira, 2008). Phosphorous wasapplied as triple superphosphate to a soil artificially contaminatedby adding 60 mg Cu kg�1, a concentration level commonly ob-served in vineyard soils in the Campanha Gaúcha, Brazil (Miottoet al., 2014). The soils of each P level were inoculated (+AMF) withspores of R. clarus, as further described, or maintained as un-inoculated (�AMF) soil.

2.2. Substrate preparation and soil analyses

A typic Hapludalf soil with a sandy texture was collected from anatural grassland area (29�43007.0400S; 53�42029.6000O). The soil pHwas adjusted to 6.0 via lime addition, then supplemented with40 or 100 mg kg�1 P and allowed to stabilize for 45 days.Subsequently, the soil was contaminated with 60 mg Cu kg�1

(CuSO4�2H2O) and allowed to stabilize for another 45 days. Finally,the soil was autoclaved twice at 120 �C for 2 h. A basal fertilizationof 100, 30, 5.0, and 0.80 mg kg�1 of N (NH4Cl), K (K2SO4), Zn(ZnSO4�7H2O) and B (H3BO3), respectively, was applied to all plots.Nitrogen fertilization was divided between two applications,delivered at 15 and 30 days after germination. The main soilchemical properties after the conditioning process are listed inTable 1.

After incubation and prior to seeding, the soil solution wasextracted to assess the effects of P addition on the chemical speciesof Cu and P. The soil solution was collected in a saturation extractfollowing the methodology described by Raij et al. (2001). The pHwas determined on an aliquot of soil solution. Another aliquot wasthen filtered through a 0.22-mm cellulose membrane filter. Thetotal soluble organic carbon (SOC) concentration was determinedaccording to Silva and Bohnen (2001).

The levels of available Cu and Zn in the soil were determinedafter extraction with an EDTA solution (0.01 mol L�1 ethylenedi-amine tetraacetic acid, 1 mol L�1 ammonium acetate, pH 7.0), andthe available Ca and Mg in the soil were extracted with 1 mol L�1

KCl. The available P and K were extracted from 5 g of dry soil with50 mL of Mehlich-1.

P. Ademar Avelar Ferreira et al. / Applied Soil Ecology 91 (2015) 37–47 39

The soil solution concentrations of Cu, Ca, Mg, Cu, and K weredetermined using an inductively coupled plasma optical emissionspectrometer (ICP-OES; PerkinElmer Optima 7000DV).

2.3. Mycorrhizal inoculation and corroboration of colonization

Mycorrhizal inoculation was performed in each pot by applying200 spores of R. clarus, which was supplied by the Laboratory ofSoil Microbiology (Laboratório de Microbiologia do Solo) of theFederal University of Santa Catarina, Brazil, to the roots of plantsafter the spores had multiplied in culture pots using Brachiariadecumbens as host plant. The spores were extracted using the wetsieving method (Gerdemann and Nicolson, 1963) followed bycentrifugation in water at 2000 rpm for 3 min in a sucrose (45%)gradient at 1500 rpm for 2 min. The numbers of spores werecounted using a stereoscopic microscope (40� magnification).Approximately 50 mL of a soil-inoculum filtrate that did notcontain any AMF propagules was applied to the non-inoculatedtreatments to balance the non-mycorrhizal soil microbiota.

A sample of roots was collected and stored in FAA [formalde-hyde (40%):alcohol (50%):acetic acid = 13 mL:200 mL:5 mL] forclearing and staining according to the Phillips and Haymanmethod (1970). The colonization rate was evaluated using a grid-plate method (Giovannetti and Mosse, 1980).

2.4. Plant growth and mineral plant nutrition

Previousstudies havedemonstratedtheC. juncea is characterizedby a tolerance to high Cu concentrations in the soil and a highpotential to accumulate Cu in the root system, which are desirabletraits for phytostabilization programs (Zancheta et al., 2011). Inaddition, C. Juncea is capable of producing large amount of drymatter, which increases the organic matter content of soils andimproves nutrient cycling.

Seeds of C. juncea were scarified with concentrated H2SO4 p.a.for 5 min and then thoroughly washed with sterile dH2O. Fourseeds were sown per pot, and ten days after sowing (DAS), potswere thinned to two plants per pot. The total production of drymatter (dry matter of shoots + roots) and the shoot levels of P, Ca,Mg, Cu, Zn, and Fe were assessed at 45 DAS. The aboveground andbelowground plant components were separated at ground level,and the shoots were washed with distilled water, oven dried, andweighed before tissue analyses. The contents of P, Ca, Mg, Fe, Zn,and Cu in the leaves were determined by ICP-OES (PerkinElmerOptima 7000 DV) after HNO3–HClO4 digestion. The total-N in thedigest was determined by Kjeldahl analysis.

2.5. Assessment of chlorophyll a fluorescence

Three plants from each treatment were collected at 45 DAS forchlorophyll a fluorescence measurements, which were conductedon fully expanded leaves from the upper third of the plants using apulse amplitude-modulated fluorometer (JUNIOR-PAM, Walz,Effeltrich, Germany). The measurements obtained to generatethe fluorescence induction curves were collected in a greenhouseduring the period between 8:00 and 9:30 AM under a meanradiation of 600 mmol m�2 s�1.

Plants were dark-adapted for 30 min, using leaf–clips designedfor this purpose. The minimal fluorescence level in the dark-adapted state (F0) was measured using a modulated pulse(<0.05 mmol m�2 s�1 for 1.8 ms) that was too small to inducesignificant physiological changes in the plant. The maximalfluorescence in this state (Fm) was measured after applying asaturating actinic light pulse of 10,000 mmol m�2 s�1 for 0.6 s. Thevalues of the variable fluorescence (Fv = Fm–F0) and the maximum

quantum efficiency of the PSII photochemistry (Fv/Fm) werecalculated from F0 and Fm.

Using the fluorescence parameters determined under both thelight- and dark-adapted states, the following variables werecalculated: (i) quantum efficiency of PSII (Fv/Fm = (Fm–F0)/Fm),which equals the proportion of light absorbed by the chlorophyllassociated with PSII that is used in photochemistry(Maxwell and Johnson, 2000), and (ii) non-photochemicalquenching (NPQ = Fm/Fm’ � 1), which is linearly related to heatdissipation (Maxwell and Johnson, 2000).

The maximum electron transport rate (ETRmax) during the pre-dawn period (5:00–6:00 AM) was assessed by generating lightcurves (ETR versus photosynthetically active radiation, PAR) bysubjecting each sample to nine levels of radiation (0; 125; 190;285; 420; 625; 820; 1,150, and 1,500 mmol electrons m�2 s�1) for10 s and fitting the measurements using the equation ETR = ETRmax

[1–e�kQ], where k is a fitting constant and Q is the light intensity(PAR); (Rascher et al., 2000).

2.6. Determination of photosynthetic pigments

The concentrations of chlorophyll a (Chl a) and chlorophyll b(Chl b), as well as carotenoid levels (Cx + c), were determinedaccording to the methodology described by Hendry and Price(1993) on three samples per treatment. For this purpose, plantmaterial of a known leaf area and fresh mass that was used in thechlorophyll fluorescence analysis were frozen in liquid N2 andstored at �80 �C until the time of quantification. The samples weremacerated in liquid N2, homogenized in 5 mL of 80% acetone, andthen transferred into 15-mL falcon tubes and centrifuged at4000 � g for 4 min at 25 �C. The absorbances of the supernatant at480, 645, and 663 nm were measured using a SF325NMspectrophotometer (Bel Engineering, Monza, Italy), and theconcentrations of Chl a, Chl b, total Chl (Chl a + b), and Cx + c werecalculated according to the methodology of Lichtenthaler (1987).

2.7. Extraction and quantification of enzymes

Crude enzyme extracts were prepared from leaf tissue samples(0.5 g fresh matter) that were macerated in liquid N2 andhomogenized in 5.0 mL of potassium phosphate buffer (100 mM,pH 7.5), supplemented with ethylenediaminetetraacetic acid(EDTA, 1 mM), dithiothreitol (DTT, 3 mM), and polyvinylpolypyr-rolidone (PVPP, 2% w/v; Azevedo et al., 1998). The homogenate wascentrifuged at 14,000 � g for 30 min at 4 �C, and the supernatantwas collected and divided into 0.5-mL aliquots that were stored at�80 �C until the time of quantification of enzyme and total proteinconcentrations. The total protein concentration of each sample wasassessed using a spectrophotometer by the absorbance at 595 nmas reported by Bradford (1976), using bovine serum albumin (BSA)as the standard.

The activity of superoxide dismutase (SOD; EC 1.15.1.1) wasassessed according to the spectrophotometric method describedby Giannopolitis and Ries (1977). One unit of SOD was defined asthe amount of enzyme that inhibits nitroblue tetrazolium (NBT) bya photoreduction of 50% (Beauchamp and Fridovich, 1971).

The activity of non-specific peroxidases (POD; EC 1.11.1.7) in theextract was assessed according to Zeraik et al. (2008) usingguaiacol as the substrate and the 26.6 mmol L�1 cm�1 molarextinction coefficient of Chance and Maehley (1955). One unit ofPOD was defined as the amount of enzyme that catalyzes theformation of 1 mmol tetraguaiacol min�1mL�1 extract at 470 nm.

The catalase (CAT; EC 1.11.1.6) activity was assessed according tothe spectrophotometric method described by Azevedo et al. (1998)using a molar extinction coefficient of 40.0 M�1 cm�1 to calculatethe activity. One unit of CAT was defined as the amount of enzyme

Table 2Effect of supplementation with P and inoculation with AMF in a soil with highcopper levels on the levels of nitrogen (N), phosphorus (P), potassium (K), calcium(Ca), magnesium (Mg), copper (Cu), iron (Fe) and zinc (Zn) in C. juncea shoots.

Inoculation 0 mg kg�1 P 40 P 100 P

N g kg�1 �AMF 38.52 ns 30.16 ns 35.45 ns+AMF 37.34 28.64 31.65

P g kg�1 �AMF 0.74 bB 0.67 bB 1.36 aA+AMF 1.63 aA 1.09 aC 1.28 aB

K g kg�1 �AMF 11.69 bA 9.26 bA 6.32 bA+AMF 15.23 aA 14.08 aA 11.62 aB

Ca g kg�1 �AMF 6.69 aB 7.80 bA 8.44 bA+AMF 7.12 aC 9.56 aB 12.42 aA

Mg g kg�1 �AMF 34.03 aA 22.98 aB 25.41 bB+AMF 35.14 aA 26.63 aB 31.49 aA

Cu mg kg�1 �AMF 79.07 aA 44.64 bB 52.52 aB+AMF 44.64 bC 63.19 aA 52.43 aB

Fe mg kg�1 �AMF 40.05 bB 52.91 bA 54.14 bA+AMF 62.41 aB 66.13 aA 65.41 aA

Zn mg kg�1 �AMF 32.57 bA 26.25 bB 25.03 bB+AMF 38.44 aB 41.00 aA 30.96 aB

Means followed by the same lowercase in columns (inoculation) and uppercaseletter in rows (doses of phosphorus), show no statistically significant differencefrom each other according to Tukey’s test at P < 0.05. ns = non-significant.

40 P. Ademar Avelar Ferreira et al. / Applied Soil Ecology 91 (2015) 37–47

that catalyzes the decomposition of 1 mmol H2O2 min�1mL�1

extract at 240 nm.The ascorbate peroxidase (APX; EC 1.11.1.11) activity was

assessed according to the method of Nakano and Asada (1981)using a molar extinction coefficient of 2800 M�1 cm�1 to calculatethe activity. The enzyme activity of ascorbate oxidase (AO; EC1.10.3.3) was also determined to correct for the oxidation ofascorbate in the absence of H2O2. One unit of APX was defined asthe amount of enzyme that catalyzes the degradation of 1 nmolascorbate min�1mL�1 extract, as determined at 290 nm.

2.8. Statistical analysis

All the data were transformed when necessary to meet theassumptions of normality and homoscedasticity. Subsequently,the data were analyzed by ANOVA, and the means werecompared using Tukey’s test when the effects of AMF inocula-tion, P supply, and/or the interaction between these factors werestatistically significant (P < 0.05). Moreover, the linear correla-tions (Pearson’s correlation coefficients) among the data weredetermined using the SigmaPlot version 12.3 software. Inaddition, the variables were subjected to a principal componentanalysis (PCA) using the CANOCO version 4.5 software (Ter Braakand Smilauer, 1998).

3. Results

3.1. Soil chemical properties and Cu2+ and P levels in the soil solution

The soil levels of available P ranged from 5.6 to 85.3 mg kg�1,and the levels of available Cu ranged from 42.5 to 45.6 mg kg�1 soil(Table 1). Supplementation with P reduced the Cu availability inthe soil solution, with decreases ranging from 31 to 54% relative tothe natural P levels. The P level in the soil solution increased withthe addition of triple superphosphate to the soil.

3.2. Mycorrhizal colonization, plant growth, and mineral plantnutrition

Neither spores nor AMF colonization were detected in any of thenon-inoculated treatments. Conversely, the mycorrhizal

Fig. 1. Total dry matter of C. juncea grown in a soil with high copper (65 mg kg�1)levels under different levels of phosphorus with and without AMF inoculation.Means followed by the same lowercase letter compare doses in the same conditioninoculation and uppercase letters compare inoculation within the same dose of P(Tukey 5%).

colonization rates in the treatments receiving AMF inoculationwere 79, 74, and 54% in the treatments with 0, 40, and 100 mg ofadded P kg�1 soil, respectively. However, the number of spores in thesoil decreased with an increase in P dose, from 86 to 25 spores per50 mL of soil in the 0 and 100 mg P kg�1 treatments, respectively(Data not shown).

AMF inoculation promoted an increase in the total plantbiomass regardless of the soil P level. However, the increase wasgreater in the soil with natural P levels, achieving 134% of thebiomass of the non-inoculated treatment (Fig. 1).

The levels of K, Mg, Fe, and Zn, but not N, in the shoots of plantsinoculated with AMF increased compared to the un-inoculatedtreatments (Table 2). In addition to these nutrients, mycorrhizalcolonization and development triggered increases of 120 and of63% in the P levels of the plants cultivated in soils containing thenatural P level and in soils supplemented with 40 mg P kg�1,respectively. This increase was less significant in the soilsupplemented with 100 mg P kg�1 (Table 2).

The first symptoms of Cu toxicity (reductions in growth, leafchlorosis, and leaf necrosis followed by apical death) were

Table 3Effect of supplementation with P and inoculation with AMF in a soil with highcopper levels on the concentration of chlorophyll (Chl a), chlorophyll b (Chl b),chlorophyll (a + b) (Chl a + b) and carotenoids (Cx + c) in C. juncea leaves.

Inoculation 0 mg kg�1 P 40P 100P

Chl a (mg g�1) �AMF 0.88 bC 1.36 aB 1.63 aA+AMF 1.14 aC 1.28 bB 1.59 aA

Chl b (mg g�1) �AMF 0.31 bC 0.48 aB 0.56 aA+AMF 0.41 aB 0.45 aB 0.60 aA

Chl a + b (mg g�1) �AMF 1.19 bC 1.83 aB 2.20 aA+AMF 1.55 aB 1.73 aB 2.19 aA

Cx + c (mg g�1) �AMF 0.22 aC 0.28 aB 0.33 aA+AMF 0.22 aC 0.26 bB 0.30 bA

Means followed by the same lowercase in columns (inoculation) and uppercaseletter in rows (doses of phosphorus), show no statistically significant differencefrom each other according to Tukey’s test at P < 0.05.

ET

R (µm

ol m

-2 s

-1)

ET

R (µm

ol m

-2 s

-1)

0

50

100

150

200

250

0

50

100

150

200

250

PAR (mmol m-2 s -1)

0

125

190

285

420

625

820

1150

1500

0

50

100

150

200

250

0 mg kg-1 P 40 mg kg-1 P

100 mg kg-1 P

-AMF +AMF

Fig. 2. Electron transport rate (ETR) relative to the photosynthetic active radiation (PAR) in leaves of C. juncea grown in a soil with high copper (65 mg kg�1) levels underdifferent levels of phosphorus with and without AMF inoculation.

P. Ademar Avelar Ferreira et al. / Applied Soil Ecology 91 (2015) 37–47 41

observed in the plants grown in the soils with the low Pconcentration at 20 DAS. Nevertheless, AMF inoculation in thesoils with natural P levels reduced the plant shoot Cu level by 44%(Table 2). This effect was not observed when 100 mg P kg�1 weresupplied to the soil.

3.3. Photosynthetic pigments

The concentrations of the assessed pigments increased with anincrease in soil P level (Table 3). Plant inoculation with R. clarus inthe soils with natural P levels triggered increases in theconcentrations of the Chl a, Chl b, and Chl a + b pigment contents.There were no significant differences in leaf carotenoid levels ofthe plants grown in the soils with natural P levels in response toAMF inoculation. However, the carotenoid levels did increase whenthe soils were supplemented with 40 and 100 mg P kg�1.

3.4. Chlorophyll fluorescence

The ETR gradually increased with an increase in PAR in theleaves of plants cultivated in soils with natural P levels and plantssubject to soil supplementation with 100 mg kg�1 P; however, theincrease in ETR is only observed above a specific PAR level (Fig. 2).In the treatment with natural P levels, there was only a statisticallysignificant difference in the ETR between the AMF-inoculated andun-inoculated plants at the highest radiation (1500 mmol m�2 s�1).This response to the AMF inoculation treatment was not observedin the soils supplemented with 40 mg P kg�1 at any PAR. However,supplementation with 100 mg P kg�1 and AMF inoculation

triggered statistically significant increases in the ETR at and above420 mmol m�2 s�1 (Fig. 2).

For the soils with natural P levels, there were no significantdifferences in the values of F0 between the inoculated and un-inoculated treatments. At the doses of 40 and 100 mg P kg�1, AMFinoculation reduced the values of F0 (Fig. 3). The value of Fv/Fm inthe soil increased in response to supplementation with P wheninoculated with AMF, and similar increases were observed in theun-inoculated soils supplemented with 40 and 100 mg P kg�1

(Fig. 3). AMF inoculation of the plants cultivated in the soils withnatural P levels resulted in an increase in the Fv/Fm of 5% comparedto the un-inoculated treatment, with the latter exhibiting a valuebelow 0.7.

The PSII effective quantum efficiency (Y(II)) of the un-inoculated plants decreased when P was supplied, whereas theY(II) of the AMF-inoculated plants increased in response to anincrease in P. On the other hand, the NPQ decreased with anincrease in soil P level. In the soils with natural P levels, the NPQ ofthe AMF-inoculated plants was 2-fold lower than that of the un-inoculated plants, which was similar to the treatment with soil thatwas supplemented with 40 mg P kg�1; the NPQ of the inoculatedplants was 3.3-fold lower than of the non-inoculated plants.

3.5. Enzyme activity

The SOD, POD, CAT, and APX enzymatic activities in the leaves ofthe plants cultivated in the soil with natural P levels and inoculatedwith AMF were 41, 15, 115 and 12% greater, respectively, comparedto the un-inoculated plants (Fig. 4). The enzymatic activities of SOD

Fig. 3. Initial fluorescence (Fo), PSII maximum quantum yield (Fv/Fm), PSII effective quantum efficiency (Y(II)) and non-photochemical quenching (NPQ) in leaves of C. junceagrown in a soil with high copper (65 mg kg�1) levels under different levels of phosphorus with and without AMF inoculation. Means followed by the same lowercase lettercompare doses in the same condition inoculation and uppercase letters compare inoculation within the same dose of P (Tukey 5%).

42 P. Ademar Avelar Ferreira et al. / Applied Soil Ecology 91 (2015) 37–47

and CAT increased with an increase in the P level in the soil, both inthe presence and absence of AMF, whereas the enzymatic activitiesof POD and APX decreased with an increase in soil P level in thetreatments with AMF.

3.6. Multivariate analysis

There were several strong relationships among parameters(Table 4). PC1 and PC2 explained 71.9% of the experimentalvariability in the PCA (52.5 and 19.4%, respectively; Fig. 5). PC1 wasprincipally correlated with the variables of TDM, CAT, SOD, Chl a,Chl b, and Fv/Fm. Moreover, the treatments supplemented with100 mg P kg�1 soil, irrespective of the presence of AMF, were highlyassociated with an increase in the above-mentioned variables.Conversely, the treatments with natural soil P levels showed agreater association with the variables of POD, NPQ, and Cu2+ in thesoil solution. The samples that were un-inoculated and supple-mented with 40 mg P kg�1 treatment tended to be positioned at anintermediate position in the PCA plot, which indicates a weakeffect of those variables.

4. Discussion

This study demonstrated that there are synergistic effectsbetween AMF inoculation and an increase in P supply that promoteplant growth in soils with high Cu levels. These effects areassociated with an improved mineral nutrition, especially in termsof P, and with a greater tolerance to Cu, as reported in other studies(Meier et al., 2011, 2015). Additionally, in our study we observed

decrease Cu levels in the soil solution upon supplementation withP. An increase in plant P uptake may promote reduced Cutranslocation to plant shoots (Christie et al., 2004; Janouskováet al., 2005; Arriagada et al., 2009) because of the Cu retention inroots attributed to the formation of insoluble compounds withmetals (Van Steveninck et al., 1994; Brown et al., 1995). In additionto the effects of an improved P nutritional status, plants mayexhibit the following attenuation mechanisms against metalphytotoxicity: (1) associations with mycorrhizal fungi (Soaresand Siqueira, 2008); (2) secretions of organic acids, especially citricacid (Meier et al., 2012a); and (3) secretions of enzymes (acidphosphatases, APases) (Nadira et al., 2014) that break the bonds oforganic forms of soil P and increase P availability, which allows P tobind to the metal ions that are free in cell compartments (includingthe cytoplasm) that are sensitive to metals. Therefore, well-nourished plants can store P-metal complexes in vacuoles or formpolyphosphate granules inside roots (Barceló and Poschenrieder,1992).

The present study demonstrated that the Cu levels in shoot drymatter can surpass the 5–30 mg kg�1 range (Kabata-Pendias andPendias, 2011), which usually is considered as normal Cu levels inplants. The inoculation with R. clarus in soils with natural P levelsreduced the plant shoot Cu levels compared to the treatmentwithout inoculation (Table 2), resulting in increased plant growth(Fig. 1). This increase may be attributed to the decreased Cu uptakecaused by Cu retention and immobilization in the cell wallcomponents of intra- and extra-radical hyphae or from metalcompartmentalization inside the fungal spores (Zhu et al., 2001;Gonzalez-Chavez et al., 2002; Meier et al., 2012b; Cornejo et al.,

Fig. 4. Enzymatic activity of superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbate peroxidase (APX) in C. juncea grown in a soil with high copper(65 mg kg�1) levels under different phosphorus levels with and without AMF inoculation. Means followed by the same lowercase letter compare doses in the same conditioninoculation and uppercase letters compare inoculation within the same dose of P (Tukey 5%).

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2013), both of which reduce the transfer of Cu to the host plant.Moreover, glomalin, a glycoprotein produced by AMF, cansequester substantial amounts of Cu in soils (Gonzalez-Chavezet al., 2004; Cornejo et al., 2008).

It is commonly recognized that an excess of Cu in plant tissuesmay affect several physiological and biochemical processes,including photosynthesis (Kabata-Pendias and Pendias, 2011).The symptoms of toxicity to an excess of Cu are the result of severalinteractions that may be expressed at the cellular and molecularlevels (Kabała et al., 2008). Cu toxicity may also result from thestrong interactions of Cu with the sulfhydryl groups of enzymesand proteins in the apoplasts of root cells, which can inhibitenzyme activities or cause changes in the structure and replace-ment of key elements, resulting in a deficiency of other nutrients(Yruela, 2005; Kabała et al., 2008). Morphological changes derivedfrom an excess of Cu in plant tissues are partially attributed to thecapacity of Cu to remove other ions from uptake sites, particularlyFe; therefore, Cu toxicity resembles Fe deficiency (Mengel andKirkby, 1987).

There was a reduction in the plant Chl a and b levels in thetreatment with natural P levels and un-inoculated plants. Theexcess of Cu was associated with the degradation of innerstructures of the chloroplasts, thus reducing the levels of pigments(Ciscato et al., 1997). In this treatment, the tissue Fe levels werebelow the critical levels (Table 2), which range from 50 to 250 mgFe kg�1 dry matter (Kabata-Pendias and Pendias, 2011; Abdel Latef,2011). Fe deficiency causes a drastic reduction in chloroplastprotein synthesis, especially for structural proteins. The main

symptom is the inhibition of chloroplast development (Rüdiger,1997; Beale, 1999). The additions of soil P and AMF inoculationpromoted increases in the Chl a and b levels. The Chl a and b levelswere positively correlated with the total dry matter production(r = 0.90, P < 0.001; r = 0.89, P < 0.001); the increases in the Chl aand b increase the amount of light capture by leaves whilesimultaneously reducing the possibility of further damage to thephotosynthetic apparatus attributed to the formation of ROS underhigh radiation (Munn-Bosch and Alegre, 2000; Kranner et al.,2002; Bagheri et al., 2011). Furthermore, the results demonstratesthat mycorrhization with AMF increased the uptake of variousnutrients, including Mg and Fe, which are key components of thephotosynthetic process, thus demonstrating a positive contribu-tion of AMF to the photosynthetic apparatus, primarily that ofphotosystem II (PSII), to C. juncea plants exposed to high soil Culevels.

Several studies have reported an effect of excess Cu on theelectron transport chain (Jegerschöld et al., 1995; Ouzounidouand Ilias, 2005) as the result of the indirect effect of Cu on Fenutrition. In this regard, the level of NPQ was negativelycorrelated with the total dry matter production, as well as thetotal chlorophyll and carotenoid levels (r = �0.83, P < 0.001;r = �0.76, P < 0.001, r = �0.62, P < 0.001). The PSII Fv/Fm andmaximum ETR1500 values of the non-mycorrhizal treatmentswere low, which characterizes a state of chronic photoinhibition(Alves et al., 2002). This reduction may result from an increase inNPQ, suggesting that plants dissipate light in the form of heat, thusprotecting leaves from the damage induced by solar radiation

Table 4Correlation matrix of some selected variables studied and the principal components (PC) obtained.

Variables SODa POD CAT APX TDM Fo Fv/Fm ETR Y(II) qP NPQ N P K Ca Mg Cu Fe Zn Cu Sol P Sol Chl a Chl b Ch a +b

PODb �0,70 nsCATc 0.85* �0.68

nsAPXd �0.22ns 0.02ns �0.19nsTDMe 0.83* �0.88* 0.89* �0.24nsFof �0.35ns 0.60ns �0.58ns �0.09ns �0.67nsFv/Fmg 0.87* �0.71* 0.86* �0.06 ns 0.90* �0.59nsETRh �0.17ns �0.09ns �0.02ns �0.01ns 0.13 ns �0.62ns 0.09nsY(II)i �0.13ns �0.15ns 0.00 ns �0.01ns 0.17ns �0.62ns 0.13 ns 0.99*qPj �0.66 ns 0.48 ns �0.41ns 0.20ns �0.43ns �0.18ns �0.42ns 0.65ns 0.58nsNPQk �0.69ns 0.63ns �0.93* 0.27 ns �0.83* 0.47 ns �0.70 ns 0.08ns 0.05ns 0.42nsNl �0.46ns 0.67ns �0.59ns �0.26ns �0.68 ns 0.80* �0.58ns -0.35ns �0.33ns �0.07ns 0.51 nsPm 0.28ns 0.27 ns 0.42ns �0.57ns 0.14 ns 0.20ns 0.21 ns �0.34ns �0.37ns �0,15ns �0.46ns 0.21 nsKn �0.42ns 0.39ns �0.16ns 0.09ns �0.42ns 0.25ns �0.44 ns �0.26ns �0.30ns 0.24 ns 0.08ns 0.13 ns 0.25nsCao 0.64 ns �0.57ns 0.69ns �0.40ns 0.82* �0.67ns 0.80* 0.48 ns 0.50ns �0.12ns �0.61ns �0.51ns 0.22ns �0.50nsMgp �0.54ns 0.76* �0.67ns �0.08ns �0.62ns 0.44 ns �0.43ns 0.08ns 0.03ns 0.40ns 0.68 ns 0.56ns 0.10 ns �0.04 ns �0.20nsCuq �0.58ns 0.16 ns �0.59ns �0.27ns �0.37ns 0.06 ns �0.60ns 0.46ns 0.45ns 0.35 ns 0.48 ns 0.24 ns �0.43ns 0.09ns �0.14ns 0.19 nsFer 0.10 ns �0.31ns 0.48 ns �0.09ns 0.36ns �0.47ns 0.14 ns 0.13 ns 0.12 ns 0.15 ns �0.56ns �0.48 ns 0.23ns 0.45ns 0.18 ns �0.61ns 0.02nsZns �0.64 ns 0.27 ns �0.32ns �0.07ns �0.28ns �0.28ns �0.46ns 0.64 ns 0.60ns 0.76 ns 0.21 ns -0.06 ns �0.16ns 0.35 ns �0.03ns 0.14 ns 0.68 ns 0.46nsCu Solt �0.87* 0.86* �0.78* 0.36ns �0.93* 0.43ns �0.84* 0.05ns 0.00 ns 0.66 ns 0.73* 0.47 ns �0.13ns 0.48 ns �0.74* 0.54ns 0.32ns �0.11ns 0.48 nsP Solu 0.85* �0.80* 0.74* �0,45ns 0.89* �0.35ns 0.81* �0.08ns �0.03ns �0.67ns �0.70 ns �0.38ns 0,20 ns �0.49 ns 0.74* �0.46ns �0.30ns 0.04 ns �0.50ns �0.99*Chl av 0.92* �0.72* 0.85* �0.31ns 0.88* �0.34ns 0.87* �0.22ns �0.18ns �0.68 ns �0.77* �0.40ns 0.34ns �0.42ns 0,66 ns �0.50ns �0.58ns 0.07 ns �0.64 ns �0.94* 0.94*Chl bw 0.89* �0.67ns 0.83* �0.38ns 0.86* �0.34ns 0.85* �0.17ns �0.14ns �0.59ns �0.74* �0.37ns 0.37 ns �0.46ns 0,68 ns �0.44 ns �0.55ns 0.04 ns �0.58ns �0.91* 0.92* 0.96nsChl a +bx 0.91* �0.70 ns 0.84* �0.32ns 0.85* �0.32ns 0.85* �0.24ns �0.20ns �0.68 ns �0.76* -0.37ns 0.35 ns �0.42ns 0,62ns �0.50ns �0.59ns 0.06 ns �0.66 ns �0.92* 0.92* 1.00ns 0.97 nsCx + cy 0.74* �0.72* 0.62 ns �0.31ns 0.71 ns �0.18ns 0.60ns �0.28ns �0.21ns �0.74* �0.62* �0.21ns 0.08ns �0.41ns 0.39ns �0.57ns �0.35ns �0.05ns �0.65ns �0.85* 0.85* 0.86ns 0.84ns 0.87ns

Pearson correlation coefficients (r) were calculated from four replicates of each sampling situation (n =18). Significance conventions: ns = not significant; *P<0.001.a Superoxide dismutase.b Peroxidase.c Catalase.d Ascorbate peroxidase.e Total dry matter, f Initial fluorescence.g PSII maximum quantum yield.h Electron transport rate.i PSII effective quantum efficiency.j Photochemical.k Non-photochemical quenching.l Total nitrogen in tissue (g kg�1).m Total phosphorus in tissue (g kg�1).n Total potassium in tissue (g kg�1).o Total calcium in tissue (g kg�1).p Total magnesium in tissue (g kg�1).q Total copper in tissue (mgkg�1).r Total iron in tissue (mgkg�1).s Total zinc in tissue (mgkg�1).t Soil solution Cu (mg L�1).u Soil solution P(mg L�1).v Chlorophyll a (mgg�1).w Chlorophyll b (mgg�1).x Chlorophyll a + b (mgg�1).y Carotenoids (mgg�1).

44

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ar Avelar

Ferreira et

al. /

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Soil Ecology

91 (2015)

37–47

Fig. 5. Principal component (PC) analysis of the (a) studied variables, and (b) distribution of experimental units according the axis obtained, for plants and rhizosphere of C.juncea plants growing in a soil with high Cu levels. PC1 and PC2 account for a 71.9% of the total experimental variance. Conventions: tissue concentration of (in mg kg�1) zinc(Zn), copper (Cu), iron (Fe), (in g kg�1) calcium (Ca), magnesium (Mg), nitrogen (N) and phosphorus (P); soil solution Cu and P, total dry matter (TDM), superoxide dismutase(SOD), catalase (CAT), ascorbate peroxidase (APX) peroxidase (POD), initial fluorescence (Fo), photochemical (qP), PSII maximum quantum yield (Fv/Fm), PSII effectivequantum efficiency (Y(II)), non-photochemical quenching (NPQ), electron transport rate (ETR), and chlorophyll a (Cha), chlorophyll b (Chb), chlorophyll (a + b) and carotenoids(Cx + c) for C. juncea grown in a soil with high copper (65 mg kg�1) levels under different levels of phosphorus with and without AMF inoculation.

P. Ademar Avelar Ferreira et al. / Applied Soil Ecology 91 (2015) 37–47 45

(Maxwell and Johnson, 2000; Cambrollé et al., 2012). Thisdissipation of chlorophyll excitation energy may prevent theformation of ROS, which can irreversibly damage proteins, lipidsand pigments of photosynthetic membranes (Horton and Ruban,2004). The removal of ROS following their formation depends onthe activation of enzymatic antioxidant defenses, including theSOD, POD and CAT enzymes, as a component of the most commondetoxification mechanism in response to oxidative stress (Mittler,2002). The enzymatic activity of SOD and CAT were positivelycorrelated with the contents of Chl a (r = 0.92, P < 0.001; r = 0.85,P < 0.001) and Chl b (r = 0.89, P < 0.001; r = 0.83, P < 0.001).

The additions of P and inoculation with AMF increased theactivity of the SOD and CAT antioxidant enzymes (Fig. 4), whichare present in various cell compartments and operate at variousstages of degradation and removal of ROS (Mishra et al., 2006).ROS, including the O2

� radical, H2O2, and the HO� radical, arenaturally formed inside cells, primarily in chloroplasts andmitochondria. However, the production of ROS may be dramati-cally increased under conditions of high soil Cu levels; in suchcases, ROS may be cytotoxic via their reaction with othermolecules and may cause significant changes in the selectivepermeability of bio-membranes (Hernandez et al., 2001; De Garaet al., 2003) and in the activity of membrane-bound enzymes (DelRío et al., 2006).

Plants that increase the production of antioxidant enzymesexhibit higher tolerances to high soil Cu levels (Andrade et al.,2010), and AMF may induce an increase in the activity ofantioxidant enzymes (Alguacil et al., 2003; Lambais et al., 2003;Abdel Latef and Chaoxing, 2011, 2014; Abdel Latef, 2011). Inmycorrhizal associations, ROS act as secondary messengers,influencing the expression of antioxidant enzymes (includingperoxidases) that are related to the development of the AMsymbiosis (Borde et al., 2011). Therefore, the inoculation with the R.clarus and the subsequent mycorrhization increased the ability ofC. juncea plants to tolerate high soil Cu levels, particularly bysupporting the maintenance of the balance between ROS and theantioxidant defense system, which is crucial for the survival andadaptation of plants cultivated in soils with high levels of toxicmetals (Słomka et al., 2008).

5. Conclusions

Soil supplementationwithPand inoculationwithR. clarus provedbe an effective method for reducing Cu phytotoxicity, and thisfinding is related to the improved nutritional status of plantsattributedtothe consequent increases in the levels of P, K, Mg, FeandZn, and chlorophyll a fluorescence parameters. Furthermore,mycorrhization and P fertilization increase the activities of theSOD and CATenzymes, which are responsible for the removal of ROSin plants exposed to Cu. The results of this study demonstrate thatthe synergistic effect between two agronomic practices, soil Psupplementation and the use of beneficial microorganisms (asAMF), provides an effective tool for promoting C. juncea growth insoils with high Cu levels.

Acknowledgments

We are grateful to the Fundação de Amparo à Pesquisa doEstado do Rio Grande do Sul (FAPERGS), the Conselho Nacionade Pesquisa (CNPq), and the Coordenação de Aperfeiçoamentode Pessoal de Nível Superior (CAPES) for the scholarshipsand the financial resources provided for this study. P. Cornejoacknowledges the support of FONDECYT1120890 grant(CONICYT, Chile).

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