Chemosphere 104 (2014) 97–104

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Chemosphere

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Polyaromatic hydrocarbons impair phosphorus transportby the arbuscular mycorrhizal fungus Rhizophagus irregularis

0045-6535/$ - see front matter � 2013 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.chemosphere.2013.10.070

Abbreviations: AMF, arbuscular mycorrhizal fungi; B[a]P, benzo[a]pyrene; BAS,branched absorbing structure; F., Funneliformis; HC, hyphal compartment; MDA,malondialdehyde; P, phosphorus; PAH, polycyclic aromatic hydrocarbons; Poly-P,polyphosphate granules; R., Rhizophagus; RC, root compartment; ROC, root organcultures; %A, arbuscular root colonization; %T, total root colonization; %V,intraradical spores/vesicles root colonization.⇑ Corresponding author at: Université du Littoral Côte d’Opale, Unité de Chimie

Environnementale et Interactions sur le Vivant (UCEIV), F-62228 Calais, France. Tel.:+33 3 21 46 36 58; fax: +33 3 21 46 36 42.

E-mail address: lounes@univ-littoral.fr (A. Lounès-Hadj Sahraoui).

Maryline Calonne a,b, Joël Fontaine a,b, Benoît Tisserant a,b, Hervé Dupré de Boulois c,Anne Grandmougin-Ferjani a,b, Stéphane Declerck c, Anissa Lounès-Hadj Sahraoui a,b,⇑a Univ Lille Nord de France, F-59000 Lille, Franceb Université du Littoral Côte d’Opale, Unité de Chimie Environnementale et Interactions sur le Vivant (UCEIV), F-62228 Calais, Francec Université catholique de Louvain, Earth and Life Institute, Applied Microbiology, Mycology, Louvain-la-Neuve, Belgium

h i g h l i g h t s

� Polycyclic aromatic hydrocarbons (PAH) affect phosphorus transport by the fungus.� This was evidenced by the decrease in 33P quantity recovered in the roots.� A decrease in acid phosphatase activity in the mycorrhizal roots was also observed.� PAH have no effect on the extraradical fungal alkaline phosphatase activities.� GintPT and GiALP gene expression was not affected by PAH in the extraradical fungus.

a r t i c l e i n f o

Article history:Received 5 July 2013Received in revised form 15 October 2013Accepted 18 October 2013Available online 25 November 2013

Keywords:Arbuscular mycorrhizal fungiGintPT and GiALP gene expressionPhosphatasesPhosphorus transportPolyaromatic hydrocarbons

a b s t r a c t

Phosphate uptake by plant roots is mainly mediated by arbuscular mycorrhizal fungi (AMF). However,the impact on phosphorus (P) transport of polycyclic aromatic hydrocarbons (PAH), persistent organicpollutants widely found in altered soils, is not known up today.

Here, we monitored the Rhizophagus irregularis fungal growth and the fungal P transport ability fromthe extraradical mycelium to the host transformed chicory roots in the presence of anthracene and ben-zo[a]pyrene (B[a]P) and the combination of both PAH, under in vitro conditions.

Firstly, our findings showed that PAH have detrimental effect on the fungal growth. The combination ofboth PAH was more toxic than each of the PAH individually due to synergistic effects. Secondly, PAHaffected the P transport by the fungus from the medium to the roots. This was evidenced by either thedecrease in 33P quantity transported in the roots as well as the decrease in acid phosphatase activityin the mycorrhizal roots. Moreover, the fungal alkaline phosphatase activities remained constant in theextraradical mycelium as well as in the roots in the absence and in the presence of PAH. The GintPTand GiALP (encoding a P transporter and an alkaline phosphatase respectively) gene expressions werealso found to be similar in the extraradical mycelium treated with PAH or not (control). These findingssuggested that the P uptake by R. irregularis was not affected by PAH but probably the transport fromthe extraradical mycelium to the intraradical mycelium.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

While many soils have a high organic and inorganic phosphorus(P) content (Sanyal and De Datta, 1991), only a small proportion(generally <1%) is immediately available to plants (Richardsonet al., 2009). Although P is critical for plant growth, its availabilityto plants is limited (Smith et al., 2011). Plants have thus evolved arange of strategies to increase either P uptake capacity or P avail-ability in the soil (Marschner, 1995; Van Aarle and Olsson, 2008).The most widespread strategy to increase P uptake is the associa-tion of plants to arbuscular mycorrhizal fungi (AMF) (Smith et al.,

98 M. Calonne et al. / Chemosphere 104 (2014) 97–104

2011). In this symbiotic association, the plant provides sugars tothe AMF, in exchange of which it receives essential mineral nutri-ents, such as P, taken up from the soil by the extraradical myceliumof the AMF (Bago et al., 2003; Pfeffer et al., 2004).

In extraradical hyphae, P enters the fungal cytoplasm via high-affinity Pi:H+ symporters (Harrison and Van Buuren, 1995; Maldona-do-Mendoza et al., 2001; Benedetto et al., 2005). Phosphorus is thenrapidly condensed into polyphosphate granules (poly-P) (Ezawaet al., 2004). In intraradical hyphae, poly-P are hydrolyzed by phos-phatases in order to release P to the plant. Two genes encoding alka-line phosphatases (GiALP and GmALP) in Rhizophagus irregularis andGigaspora margarita, respectively, were expressed both in intraradi-cal and extraradical hyphae (Aono et al., 2004). The P is finally trans-ferred to the peri-arbuscular apoplastic compartment, probablyunder the control of the AMF and removed from the peri-apoplasticcompartment by plant P transporters (Benedetto et al., 2005; Bales-trini et al., 2007; Javot et al., 2007a,b). Three P transporters: GvPT,GintPT and GmosPT were respectively identified in R. versiforme,R. irregularis and Funneliformis mosseae (Harrison and Van Buuren,1995; Maldonado-Mendoza et al., 2001; Benedetto et al., 2005).

Even if the knowledge on P transport by AMF to the roots is inprogress, the impact of organic pollutants on this essential physio-logical activity is poorly documented. The radiolabelled 32P trans-port inhibition from R. caledonius hyphae to Cucumis sativus L.roots was reported in the presence of the fungicide benomyl(Larsen et al., 1996). More recently, it was observed that the capac-ity of Rhizophagus sp. to take up and translocate labelled 33P toMedicago truncatula roots was inhibited in the presence of the fun-gicide fenpropimorph (Zocco et al., 2011). Nevertheless, the impactof persistent organic pollutants, such as polycyclic aromatic hydro-carbons (PAH) on this essential mechanism is not described to date.

Polycyclic aromatic hydrocarbons are of major environmentand human health concern due to their widespread occurrence,toxicity and persistence in ecosystems, particularly in soils. Sofar, several management and remediation methods have been pro-posed and tested (Gan et al., 2009). Among these, phytoremedia-tion assisted by AMF may represent a cost-effective andenvironmental-friendly approach (Leyval et al., 2002). Indeed, anincreased dissipation of PAH was observed in mycorrhizal plantsas compared to non-mycorrhizal ones (Verdin et al., 2006; Liuand Dalpé, 2009; Gao et al., 2011). Recently, it was demonstratedthat low-phosphorus and high-water treatments were importantparameters for the dissipation of the PAH, dibenzo[a,h]anthracene(Zhou et al., 2009). No effect was observed in high-water high-phosphorus and low-water low-phosphorus treatments, wherearbuscular mycorrhizal colonization was very low. Moreover, a po-sitive linear relationship was detected between pyrene dissipationand the percentage of Gram-positive PAH-ring hydroxylating diox-ygenase genes in high-water high-phosphorus treatments, but notin the other two treatments with lower phosphorus concentrationsand water contents (Zhou et al., 2009). An enhanced toleranceagainst these pollutants was also reported with mycorrhizal plants(Joner and Leyval, 2001, 2003; Debiane et al., 2008, 2009). Debianeet al. (2008, 2009) demonstrated with root organ cultures (ROC) ofchicory, that root colonization by AMF may alleviate the oxidativestress caused by anthracene and benzo[a]pyrene (B[a]P). Cellmembrane damage (evaluated via malondialdehyde (MDA), a lipidperoxidation biomarker) and DNA oxidative damage (evaluatedthrough 8-hydroxy-20-deoxyguanosine, a DNA alteration bio-marker) were lower in mycorrhizal roots as compared to non-mycorrhizal ones. An induction of the antioxidant enzyme activitysuperoxide dismutase was further revealed in the mycorrhizalroots grown in the presence of anthracene and B[a]P (Debianeet al., 2008, 2009).

To date, several studies have reported the detrimental impact ofPAH on AMF spore germination (Alarcón et al., 2006; Verdin et al.,

2006; Franco-Ramírez et al., 2007), root colonization (Leyval andBinet, 1998; Joner and Leyval, 2001; Gaspar et al., 2002) and extra-radical mycelium development (Debiane et al., 2008, 2009, 2011),while no study focused on some essential physiological character-istics. In particular PAH impact on the uptake of nutrients (i.e. P) byAMF, their subsequent translocation within the hyphae and trans-fer from fungal cell to plant cell remains unknown. It is obviousthat any change with respect to the capacity of AMF to establisha functional symbiosis would partly compromise their biotechno-logical value.

Here, we aimed to investigate, under strict in vitro ROC condi-tions, the ability of R. irregularis to take up 33P from the mediumand to translocate this element to chicory roots in the presenceof PAH (anthracene, B[a]P) and the combination of both PAH. Inaddition, the alkaline and acid phosphatase activities in the mycor-rhizal chicory roots as well as in R. irregularis extraradical myce-lium and the expression of two genes involved in P transport inR. irregularis extraradical mycelium (GintPT, encoding a P trans-porter and GiALP, encoding an alkaline phosphatase) were studied.

2. Materials and methods

2.1. Biological material

All the experiments were conducted in vitro with Ri T-DNA-transformed chicory roots (Cichorium intybus L.) (Fontaine et al.,2004) associated or not with the arbuscular mycorrhizal fungusR. irregularis MUCL 43194 (DAOM 197198) (Schüßler and Walker,2010).

2.2. Anthracene and B[a]P preparation

The two PAH (anthracene and B[a]P) used in this work are fre-quently found in polluted soils (Juhasz and Naidu, 2000; Skupinskaet al., 2004). They are representative of a low (3 aromatic cycles)and a high (5 aromatic cycles) molecular weight PAH respectively.They possess distinctive physico-chemical properties: B[a]P pre-sents a lower solubility and higher adsorption and persistence inthe environment than anthracene. Moreover, whereas it was dem-onstrated that the high molecular weight PAH, B[a]P, presentsmutagenic and/or carcinogenic effects on Human (Mastrangeloet al., 1996), such effect was not reported for anthracene.

The ultrasonic dispersion of anthracene and B[a]P, used eithersingle or in combination, was realized in a flask containing sterilegelatine [(gelatine (0.5%) and NaCl (0.9%)] during 5 min at ambienttemperature. PAH were then added in the minimal (M) medium(Bécard and Fortin, 1988) [solidified with Phytagel 0.25% (p/v)(Sigma, St. Louis, MO, USA)] autoclaved at 121 �C during 20 min,to obtain 280 lM final concentrations of anthracene, B[a]P andthe combination of both PAH [140 lM of anthracene plus140 lM of B[a]P (50/50, v/v)].

2.3. Experimental procedures

2.3.1. Determination of the PAH impact on the arbuscular mycorrhizalfungus growth

The M medium supplemented or not with PAH was distributedin mono-compartmented Petri dishes of 9 cm diameter (25 mL/Petri dish). A piece of gel (1.5 cm2) containing roots isolated froma 2-month-old ROC of Ri T-DNA transformed chicory roots colo-nized or not by R. irregularis was placed in the middle of the Petridishes. The Petri dishes were incubated at 27 �C in the dark in aninverted position during 9 weeks. The treatments thus consistedof mycorrhizal and non-mycorrhizal roots grown in presence of

M. Calonne et al. / Chemosphere 104 (2014) 97–104 99

anthracene, B[a]P or both in combination or in absence of PAH(control).

After 9 weeks of incubation, the extraradical hyphal length andthe spore number of R. irregularis were determined from fivemono-compartmented Petri dishes under a low power microscopeat 10–40�magnification using the gridline intersects technique asdescribed by Declerck et al. (2001) and using the Newman’sformula (1966) to determinate the hyphal length.

Root colonization was also quantified after root collection by fil-tration and rinsing with sterile water after solubilization of themedium in 1 vol. of Tris–HCl buffer (50 mM – EDTA 10 mM pH7.5) (Nagahashi et al., 1993). Roots were cleared in KOH 10% for1 h at 70 �C and stained with Trypan Blue (Trypan Blue 0.5 g in32:32:467 of water:glycerol:lactic acid (v:v:v) for 1 h at 70 �C(Phillips and Hayman, 1970) and the total (%T), arbuscular (%A)and intraradical spores/vesicles (%V) root colonizations were esti-mated according to the method of McGonigle et al. (1990). For eachtreatment, three slides containing each 20 stained root fragmentswere made and 3 sections per root fragment were observed undermicroscope at 400� magnification.

2.3.2. Impact of the PAH on P transport, enzymatic activities and geneexpression in hyphae and in roots

R. irregularis cultures were set up in bi-compartmented Petridishes (9 cm) with a watertight plastic wall separating the rootcompartment (RC) containing the chicory root associated to thefungus, from the hyphal compartment (HC) in which only the fun-gus was allowed to grow (St-Arnaud et al., 1996). The RC was filledwith 25 mL M medium without PAH. A standardized 1.5 cm2 pieceof gel containing root inoculum of a 2-month-old monoxenic cul-tures of Ri T-DNA transformed chicory roots colonized by R. irreg-ularis was placed on the M medium. After 3 weeks, the HC wasfilled with 30 mL of modified M medium without vitamins and su-crose (named M1 medium) and supplemented or not (control)with PAH. Roots that grew over the plastic wall in the HC weretrimmed and removed. The Petri dishes were incubated at 27 �Cin the dark in an inverted position during 6 weeks.

After 9 weeks of incubation, the %T, %A and %V root coloniza-tions in the RC was estimated from five Petri plates per treatment(control, anthracene, B[a]P and the combination of both). In addi-tion, the number of Branched Absorbing Structure (BAS) (Bagoet al., 1998) formed in the HC of the bi-compartmented Petri dishesand the total number of hyphae crossing the plastic wall separatingthe RC from the HC were measured under a low power microscopeat 10–40� magnification.

2.3.2.1. 33P transport. The fungal transport of radiolabelled 33P wasevaluated following the method described by Zocco et al. (2011).Eight bi-compartmented Petri dishes were considered per treat-ment (control, anthracene, B[a]P and the combination of both).After 6 weeks of growth in the HC, a source of 33P was added to thiscompartment. The source of 33P was orthophosphate in dilutedhydrochloric acid (<0.1 M) as supplied by PerkinElmer (Zaventem,Belgium). The source of 33P was filter-sterilized (Acrodisc Syringefilters, PALL Corporation, Ann Arbor, MI, USA) and 200 lL was ap-plied on the surface of M1 medium in the HC of each Petri dish.The final activity in the HC at the start of the experiment was1666.7 Bq mL�1 of M1 medium. A formaldehyde-control treatment(a control in which the fungal transport is inactivated) was includedby addition of formaldehyde (2%; v/v), on the surface of M1 mediumin the HC, 2 days before the 33P addition (Dupré de Boulois et al.,2005). Five replicates were considered for this control. After 72 hof incubation in the presence of 33P, roots were removed from theRC and gently washed-clean from the M medium. The M mediumin the RC and M1 medium in the HC (containing the AMF) were alsorecovered. 33P transport was measured by counting the activity in

the RC and HC medium (on a piece of gel of 2 cm3, correspondingto 2 mL medium, solubilized in the same volume of citrate buffer(10 mM, pH 6) (Doner and Bécard, 1991)) and in the chicory roots.

Root were first digested in perchloric acid/nitric acid (70/30;v/v) as described by Becker et al. (1992).

An aliquot of 10 mL of liquid scintillation cocktail was added tothe root and the medium samples (Ultima GoldTM, Packard BioSci-ence, Groningen, Netherlands). 33P activity in each sample wasthen counted on a Packard TR2500 Liquid Scintillation Analyser(Packard Instruments, Meriden, CT, USA).

2.3.2.2. Alkaline and acid phosphatase assays. Forty bi-compart-mented Petri dishes per treatment (control, anthracene, B[a]Pand the combination of both) were used to study the impact ofanthracene, B[a]P and the combination of both on the alkalineand acid phosphatase activities. The media containing R. irregularis(about 25 mg) in the HC and the mycorrhizal roots (about 100 mg)in the RC of the bi-compartmented Petri dishes were dissolved in 1vol. of Tris–HCl. The biological material was ground in liquid N2

and extracted in 1 mL of sucrose-triton extraction buffer (STEB)(Sen and Hepper, 1986). After centrifugation at 16000 rpm during20 min at 4 �C, the upper phase was recovered and phosphataseactivities were assayed using the p-nitrophenylphosphate as thesubstrate (Wako LabAssay™ ALP, Wako Pure Chemical Industries,Osaka, Japan). Carbonate buffer (0.1 M, pH 8.8) was prepared as de-scribed by Delory and King (1945) to measure alkaline phospha-tase activities and sodium acetate buffer (0.1 M, pH 5.5) wasprepared as described by Green (1933) to measure acid phospha-tase activities. Supernatants recovered previously were added to400 lL of each buffer containing the p-nitrophenylphosphate(6.7 mM) and incubated during 15 min in a water bath at 37 �C.The enzymatic reaction was stopped by adding 320 lL of NaOH(0.2 M) to each sample. Activities were quantified by comparingthe absorption at 405 nm to a standard curve of diluted p-nitro-phenol solutions and NaOH. They were expressed in U/mg of pro-teins. Total protein concentrations were determined insupernatants using the Total Protein Kit, Micro Lowry, Peterson’sModification (Sigma–Aldrich, Saint Louis, Missouri, USA).

2.3.2.3. P transporter (GintPT) and alkaline phosphatase (GiALP) geneexpression analysis. Fifty bi-compartmented Petri dishes per treat-ment (control, anthracene, B[a]P and the combination of both)were used to investigate the effect of PAH on the P transporter(GintPT) and an alkaline phosphatase (GiALP) gene expressions inR. irregularis. The medium containing the arbuscular mycorrhizalfungus (about 100 mg) in the HC was dissolved in 1 vol. of Tris–HCl. RNA extraction was conducted on 3 replicates for each treat-ment in two independent experiments for anthracene and thecombination of anthracene and B[a]P and three independentexperiments for B[a]P. The biological material was grinded with li-quid N2 and using ‘‘RNeasy Plant Mini Kit’’ (Qiagen, Hilden, Ger-many), according to manufacturer’s instructions, including atreatment with a deoxyribonuclease I enzyme in order to eliminateresidual DNA. RNA was routinely quantified by spectrophotometry.cDNA corresponding to isolated RNA were then synthesized using‘‘High Capacity cDNA Reverse transcription Kit’’ (Applied Biosys-tem, Life Technologies, Carlsbad, CA, USA), according to the fabri-cant’s protocol. The enzymatic reaction performed on 1 lg ofRNA which allows the cDNA synthesis double-strand was inducedin a PTC-200 thermocycler (Peltier Thermal Cycler).

A real-time PCR was done in 96 well plates on a 7300 Real TimePCR system apparatus (Applied Biosystems), in order to determinegene transcription level. Each real-time PCR individual reactionwas conducted in a total volume of 25 lL containing 12.5 lL ofPower Syber Green Master Mix (Applied Biosystems), 400 nM ofeach forward or reverse primer (1 lL), 5.5 lL H2O and 25 ng of

100 M. Calonne et al. / Chemosphere 104 (2014) 97–104

cDNA (5 lL) from the reverse transcription following the pro-gramme: incubation at 95 �C during 10 min, followed by 40 cycleseach composed with a period at 95 �C during 30 s, 60 �C during 45 sand 72 �C during 45 s, when the fluorescence was measured. A dis-sociation curve was done after each run in order to check the ab-sence of non specific PCR products. Each reaction contained 3independent technical repetitions for each treatment (control,anthracene alone, B[a]P alone, as pure molecules and the (anthra-cene/B[a]P) mixture), for each independent experiment.

The following primers were used to amplify GintPT: Glomusintraradices GintPT forward (50-CCG TTC CTG GTT ATT GGG TTACT-30) and reverse (50-GGC AGC ACC CAT GAC AAT AAA-30), the Glo-mus intraradices GiALP forward (50-TGT CAC GAG CCG TAT CACTCA-30) and reverse (50-TTC CAA GAG GAT AAT CGC CAA TT-30).

Transcription levels were normalized using the housekeepinggene Glomus intraradices 18S, that forward primer was (50-GGAACA ATT GGA GGG CAA GTC-30) and reverse primer was (50-CAACCT ACT AAC CCC GAA ATT CAA C-30).

2.4. Statistical analysis

Hyphal length and number of spores in the HC, rootcolonization percentages, 33P activity in roots and medium of RCand HC, root phosphatase activities were analyzed for the differenttreatments (control, anthracene, B[a]P and the combination ofboth) by ANOVA, using Statgraphics release 5.1 (Manugistic, Inc.,Rockville, MD, USA). The method used to discriminate betweenthe means was the LSD test (p 6 0.05). Levene’s test of variancehomogeneity was checked before the use of the multiplecomparison procedure. The AMF enzymatic alkaline phos-phatase activities were analyzed by the Mann–Whitney U test(p 6 0.05).

Data of root colonization and 33P percentages were converted toarcsine of the square root values before the analysis of ANOVA andLSD test.

The comparative threshold cycle (Ct) method (Rasmussen,2001) was used to calculate the GintPT and GiALP relative expres-sion level and data were analyzed by ANOVA (Statgraphics release5.1).

3. Results

3.1. PAH impact on the fungal growth

Firstly, the %T root colonization was significantly reduced ascompared to the control whatever the PAH added in the culturemedium contained in the mono-compartmented Petri dishes(Table 1). The relative decrease was about 41% and 32% in the pres-ence of anthracene and B[a]P respectively. The decrease was evenmore important in the presence of the combination of both PAHand was significantly lower as compared to each of PAH alone.The relative decrease was close to 69% as compared to the control.The %A and %V were significantly decreased by 77% and 59%respectively in the presence of the combination of both PAH, ascompared to the control whereas no significant differences werenoticed for these parameters between the control and each PAHalone.

Secondly, R. irregularis hyphae length in the mono-compart-mented Petri dishes was significantly higher in the controltreatment as compared to the PAH treatments (Table 1). Therelative decrease was about 62%, 67% and 72% with anthracene,B[a]P and the combination of both PAH, respectively, as comparedto the control. The number of spores produced was alsosignificantly lower as compared to the control (i.e. a relativedecrease of 75%, 65% and 85% in the presence of anthracene,

B[a]P and the combination of both, respectively). The anthracene,B[a]P and the combination of both PAH treatments did not showsignificant differences for hyphal length and number of spores pro-duced. Interestingly, no visible impact was observed on the myce-lium morphology in presence of PAH as compared to the control.

In the bi-compartmented Petri dishes, the root colonizationgrown in the RC remains unchanged whatever the PAH appliedin the HC (Table 1). In addition, the number of hyphae crossingthe plastic barrier separating the RC from the HC was similar be-tween the different treatments. Similarly, the number of BAS, esti-mated per cm of hyphae in the HC did not differ significantly in theabsence and in the presence of PAH (Table 1).

3.2. PAH impact on the 33P transport, the enzymatic activities and theGintPT and GiALP gene expression

3.2.1. PAH impact on 33P transportIn the absence of PAH (control), the mean 33P activity measured

in the chicory roots was close to 32200 Bq which corresponds to67.7% of the initial 33P activity in the HC (Table 2). The 33P activitymeasured in the roots in presence of anthracene did not differ fromthe control, while it decreased significantly in the B[a]P (by 26%)and the combination of both PAH (by 24%) treatments, as com-pared to the control and corresponded to 47% and 48% of the initial33P activity in the HC in the presence of B[a]P and the combinationof both PAH respectively. The 33P activity measured in roots perfungal biomass in the HC remained unchanged in the presence ofanthracene, whereas it decreased significantly in the presence ofB[a]P and the combination of both PAH (Table 2). When the extra-radical fungus was grown in the HC containing B[a]P or the combi-nation of both PAH, significant 33P accumulations by about 2.4 and2 folds respectively were measured in the RC medium, when com-pared to the control. On the other hand, in the medium of the HC,the recovered 33P remained unchanged whatever the PAH con-tained in the medium.

3.2.2. PAH impact on the enzymatic phosphatase activitiesThe alkaline phosphatase activities in the extraradical myce-

lium of R. irregularis reached 29.3 U mg�1 of protein in the control.No significant difference was noted in the enzymatic activity be-tween the control and the treatments having received anthracene,B[a]P and the combination of both molecules (Fig. 1). Similarly, nosignificant differences were observed in the alkaline phosphataseactivities measured in the chicory roots grown in the RC whateverthe treatment (Fig. 2).

On the other hand, the acid phosphatase activities measured inroots in the RC significantly decreased by 39% and 24% in the treat-ments containing B[a]P and the combination of anthracene andB[a]P, as compared to the control, whereas it did not differ in theanthracene treatment (Fig. 2).

3.2.3. PAH impact on GintPT and GiALP gene expressionThe expression level of GintPT and GiALP analyzed in the extra-

radical mycelium after 6 weeks exposure to anthracene, B[a]P andthe combination of both PAH did not differ significantly whateverthe PAH treatment as compared to the control (Fig 3).

4. Discussion

The knowledge on the relationships between AMF and theirhosts in PAHs-polluted habitats is poorly documented. Little infor-mation is available on the impact of these organic pollutants on themorpho-anatomy and life cycle of AMF (Leyval and Binet, 1998;Alarcón et al., 2006; Verdin et al., 2006; Debiane et al., 2008,2009, 2011). In addition, no study, to our knowledge, has been

Table 1Rhizophagus irregularis development after 9 weeks of growth in mono-compartmented [extraradical hyphal length (cm/Petri dish), spore number and chicory root colonization:total (%T), arbuscular (%A) and intraradical spores/vesicles (%V)] and after 6 weeks of growth in bi-compartmented Petri dish [number of hyphae crossing the plastic wallseparating the RC from HC, BAS number/cm of hyphae of R. irregularis grown in the HC and colonization of chicory roots grown in the absence of PAH in the RC] in absence(control) or presence of PAH [anthracene, benzo[a]pyrene (B[a]P) and a combination of both (anthracene + B[a]P)] at the concentration of 280 lM. Data are presented asmeans ± SD. Values in the same row followed by identical letter do not differ significantly, as determined by ANOVA followed by a multiple range test (LSD) (p 6 0.05).

Control Anthracene B[a]P Anthracene + B[a]P

Mono-compartmented Petri dishHyphal length (cm by Petri dish) 2701 ± 229a 1038.4 ± 303.9b 889.3 ± 109.4b 749.1 ± 37.5b

Spore number 3223 ± 696a 805 ± 389b 1129 ± 587b 492 ± 152b

%T 65 ± 8a 39 ± 3b 45 ± 12b 20 ± 11c

%A 26 ± 2a 17 ± 2ab 18 ± 14ab 6 ± 5b

%V 20 ± 5a 17 ± 3ab 16 ± 6ab 8 ± 7b

Bi-compartmented Petri dishCrossing hyphae number 84 ± 23a 69 ± 15a 83 ± 39a 62 ± 18a

HCBAS number cm�1 of hyphae 0.66 ± 0.1a 0.7 ± 0.26a 0.6 ± 0.16a 0.58 ± 0.12a

RC%T 51 ± 21a 53 ± 8a 53 ± 5a 46 ± 15a

%A 27 ± 15a 26 ± 8a 23 ± 6a 26 ± 16a

%V 27 ± 16a 32 ± 10a 28 ± 6a 24 ± 16a

Table 2Phosphorus (33P) activity (Bq) measured (1) in the roots (2) in the roots per mg of mycelium dry weight in the HC, (3) in the solid medium of the RC containing the mycelium and(4) in the solid medium of the HC containing the mycelium of Petri dish in absence (control) or presence of PAH [anthracene, benzo[a]pyrene (B[a]P) and a combination of both(anthracene + B[a]P)] at the concentration of 280 lM added to the HC. The AMF (R. irregularis) was allowed to grow for 6 weeks in the HC before 33P addition. The measure ofactivity was done 72 h after addition of 33P. Data are presented as means ± SD. Values in the same row followed by identical letter do not differ significantly, as determined byANOVA followed by a multiple range test (LSD) (p 6 0.05).

Activity (Bq) measured in the compartment Control Anthracene B[a]P Anthracene + B[a]P

RC(1) 33P activity in the roots 32181 ± 6966a 30286 ± 4560ab 23902 ± 4665b 24404 ± 4059b

(2) Roots (per mg of mycelium in the HC) 13753 ± 2977a 13642 ± 2244a 10171 ± 1985b 10895 ± 1812b

(3) 33P activity in the solid medium with fungal mycelium 1409 ± 572a 1235 ± 437a 3419 ± 2098b 2833 ± 1266b

HC(4) 33P activity in the solid medium with fungal mycelium 19759 ± 6875a 28578 ± 10428a 21910 ± 3422a 24579 ± 8320a

Fig. 1. Alkaline phosphatase activities (U mg�1 of protein) measured in theextraradical hyphae of R. irregularis after 6 weeks of growth in absence (control)or presence of PAH [anthracene, benzo[a]pyrene (B[a]P) and a combination of both(anthracene + B[a]P)] at the concentration of 280 lM, in the HC of bi-compart-mented Petri dish. Data are presented as means ± SD. Different letters (a and b)indicate significant differences between the respective controls and the PAHtreatments, as determined by the Mann–Whitney U test (p 6 0.05).

M. Calonne et al. / Chemosphere 104 (2014) 97–104 101

reported on the impact of PAH on the transport of essential nutri-ents (e.g. P). Here, we aimed to study the impact of two PAH(anthracene and B[a]P), tested individually or in combination, onthe fungal growth and for the first time, on the ability of the arbus-cular mycorrhizal fungus (i.e. R. irregularis DAOM 197198) to trans-port P from the medium to the intraradical hyphae under strictin vitro culture conditions.

Our results showed that the total root colonization was reduced inthe presence of the PAH. This reduction was particularly marked withthe combination of both PAH. Furthermore, our results exhibited sig-nificant decreases in the percentage of arbuscules and intraradical

spores/vesicles in the presence of the combination of both PAH, whileno significant effect was observed with either PAH alone. This sug-gested a synergistic effect between both pollutants. Polluted soilsbeing generally composed of a cocktail of pollutants, their detrimen-tal effects on telluric organisms, as AMF, could thus be increased.

The PAH detrimental impact on the total root colonization, wasparalleled with a strong decrease in the extraradical myceliumdevelopment. This decrease in hyphae and spore production couldbe attributed to damages caused in membrane lipid constituents.Decrease in total phospholipid and sterol contents in the extraradi-cal mycelium of R. irregularis could be attributed to a membrane lipidperoxidation (evidenced by a MDA overproduction) as well as a slowdown in sterol and phospholipid biosyntheses (Debiane et al., 2011;Calonne et al., 2013). We may speculate that this disturbance may inturn affect the nutrient uptake and transport across this fungalmembrane. Thus, the detrimental impact of PAH on the fungusgrowth and the symbiosis could presumably result in a decrease inthe arbuscular mycorrhizal fungus capacity to transport mineralnutrients, such as P, from the extraradical mycelium to the roots.

Using bi-compartmented Petri dishes, the transport of P fromthe HC to the RC in the PAH-free control as well as in the presenceof the different PAH treatments, was measured 72 h after 33P addi-tion in the HC. The activity of 33P was detected in the roots of eachtreatment demonstrating the ability of the arbuscular mycorrhizalfungus to take up, translocate and transfer P from the HC to thehost roots in the RC even in the presence of PAH. The translocationof P from the HC to RC occurred mainly through active mechanismsince almost no P translocation (less than 1%) was detected in theformaldehyde controls (data not shown). However, a significantdecrease in the 33P activity measured in the roots was observed

Fig. 2. Alkaline and acid phosphatase activities (U mg�1 of protein) in the chicory roots after 9 weeks of growth in absence of PAH in the RC of bi-compartmented Petri dishesand colonized by the AMF R. irregularis grown in the absence (control) or presence of PAH [anthracene, benzo[a]pyrene (B[a]P) and a combination of both(anthracene + B[a]P)] at the concentration of 280 lM in the HC of bi-compartmented Petri dish. Data are presented as means ± SD. Different letters (a, b, a0 , b0 , c0) indicatesignificant differences between the respective controls and the PAH treatments, as determined by ANOVA followed by a multiple range test (LSD) (p 6 0.05).

Fig. 3. Real-time RT-PCR analysis of the GiALP and GiPT gene expression of R. irregularis, after 6 weeks of growth in absence (control) or presence of PAH [anthracene,benzo[a]pyrene (B[a]P) and a combination of both (anthracene + B[a]P)] at the concentration of 280 lM, in the HC of bi-compartmented Petri dish. Data are presented asmeans ± SD. Different letters (a, b, a0 , b0) indicate significant differences between the respective controls and PAH treatments, as determined by ANOVA followed by a multiplerange test (LSD) (p 6 0.05). Relative expression levels were obtained with the Ct method and were normalized with the 18S gene expression.

102 M. Calonne et al. / Chemosphere 104 (2014) 97–104

when the extraradical mycelium was grown in the presence ofB[a]P and the combination of both PAH, whereas the myceliumdry weight remained unchanged in the HC. Thus, considering the33P quantity in the roots which decreased in the presence ofB[a]P and the combination of both PAH, we suggest that thesePAH can alter evenly the P uptake by the AMF from the medium,its translocation to the intraradical mycelium and/or its transferto the roots.

Our results further demonstrated that the alkaline phosphataseactivity of the extraradical mycelium was not altered in the pres-ence of PAH. It was reported in the literature that some phospha-tases are associated with the vacuoles of G. intraradices (Olssonet al., 2002) while others are associated with the hyphal wallwhere it is supposed to be involved in P uptake from the soil(Van Aarle and Olsson, 2008). Our result thus suggested that P up-take was not affected by PAH and corroborates the result of theGiALP expression, a gene encoding an alkaline phosphatase familyin R. irregularis. The expression level of this gene remained un-changed in the presence of PAH as compared to the controls. Sim-ilarly, the GintPT transcript levels (a gene encoding a P transporteridentified in R. irregularis (Maldonado-Mendoza et al., 2001)) didnot show any change in the expression level between the PAHand control treatments. These results fit with the residual P activityin the HC and the BAS number per hyphae length which werefound to be similar whatever the PAH applied in the culture. Zoccoet al. (2008) demonstrated that the extraradical mycelium net-work, supporting the BAS, was impacted in the presence of two ste-rol biosynthesis inhibitor fungicides (fenpropimorph andfenhexamid) leading to a decrease in the capacity of the AMF totake up P (Zocco et al., 2011). Moreover, AMF possess not only anactive P transport system, via the P transporters, but also a passive

low affinity system for P transport across the membrane, as thosehighlighted in G. margarita germ-tubes (Thomson et al., 1990). Thispassive transport system may possibly not be affected by environ-mental disturbances. This could be the reason why R. irregulariscapacity to take up P was not affected by PAH in our experimentalconditions. This hypothesis corroborates the study of Ezawa et al.(2004) which demonstrated that P uptake and subsequent poly-Pbiosynthesis in AMF was fast, suggesting that the rate-limiting stepof P delivery is the translocation to the host.

After P acquisition by the extraradical hyphae via the P trans-porters, the element is translocated from the extraradical hyphaeto the intraradical hyphae. Our results showed that the total num-ber of hyphae crossing the plastic wall separating the RC from theHC remained unchanged in the presence of PAH. Nielsen et al.(2002) reported a logarithmic correlation between transported32P from the HC to the RC and the number of hyphae crossingthe barrier. This correlation was further enhanced if only living hy-phae were considered. The number of hyphae crossing the plasticwall remaining constant, it can thus be hypothesized that 33Ptranslocation from the HC to the intraradical hyphae was not af-fected by the PAH, even if it will be interesting to determine thenumber of active crossing hyphae by assessing the succinate dehy-drogenase activity for instance.

Once in the intraradical hyphae, poly-P are hydrolyzed byphosphatases, which results in P ions released into the periarbus-cular space (Ezawa et al., 2001). In the current work, the acidphosphatase activities in mycorrhizal roots decreased in the pres-ence of B[a]P and the combination of both PAH as compared tothe control, suggesting that these pollutants affected these enzy-matic activities. According to our results, it seems that this deple-tion concerns only the intraradical hyphae enzymatic activities

M. Calonne et al. / Chemosphere 104 (2014) 97–104 103

but not the root enzymatic activities. This suggestion could beexplained by the fact that the roots were grown on a PAH-freemedium in the RC. The location of acid phosphatases in the intra-radical mycelium suggested their involvement in the P transfer tothe plant (Van Aarle and Olsson, 2008). Indeed, Ezawa et al.(2001) reported that acid phosphatases could be involved in thehydrolysis of vacuolar poly-P in intraradical hyphae upon whichthe inorganic P is released into the apoplast of the plant-fungusinterface. Since the hydrolysis of poly-P is linked to the acid phos-phatase activity, a high P transfer from AMF to the plant could berelated to a high acid phosphatase activity in the intraradicalmycelium (Van Aarle and Olsson, 2008) and vice versa. The de-crease of the intraradical acid phosphatase activities observed inthe presence of B[a]P and the combination of both PAH suggestsa drop in poly-P hydrolysis in intraradical hyphae. Subsequently,a lower amount of P will be available to the transfer from theintraradical hyphae to the root cell via the arbuscule. This de-crease in acid phosphatase activities could thus explain the dropin 33P quantity recovered in the roots. This result is also in accor-dance with the 33P accumulation observed in the medium con-taining the arbuscular mycorrhizal fungus in the RC. The poly-Pcould be less hydrolyzed and thus accumulated in hyphae. Takentogether, these results suggest that the decrease in intraradicalmycelium acid phosphatase activity resulted in a poly-P accumu-lation in hyphae, when the extraradical fungus developed in thepresence of B[a]P in the HC. On the other hand, the alkaline phos-phatase activities in the roots did not seem to be correlated withthe P transfer decrease. This hypothesis matches with the resultexpected since these enzymes are not involved in poly-P hydroly-sis but in glycerolphosphate and, to a lesser extent of glucose-1- and 6-P and trehalose-6-P hydrolyzes, as demonstrated inClaroideoglomus etunicatum and F. mosseae (Ezawa et al., 1999;Liu et al., 2013).

In conclusion, our findings showed firstly that PAH have detri-mental effect on the fungal growth. The combination of both PAHwas more toxic than each of the PAH individually due probablyto synergistic effects. Secondly, PAH affected the P transport bythe AMF from the medium to the roots. This was evidenced byeither the decrease in 33P quantity transported in the roots as wellas the decrease in acid phosphatase activity in the mycorrhizalroots. Moreover, the fungal alkaline phosphatase activity as wellas the GintPT and GiALP gene expression remained constant inthe presence and in the absence of PAH.

These findings suggested that it is not the P uptake by the AMFwhich is altered but probably another transport step, like the poly-P hydrolysis occurring just before the P transfer to the plant. Toconfirm the non-impairment of P uptake, it will be interesting inthe future to quantify the 33P contained in the extraradical arbus-cular mycorrhizal fungus grown in the HC.

Taken together, our results provide the first evidence that PAHaffect one of the major benefit of the symbiosis, P transport byR. irregularis.

Additionally, it is worth to note that the ROC allow not only tostudy the pollutants impact on the symbiosis but represent also astandardized culture method well suited for the understanding ofthe mechanisms involved in the symbiosis functioning.

Finally, in a future experiment, it could be interesting to inves-tigate the 33P transport by AMF to the roots in PAH-spiked soil. Theuse of rhizoboxes containing polluted soils, composed of one com-partment where roots and mycelium can grow and a second com-partment for fungal growth only, will allow the spatial separationof root and fungal growing zones in the soil (Gao et al., 2010; Wuet al., 2011). This culture condition could improve our understand-ing concerning PAH impact on P transport by AMF to the rootswhile taking into account soil microorganisms and soil physico-chemical properties influence.

Acknowledgements

The authors wish to thank the Syndicat Mixte de la Côte d’Opale(SMCO) for providing financial support to M. Calonne thesis. Thelaboratory participates in the Institut de Recherche en ENvironne-ment Industriel (IRENI) which is financed by the CommunautéUrbaine de Dunkerque, the Région Nord Pas-de-Calais, theMinistère de l’Enseignement Supérieur et de la Recherche, theCNRS and European Regional Development Fund (ERDF). We thankMrs. Natacha Bourdon for her technical assistance.

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