Water Air Soil Pollut (2012) 223:399–410 DOI 10.1007/s11270-011-0868-8

Effects of Endo- and Ectomycorrhizal Fungi on Physiological Parameters and Heavy Metals Accumulation of Two Species from the Family Salicaceae

Libor Mrnka & Michal Kuchár & Zuzana Cieslarová & Pavel Matějka & Jiřina Száková & Pavel Tlustoš & Miroslav Vosátka

Received: 31 December 2010 /Accepted: 13 June 2011 /Published online: 6 July 2011 # Springer Science+Business Media B.V. 2011

Abstract There is increasing interest in poplars and inoculated separately and in combination to a soil willows due to their biomass production and phytor- substrate polluted by a mixture of heavy metals emediation potential. They host two major types of (mainly Cd, Pb, and Zn). Tree species differed in mycorrhizal fungi that can substantially modulate the their mycorrhizal affinities, with poplar being colo- physiology of their hosts. In this study, the effects of nized predominantly by Glomus intraradices and endo- and ectomycorrhizal fungi on growth, physio- willow by Hebeloma mesophaeum. H. mesophaeum logical parameters, and heavy metals accumulation increased willow height and biomass, while G. intra- were studied in a pot experiment using Salix alba L. radices decreased poplar height. The photosynthetic and Populus nigra L. The mycorrhizal fungi were rate remained unchanged, and only minor changes were observed in the relative composition of photo- synthetic pigments. Poplar photosynthetic rates and Electronic supplementary material The online version of this levels of photosynthetic pigments declined, while the article (doi:10.1007/s11270-011-0868-8) contains epicuticular waxes in leaves increased toward the end supplementary material, which is available to authorized users. of the experiment, irrespective of the inoculation. H. : : L. Mrnka M. Kuchár M. Vosátka mesophaeum strongly reduced the accumulation of Department of Mycorrhizal Symbioses, Institute of Botany, Cd and Fe in willow and poplar shoots, respectively. Academy of Sciences of the Czech Republic, Our results support the use of selected mycorrhizal Zámek 1, 252 43 Průhonice, Czech Republic strains to tune phytoremediation outcomes in their : Z. Cieslarová P. Matějka plant hosts. Department of Analytical Chemistry, Institute of Chemical Technology, Prague, Keywords Photosynthesis . Photosynthetic pigments . Technická 5, 166 28 Prague 6-Dejvice, Czech Republic Phytoremediation . Populus . Raman spectroscopy. : J. Száková P. Tlustoš Salix Department of Agroenvironmental Chemistry and Plant Nutrition, Faculty of Agrobiology, Food and Natural Resources, Czech University of Life Sciences Prague, Kamýcká 129, 165 21 Prague 6-Suchdol, Czech Republic 1 Introduction

L. Mrnka (*) Poplars and willows have recently been extensively Department of Mycorrhizal Symbioses, Institute of Botany, tested for phytoremediation of land contaminated by Academy of Sciences of the Czech Republic, Lesní 322, 252 43 Průhonice, Czech Republic heavy metals (HMs) (Dickinson 2006; French et al. e-mail: [email protected] 2006). Their potential resides in large biomass 400 Water Air Soil Pollut (2012) 223:399–410 production, extensive root systems, considerable Wentworth 1990). The ability of a plant host to tolerance to HMs, and high accumulation of HMs in develop dual mycorrhizae may enable the plant to the biomass (Pulford and Watson 2003). Although not adapt to a wider range of edaphic or climatic without risks, the contaminated biomass can putative- conditions compared with exclusive mycorrhizal ly be used for energetic purposes (Keller et al. 2005). hosts. A dual mycorrhizal plant may also benefit The fast-growing clones of poplars and willows differ from different abilities provided by the distinct in transport and accumulation of particular HMs, groups of fungi of both mycorrhizal types (van der production of leaves/wood biomass, root architecture, Heijden 2001). interaction with soil microorganisms, and other It was hypothesized that the efficiency of phytoex- parameters (Castiglione et al. 2009; Negri et al. traction of HMs by willows can be increased through 2003; Tlustoš et al. 2007). All of the abovementioned growth promotion caused by EM fungi (Baum et al. differences have impacts on the final outcome of the 2006). Similarly, the potential of AM fungi to enhance phytoremediation process, and careful selection of phytoremediation by poplars was underscored by appropriate tree species/clones seems to be a prereq- Lingua et al. (2008). Yet, the effects of mycorrhizal uisite for any field-based phytoremediation attempts fungi are strongly species and strain dependent, and (Pulford and Watson 2003). both enhancement and attenuation of HMs accumu- Widespread aspirations to decrease the use of lation in plants due to mycorrhizal fungi have been inorganic fertilizers and pursue sustainable and reported (Baum et al. 2006; Leyval et al. 1997). Thus, environmentally friendly technologies provide strong when introducing mycorrhizal fungi by inoculation, incentives to optimize the phytoremediation process, the selection of optimal fungal strain–plant clone including its below-ground aspects. Adjusting con- combinations is necessary (Baum et al. 2006; Sudová sortia of soil microorganisms seems to be one et al. 2008). Even with pre-selection, the uptake of conceivable method (Zimmer et al. 2009). Among HMs by mycorrhizal plants is modulated by environ- soil microorganisms, mycorrhizal fungi are a logical mental factors such as the level of soil contamination choice, as they consume substantial amounts of plant- (Audet and Charest 2007). fixed carbon and provide plants with essential Despite growing knowledge of molecular mecha- nutrients (Smith and Read 1997). Mycorrhizal fungi nisms driving plant and fungal tolerance to HMs have also been repeatedly shown to increase plant (Bellion et al. 2006; Schützendübel and Polle 2002), tolerance to various abiotic and biotic stresses, there are numerous gaps in our understanding and including HMs (Adriaensen et al. 2006; Cicatelli et ability to practically use these processes. Moreover, al. 2010). A variety of mechanisms have been information about impacts of interactions of endo- proposed to explain the observed enhancement of and ectomycorrhizal fungi on dual mycorrhizal plants plant tolerance to HMs conferred by mycorrhizal that are stressed by HMs is missing. The aim of the fungi, including enhanced HM chelation, adsorption, present paper was to test the impacts of arbuscular intracellular detoxification, and alteration of plant– and ectomycorrhizal fungi (both separately and in host transcriptomic responses (Jentschke and Godbold combination) on basic physiological processes and 2000; Leyval et al. 1997). The trees of the family heavy metals transport of Populus nigra L. and Salix Salicaceae, including poplars and willows, are able to alba L. These species play important roles in form so-called dual mycorrhizae (i.e., to simulta- riparian forests (Schnitzler 1997), where dual my- neously associate with arbuscular mycorrhizal (AM) corrhizal hosts generally dominate. Two generalist and ectomycorrhizal (EM) fungi) (Vozzo and Hacskaylo ectomycorrhizal species (Hebeloma mesophaeum 1974;Chilversetal.1987). The extent of particular (Pers.) Quél. and Paxillus involutus (Batsch) Fr.) plant clone colonization by fungi of either mycorrhizal and two arbuscular mycorrhizal species (Glomus type depends on both the clone genotype and on intraradices N.C. Schenck & G.S. Sm and Glomus environmental factors, with the latter being more claroideum N.C. Schenck & G.S. Sm.) were select- important (Gehring et al. 2006; Khasa et al. 2002). ed. We hypothesized that growth, physiological Ontogenic development should also not be neglected, parameters, and HMs accumulation would be affect- as shifts between the mycorrhizal types were frequent- ed in different ways depending on the tree species ly observed in aging poplar seedlings (Lodge and and fungal types used. Water Air Soil Pollut (2012) 223:399–410 401

2 Material and Methods ula stand in the Czech Republic. We expected that PH5 would exhibit a higher tolerance to HMs 2.1 Experimental Setup: Plant and Fungal Material compared to BEG96 and wanted to test whether the tolerance would be conferred to the host plant A pot experiment was established using soil sampled (Sudová et al. 2008). at multiple heavy metal-polluted site near Příbram The EM fungal inoculum consisted of a mixture of City, Czech Republic. Basic soil parameters were as mycelium grown for 2 to 3 months simultaneously in follows: silt loam kambisol Corg 2.4%, N 0.3%, K a non-aerated liquid PDA medium and an aerated

9576 ppm, Ca 17,721 ppm, Mg 354 ppm, pH (H2O) perlite-based MMN medium (ratio 1:2). The AM 6.5, pH (KCl) 5.7 with total/exchangeable concen- fungal inoculum consisted of root fragments of trations of HMs (in parts per million: Cd 10/3.5, Pb colonized maize (4 months), extra-radical mycelium, 2,172/734, Zn 318/28). The soil substrate was sieved and spores. All of the treatments received the same through a 2-mm sieve and gamma-sterilized (25 kGy). dose of both AM inoculum (10 mL) and EM Filtered eluate of uncontaminated kambisol (1/10 v/v inoculum (20 mL). In single-strain treatments, irrele- with distilled water) was added to the soil 2 weeks vant inoculum was autoclaved prior to use. In control prior to commencing the experiment to restore treatments, both inocula were autoclaved. This ap- bacterial populations. proach ensured that all experimental treatments The experiment had a two-factorial design, with received the same amount of inoculum, dead or alive. two Salicaceae species (the first factor) and eight Tree cuttings (20 cm long) were kept in a darkened inoculation treatments (the second factor). Two fast- moist room at 4°C until the start of the experiment. growing clones were used in the experiment: S. alba Three days prior to commencing the experiment, the L. clone S-117, autochthonous in the Czech Republic, cuttings were soaked in water at room temperature. At and P. nigra L. clone Wolterson, originating from the the start of the experiment, the cuttings were Netherlands. Eight experimental treatments comprised shortened to the 5-cm length with at least two buds, of either individual mycorrhizal fungi or their combi- and their surfaces were sterilized in 6% H2O2. nations were as follows [variant code]: [AMI] G. Following the filling and mounting of containers intraradices strain PH5, [AMC] G. claroideum strain made from Petri dishes (15 cm in diameter), applica- BEG96, [AMIC] mixed AMI and AMC inoculum tion of inocula and the placement of the cuttings, each 1:1, [EMH] H. mesophaeum strain HME-1, [EMP] P. Petri dish received 5 mL of additional filtered soil involutus strain Maj, [EMHP] mixed EMH and EMP eluate (obtained from soil substrates in equal amounts inoculum 1:1, [AMEM] mixed AM and EM inoculum of AMI and AMC greenhouse cultures and S. alba L. 1:1, and [CON] control variant with no fungal and P. nigra L. rhizosphere soil). The containers were treatment. All of the treatments had 10 replicates. wrapped with 3M Micropore surgical paper tape (3M, Two EM strains were selected based on an in vitro St. Paul, Minnesota), and the cutting holes were screening test using two different growth media sealed with gamma-sterilized gardening wax. The amended with HMs (unpublished data): the tolerant wax was also deposited on the top of the cuttings to P. involutus strain Maj (derived from a fruitbody prevent excessive drying. All of the containers were growing under a poplar tree, France) which highly then wrapped with aluminum foil to keep the roots in accumulated HMs in the mycelium and the tolerant darkness. yet low accumulating H. mesophaeum strain HME-1 (derived from a fruitbody, Slovenia). These particular 2.2 Growth Conditions characteristics were targeted to assess whether the level of HMs accumulation in the mycelium is The cultivation was conducted in a growth chamber reflected in the HMs level in host plant tissues. As (photon flux approx. 500 μmol m2 s−1, daylight/ for AM fungi, we selected two different strains: G. darkness 16 h/8 h at 24°C and 18°C, respectively; intraradices PH5 originated from a site polluted by relative air humidity 50% to 70%) for 6 months multiple HMs (As, Cd, Pb, Zn) near the municipality (May–October). The pots were randomized and of Příbram and G. claroideum BEG96 originated from watered with deionized H2O once a week. The plants an unpolluted Calamagrostis epigejos/Populus trem- exhibited water stress symptoms 8 weeks after the 402 Water Air Soil Pollut (2012) 223:399–410 start of the experiment, and thereafter, they were of each poplar plant was cut and kept refrigerated in irrigated more frequently (two to three times per week darkness until the measurements were conducted (less according to the requirements). The position of than 3 days). The sampling dates were analogous to containers was randomized every 4 weeks. the dates of the photosynthetic measurements. Raman spectra were obtained using a Fourier transform (FT) 2.3 Measurement of Biometric Parameters near-infrared spectrometer Equinox 55/S with an FT- and Assessment of Photosynthetic Activity Raman module FRA 106/S (Bruker, Germany). Two spectra per plant were collected from the upper side of The number and lengths of plants’ shoots, together the leaf. The fixed leaf was irradiated by the focused with plant mortality, were monitored monthly. Photo- laser beam with a laser power of 50 mW using a Nd: synthetic activity was measured twice, once in the YAG laser (1,064 nm, Coherent). The scattered light 16th week after the start of the experiment and once at was collected in backscattering geometry. A quartz the end of the experiment (the 24th week). Measure- beam splitter and Ge detector (using cooled liquid ments were carried out using a Li 6400 System N2) were used to obtain interferograms. A total of portable gas-exchange measuring system (Li 6400 1,024 scans were collected for every individual System, Li Cor, Lincoln, NE). Actual net photosyn- spectrum. A standard 4 cm−1 spectral resolution was thetic rates and stomatal conductance were used for used for all data accumulation. The spectra were comparisons of the experimental groups. Only poplar acquired using the software package OPUS (Bruker, plants were used for measurements due to the poor Germany) and were exported to JCAMP-DX format quality of willow leaves. Measurements were carried for chemometric evaluation. A principal component out under constant conditions (800 μmol m2 s−1, analysis (PCA) of the FT-Raman spectra was per- ambient CO2 concentration 370 ppm) using the fifth formed on spectral data in the Stokes range (3,600– unfolded leaf from the shoot apex. Each measurement 100 cm−1) using the Unscrambler 9.2 (Camo, Nor- lasted 20 min to allow the leaf to adapt to the way). A full cross validation procedure was applied. measurement chamber and to obtain steady values of conductivity and photosynthetic rates. Only values 2.6 Microscopy Analysis of Root Colonization obtained at the 20th minute were compared. At harvest (25 weeks after the start of the experi- 2.4 Assessment of Photosynthetic Pigments ment), the containers were dismounted, and the roots separated, carefully washed, and stored in 25% The concentrations of photosynthetic pigments ethanol for microscopy analysis. The EM colonization were measured spectrophotometrically according to of root tips was assessed under a stereomicroscope. Lichtenthaler (1987). The measurements were per- On average, 250 root tips were examined for each formed using the same leaves and on the same dates plant. Morphotyping was performed based on the EM as the photosynthetic measurements. Dimethylforma- root tips color, thickness, texture, branching patterns, mide was used as an extraction agent. The extracts’ presence of emanating hyphae, rhizomorphs, and/or absorptions were measured at 480, 647, 664, and cystidia. The relative abundance of each morphotype 750 nm on a Hach DR 4000 U spectrophotometer was calculated for each sample. The AM root (Hach Company, Loveland, CO). The concentrations colonization was determined after cleaning the roots of photosynthetic pigments (chlorophyll A, chlorophyll in KOH for 40 min at 80°C and subsequently staining B, and total carotenoids) were calculated according to them with 0.05% trypan blue in lactoglycerol (Koske Lichtenhaler’s equations (Lichtenthaler 1987). and Gemma 1989) using the gridline intersect method (Giovannetti and Mosse 1980). 2.5 Raman Spectroscopy of Leaves 2.7 Assessment of Biomass Yield and Heavy Metals The surface chemistry of the leaves was assessed by in Biomass Raman spectroscopy in a similar method as used for analysis of Norway spruce needles (Matějka et al. In addition to the roots, the aboveground biomass was 2001). The fourth unfolded leaf from the shoot apex processed. Fresh and oven-dried (65°C) weights were Water Air Soil Pollut (2012) 223:399–410 403 assessed for leaves and stems separately. The dried intraradices and G. claroideum) were able to suc- plant material was ground using a Fritsch Pulverisette cessfully colonize at least three replicates/treatment. 3-ball mill (Fritsch GmbH, Germany) to a particle Of the introduced ectomycorrhizal fungi (H. meso- size of <0.1 mm and was mineralized using the dry phaeum and P. involutus), H. mesophaeum was ashing procedure in a mixture of oxidizing gases (O2, detected in the EMH, EMHP, and AMEM willow O3, and NOx) at 400°C for 10 h in a Dry Mode treatments, while P. involutus did not colonize willow Mineralizer Apion. The ash was dissolved in 1.5% roots. The relative abundance of willow root tips

HNO3. Metal concentrations (Cd, Fe, Mn, Pb, and associated with H. mesophaeum in individual pots Zn) in digests were determined using ICP-OES ranged from 20% to almost 70%. It was significantly (Varian VistaPro). The standard reference material lower in the AMEM treatment (33.9±7.8%; mean DC73350 Leaves of Poplar (China National Analysis ±SEM) compared to the EMH treatment (53.7± Centre for Iron and Steel, China) was used for 3.3%). There was no significant difference in coloni- evaluating the measurement precision. zation by H. mesophaeum between the EMH and EMHP (53.2±7.9%) willow treatments. No arbuscu- 2.8 Statistical Analyses lar colonization was present in the willow AMEM group at the end of the experiment, suggesting that The data were analyzed using the STATISTICA 9.0 either the inoculated arbuscular fungi did not associ- statistical package (Statsoft, USA). Firstly, the data were ate with willows or that they were out-competed by subjected to a Shapiro–Wilks test of normality and to H. mesophaeum throughout the experiment. Hartley’s, Cochran’s, and Bartlett’shomoscedasticity In poplars, G. intraradices colonized the majority tests. The data were transformed whenever they of replicates in the AMI and AMIC treatments. The exhibited non-normal distributions. If the presumption colonization frequency in individual pots ranged from of homoscedasticity was not violated, the data were 55% to 93% and showed no significant difference analyzed using ANOVA followed by the Fisher least between the AMI (84.8±2.1%) and AMIC (78.5±1.8%) significance difference post hoc test. Otherwise, the data treatments. Similarly to willow, G. claroideum was were analyzed using the Kruskal–Wallis ANOVA test. not detected in the poplar AMC and AMIC treat- Correlations were assessed by the non-parametric ments. Colonization of poplar roots by H. meso- Spearman correlation test. In all cases, statistical phaeum ranged from less than 2% to almost 12% in probability at p<0.05 was considered as significant. individual pots and showed no significant difference between the EMH (26.6±20.7%), EMHP (5.5±1.7%), and AMEM (8.2±3.6%) treatments. Poplar inoculation 3 Results by P. involutus was successful in only two pots of the EMP treatment (3.45±0.05%). Similarly, only two 3.1 Mortality and Mycorrhizal Colonization replicates of the poplar AMEM treatment were colonized simultaneously with H. mesophaeum (8.2± An overview of mortality and inoculation success is 3.6%) and G. intraradices (85.6±2.0%). Due to the low summarized in Table 1. Overall mortality reached number of these replicates, the poplar EMP treatment 17.5% and 22.5% at the end of the experiment for was omitted, and the poplar AMEM treatment was willows and poplars, respectively. Only plants that reduced to six replicates with solely arbuscular (G. were successfully inoculated by at least one of the intraradices) colonization. The frequency of colonized introduced fungal strains and that were free from roots in these AMEM replicates reached 68.8±5.0% contamination were analyzed further. Moreover, treat- and was significantly lower than frequencies found in ments with less than three replicates were omitted the poplar AMI and AMIC groups. from further analyses. The treatments suitable for Both EM and AM contaminations occurred in the further analyses were reduced to CON, EMH, EMHP, experiment and reached 18.1% of the containers. All and AMEM for willows and to CON, AMI, AMIC, EM contaminations were extrinsic, that is, no cross- EMHP, and AMEM for poplars. contamination by the inoculated EM fungi (either H. Microscopy analyses of willow roots revealed that mesophaeum or P. involutus) occurred. The most neither of the two introduced arbuscular fungi (G. abundant contaminating morphotype occurred occa- 404 Water Air Soil Pollut (2012) 223:399–410

Table 1 Overview of mortality and inoculation success

Clone Salix alba L. Populus nigra L.

Treatment M SC FDM M SC FDM

CON 3 4 – 19– AMI 2 1 G. intraradices 09G. intraradices AMC 3 0 – 40– AMIC 0 2 G. intraradices 37G. intraradices EMH 0 8 H. mesophaeum 32H. mesophaeum EMP 4 0 – 32P. involutus EMHP 2 3 H. mesophaeum 24H. mesophaeum AMEM 0 3 H. mesophaeum 28a H. mesophaeum, G. intraradices

The data are expressed as the number of replicates for the particular experimental treatment (willows and poplars are separate). The original number of replicates per treatment was 10. For explanation of the experimental treatments codes and fungal identities, see Section 2 M mortality, SC successful colonization (i.e., the presence of at least one of the introduced fungal strains was confirmed in the given replicate; in CON treatment, no fungi were present), FDM fungi detected microscopically in the treatment replicates a Only two replicates were colonized by both fungal isolates, and the rest of the pots were colonized only by G. intraradices sionally in the willow EMP and AMEM treatments 3.3 Photosynthetic Activity and Concentrations and massively contaminated almost the whole AMC of Photosynthetic Pigments treatment (both poplars and willows). We suppose that most of the EM contaminants were introduced with Both photosynthetic activity and the concentrations of the AMC inoculum and subsequently spread to other photosynthetic pigments were measured in P. nigra L. treatments. twice during the experiment, and the data are summarized in Table 2. We observed no significant 3.2 Biometric Parameters effects of treatments on photosynthetic activity, stomatal conductivity, or the concentrations of chlo- The S. alba treatments EMH, EMHP, and AMEM rophyll A or B or total carotenoids. The effects of with successfully introduced H. mesophaeum HME-1 sampling date were apparent in all of the abovelisted exhibited enhanced willow growth compared to parameters except for stomatal conductivity, which controls as reflected by the maximum shoot length and dry shoot weight (Fig. 1). The total shoot length and dry leaf weight were not statistically different. Significantly lower relative water content in the shoots, but not in the leaves, was observed in the abovementioned three treatments (dry weight (DW)/ fresh weight (FW) in percent: 33.0±1.4%, 33.6± 1.2%, and 34.0±0.7% for EMH, EMHP, and AMEM, respectively) compared with control (25.6±1.4%). In poplars, H. mesophaeum HME-1 (treatment EMHP) did not significantly influence any biometric parameters. On the other hand, two biometric param- eters (max. shoot length and the sum of all shoot Fig. 1 Biometric parameters measured in the end of the lengths) were significantly lower in the AMI treat- experiment are expressed as percents of control (means ±SEM). Max_l maximum shoot length, Dw_s dry weight of ment colonized by G. intraradices compared to the shoots. *p<0.05, significant difference from the control group control group (Fig. 1). at this probability level Water Air Soil Pollut (2012) 223:399–410 405

Table 2 Photosynthetic activity and photosynthetic pigments in P. nigra L.

Var Samp Phot Cond CH_A CH_B CAR

CON 1 10.6±0.6 0.19±0.02 2.0±0.2 0.63±0.06 0.38±0.03 EMHP 1 9.9±1.1 0.17±0.04 1.7±0.2 0.49±0.07 0.33±0.04 AMI 1 9.6±1.0 0.13±0.02 1.9±0.2 0.54±0.05 0.38±0.03 AMIC 1 9.8±0.9 0.14±0.02 1.7±0.2 0.50±0.05 0.35±0.03 AMEM 1 9.4±0.8 0.12±0.02 1.9±0.1 0.56±0.04 0.35±0.02 C 2 8.6±0.7 0.17±0.02 1.5±0.1 0.43±0.04 0.32±0.03 EMHP 2 6.5±1.2 0.09±0.03 1.1±0.2 0.33±0.05 0.26±0.03 AMI 2 8.3±0.6 0.12±0.01 1.4±0.1 0.41±0.02 0.31±0.01 AMIC 2 8.3±0.8 0.12±0.02 1.3±0.2 0.34±0.04 0.27±0.03 AMEM 2 9.4±0.8 0.15±0.03 1.5±0.1 0.44±0.04 0.32±0.02 Sign 1 vs. 2 0.007 0.212 0.000 0.000 0.001

Photosynthetic activity and concentrations of photosynthetic pigments were measured in poplars on two sampling dates (Samp) as specified in Section 2. The data are expressed as the means±SEM. The number of observations in a particular treatment (Var) corresponds to the number of SC given in Table 1. Sign 1 vs. 2, significant difference of data between sampling dates (probability level p is given)

Phot intensity of photosynthesis [micromoles of CO2 per square meter per second], Cond stomatal conductivity [moles of H2O per square meter per second], CH_A concentration of chlorophyll A [micrograms per milligram of FW], CH_B concentration of chlorophyll B [micrograms per milligram of FW], CAR concentration of carotenoids [micrograms per milligram of FW] remained unaltered throughout the course of the (Fig. 2) clearly showed the primary effects of the experiment. All of the other parameters significantly sampling date on data grouping. The 24th week decreased toward the end of the experiment. We also results were shifted to lower values of PC1 and calculated derived parameters, namely the total higher values of PC2 compared to those from the 16th chlorophylls (tot_CH), the ratio of chlorophyll A to week (Fig. 2). As revealed by x-loading graphs chlorophyll B (CH_A/B), and the ratio of total (Supplementary Material Fig. S1), the PC1 decrease chlorophylls to total carotenoids (tot_CH/CAR). The (mostly contributing to data variability) was caused effects of the sampling date were significant in all mainly by the strong decrease of carotenes and three derived parameters. Parameters tot_CH and xanthophylls bands (e.g., 1,158 and 1,526 cm−1 tot_CH/CAR significantly increased from sampling maxima with shoulders) and the weak decrease of date 1 to 2, while parameter CH_A/B decreased chlorophylls bands (e.g., 1,327 and 747 cm−1), while during that time. There were slight, yet significant, PC2 was affected by the pigment bands only effects of the treatments detected in parameters marginally. Furthermore, there was a noticeable CH_A/B and tot_CH/CAR. The ratio CH_A/B was increase in the content of epicuticular waxes (e.g., significantly higher in the AMIC (3.55±0.01) com- 2,915 cm−1) and water (3,260 and 1,630 cm−1), pared to the CON treatment (3.39±0.02) but not related dominantly to PC2 (Fig. S1). compared to other treatments (AMEM, 3.45±0.06; The score values related to centers of Raman AMI 3.48±0.03; EMHP 3.48±0.02). The ratio models for individual treatments exhibited similar tot_CH/CAR was significantly higher in the CON mutual arrangement (approximately in the diagonal (6.45±0.08) and AMEM (6.56±0.08) treatments direction from AMI to EMHP) for both sampling compared to other treatments (EMHP, 6.06±0.12; terms (Fig. 2), confirming the evidence of data AMIC 6.11±0.06; AMI 6.17±0.07). clustering related to the colonizing fungi. Neverthe- less, as apparent from both score (Fig. 2)andx- 3.4 FT-Raman Spectroscopy of Leaves loading (Fig. S1) graphs, the relatively small data shifts assigned to the effect of treatment cannot be To analyze the data structure of a multivariate FT- easily attributed to specific chemical components Raman spectral data matrix, PCA of all spectra was without detailed band assignment based on analysis performed to find latent variables. The score graph of many reference compounds. 406 Water Air Soil Pollut (2012) 223:399–410 Fig. 2 Principal component analysis of FT-Raman spec- tra. The data clusters are related to the sampling terms (J—16th week and O—24th week), and the inoculation treatments are depicted in the score graph. The x-loadings for PC1 and PC2 are shown in the Supplementary Mate- rial (Fig. S1)

3.5 Concentrations of Heavy Metals in Plant Biomass colonized by H. mesophaeum) were nearly half of the levels shown by the control group (Table 3). Similar The concentrations of heavy metals were higher in tendencies (though not significant) were observed for leaves than in shoots, except for Pb (Table 3). Tree other metals. In addition, the poplar EMHP treatment species had a significant effect on the concentrations of colonized by the same fungal strain exhibited signif- all HMs in leaves. In all metals except for Pb, willow icantly lower concentrations of Fe in shoots than did concentrations exceeded poplar concentrations. In all of the other treatments, including controls. Poplar shoots, only Cd and Zn concentrations were signifi- treatments inoculated by G. intraradices (AMI, AMIC, cantly affected by clones. Metals concentrations in and AMEM) did not show altered levels of HMs in willow shoots were higher compared to poplar shoots. shoots. The elevation of Pb in shoots by 57% in the The inoculation treatment significantly influenced Cd AMI treatment compared to controls was not signifi- concentrations in willow shoots. Cd levels of the cant. There was no effect of inoculation on HMs experimental variants EMH, EMHP, and AMEM (all concentrations in the leaves of either clone (Table 3).

Table 3 Concentrations of heavy metals in shoots and leaves of S. alba L. and P. nigra L.

HMs Cd Fe Pb Zn

Clone Var Shoots Leaves Shoots Leaves Shoots Leaves Shoots Leaves

S. alba CON 72.5±7.8a 81.7±4.7a 22.4±6.2a 61.5±9.4a 35.6±5.8a 4.1±0.9a 163.6±13.9a 412.9±42.5a EMH 39.8±8.2b 60.3±7.8a 10.7±2.0a 53.5±11.3a 20.2±4.4a 5.9±1.1a 108.8±20.8a 367.0±48.1a EMHP 38.7±6.8b 56.0±22.6a 9.8±1.0a 31.2±12.6a 16.9±3.2a 3.6±1.5a 108.3±5.1a 284.2±131.6a AMEM 35.0±3.2b 54.9±10.6a 9.7±1.8a 33.5±7.8a 19.7±2.2a 3.8±1.5a 109.6±4.4a 277.3±30.8a P. nigra CON 26.9±3.5x 33.7±2.2x 13.3±2.6x 22.8±3.3x 18.7±2.7x 6.6±0.7x 62.8±6.2x 177.5±8.7x EMHP 13.3±5.3x 28.2±5.6x 3.6±1.3y 16.0±4.9x 10.3±3.6x 6.8±1.8x 44.3±17.1x 199.3±47.7x AMI 22.3±3.4x 31.1±4.0x 21.8±5.7x 23.1±4.1x 29.3±5.4x 9.8±1.6x 65.0±9.8x 200.6±24.5x AMIC 22.0±4.0x 25.1±4.3x 15.0±2.0x 15.6±3.1x 21.6±3.4x 6.8±1.2x 63.2±5.6x 143.8±24.5x AMEM 19.6±1.2x 28.4±3.1x 14.0±2.1x 17.8±1.9x 19.8±1.6x 7.6±0.8x 65.8±4.5x 192.2±16.6x

Concentrations of metals are given in milligrams per kilogram of DW. The data are expressed as the means±SEM. The number of observations in the particular treatment (Var) corresponds to the number of SC given in Table 1. Different letters denote significant difference at the probability level p<0.05 (evaluated separately for each clone) Water Air Soil Pollut (2012) 223:399–410 407

4 Discussion Populus tremuloides Michx. The EM root tips dominated in shallow organic soils (<10 cm), while There are numerous reports on co-occurrence of both AM colonization was more abundant in deeper mycorrhizal types in Salicaceae (e.g., Baum and mineral soils. Lodge (1989) postulated that EM Makeschin 2000, Harley and Harley 1987; Hashimoto fungi can withstand only a narrow range of soil and Higuchi 2003; Khasa et al. 2002; van der Heijden water potential, while AM fungi prevail in drier and and Vosátka 1999). It has been shown that proportion wetter (flooded) soils. The abovementioned observa- of AM and EM fungi depends on the host genotype as tion of Parádi and Baar (2006) indicates that such a well as environmental factors (Gehring et al. 2006; claim might not be universal. Khasa et al. 2002). In the present paper, the The potential of mycorrhizal fungi to increase the frequencies of colonization of poplar and willow biomass (both stem and root) of host trees in pot roots by individual fungi were in general agreement experiments has been repeatedly observed, although it with prior observations (e.g., Khasa et al. 2002; depends on many variables, including light intensity, Lingua et al. 2008; Parádi and Baar 2006; van der nutrient content of substrate, rooting volume, and Heijden and Vosátka 1999). Simultaneous formation rooting density. In our experiment, the maximum of both arbuscular mycorrhiza and ectomycorrhiza height of plants (together with the dry weight of was limited to only two experimental replicates which shoots) was increased by H. mesophaeum. Similarly prevented us from assessing possible effects of the to our results, Hebeloma leucosarx increased the interaction of the two mycorrhizal types. Such mean shoot length of Salix repens after 30 weeks of differentiated and host-dependent colonization sug- growth in an unpolluted substrate (van der Heijden gests rather strong host-mycobiont preferences. 2001), and Hebeloma crustuliniforme increased the The ontogenetic shift from AM to EM fungi in biomass of stem and leaves of Populus×canadensis poplar has been already reported (Aguillon and after 11 weeks of growth in Cd-polluted soil (Sell et Garbaye 1989, Lodge and Wentworth 1990). In 3- al. 2005). However, we observed significantly de- year-old P. nigra, AM still slightly prevailed over EM creased shoot lengths in the poplar clone caused by G. (Lukac et al. 2003). However, in the extensive study intraradices. Usually, the growth depression is sup- by Khasa et al. (2002) inspecting 28 poplar clones at posed to be caused by the fact that fungal demand for the age of 5 years, the vast majority exhibited plant carbon outweighs any benefits provided by P prevalence of EM. We suppose that dominance of transfer via the AM. Nevertheless, there is an AM over EM in early ontogenetic stages contributed increasing body of evidence suggesting that the cost/ to the limited success of the poplar EM inoculation in benefit balance is not so straightforward and that our experiment. differences in C demand and balance between P Contrary to poplars, willows were shown to host uptake via the AM fungal pathway and directly via mostly EM fungi (Harley and Harley 1987; Hrynkiewicz the roots may play important roles. The importance of et al. 2008; Parádi and Baar 2006). The selected willow such factors increases under conditions of small species, S. alba L., grows in regularly flooded riparian rooting volumes and high rooting densities (Li et al. forests and exhibits high flood tolerance (Schnitzler 2008). 1997). Only ectomycorrhizae have been reported in It has been well documented that heavy metals this host tree by Harley and Harley (1987). The EM affect various plant physiological processes, including preference of this species is also supported by a photosynthetic rates, respiration, enzymatic activities, recent study of three S. alba L.-dominated riparian synthesis of photosynthetic pigments, and stomatal stands of different ages (Parádi and Baar 2006). No functions (Lunáčková et al. 2003; Schützendübel and arbuscular mycorrhizae were found within the roots Polle 2002). In general, Salicaceae species exhibit of this species in any of these habitats. Nevertheless, good tolerance to HMs, but inter- and intra-specific such inspection of the root mycorrhizae can be differences do exist. Decreased photosynthetic rates biased due to uneven distribution of mycorrhizal after exposure to Cd were observed in several Salix fungi along the vertical axis. Neville et al. (2002) and Populus species but not in others (including S. observed that EM and AM were preferentially alba L.) (Lunáčková et al. 2003). No significant partitioned at different soil depths on the roots of impacts on total chlorophylls (a+b) or carotenoids 408 Water Air Soil Pollut (2012) 223:399–410 were detected in either of the inspected clones. inter-treatment differences indicated by the PCA are Regvar et al. (2010) observed no differences in based on the whole spectral range (which is com- chlorophyll or carotenoid content among Salix caprea prised of features of numerous chemical substances of L. growing in soils differentially polluted by Cd and leaves, only few of which have been reliably Pb. By contrast, total chlorophyll declined markedly identified so far). Thus, for detailed information, upon Zn treatment of Populus alba L. in vitro thorough research exploring complete spectral band (Castiglione et al. 2007). After 10 days of exposure assignments would be desirable. In summary, (1) at the highest Zn levels, a reduced chlorophyll A/B Raman spectroscopy allowed identification of leaf ratio was observed. Di Baccio et al. (2009) explored changes in the course of the experiment and (2) it responses of the Populus×euramericana clone I-214 reflected tiny chemical differences of leaves influ- to excess Zn. They found elevated chlorophyll A/B enced by fungal treatment using a holistic approach ratios at low Zn concentrations (1 mM) and remark- based on PCA of original spectral data in the whole able decreases of the ratios at high concentrations of spectral range without any band selection and/or pre- Zn (≥5 mM). Our chlorophyll A/B ratios comply with treatment. the values of Di Baccio et al. (2009) and do not Few papers have demonstrated the ability of indicate strong plant stress. Adriaensen et al. (2006) mycorrhizal fungi to modulate translocation of HMs reported that a Zn-tolerant Suillus bovinus isolate was to fast-growing trees and their distribution within able to prevent a drop in total chlorophyll content plant tissues. The enhancement or attenuation of HMs caused by Zn in needles of Scots pines. Analogously, transport to the plant has been shown to depend on a two Glomus species were able to improve nutrition particular plant–fungus–heavy metal combination and increase chlorophyll content in the leaves of (Sell et al. 2005). Moreover, mycorrhizal fungi might Eucalyptus globulus exposed to high Pb (Arriagada et selectively influence HM transport to particular plant al. 2005). In our experiment, only minor changes in compartments (Baum et al. 2006; Dos Santos Utmazian chlorophyll A/B and total chlorophylls/carotenoids et al. 2007). The effect of mycorrhizal fungi is also ratios were detected due to fungal treatments. We influenced by other soil microbes (Dos Santos Utmazian assume that putative beneficial effects of the isolates et al. 2007; Zimmer et al. 2009). In our study on used in this study remained masked because of the moderately polluted soils, H. mesophaeum exhibited a low overall levels of plant stress. Generally, it seems tendency to retard heavy metals accumulation in the that photosynthetic pigments are neither specific nor shoots of both clones, but the effect was only significant sensitive biomarkers of heavy metal stress (Lunáčková in Fe (poplar shoots) and Cd (willow shoots). Arbus- et al. 2003). cular fungi were more often found to retard heavy In accordance with the chemical analyses and metals accumulation in Salix or Populus species than measured photosynthetic activity, the Raman spec- were ectomycorrhizal fungi (Lingua et al. 2008; troscopy of poplar leaves indicated decreased carote- Bissonnette et al. 2010) Nevertheless, taking into noids and chlorophylls toward the end of the account other host species and a wider range of polluted experiment. Furthermore, there was a noticeable substrates, AM fungi appear to generally enhance the increase in epicuticular waxes and water content. On accumulation of many HMs at low soil concentrations the contrary, the Raman spectroscopy results sug- and to attenuate HMs accumulation at high soil gested significant shifts in the AMI treatment com- concentrations (Audet and Charest 2007). This threshold pared to the controls that were not detected by the effect should not be overlooked when published results chemical analyses of photosynthetic pigments. Nev- on the modulation of translocation of HMs to plants by ertheless, it should be noted that the Raman spectros- mycorrhizal fungi are compared. copy monitored pigments and many other chemical Based on the present study, we conclude that H. species directly in the leaf matrix, while spectropho- mesophaeum strain HME1 is a good candidate for tometry determined only extracted fractions of pig- phytostabilization strategies on moderately polluted ments. The latter result is influenced by the polarity of sites, as the treatments with this fungus produced the the solvent and the procedure applied (low-polar largest biomass in both clones examined and showed carotenes could be less efficiently extracted by DMF an overall tendency to retard accumulation of several than xanthophylls and chlorophylls). Furthermore, the HMs. It is known that H. mesophaeum, together with Water Air Soil Pollut (2012) 223:399–410 409 some other phylogenetic clades of Hebeloma species, tremuloides). Journal of Plant and Nutrition Soil Science, – preferentially grows with Salicaceae and often occur 163, 491 497. Baum, Ch, Hrynkiewicz, K., Leinweber, P., & Meißner, R. (2006). at polluted or early successional sites (Krpata et al. Heavy-metal mobilization and uptake by mycorrhizal and 2008; Hrynkiewicz et al. 2008). Moreover, these nonmycorrhizal willows (Salix × dasyclados). Journal of species are apparently able to withstand periods of Plant and Nutrition Soil Science, 169,516–522. flooding (Parádi and Baar 2006), suggesting desirable Bellion, M., Courbot, M., Jacob, Ch, Blaudez, D., & Chalot, M. (2006). Extracellular and cellular mechanisms sustaining resistance capacity to that disturbance. G. intraradices metal tolerance in ectomycorrhizal fungi. FEMS Microbi- PH5 decreased poplar biomass in this study but ology Letters, 254, 173–181. indicated potential to enhance shoot Pb accumulation, Bissonnette, L., St-Arnaud, M., & Labrecque, M. (2010). which is of interest considering the low mobility and Phytoextraction of heavy metals by two Salicaceae clones in symbiosis with arbuscular mycorrhizal fungi during the uptake of this element. Sudová et al. (2008) reported second year of a field trial. Plant and Soil, 332,55–67. unchanged or even higher Pb concentrations in shoots Castiglione, S., Franchin, C., Fossati, T., Lingua, G., Torrigiani, of Agrostis capillaris in response to AM inoculation. P., & Biondi, P. (2007). High zinc concentrations reduce Further trials with a broader range of AM isolates in rooting capacity and alter metallothionein gene expression in white poplar (Populus alba L. cv. Villafranca). Chemo- less artificial conditions would be valuable. Neither S. sphere, 67, 1117–1126. alba L. or P. nigra L. exhibited stress symptoms due Castiglione, S., Todeschini, V., Franchin, C., Torrigiani, P., to HMs in the soil substrate, and we consider them Gastaldi, D., Cicatelli, A., et al. (2009). Clonal differences tolerant for moderately polluted soil sites with in survival capacity, copper and zinc accumulation, and correlation with leaf polyamine levels in poplar: A large- mixtures of Cd, Pb, and Zn. Although the particular scale field trial on heavily polluted soil. Environmental aim of this study was to reveal the effects of Pollution, 157, 2108–2117. interactions of two fundamental types of mycorrhizal Chilvers, G. A., Lapeyrie, F. F., & Horan, D. P. (1987). fungi, this outcome was prevented by the host Ectomycorrhizal vs endomycorrhizal fungi within the same root system. The New Phytologist, 107, 441–448. mycorrhizal exclusivity. Further research should be Cicatelli, A., Lingua, G., Todeschini, V., Biondi, S., Torrigiani, conducted on this topic using less exclusive hosts. P., & Castiglione, S. (2010). Arbuscular mycorrhizal fungi restore normal growth in a white poplar clone grown on Acknowledgments We greatly acknowledge the support of the heavy metal-contaminated soil, and this is associated with research by a grant from Norway through the Norwegian Financial upregulation of foliar metallothionein and polyamine Mechanism (project no. CZ0092), by the Ministry of Education, biosynthetic gene expression. Annals of Botany. Youth and Sports of the Czech Republic (MSM 6046137307), and doi:10.1093/aob/mcq170. by the Academy of Sciences of the Czech Republic (grant Di Baccio, D., Tognetti, R., Minnocci, A., & Sebastiani, L. AV0Z60050516). We also thank Dr. Radka Sudová for the (2009). Responses of the Populus × euramericana clone I- selection of the AM strains, Mgr. Pavla Doubková for the 214 to excess zinc: Carbon assimilation, structural preparation of the AM inocula, and Dr. Andrea Polle for kindly modifications, metal distribution and cellular localization. providing us the isolate P. involutus Maj. Environmental and Experimental Botany, 67, 153–163. Dickinson, N. M. (2006). Phytoremediation of industrially- contaminated sites using trees. NATO Science Series, 68, – References 229 240. Dos Santos Utmazian, M. N., Schweiger, P., Sommer, P. M., Gorfer, M., Strauss, J., & Wenzel, W. W. (2007). Influence Adriaensen, K., Vangronsveld, J., & Colpaert, J. V. (2006). Zinc- of Cadophora finlandica and other microbial treatments tolerant Suillus bovinus improves growth of Zn-exposed on cadmium and zinc uptake in willows grown on polluted Pinus sylvestris seedlings. Mycorrhiza, 16,553–558. soil. Plant and Soil Environment, 53, 158–166. Aguillon, R. L., & Garbaye, J. (1989). Some aspects of a French, Ch. J., Dickinson, N. M., & Putwain, P. D. (2006). double symbiosis with ectomycorrhizal and VAM fungi. Woody biomass phytoremediation of contaminated brown- Agriculture, Ecosystem and Environment, 29, 263–266. field land. Environmental Pollution, 141, 387–395. Arriagada, C. A., Herrera, M. A., & Ocampo, J. A. (2005). Gehring, C. A., Mueller, C., & Whitham, T. G. (2006). Contribution of arbuscular mycorrhizal and saprobe fungi Environmental and genetic effects on the formation of to the tolerance of Eucalyptus globulus to Pb. Water, Air, ectomycorrhizal and arbuscular mycorrhizal associations and Soil Pollution, 166,31–47. in cottonwoods. Oecologia, 149, 158–164. Audet, P., & Charest, Ch. (2007). Heavy metal phytoremedia- Giovannetti, M., & Mosse, B. (1980). An evaluation of tion from a meta-analytical perspective. Environmental techniques for measuring vesicular arbuscular mycorrhi- Pollution, 147, 231–237. zalinfectioninroots.The New Phytologist, 84,489– Baum, Ch, & Makeschin, F. (2000). Effects of nitrogen and 500. phosphorus fertilization on mycorrhizal formation of two Harley, J. L., & Harley, E. L. (1987). A check-list of mycorrhiza in poplar clones (Populus trichocarpa and P. tremula x the British Flora. The New Phytologist, 105(Suppl), 1–120. 410 Water Air Soil Pollut (2012) 223:399–410

Hashimoto, Y., & Higuchi, R. (2003). Ectomycorrhizal and McCutcheon & J. L. Schnoor (Eds.), Phytoremediation arbuscular mycorrhizal colonization of two species of transformation and control of contaminants (pp. 233– floodplain willows. Mycoscience, 44, 339–343. 262). Hoboken: Wiley. Hrynkiewicz, K., Haug, I., & Baum, Ch. (2008). Ectomycor- Neville, J., Tessier, J. L., Morrison, I., Scarratt, J., Canning, B., rhizal community structure under willows at former ore & Klironomos, J. N. (2002). Soil depth distribution of mining sites. European Journal of Soil Biology, 44,37–44. ecto- and arbuscular mycorrhizal fungi associated with Jentschke, G., & Godbold, D. L. (2000). Metal toxicity and Populus tremuloides within a 3-year-old boreal forest ectomycorrhizas. Physiologia Plantarum, 109, 107–116. clear-cut. Applied Soil Ecology, 19, 209–216. Keller, C., Ludwig, Ch, Davoli, F., & Wochele, J. (2005). Parádi, I., & Baar, J. (2006). Mycorrhizal fungal diversity in Thermal treatment of metal-enriched biomass produced willow forests of different age along the river Waal, The from heavy metal phytoextraction. Environmental Science Netherlands. Forest Ecology and Management, 237,366– and Technology, 39, 3359–3367. 372. Khasa, P. D., Chakravarty, P., Robertson, A., Thomas, B. R., & Pulford, I. D., & Watson, C. (2003). Phytoremediation of heavy Dancik, B. P. (2002). The mycorrhizal status of selected metal-contaminated land by trees-review. Environment poplar clones introduced in Alberta. Biomass and Bioen- International, 29, 529–540. ergy, 22,99–104. Regvar M., Likar M., Piltaver A., Kugonič N., & Smith J. E. Koske, R. E., & Gemma, J. N. (1989). A modified procedure (2010) Fungal community structure under goat willows for staining roots to detect VA mycorrhizas. Mycological (Salix caprea L.) growing at metal polluted site: the Research, 92, 486–488. potential of screening in a model of phytostabilisation Krpata, D., Peinter, U., Langer, I., Fitz, W. J., & Schweiger, P. study. Plant and Soil, 330, 345–356. (2008). Ectomycorrhizal communities associated with Schnitzler, A. (1997). River dynamics as a forest process: Populus tremula growing on a heavy metal contaminated interaction between fluvial systems and alluvial forests in site. Mycological Research, 112, 1069–1079. large European river plains. The Botanical Review, 63,40– Leyval, C., Turnau, K., & Haselwandter, K. (1997). Effect of 64. heavy metal pollution on mycorrhizal colonization and Schützendübel, A., & Polle, A. (2002). Plant responses to function: physiological, ecological and applied aspects. abiotic stresses: heavy metal-induced oxidative stress and Mycorrhiza, 7, 139–153. protection by mycorrhization. Journal of Experimental Li, H., Smith, F. A., Dickson, S., Holloway, R. E., & Smith, S. Botany, 53, 1351–1365. E. (2008). Plant growth depressions in arbuscular mycor- Sell, J., Kayser, A., Schulin, R., & Brunner, I. (2005). rhizal symbioses: not just caused by carbon drain? The Contribution of ectomycorrhizal fungi to cadmium uptake New Phytologist, 178, 852–862. of poplars and willows from a heavily polluted soil. Plant Lichtenthaler, H. K. (1987). Chlorophylls and carotenoids: and Soil, 277, 245–253. pigments of photosynthetic biomembranes. Methods in Smith, S. E., & Read, D. J. (1997). Mycorrhizal symbiosis (2nd Enzymology, 148, 350–382. ed.). London, UK: Academic Press Ltd. Lingua, G., Franchin, C., Todeschini, V., Castiglione, S., Sudová, R., Doubková, P., & Vosátka, M. (2008). Mycorrhizal Biondi, S., Burlando, B., et al. (2008). Arbuscular association of Agrostis capillaris and Glomus intraradices mycorrhizal fungi differentially affect the response to high under heavy metal stress: combination of plant clones and zinc concentrations of two registered poplar clones. fungal isolates from contaminated and uncontaminated Environmental Pollution, 153, 137–147. substrates. Applied Soil Ecology, 40,19–29. Lodge, D. J. (1989). The influence of soil moisture and Tlustoš, P., Száková, J., Vysloužilová, M., Pavlíková, D., Weger, flooding on formation of VA-endo- and ectomycorrhizae J., & Javorská, H. (2007). Variation in the uptake of in Populus and Salix. Plant and Soil, 117, 243–253. arsenic, cadmium, lead, and zinc by different species of Lodge, D. J., & Wentworth, T. R. (1990). Negative associations willows Salix spp. grown in contaminated soils. Central among VA-mycorrhizal fungi and some ectomycorrhizal European Journal of Biology, 2, 254–275. fungi inhabiting the same root system. Oikos, 57, 347– van der Heijden, E. W. (2001). Differential benefits of 356. arbuscular mycorrhizal and ectomycorrhizal infection of Lukac,M.,Calfapietra,C.,&Godbold,D.L.(2003). Salix repens. Mycorrhiza, 10, 185–193. Production, turnover and mycorrhizal colonization of root van der Heijden, E. W., & Vosátka, M. (1999). Mycorrhizal systems of three Populus species grown under elevated associations of Salix repens L. communities in succession CO2 (POPFACE). Global Change Biology, 9, 838–848. of dune ecosystems. II. Mycorrhizal dynamics and Lunáčková, L., Masarovičová, E., Kráľová, K., & Streško, V. interactions of ectomycorrhizal and arbuscular mycorrhizal (2003). Response of fast growing woody plants from family fungi. Canadian Journal of Botany, 77, 1833–1841. Salicaceae to cadmium treatment. Bulletin of Environmental Vozzo, J. A., & Hacskaylo, E. (1974). Endo- and ectomycor- Contamination and Toxicology, 70,576–585. rhizal associations in five Populus species. Bulletin of the Matějka, P., Plešerová, L., Budínová, G., Havířová, K., Mulet, X., Torrey Botanical Club, 101, 182–186. Skácel, F., et al. (2001). Vibrational biospectroscopy: what can Zimmer, D., Baum, Ch, Leinweber, P., Hrynkiewicz, K., & we say about the surface wax layer of Norway spruce needles? Meissner, R. (2009). Associated bacteria increase the Journal of Molecular Structure, 565–566,305–310. phytoextraction of cadmium and zinc from metal- Negri, M. C., Gatliff, E. G., Quinn, J. J., & Hinchman, R. R. contaminated soil by mycorrhizal willows. International (2003). Root development and rooting at depths. In S. C. Journal of Phytoremediation, 11, 200–213.