Effects of Endo- and Ectomycorrhizal Fungi on Physiological ...

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 willows due to their biomass production and phytoremediation potential. They host two major types of mycorrhizal fungi that can substantially modulate the physiology of their hosts. In this study, the effects of endo- and ectomycorrhizal fungi on growth, physiological parameters, and heavy metals accumulation were studied in a pot experiment using Salix alba L. and Populus nigra L. The mycorrhizal fungi were

Electronic supplementary material The online version of this article (doi:10.1007/s11270-011-0868-8) contains supplementary material, which is available to authorized users. L. Mrnka : M. Kuchár : M. Vosátka Department of Mycorrhizal Symbioses, Institute of Botany, Academy of Sciences of the Czech Republic, Zámek 1, 252 43 Průhonice, Czech Republic Z. Cieslarová : P. Matějka Department of Analytical Chemistry, Institute of Chemical Technology, Prague, Technická 5, 166 28 Prague 6-Dejvice, Czech Republic J. Száková : P. Tlustoš 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 L. Mrnka (*) Department of Mycorrhizal Symbioses, Institute of Botany, Academy of Sciences of the Czech Republic, Lesní 322, 252 43 Průhonice, Czech Republic e-mail: [email protected]

inoculated separately and in combination to a soil substrate polluted by a mixture of heavy metals (mainly Cd, Pb, and Zn). Tree species differed in their mycorrhizal affinities, with poplar being colonized predominantly by Glomus intraradices and willow by Hebeloma mesophaeum. H. mesophaeum increased willow height and biomass, while G. intraradices decreased poplar height. The photosynthetic rate remained unchanged, and only minor changes were observed in the relative composition of photosynthetic pigments. Poplar photosynthetic rates and levels of photosynthetic pigments declined, while the epicuticular waxes in leaves increased toward the end of the experiment, irrespective of the inoculation. H. mesophaeum strongly reduced the accumulation of Cd and Fe in willow and poplar shoots, respectively. Our results support the use of selected mycorrhizal strains to tune phytoremediation outcomes in their plant hosts. Keywords Photosynthesis . Photosynthetic pigments . Phytoremediation . Populus . Raman spectroscopy . Salix

1 Introduction Poplars and willows have recently been extensively tested for phytoremediation of land contaminated by heavy metals (HMs) (Dickinson 2006; French et al. 2006). Their potential resides in large biomass

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production, extensive root systems, considerable tolerance to HMs, and high accumulation of HMs in the biomass (Pulford and Watson 2003). Although not without risks, the contaminated biomass can putatively be used for energetic purposes (Keller et al. 2005). The fast-growing clones of poplars and willows differ in transport and accumulation of particular HMs, production of leaves/wood biomass, root architecture, interaction with soil microorganisms, and other parameters (Castiglione et al. 2009; Negri et al. 2003; Tlustoš et al. 2007). All of the abovementioned differences have impacts on the final outcome of the phytoremediation process, and careful selection of appropriate tree species/clones seems to be a prerequisite for any field-based phytoremediation attempts (Pulford and Watson 2003). Widespread aspirations to decrease the use of inorganic fertilizers and pursue sustainable and environmentally friendly technologies provide strong incentives to optimize the phytoremediation process, including its below-ground aspects. Adjusting consortia of soil microorganisms seems to be one conceivable method (Zimmer et al. 2009). Among soil microorganisms, mycorrhizal fungi are a logical choice, as they consume substantial amounts of plantfixed carbon and provide plants with essential nutrients (Smith and Read 1997). Mycorrhizal fungi have also been repeatedly shown to increase plant tolerance to various abiotic and biotic stresses, including HMs (Adriaensen et al. 2006; Cicatelli et al. 2010). A variety of mechanisms have been proposed to explain the observed enhancement of plant tolerance to HMs conferred by mycorrhizal fungi, including enhanced HM chelation, adsorption, intracellular detoxification, and alteration of plant– host transcriptomic responses (Jentschke and Godbold 2000; Leyval et al. 1997). The trees of the family Salicaceae, including poplars and willows, are able to form so-called dual mycorrhizae (i.e., to simultaneously associate with arbuscular mycorrhizal (AM) and ectomycorrhizal (EM) fungi) (Vozzo and Hacskaylo 1974; Chilvers et al. 1987). The extent of particular plant clone colonization by fungi of either mycorrhizal type depends on both the clone genotype and on environmental factors, with the latter being more important (Gehring et al. 2006; Khasa et al. 2002). Ontogenic development should also not be neglected, as shifts between the mycorrhizal types were frequently observed in aging poplar seedlings (Lodge and

Water Air Soil Pollut (2012) 223:399–410

Wentworth 1990). The ability of a plant host to develop dual mycorrhizae may enable the plant to adapt to a wider range of edaphic or climatic conditions compared with exclusive mycorrhizal hosts. A dual mycorrhizal plant may also benefit from different abilities provided by the distinct groups of fungi of both mycorrhizal types (van der Heijden 2001). It was hypothesized that the efficiency of phytoextraction of HMs by willows can be increased through growth promotion caused by EM fungi (Baum et al. 2006). Similarly, the potential of AM fungi to enhance phytoremediation by poplars was underscored by Lingua et al. (2008). Yet, the effects of mycorrhizal fungi are strongly species and strain dependent, and both enhancement and attenuation of HMs accumulation in plants due to mycorrhizal fungi have been reported (Baum et al. 2006; Leyval et al. 1997). Thus, when introducing mycorrhizal fungi by inoculation, the selection of optimal fungal strain–plant clone combinations is necessary (Baum et al. 2006; Sudová et al. 2008). Even with pre-selection, the uptake of HMs by mycorrhizal plants is modulated by environmental factors such as the level of soil contamination (Audet and Charest 2007). Despite growing knowledge of molecular mechanisms driving plant and fungal tolerance to HMs (Bellion et al. 2006; Schützendübel and Polle 2002), there are numerous gaps in our understanding and ability to practically use these processes. Moreover, information about impacts of interactions of endoand ectomycorrhizal fungi on dual mycorrhizal plants that are stressed by HMs is missing. The aim of the present paper was to test the impacts of arbuscular and ectomycorrhizal fungi (both separately and in combination) on basic physiological processes and heavy metals transport of Populus nigra L. and Salix alba L. These species play important roles in riparian forests (Schnitzler 1997), where dual mycorrhizal hosts generally dominate. Two generalist ectomycorrhizal species (Hebeloma mesophaeum (Pers.) Quél. and Paxillus involutus (Batsch) Fr.) and two arbuscular mycorrhizal species (Glomus intraradices N.C. Schenck & G.S. Sm and Glomus claroideum N.C. Schenck & G.S. Sm.) were selected. We hypothesized that growth, physiological parameters, and HMs accumulation would be affected in different ways depending on the tree species and fungal types used.


2 Material and Methods 2.1 Experimental Setup: Plant and Fungal Material A pot experiment was established using soil sampled at multiple heavy metal-polluted site near Příbram City, Czech Republic. Basic soil parameters were as follows: silt loam kambisol Corg 2.4%, N 0.3%, K 9576 ppm, Ca 17,721 ppm, Mg 354 ppm, pH (H2O) 6.5, pH (KCl) 5.7 with total/exchangeable concentrations of HMs (in parts per million: Cd 10/3.5, Pb 2,172/734, Zn 318/28). The soil substrate was sieved through a 2-mm sieve and gamma-sterilized (25 kGy). Filtered eluate of uncontaminated kambisol (1/10 v/v with distilled water) was added to the soil 2 weeks prior to commencing the experiment to restore bacterial populations. The experiment had a two-factorial design, with two Salicaceae species (the first factor) and eight inoculation treatments (the second factor). Two fastgrowing clones were used in the experiment: S. alba L. clone S-117, autochthonous in the Czech Republic, and P. nigra L. clone Wolterson, originating from the Netherlands. Eight experimental treatments comprised of either individual mycorrhizal fungi or their combinations were as follows [variant code]: [AMI] G. intraradices strain PH5, [AMC] G. claroideum strain BEG96, [AMIC] mixed AMI and AMC inoculum 1:1, [EMH] H. mesophaeum strain HME-1, [EMP] P. involutus strain Maj, [EMHP] mixed EMH and EMP inoculum 1:1, [AMEM] mixed AM and EM inoculum 1:1, and [CON] control variant with no fungal treatment. All of the treatments had 10 replicates. Two EM strains were selected based on an in vitro screening test using two different growth media amended with HMs (unpublished data): the tolerant P. involutus strain Maj (derived from a fruitbody growing under a poplar tree, France) which highly accumulated HMs in the mycelium and the tolerant yet low accumulating H. mesophaeum strain HME-1 (derived from a fruitbody, Slovenia). These particular characteristics were targeted to assess whether the level of HMs accumulation in the mycelium is reflected in the HMs level in host plant tissues. As for AM fungi, we selected two different strains: G. intraradices PH5 originated from a site polluted by multiple HMs (As, Cd, Pb, Zn) near the municipality of Příbram and G. claroideum BEG96 originated from an unpolluted Calamagrostis epigejos/Populus trem-

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ula stand in the Czech Republic. We expected that PH5 would exhibit a higher tolerance to HMs compared to BEG96 and wanted to test whether the tolerance would be conferred to the host plant (Sudová et al. 2008). The EM fungal inoculum consisted of a mixture of mycelium grown for 2 to 3 months simultaneously in a non-aerated liquid PDA medium and an aerated perlite-based MMN medium (ratio 1:2). The AM fungal inoculum consisted of root fragments of colonized maize (4 months), extra-radical mycelium, and spores. All of the treatments received the same dose of both AM inoculum (10 mL) and EM inoculum (20 mL). In single-strain treatments, irrelevant inoculum was autoclaved prior to use. In control treatments, both inocula were autoclaved. This approach ensured that all experimental treatments received the same amount of inoculum, dead or alive. Tree cuttings (20 cm long) were kept in a darkened moist room at 4°C until the start of the experiment. Three days prior to commencing the experiment, the cuttings were soaked in water at room temperature. At the start of the experiment, the cuttings were shortened to the 5-cm length with at least two buds, and their surfaces were sterilized in 6% H2O2. Following the filling and mounting of containers made from Petri dishes (15 cm in diameter), application of inocula and the placement of the cuttings, each Petri dish received 5 mL of additional filtered soil eluate (obtained from soil substrates in equal amounts of AMI and AMC greenhouse cultures and S. alba L. and P. nigra L. rhizosphere soil). The containers were wrapped with 3M Micropore surgical paper tape (3M, St. Paul, Minnesota), and the cutting holes were sealed with gamma-sterilized gardening wax. The wax was also deposited on the top of the cuttings to prevent excessive drying. All of the containers were then wrapped with aluminum foil to keep the roots in darkness. 2.2 Growth Conditions The cultivation was conducted in a growth chamber (photon flux approx. 500 μmol m2 s−1, daylight/ darkness 16 h/8 h at 24°C and 18°C, respectively; relative air humidity 50% to 70%) for 6 months (May–October). The pots were randomized and watered with deionized H2O once a week. The plants exhibited water stress symptoms 8 weeks after the

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start of the experiment, and thereafter, they were irrigated more frequently (two to three times per week according to the requirements). The position of containers was randomized every 4 weeks. 2.3 Measurement of Biometric Parameters and Assessment of Photosynthetic Activity The number and lengths of plants’ shoots, together with plant mortality, were monitored monthly. Photosynthetic activity was measured twice, once in the 16th week after the start of the experiment and once at the end of the experiment (the 24th week). Measurements were carried out using a Li 6400 System portable gas-exchange measuring system (Li 6400 System, Li Cor, Lincoln, NE). Actual net photosynthetic rates and stomatal conductance were used for comparisons of the experimental groups. Only poplar plants were used for measurements due to the poor quality of willow leaves. Measurements were carried out under constant conditions (800 μmol m2 s−1, ambient CO2 concentration 370 ppm) using the fifth unfolded leaf from the shoot apex. Each measurement lasted 20 min to allow the leaf to adapt to the measurement chamber and to obtain steady values of conductivity and photosynthetic rates. Only values obtained at the 20th minute were compared. 2.4 Assessment of Photosynthetic Pigments The concentrations of photosynthetic pigments were measured spectrophotometrically according to Lichtenthaler (1987). The measurements were performed using the same leaves and on the same dates as the photosynthetic measurements. Dimethylformamide was used as an extraction agent. The extracts’ absorptions were measured at 480, 647, 664, and 750 nm on a Hach DR 4000 U spectrophotometer (Hach Company, Loveland, CO). The concentrations of photosynthetic pigments (chlorophyll A, chlorophyll B, and total carotenoids) were calculated according to Lichtenhaler’s equations (Lichtenthaler 1987).


of each poplar plant was cut and kept refrigerated in darkness until the measurements were conducted (less than 3 days). The sampling dates were analogous to the dates of the photosynthetic measurements. Raman spectra were obtained using a Fourier transform (FT) near-infrared spectrometer Equinox 55/S with an FTRaman module FRA 106/S (Bruker, Germany). Two spectra per plant were collected from the upper side of the leaf. The fixed leaf was irradiated by the focused laser beam with a laser power of 50 mW using a Nd: YAG laser (1,064 nm, Coherent). The scattered light was collected in backscattering geometry. A quartz beam splitter and Ge detector (using cooled liquid N2) were used to obtain interferograms. A total of 1,024 scans were collected for every individual spectrum. A standard 4 cm−1 spectral resolution was used for all data accumulation. The spectra were acquired using the software package OPUS (Bruker, Germany) and were exported to JCAMP-DX format for chemometric evaluation. A principal component analysis (PCA) of the FT-Raman spectra was performed on spectral data in the Stokes range (3,600– 100 cm−1) using the Unscrambler 9.2 (Camo, Norway). A full cross validation procedure was applied. 2.6 Microscopy Analysis of Root Colonization At harvest (25 weeks after the start of the experiment), the containers were dismounted, and the roots separated, carefully washed, and stored in 25% ethanol for microscopy analysis. The EM colonization of root tips was assessed under a stereomicroscope. On average, 250 root tips were examined for each plant. Morphotyping was performed based on the EM root tips color, thickness, texture, branching patterns, presence of emanating hyphae, rhizomorphs, and/or cystidia. The relative abundance of each morphotype was calculated for each sample. The AM root colonization was determined after cleaning the roots in KOH for 40 min at 80°C and subsequently staining them with 0.05% trypan blue in lactoglycerol (Koske and Gemma 1989) using the gridline intersect method (Giovannetti and Mosse 1980).

2.5 Raman Spectroscopy of Leaves The surface chemistry of the leaves was assessed by Raman spectroscopy in a similar method as used for analysis of Norway spruce needles (Matějka et al. 2001). The fourth unfolded leaf from the shoot apex

2.7 Assessment of Biomass Yield and Heavy Metals in Biomass In addition to the roots, the aboveground biomass was processed. Fresh and oven-dried (65°C) weights were


assessed for leaves and stems separately. The dried plant material was ground using a Fritsch Pulverisette 3-ball mill (Fritsch GmbH, Germany) to a particle size of