Elsevier

Environmental Pollution

Volume 218, November 2016, Pages 176-185
Environmental Pollution

Invited paper
Bioaccumulation of heavy metals, metalloids, and chlorine in ectomycorrhizae from smelter-polluted area

https://doi.org/10.1016/j.envpol.2016.08.009Get rights and content

Abstract

Ectomycorrhizal (ECM) fungi contribute to the survival of host trees on metal-rich soils by reducing the transfer of toxic metals into roots. However, little is known about the ability of ECM fungi to accumulate elements in ectomycorrhizae (ECMs). Here we report Ag, As, Cd, Cl, Cu, Sb, V, and Zn contents in wild-grown Norway spruce ECMs collected in a smelter-polluted area at Lhota near Příbram, Czech Republic. The ECMs data were compared with the element concentrations determined in the corresponding non-mycorrhizal fine roots, soils, and soil extracts. Bioaccumulation factors were calculated to differentiate the element accumulation ability of ECMs inhabited by different mycobionts, which were identified by ITS rDNA sequencing. Among the target elements, the highest contents were observed for Ag, Cl, Cd, and Zn; Imleria badia ECMs showed the highest capability to accumulate these elements. ECMs of Amanita muscaria, but not of other species, accumulated V. The analysis of the proportions of I. badia and A. muscaria mycelia in ECMs by using species-specific quantitative real-time PCR revealed variable extent of the colonization of roots, with median values close to 5% (w/w). Calculated Ag, Cd, Zn and Cl concentrations in the mycelium of I. badia ECMs were 1 680, 1 510, 2 670, and 37,100 mg kg−1 dry weight, respectively, indicating substantial element accumulation capacity of hyphae of this species in ECMs. Our data strengthen the idea of an active role of ECM fungi in soil-fungal-plant interactions in polluted environments.

Introduction

Ectomycorrhizal (ECM) fungi significantly interfere in biogeochemical cycles of elements in forest soils (Gadd, 2007). The major active roles of ECM fungi in ecosystems involve, besides the degradation and cycling of soil organic matter (Clemmensen et al., 2013, Phillips et al., 2014), contribution to weathering processes through excretion of organic acids and subsequent transfer of mobilized nutrients towards the colonized plant roots (Gadd, 2007, Schmalenberger et al., 2015), and trace element translocation and accumulation (Clarholm and Skyllberg, 2013, Falandysz and Borovička, 2013).

High concentrations of elements accumulated in fruit-bodies suggest that ECM macrofungi substantially contribute to their sequestration and cycling, Ag, As, Cd, Cl, Cu, Se, V, and Zn in particular (Vetter, 2005, Falandysz and Borovička, 2013). Ectomycorrhizae (ECMs) represent organs where the fungal-plant nutrient exchange takes place; studies have also suggested that they benefit the host by forming a protective barrier against heavy metal toxicity (Krupa and Kozdrój, 2004, Krupa and Kozdrój, 2007, Gadd, 2007, Colpaert et al., 2011). Extraradical mycelium is hardly separable from soil under the natural conditions. However, analysis of ECMs might represent an interesting tool for inspecting the soil-fungal-plant interactions in situ.

Vinichuk (2013) recently reported a stepwise increase in the concentration of Cd, Zn, and Cu from unpolluted soil to extraradical mycelia (5, 2.4 and 2-fold increase relative to bulk soil, respectively) and to the fruit-bodies of ECM fungi (1.8, 1.4 and 1.8-fold compared to mycelia, respectively). However, the information about the element content in ECMs and the mycelia directly colonizing plant roots is scarce. To our knowledge, the largest dataset published so far was a study by Kottke et al. (1998) who focused on nutrients and essential metals P, K, Mg, Ca, Fe, Zn, Al, and Mn. Berthelsen et al. (1995) and Krupa and Kozdrój, 2004, Krupa and Kozdrój, 2007 have reported substantially elevated concentrations of Cu, Cd, Pb and Zn in ECMs. In contrast, the concentrations of Au (Borovička et al., 2010a) and U (Kubrová et al., 2014) were found to be low even in ECMs from auriferous and U-polluted areas.

The fact that the specific ability of fungal species to accumulate elements in ECMs has never been investigated prompted us to perform a systematic investigation of element concentrations in ECMs, fine roots and soils at Norway spruce forest plantation in a smelter-polluted area in the Czech Republic. Element concentrations in the ECMs were compared to those in the fine roots and accumulation of elements in the ECMs was accessed by calculation of the bioaccumulation factor (BAF). Additionally, fungal biomass concentration was quantified by real-time PCR (qRT-PCR) approach in ECMs of two species, Imleria badia and Amanita muscaria; it enabled us to semi-quantify the element concentrations in fungal hyphae. This is the first example of the use of the qRT-PCR-based molecular approach to determine the mycobiont proportions in ECMs.

Section snippets

Investigated area and sample collection

Norway spruce (Picea abies) forest plantation above sedimentary bedrock (greywacke) at Lhota near Příbram was selected for this study; most of the trees were circa 70 years old. Long-term Ag-Cu-Pb-Zn ore mining activities in the region and decades of lead smelting (Sucharová and Suchara, 2003) resulted in elevated levels of Ag, As, Cd, Cu, Pb, Sb, and Zn levels in soils (Ettler et al., 2004, Ettler et al., 2007, Komárek et al., 2007). Samples of ECMs, roots and soils were collected from Oe

Ectomycorrhizae

Identification of the analyzed ECMs based on the obtained ITS rDNA sequences, including the identities they share with GenBank entries, is presented in the Supplementary Table S2; 6 samples remained unidentified. Among the identified ones, 11 distinct operational taxonomic units (OTUs) were observed from which those corresponding to Thelephora terrestris, I. badia, and Paxillus involutus occurred with the highest frequency. The quantities of fungal biomass in ECMs of I. badia and A. muscaria

Fungal biomass in ectomycorrhizae

Quantitative real-time PCR is a useful tool to determine the biomass of individual fungal species in soil (Landeweert et al., 2003). This approach has been successfully used for quantification of soil mycelia of ECM and saprotrophic fungi both in field studies (Hortal et al., 2008, de la Varga et al., 2012, Borovička et al., 2014) and under laboratory conditions (Parladé et al., 2007, Kurth et al., 2013). Recently, Gryndler et al. (2013) detected and quantified mycelium of Tuber aestivum in

Conclusions

ECM fungi were found to highly accumulate trace elements in their ECMs, particularly Ag, Cd, Cl, and Zn, which are also known to be accumulated in fruit-bodies. Among the inspected fungal species, Imleria badia was the most efficient accumulator, followed by Thelephora terrestris. Amanita muscaria was the only species accumulating V in ECMs. Concentrations of As in ECMs were significantly higher than those in roots and might possibly allow inspecting chemical forms of As.

The proportions of

Acknowledgements

We thank four anonymous reviewers for valuable and constructive comments which helped us to improve our manuscript. This research was supported by the project GF16-34839L (Czech Science Foundation); the results for Cl concentrations in macrofungi were obtained within the frame of the project GAUK 200415 (The Charles University Grant Agency). INAA measurements were carried out at the CANAM infrastructure of the NPI CAS Řež supported through the project No. LM2011019 (Ministry of Education, Youth

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