Trace Elements

Fungi have important biogeochemical roles in the biosphere and are intimately involved in the cycling of elements and transformations of both organic and inorganic substrates (Gadd, 2007; Fig. 1).  The research area of geomycology is focused on the interactions of fungi with their geological environment.

Figure 1. Proton- and organic acid ligand-mediated dissolution of metals of soils componets and minerals (from Gadd, 2004).  Proton release results in cation exchange with sorbed metal ions on clay particles, colloids etc. and metal displacement from mineral surfaces.  Released metals can interact with biomass and also be taken up by other biota, and react with other environmental components.  Organic acids anions, e.g. citrate, may cause mineral dissolution or removal by complex formation.  Metal complexes can interact with biota as well as environmental constituents.  In some circumstances, complex formation may be followed by crystallization, e.g. metal oxalate formation.

Many macrofungal species are capable of accumulating high concentrations of various trace elements including toxic heavy metals (Hg, Cd), alkali metals (Rb, Cs), noble metals (Ag, Au), and metalloids (As, Se) in fruit-bodies and thereby affect elemental geochemical cycling.

It has been repeatedly demonstrated that a specific element accumulation by particular fungus is often species-dependent and represents a distinctive feature of particular species (or closely related group of species).  Amanitas represent an interesting group of fungi with a notable ability to accumulate some elements in fruit-bodies.  A multi-element study of 7 European Amanita species was published by Vetter (2005), see Table 1.

Table 1. (from Vetter 2005).

Fig. 2. Amavadine

Vanadium

Concentrations of vanadium in common macrofungi are very low, they are often below 1 mg/kg dry weight ("DW") (Meisch et al. 1978, Řanda et al. 2005).  However, an exceptional ability to accumulate vanadium was found in Amanita muscaria and related species A. regalis and A. velatipes (Koch et al. 1987).  Concentrations of vanadium range mostly from the tens to the lower hundreds of mg/kg DW (Koch et al. 1987, Meisch et al. 1978).  Investigation on the chemical form of vanadium in fruit-bodies has revealed that vanadium is present in the coordination complex (organometallic compound) that was called amavadin (Kneifel and Bayer 1986, Fig. 2).

Despite the relatively high interest of scientists, the biological importance of the accumulation process and function of amavadin in the physiology of V-accumulating species are still unknown. For a review see (Rehder 1992).

Silver

The ability of macrofungi to accumulate silver has been known since the 1970’s (Schmitt et al. 1978).  A thorough review supported by original data (Borovička et al. 2010, 1369 results) has revealed that in pristine areas, saprobic macrofungi, with the median Ag concentration of 2.94 mg/kg, are more effective Ag-accumulators than ectomycorrhizal species (0.79 mg/kg).

However, Borovička et al. (2007) reported an extraordinarily high accumulation ("hyperaccumulation", for discussion see also Borovička et al. 2010) in two species of Amanita [subgenus Lepidella] section Lepidella: A. strobiliformis and A. solitaria (Table. 2)  Both Amanita species were collected in non-argentiferous areas with background silver content in soils (0.07 to 1.01 mg kg-1 Ag). Silver concentrations in these collections of both taxa were mostly in the range of 200-700 mg kg-1 with the highest content of 1253 mg kg-1 in one sample of A. strobiliformis.  Silver concentrations in macrofungal fruit-bodies were commonly 800-2500 times higher than in underlying soils.

Table 2. Silver concentrations (mg/kg) in various Amanita species of section Lepidella (mostly from the Czech Republic, Europe) and underlying soils (Ag SC); from Borovička et al. (2007).

The recent preliminary studies on the chemical form of Ag in Amanita spp. (A. strobiliformis and A. cf. submembranacea) have suggested that Ag in fruit-bodies is sequestered on cysteine-rich, low molecular weight proteins – metallothionens (Urban et al. 2008a, 2008b, Borovička et al. 2008, Kotrba et al. 2009).

Fig. 3. Cell-free extracts in 50 mM HEPES (pH 7.3) prepared from nitrogen frozen fruit-bodies of Amanita strobiliformis by grinding were subjected to (NH₄)₂SO_{4 precipitation.  Fraction remaining soluble at 80% (NH4)2}SO₄ saturation was desalted, concentrated by ultrafiltration and resolved on Superdex Peptide GL column (1 x 30 cm; GE-Healthcare) with 50 mM HEPES, 100 mM KNO3 (pH 7.3) as a mobile phase at a flow rate of 0.5 ml/min. For further details see Fig. 4.



Fig. 4. Upper panel: The sulphydryl-containing molecules from 0.5 ml of individual fractions were labeled with 7-fluorobenzofurazan-4-sulfonic acid (SBD-F) by using the standard protocol, resolved by using 16% acrylamide sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in Tris-Tricine buffer system with 6M urea under reducing conditions.  Fluorescent signal was scanned by using LAS1000 (Fuji). MTF: SBD labeled rabbit liver metallothionein I (5.6 kDa).  Lower panel: 10 ml aliquots from individual fractions were resolved by SDS-PAGE under reducing conditions.  The Ag content of each fraction is indicated below.  The electrophoretic mobility of material contained in fractions 28 to 35 with significant shift to higher molecular weights is characteristic of metallothioneins and was observed also with non-labeled rabbit metallothionein (not shown).


Chlorine and Bromine

There has not been a large interest in halogen element content in macrofungi.  However, several elements, especially chlorine (Cl) and bromine (Br), can be easily determined by using instrumental neutron activation analysis (Hedrich 1988, Řanda et al. 2005).

According to current knowledge, Cl concentrations in common macrofungi usually range from the hundreds to the lower thousands of mg/kg.  However, high concentrations are commonly found in various Amanita species where levels up to 29,600 mg/kg have been reported (Stijve 1984, Borovička et al. 2007), with the highest values in Amanita phalloides.

Concentrations of Br in macrofungi are mostly in units of mg/kg (Stijve 1984; Borovička, unpublished results); for detailed data see Stijve (1984, 1985, 1997) and Stijve et al. (1994).  Apparently, the highest levels of Br have been found in Amanita species of various sections, but highest in section Phalloideae, namely in A. phalloides – up to 113 mg/kg.

Arsenic

Some macrofungi are effective accumulators of arsenic, e.g., Sarcosphaera coronaria or some Laccaria species (Stijve et al. 1990, Stijve and Bourqui 1991).  In the genus Amanita, no specific As-accumulators are known.  However, in areas with polluted soils, elevated As concentrations can be found in various fungal species, including Amanita spp.; Kuehnelt et al. (1997a) reported 21.9 mg/kg in Amanita muscaria from a arsenic smelter site in Austria (Europe).  Macrofungi are known to contain various methylated arsenic species, e.g., methylarsonic acid (MA), dimethylarsinic acid (DMA), tetramethylarsonium cation (TMA), and arsenobetaine (AB) (Byrne et al. 1995).

Fig. 5. Chemical structure of arsenocholine.

Detailed investigation of the chemical form of arsenic in A. muscaria from a As-polluted site (Kuehnelt et al. 1997b) has revealed the occurrence of arsenite, arsenate, DMA and TMA as minor As compounds (~ 2% each of the total 22 mg/kg dry mass).  AB, arsenocholine (~ 15% each) and several unidentified As compounds (~ 60%) were the major As compounds.  Arsenocholine (Fig. 5), which had been known only from marine biota, was identified for the first time in a terrestrial sample.  However, A. muscaria contains choline (Michelot and Melendez-Howell 2003); and, therefore, the occurrence of arsenocholine is not a big surprise.

Acknowledgements

The phenomenon of Ag-accumulation is currently investigated within the scope of the project IAA600480801: Silver and zinc content of macrofungi: chemical form in fruit bodies, geochemical and environmental aspects and chemotaxonomic significance (Grant agency of the Academy of Sciences of the Czech Republic). This overview was indirectly supported by this project.