Jan Borovička

Macro and trace mineral constituents and radionuclides in mushrooms: health benefits and risks.

This article reviews and updates data on macro and trace elements and radionuclides in edible wild-grown and cultivated mushrooms. A huge biodiversity of mushrooms and spread of certain species over different continents makes the study on their multi-element constituents highly challenging. A few edible mushrooms are widely cultivated and efforts are on to employ them (largely Agaricus spp., Pleurotus spp., and Lentinula edodes) in the production of selenium-enriched food (mushrooms) or nutraceuticals (by using mycelia) and less on species used by traditional medicine, e.g., Ganoderma lucidum. There are also attempts to enrich mushrooms with other elements than Se and a good example is enrichment with lithium. Since minerals of nutritional value are common constituents of mushrooms collected from natural habitats, the problem is however their co-occurrence with some hazardous elements including Cd, Pb, Hg, Ag, As, and radionuclides. Discussed is also the problem of erroneous data on mineral compounds determined in mushrooms.

Bioaccumulation of silver in ectomycorrhizal and saprobic macrofungi from pristine and polluted areas

Macrofungi are effective accumulators of Ag. This study provides a comprehensive review of this phenomenon supported by original data on the Ag concentrations of macrofungi from pristine and Ag-polluted areas. In pristine areas, the median Ag concentrations of ectomycorrhizal (ECM) and saprobic (SAP) macrofungi were 0.79 and 2.94 mg kg(-1), respectively. In these areas, hyperaccumulation thresholds for Ag in ECM and SAP macrofungi are proposed as 100 and 300 mg kg(-1), respectively. In a Ag-polluted area, the Ag concentrations in macrofungi (ECM and SAP) were significantly elevated with the median value of 24.7 mg kg(-1) and the highest concentrations in Amanita spp. of the section Vaginatae (304-692 mg kg(-1)). The intracellular speciation of Ag in fruit-bodies of the Ag-accumulator Amanita submembranacea was inspected by size exclusion chromatography followed by sulfhydryl-specific fluorimetric assays of ligands using reverse phase high-performance liquid chromatography and improved polyacrylamide gel electrophoresis. Virtually all Ag was found to be intracellular and sequestered in the major 7 kDa and minor 3.3 kDa complexes. The lack of glutathione and phytochelatins and the presence of a single 3 kDa sulfhydryl-containing peptide in the isolated Ag-complexes suggest that detoxification of Ag in A. submembranacea may rely on metallothionein. Vertical distribution of Ag in a polluted forest soil profile has shown substantial enrichment in organic horizons; in polluted technosol, the highest Ag concentrations were found in surface layers. Standardized EDTA extraction of Ag in both the investigated soil profiles showed relatively low Ag extractibility, generally within the range of 2.2-7.7% of total Ag content.

Three metallothionein isoforms and sequestration of intracellular silver in the hyperaccumulator Amanita strobiliformis

Metallothioneins (MTs) are cysteine-rich peptides involved in heavy metal tolerance of many eukaryotes. Here, we examined their involvement in intracellular binding of silver (Ag) in the ectomycorrhizal fungus Amanita strobiliformis. The Ag complexes and their peptide ligands were characterized using chromatography and mass spectrometry. The full-length coding sequences obtained from a cDNA library were used for complementation assays in yeast mutant strains. Abundance of respective transcripts in A. strobiliformis was measured by quantitative real-time reverse-transcribed polymerase chain reaction (qRT-PCR). Ag-speciation analyses showed that intracellular Ag was in wild-grown fruit bodies and cultured extraradical mycelia of A. strobiliformis sequestered by metallothioneins. The determined sequence of the peptide facilitated isolation of three cDNA clones, AsMT1a, AsMT1b and AsMT1c. These encode isomorphic MTs consisting of 34 amino acid residues and sharing 82% identity. In mycelia the expression of AsMT1s is induced by Ag. All AsMT1s expressed in yeasts complemented hypersensitivity of mutants to cadmium (Cd) and copper (Cu) and formed Ag complexes. Only the Ag-AsMT1a complex was detected in the A. strobiliformis fruit body in which AsMT1a was the prevailing transcript. The present study identified the existence of metallothionein isoforms in ectomycorrhizal fungi. We demonstrated that intracellular sequestration of Ag in fruit bodies and mycelia of hyperaccumulating A. strobiliformis is dominated by metallothioneins.

Detection of summer truffle (Tuber aestivum Vittad.) in ectomycorrhizae and in soil using specific primers

Tuber aestivum is becoming an important commodity of great economical value in some European countries. At the same time, it is a highly protected organism in other countries, where it needs careful treatment. A reliable method of detection in roots and soil is thus needed for assessment of geographic distribution, ecological studies and inoculation efficiency testing in man-made experiments. A PCR-based method of detection of T. aestivum using specific primers was therefore developed. A pair of PCR primers Tu1sekvF/Tu2sekvR selective for T. aestivum and some genotypes of Tuber mesentericum was designed on the basis of the known internal transcribed spacer T. aestivum sequences. TaiI restriction cleavage was then used to distinguish the two species. The selectivity of the designed primer pair was evaluated using DNA extracted from specimens of a further 13 Tuber spp. Subsequently, the selectivity and robustness to false-positive results with nontarget DNA of the designed primers was compared with two other primer pairs (UncI/UncII and BTAE-F/BTAEMB-R). The occurrence of T. aestivum in soil and ectomycorrhizae collected in its native habitat has been successfully detected using the designed primers and nested PCR. The method is reliable and thus suitable for detection of T. aestivum in the field.

A quest for indigenous truffle helper prokaryotes

Tuber aestivum is the most common European truffle with significant commercial exploitation. Its production originates from natural habitats and from artificially inoculated host tree plantations. Formation of Tuber ectomycorrhizae in host seedling roots is often inefficient. One possible reason is the lack of indigenous associative microbes. Here we aimed at metagenetic characterization and cultivation of indigenous prokaryotes associated with T. aestivum in a field transect cutting through the fungus colony margin. Several operational taxonomic units (OTUs) showed close association with the T. aestivum in the ectomycorrhizae and in the soil, but there was no overlap between the associative prokaryotes in the two different habitats. Among those positively associated with the ectomycorrhizae, we identified several bacterial genera belonging to Pseudonocardineae. Extensive isolation efforts yielded many cultures of ectomycorrhizae-associative bacteria belonging to Rhizobiales and Streptomycineae, but none belonging to the Pseudonocardineae. The specific unculturable Tuber-associated prokaryotes are likely to play important roles in the biology of these ectomycorrhizal fungi, including modulation of competition with other symbiotic and saprotrophic microbes, facilitation of root penetration and/or accessing mineral nutrients in the soil. However, the ultimate proof of this hypothesis will require isolation of the microbes for metabolic studies, using novel cultivation approaches.

Intracellular sequestration of zinc, cadmium and silver in Hebeloma mesophaeum and characterization of its metallothionein genes

Sequestration of intracellular heavy metals in eukaryotes involves compartmentalization and binding with cytosolic, cysteine-rich metallothionein (MT) peptides. We examined the roles of these processes in handling of zinc (Zn), cadmium (Cd) and silver (Ag) in sporocarps and a metal-exposed extraradical mycelium of Hebeloma mesophaeum, the Zn-accumulating ectomycorrhizal (EM) species frequently associated with metal disturbed sites. Size exclusion chromatography revealed that the majority of Zn and Cd in the sporocarps and mycelium was contained in a low molecular mass fraction attributable to compartmentalized metal. The staining of hyphal cells with the Zn-specific Zinquin and Cd-specific Leadmium fluorescent tracers labeled Zn and Cd in small, punctuated vesicles and vacuoles, respectively. By contrast, the sporocarp and mycelium Ag was associated with cysteine-rich, 5-kDa peptides. The peptides of the same size were also identified in minor Zn and Cd complexes from the metal-exposed mycelium. We have further isolated and characterized HmMT1, HmMT2 and HmMT3 genes coding for different 5-kDa MTs of H. mesophaeum collected at a lead smelter site. Heterologous complementation assays in metal-sensitive yeast mutants indicated that HmMTs encode functional, metal-specific peptides: only HmMT1 was able to complement sensitivity to Zn; HmMT1 conferred higher tolerance to Cd and Cu than HmMT2 or HmMT3; and both HmMT2 and HmMT3, but not HmMT1, conferred increased tolerance to Ag. The presence of HmMT1 and HmMT3, but not HmMT2, was also confirmed in a H. mesophaeum isolate from an unpolluted site. Gene expression analysis in the extraradical mycelium of this isolate revealed that the transcription of HmMT1 was preferentially induced in the presence of Zn and Cd, while Ag was a stronger inducer of HmMT3. Altogether, these results improve our understanding of the handling of intracellular Zn, Cd and Ag in Hebeloma and represent the first evidence suggesting involvement of MTs in sequestration of Zn in EM fungi.

Gold content of ectomycorrhizal and saprobic macrofungi from non-auriferous and unpolluted areas

Ectomycorrhizal and saprobic macrofungi growing in the wild were collected from non-auriferous and unpolluted areas and analyzed for gold. Gold was determined using long-term instrumental neutron activation analysis (INAA). In total, 154 samples, including 67 species of ectomycorrhizal fungi and 22 species of terrestrial saprobes, were examined. Gold contents of the both groups were mostly less than 20 ng g(-1) of D.W. The highest concentrations (expressed in D.W.) were found in the ectomycorrhizal species Amanita strobiliformis (136 ng g(-1)), Russula claroflava (148 ng g(-1)), Cantharellus lutescens (156 and 210 ng g(-1)), and Boletus edulis (235 ng g(-1)). Among the saprobic fungi, the highest values were found in Langermannia gigantea (160 ng g(-1)) and Morchella esculenta (189 ng g(-1)). Species of Agaricus commonly had relatively high gold values, 10s of ng g(-1). The gold content of macrofungal fruit bodies was considerably higher than that of vascular plants, and parallels concentrations found in plants growing in auriferous areas.

Molecular phylogeny of Psilocybe cyanescens complex in Europe, with reference to the position of the secotioid Weraroa novae-zelandiae

A phylogenetic analysis with three molecular markers was undertaken to test the hypothesis that the complex of Psilocybe cyanescens in Europe consists of several, morphologically distinct species. The results support the existence of two molecularly well-supported morphological groups, that of Psilocybe cyanescens and P. azurescens on the one hand, and the complex of P. serbica on the other. However, in the last group, no sequence variability within the three molecular markers from P. serbica and related taxa P. bohemica, P. arcana, and P. moravica was found. It was decided, therefore, to merge these taxa into P. serbica, and to distinguish them below species level. It was also demonstrated that the secotioid Weraroa novae-zelandiae belongs to the P. cyanescens species complex. Accordingly, it was transferred to Psilocybe as P. weraroa, nomen novum.

On the possible role of macrofungi in the biogeochemical fate of uranium in polluted forest soils.

Interactions of macrofungi with U, Th, Pb and Ag were investigated in the former ore mining district of Příbram, Czech Republic. Samples of saprotrophic (34 samples, 24 species) and ectomycorrhizal (38 samples, 26 species) macrofungi were collected from a U-polluted Norway spruce plantation and tailings and analyzed for metal content. In contrast to Ag, which was highly accumulated in fruit-bodies, concentrations of U generally did not exceed 3mg/kg which indicates a very low uptake rate and efficient exclusion of U from macrofungi. In ectomycorrhizal tips (mostly determined to species level by DNA sequencing), U contents were practically identical with those of the non-mycorrhizal fine spruce roots. These findings suggest a very limited role of macrofungi in uptake and biotransformation of U in polluted forest soils. Furthermore, accumulation of U, Th, Pb and Ag in macrofungal fruit-bodies apparently does not depend on total content and chemical fractionation of these metals in soils (tested by the BCR sequential extraction in this study).

Arsenic hyperaccumulation and speciation in the edible ink stain bolete (Cyanoboletus pulverulentus)

The edible ink stain bolete (Cyanoboletus pulverulentus) was found to hyperaccumulate arsenic. We analyzed 39 individual collections determined as C. pulverulentus, mostly from the Czech Republic. According to our results, concentrations of arsenic in C. pulverulentus fruit-bodies may reach 1300mgkg-1 dry weight. In most collections, data for total and bioavailable arsenic in underlying soils were collected but no significant correlation between the soil arsenic content and arsenic concentrations in the associated fruit-bodies was found. Within the fruit-bodies, we found the majority of arsenic accumulated in the hymenium. Besides occasional traces of methylarsonic acid (MA), the arsenic speciation in all mushroom samples consisted solely of dimethylarsinic acid (DMA) and no inorganic arsenic was detected. Because of the carcinogenic potential of DMA, C. pulverulentus should not be recommended as an edible mushroom and its consumption should be restricted.