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compounds originating from fungal biomass, or substances synthesized on the basis of

the structure of the fungal substances, was developed.

Metallic elements of specific gravity close to 5 or above are termed heavy metals.

Despite their general feature of being potentially toxic to living biota, certain ones belong

to trace elements essential for growth and metabolism, while others have no known

function (Woolhouse, 1983). The tolerance to a given toxicity level, the mechanisms of

adaptation, and the ability to sequester and accumulate metals from the external environment vary not only among different strains and species of fungi, but also at different stages

of fungal development. Reliable knowledge on the developmental and physiological state

of the investigated material will improve our understanding of the mechanisms involved

in fungal interaction with metals.

The knowledge of the above exampled subjects is based on the use of different

techniques. Atomic absorption spectroscopy (AAS), inductively coupled plasma–atomic

emission spectrometry (ICP-AES), and nuclear magnetic resonance (NMR) spectroscopy

were used to estimate the element content in the mycelium and to characterize the general

response of a given species or strain to heavy metals. Microanalytical tools related to

optical or electron microscopy have significantly enhanced our understanding of the

interactions with the environment. The localization of elements on the cellular and subcellular levels using one of the microanalytical systems gives important data on element

distribution and, by this, on the role of elements in physiological processes, interactions

between elements, and reasons for their deficiency or toxicity. It also provides the link

between physiological and anatomical studies, which is especially important when studying responses of organisms to environmental stress, such as the presence of naturally

occurring heavy metals or metals introduced by pollution.

This chapter discusses the use of microanalytical tools to study microscale interactions of fungi with soil or other diverse substrata and with plants. It covers the diversity

of techniques and the criteria that must be met for successful application of the methods.



14.2



CELLULAR AND SUBCELLULAR IDENTIFICATION,

LOCALIZATION, AND MAPPING OF ELEMENTS



The distribution of elements in biological specimens on the cellular and subcellular

levels may be determined by several methods. Detection of characteristic x-rays generated during the interaction of electrons with distinctive elements (Z > 9 mostly) in

a specimen is an extension of the capabilities of a scanning or transmission electron

microscope (SEM or TEM). X-rays are more often detected in energy-dispersive (EDS)

than in wavelength-dispersive (WDS) mode. Electron energy loss spectroscopy (EELS)

and electron spectroscopic imaging (ESI) is another technique exploiting the interactions with the inner shell electrons of distinctive elements (Z = 3 to 92). New insight

into interactions between elements, interatomic distances, bond angles, and types and

numbers of neighboring atoms can be gained when extended x-ray absorption finestructure spectroscopy (EXAFS) or near-edge fine-structure electron energy loss spectroscopy (ELNES) is applied (Teo, 1986; Koningsberger and Prins, 1988; Williams and

Carter, 1996).

The use of focused protons instead of electrons for the generation of characteristic

x-rays is the basis of particle-induced x-ray emission (PIXE). Quantitative PIXE analysis

benefits from the possibility of simultaneous use of proton backscattering (BS) or scanning

transmission ion microscopy (STIM) techniques (Johansson et al., 1995; Mesjasz-Przybylowicz and Przybylowicz, 2002). These complementary techniques are used for accurate



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determination of local changes of the specimen matrix (composition and thickness/areal

density). In secondary ion mass spectrometry (SIMS) a specimen is eroded with the use

of low-energy ions. In addition to electron and proton microscopy, the microprobe analysis

might also be carried out with the laser microprobe mass analyzer (LAMMA) fitted to a

laser light microscope with a high-energy pulse laser (Kuhn et al., 1995). Each method

has its advantages and limitations. The differences involve the spatial resolution, detection

limit, elements that can be detected, and access to qualitative and quantitative standards.

Detailed information is given by Hall and Gupta (1984), Benninghoven et al. (1987),

Kottke (1994), Johansson et al. (1995), Egerton (1996), Williams and Carter (1996),

Bücking et al. (1998), van Steveninck and van Steveninck (1991), Leapman and Rizzo

(1999), and Kuhn et al. (2000). A general advantage of microscale methods is the low

amount of material necessary for the analysis and the possibility to control the physiological and developmental states on the ultrastructural level.



14.3



PREPARATION OF SAMPLES FOR ELEMENT

ANALYSIS



A critical point of using microanalytical tools on biological material is the preparation of

the samples, which should be properly cleaned and washed in ice-cooled water to avoid

losses of elements such as K. The material should be lyophilized or chemically fixed as

soon as possible. Drying/rehydrating and freezing/thawing of soil samples containing

fungal hyphae might result in large decreases of metal concentration in the hyphae, in

comparison from moist samples transported directly from the field and analyzed without

delay (Berthelsen et al., 2001). However, drying and rehydrating occur frequently under

natural conditions; therefore, results obtained on both dry and wet material are of biological interest.

Fungal ultrastructure is by now well documented. Artifacts resulting from insufficient preparation can be recognized by TEM. Changes are observed mainly in senescent

and dead cells. Distinguishing between fixation-induced and natural changes usually

requires experience. Methods accompanying the microanalytical tools are vital for the

evaluation of the element localization. Without the guidance of some reliable microscopical observations, misleading interpretations can be made. Primary observations should

be carried out on living mycelia of interest under a light microscope, often underestimated

by modern researchers. A skillful scientist will be able to observe, e.g., cisternae 200 to

300 Å thick, far below the expected resolution of light optics, simply using phase contrast

(Girbardt, 1965). The Nomarski contrast is another tool that is very useful in such

research. The observations should be supported by data obtained from physiological

studies.

Among available preparation techniques the best option is cryofixation. When working at lower magnifications of the SEM or proton microscope, the best solution is to cryofix

the sample by plunge cooling or cryopunching, followed by freeze drying. A more sophisticated method is high-pressure cryofixation followed by freeze substitution. The most

adequate protocol to study element distribution in cell walls seems to be anhydrous freeze

substitution (Orlovich and Ashford, 1995). Cryosectioning of such material is, however,

extremely difficult, and embedding in nonpolar resins seems to be a better option to obtain

dry-cut sections of material for elemental analysis (Fritz, 1989). Ultrathin sections of 30

to 40 nm, as required for EELS microanalysis, are even more difficult to obtain, and wet

sectioning is necessary. In the latter case, only the distribution of precipitated elements

can be studied.



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MICROANALYTICAL STUDIES ON STRUCTURAL

AND BIOCHEMICAL DIFFERENTIATION DURING

MORPHOGENESIS OF FUNGI AND FUNGAL–PLANT

INTERACTION



The development of electron microscopy techniques resulted in detailed descriptive characterization of the ultrastructure of many groups of fungi and showed the diversity of this

group of organisms. It is a challenge to resolve the significance of ultrastructural patterns,

which would lead to understanding the role of particular elements in physiological processes,

nutrient requirement, and deficiency. Qualitative and quantitative elemental analysis using

microanalytical techniques can constitute an important step in understanding fungal differentiation and morphogenesis, at least by drawing our attention to particular events. PIXE

has been used to determine the distribution and element concentration of macroelements

such as K, P, S. and trace metals like Cu, Mn, Fe, and Zn in germ tubes of Aureobasidium

pullulans and Ophiostoma ulmi (Brunton et al., 1988; Gadd et al., 1988). The highest

concentrations of K, P, Na, and Mg were shown at the tip and in older parts of the mycelium,

where new branches of the hyphae or the yeast-like cells develop. A different pattern of

element distribution was found in the mycelium of Aspergillus niger, with the concentration

gradient decreasing from the hyphal tip toward the older regions. Combined with the use

of fluorescent indicator dyes distinguishing biologically available ions from the bound pool,

this method may enrich our knowledge (Gadd et al., 1988). The accumulation of nitrogencontaining compounds within vacuoles of Cenococcum geophilum–Pinus sylvestris has been

measured by use of EELS and ESI (Kottke et al., 1995a). In the hyphal sheath of Amanita

muscaria mycorrhizas cultivated at two different atmospheric CO2 concentrations and two

different levels of nitrogen, the interaction between storage of glycogen in the cytosol and

nitrogen compounds in the fungal vacuoles was shown (Turnau et al., 2001a).

Differences in element composition have been found between elongated and spherical bodies associated with septae of ascomycetes. P, N, and S are the most common

elements of spherical bodies of Sarcosphaera crassa, while Ca is dominant in elongated

bodies of Disciotis venosa (Turnau et al., 1993a). Different types of septal bodies can be

present in the same species — elongated bodies within ascogenic hyphae and spherical

bodies within vegetative mycelium (Turnau, unpublished material).

Long-distance transport has been studied using lanthanum (La) or cerium (Ce) as

tracers and x-ray microanalysis or EELS/ESI to map the element distribution at the

ultrastructural level in mycorrhizas. Results show that lanthanum is primarily transported

apoplastically in hyphal and plant cell walls, arrested only by the Casparian band. It is

occasionally taken up by the plant cells via endocytosis (Pargney and Le Disquet, 1994;

Kottke et al., 1995b; Carnero-Diaz Le Disquet, 1996; Vesk et al., 2000). High-resolution

ESI reveals the presence of La and Ce in the fungal vacuoles and its passage through the

fungal porus (Kottke, 1991; Carnero-Diaz Le Disquet, 1996). The latter results obtained

from chemically fixed material were supported by studies on material prepared by highpressure freezing using Cs and Sr as tracers (Frey et al., 1997). It is obvious that plants

and fungi interact in different ways with metals.

Microanalytical tools can also be useful to study infection processes by fungi and

the reaction of plants to pathogens. PIXE reveals a depletion of K in regions of Pisum leaf

infection by Erysiphe pisi. At the same time, an increase of Ca content has been observed

in thickened walls around infected cells (Watt and Grime, 1988). The accumulation of Ni,

Zn, Cu, Mn, Fe, Ca, Ti, As, and Sr accompanied by a drastic depletion of P, S, and K has

been demonstrated in leaf areas of a resistant genotype of Lagenaria sphaerica (Cucurbitaceae) infected by a foliar pathogen, the powdery mildew Sphaerotheca fuliginea (Weiers-



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bye-Witkowski et al., 1997; Mesjasz-Przybylowicz, 2001). The metal content increased

within the first 4 days after infection, and actually, the data obtained were the first to show

the concentration of heavy metals that can be reached within plant cells in response to a

pathogenic fungus. The infected cells were obviously killed by the toxicity of the metals,

followed by the deposition of Si reaching 23% of dry weight within necrotic lesions,

suggesting the formation of a barrier protecting the surrounding tissues from the fungus.



14.5



TRANSFORMATION OF SOIL MINERALS BY FUNGI



The involvement of fungi in rock weathering and soil formation has been known for a

long time. Especially lichen-forming fungi have received much attention (Schatz, 1963;

Seyers and Iskandar, 1974). The weathering phenomena occurring at the lichen/basalt and

granite rock interface have been studied with SEM accompanied by an EDS analyzer

(Jones et al., 1980; Ascaso et al., 1995). Extensive etching and degradation of minerals

due to the production of oxalic acid have been observed. Similar changes have been found

when Aspergillus niger was cultivated in liquid growth medium supplemented with labradorite and clay material separated from basalt (Jones et al., 1980). This “mining” ability

has been suggested as well for mycorrhizal fungi (Jongmans et al., 1997; van Breemen et

al., 2000).

The production of oxalate crystals occurs in a wide range of ectomycorrhizal fungi

(Graustein et al., 1977; Cromack et al., 1979; Paris et al., 1995; Unestam and Sun, 1995;

Arocena et al., 2001) and has been suggested to play various roles, such as avoiding

calcium and oxalate toxicity (Snetselaar and Whitney, 1990) and providing a hydrophobic

coating that prevents hyphae from becoming hydrated, which could result in reduced

microbial attack (Whitney and Arnott, 1987; Arocena et al., 2001). Encrustation of the

mycelium with oxalate crystals may also serve as protection against grazing soil animals.

Finally, oxalate crystals may play a role in water regulation, as has been suggested in the

case of lichen-forming fungi (Clark et al., 2001). TEM/EDS analysis accompanied by

several other techniques, such as NMR spectroscopy, and gas-liquid chromatography–mass

spectrometry, has been used in an elegant study of exudation–reabsorption in a mycorrhizal

fungus Suillus bovinus (Sun et al., 1999). X-rays were used to identify the released ions

within the fluid droplets exudated on the surface of hydrophobic mycelium. The interaction

of rhizomorphs with minerals has been studied using PIXE by Wallander et al. (2002).

Ca originating from apatite, the least soluble calcium phosphate from aerated soils, has

been shown on the surface of rhizomorphs in the form of calcium oxalate crystals. Some

rhizomorphs are rich in K, which suggests that these fungi might be good accumulators

of these elements and, as claimed by the authors, might play an important role in transferring K to trees. Oxalate crystals have also been occasionally reported on the mycelium

and spores of arbuscular mycorrhizal (AM) fungi (Boyetschko and Tewari, 1986; Jurinak

et al., 1986), but their presence has not been confirmed in subsequent studies carried out

in pot cultures (Knight et al., 1992) and in field studies performed on soil samples from

diverse vegetation types of the arid zone of California (Allen et al., 1996). These studies

do not exclude oxalate formation in other species of AM fungi or under conditions other

than those studied so far. Such studies are not easy without techniques involving scanning

electron microscopy accompanied by microanalytical tools. As AM fungi cannot be grown

separately in aseptic conditions, the production of oxalate by the plant cannot be excluded.

It is also important to confirm the oxalate nature of the crystals. The data should be verified

with other techniques such as ELNES (Lichtenberger and Neuman, 1997), as the presence

of a strong Ca peak does not exclude other chelating agents.



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Fungi also form cation-binding polyphosphate, stored in vacuoles. Al–polyphosphate

complexes have been demonstrated by in vivo NMR spectroscopy of Laccaria laccata

(Martin et al., 1994). EELS enables precise localization of the position of elements in the

small, osmophilic vacuolar bodies of Laccaria amethystea mycelium cultured in the same

conditions as L. laccata (Kottke and Martin, 1994). Al has also been found within the

fungal wall (Turnau et al., 1994a, 1994b), often bound by phosphate-containing pigments.

Although the galactosamine polymer of the cell wall has been implicated as offering a

potential site for binding polyphosphates (Harold, 1962; Marschner, 1997), the description

of Al localization would suggest pigments to be binding sites for Al (Turnau et al., 1994a,

1994b) rather than polyphosphates, as suggested by Väre (1990) and Tam (1995).



14.6



TRANSFORMATION AND COMPARTMENTALIZATION

OF HEAVY METALS WITHIN THE FUNGAL MYCELIUM



Many fungi can adapt to growth in polluted sites. However, the mechanisms of resistance

or tolerance to heavy metals are still rather fragmentarily elucidated. Saprobic fungi are

particularly interesting, as they grow relatively easily in laboratory cultures and can be

used as biosorbents for metals, e.g., in aqueous waste stream (Galun et al., 1983; Gadd

and White, 1989; Huang et al., 1990). The first step of research involves the assessment

of the ability of the selected fungus to accumulate the metals. Such data are usually

obtained with atomic absorption spectrophotometry, but this gives no information on the

distribution or compartmentalization of elements within the fungal hyphae or on the surface

of the fungal wall. Microanalysis is important to explain the mechanisms of action, and

in recognition of conditions under which the metals are not efficiently chelated, they may

become cytotoxic.

14.6.1

Extracellular Sequestration of Heavy Metals

Elemental microbeam investigations indicated that heavy metals may be chelated on the

surface or within the cell wall, or intracellularly, within the cytoplasm or the vacuoles.

However, the distribution varies strongly among fungal strains and species, and among

individual metal ions. Relatively much is known on substances such as organic acids that

are exudated by the fungi and sequestrate heavy metals (Figure 14.1). The ability of

Aspergillus niger to produce various organic acids that mobilize inorganic phosphates

resulting in immobilization of Co and Zn has been demonstrated. The identification of the

substances present in culture media has been carried out by various techniques, such as

differential pulse polarography, ion chromatography, and gas chromatography–mass spectrometry. X-ray microprobe analysis has been very useful as a complementary tool that

clearly demonstrates the deposition of crystals containing the different elements (Sayer

and Gadd, 2001).

The activity of fungi in heavy metal-rich soils leads to the transformation of metals

and metalloids by processes such as oxidation, reduction, and methylation, resulting in

changed mobility and toxicity of heavy metals toward other living organisms (Gadd, 1993,

1996; Morley and Gadd, 1995; White et al., 1995; Morley et al., 1996; Jacobs et al., 2002).

This process can also play a role in the leaching of contaminants from the soil and polluting

of the groundwater. Microanalysis can be efficient in evaluation and development of

bioremediation techniques. Pyromorphite (Pb5(PO4)3Cl), which was believed to be a safe

and stable mineral in urban and polluted soils, has been demonstrated to be susceptible

to transformation by fungal phosphatases and to the activity of organic acids. This resulted



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p



2.5,µm



(a)



1 µm



(b)



p

c



p



p



0.6 µm



(c)



(d)



0.3 µm



c



(e)



0.6 µm



(f)



50 µm



Figure 14.1 SEM and TEM micrographs showing various extracellular and intracellular depositions where heavy metals were detected. (a) Pigments excreted on the surface of mycelium. (b)

Crystals produced on the mycelium. (c) Pigment material visible on the surface and within the

mycelium. (d) Production of crystals underneath the pigment layer. (e) Disappearance of crystals

after treatment with uranyl acetate. (f) Crystaloid structure of the mycelium wall of Acremonium

sp. cultivated on medium with excess Cu. p, pigments; c, crystals. (Most images from Turnau et al.,

Acta Soc. Bot. Pol., 71, 253–261, 2002. With permission.)



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in significant alteration of mobility, toxicity, and transfer of Pb into other organisms (Sayer

et al., 1999).

14.6.2

Binding Properties of the Hyphal Walls

The heavy metal-binding properties of fungal cell walls rely on the presence of the

carboxyl, hydroxyl, and amino groups of the cell wall carbohydrates, proteins, and

chitin/chitosan (Zimmermann and Wolf, 2002). The distribution of metals within cell walls

has often been demonstrated by microanalytical tools and confirmed by other techniques,

e.g., by analyzing cellular subfractions (Ono et al., 1988; Brady and Duncan, 1994a, 1994b;

Yazgan and Özcengiz, 1994, Krantz-Rülcker et al., 1995; Gorovoi and Kosyakov, 1996;

Zhou, 1999). In some cases, the metals are uniformly spread within the cell walls, which

have been found to be the main storage pool of Cd and Zn (Blaudez et al., 2000; Frey et

al., 2000). In other cases, they are localized in special structures. Volesky and May-Phillips

(1995) found that uranium was precipitated within the cell walls in the form of fine, needleshaped crystals. Research on the sorption of heavy metals by Aspergillus niger and Mucor

rouxii has been carried out mainly with inductively coupled argon plasma spectrometry

and complemented by a demonstration of Ag precipitations on the cell walls as colloidal

silver using TEM/EDS (Mullen et al., 1992). Such studies can be further elaborated by

the use of EXAFS, as has been done in the case of Penicillium chrysogenum (Sarret et

al., 1998, 1999). In this work, Pb was shown to be bound to carboxyl and phosphoryl

groups within the cell wall. The carboxyl groups had a high affinity for Pb, but were

present in low amounts, while the phosphoryl groups of lower affinity were more abundant.

SEM/EDS microanalysis is valuable to understand why fungi such as Armillaria

spp. can occur in extremely polluted places, although high concentrations of heavy metals

can be toxic to mycelia in culture. Abundant fruit bodies and rhizomorphs have been noted

on experimental plots treated with up to 5000 tons ha–1 of cadmium dust (Turnau, 1990).

Both fruit bodies and rhizomorphs were often found in direct contact with pure industrial

dust. Using EDS, Rizzo et al. (1992) found elements such as Al, Zn, Fe, Pb, and Cu

located only on the outer, melanized parts of the rhizomorphs, but not in the interior. The

accumulation of toxic levels of heavy metals may also serve as protection from antagonistic

microorganisms in both polluted and nonpolluted environments.

Some fungi may become bluish while grown in the presence of excess Cu. In most

cases, abundant production of oxalate is responsible for this alteration. However, this was

not the case of Acremonium pinkertoniae (Figure 14.1). Observations carried out with

SEM revealed crystals occurring within the cell walls. Differences in shape, localization,

and effectiveness of Cu binding within the crystals led to further research with infrared

spectroscopy, suggesting the involvement of cell wall components such as chitin (Zapotoczny, unpublished data). This information may be useful for the development of new

soil-cleaning technologies.

During studies on compartmentalization of heavy metals within the mycelium, it is

extremely important to control precisely the vitality of the fungal hyphae because when

the metabolic activity is inhibited or absent, the accumulation of metal is mostly due to

biosorption, resulting in its binding on the surface or within the cell wall (Tobin et al.,

1984; Avery and Tobin, 1992). In addition, if the metal-to-biomass ratio is below 100 nmol

g–1, then metal accumulation is almost entirely dependent on biosorption of metal ions to

the cell wall (Brady and Duncan, 1994a).

14.6.3

Metal Chelation within Cytosol and Vacuoles

Heavy metals that have entered the cytosol of the hyphae due to energy-dependent cellular

transporters are immobilized by chelators such as phytochelatins and metallothioneins



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(Lerch, 1980; Munger and Lerch, 1985; Howe et al., 1997). The nature of these compounds

was studied in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida glabrata, Kluyveromyces marxianus, and Neurospora crassa (Wemmie et al., 1994; Yazgan

and Özcengiz, 1994; Li et al., 1997; Sajani and Mohan, 1998). Microscopic observations

are useful to study this subject. The method adopted by Morselt et al. (1986) is the one

most often cited. There are, however, several limitations to this technique; because no

visible reaction occurs in dark pigments producing fungi, change of staining within the

cell wall or a positive reaction in control samples was observed. In the study of extraradical

mycelium of several mycorrhizas collected from heavy metal-rich sites, the positive

staining reaction was shown only in Hebeloma spp. that have been found to act as

bioexcluders of heavy metals. No reaction was observed in the case of Rhizopogon

roseolus and Suillus luteus. The latter was shown to produce a positive reaction to the

Gomori–Swift technique that visualizes S-S groups in proteins at the ultrastructural level.

In the same fungi, the distribution of heavy metals was studied by EELS/TEM, revealing

the presence of the heavy metals within the cytoplasm and vacuoles (Turnau, unpublished

results). This might suggest that the staining according to Morselt et al. (1986) is specific

for few substances.

Heavy metals that are taken up in the cytosol are subsequently transported into the

vacuoles. Yeast tonoplast-located transporters are already known (Li et al., 1997; Klionsky,

1998). The presence of heavy metals such as Cd, Zn, and Cu within vacuoles has been

originally indicated using microanalytical techniques in chemically fixed fungal material

(Turnau et al., 1993b, 1993c). In freeze-dried mycelium studied with PIXE, these elements

are usually nonuniformly distributed, and the elemental maps suggest the involvement of

intracellular granular material in heavy metal sequestration (Figure 14.2 and Figure 14.3).

Mycelia of Fusarium sp. and Penicillium citrinum have been found by means of TEM/EDS

microanalysis to transform tellurium, leading to possible destruction of semiconductor

thermoelectric cells, pipes, and protective sheathing for electric cables. The deposition of

large black granules, apparently in vacuoles, which corresponded with the reduction of

tellurite to amorphous elemental tellurium, has been demonstrated (Gharieb et al., 1999).

The cultivation of fungi in axenic conditions on media enriched with heavy metals

such as Pb or Cu leads to an increase of the number and size of metachromatic granules

observed with Nomarski DIC optics, as noted in the case of Paxillus involutus and Suillus

luteus (Turnau, unpublished data). The first report on Zn localization within the mycelium

of freeze-substituted mycorrhizas demonstrated the presence of this element only in the

hyphal cell wall and extrahyphal, polysaccharide slime (Denny and Wilkins, 1987). On

the contrary, Bücking and Heyser (1999) have shown that the distribution of Zn depends

on the strain of the fungus. Mycelia of Suillus bovinus cultivated on media with high levels

of Zn accumulated more Zn within the vacuoles, or a similar level of Zn was detected in

the vacuolar polyphosphates and within the cytoplasm. In addition, other techniques proved

that vacuoles are important in the sequestration of potentially toxic metals; most of these

data concern yeasts. Seventy percent of Sr2+, 90% of Mn2+, and 60% of Zn2+ accumulated

intracellularly by Saccharomyces cerevisiae were compartmented in the vacuoles (Nieuwenhuis et al., 1981; Lichko et al., 1982; White and Gadd, 1987; Blackwell et al., 1995).

Studies on the kinetics of Cd uptake by Paxillus involutus, a mycorrhizal fungus, clearly

demonstrated the accumulation of Cd within the vacuoles (Blaudez et al., 2000). In

previous studies on this species originating from Cd-rich substratum, carried out on

chemically fixed material, this element was most prominently detected within the vacuoles

(Turnau et al., 1993b, 1993c).

Microanalytical tools often indicate the co-occurrence of P and heavy metals within

the vacuoles. This does not necessarily mean that metals are bound to polyphosphate; they



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Zn



250

230

200

180

150

130

100

75



d



50

0.3 µm



(a)



p



25

(b)



0



Cd



800



3000



720



2400



560



2100



480



1800



400



1500



320



1200



240



900



160

(c)



2700



640



600



80

0



300



(d)



0

0.3 µm



Figure 14.2 (See color insert following p. 460.) Metal localization in spores of Glomus sp. (a)

Spore stained with sodium rhodizonate, suggesting the deposition (d) of heavy metals on the inner

surface of the cell wall. (From Turnau, Acta Soc. Bot. Pol., 67, 105–113, 1998. With permission.)

(b–d) PIXE elemental maps of spore isolated from polluted soil; concentrations given in mg kg–1

(Turnau, Mesjasz-Przybylowicz, and Przybylowicz, unpublished material).



may be bound to OH or SH groups as well. This subject can be further elaborated by

EXAFS and ELNES.



14.7



METAL-ACCUMULATING PROPERTIES OF

LICHEN-FORMING FUNGI



Epiphytic lichen thalli are commonly used for biomonitoring of air pollution, generating

data useful in interpretation of epidemiological patterns of respiratory diseases, due to the

fact that they accumulate high concentrations of metals originating almost entirely from

the air. Most data on metal content in thalli have been obtained using conventional

spectroscopy (see recent reviews: Bennett, 2000; Garty, 2001) or particle-induced x-ray

emission (Hrynkiewicz et al., 1979; Olech et al., 1998). In the first case, the metal content

is evaluated after acid digestion of samples, while in the second case, the thalli are dried

and ground, and a known amount is pressed into a pellet. The lichens are collected from



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Fungal Activity as Determined by Microscale Methods

P



(b)



2000

1800

1600

1400

1200

1000

800

600

400

200

0



297

Al



(c)



Zn



(a)



(d)

200 µm



250

230

200

100

150

130

100

75

50

25

0



Ca



(e)

50 µm



10000

9000

8000

7000

6000

5000

4000

3000

2000

1000

0

2500

2300

2000

1800

1500

1300

1000

750

500

250

0



Figure 14.3 (See color insert following p. 460.) Element distribution in orchid mycorrhizas. (a)

SEM micrograph of fungal coils. (b–e) PIXE elemental maps of coils separated from the plant

material; concentrations given in mg kg–1 (Turnau, Mesjasz-Przybylowicz, and Przybylowicz, unpublished maps; for more information, see Jurkiewicz et al., 2001).



the site of interest and directly analyzed, or the thalli are collected from relatively nonpolluted areas and transplanted for a certain period to the area of interest. Large, agerelated differences between peripheral and inner zones of the thalli are often visible.

Furthermore, layers of thalli observed in transverse sections differ in compaction, level of

gelatinization of the cementing material, thickness of the fungal cell walls, and distribution

of various crystals, crystalloids, and hydrophilic wall material (Chiarenzelli et al., 1997).

The impact of these differences on element accumulation still remains unresolved. The

first attempts were done using the sequential elution procedure to distinguish between

extracellular and intracellular uptake (as reviewed by Garty, 2001). These findings were

followed by studies with SEM accompanied by EDS (e.g., Garty et al., 1979), x-ray

emission spectrography (SEMEX) (Johnsen, 1981), and x-ray fluorescence (XRF) (Olmez

et al., 1985). A combination of methods such as x-ray mapping, field emission scanning

electron microscopy, light microscopy, chemical spot test, thin-layer chromatography, and

Fourier transform infrared spectroscopy allowed the identification of specific accumulation

of Pb derived from smelter particles within the fungal tissues of Acarospora smagardula,

while no Pb was found in the algal zone (Purvis et al., 2000).

Recently, PIXE spectrometry utilizing a focused proton beam has been used to obtain

the distribution of elements within thalli of Xanthoparmelia chlorochroa (Clark et al.,

2001) and Hypogymnia physodes (Budka et al., 2002). In both cases, quantitative, twodimensional element distribution maps for a wide range of elements, including Fe, Ni,

Cu, Zn, and As, have been generated. The distribution of oxalates accompanied by

increased levels of heavy metals was shown, and the evidence for the transfer of inorganic

nutrients across the thalli was presented. In addition, Clark et al. (2001) used SEM and

thermogravimetric analyses to characterize the calcium oxalate region, and provided further evidence for the functional role of the oxalate layer, localized between the medulla

and the algal layer, in regulation of the water and light regimes.



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Turnau and Kottke



HEAVY METAL/MYCORRHIZA INTERACTIONS



The role of mycorrhiza in the uptake and translocation of nutrients has been the subject

of a broad range of investigations demonstrating the improved nutritional status of mycorrhizal plants (Harley and Smith, 1983; Jeffries et al., 2003). In several cases, the

attenuation of the effect of heavy metals on plants has also been shown (for review, see

Gadd, 1993; Hartley et al., 1997; Leyval et al., 1997; Jentschke and Godbold, 2000; Meharg

and Cairney, 2000). Developing restoration techniques requires extensive knowledge of

the function of mycorrhiza. Relatively much interest has been paid to metal sorption by

extraradical mycelia, restricting metal translocation to the host tissues (Jentschke and

Godbold, 2000; Joner et al., 2000). Although metal sorption by mycorrhizal mycelia in

artificial substrates has been well documented, the significance of this phenomenon under

field conditions has not yet been investigated.

Most of the data concerning metal distribution originate from ectomycorrhizal fungi.

The individual ectomycorrhizas interact with the soil solution that might contain potentially

toxic compounds, and the ability of the fungal mantle to act as a barrier for their penetration

is important for root protection. The estimation of the metal content by conventional

methods suggests differences in the efficiency of ligand production and heavy metal

immobilization among fungal species (Berthelsen et al., 1995; Kottke et al., 1998). Studies

involving microanalysis carried out on freeze-dried (Turnau et al., 2001b, 2002) and

chemically fixed (Turnau et al., 1996) material have confirmed the diversity among different types of mycorrhizas and demonstrated not only the presence of heavy metals within

polysaccharides and pigments of the surface layer of fungal mantle, but also the occurrence

of the filtering effect in mycorrhizas such as Rhizopogon roseolus and Suillus luteus

associated with Pinus sylvestris. Maps of elemental distribution of freeze-dried mycorrhizas of S. luteus, obtained with micro-PIXE, presented quantitative data of metal accumulation. Mycorrhizas of both species are hydrophobic. It is therefore interesting to find out

how the metals are able to enter the fungal sheath. Research carried out on Pisolithus

tinctorius/Eucalyptus pilularis mycorrhizas, in which a fluorochrome, 8-hydroxypyrene1,3,6-trisulphonate (PTS), and lanthanum were traced by x-ray microanalysis, showed that

despite the presence of hydrophobins, both the fluorochrome and lanthanum were able to

penetrate the mycorrhizas (Vesk et al., 2000). The filtering capacity of R. roseolus and S.

luteus makes them potentially useful for the restoration of industrial wastes. However,

further studies are needed to show whether they can indeed be used under field conditions.

Both mycorrhizas usually do not make up the most common morphotypes. Although they

form very abundant fruit bodies, they are often outcompeted by fungi, which are less

effective in metal filtering (Turnau et al., 2002).

Heavy metal distribution has also been studied within endomycorrhizas. The first

attempt was done on chemically fixed Pteridium aquilinum roots (Turnau et al., 1993b).

Heavy metals were detected within the arbuscules. This was followed by observations of

freeze-dried arbuscular mycorrhizas of Plantago lanceolata (Orowska et al., 2002) and

freeze-substituted roots of Zea mays (Kaldorf et al., 1999). In the last case, cryosections

were studied by laser microprobe and EDS analysis. Secondary ion mass spectrometry

has been used to generate data on the distribution of elements within mycorrhizal and

nonmycorrhizal roots of maize. The Glomus strain used for inoculation was isolated from

Viola calaminaria and has been shown to alleviate metal toxicity to the host (Hildebrandt

et al., 1999). The maps clearly showed the accumulation of Ni, Zn, and Fe within cortical

cells in which the arbuscules are usually formed. Furthermore, fewer heavy metals were

found within the stele than in the cortex, again suggesting a filtering effect. Similar results

have been obtained from roots of P. lanceolata cultivated on industrial wastes examined