**1.2.2 Study design**

282 Radioisotopes – Applications in Physical Sciences

bioavailable fraction of soils within forest ecosystems is reported Karadeniz & Yaprak (2007) but in cultivated soils, equilibrium between fallout 137Cs and stable 133Cs among exchangeable, organic bound and strongly bound fractions has not reached, even though

The important roles fungi play in nutrient uptake in forest soils, in particular its role in 137Cs transfer between soil and fungi, requires better understanding of the mechanisms involved. Although transfer of radioactive cesium from soils to plants through fungi is well researched, there is still limited knowledge on natural stable 133Cs and other alkali metals (K and Rb) and the potential role as a predictor for radiocesium behavior, and less is known about the

To explore mechanisms governing the uptake of radionuclides (137Cs) data on uptake of stable isotopes of alkali metals (K, Rb, 133Cs) by fungal species, and the behavior of the three alkali metals K, Rb and 133Cs in bulk soil, fungal mycelium and sporocarps are required. Therefore, an attempt was made to quantify the uptake and distribution of the alkali metals in the soil–mycelium–sporocarp compartments and to study the relationships between K, Rb and 133Cs in the various transfer steps. Additionally, the sporocarps of ectomycorrhizal fungi *Suillus variegatus* were analyzed to determine whether i) Cs (133Cs and 137Cs) uptake was correlated with K uptake; ii) intraspecific correlation of these alkali metals and 137Cs activity concentrations in sporocarps was higher within, rather than among different fungal

most 137Cs was deposited on the soils more than 20 years before (Tsukada, 2006).

relationships between 133Cs and other alkali metals (K and Rb) during uptake by fungi.

species; and, iii) the genotypic origin of sporocarps affected uptake and correlation.

al., 2004; 2010a; 2010b; 2011).

methods used is presented (section 1.2).

**1.2.1 Study area** 

**1.2 Study area, study design and methods for results presented** 

Substantial research in this area has been conducted in Sweden after the fallout from nuclear weapons tests and the Chernobyl accident. Some results are published in a series of several articles in collaboration with Profs K.J. Johanson, H. Rydin and Dr. A. Taylor (Vinichuk et

This chapter aims to summarize the acquired knowledge from studies in Sweden and to place them in a larger context. The results are summarized and discussed and address the issues of K, Rb and 133Cs concentrations in soil fractions and fungal compartments (Section 1.3.); concentration ratios of K, Rb and 133Cs in soil fractions and fungi (Section 1.4); relationships between K, Rb and 133Cs in soil and fungi (Section 1.5); the isotopic (atom) ratios 137Cs/K, 137Cs/Rb and 137Cs/133Cs in fungal species (Section 1.6); K, Rb and Cs (137Cs and 133Cs) in sporocarps of a single species (Section 1.7); mechanisms of 137Cs and alkali metal uptake by fungi (Section 1.8); Cs (137Cs and 133Cs), K and Rb in *Sphagnum* plants (Section 2); distribution of Cs (137Cs and 133Cs), K and Rb within *Sphagnum* plants (Section 2.3); mass concentration and isotopic (atom) ratios between 137Cs, K, Rb and 133Cs in segments of *Sphagnum* plants (Section 2.4); relationships between 137Cs, K, Rb and 133Cs, in segments of *Sphagnum* plants (Section 2.5); mechanisms of 137Cs and alkali metal uptake by *Sphagnum* plants (Section 2.6); and conclusions from the Swedish studies (Section 3). Before presenting and discussing results a short description of study area, study design and

The K, Rb and 133Cs concentrations in soil fractions and fungal compartments were studied in an area located in a forest ecosystem on the east coast of central Sweden (60°22′N, 18°13′E). The soil was a sandy or clayey till and the humus mainly occurred in the form of mull. A more detailed description of the study area is presented by Vinichuk et al. (2010b).

For studies of K, Rb and 133Cs concentrations in soil fractions and fungal compartments, samples of soil and fungal sporocarps were collected from 10 sampling plots during September to November 2003. Four replicate soil samples were taken, with a cylindrical steel tube with a diameter of 5.7 cm, from around and directly underneath the fungal sporocarps (an area of about 0.5 m2) and within each 10 m2 area to a depth of 10 cm. Soil cores were divided horizontally into two 5-cm thick layers. Sporocarps of 12 different fungal species were collected and identified to species level, and the 137Cs activity concentration in fresh material was determined. The sporocarps were dried at 35°C to constant weight and concentrations of 133Cs, K and Rb were determined.

A selection of dried sporocarps of *S. variegatus* (n*=*51), retained from a study by Dahlberg et al. (1997) on the relationship between 137Cs activity concentrations and genotype identification, was used. The sporocarps were collected once a week during sporocarp season (end of August through September) in 1994 and were taken from five sampling sites (100 to 1600 m2 in size) within an area of about 1 km2. Eight genotypes with 2 to 8 sporocarps each were tested (in total 32 sporocarps) and are referred to here as individual genotypes. Sporocarps within genotypes were spatially separated by up to 10-12m. All genotypes were used for the estimation of correlation coefficients, but only genotypes with at least four sporocarps were included in the alkali metal analysis. In addition, 19 individual sporocarps with unknown genotype (i.e. not tested for genotype identity) were included: these sporocarps consisted of both the same and different genotypes. The combined set of sporocarps refers to all sporocarps: for further details about the sampling and identification of genotypes see Dahlberg et al. (1997). The 137Cs activity concentration values corrected to sampling date and expressed as kBq kg−1 dry weight (DW) for each sporocarp, as reported by Dahlberg et al. (1997), were used.

#### **1.2.3 Methods**

For the studies of K, Rb and 133Cs concentrations in soil fractions and fungal compartments, fungal mycelia were separated from the soil samples (30–50 g, 0–5 cm layer depth) under a dissection microscope (magnification X64) with forceps and by adding small amounts of distilled water to disperse the soil. The prepared fraction of mycelium (30−60 mg DW g–1 soil) was not identified to determine of the mycelia extracted from the soil samples and the sporocarps belonged to the same species, as it assumed a majority of the prepared mycelia belonged to the same species as the nearby sporocarps. The method for mycelium preparation is described in Vinichuk & Johanson (2003). Mycelium samples were dried at 35°C to constant weight for determination of K, Rb and 133Cs.

Cesium (137Cs and 133Cs), Potassium

fractions and fungi1.

about 7-fold higher (Table 1).

found in soil mycelium.

and Rubidium in Macromycete Fungi and *Sphagnum* Plants 285

K 642.6 (214.6)a 899.3 (301.4)a 3215 (842.8)b 2 867(727.5)b 43 415 (20 436)b Rb 3.9 (2.7)a 5.4 (4.4)a 6.8 (1.7)a 13.8 (6.9)b 253.9 (273.6)b 133Cs 0.3 (0.2)a 0.4 (0.3)a 0.2 (0.05)a 0.8 (0.8) a 5.65 (7.1)b

Table 1. Mean concentrations of K, Rb and 133Cs (mg kg−1 DW (standard deviation)) in soil

Potassium concentrations were higher in both the soil–root interface and fungal mycelium fractions than in the bulk soil and rhizosphere fraction. A comparison of K, Rb and 133Cs concentrations revealed fungal sporocarps accumulated much greater amounts of these elements than mycelium. For example, K concentrations in fungal sporocarps collected from the same plots where soil samples and mycelium were extracted were about 15 times higher than K concentrations found in mycelium. The concentrations of Rb in fungal sporocarps were about 18-fold higher than in corresponding fungal mycelium, and those of 133Cs were

Thus, potassium concentration increased in the order bulk soil<rhizosphere<fungal mycelium<soil–root interface<fungal sporocarps and was higher in the soil–root interface fraction and fungi than in bulk soil. The high concentrations of K in fungal sporocarps may reflect a demand for this element as a major cation in osmoregulation and that K is an important

Rb in mycelium was 3.5-fold higher than in bulk soil and 2.5-fold higher than in rhizosphere, and concentrations increased in the order bulk soil<rhizosphere<soil–root interface<fungal mycelium<fungal sporocarps. The concentrations of Rb were slightly higher in the soil–root interface fraction than in bulk soil; thus, fungi appeared to have high preference for this element, as the accumulation of Rb by fungi, and especially fungal sporocarps, was pronounced. Rubidium concentrations in sporocarps were more than one order of magnitude higher than those in mycelium extracted from soil of the same plots where fungal sporocarps were sampled. The ability of fungi to accumulate Rb is documented: mushrooms accumulate at least one order of magnitude higher concentrations

Concentrations of stable cesium varied considerably among samples but no significant differences were found among the different fractions analyzed. Cesium concentrations increased in the order soil–root interface<bulk soil<rhizosphere<fungal mycelium<fungal sporocarps, and were only significantly higher in fungal sporocarps, compared with bulk soil. Stable 133Cs was generally evenly distributed within bulk soil, rhizosphere and soil–root interface fractions, indicating no 133Cs enrichment in those forest compartments. However, 133Cs concentrations in sporocarps were nearly one order of magnitude higher than those

Radioactive 137Cs presented similar to 133Cs behavior, where 137Cs activity increased in the order soil<mycelium<fungal sporocarps (Vinichuk & Johanson, 2003; Vinichuk et al., 2004). The differences between fungal species in their preferences for uptake of 137Cs or stable 133Cs appear to reflect the location of the fungal mycelium relative to that of cesium within the soil profile (Rühm et al., 1997). Unlike 137Cs, stable 133Cs originates from soil; therefore, the

element in regulating the productivity of sporophore formation in fungi (Tyler, 1982).

of Rb than plants growing in the same forest (Yoshida & Muramatsu, 1998).

Element Bulk soil Rhizosphere Soil root-interface Fungal mycelium Fruit bodies

1Means within rows with different letters (a or b) are significantly different (p < 0.001).

The soil samples (0–5 cm layer) were partitioned by the method described in Gorban & Clegg (1996). First, soil was gently sieved through a 2 mm mesh giving a bulk soil fraction. The remaining soil aggregates containing roots were further crumbled and gently squeezed between the fingers: this was called the rhizosphere fraction. The residue (finest roots with adhering soil particles) was called the soil–root interface fraction. Nine samples of bulk soil fraction and mycelium, 12 samples of fungal sporocarps, and six samples of rhizosphere and soil–root interface fraction were analyzed for K, Rb and 133Cs.

The 137Cs activity concentrations in the bulk soil samples and sporocarps were determined with calibrated HP-Ge detectors, corrected to sampling date and expressed as Bq kg−1 DW. The measuring time employed provided a statistical error ranging between 5 and 10%. For element analyses, a 2.5 g portion of each sample was analyzed by inductively coupled plasma in the laboratories of ALS Scandinavia (Luleå, Sweden) with recoveries 97–101% for K; 97.5–99.4% for Rb, and 93.7–102.5% for, 133Cs. For soil, CRM SO-2 (heavy metals in soil) was used which had no certified values for K, Rb or 133Cs. Element concentrations in the analyzed fractions are reported as mg kg−1 DW.

For element analyses (K, Rb and 133Cs) of *S. variegatus* sporocarps, aliquots of about 0.3 g of each sample were analyzed by the same technique. Element concentrations are reported as mg kg<sup>−</sup>1 DW and the isotopic ratio of 137Cs/133Cs was calculated with Equations 1 and 2 (Chao et al., 2008):

$$\frac{\lambda^{137}\text{Cs}}{\lambda^{133}\text{Cs}} = \frac{A}{\mathcal{C}} \times \frac{a}{\lambda \times \mathcal{N}} \times 10^3\tag{1}$$

where: A is the 137Cs radioactivity (Bq kg−1); λ is the disintegration rate of 137Cs 7.25 x10−<sup>10</sup> s−1; a is the atomic weight of cesium (132.9); N is the Avogadro number, which is 6.02 x1023; and, 133C and C are the 133Cs concentration (mg g−1). Eq. (1) can be simplified to Eq. 2:

$$\frac{1^{137}\text{Cs}}{1^{133}\text{Cs}} = 3.05 \times 10^{-10} \times \frac{A}{c} \tag{2}$$

where: A is the 137Cs activity concentration in Bq kg−1 and 133C is the 133Cs concentration in mg kg−1. Thus, the units of the isotope ratio are dimensionless.

Relationships between K, Rb, 133Cs and 137Cs concentrations in different fractions and sporocarps of *S. variegatus* were identified by Pearson correlation coefficients. Correlation coefficients were analyzed in five separate sets of samples: in four sets, all samples had known genotype identity, and in the last set, there was a combined set of samples containing both genotypes that had been tested by somatic incompatibility sporocarps and genotypes that had not been tested. Correlation analyses for genotypes with three or less sporocarps were omitted. All statistical analyses were run with Minitab® 15.1.1.0. (© 2007 Minitab Inc.) software, with level of significance of 5% (0.05), 1% (0.01) and 0.1% (0.001).

#### **1.3 K, Rb and 133Cs concentrations in soil fractions and fungal compartments**

K, Rb and 133Cs concentrations values in soil fractions and fungal compartments are necessary for calculating the concentration ratio at each step of its transfer in the soil-fungi system, differences in the uptake between elements and the relationships. This in turn will be the main reason for the different K, Rb and 133Cs concentrations observed in sporocarps of various fungal species. Concentrations of K, Rb and 133Cs in bulk soil were not significantly different from those in the rhizosphere, although the values for all three elements were slightly higher in the rhizosphere fraction (Table 1).

#### Cesium (137Cs and 133Cs), Potassium and Rubidium in Macromycete Fungi and *Sphagnum* Plants 285

284 Radioisotopes – Applications in Physical Sciences

The soil samples (0–5 cm layer) were partitioned by the method described in Gorban & Clegg (1996). First, soil was gently sieved through a 2 mm mesh giving a bulk soil fraction. The remaining soil aggregates containing roots were further crumbled and gently squeezed between the fingers: this was called the rhizosphere fraction. The residue (finest roots with adhering soil particles) was called the soil–root interface fraction. Nine samples of bulk soil fraction and mycelium, 12 samples of fungal sporocarps, and six samples of rhizosphere and

The 137Cs activity concentrations in the bulk soil samples and sporocarps were determined with calibrated HP-Ge detectors, corrected to sampling date and expressed as Bq kg−1 DW. The measuring time employed provided a statistical error ranging between 5 and 10%. For element analyses, a 2.5 g portion of each sample was analyzed by inductively coupled plasma in the laboratories of ALS Scandinavia (Luleå, Sweden) with recoveries 97–101% for K; 97.5–99.4% for Rb, and 93.7–102.5% for, 133Cs. For soil, CRM SO-2 (heavy metals in soil) was used which had no certified values for K, Rb or 133Cs. Element concentrations in the

For element analyses (K, Rb and 133Cs) of *S. variegatus* sporocarps, aliquots of about 0.3 g of each sample were analyzed by the same technique. Element concentrations are reported as mg kg<sup>−</sup>1 DW and the isotopic ratio of 137Cs/133Cs was calculated with Equations 1 and 2

where: A is the 137Cs radioactivity (Bq kg−1); λ is the disintegration rate of 137Cs 7.25 x10−<sup>10</sup> s−1; a is the atomic weight of cesium (132.9); N is the Avogadro number, which is 6.02 x1023;

�� ��� = 3.05 × 10��� <sup>×</sup>

where: A is the 137Cs activity concentration in Bq kg−1 and 133C is the 133Cs concentration in

Relationships between K, Rb, 133Cs and 137Cs concentrations in different fractions and sporocarps of *S. variegatus* were identified by Pearson correlation coefficients. Correlation coefficients were analyzed in five separate sets of samples: in four sets, all samples had known genotype identity, and in the last set, there was a combined set of samples containing both genotypes that had been tested by somatic incompatibility sporocarps and genotypes that had not been tested. Correlation analyses for genotypes with three or less sporocarps were omitted. All statistical analyses were run with Minitab® 15.1.1.0. (© 2007 Minitab Inc.) software, with level of significance of 5% (0.05), 1% (0.01) and 0.1% (0.001).

� �

and, 133C and C are the 133Cs concentration (mg g−1). Eq. (1) can be simplified to Eq. 2:

**1.3 K, Rb and 133Cs concentrations in soil fractions and fungal compartments** 

K, Rb and 133Cs concentrations values in soil fractions and fungal compartments are necessary for calculating the concentration ratio at each step of its transfer in the soil-fungi system, differences in the uptake between elements and the relationships. This in turn will be the main reason for the different K, Rb and 133Cs concentrations observed in sporocarps of various fungal species. Concentrations of K, Rb and 133Cs in bulk soil were not significantly different from those in the rhizosphere, although the values for all three

�� × 10� (1)

(2)

�� ��� �� ��� <sup>=</sup> � � <sup>×</sup> �

�� ���

mg kg−1. Thus, the units of the isotope ratio are dimensionless.

elements were slightly higher in the rhizosphere fraction (Table 1).

soil–root interface fraction were analyzed for K, Rb and 133Cs.

analyzed fractions are reported as mg kg−1 DW.

(Chao et al., 2008):


1Means within rows with different letters (a or b) are significantly different (p < 0.001).

Table 1. Mean concentrations of K, Rb and 133Cs (mg kg−1 DW (standard deviation)) in soil fractions and fungi1.

Potassium concentrations were higher in both the soil–root interface and fungal mycelium fractions than in the bulk soil and rhizosphere fraction. A comparison of K, Rb and 133Cs concentrations revealed fungal sporocarps accumulated much greater amounts of these elements than mycelium. For example, K concentrations in fungal sporocarps collected from the same plots where soil samples and mycelium were extracted were about 15 times higher than K concentrations found in mycelium. The concentrations of Rb in fungal sporocarps were about 18-fold higher than in corresponding fungal mycelium, and those of 133Cs were about 7-fold higher (Table 1).

Thus, potassium concentration increased in the order bulk soil<rhizosphere<fungal mycelium<soil–root interface<fungal sporocarps and was higher in the soil–root interface fraction and fungi than in bulk soil. The high concentrations of K in fungal sporocarps may reflect a demand for this element as a major cation in osmoregulation and that K is an important element in regulating the productivity of sporophore formation in fungi (Tyler, 1982).

Rb in mycelium was 3.5-fold higher than in bulk soil and 2.5-fold higher than in rhizosphere, and concentrations increased in the order bulk soil<rhizosphere<soil–root interface<fungal mycelium<fungal sporocarps. The concentrations of Rb were slightly higher in the soil–root interface fraction than in bulk soil; thus, fungi appeared to have high preference for this element, as the accumulation of Rb by fungi, and especially fungal sporocarps, was pronounced. Rubidium concentrations in sporocarps were more than one order of magnitude higher than those in mycelium extracted from soil of the same plots where fungal sporocarps were sampled. The ability of fungi to accumulate Rb is documented: mushrooms accumulate at least one order of magnitude higher concentrations of Rb than plants growing in the same forest (Yoshida & Muramatsu, 1998).

Concentrations of stable cesium varied considerably among samples but no significant differences were found among the different fractions analyzed. Cesium concentrations increased in the order soil–root interface<bulk soil<rhizosphere<fungal mycelium<fungal sporocarps, and were only significantly higher in fungal sporocarps, compared with bulk soil. Stable 133Cs was generally evenly distributed within bulk soil, rhizosphere and soil–root interface fractions, indicating no 133Cs enrichment in those forest compartments. However, 133Cs concentrations in sporocarps were nearly one order of magnitude higher than those found in soil mycelium.

Radioactive 137Cs presented similar to 133Cs behavior, where 137Cs activity increased in the order soil<mycelium<fungal sporocarps (Vinichuk & Johanson, 2003; Vinichuk et al., 2004). The differences between fungal species in their preferences for uptake of 137Cs or stable 133Cs appear to reflect the location of the fungal mycelium relative to that of cesium within the soil profile (Rühm et al., 1997). Unlike 137Cs, stable 133Cs originates from soil; therefore, the

Cesium (137Cs and 133Cs), Potassium

Plot Species

fungi for fungal sporocarps.

not significant (r=0.602, ns: Figure1b).

and Rubidium in Macromycete Fungi and *Sphagnum* Plants 287

*Boletus edulis* 62.7 77.4 37.4 *Cantharellus tubaeformis* 104.7 109.7 15.5 *Cortinarius armeniacus* 67.5 69.6 19.2 *C. odorifer* 71.8 70.9 34.7 *C. spp.* 90.9 157.2 14.8 8-10 *Hypholoma capnoides1* 26.6 13.1 6.9 *Lactarius deterrimus* 29.9 17.2 2.6 *L. scrobiculatus* 67.8 26.2 3.7 *L. trivialis* 77.5 126.9 52.2 5-7 *Sarcodon imbricatus* 101.7 675.7 258.8 *Suillus granulates* 58.6 41.4 14.7 10-11 *Tricholoma equestre* 66.6 75.4 15.4

Table 3. Element concentration ratios (mg kg−1 DW in fungi)/(mg kg−1 DW in bulk soil) in

Although correlation analysis may be not definitive, it is a useful approach for elucidating similarities or differences in uptake mechanisms of cesium (137Cs and 133Cs), K and Rb: close correlation between elements indicates similarities in their uptake mechanisms. No significant correlations between K in soil and in either mycelium (r=0.452, ns) or in sporocarps (r=0.338, ns) has been identified and sporocarp Rb and 133Cs concentrations were unrelated to soil concentrations, however, in mycelium both elements were correlated with soil concentrations (Rb: r=0.856, p=0.003; Cs: r=0.804, p=0.009). There was a close positive correlation (r=0.946, p=0.001) between the K:Rb ratio in soil and in fungal mycelium (Figure 1b) and this relationship was also apparent between soil and sporocarps, but was weak and

The K:133Cs ratio in soil and fungal components had a different pattern: the K:Cs ratio in mycelium was closely positively correlated (r=0.883, p=0.01) to the K:133Cs ratio in soil (Figure 1a), but was relatively weakly and non-significantly correlated to soil in fungal sporocarps. No significant correlations were found between the concentrations of the three

The competition between K, Rb and 133Cs in the various transfer steps was investigated in an attempt to estimate the relationships between the concentrations of these three elements in soil, mycelia and fungal sporocarps. The lack of a significant correlation between K in soil and in either mycelium or sporocarps indicated a demand for essential K in fungi, regardless of the concentration of this element in soil. Regardless of fungal species, K concentration in fungi appears to be controlled within a narrow range, (Yoshida & Muramatsu, 1998), and supports the claim K uptake by fungi is self-regulated by the internal

elements in fungi, soil pH or soil organic matter content (data not shown).

nutritional requirements of the fungus (Baeza et al., 2004).

1Saprophyte, all other analyzed fungal species are ectomycorrhizal

**1.5 Relationships between K, Rb and 133Cs in soil and fungi** 

Concentration ratios

K Rb 133Cs

amount of unavailable 133Cs, compared to the total amount of 133Cs, in soil presumably higher than that of 137Cs. As a result, stable 133Cs is considered less available for uptake as it is contained in mineral compounds and is difficult for fungi or plants to access: the concentration ratio of stable 133Cs in mushrooms is lower than for 137Cs (Yoshida & Muramatsu, 1998). The differing behavior of the natural and radioactive forms of 133Cs may derive from their disequilibrium in the ecosystem (Horyna & Řanad, 1988).
