Scholarly article on topic 'Arbuscular mycorrhiza of Deschampsia cespitosa (Poaceae) at different soil depths in highly metal-contaminated site in southern Poland'

Arbuscular mycorrhiza of Deschampsia cespitosa (Poaceae) at different soil depths in highly metal-contaminated site in southern Poland Academic research paper on "Biological sciences"

CC BY
0
0
Share paper
Academic journal
Acta Societatis Botanicorum Poloniae
OECD Field of science
Keywords
{""}

Academic research paper on topic "Arbuscular mycorrhiza of Deschampsia cespitosa (Poaceae) at different soil depths in highly metal-contaminated site in southern Poland"

Acta Societatis Botanicorum Poloniae

Journal homepage: pbsociety.org.pl/journals/index.php/asbp

ORIGINAL RESEARCH PAPER Received: 2013.06.18 Accepted: 2013.11.22 Published electronically: 2013.12.19 Acta Soc Bot Pol 82(4):251-258 DOI: 10.5586/asbp.2013.033

Arbuscular mycorrhiza of Deschampsia cespitosa (Poaceae) at different soil depths in highly metal-contaminated site in southern Poland

Ewa Gucwa-Przepióra1*, Janusz Btaszkowski2, Renata Kurtyka3, tukasz Matkowski1, Eugeniusz Matkowski3

1 Department of Plant Systematics, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland

2 Department of Plant Pathology, West Pomeranian University of Technology, Stowackiego 17, 71-434 Szczecin, Poland

3 Department of Plant Physiology, University of Silesia, Jagiellonska 28, 40-032 Katowice, Poland

Abstract

This study presents root colonization of Deschampsia cespitosa growing in the immediate vicinity of a former Pb/Zn smelter by arbuscular mycorhizal fungi (AMF) and dark septated endophytes (DSE) at different soil depths. AMF spores and species distribution in soil profile were also assessed. Arbuscular mycorrhiza (AM) and DSE were found in D. cespitosa roots at all investigated soil levels. However, mycorrhizal colonization in topsoil was extremely low with sporadically occurring arbuscules. AM parameters: frequency of mycorrhization of root fragments (F%), intensity of root cortex colonization (M%), intensity of colonization within individual mycorrhizal roots (m%), and arbuscule abundance in the root system (A%) were markedly higher at 20-40, 40-60 cm soil levels and differed in a statistically significant manner from AM parameters from 0-10 and 10-20 cm layers. Mycorrhizal colonization was negatively correlated with bioavailable Cd, Pb and Zn concentrations. The number of AMF spores in topsoil was very low and increased with soil depth (20-40 and 40-60 cm). At the study area spores of three morphologically distinctive AMF species were found: Archaeospora trappei, Funneliformis mosseae and Scutellospora dipurpurescens. The fourth species Glomus tenue colonized roots of D. cespitosa and was observed in the root cortex at 20-40 and 40-60 soil depth, however, its spores were not found at the site.

Keywords: arbuscular mycorrhiza (AM), soil depth, heavy metals, Glomeromycota, DSE, grasses, Glomus tenue

Introduction

Arbuscular mycorrhizal fungi (AMF) are a natural constituent of soil in most ecosystems. It has been estimated that over 80% of all vascular plants form arbuscular mycorrhizas [1]. AMF have a positive effect on plant mineral nutrition and increase tolerance of plants to drought, heavy metals and other environmental stress factors [2-5]. These fungi are a crucial contributor to the formation and maintenance of soil structure as well as constitute a significant component of soil organic matter [6].

Heavy metal contamination of soil is a worldwide problem that affects large number of sites [7-10]. Particularly high concentrations of Cd, Pb, Zn were recorded in soils contaminated by nonferrous mining and smelting activities [11-13]. At such sites in Silesia, an industrial region of southern Poland, total

* Corresponding author. Email: ewa.gucwa-przepiora@us.edu.pl Handling Editor: Aleksandra Samecka-Cymerman

This is an Open Access digital version of the article distributed under the terms of the Creative Commons Attribution 3.0 License (creativecommons.org/licenses/by/3.0/), which permits redistribution, commercial and non-commercial, provided that the article is properly cited.

concentration of Cd, Pb and Zn in soil exceeds 500, 14000, 12000 mg kg-1 respectively [14-16].

A very high concentration of these metals in soil results in poor development of natural vegetation [17] and has a negative influence on the development of AMF [18-20]. Nevertheless, mycorrhizal plant species, the most common of which are grasses, are capable of growing on highly contaminated soils, e.g. Agropyron repens [21,22], Festuca ovina sensu lato [23] or Deschampsia cespitosa [16,17,22,24]. These mycorrhizal plants and the associated with their roots AM fungi play an important part in areas affected by industry, such as former mining areas or waste sites, since they are able to accelerate their revegetation [25-29].

Most mycorrhizal studies are generally restricted to the main rooting zone (0-20 cm soil depth) [30-32]. There are a few studies where AMF have been examined at different soil levels. It was found that the percentage of roots colonized by AM fungi [33-35], the amount of their extraradical hyphae [36] as well as their spore abundance and species richness decreased [37] with increasing soil depth. However, there is a dearth of data on vertical distribution of AM at the sites contaminated with heavy metals.

Recently Gucwa-Przepiora et al. [16] studied the effect of phytostabilization practices on the AM development in the roots of Deschampsia cespitosa (L.) PB in soils contaminated by nonferrous mining and smelting activities. In addition, they investigated the colonization at different soil depths. They

demonstrated that mycorrhizal colonization was very limited in the upper soil layers, where bioavailable concentrations of Cd, Pb and Zn were high, and increased with soil depth. In contrast, in non-metal contaminated soils a decrease in mycorrhizal colonization with soil depth was observed [33-35].

In this paper we focused on evaluating the effect of soil depth on AMF colonization parameters in Deschampsia cespitosa roots growing spontaneously in the immediate vicinity of the former Zn/Pb smelter, where interactions among heavy metals (Zn, Pb and Cd) and soil constituents were long-lasting. The abundance of AMF spores and AMF species in soil were also investigated. In addition the colonization of roots by dark septated endophytes (DSE) was assessed.

Material and methods

Site description

The experimental site is located at a former mine and smelter area, situated between the towns Bytom and Piekary Sl^skie, in the Upper Silesia industrial region of southern Poland (N 50°21'59.64" E 18°58'17,90"). The mine and smelter operated for approximately 70 years. In 1989 the production stopped, all the facilities were closed down and dismantled. The study area, where D. cespitosa grew spontaneously in large numbers is located in the immediate vicinity of the former smelter.

Collection of soil and root samples

On 3 August 2005 root samples of D. cespitosa were taken from the investigated area in order to check its mycorrhizal status and to examine the vertical distribution of AM development. An approx. 70 cm deep trench was dug along three large D. cespitosa specimens. Four blocks of soil approx. 1 m long and 15 cm wide containing roots of D. cespitosa were taken, each at different soil level: 0-10, 10-20, 20-40 and 40-60 cm. From a single block of soil four samples of plant roots for mycorrhizal studies were collected. From the same block of soil also three samples were taken to conduct chemical analyses of soil and to determine arbuscular fungi spores and species. At the laboratory the soil samples were air-dried and sieved through 2 mm screen for chemical analyses and for identification of spores.

Mycorrhizal studies

EVALUATION OF ROOT COLONIZATION PARAMETERS. For the estimation of mycorrhizal development D. cespitosa roots were prepared according to the modified method of Phillips and Hayman [38]. After careful washing with tap water, the roots were softened in 7% KOH for 24 h, washed in water, acidified in 5% lactic acid in water for 1-24 h, and stained with 0.01% aniline blue in 5% lactic acid for 24 h at room temperature. The stained roots were stored in lactoglycerol until they were used for slide preparation. Five parameters of mycorrhizal colonization were evaluated microscopically using thirty 1-cm root fragments per sample and calculated as percentages: frequency of mycorrhization of root fragments (F%), intensity of root cortex colonization (M%), intensity of colonization within individual mycorrhizal roots (m%), arbuscule abundance in the root system (A%) and arbuscule richness in root fragments where the arbuscules were present (a%; http://www.dijon.inra. fr/mychintec/Mycocalc-prg/downlo ad.html) [39].

ISOLATION AND IDENTIFICATION OF AMF SPORES. AMF spores were extracted from 50 g of sieved (<2 mm)

air-dried soil from rooting zone by wet sieving and decanting [40]. Morphological properties of spores and their subcellular structures were determined on spores mounted in polyvinyl alcohol/lactic acid/glycerol (PVLG) [41] and a mixture of PVLG and Melzer's reagent (1:1, v/v). Spores were crushed to varying degrees by applying pressure to the cover slip and then stored at 65°C for 24 h to clear their contents from oil droplets. These were examined under an Olympus BX 50 compound microscope equipped with Normarski differential interference contrast optics [42].

Soil analyses

Physical and chemical properties of the soil samples collected from four levels were analyzed. Soil texture was determined by hydrometer method developed by Proszynski [43]. Soil pH (H2O) and electrical conductivity (EC) were measured in water suspension (soil to solution ratio 1:2.5) after 24 h of equilibration. Cation exchange capacity [cmol(+)/kg] was determined according to Houba et al. [44]. The fraction of bioavailable metals was obtained by extraction of 3 g of air-dried soil (ground to <0.25 mm) with 30 ml 0.01 M CaCl2 for 5 h; total metal content was determined after extraction of air-dried soil ground to <0.25 mm with concentrated HClO4 and HF. Concentration of metals was analyzed by using flame atomic absorption spectrophotometry (Varian Spectra AA300).

Statistical analysis

Normality tests Kolmogorov-Smirnov with the Lilliefors correction for the mycorrizal colonization and soil parameters data were run. The applied normality tests showed that the distribution of most of the variables was normal. Data, which did not meet test requirements, were transformed. Then data were subjected to one-way ANOVA. Significant differences among mean values were calculated using the LSD test. Pearson's correlation coefficients were calculated between particular mycorrhizal parameter (F%, M%, m%, a%, A%) and the concentration of metals (Cd, Zn, Pb, Cu). The probability level of 0.05 or less value was considered to be statistically significant. The statistical analysis was performed using the computer software Statistica version 7.1 (StatSoft Inc., Tulsa, OK, USA).

Results

Soil properties

The substrate sandy silt loam was characterized by high content of organic matter and relatively low cation exchange capacity (Tab. 1). Soil pH was close to 7.0 in the upper layer and decreased with increasing soil depth with the lowest value of 5.18 in the 40-60 cm layer (Tab. 2). Soil EC was low and did not exceed 130.5 ^S cm-1. Highest EC levels were found in the top 10 cm of soil and they decreased with soil depth. The soil pH showed a similar trend (Tab. 2). The total concentrations of Cd, Pb and Zn were very high in the topsoil (0-10 cm) and decreased with the increasing soil depth. The same trend was observed in concentrations of bioavailable Cd, Pb and Zn (Tab. 2). The concentrations of bioavailable Cd were very high (almost 90 mg kg-1) in the topsoil (0-10 cm) and decreased with the increasing soil depth, being 14-fold lower in the deepest layer (40-60 cm). Statistically significant differences were found in bioavailable Cd concentrations among all investigated soil layers (Tab. 2). The concentrations of bioavailable Zn were

Tab. 1 General properties of the soil.

Property Value

Organic matter content (%) 8.52 ±0.12

Cation exchange capacity [cmol(+)/kg] 6.67 ±0.24 Sand [1-0.05 mm; (%)] 37.3 Silt [0.05-0.002 mm; (%)] 56.3 Clay [< 0.002 mm; (%)] 6.4

Values represent mean of three replicate samples ±SE. Soil was collected from the top 30 cm layer.

also markedly higher in the upper soil layers and differed in a statistically significant manner from the content of the two deeper soil layers. The concentration of bioavailable forms of Pb decreased with soil depth, however, it was very low even in the 0-10 cm layer (7.0 mg kg-1).

AM root colonization

Arbuscular mycorrhiza was found in D. cespitosa roots in all soil levels investigated. However, in topsoil the mycorrhizal colonization was extremely low with sporadically occurring arbuscules (Fig. 1). Vesicles were absent at the 0-10 cm soil level, while in the 10-20 cm layer they were noticed only in one sample. There was no AM coils detected in the roots from two upper soil levels. The relative arbuscule richness (A%) did

not exceeded 3% and the intensity of root cortex colonization (M%) was also very low (below 5%; Fig. 1). On the contrary, D. cespitosa roots from the depth below 20 cm comprised properly developed AM structures, like arbuscules, vesicles and coils. Roots were heavily mycorrhizal, with more than 35% of root cortex colonized by AMF. Almost all AM parameters (F%, M%, m%, and A%) increased considerably and were markedly higher at the 20-40, 40-60 cm soil levels when compared to the upper levels (0-10, 10-20 cm). These differences were statistically significant (Fig. 1).

Coarse AMF (mycelium above 2 ^m diameter) dominated in D. cespitosa roots. The fine endophyte, usually considered Glomus tenue (Greenall) I. R. Hall, characterized by mycelium of ca. 1 ^m diameter, was observed in the Deschampsia root cortex only in the two deeper layers (20-40 and 40-60 cm) where it formed well-shaped arbuscules (Fig. 2).

Dark septated endophytes were found in all soil levels investigated. The hyphae of DSE could be readily distinguished from AM hyphae by their dark brown colour, thicker lateral wall, and frequent septa. However they were not abundantly developed. The regularly septated hyphae were sporadically accompanied by sclerotia. The mycelium did not stain with aniline blue and remained brownish. DSE were observed in the cortex together with AMF but mainly in the root fragments where arbuscules were absent.

Relationships between AM colonization and soil properties

The interrelations between the mycorrhizal parameters (F%, M%, m%, a%, A%) and bioavailable metal concentrations, total

Tab. 2 Chemical and physical soil properties in different soil layers.

Soil depth Bioavailable metal concentrations (mg kg ') Total metal concentrations (mg kg ')

(cm) EC (^S cm-1) pH (H2O) Cd Zn Pb Cd Zn Pb

0-10 121.1 ±4.21 a 6.47 ±0.026 a 89.89 ±1.745 a 598.7 ±32.3 a 7.03 ±0.37 a 598.75 ±125.7 a 7770.8 ±2276 a 9785.4 ±2422 a

10-20 66.7 ±3.46 b 6.23 ±0.005 a 55.61 ±2.735 b 463.0 ±24.95 c 1.39 ±0.12 ac 155.85 ±39.20 a 2049.8 ±469 b 1719.58 ±428 b

20-40 45.5 ±4.21 c 5.66 ±0.150 b 13.00 ±0.46 c 167.3 ±11.75 b BDL b 22.38 ±12.93 b 359.5 ±91.9 b 128.21 ±17.8 b

40-60 42.3 ±1.09 c 5.18 ±0.049 c 6.42 ± 0.455 d 117.0 ± 4.03 b 0.46 ±0 07 bc 9.53 ±4.09 b 201.2 ±61.8 b 85.10 ±14.56 b

Values are means ±SE (n = 3, except for bioavailable metal concentration where n = 4). Means followed by the same letter in a column are not significantly different from each other using LSD test (P < 0.05). BDL - below detection limit.

Tab. 3 Pearson's correlation coefficients between mycorrhizal parameters and soil properties.

Correlation coefficient

Mycorrhizal Bioavailable metal concentration (mg kg-1) Total metal concentration (mg kg-1)

parameters Cd Zn Pb Cd Zn Pb pH (H2O) EC (^S cm-1)

F% -0.79* -0.81* -0.80* -0.80* -0.76* -0.53 -0.74* -0.83*

M% -0.83* -0.88* -0.70* -0.68* -0.64* -0.63* -0.80* -0.75*

m% -0.27 -0.45 -0.31 -0.72* -0.67* -0.66* -0.77* -0.76*

a% -0.07 -0.20 -0.16 -0.67* -0.61* -0.60* -0.89* -0.75*

A% -0.75* -0.82* -0.62* -0.36 -0.30 -0.01 -0.46 -0.36

a% - arbuscule richness in root fragments where the arbuscules were present; A% - arbuscule abundance in the root system; F% - frequency of mycorrhization; m% - intensity of colonisation within individual mycorrhizal roots; M% - intensity of root cortex colonization. * Correlation coefficient was statistically significant at P < 0.05.

Fig. 1 Differences in frequency of mycorrhization (F%), intensity of root cortex colonization (M%) intensity of colonisation within individual mycorrhizal roots (m%), arbuscule abundance in the root system (A%) and arbuscule richness in root fragments where the arbuscules were present (a%) in Deschampsia cespitosa roots at different soil depth. Values are means (n = 4). Means followed by different letters are significantly different from each other using LSD test (P < 0.05). Lack of letters denotes a lack of statistical differences among the means.

metal concentrations, the soil pH (H2O) or EC were assessed (Tab. 3). All mycorrhizal parameters were negatively correlated with bioavailable Cd, Pb and Zn concentrations. However in the case of m% and a% the correlations were low and were not statistically significant. Negative correlation coefficients were also found between particular mycorrhizal parameter (F%, M%, m%, a%, A%) and the total concentration of metals (Cd, Zn, Pb) but for parameter A% they were statistically insignificant. The same trend was observed between all mycorrhizal parameters and soil pH or EC (Tab. 3).

AMF spore abundance and species diversity

Spore numbers were very low and their vertical distribution differed considerably among soil layers in metal contaminated soil. The abundance of spores was highest in deeper soil layers, where 15 and 11 spores per 150 g of soil were found at 20-40 and 40-60 cm soil depth respectively, whereas AMF spores were absent in the 0-10 cm soil layer (Tab. 4).

Spores of only three morphologically distinctive AMF species were found at the site (Archaeospora trappei, Funneliformis mos-seae, Scutellospora dipurpurescens), of which S. dipurpurescens

Fig. 2 Fine endophyte Glomus tenue mycelium in the cortex of De-schampsia cespitosa roots from 40-60 cm soil layer. Ar - arbuscules; At - arbuscule trunk; Sh - swollen hyphae. Scale bar: 20 |im.

occurred most frequently (Tab. 4). However, S. dipurpurescens was recorded only in the two deeper and moderately polluted soil layers. In addition, in roots of D. caespitosa mycorrhizal structures of Glomus tenue, the so-called fine endophyte, were identified. Most likely the fungus rarely produces extraradical spores or they are difficult to extract due to their small size [43]. Glomus tenue was observed in root cortex at 20-40 and 40-60 cm soil depths. At present the systematic position of the fine endophyte is not clear [45].

Discussion

The results presented in the current study show that AM colonization level of Deschampsia cespitosa roots was low in topsoil (0-20 cm) and increased with soil depth (20-60 cm; Fig. 1). Among the examined soil properties EC is not likely to have an effect on the above mentioned differences in mycor-rhizal colonization as its decline is observed when EC exceeds 500 ^S cm-1 [46] whereas, the highest EC was found in the 0-10 cm soil level and it was 121 ^S cm-1 (Tab. 2).

The soil reaction, which decreased with depth in a statistically significant manner (Tab. 2), also could not be the factor that would cause substantial differences in mycorrhizal colonization. Since experiments have shown that in soil with pH between 5.5 and 6.5 spores of most of AM fungi species are able to germinate [47]. There is also lack of data on inhibition of mycelium growth or root colonization in this pH range [2].

Data presented in Tab. 3 also shows statistically significant negative correlations between total Cd, Zn and Pb concentrations and most of the mycorrhizal parameters (F%, M%, m% and a%). However, research data indicates that the link between total metal concentrations in soil and their toxic effect on plants and mycorrhizal fungi is considerably less distinct than the concentrations of bioavailable metal forms [48-51]. Thus the main reason for the low value of F%, A%, M% and m% found at the depth of 0-20 cm might be the high concentration of bioavailable Cd and Zn, which was observed in this soil layer (Tab. 2). The negative correlation between bioavailable Cd, and Zn concentrations in soil and mycorrhizal parameters (F%, M%, A%; Tab. 3) support the notion that metal content is the main factor behind the low level of AM colonization in topsoil. Also a negative correlation between the concentration of bioavailable forms of Pb and F%, M% and A% (Tab. 3) has been found. However, taking into account very low concentrations of bioavailable forms of Pb (max. 7 mg kg-1) when compared to Cd (90 mg kg-1) and Zn (599 mg kg-1) Pb could not have had a significant effect on mycorrhizal colonization of the roots of D. cespitosa.

A toxic effect of Cd, Zn and Pb on mycorrhizal fungi as well as their resistance to these metals was also reported by other authors [26,52-55]. In the present field experiment with spontaneous vegetation mycorrhizal colonization was high in soil layers with the concentration of bioavailable Cd lower than 20 mg kg-1 (Fig. 1, Tab. 2). This observation agrees well with the results obtained in laboratory experiments. Joner and Leyval [56] observed that the AM fungi hyphal growth was reduced at extractable Cd soil concentration higher than 20 mg Cd kg-1 of soil, while Repetto et al. [57] and Rivera-Becerril et al. [58] observed a decrease in mycorrhizal parameters (F%, M%, A%) even at the concentration of 2-3 mg of bioavailable Cd kg-1 of substrate. Chen et al. [59], in turn, recorded a toxic effect of Zn on Z. mays mycorrhizal root colonization.

In the present study root samples were generally co-colonized by AMF and DSE. However DSE were rare and occurred mainly in root fragments where AM colonization was poorly developed. DSE are ubiquitous root-associated fungi common in stressful environments [60]. Some DSE have been reported to be either weak or serious pathogens, whereas others appeared to improve host growth. This range of host responses is attributable to the great diversity of fungal taxa and strains of these endophytes [60-62]. It is possible that DSE may improve D. cespitosa growth at the investigated area, particularly in case where the AM colonization in roots was poorly developed, but their role has to be experimentally studied.

It was documented that in agroecosystems, non-contaminated by heavy metals, such as grasslands, vineyards and maize fields the largest number of AM fungi spores and species

Tab. 4 AMF species (Glomeromycota) associated with Deschampsia cespitosa rooting zone at different soil depth.

Soil depth (cm) AMF species Number of spores per 150 g dry soil

10-20 Archaeospora trappei (R. N. Ames & Linderman) J. B. Morton & D. Redecker 1

20-40 Scutellospora dipurpurescens J. B. Morton & Koske 15

40-60 Scutellospora dipurpurescens J. B. Morton & Koske 9

40-60 Funneliformis mosseae (T. H. Nicolson & Gerd.) C. Walker & A. Schüßler 2

For each soil level 3 soil samples were taken, 50 g of dry soil per sample, but they were pooled together for each soil depth because of very low number of spores.

richness occurred in the topsoil layers (0-20 cm) and they decreased with the increasing soil depth [37]. By contrast, in D. cespitosa rooting zone AMF spore numbers and species richness increased with the increasing soil depth and this increase was positively correlated with the diminishing concentration of Cd and Zn in the deeper layers of soil (Tab. 2, Tab. 4).

The maximum spore number observed in D. cespitosa rooting zone was low (15 spores per 150 g of soil) when compared with the spore abundance reported from metal contaminated sites by other authors. Zarei et al. [55,63,64] found from 60 to 131 spores per 150 g of dry soil at a site adjacent to a Zn and Pb open pit mine. Pawlowska et al. [65] determined 30-38 spores per 150 g of substrate from calamine spoil mound, del Val et al. [19] 45-345 spores per 150 g of soil contaminated by addition of sewage sludge and Wu et al. [66] 132-492 spores per 150 g of soil contaminated by As/Pb/Zn mines. Only Ortega-Larrocea et al. [67] reported low spore abundance (0-19 spores per 150 g) at a metal contaminated site with very high concentrations of Cd, which was similar to the metal content measured by us in D. cespitosa rooting zone (Tab. 2). These data suggests that Cd and Zn concentrations are a major factor contributing to the low abundance of spores in soil.

Scutellospora dipurpurescens was the most abundant AMF species. Its spores were found in deeper soil layers (20-40, 40-60 cm) where the concentration of bioavailable Cd and Zn was still high when compared to the concentration of bioavailable metals reported by other authors [19,48,55,64]. Two other species Funneliformis mosseae and Archaeospora trappei were represented by 2 and 1 spore respectively (Tab. 4). Of the three morphologically distinctive AMF species found in the rooting zone of D. cespitosa only Funneliformis mosseae was detected many times in metal contaminated soils [19,55,64,66-70].

S. dipurpurescens was capable of growing and sporulating in soil highly contaminated with metals, which might suggest that this species is particularly resistant to Cd, Pb and/or Zn. However, Oehl et al. [37] observed that AMF species from genus Scutellospora in grasslands preferentially occurred in deeper soil layers in contrast to other AMF species. On the basis of this data it is possible to conclude that the presence of S. dipurpurescens at the investigated area is not connected with an exceptional resistance of this species to the metals, but with its ability to grow and sporulate in deeper soil layers, where the concentration of bioavailable Cd and Zn was considerably lower than in topsoil (Tab. 2, Tab. 4). This preference of S. dipurpurescens to lower soil depth could be the main reason for a dearth of data on the presence of this species in metal contaminated soils. To the best of the authors' knowledge there are only few papers, which documented the presence of S. dipurpurescens in metal contaminated soils from rhizosphere of Agrostis capillaris [71-73] and Molinia caerulea [74].

No spore of the fine endophyte Glomus tenue was found at the investigated site. Glomus tenue rarely or never produces extraradical spores [42] (Tab. 4). However, Glomus tenue colonized the root cortex of D. cespitosa in two deeper soil layers (20-40 and 40-60 cm; Fig. 2). It was reported that Glomus tenue is a common AMF in grassland soils especially those which are very low in phosphorus and it was frequently found in association with plant species colonizing primary habitats [75]. In our study the fine endophyte occurred along with a coarse AMF in Deschampsia roots (Fig. 1, Fig. 2), much as in the roots of the numerous cultivated and uncultivated plant species examined by Blaszkowski [76]. There are a few papers on G. tenue colonizing roots of plants growing in soils

contaminated by heavy metals [71,72,77]. Most studies dealing with the AMF at such sites did not consider the fungus [19,55,64,66-70]. Therefore further studies on the occurrence and role of G. tenue occurring alone and with other AM fungi in the roots of plants colonizing contaminated sites are needed.

Acknowledgments

The presented research was funded by the Department of Biology and Environmental Protection, University of Silesia.

Authors' contributions

The following declarations about authors' contributions to the research have been made: study conception: EGP, EM; field research: EGP, JB, LM; writing the manuscript: EGP, JB, RK, LM

References

1. Harrison MJ. Molecular and cellular aspects of the arbuscular mycorrhizal symbiosis. Annu Rev Plant Physiol Plant Mol Biol. 1999;50(1):361-389. http://dx.doi.org/10.1146/annurev.arplant.50.L361

2. Smith SE, Read DJ. Mycorrhizal symbiosis. San Diego CA: Academic Press; 2008.

3. Zubek S, Turnau K, Blaszkowski J. Arbuscular mycorrhiza of endemic and endangered plants from the Tatra Mts. Acta Soc Bot Pol. 2008;77(2):149-156. http://dx.doi.org/10.5586/asbp.2008.019

4. Bothe H, Regvar M, Turnau K. Arbuscular mycorrhiza, heavy metal, and salt tolerance. In: Sherameti I, Varma A, editors. Soil heavy metals. Berlin: Springer; 2010. p. 87-111. (vol 19). http://dx.doi. org/10.1007/978-3-642-02436-8_5

5. Mardukhi B, Rejali F, Daei G, Ardakani MR, Malakouti MJ, Miransari M. Arbuscular mycorrhizas enhance nutrient uptake in different wheat genotypes at high salinity levels under field and greenhouse conditions. C R Biol. 2011;334(7):564-571. http://dx.doi.org/10.1016/j.crvi.2011.05.001

6. Rillig MC. Arbuscular mycorrhizae and terrestrial ecosystem processes. Ecol Lett. 2004;7(8):740-754. http://dx.doi. org/10.1111/j.1461-0248.2004.00620.x

7. Hashimoto Y, Matsufuru H, Sato T. Attenuation of lead leachability in shooting range soils using poultry waste amendments in combination with indigenous plant species. Chemosphere. 2008;73(5):643-649. http:// dx.doi.org/10.1016/j.chemosphere.2008.07.033

8. Zheljazkov VD, Craker LE, Xing B, Nielsen NE, Wilcox A. Aromatic plant production on metal contaminated soils. Sci Total Env. 2008;395(2-3):51-62. http://dx.doi.org/10.1016/j.scitotenv.2008.01.041

9. Yang P, Mao R, Shao H, Gao Y. The spatial variability of heavy metal distribution in the suburban farmland of Taihang Piedmont Plain, China. C R Biol. 2009;332(6):558-566. http://dx.doi.org/10.1016/j.crvi.2009.01.004

10. Plaza GA, Nal^cz-Jawecki G, Pinyakong O, Illmer P, Margesin R. Ecotoxico-logical and microbiological characterization of soils from heavy-metal- and hydrocarbon-contaminated sites. Env Monit Assess. 2010;163(1-4):477-488. http://dx.doi.org/10.1007/s10661-009-0851-7

11. Száková J, Tlustos P, Pavlíková D, Hanc A, Batysta M. Effect of addition of ameliorative materials on the distribution of As, Cd, Pb, and Zn in extractable soil fractions. Chem Pap. 2007;61(4):276-281. http://dx.doi. org/10.2478/s11696-007-0033-4

12. Epelde L, Becerril JM, Barrutia O, González-Oreja JA, Garbisu C. Interactions between plant and rhizosphere microbial communities in a metalliferous soil. Env Pollut. 2010;158(5):1576-1583. http://dx.doi. org/10.1016/j.envpol.2009.12.013

Kapusta P, Szarek-Lukaszewska G, Stefanowicz AM. Direct and indirect effects of metal contamination on soil biota in a Zn-Pb post-mining and smelting area (S Poland). Env Pollut. 2011;159(6):1516-1522. http://dx.doi. org/10.1016/j.envpol.2011.03.015

Karczewska A, Bogda A, Galka B, Szulc A, Czwarkiel D, Duszynska D. Natural and antropogenic soil enrichment in heavy metals in areas of former metallic ore mining in the Sudety Mts. Pol J Soil Sci. 2006;39:131-142. Siebielec G, Stuczynski T, Korzeniowska-Puculek R. Metal bioavailability in long-term contaminated Tarnowskie Gory Soils. Pol J Env. Stud. 2006;15:121-129.

Gucwa-Przepióra E, Malkowski E, Sas-Nowosielska A, Kucharski R, Krzyzak J, Kita A, et al. Effect of chemophytostabilization practices on arbuscular mycorrhiza colonization of Deschampsia cespitosa ecotype Warynski at different soil depths. Env Pollut. 2007;150(3):338-346. http:// dx.doi.org/10.1016/j .envpol.2007.01.024

Kucharski R, Sas-Nowosielska A, Malkowski E, Japenga J, Kuperberg JM, Pogrzeba M, et al. The use of indigenous plant species and calcium phosphate for the stabilization of highly metal-polluted sites in southern Poland. Plant Soil. 2005;273(1-2):291-305. http://dx.doi.org/10.1007/ s11104-004-8068-6

Gildon A, Tinker PB. Interactions of vesicular-arbuscular mycorrhizal

infection and heavy metals in plants. New Phytol. 1983;95(2):247-261.

http://dx.doi.org/10.1111/j.1469-8137.1983.tb03491.x

del Val C, Barea JM, Azcon-Aguilar C. Diversity of arbuscular mycor-

rhizal fungus populations in heavy-metal-contaminated soils. Appl Env

Microbiol. 1999;65(2):718-723.

Tullio M, Pierandrei F, Salerno A, Rea E. Tolerance to cadmium of vesicular arbuscular mycorrhizae spores isolated from a cadmium-polluted and unpolluted soil. Biol Fertil Soils. 2003;37(4):211-214. http://dx.doi. org/10.1007/s00374-003-0580-y

Brej T. Heavy metal tolerance in Agropyron repens (L.) P. Bauv. populations from the Legnica copper smelter area, Lower Silesia. Acta Soc Bot Pol. 1998;67(3-4):325-333. http://dx.doi.org/10.5586/asbp.1998.041 Gucwa-Przepióra E, Turnau K. Arbuscular mycorrhiza and plant succe-sion on zinc smelter spoil heap in Katowice-Welnowiec. Acta Soc Bot Pol. 2001;70(2):153-158. http://dx.doi.org/10.5586/asbp.2001.020 Turnau K, Orlowska E, Ryszka P, Zubek S, Anielska T, Gawronski S, et al. Role of micorrhizal fungi in phytoremediation and toxicity monitoring of heavy metal rich industrial wastes in southern Poland. In: Twardowska I, Allen HE, Haggblom MM, Stefaniak S, editors. Soil and water pollution monitoring, protection and remediation. Netherlands: Springer; 2006. p. 533-551. (vol 69). http://dx.doi.org/10.1007/978-1-4020-4728-2_35 von Frenckell-Insam BAK, Hutchinson TC. Occurrence of heavy metal tolerance and co-tolerance in Deschampsia cespitosa (L.) Beauv. from european and canadian populations. New Phytol. 1993;125(3):555-564. http://dx.doi.org/10.1111/j.1469-8137.1993.tb03903.x Gaur A, Adholeya A. Prospects of arbuscural mycorrhizal fungi in phytoremediation of heavy metal contaminated soils. Curr Sci. 2004;86:528-534. Regvar M, Vogel-Mikus K, Kugonic N, Turk B, Batic F. Vegetational and mycorrhizal successions at a metal polluted site: Indications for the direction of phytostabilisation? Env Pollut. 2006;144(3):976-984. http://dx.doi. org/10.1016/j.envpol.2006.01.036

Turnau K, Jurkiewicz A, Lingua G, Barea JM, Gianinazzi-Pearson V. Role of arbuscular mycorrhiza and associated microorganisms in phytoremediation of heavy metal-polluted sites. In: Prasad MNV, Sajwan KS, Naidu R, editors. Trace elements in the environment. London: CRC Press; 2005. p. 235-252. http://dx.doi.org/10.1201/9781420032048.ch13 Turnau K, Anielska T, Ryszka P, Gawronski S, Ostachowicz B, Jurkiewicz A. Establishment of arbuscular mycorrhizal plants originating from xero-thermic grasslands on heavy metal rich industrial wastes - new solution for waste revegetation. Plant Soil. 2008;305(1-2):267-280. http://dx.doi. org/10.1007/s11104-008-9563-y

Turnau K, Ryszka P, Wojtczak G. Metal tolerant mycorrhizal plants: a

review from the perspective on industrial waste in temperate region. In: Koltai H, Kapulnik Y, editors. Arbuscular mycorrhizas: physiology and function. Netherlands: Springer; 2010. p. 257-276. http://dx.doi. org/10.1007/978-90-481-9489-6_12

30. Guadarrama P, Álvarez-Sánchez FJ. Abundance of arbuscular mycorrhizal fungi spores in different environments in a tropical rain forest, Veracruz, Mexico. Mycorrhiza. 1999;8(5):267-270. http://dx.doi.org/10.1007/ s005720050244

31. Joner E, Leyval C. Time-course of heavy metal uptake in maize and clover as affected by root density and different mycorrhizal inoculation regimes. Biol Fertil Soils. 2001;33(5):351-357. http://dx.doi.org/10.1007/s003740000331

32. Orlowska E, Zubek S, Jurkiewicz A, Szarek-Lukaszewska G, Turnau K. Influence of restoration on arbuscular mycorrhiza of Biscutella laevigata L. (Brassicaceae) and Plantago lanceolata L. (Plantaginaceae) from calamine spoil mounds. Mycorrhiza. 2002;12(3):153-159. http://dx.doi.org/10.1007/ s00572-001-0155-4

33. Mejstrik VK. Vesicular-arbuscular mycorrhizas of the species of a Molin-ietum coeruleae L. I. association: the ecology. New Phytol. 1972;71(5):883-890. http://dx.doi.org/10.1111/j.1469-8137.1972.tb01968.x

34. Rillig MC, Field CB. Arbuscular mycorrhizae respond to plants exposed to elevated atmospheric CO2 as a function of soil depth. Plant Soil. 2003;254(2):383-391. http://dx.doi.org/10.1023/A:1025539100767

35. Asghari HR, Chittleborough DJ, Smith FA, Smith SE. Influence of arbuscular mycorrhizal (AM) symbiosis on phosphorus leaching through soil cores. Plant Soil. 2005;275(1-2):181-193. http://dx.doi.org/10.1007/ s11104-005-1328-2

36. Kabir Z, O'Halloran IP, Widden P, Hamel C. Vertical distribution of arbuscular mycorrhizal fungi under corn (Zea mays L.) in no-till and conventional tillage systems. Mycorrhiza. 1998;8(1):53-55. http://dx.doi. org/10.1007/s005720050211

37. Oehl F, Sieverding E, Ineichen K, Ris EA, Boller T, Wiemken A. Community structure of arbuscular mycorrhizal fungi at different soil depths in extensively and intensively managed agroecosystems. New Phytol. 2005;165(1):273-283. http://dx.doi.org/10.1111/j.1469-8137.2004.01235.x

38. Phillips JM, Hayman DS. Improved procedures for clearing roots and staining parasitic and vesicular-arbuscular mycorrhizal fungi for rapid assessment of infection. Trans Br Mycol Soc. 1970;55(1):158-160. http:// dx.doi.org/10.1016/S0007-1536(70)80110-3

39. Trouvelot A, Kough JL, Gianinazzi-Pearson V. Mesure du taux de mycorhization VA d'un systéme radiculaire. Recherche de méthodes d'estimationayant une signification fonctionelle. In: Gianinazzi-Pearson V, Gianinazzi S, editors. Physiological and genetical aspects of mycorrhizae. Paris: INRA; 1986. p. 217-221.

40. Gerdemann JW, Nicolson TH. Spores of mycorrhizal Endogone species extracted from soil by wet sieving and decanting. Trans Br Mycol Soc. 1963;46(2):235-244. http://dx.doi.org/10.1016/S0007-1536(63)80079-0

41. Omar MB, Bollan L, Heather W. A permanent mounting medium for fungi. Bull Br Mycol Soc. 1979;13:31-32.

42. Blaszkowski J. Glomeromycota. Cracow: W. Szafer Institute of Botany, Polish Academy of Sciences; 2012.

43. Litynski T, Jurkowska H, Gorlach E. Analiza chemiczno-rolnicza. Warsaw: Polish Scientific Publishers PWN; 1976.

44. Houba VJG, Van der Lee JJ, Novozamsky I. Soil analysis procedures, other procedures (soil and plant analysis, part 5b). Wageningen: Wageningen Agricultural University; 1995.

45. Schüfiler A, Walker C. The Glomeromycota: a species list with new families and genera [Internet]. 2013 [cited 2013 Apr 10]; Available from: http:// www.amf-phylogeny.com

46. Pattinson GS, Sutton BG, McGee PA. Leachate from a waste disposal centre reduces the initiation of arbuscular mycorrhiza, and spread of hyphae in soil. Plant Soil. 2000;227(1-2):35-45. http://dx.doi. org/10.1023/A:1026519527211

47. Giovannetti M, Avio L, Sbrana C. Fungal spore germination and

pre-symbiotic mycelial growth - physiological and genetic aspects. In: Koltai H, Kapulnik Y, editors. Arbuscular mycorrhizas: physiology and function. Berlin: Springer; 2010. p. 3-32. http://dx.doi. org/10.1007/978-90-481-9489-6_1

Tonin C, Vandenkoornhuyse P, Joner EJ, Straczek J, Leyval C. Assessment of arbuscular mycorrhizal fungi diversity in the rhizosphere of Viola calaminaria and effect of these fungi on heavy metal uptake by clover. Mycorrhiza. 2001;10(4):161-168. http://dx.doi.org/10.1007/s005720000072 Nadgórska-Socha A, Kafel A, Kandziora-Ciupa M, Gospodarek J, Zawisza-Raszka A. Accumulation of heavy metals and antioxidant responses in Vicia faba plants grown on monometallic contaminated soil. Env Sci Pollut Res. 2013;20(2):1124-1134. http://dx.doi.org/10.1007/s11356-012-1191-7 Pourrut B, Shahid M, Dumat C, Winterton P, Pinelli E. Lead uptake, toxicity, and detoxification in plants. Rev Env Contam Toxicol. 2011;213:113-136. http://dx.doi.org/10.1007/978-1-4419-9860-6_4

Trigueros D, Mingorance MD, Rossini Oliva S. Evaluation of the ability of Nerium oleander L. to remediate Pb-contaminated soils. J Geochem Explor. 2012;114:126-133. http://dx.doi.org/10.1016/j.gexplo.2012.01.005 Colpaert JV, Muller LAH, Lambaerts M, Adriaensen K, Vangrons-veld J. Evolutionary adaptation to Zn toxicity in populations of Suilloid fungi. New Phytol. 2004;162(2):549-559. http://dx.doi. org/10.1111/j.1469-8137.2004.01037.x

Pawlowska TE, Charvat I. Heavy-metal stress and developmental patterns of

arbuscular mycorrhizal fungi. Appl Env Microbiol. 2004;70(11):6643-6649.

http://dx.doi.org/10.1128/AEM.70.11.6643-6649.2004

Orlowska E, Ryszka P, Jurkiewicz A, Turnau K. Effectiveness of arbuscular

mycorrhizal fungal (AMF) strains in colonisation of plants involved in

phytostabilisation of zinc wastes. Geoderma. 2005;129(1-2):92-98. http://

dx.doi.org/10.1016/j .geoderma.2004.12.036

Zarei M, Konig S, Hempel S, Nekouei MK, Savaghebi G, Buscot F. Community structure of arbuscular mycorrhizal fungi associated to Veronica rechingeri at the Anguran zinc and lead mining region. Env Pollut. 2008;156(3):1277-1283. http://dx.doi.org/10.1016/j.envpol.2008.03.006 Joner EJ, Leyval C. Uptake of 109Cd by roots and hyphae of a Glomus mos-seae/Trifolium subterraneum mycorrhiza from soil amended with high and low concentrations of cadmium. New Phytol. 1997;135(2):353-360. http://dx.doi.org/10.1046/j.1469-8137.1997.00633.x Repetto O, Bestel-Corre G, Dumas-Gaudot E, Berta G, Gianinazzi-Pearson V, Gianinazzi S. Targeted proteomics to identify cadmium-induced protein modifications in Glomus mosseae-inoculated pea roots. New Phytol. 2003;157(3):555-567. http://dx.doi.org/10.1046/j.1469-8137.2003.00682.x Rivera-Becerril F, van Tuinen D, Martin-Laurent F, Metwally A, Dietz KJ, Gianinazzi S, et al. Molecular changes in Pisum sativum L. roots during arbuscular mycorrhiza buffering of cadmium stress. Mycorrhiza. 2005;16(1):51-60. http://dx.doi.org/10.1007/s00572-005-0016-7 Chen B, Shen H, Li X, Feng G, Christie P. Effects of EDTA application and arbuscular mycorrhizal colonization on growth and zinc uptake by maize (Zea mays L.) in soil experimentally contaminated with zinc. Plant Soil. 2004;261(1-2):219-229. http://dx.doi.org/10.1023/ B:PLSO.0000035538.09222.ff

Mandyam K, Jumpponen A. Seeking the elusive function of the root-colonising dark septate endophytic fungi. Stud Mycol. 2005;53(1):173-189. http://dx.doi.org/10.3114/sim.53.L173

Jumpponen A. Dark septate endophytes - are they mycorrhizal? Mycorrhiza. 2001;11(4):207-211. http://dx.doi.org/10.1007/s005720100112 Usuki F, Narisawa K. A mutualistic symbiosis between a dark septate endophytic fungus, Heteroconium chaetospira, and a nonmycorrhizal

plant, Chinese cabbage. Mycologia. 2007;99(2):175-184. http://dx.doi. org/10.3852/mycologia.99.2.175

63. Zarei M, Saleh-Rastin N, Jouzani GS, Savaghebi G, Buscot F. Arbuscular mycorrhizal abundance in contaminated soils around a zinc and lead deposit. Eur J Soil Biol. 2008;44(4):381-391. http://dx.doi.org/10.1016/). ejsobi.2008.06.004

64. Zarei M, Hempel S, Wubet T, Schäfer T, Savaghebi G, Jouzani GS, et al. Molecular diversity of arbuscular mycorrhizal fungi in relation to soil chemical properties and heavy metal contamination. Env Pollut. 2010;158(8):2757-2765. http://dx.doi.org/10.1016/j.envpol.2010.04.017

65. Pawlowska TE, Blaszkowski J, Rühling Ä. The mycorrhizal status of plants colonizing a calamine spoil mound in southern Poland. Mycorrhiza. 1997;6(6):499-505. http://dx.doi.org/10.1007/s005720050154

66. Wu FY, Bi YL, Leung HM, Ye ZH, Lin XG, Wong MH. Accumulation of As, Pb, Zn, Cd and Cu and arbuscular mycorrhizal status in populations of Cynodon dactylon grown on metal-contaminated soils. Appl Soil Ecol. 2010;44(3):213-218. http://dx.doi.org/10.1016/j.apsoil.2009.12.008

67. Ortega-Larrocea MP, Xoconostle-Cázares B, Maldonado-Mendoza IE, Carrillo-González R, Hernández-Hernández J, Garduño MD, et al. Plant and fungal biodiversity from metal mine wastes under remediation at Zimapan, Hidalgo, Mexico. Env Pollut. 2010;158(5):1922-1931. http:// dx.doi.org/10.1016/j.envpol.2009.10.034

68. Turnau K, Ryszka P, Gianinazzi-Pearson V, van Tuinen D. Identification of arbuscular mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland. Mycorrhiza. 2001;10(4):169-174. http:// dx.doi.org/10.1007/s005720000073

69. Whitfield L, Richards AJ, Rimmer DL. Relationships between soil heavy metal concentration and mycorrhizal colonisation in Thymus polytrichus in northern England. Mycorrhiza. 2004;14(1):55-62. http://dx.doi. org/10.1007/s00572-003-0268-z

70. Orlowska E, Przybylowicz W, Orlowski D, Turnau K, Mesjasz-Przybylowicz J. The effect of mycorrhiza on the growth and elemental composition of Ni-hyperaccumulating plant Berkheya coddii Roessler. Env Pollut. 2011;159(12):3730-3738. http://dx.doi.org/10.1016/j.envpol.2011.07.008

71. Ietswaart JH, Griffioen WAJ, Ernst WHO. Seasonality of VAM infection in three populations of Agrostis capillaris (Gramineae) on soil with or without heavy metal enrichment. Plant Soil. 1992;139(1):67-73. http:// dx.doi.org/10.1007/BF00012843

72. Griffioen WAJ. Characterization of a heavy metal-tolerant endomycor-rhizal fungus from the surroundings of a zinc refinery. Mycorrhiza. 1994;4(5):197-200. http://dx.doi.org/10.1007/BF00206780

73. Griffioen WAJ, Ietswaart JH, Ernst WHO. Mycorrhizal infection of an Agrostis capillaris population on a copper contaminated soil. Plant Soil. 1994;158(1):83-89. http://dx.doi.org/10.1007/BF00007920

74. Blaszkowski J. Polish Glomales. Mycorrhiza. 1994;4(4):173-182. http:// dx.doi.org/10.1007/s005720050017

75. Rabatin SC. Seasonal and edaphic variation in vesicular-arbuscular mycorrhizal infection of grasses by Glomus tenuis. New Phytol. 1979;83(1):95-102. http://dx.doi.org/10.1111/j.1469-8137.1979.tb00730.x

76. Blaszkowski J. Comparative studies of the occurrence of arbuscular fungi and mycorrhizae (Glomales) in cultivated and uncultivated soils of Poland. Acta Mycol. 1993;28:93-140.

77. Turnau K, Mesjasz-Przybylowicz J. Arbuscular mycorrhiza of Berkheya coddii and other Ni-hyperaccumulating members of Asteraceae from ultramafic soils in South Africa. Mycorrhiza. 2003;13(4):185-190. http:// dx.doi.org/10.1007/s00572-002-0213-6

Copyright of Acta Societatis Botanicorum Poloniae is the property of Polish Botanical Society and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use.