Fungal abundance and diversity as influenced by properties of Technosols developed from mine wastes containing iron sulphides: A case study from abandoned iron sulphide and uranium mine in Rudki, south-central Poland

Applied Soil Ecology 145 (2020) 103349

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Fungal abundance and diversity as influenced by properties of Technosols developed from mine wastes containing iron sulphides: A case study from abandoned iron sulphide and uranium mine in Rudki, south-central Poland

T

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Hanna Stępniewskaa, Łukasz Uzarowiczb, , Ewa Błońskac, Wojciech Kwasowskib, Zuzanna Słodczykb, Daria Gałkab, Anna Hebdad a

University of Agriculture in Krakow, Faculty of Forestry, Department of Forest Pathology, Mycology and Tree Physiology, Al. 29 Listopada 46, 31-425 Kraków, Poland Warsaw University of Life Sciences – SGGW, Faculty of Agriculture and Biology, Department of Soil Environment Sciences, ul. Nowoursynowska 159, building no. 37, 02776 Warszawa, Poland c University of Agriculture in Krakow, Faculty of Forestry, Department of Forest Soil Science, Al. 29 Listopada 46, 31-425 Kraków, Poland d Jagiellonian University, Faculty of Biochemistry, Biophysics and Biotechnology, Department of Plant Biotechnology, ul. Gronostajowa 7, 30-387 Kraków, Poland b

A R T I C LE I N FO

A B S T R A C T

Keywords: Technosols Fe sulphides Soil properties Soil fungi Acidiella Reclamation

Reclamation of mine spoils is a process leading to initiation of soil-forming and biological processes in former mining areas. The aim of the present study was to demonstrate the change in soil fungi abundance and diversity depending on Technosols properties as an effect of reclamation in 1970s. The investigation was carried out in the area of the abandoned “Staszic” mine in Rudki village (south-central Poland). Three soil profiles developed from mine wastes were investigated. Basic physicochemical properties and fungal abundance and diversity were determined. The results showed that fungal abundance and species composition is diverse in the studied Technosols, which is closely related to soil properties, in particular with pH, total organic carbon (TOC) and total nitrogen (TN) content. Profile R1 represented a site where reclamation (i.e. neutralization of high acidity) did not succeed, therefore the pH of soil was low (3.0–3.9), plant cover was very scarce, and there was a very low concentration of TOC (up to 0.6%) and TN (up to 0.06%). Profile R1 was the most specific in terms of fungal diversity. The most frequent fungal species was acidophilous Acidiella sp. Profile R2 was located in close vicinity of R1 profile, though it represented a site where neutralization succeeded. Therefore, pH was ~7, and high contents of TOC and TN (up to 8% and 0.65% respectively) occurred in the topsoil of profile R2, whereas the subsoil was acidic (pH 3.9) and poor in TOC and TN. Profile R3 was characterized by high pH (7.2–7.6) throughout the profile, high contents of TOC and TN (up to 4.8% and 0.41% respectively) in the topsoil as well as high concentrations of trace elements (e.g. Pb, Zn, Cu). Our analysis showed that the highest fungal diversity was noted in surface horizons. The study confirmed the distinction of surface horizon of profile R2 and the whole profile R3 in terms of fungal species. High fungal abundance and diversity in R2 and R3 soil profile suggests favourable changes in the mycobiota structure as a result of improvement of soil properties due to reclamation works.

1. Introduction Iron sulphide mining produces wastes the majority of which are deposited on the land surface. Once the mines are closed, mine spoils overgrow due to spontaneous plant succession or are subject to reclamation. As an effect, technogenic soils (Technosols) develop on surfaces of disposal sites (e.g. Skawina, 1959; Struthers, 1964; Barnhisel and Massey, 1969). The occurrence of iron sulphides and their weathering rate is regarded as a key factor influencing properties of

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technogenic soils developed from wastes containing sulphides. This is because sulphide weathering causes strong soil acidification unless neutralizing agents (e.g. carbonates) occur in parent material (Dixon et al., 1982; Uzarowicz and Skiba, 2011, 2013). Acidification is a negative phenomenon itself as it causes leaching of nutrients (e.g. Ca and Mg) and limits their availability for plants, inhibits the activity of biota and may lead to release of Al and trace elements from soil material (Bolan et al., 2005; Lu et al., 2005). Sulphide weathering and related acidification leads to intense mineral transformations of soil substrate

Corresponding author. E-mail address: [email protected] (Ł. Uzarowicz).

https://doi.org/10.1016/j.apsoil.2019.08.011 Received 13 April 2019; Received in revised form 23 August 2019; Accepted 28 August 2019 Available online 14 September 2019 0929-1393/ © 2019 Elsevier B.V. All rights reserved.

Applied Soil Ecology 145 (2020) 103349

Meadow; Medicago sp., Calamagrostis epigejos, Hypericum perforatum, Rubus sp. Flat area of the former flotation tank. 50°53′48.9″N 21°05′57.9″E Alt. 224 m a.s.l. Spolic Technosol (Pantoeutric, Epiloamic, Endoarenic, Endodolomitic, Hyperartefactic, Ochric, Raptic, Endohyposulfidic, Toxic) R3

Meadow; Alopecurus pratensis, Festuca sp., Urtica dioica, Artemisia vulgaris Flat, neutralized surface of the former “Serwis” mine waste dump. 50°53′36.0″N 21°04′47.9″E Alt. 254 m a.s.l. Spolic Technosol (Epihypereutric, Loamic, Hyperartefactic, Ochric, Endosalic, Endothionic) R2

Very weakly developed soil profile developed on the border of an extremely acid area with no plant cover where neutralization was unsuccessful. The entire profile was brown and rusty, and the horizons distinguished in the field (from C1 to C4) slightly differed in colour. In C3 horizon, several centimetre large rock fragments composed of iron sulphides were found. Weakly developed soil profile occurring in the place where neutralization was successful. The subsoil (C1 and C2 horizons) comprising acidic waste material was brown and rusty. In the topsoil, the A horizon was developed due to accumulation of soil organic matter. The topsoil contains patches of white carbonate-rich material. Weakly developed and bipartite soil profile. In the subsoil (2C horizon), brown post-flotation sludge containing dolomite and iron sulphides occurred. The topsoil (A, C1, and C2 horizons) consisted of brown and orange loamy material deposited on flotation tanks during reclamation. Sparse meadow; Calamagrostis epigejos Flat, not neutralized surface of the former “Serwis” mine waste dump. 50°53′36.0”N 21°04′50.0″E Alt. 253 m a.s.l. Spolic Technosol (Loamic, Hyperartefactic, Salic, Thionic, Toxic) R1

General description of parent material and morphology of soil profiles Vegetation type and the dominant plant species Field setting and topography Geographical position (GPS) Soil classification according to WRB (IUSS Working Group WRB, 2015) Profile

Table 1 Location, classification and description of the investigated soils.

H. Stępniewska, et al.

(Šucha et al., 2002; Uzarowicz et al., 2012; Mangová and Lintnerová, 2015). Although the properties and mineral transformations in Technosols developed from mine wastes containing iron sulphides are recognized, little is still known about biological activity (e.g. Johnson, 2003), including fungal diversity and activity of these soils. Fungi are eukaryotic microorganisms widely distributed in all terrestrial ecosystems (Jeffery et al., 2010). They play a major role in ecosystem processes, such as carbon cycling and plant mineral nutrition. Fungi contribute to the organic matter decomposition, nutrient release, formation of soil aggregates and creation of mycorrhizae with plant roots (Taylor and Sinsabaugh, 2015; Tedersoo et al., 2014). The majority of fungi inhabiting soil are regarded as having a cosmopolitan distribution. They may colonize various environments even as extreme as deserts, high mountains, saline substrates as well as highly alkaline and highly acidic sites (Selbmann et al., 2013). Fungi may also occur in extreme environments created by anthropogenic activities, i.e. soils polluted with heavy metals or technogenic soils with extreme chemical and physical properties (Stefanowicz et al., 2008; Wang, 2017). Mining and other industrial activities cause change in soil properties and soil microbial activity (Pająk et al., 2016, 2018). Fungi shows high plasticity and capacity to adopt various forms under varied soil conditions (Frąc et al., 2018) and can be found in different environments and live in a wide range of temperature, moisture and pH (Rousk and Bååth, 2011). According to Hirose et al. (2009), changes in species composition and richness of fungal communities can be used as indicators of environmental change. Cutler et al. (2014) and Harantová et al. (2017) used fungi species composition as an indicator of transformations of sites during primary succession. Azevedo and Cássio (2010) and Goupil et al. (2015) demonstrated the effect of metal on fungi diversity and abundance. According to the authors, fungal tolerance to metals varied with fungal species and metal type while soil pH might also be an important factor. Zornoza et al. (2016) investigated the influence of various amendments simulating reclamation works on biological properties of soils developed from mineral materials containing Fe sulphides. They noted that bacterial and fungal growths were highly dependent on pH and labile organic C. Despite our vast knowledge of fungi behaviour in the soil environment, little is still known about the development of mycobiota in relatively young mining area Technosols following land reclamation (e.g. Goupil et al., 2015; Harantová et al., 2017; Heděnec et al., 2017). The aim of the studies presented here is to recognize fungal diversity and activity in young (~45 years) technogenic soils (Technosols) as an effect of reclamation in the area of an abandoned Fe sulphide and uranium mine in Rudki village (south-central Poland). The region is a unique place where Technosols characterized by extremely different physicochemical properties and geochemistry can be found in a small area. This enables interdisciplinary research concerning impact of reclamation works on a local environment. The influence of reclamation works on fungal abundance and diversity, as well as relationships between fungi and soil physicochemical properties were discussed in the present paper. The authors hypothesise that soil properties such as pH and content of soil organic matter (expressed by concentration of total organic carbon (TOC) and total nitrogen (TN)) are the most important factors influencing abundance and diversity of soil fungi in the investigated Technosols. 2. Materials and methods 2.1. Study area and object The study area is located in the vicinity of the abandoned Fe sulphide and uranium “Staszic” mine in Rudki village (Holy Cross Mts., south-central Poland) (Uzarowicz and Skiba, 2011; Migaszewski et al., 2015; Gałuszka et al., 2016). The mine operated between 1920s and 1971. Two types of wastes were produced during its operation: (1) 2

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loamy sulphide-rich mine tailings deposited on the so-called “Serwis” dump, and (2) carbonate and sulphide-rich post-flotation sludge sedimented as slurry in settling ponds. Both sites were reclaimed in 1970s immediately after the mine closed (Skawina et al., 1974). Therefore, the reclaimed topsoils of the soils developed in Rudki are approximately 45 years old whereas the subsoils are older. Three soil profiles (R1, R2, and R3) developed from mine wastes containing Fe sulphides were investigated. The location, classification, and description of field setting are shown in Table 1. Profiles R1 and R2 represent a reclaimed “Serwis” dump where sulphide-rich spoils were deposited. The main issue during reclamation of that area was the strong acidity of mine wastes (pH ~2). Therefore, reclamation involved a neutralization of the ground by means of Ca and Mg oxides, carbonates, and phosphates and subsequent sowing grasses and legumes (Skawina et al., 1974), however reclamation did not fully succeeded in the area of the dump. Until now, there are small areas (several dozen of m2) with strongly acidic soil and lack of vegetation (Warda, 2007; Uzarowicz, 2011, 2013; Uzarowicz and Skiba, 2011) occurring in places where the dose of neutralizing agent used during reclamation was too low as represented by profile R1 (Table 1). On the other hand, there are areas on the former “Serwis” dump where neutralization was successful. Nowadays, they are covered with luxuriant meadow communities as represented by profile R2 (Table 1). Finally, profile R3 represents soils developed in a former settling pond. The main issue in that area after mine closure was to isolate very compact sludge material rich in dolomite and Fe sulphides from the environment (Skawina et al., 1974). This was done by covering settling ponds with a layer (thickness of about 40–50 cm) of loamy material. The effect of reclamation are bipartite Technosols with loamy topsoil and sandy loamy sludge material in the subsoil (Table 1) (Uzarowicz and Skiba, 2011).

2.2.1. Determination of soil properties and soil classification In order to determine soil properties, roots were removed from subsamples followed by air drying at room temperature. Dry soil samples were sieved (< 2 mm). The properties of the fine earths (< 2 mm) were determined using common pedological methods (van Reeuwijk, 2002; Pansu and Gautheyrou, 2006). Texture was determined using the Bouyoucos-Casagrande method. Soil textural classes were defined according to U.S.D.A. classification (Soil Survey Division Staff, 1993). The pH (in deionized water and 1 mol·dm−3 KCl) was analysed potentiometrically using a soil/solution ratio of 1:2.5 (w/v) (for samples from organic horizons the ratio was 1:10). The content of carbonates was determined using the Scheibler volumetric method (reagent: 10% HCl). Electrical conductivity (ECe) was measured based on water extracts from saturated soil pastes (van Reeuwijk, 2002) and soil salinity classes were defined according to FAO classification (Website 1). Total organic carbon (TOC), total nitrogen (TN), and total sulphur (TS) was determined using an elemental analyzer (LECO CNS TrueMac Analyzer, Leco, St. Joseph, MI, USA). The C/N ratio was calculated based on TOC and TN contents. Exchangeable aluminium (Alex) and hydrogen (Hex) was determined using the Sokolov method (extraction using 1 mol·dm−3 KCl and titration using 0.05 mol·dm−3 NaOH). Exchangeable acidity (EA) was calculated as a sum of Alex and Hex. Exchangeable bases (Ca2+, Mg2+, K+, and Na+) were extracted using ammonium chloride (pH = 8.2) for soil samples containing carbonates or ammonium acetate (pH = 7) for samples without carbonates. Contents of Ca2+ and Mg2+ in extracts were determined using flame atomic absorption spectrometry, and amounts of K+ and Na+ were measured using flame emission spectroscopy. The sum of exchangeable bases (EB) was then calculated. Cation exchange capacity (CEC) was obtained as a sum of EA and EB. Base saturation (BS) was calculated as a percentage of EB in CEC. The soil profiles were classified according to the World Reference Base for Soil Resources (IUSS Working Group WRB, 2015).

2.2. Analytical methods Soil samples were taken from soil horizons/layers distinguished in the studied profiles during field works on June 18, 2016. The samples from each horizon were placed in a plastic container, mixed, and divided into two subsamples on which (1) soil properties and (2) biological properties (i.e. fungal abundance and diversity) were determined. The container and tools for taking samples from profile were washed with water and ethanol (> 99% w/w) each time before the next sample was taken. In total, 16 samples were taken from soils. Fungal abundance and diversity was investigated in 10 selected samples (Table 2).

2.2.2. Determination of total contents of Fe, Mn, and trace elements in soils Fine earths (< 2 mm) from soils were subject to sequential extraction analyses. Sequential extraction procedure by Zeien and Brümmer (1989) was used in order to determine forms of selected metals (Fe, Mn, Zn, Pb, Cu, Cd, Co, Ni, Cr). The analyses were performed in duplicates. That procedure includes seven steps of sequential extraction. The sum of concentrations of each metal from each step was considered as a total content of a metal.

Table 2 Selected physical and chemical properties of the studied soils. Profile

Horizon

Depth (cm)

% of fraction 2.0–0.05 mm

0.05–0.002 mm

< 0.002 mm

R1

Oi AC† C1† C2 C3† C4

0–1 1–10 10–20 20–35 35–70 70–90

– 51 45 34 48 14

– 34 37 41 39 48

– 15 18 25 13 38

R2

Oi A† AC† C1 C2† Oi A† C1† C2† 2C†

0–2 2–10 10–20 20–40 40–70 0–1 1–3 3–15 15–45 45–85

– 54 68 64 61 – 53 42 40 79

– 34 29 21 26 – 29 30 28 20

– 12 3 15 13 – 18 28 32 1

R3

- not analysed, n – lack of carbonates, 0 – below detection limit,

†

Soil textural class (USDA) – Loam Loam Loam Loam Silty Clay Loam – Sandy Loam Sandy Loam Sandy Loam Sandy Loam – Sandy Loam Clay Loam Clay Loam Loamy Sand

pH (H2O)

pH (KCl)

6.0 3.8 3.2 3.0 3.7 3.9

6.0 3.7 3.0 2.7 3.6 3.3

5.5 6.7 7.5 3.9 3.9 4.6 7.5 7.4 7.2 7.6

5.3 6.4 7.4 3.2 3.0 4.5 6.8 6.7 6.6 7.5

ECe (dS∙m−1)

% TOC

%TN

C/N

– n n n n n

– 13.0 12.6 13.9 19.6 9.5

– 0.6 0.4 0.4 0.5 0.1

– 0.06 0.04 0.05 0.04 0.06

– 10.6 8.7 7.0 13.2 1.2

– 0.79 0.61 0.73 2.03 0.18

– 0.1 2.6 n n – 0.1 0.1 0.2 48.8

– 3.3 11.7 12.3 16.3 – 1.1 1.0 0.7 13.7

– 8.0 0.3 0.1 0.1 – 4.8 2.7 0.6 0.6

– 0.65 0.02 0.02 0.02 – 0.41 0.23 0.05 0.08

– 12.4 14.9 2.9 3.2 – 11.8 11.7 14.0 7.5

– 0.19 0.17 0.14 0.10 – 0.48 0.37 0.22 16.78

% eq. CaCO3

– the horizons for which fungal abundance and diversity was determined. 3

%TS

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gel stained with Midori Green. Amplified products were sequenced with the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) using the same primers used for the PCR. Sequences were compared with data from NCBI GenBank using the BLASTn algorithm. All sequences generated in this study were deposited in NCBI GenBank.

2.2.3. Determination of fungal diversity Subsamples for analysis of fungal diversity were immediately transported to the laboratory. The samples were stored in the darkness at 4 °C before the analyses. Altogether 10 soil samples were collected from profile R1 (AC, C1 and C3 horizons), profile R2 (A, AC and C2 horizons) as well as profile R3 (A, C1, C2 and 2C horizons). The culturable microfungi were isolated by soil dilutions. Ten grams of a soil sample was suspended in 90 ml of sterile distilled water and thoroughly mixed for three minutes (10−1 suspension), and then serial dilutions 10−2, 10−3, 10−4 and 10−5 were prepared. Portions of 0.1 ml of each dilution were added to sterile plates (five replicates) containing Rose Bengal Agar medium (Martin, 1950) supplemented with tetracycline hydrochloride to avoid bacterial contamination. Inoculated plates were incubated up to 7–10 days at 22 °C in the darkness. Subsequently, individual fungal colonies were transferred on malt extract agar medium (MEA, 2% malt extract, 1.5% agar) for identification purpose. Finally, only three selected dilutions have been considered for determination of culturable microfungi on each sample depending on the number of fungal colonies per plate (up to 50 colonies). The following dilutions were selected: (1) profile R1: AC horizon – 10−3/−4/−5, C1 and C3 horizons – 10−1/−2/−3; (2) profile R2: A horizon – 10−3/−4/−5, AC horizon – 10−2/−3/−4, C2 horizon – 10−1/−2/−3; (3) profile R3: A and C1 horizons – 10−3/−4/−5, C2 horizon – 10−2/−3/−4, 2C horizon – 10−1/−2/−3. Morphological characteristics were investigated and used in order to divide the fungal isolates into distinct morphological groups. Representative isolates of each morphological group were subjected to molecular identification.

2.2.5. Fungal data analyses For each soil sample, the number of colony forming units (CFU) per gram of soil was determined based on 10−3 dilution isolation results. For the taxa isolated, the frequency of occurrence in a single sample (soil horizon) and at an individual site (soil profile) was calculated as the percent of all plates for an individual site (n = 45 in profile R1, n = 45 in profile R2, n = 60 in profile R3) in which a particular taxon was identified. Moreover, the total frequency of occurrence was calculated as the percent of all plates used (n = 150) in which a particular taxon was identified. The Shannon (Shannon and Weaver, 1963) and Simpson (Simpson, 1949) diversity indices and dominance index were estimated for all fungal taxa. 2.3. Statistical analysis Principal Components Analysis (PCA) method was used to evaluate the relationships between soil properties and fungal species composition. Pearson correlation coefficients between soil properties and fungal diversity index were also calculated. On the basis of Ward's method (Everrit, 1980), agglomeration of the soils samples into groups differing in the fungal species composition was conducted. Differences with P < 0.05 were considered statistically significant. All the statistical analyses were performed with Statistica 12 software (2012).

2.2.4. Molecular examination of fungal isolates DNA was extracted using the Genomic Mini AX Plant Kit (A&A Biotechnology, Gdynia, Poland) according to the manufacturer's protocols. The ITS rDNA region (ITS1–5.8 S-ITS2) was amplified using the primers ITS1F (Gardes and Bruns, 1993) and ITS4 (White et al., 1990). Gene fragments were amplified in a 25-μL reaction mixture containing 0.25 μL of Phusion High-Fidelity DNA polymerase (Finnzymes, Espoo, Finland), 5 μL Phusion HF buffer (5×), 0.5 μL of dNTPs (10 mM), 0.75 μL DMSO (100%), 1 μL of DNA template at concentration of 20 μg·μL−1 and 0.5 μL of each primer (25 μM). Amplification reactions were performed using a Biometra TPersonal 48 Thermocycler (Biometra GmbH, Goettingen, Germany). The PCR (polymerase chain reaction) conditions for the ITS gene region were an initial denaturation step at 98 °C for 30 s followed by 35 cycles of 5 s at 98 °C, 10 s at 57 °C and 30 s at 72 °C and a final chain elongation at 72 °C for 8 min. The PCR products were visualised under UV light on a 2% agarose

3. Results 3.1. Soil properties Profile R1 was a uniform formation built of loamy material (Table 2). The reaction was strongly acidic (pHH2O from 3.0 to 3.9, excluding Oi horizon) in the entire profile. Carbonates were not present in the profile. The soil was strongly and very strongly saline (ECe from 9.5 to 19.6 dS·m−1), and the salinity was diverse throughout the profile. Profile R1 was poor in TOC (up to 0.6%) and TN (up to 0.06%), and contained up to about 2% of TS. The profile exhibited the highest EA in comparison with other studied soils (Table 3). Al was a predominating exchangeable acidic cation, whereas Ca and Mg were dominant

Table 3 Sorption properties of the studied soils. Profile

Horizon

Depth (cm)

Alex

Hex

cmol(+)·kg R1

R2

R3

Oi AC† C1† C2 C3† C4 Oi A† AC† C1 C2† Oi A† C1† C2† 2C†

0–1 1–10 10–20 20–35 35–70 70–90 0–2 2–10 10–20 20–40 40–70 0–1 1–3 3–15 15–45 45–85

– 2.5 6.2 9.5 5.2 1.4 – 0 0 3.2 3.9 – 0 0 0 0

EA (Alex + Hex)

Ca2+

Mg2+

K+

Na+

– 2.5 6.2 9.6 5.2 1.4 – 0 0 3.2 3.9 – 0 0 0 0.04

– 38.5 19.7 21.5 24.3 9.6 – 42.4 83.6 7.0 8.5 – 27.1 14.1 5.8 613.8

– 4.2 9.8 19.0 24.8 62.4 – 95.7 33.4 14.4 26.9 – 62.2 39.5 25.7 1711.9

– 0.1 0.1 0.1 0.1 0.3 – 3.1 1.6 0.1 0.1 – 3.5 4.1 3.1 0.8

– 0.1 0.1 0.1 0.2 0.5 – 0.1 0.1 0.1 0.2 – 0.7 0.7 0.4 0.8

EB

CEC (EA + EB)

BS (%)

– 45.3 35.9 50.2 54.5 74.2 – 141.3 118.7 24.9 39.5 – 93.6 58.4 35.1 2327.3

– 94.6 82.7 81.0 90.5 98.1 – 100.0 100.0 87.0 90.3 – 100.0 100.0 100.0 100.0

−1

– 0 0 0.1 0 0 – 0 0 0 0 – 0 0 0 0.04

- not analysed, 0 – below detection limit, † − the horizons for which fungal abundance and diversity was determined. 4

– 42.9 29.7 40.7 49.4 72.8 – 141.3 118.7 21.6 35.6 – 93.6 58.4 35.1 2327.3

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remained undetermined. Taxonomic identification of all taxa was based on morphological characteristics and ITS rDNA sequencing. For 42 taxa, a Blast search ITS sequence similarity of ≥99% was found with GenBank sequences, enabling identification to the species level, whereas for seven taxa ˃95% ITS region sequence similarity was found, enabling identification to the genus level according to Landeveert et al. (2003). Fourteen taxa of Fusarium, Ilyonectria, and Penicillium were linked with two or more known species (Table 5), thus they were determined to the genus level only. Nine taxa studied by ITS sequencing were not determined neither to species nor genus level and remained identified only by morphological approach (Table 5). The prevailing genus was Penicillium (21 species), which was detected on almost all plates (97.3%). High frequencies were determined also for Fusarium (60.7%), Absidia (45.3%), Sarocladium (40.7%), and Cladosporium (36.7%). In total, 14 plates (9.3%) of the 150 plates remained sterile (Table 5).

exchangeable base cations. Contents of exchangeable Ca decreased downward, and contents of exchangeable Mg showed an opposite tendency (Table 3). Although the pH was low, BS in the entire profile was high (81–98%). Profile R2 had sandy loamy texture throughout the profile (Table 2). The pHH2O was high (up to 7.5) in the topsoil due to occurrence of carbonates (up to 2.6%) added to the soil during reclamation works in 1970s. On the other hand, the pHH2O was low (3.9) in the subsoil, where there were no carbonates. Soil substrate was slightly saline in A horizon and strongly saline in the remaining horizons. The highest contents of TOC, TN, and TS occurred in the topsoil where soil organic matter was accumulated, and they decreased downward (Table 2). Carbonate-bearing topsoil exhibited no EA and contained high contents of EB, among which Ca and Mg predominated (Table 3). The subsoil of profile R2 contained up to 3.9 cmol(+)·kg−1 of exchangeable Al together with Mg and Ca (up to 26.9 and up to 8.5 cmol(+)·kg−1, respectively) which were dominant exchangeable base cations. BS was 100% in the topsoil, whereas it was about 90% in the subsoil (Table 3). Profile R3 was a bipartite soil formation with a firm sandy layer (2C horizon) of post-flotation sludge in the subsoil and loamy material in the topsoil (horizons A, C1, and C2) (Table 2). The pHH2O was high throughout the profile as soil substrate contained carbonates (about 49% in 2C horizon and up to 0.2% in the topsoil). Soil material was not saline (ECe < 2 dS m−1) in the topsoil, whereas it was strongly saline in 2C horizon. The highest concentrations of TOC and TN were present in A and C1 horizon where soil organic matter was accumulated. Contents of TOC and TN decreased downwards (Table 2). Concentration of TS was the highest in 2C horizon. Profile R3 exhibited no EA (excluding trace contents of Hex in 2C horizon) (Table 3) therefore BS was 100% throughout the profile. Mg and Ca were dominant exchangeable base cations.

3.4. Diversity of fungi in soil profiles and soil horizons The number of microfungi expressed as the total number of isolates was the highest in the R3 profile (Table 6). The lowest number of microfungi was observed in the R1 profile. The number of fungal genera isolated and identified from R1, R2, and R3 profile was 21, 22, and 17 genera respectively (Table 6). Only eight genera, i.e. Absidia, Cladosporium, Clonostachys, Cuninghamella, Fusarium, Mortierella, Penicillium, and Sarocladium were recorded in all soil profiles. All these genera were present in all soil horizons, both top A and AC horizons, and deep C2, C3 and 2C horizons (Table 6). Remaining fungal genera were specific to only one profile. Nine genera Acidea, Acidiella, Apiotrichum, Cladophialophora, Didymosphaeria, Lecanicillium, Metapochonia, Pyrenopeziza, and Venturia were specific to R1 profile and make up 42.9% of all fungal genera detected in this profile. Seven genera, i.e. Arthrinium, Cylindrocarpon, Gliomastix, Microdochium, Plectosphaerella, Umbelopsis, and Volutella were specific to R2 profile (31.8% of all fungal genera detected in the profile), whereas Doratomyces, Exophiala, Ilyonectria, Phialemonium, and Purpureocillium were specific to R3 profile (29.4% of all fungal genera detected in the profile). The variety of fungal species isolated from profiles R1 and R3 was similar (37 and 36 respectively) and it was lower in comparison with profile R2 (47 species) (Table 6). Eight species were found in each soil profile. Seven species were isolated from profile R1 and R2 only, 12 species were isolated from profiles R2 and R3 only, whereas only two species were isolated from both R1 and R3 profiles (Table 6). A total of 50 species were specific to only one profile: 19 species to R1, 18 species to R2 and 13 species to R3 (Table 6). Among fungal species specific to

3.2. Total concentrations of Fe, Mn, and trace elements in soils Contents of Fe, Mn, and selected trace elements were very variable among soils (Table 4). Profile R3 contained high concentrations of Fe and Mn in the whole profile, Pb and Cu in the topsoil (i.e. A, C1, and C2 horizons) but also contents of Zn, Cd, Ni, and Co were high in certain horizons in the topsoil (Table 4). High concentrations of Zn, Cd, and Co were found in the topsoil (A and AC horizons) of profile R2. High contents of Cr were identified in C4 horizon, profile R1. 3.3. Fungal species composition and identification In total, 80 fungal taxa, belonging to 37 genera were identified from 1006 isolates obtained from 10 soil samples (Table 5). Three taxa

Table 4 Total concentration of Fe, Mn, and selected trace elements in the studied soils (in mg·kg−1). Profile

Horizon

R1

Oi AC† C1† C2 C3† C4 Oi A† AC† C1 C2† Oi A† C1† C2† 2C†

R2

R3

- not determined,

†

Depth (cm)

Fe

Mn

Zn

Pb

Cu

Cd

Ni

Co

Cr

0–1 1–10 10–20 20–35 35–70 70–90 0–2 2–10 10–20 20–40 40–70 0–1 1–3 3–15 15–45 45–85

– 71,684 57,617 64,431 70,727 46,057 – 34,613 29,108 33,750 48,973 – 200,266 161,829 172,566 150,845

– 159.2 128.1 145.2 233.2 569.6 – 1125.5 2016.7 182.4 342.1 – 2789.0 2389.9 2376.6 3242.1

– 109.0 88.8 115.5 94.9 178.4 – 541.9 114.1 112.2 136.5 – 537.4 342.0 298.7 311.1

– 115.4 97.5 44.2 100.2 17.1 – 13.8 21.5 33.2 81.9 – 1546.3 1822.6 1557.4 277.4

– 23.5 77.6 11.8 8.8 32.3 – 17.6 18.1 13.8 20.7 – 75.8 91.9 87.2 27.0

– 0.1 1.1 0.1 0.6 0.1 – 2.4 1.0 0.3 2.0 – 0.9 0.9 2.3 0.9

– 0.2 1.5 1.0 7.2 9.1 – 6.5 16.4 3.9 7.2 – 5.2 18.6 11.0 3.4

– 25.8 33.7 20.2 12.3 27.6 – 11.8 67.5 63.5 59.1 – 30.8 28.8 61.6 49.1

– 14.0 24.7 30.6 17.2 89.6 – 20.1 16.1 12.5 14.8 – 33.9 32.5 38.0 3.7

– the horizons for which fungal abundance and diversity was determined. 5

Applied Soil Ecology 145 (2020) 103349

H. Stępniewska, et al.

Table 5 Fungal taxa identified in soil profiles and frequencies (F%) of their occurrence. Taxon

GenBank Accession No. ITS

Blast

Absidia glauca Hagem† HSP§144 Absidia cylindrospora Hagem† HSP145; HSP148 Acidea sp. HSP96 Acidiella sp. HSP95; HSP213 Acremonium sp. HSP177 Alternaria sp. HSP110 Apiotrichum porosum Stautz HSP137 Arthrinium arundinis (Corda) Dyko & B. Sutton HSP171 Cladophialophora sp. HSP134 Cladosporium cladosporioides (Fresen.) G.A. de Vries HSP77 Cladosporium herbarum (Pers.) Link HSP102 Cladosporium sp. 1 HSP106 Cladosporium sp. 2 HSP149 Clonostachys rosea (Link) Schroers, Samuels, Seifert & W. Gams HSP117 Cunninghamella elegans Lendn. HSP153 Cylindrocarpon s. l.† HSP181 Didymosphaeria futilis (Berk. & Broome) Rehm HSP98 Doratomyces sp.† HSP120 Epicoccum nigrum Link HSP143 Exophiala sp. HSP86 Fusarium acuminatum Ellis & Everh. HSP80 Fusarium oxysporum Ellis & Everh. HSP75; HSP99

– – MK280763 MK271751; MK271752 MN056422 MK433617 MK272957 MK543238 MK272958 MK433618 MK433619 MK433620 MK543239 MK433621

– – Acidea extrema FJ430779, 96% ITS Acidiella bohemica JQ172757, 95% ITS Acremonium sp. KR935837, 99% ITS Alternaria sp. JN038476, 100% ITS Apiotrichum porosum KY558352, 100% ITS Arthrinium arundinis KF144889, 99% ITS Cladophialophora sp. JX171165, 99% ITS Cladosporium cladosporioides KT898584, 99% ITS Cladosporium herbarum KX611004, 99% ITS Cladosporium sp. KF128859, 99% ITS Cladosporium sp. LN809012, 99% ITS Clonostachys rosea KM519669, 100% ITS

13.3 32 0.7 10 3.3 3.3 2 1.3 0.7 2 10.7 0.7 23.3 16

MK543240 – MK272959 – MK543241 MK433622 MK433623 MK433624; MK272960 MK433626 MK543242 MN056421 MK488053 MK543236 – MK433627 MK488054 MK488055 MK272961 MK272962 MK307853

Cuninghamella elegans FJ792589, 99% ITS – Didymosphaeria futilis KM246243, 99% ITS – Epicoccum nigrum KT898567, 99% ITS Exophiala clone HG935703 (uncultured), 99% ITS Fusarium acuminatum KJ082098, 99% ITS Fusarium oxysporum KJ699122, 99% ITS; HG423346, 100% ITS

14 0.7 0.7 1.3 1.3 0.7 1.3 24

Fusarium solani GQ229075, 99% ITS Fusarium sporotrichioides KU516465, 99% ITS Fusarium solani KP992939, 98% ITS Fusarium solani GQ229075, F. striatum KM231798, 99% ITS Fusarium oxysporum MK424838, 98% ITS – Gliomastix murorum HG008752, 99% ITS Ilyonectria pseudodestructans JF735291, I. mors-panacis KR019870 99% ITS; Ilyonectria robusta JF735264, Uncultured Ilyonectria MG670419 99% ITS Lecanicillium primulinum AB712268, 99% ITS Lecanicillium sp. LC145294, 99% ITS Pochonia bulbillosa AB378552, 99% ITS

18 8.7 1.3 6 0.7 0.7 2.7 3.3 1.3 1.3 4.7 0.7

MK433629

Paecilomyces carneus AB258369, 99% ITS

MK543243 MK543244

Metarhizium flavoviride EF113337, 99% ITS Microdochium lycopodinum NR_145223, 99% ITS

MK433628 MK543245 MK272963 MK272964 MK543246 – – MK543247 MK543248 MK543249 MK543250 MK543251 MK543252 MK307847; MK543253; MK307848 MK543254 MK543255 MK307849 MK543256; MK543257 MK543259 MK543260 MK307850; MK307851 MK307852 MK543261 MK543262 MK543263 MK543264 MK543265

Mortierella alpina KJ469836, 99% ITS Mortierella elongata JF439485, 99% ITS Mortierella pulchella JX976031, 99% ITS Mortierella sp. JX270364, 96% ITS Mucor hiemalis KP942918, 99% ITS – – Penicillium atramentosum KJ775602, 100% ITS Penicillium brasilianum KX096683, 99% ITS Penicillium camemberti KF285997, 99% ITS Penicillium canescens JF311911, 99% ITS Penicillium clavigerum DQ339555, 99% ITS Penicillim coprobium FJ439772, 100% ITS Penicillium janthinellum GU212865, 99% ITS

12.7 0.7 2 6 3.3 1.3 6.7 5.3 1.3 4 9.3 2 1.3 5.3

Penicillium Penicillium Penicillium Penicillium

scabrosum KM023349, 99% ITS spinulosum KX090293, 99% ITS janthinellum KM268694, 97% ITS ochrochloron AF178516, Penicillium sp. LC133788 97% ITS

7.3 12.7 4 1.3

Penicillium canescens KY684281, P. murcianum NR_138358, 100% ITS Penicillium murcianum KP016843, P. janczewskii, KP016839, 99% ITS Penicillium melinii KP132492, Penicillium sp. KU556542, 99% ITS

0.7 18 8.7

Penicillium citreonigrum KU847865, P. citreosulfuratum KP016814, 99% ITS Penicillium manginii JN617662, P. ubiquetum JN617680, 99% ITS Penicillium pasqualense JN617676, P. vancouverense JN617675, 99% ITS Penicillium polonicum KX610157, P. cordubense KJ191427, 99% ITS Penicillium quebecense JN617661, P. cairnsense KF624802, 99% ITS Penicillium sanguifluum KJ191428, P. roseopurpureum JN246025, 99% ITS

0.7 4 11.3 2.7 0.7 3.3

Fusarium solani (Mart.) Sacc. HSP92 Fusarium sporotrichioides Sherb. HSP156 Fusarium sp. 1 HSP79 Fusarium sp. 2 HSP90 Fusarium sp. 3 HSP107 Fusarium sp. 4† HSP190 Gliomastix murorum (Corda) S. Hughes HSP114 Ilyonectria sp. 1 HSP87 Ilyonectria sp. 2 HSP88 Lecanicillium primulinum Kaifuchi, Nonaka & Masuma HSP131 Lecanicillium sp. HSP129 Metapochonia bulbillosa (W. Gams & Malla) Kepler, S.A. Rehner & Humber HSP121 Metarhizium carneum (Duche & R. Heim) Kepler, S.A. Rehner & Humber HSP112 Metarhizium flavoviride W. Gams & Rozsypal HSP147 Microdochium lycopodinum (Jaklitsch, Siepe & Voglmayr) Hern.Restr. & Crous HSP139 Mortierella alpina Peyronel HSP119 Mortierella elongata Linnem. HSP142 Mortierella pulchella Linnem. HSP126 Mortierella sp. HSP125 Mucor hiemalis Wehmer HSP150 Mucor sp. 1† HSP165 Mucor sp. 2† HSP152; HSP151 Penicillium atramentosum Thom HSP219 Penicillium brasilianum Bat. HSP206 Penicillium camemberti Thom HSP173 Penicillium canescens Sopp HSP207 Penicillium clavigerum Demelius HSP220 Penicillium coprobium Frisvad HSP172 Penicillium janthinellum Biourge HSP168; HSP174; HSP212

Penicillium Penicillium Penicillium Penicillium

scabrosum Frisvad, Samson & Stolk HSP221 spinulosum Thom HSP154 sp. 1 HSP169 sp. 2 HSP161; HSP223

Penicillium sp. 3 HSP208 Penicillium sp. 4 HSP175 Penicillium sp. 5 HSP167; HSP211 Penicillium Penicillium Penicillium Penicillium Penicillium Penicillium

sp. sp. sp. sp. sp. sp.

6 HSP214 7 HSP155 8 HSP157 9 HSP176 10 HSP210 11 HSP217

Total F%‡

0.7 0.7

(continued on next page) 6

Applied Soil Ecology 145 (2020) 103349

H. Stępniewska, et al.

Table 5 (continued) Taxon

GenBank Accession No. ITS

Blast

Penicillium sp. 12 HSP218

MK543266

Phialemonium inflatum (Burnside) Dania García, Perdomo, Gené, Cano & Guarro HSP111 Plectosphaerella cucumerina (Lindf.) W. Gams HSP115 Purpureocillium lilacinum (Thom) Luangsa-ard, Houbraken, HywelJones & Samson HSP93 Pyrenopeziza cf. revincta (P. Karst.) Gremmen HSP94 Sarocladium strictum (W. Gams) Summerb. HSP72 Sarocladium summerbelli A. Giraldo, Gené & Guarro HSP135 Septoriella poae (Crous & Quaedvl.) Hern.-Restr., J.Z. Groenew. & Crous HSP164 Trichoderma asperellum Samuels, Lieckf. & Nirenberg HSP127 Trichoderma harzianum Rifai HSP136 Trichoderma viride Pers. HSP141 Trichoderma sp. HSP123 Truncatella sp.† HSP73 Umbelopsis nana (Linnem.) Arx † HSP2050.16 Venturia sp. HSP97 Volutella ciliata (Alb. & Schwein.) Fr. HSP113

MK433630

Penicillium crustosum KU847869, P. chrysogenum LT603034, P. griseofulvum KU612371, 98% ITS Phialemonium inflatum KU702692, 99% ITS

MK433631 MK433632

Plectosphaerella cucumerina KJ5730077, 99% ITS Purpureocillium lilacinum KJ207400, 99% ITS

MK307854 MK433633 MK307855 MK275265

Axenic grass root isolate (cf. Pyrenopeziza revincta) AJ430225, 99% ITS Sarocladium strictum JQ676174, 100% ITS Sarocladium summerbelli HG965035, 99% ITS Septoriella poae KJ869111, 99% ITS

MK275266 MK275267 MK543267 MK275268 – – MK275269 MK433634

Trichoderma asperellum KR868286, 99% ITS Trichoderma harzianum KR868358, 99% ITS Trichoderma viride FJ872073, 99% ITS Trichoderma sp. KU556489, 99% ITS – – Venturia hystrioides EU035459, 96% ITS Volutella ciliata AJ301966, 99% ITS

6.7 1.3 1.3 6.7 0.7 1.3 2 0.7

UNCLASSIFIED Dothideomycetes sp. HSP101 Unidentified HSP100 Unidentified HSP140

MK275270 MK275271 MK543268

Dothideomycetes sp. LN901102, 98% ITS Uncultured fungus KM504438, 99% ITS Uncultured Phaeosphaeria HG936175, Phoma sp. KF646102, 99% ITS; Uncultured fungus KF800134, 98% ITS

8.7 2 0.7

Sterile plates Total no. of identified taxa Total no. of isolates † ‡ §

Total F%‡ 12.7 6.7 3.3 1.3 2 40 0.7 1.3

9.3 80 1006

Species identified only by morphological approach. Frequency was calculated as percent of the total number of plates for all soil profiles (150 plates). HSP – personal culture collection of H. Stępniewska.

Didymosphaeria futilis, Lecanicillum sp., Mortierella pulchella, Trichoderma asperellum, Venturia sp. found only in profile R1, and Epicoccum nigrum, Mortierella sp. and Penicillum spp. occurring both in profile R1 and C2 horizon in profile R2. The higher CaCO3 content was noted in profile R2 (horizon AC) and R3 (horizon 2C). Cladosporium cladosporioides, Gliomastix murorum, Fusarium sp., Metarhizium flavoviride, Penicillium spp., Phialemonium inflatum, Purpureocillium lilacinum were found in these horizons. The agglomeration analysis results showed that the fungi species composition is diverse in the studied soils (Fig. 3). The surface horizons from profiles R2 and R3, the properties of which were the consequence of reclamation works, clearly differs from other horizons in terms of fungi species composition.

R1 profile, the most frequent species (33.3%) was Acidiella sp., which was detected in each studied soil horizons (Table 6). When comparing fungal abundance and diversity throughout soil profiles, it was found that minimum number of fungal taxa were isolated from the subsoil (i.e. C2, C3, and 2C horizons) (Table 6). Also the number of fungal isolates as well as the number of fungal CFUs (colony forming units) were the lowest in these horizons (Table 6). Shannon's and Simpson's diversity indices varied in the studied soils (Table 7). The highest fungal diversity was noted in surface horizons. The highest value of Shannon diversity index (1.34) was noted in R2 profile in A horizon. Deeper horizons were characterized by the greater dominance index (0.129–0.148) (Table 7). Statistical significance correlations were noted between fungal species richness and TOC content (Table 8). Similar dependences were noted between fungal species richness and TN content. Factors 1 and 2 distinguished through PCA analysis explain 43.28% of the variance of the determined properties (Fig. 1). Factor 1 is associated with acidification and salinity of soils. With regard to factor 2, the high value of factor loading is held by the total carbon and nitrogen content and by the selected trace elements. Fig. 2 demonstrates how the studied samples representing different soil horizon differ in terms of physicochemical properties and fungi species composition. Surface horizon in R2 profile was characterized by the highest total organic carbon and nitrogen content and by the presence of fungi such as Arthrinium arundinis, Cladosporium herbarum, Fusarium sporotrichioides, Microdochium lycopodinum, Mucor hiemalis, Mucor sp. 2, Metarhizium carneum, Penicillium atramentosum, P. camemberti, P. coprobium, P. scabrosum, Penicillium sp. 11, Trichoderma viride, Umbelopsis nana, and Volutella ciliata. Soil horizons A, C1, and C2 from profile R3 were characterized by high pH, clay and heavy metals content (Figs. 1 and 2). Soil horizons AC, C1, and C3 from profile R1 and deep horizon C2 from profile R2 were characterized by high electrical conductivity, low pH, and a presence of fungi such as Acidea sp., Acidiella sp.,

4. Discussion Mining activity in the area of Rudki village, south-central Poland, led to the deposition of Fe diverse sulphide-rich mine wastes on land surface. Some wastes (e.g. mine tailings from the so-called “Serwis” dump) are strongly acidic (pH about 3–4) due to occurrence of high contents of iron sulphides and their weathering products. Others (e.g. post-flotation sludge) are originally characterized by neutral reaction (pH about 7) despite the occurrence of sulphides. This is due to the presence of carbonates which neutralize acidity generated during sulphide weathering. The sites located within the study area differ in terms of contents of soil organic matter depending on the degree of development of plant cover after reclamation conducted in 1970s. Moreover, some sites in the study area contain high amounts of trace elements. Therefore, the study area seems to be a perfect place to examine the influence of properties (e.g. pH, content of soil organic matter) and contents of trace elements in Spolic Technosols on fungal abundance and diversity in soils after reclamation. Investigated soil profiles (R1, R2 and R3) differ in terms of fungal diversity and abundance as expressed by species richness and number 7

Applied Soil Ecology 145 (2020) 103349

H. Stępniewska, et al.

Table 6 Frequencies (%) of fungal taxa identified in each soil horizons (A, AC, C1, C2, C3, 2C) of the studied soil profiles (R1, R2, R3). Taxon

R1 AC

Absidia glauca Absidia cylindrospora Acidea sp. Acidiella sp. Acremonium sp. Alternaria sp. Apiotrichum porosum Arthrinium arundinis Cladophialophora sp. Cladosporium cladosporioides Cladosporium herbarum Cladosporium sp. 1 Cladosporium sp. 2 Clonostachys rosea Cunninghamella elegans Cylindrocarpon s.l. Didymosphaeria futilis Doratomyces sp. Epicoccum nigrum Exophiala sp. Fusarium acuminatum Fusarium oxysporum Fusarium solani Fusarium sporotrichioides Fusarium sp. 1 Fusarium sp. 2 Fusarium sp. 3 Fusarium sp. 4 Gliomastix murorum Ilyonectria sp. 1 Ilyonectria sp. 2 Lecanicillium primulinum Lecanicillium sp. Metapochonia bulbillosa Metarhizium carneum Metarhizium flavoviride Microdochium lycopodinum Mortierella alpina Mortierella elongata Mortierella pulchella Mortierella sp. Mucor hiemalis Mucor sp. 1 Mucor sp. 2 Penicillium atramentosum Penicillium brasilianum Penicillium camemberti Penicillium canescens Penicillium clavigerum Penicillium coprobium Penicillium janthinellum Penicillium scabrosum Penicillium spinulosum Penicillium sp. 1 Penicillium sp. 2 Penicillium sp. 3 Penicillium sp. 4 Penicillium sp. 5 Penicillium sp. 6 Penicillium sp. 7 Penicillium sp. 8 Penicillium sp. 9 Penicillium sp. 10 Penicillium sp. 11 Penicillium sp. 12 Phialemonium inflatum Plectosphaerella cucumerina Purpureocillium lilacinum

2.2 2.2 11,1

R2 C1

C3

2.2 15.6

6.7

Total

4.4 2.2 33.3

A

R3 AC

2.2 15.6

4.4 24.4

2.2

2.2

C2

6.7 40

2.2 6.7

6.7

2.2 4.4

2.2 4.4 2.2

11,1

2.2 2.2

15.6 2.2 2.2

2.2

2.2

2.2

2.2

2.2

6.7

2.2

6.7

4.4 2.2

A

C1

C2

2C

Total

18.3 5

10 18.3

18.3

5

28.3 46.7

5 5

1.7

5 6.7

1.7

1.7 13.3

6.7 2.2

2.2 2.2

†

4.4

4.4

2.2

Total

2.2 3 2.2

4.4 8.9 2.2

4.4

2.2

4.4 4.4 17.8

6.7 4.4

4.4

20 2.2 2.2 15.6 11.1 2.2

1.7

8.3

18.3 8.3 5

11.7 10 8.3

10 11.7 6.7 11.7

3.3

3.3

17.8 8.9 17.8

1.7 15 5 8.3 1.7

1.7 1.7 6.7 8.3

1.7 3.3 38.3 38.3 8.3 3.3 6.7

16.7 8.3

16.7

1.7 6.7

1.7

1.7 6.7

2.2

8.9 1.7

6.7 13.3

4.4

4.4 2.2

4.4 2.2

2.2

2.2

2.2

2.2

2.2

2.2 11,1

4.4 2.2

4.4 2.2

4.4

6.7 11.1

20 4.4

20 4.4

13.3

13.3 8.9 6.7 4.4 13.3 13.3

13.3

11,1

8.9 2.2 4.4 2.2

4.4

2.2

4.4 11,1

2.2

13.3 4.4

4.4

2.2

11.1

2.2 2.2

13.3

8.9 2.2

2.2 6.7

2.2 28.9 2.2

4.4

8.9

2.2 8.9 2.2

8.3 3.3

6.7 3.3

4.4 15.6 2.2

4.4 15.6 2.2

2.2 13.3

45 26.7 25

2.2

6.7 2.2

11,1 2.2

3.3 1.7

2.2

3.3

2.2 8.9

1.7

3.3

28.3

13.3

8.3

1.7

1.7

3.3

10

6.7

16.7

1.7 23.3

5 8.3

8.3 31.7

18.3

1.7 10

10 13.3

10

3.3

15

13.3

2.2 2.2 15.6

1.7

1.7 3.3

1.7 1.7 31.7

10 26.7

2.2 6.7 4.4

4.4

11,1

11.1 4.4

16.7

28.3 16.7

3.3

3.3

11.1

(continued on next page) 8

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H. Stępniewska, et al.

Table 6 (continued) Taxon

R1

R2

AC Pyrenopeziza cf. revincta Sarocladium strictum Sarocladium summerbelli Septoriella poae Trichoderma asperellum Trichoderma harzianum Trichoderma viride Trichoderma sp. Truncatella sp. Umbelopsis nana Venturia sp. Volutella ciliata UNCLASSIFIED Dothideomycetes sp. HSP101 Unidentified HSP100 Unidentified HSP140 Sterile plates Total no. of identified taxa No. of isolates No. of CFU g−1 †

C1

6.7 8.9

C3

Total 6.7 24.4 2.2 2.2 22.2 4.4

15.6 2.2

2.2 15.6

6.7 4.4

4.4

2.2

8.9

2.2

R3

A

AC

C2

Total

A

C1

22.2

24.4

15.6

62.2

15

18.3

2.2

2.2

4.4 6.7

C2

2C

Total

1.7

35

4.4 13.3

6.7

1.7

1.7 4.4

4.4

2.2

2.2

2.2

2.2 11.1 48

6.7

6.7

17.8

11,1

28.9

6.7

6.7 8.9 37

27

17

10

89 2.83 × 105

80 0.66 × 105

22 0.1 × 105

191

32

24

12

145 5.34 × 105

108 1.08 × 105

39 0.08 × 105

291

8.3 36

24

21

14

12

199 6.48 × 105

143 3.9 × 105

109 1.12 × 105

73 0.12 × 105

524

Frequency was calculated as percent of the total number of plates for each profile (45 plates for profile R1, 45 plates for profile R2, 60 plates for profile R3).

studied by Baldrian et al. (2012), the soil horizons were significantly different with respect to organic matter and C and N contents, and decreasing nutrient availability was reflected by a decrease in both bacterial and fungal biomass contents with depth. Moreover, scarification studies on forest soils demonstrated that lower total C and organic matter contents lead to lower fungal and bacterial biomass in soils (Jimenez Esquilin et al., 2008). In our study, correlation analysis and PCA analysis showed that TOC was closely related to fungal diversity. Similar results were obtained by Yang et al. (2017), who suggested that soil organic carbon content is the primary factor influencing soil fungal diversity regardless of land use type. Narendrula-Kotha and Nkongolo (2017) suggested that organic matter and CEC are key factors involved in changes in microbial communities. The variation of the soil nitrogen quantities has impact on the organism distribution, as well as on the fungal community richness and abundance (Cline et al., 2018). Relationships between fungal diversity index and total nitrogen were noted in our study (Table 8). The A horizon in profile R2 was characterized by the highest content of TOC and TN (Table 2). Cluster analysis confirmed the distinctiveness of this part of profile R2 in terms of fungal species (Fig. 3). Profile R2 occurred in distance of several dozen of meters from acidic profile R1, however profile R2 represented an area where neutralization of high acidity succeeded during reclamation works in 1970s. Therefore the pH in the topsoil (A and AC horizons) of profile R2 is about 7, whereas the reaction in the subsoil (C1 and C2 horizons) is still acidic (pH about 4) as neutralization did not reach deeper parts of the profile. The most abundant fungi community in profile R2 (Table 6) was found in the topsoil (A and AC horizons) which was favourable for development of fungi due to the high TOC contents (Table 2) and high pH (about 7) (Table 2). The aforementioned fungi tolerate a wide pH range, from

Table 7 Fungal diversity, richness and dominance index in the studied soils. PROFILE

Horizon

R1

AC C1 C3 A AC C2 A C1 C2 2C

R2

R3

Depth (cm) 1–10 10–20 35–70 2–10 10–20 40–70 1–3 3–15 15–45 45–85

H

D

C

Species richness

1.2900 1.1587 0.8388 1.3395 1.2522 0.9618 1.2640 1.2362 1.0509 0.9183

0.0563 0.0740 0.1354 0.0533 0.0647 0.1296 0.0598 0.0596 0.0947 0.1480

0.0629 0.0822 0.1679 0.0580 0.0701 0.1467 0.0643 0.0648 0.1038 0.1611

27 17 10 32 24 12 24 21 14 12

H – Shannon diversity index, D – Simpson diversity index, C – dominance index.

of isolates and colony forming units (CFU) respectively. Only eight fungal genera of the 37 identified were present in all soils (Table 6). All are cosmopolitan saprotrophic fungi, typical for soil habitat and dead organic matter (Domsch et al., 1980). 4.1. Influence of soil organic matter contents on fungal abundance and diversity in studied Technosols In all investigated soils, fungal abundance and diversity was the highest in topsoil where soil organic matter (expressed by TOC and TN contents) was accumulated (Table 2) and was shown to decrease downwards (Table 6). Organic matter is one of the most important factors determining the growth of saprotrophic soil-inhabiting fungi as they are heterotrophic organisms depending on organic carbon from organic matter decomposition (Rousk and Bååth, 2011). In forest soils

Table 8 Pearsons's correlations between soil properties and fungal diversity index in the studied soils.

H D C Specie richness ⁎

Sand(2–0.05 mm)

Silt (0.05–0.002 mm)

Clay (< 0.002 mm)

−0.2099 0.3541 0.4158 −0.0803

0.1972 −0.2531 −0.3586 0.2288

0.1516 −0.3019 −0.3189 −0.0310

P < 0.05; H – Shannon diversity index, D – Simpson diversity index, C – dominance index. 9

pHH2O

ECe

TOC

TN

0.2759 −0.2640 −0.1975 0.2623

−0.5964 0.6544⁎ 0.6154 −0.4875

0.5791 −0.5128 −0.5148 0.6946⁎

0.5697 −0.5005 −0.4988 0.6844⁎

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Fig. 1. PCA diagram with projection of variables of soil properties and fungal taxa on a plane of the first and second factor.

communities (Zhang et al., 2016). Mycobiota of highly acidic substrates (pH < 3) may be relatively rich but species specific (Hujslová et al., 2010, 2014). Also in our studies it was shown that fungal community of highly acidic R1 soil profile was the most specific (Table 6). Profile R1 represents an area with strongly acidic parent material where reclamation (neutralization) works in 1970s did not succeed. High acidity in the profile R1 is among the factors contributing to the lowest number of isolates and the lowest number of CFU g−1 in profile R1 in comparison with profiles R2 and R3. Other factors influencing fungal abundance could be high salinity and lack of vegetation. It was found that in extreme environments, the complex of environmental factors such as pH, moisture, salinity, and vegetation cover, affects the soil mycobiota (Hujslová et al., 2010). These environments may be suitable for some fungi, particularly dematiaceous fungi belonging to Capnodiales (Ascomycota) (Baker et al., 2004; Hujslová et al., 2014). Among them, some species are strictly acidophilic as Acidomyces acidophilus, A. acidothermus, Hortaea acidophila or acidotolerant as Acidiella bohemica. In our study, Acidiella sp. was recorded in strongly acidic profile R1 as a dominant species throughout that profile. ITS rDNA sequence of this fungus was the most similar (95% identity) to Acidiella bohemica,

slightly acid or neutral to slightly alkaline (Domsch et al., 1980). The low pH in the subsoil (pH 4) (Table 2) has limited their occurrence. These fungi were much less abundant or even absent in strongly acidic profile R1, whereas most of these fungi were found in slightly alkaline profile R3 (pH 7.2–7.6) and they were as frequent as in the topsoil of profile R2.

4.2. Influence of soil pH on fungal abundance and diversity in studied Technosols The diversity of fungal communities is known to be influenced by a range of environmental factors (Liu et al., 2018; Thiem et al., 2018) especially by pH (Bååth and Anderson, 2003; Wang et al., 2015). However, studies by Rousk et al. (2010) showed that the abundance of soil fungi was unaffected by pH and fungal diversity was only weakly related with pH 4.0–8.3 in a long-term liming experiment. Fungi are more tolerant to acidic conditions than bacteria and prevalent in acidic soils. Along with the decrease in soil pH, the fungal biomass increases whereas bacterial biomass decreases (Rousk et al., 2009). However, extremely low soil pH determines the structure of soil fungal 10

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Fig. 2. PCA diagram showing the position of the samples in the plane formed by first two axes. The figure shows tendency to split the samples into groups. The soil properties and fungal species composition were used for diagram preparation. R1, R2, and R3 – soil profiles; A, AC, C1, C2, C3, 2C – soil horizons.

Fig. 3. Dendrogram with groups identified in the cluster analysis. The fungal species composition was used for diagram preparation. R1, R2, and R3 – soil profiles; A, AC, C1, C2, C3, 2C – soil horizons. 11

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described from highly acidic soils in Czech Republic (Hujslová et al., 2013). Fungi of the genus Acidiella were also found in uranium mine in northern Australia (Vázquez-Campos et al., 2014). That habitat is similar to our study area located near the mine in which both Fe sulphides and uranium was exploited. From acid uranium mine water (raffinate) (pH 1.7–1.8) in Australia's mine, Fodinomyces uranophilus was isolated and described by Vázquez-Campos et al. (2014) as a new genus and species. According to Kolařík et al. (2015) the genus Fodinomyces is a synonym of Acidiella. Mycobiota of extremely acidic soils (pH < 3) are represented also by hyaline fungi from among which three new genera were described recently (Hujslová et al., 2014): Acidothrix, Acidea, and Soosiella. In our study one isolate close to Acidea extrema (Hujslová et al., 2014) (ITS rDNA similarity 95%) was found in strongly acidic R1 profile. Extremely acidic environments are often a habitat for a small group of specialized organisms (e.g. algae and rotifers) adapted to living in such conditions (Wołowski et al., 2013; Pociecha et al., 2018).

of Technosols as an effect of reclamation were studied in the area of an abandoned “Staszic” mine in Rudki village (south-central Poland). Three soil profiles were examined. Profile R1 represented a site where reclamation (i.e. neutralization of high acidity) did not succeed, therefore the pH of soil was low (3.0–3.9), and the soil was poor in TOC and TN. Profile R2 was located in close vicinity of R1 profile, however represented a site where neutralization succeeded. Therefore, pH was ~7, carbonates were present, and high contents of TOC and TN occurred in the topsoil of profile R2, whereas the subsoil was acidic (pH 3.9) and poor in TOC and TN. Profile R3 was characterized by high pH (7.2–7.6) throughout the profile, high contents of TOC and TN in the topsoil, as well as high concentrations of trace elements (e.g. Pb, Zn, Cu). In total, 80 fungal taxa, belonging to 37 genera were identified from 1006 isolates. The prevailing genus was Penicillium (21 species) which was detected on almost all plates (97.3%). High frequencies were determined also for Fusarium (60.7%), Absidia (45.3%), Sarocladium (40.7%), and Cladosporium (36.7%). Soil properties, especially pH and soil organic matter content (expressed by TOC and TN contents), are the most important factors influencing abundance and diversity of fungi in the investigated Technosols. These properties influenced community variance more than other factors (e.g. soil texture and trace elements content). Analysed profiles were different according to fungal diversity and abundance as expressed by species richness and number of isolates and colony forming units (CFU) respectively. It was shown that fungal community of strongly acidic R1 soil profile was the most specific with acidophilous Acidiella sp. as the most frequent fungal species. Profile R3 and surface horizons of profile R2 which were slightly alkaline, were characterized by different and more abundant mycobiota compared to profile R1. Simultaneously, profile R2 and R3 were characterized by the highest concentrations of TOC. The results show that reclamation of Fe sulphide-bearing technogenic substrates improved the properties of soils which resulted in favourable changes of structure and abundance of mycobiota in Technosols.

4.3. Influence of trace element contents on fungal abundance and diversity in studied Technosols The most abundant fungi in profile R3 (Table 6) containing high concentrations of trace elements represent taxons common in various soil types all around the world. These fungi (in particular Absidia cylindrospora, A. glauca, and Penicillium spinulosum) have been frequently isolated from grasslands, arable lands, and forest soils (Domsch et al., 1980). All aforementioned species tolerate a wide pH range, except for Mortierella alpina and Clonostachys rosea which prefer neutral to alkaline soil (Domsch et al., 1980). High abundance of these fungi in profile R3 (Table 6) suggests favourable conditions for the mycobiota structure. High concentrations of Fe, Mn as well as Pb, Cu, Zn, Cd, Ni and Co have been found in profile R3, especially in the topsoil (0–45 cm). Trace elements can be toxic to fungi. They can affect fungal enzymes, destroy membranes or cause oxidative stress and, as a result, the growth of fungi is reduced, and their metabolic processes and biology may be disturbed (Baldrian, 2010). The studies by Pennanen et al. (1996) have shown that fungi appear to be more tolerant to metals than bacteria. As found by Kouchou et al. (2017), long-term contamination of soil by trace elements is negatively correlated with quantity of fungal population. According to Narendrula-Kotha and Nkongolo (2017), metal toxicity varies depending on fungal species, metal type and concentration, environmental factors such as soil pH or nutrient availability. Metal contamination of soils influences fungus community structure as well. Some species are eliminated but the others become more abundant. Dirginčiutė-Volodkienė and Pečiulytė (2011) have demonstrated in their laboratory research that saprotrophic fungi such as Absidia glauca, Acremonium kiliense, Aspergillus fumigatus, and Alternaria alternata were eliminated in a soil with higher content of Cu, Zn, and Pb, whereas fungi of the genus Paecilomyces, Clonostachys, Penicillium, Lecanicillium and Cunninghamella echinulata and Mucor hiemalis were more abundant, compared with control soil. Similarly, in our studies some of the aforementioned fungi (e.g. some species of Penicillium and Clonostachys rosea) seem to be resistant to high trace element contents, as they were more abundant in profile R3 in comparison with profile R2. Both profiles have similar chemical properties (e.g. pH ~7) in the topsoil, however the latter profile did not contain high concentrations of trace element (Table 4). In contrast to results by Dirginčiutė-Volodkienė and Pečiulytė (2011), in our study A. glauca was more abundant in R3 profile compared with R2 indicating that species is tolerant to trace elements concentration in soils. This indicates, according to Baldrian (2010), species or even strain sensitivity of soil saprotrophic fungi to trace elements.

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5. Conclusions The abundance and diversity of soil fungi depending on properties 12

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