Identification of Bacterial and Fungal Communities in the Roots of Orchids and Surrounding Soil in Heavy Metal Contaminated Area of Mining Heaps

Orchids represent a unique group of plants that are well adapted to extreme conditions. In our study, we aimed to determine if different soil contamination and pH significantly change fungal and bacterial composition. We identified bacterial and fungal communities from the roots and the surrounding soil of the family Orchidaceae growing on different mining sites in Slovakia. These communities were detected from the samples of Cephalanthera longifolia and Epipactis pontica from Fe deposit Sirk, E. atrorubens from Ni-Co deposit Dobšiná and Pb-Zn deposit Jasenie and Platanthera bifolia by 16S rRNA gene and ITS next-generation sequencing method. A total of 171 species of fungi and 30 species of bacteria were detected from five samples of orchids. In summary, slight differences in pH of the initial soils do not significantly affect the presence of fungi and bacteria and thus the presence of the studied orchids in these localities. Similarly, the toxic elements in the studied localities, do not affect the occurrence of fungi, bacteria, and orchids. Moreover, Cortinarius saturatus, as a dominant fungus, and Candidatus Udaeobacter as a dominant bacterium were present in all soil samples and some root samples. Finally, many of these fungal and bacterial communities have the potential to be used in the bioremediation of the mining areas.


Introduction
Orchids belonging to the family Orchidaceae are well known for their rarity and presence of mycorrhizal associations [1,2]. This specialization contributes to the diversity and rarity of orchid species [3,4]. They grow in specific habitats such as mining dumps. Mine heaps and mine wastes created by the mining industry are one of the extreme habitats made by anthropogenic activity. Interestingly, mine heaps create an environment with specific ecological conditions for plant adaptation. They are characterized by the lack of soil as well as nutrients and moisture, and the absence of a humus layer [5]. Unique species from the family Orchidaceae, such as narrow-leaved Helleborine (Cephalanthera longifolia; Figure 1D) and Pontic Helleborine (Epipactis pontica; Figure 1E) which grows on the Fe deposit in Sirk, dark-red Helleborine (E. atrorubens; Figure 1C) from the Ni-Co deposit Dobšiná and Pb-Zn deposit Jasenie (E. atrorubens; Figure 1A) and Lesser Butterfly orchid (Platanthera bifolia; Figure 1B) from the Pb-Zn deposit Jasenie which grows on the initial soils (rankers) and tolerates high concentration of The fungi provide the basic physiological function for the orchids in the uptake of needed nutrients and water, otherwise inaccessible to plants [7], facilitate the emergence and survival of vegetation under stressful conditions [8], stabilize waste material through a network of veneers and improve its structure by producing substances that bind to soil particles and lead to the formation of soil aggregates [9].
The microorganisms are known to detoxify metals by several mechanisms including ion exchange, chelation, adsorption, crystallization, valence transformation, extra-and intracellular precipitation and active absorption [10]. The sorption and accumulation of metals depend on various factors such as pH, temperature, organic matter, ion species and the presence of other ions in solutions that may be in competition. They develop adaptation by temporarily altering their developmental patterns or modifying physiological characteristics depending on the toxicity of metals, which is affected by the concentration and form of salts in which the metal exists [11].
Bacteria play a key role in the element biotransformation, biogeochemical cycling, metal and mineral transformation, biological weathering and formation of the soil and sediment [12]. They have high importance in industrial processes, bioremediation, and heavy metal tolerance [13].
To identify fungi and bacterial communities the Illumina MiSeq platform was used. It is a scalable, ultra-high-throughput, cost-effective and a powerful tool to detect soil microbial communities at greater depth and with more detailed sequence information [14,15]. It has been successfully applied to identify microbial communities almost everywhere [16,17], including heavy metal contaminated environments [18,19].
The aim of this paper is to identify the composition of bacterial and fungal communities present in the roots and the surrounding soil of the orchids growing on heavy metal contaminated areas of mining heaps. To detect these communities we used 16S rRNA gene and ITS next-generation sequencing approach. At the same time, our aim was to compare the data of fungal and bacterial communities with the pH value in the Pb-Zn mining heap Jasenie-Soviansko, Ni-Co mining heap Dobšiná-Dobrá nádej and Fe mining heap Sirk-Železník. The broader goal of the study was to determine the unique microbial content of plant root collected from contaminated soil as potential biomarker applicable in further studies or monitoring efforts.

Site Description
Plants, soils, and geological bedrock investigated in this study originated from three abandoned mining sites in Slovakia, Central Europe (Pb-Zn deposit Jasenie-Soviansko, Ni-Co deposit Dobšiná-Dobrá nádej and Fe deposit Sirk-Železník).
The Pb-Zn deposit Jasenie-Soviansko (N 48 • 89 27 , E 19 • 44 15 ) is situated in the westerň Dumbier part of the Nízke Tatry Mts., 7 km north from the village Jasenie. The deposit consists of several old mining waste dumps, but orchids occurred only in the heap of the Emil adit, which is located at an altitude of 834 m a. s.l. E. atrorubens occurs mainly on a sharp slope of a mining heap with almost no moss or lower plants. Few orchids (E. atrorubens and P. bifolia) grew on the horizontal part of the waste dump under trees (especially spruce, willow and birch) or above heap material covered with moss, lower vegetation, and trees. E. atrorubens was extremely widespread in the mining heap and more than 1000 individuals grew on an area about 500 m 2 . Technogenic substrate is formed mainly of metamorphic rocks of the crystalline basement of the Tatric unit of the Western Carpathians (mostly orthogneisses and migmatites) with fragments of mineralized rocks and also partially ore veins. Parts of ore veins and hydrothermally altered rocks formed hydrothermal carbonates (siderite, dolomite, ankerite, calcite), quartz and baryte and the sulphides are represented mainly as tetrahedrite, galena, bournonite, pyrite, chalcopyrite and sphalerite [20][21][22]. At the sampling site of the orchid and the substrate, the size of the rock fragments was up to~7 cm. E. atrorubens grew in a bright habitat, without the presence of other higher plants, mosses and lichens. The slope of the mining heap with orchids was oriented to the south (SSW).
The Ni-Co deposit Dobrá nádej in Dobšiná is located in the Spišsko-Gemerské Rudohorie Mts., in the central part of the Slovak Ore Mountains Mts. in the Inner Western Carpathians (N 48 • 84 31 , E 20 • 39 08 ). The mining heap is situated at an altitude of 807 m a.s.l. Orchids occur on the vertical part of a heap without vegetation but also in the horizontal part of a waste dump between low spruces and sparse low vegetation represented mainly by grasses. Samples of the orchid E. atrorubens and technogenic substrate were taken from the mining heap, where metamorphic rocks (phyllites, acidic metavolcanites and metasediments) dominate the Gemer superunit. Hydrothermal minerals were represented by siderite, ankerite, dolomite, quartz, hematite, Fe-Ni-Co sulphides, pyrite, chalcopyrite and minerals of tetrahedrite group [20]. Up to 4 cm was the size of the rock fragments at the sampling point of the plant and substrate. The lighting and botanical conditions at the site and the slope orientation of the mining heap were the same as in the locality Jasenie.
The Fe deposit Sirk-Železník (N 48 • 61 71 , E 20 • 10 54 ) represents a weathered zone of hydrothermal Fe deposit which is located in the Revúcka vrchovina Highlands, 10 km west of Jelšava, near the village Sirk at an altitude 416 m a.s.l. Phyllites, limestones, metaryolite tuffites and metalalydites [23] occurred mostly at this deposit site. Orchids collected for research were found in the forest of a technogenic substrate with a frequent abundance of fungi and incomplete decomposed Appl. Sci. 2020, 10, 7367 4 of 18 plant residues. This initial substrate was located between pieces of limonite-quartz iron ore~8-30 cm in size. No hydrothermal carbonates were present here. Orchids grew in a light deciduous to mixed forest, mostly on a horizontal part of a mining heap between moss-covered pieces of heap material. All individuals of E. pontica grew in one place, in an area of~15 m 2 . C. longifolia grew in a disseminated forest a few meters to tens of meters from the occurrence of E. pontica.

Sampling and DNA Extraction
The samples of the family Orchidaceae were taken from their place of growth. After the plant was collected, the surrounding soil was taken from this area (up to 30 cm in depth). The roots were washed with distilled water to remove remaining soil particles followed by sterilization, according to Cao et al. (2004) [24] with some modifications, with 70% ethanol for 5 min, sodium hypochlorite (2.5% available chlorine) for 5 min, and again with 70% ethanol for 1 min. After that, the roots were washed multiple times with distilled water to remove sterilization agents. Surface-sterilized roots were cut into 1-2 cm pieces and crushed on powder using mortar, pestle, and liquid nitrogen. Isolate II Plant DNA kit (Bioline, Toronto, ON, Canada) was used to extract DNA from these crushed roots. For DNA extraction from soil DNeasy PowerSoil kit (Qiagen, Hilden, Germany) was used.

Chemical Analysis
The pH of the soil was determined in the distilled H 2 O and NH 4 NO 3 . Oxidisable organic carbon (%CO x ) was analyzed by oxidimetry under laboratory conditions. Collected soils contained various heavy metals and potentially toxic elements above limit values as described in detail at Račko et al. (2020) [20].

Polymerase Chain Reaction and Library Preparation for Next-Generation Sequencing
Bacterial 16S rRNA gene and fungal ITS region were amplified using primer pair sequences for the V3 and V4 region [25] and ITS region [26]. We used 16S Amplicon PCR Forward Primer We used the 16S Metagenomic Sequencing Library Preparation protocol (Illumina Inc., San Diego, CA, USA) to create libraries. Index PCR was performed following the manufacturer's instructions using 2x KAPA HiFi HotStart ReadyMix (25 µL, Kapa Biosystems, Wilmington, MA, USA), Nextera XT Index Primers (5 µL per sample, Illumina Inc., San Diego, CA, USA), PCR grade water, and 5 µL of template DNA/cDNA solution in the total reaction volume of 50 µL. PCR Clean-Up 2 was performed as described in the protocol, followed by library validation using Qubit dsDNA high sensitivity assay (LifeTechnologies, Eugene, OR, USA) and Agilent ® HS DNA Chip (Agilent ® Technologies 2100 Bioanalyser, Santa Clara, CA, USA). Finally, DNA libraries were normalized to 4 nM concentration, denatured and processed to the sequencing run on MiSeq instrument (Illumina Inc., San Diego, CA, USA) using a MiSeq Reagent kit v3 (Illumina Inc., San Diego, CA, USA) with the paired-end setting of 2 × 300 bp reads.
Taxonomy of OTUs was assigned by QIIME's pre-fitted sklearn-based taxonomy classifier [32]. The pre-fitted classifier was trained by the Naive-Bayes classifier with default parameters on Silva 16S database (Silva release 132) [33] and ITS database (UNITE 8.2) [34]. Only taxons with <0.1% of all reads per sample were used for bacteria and <0.01% for fungi. Finally, Faith's phylogenetic diversity for all samples was calculated using faith-pd plugin [35]. All computational analyses were written and executed using the SnakeLines framework (v0.9.2) [36,37].

Results and Discussion
Orchidaceae is a family of rare plants and their presence in metal-rich biotopes has ecological and conservation importance [38]. Orchids colonizing mining heaps rely on the fungal mycelium to obtain the necessary nutrients since their root system is not well developed. They belong to metal excluders, and mycorrhizal fungi seem to play an important role in filtering out the heavy metals [38][39][40]. Despite these extreme conditions, the environment is sufficiently tolerable to the population of orchids, soil filamentous fungi and bacteria.
In orchidaceous mycorrhiza, fungi have the ability to support the composition of plants in stressed ecological niches and to increase the plant's condition [41]. The properties of orchidaceous mycorrhiza in mycoheterotrophic plants that allow colonization under stress conditions are changes in root morphology, orchid metabolism, such as enzymatic activities, secretion of organic acids with low molecular weight and phenols, and associations with orchideous mycorrhizal fungus adapted to the environmental conditions in which orchids occur [42,43]. To identify fungal and bacterial communities in the roots of orchids and surrounding soil in heavy metal contaminated areas we used the marker gene sequencing approach which has been the most commonly used method to analyze these communities. It is simple, fast, cost-effective, and well-tested approach [44,45] in comparison to whole-genome sequencing [45,46].
A total of 171 species of fungi were detected from five samples of orchids, belonging to seven phyla, Ascomycota, Basidiobolomycota, Basidiomycota, Chytridiomycota, Monoblepharomycota, Mortierellomycota, and Mucoromycota (Table 1). The highest biodiversity of fungi was situated in the roots of E. pontica (Sirk), and the surrounding soil of E. atrorubens (Jasenie). Moreover, the most abundant phylum of fungi was Basidiomycota in both soil and the roots of orchids. Furthermore, the most abundant species of fungi in the roots of orchids was Cortinarius saturatus, Mycena sp., Mycena citrinomarginata, Mycenella bryophila, Colletotrichum capsici, Trichophaea pseudogregaria, Phacidium sp., Russula solaris, Mortierella sp., Amphinema byssoides, Mycena sanguinolenta, Amphinema sp., and Piloderma byssinum ( Figure 2). Further, in the surrounding soil, it was Cortinarius saturatus, Cortinarius scandens, Helvellosebacina concrescens, Mortierella sp., Cortinarius sp., Helvellosebacina sp., Cuphophyllus virgineus, Tetracladium sp., Colletotrichum capsici, Cortinarius vernus, Penicillium sp., Saitozyma podzolica, and Solicoccozyma terricola ( Figure 3). In addition, we observed that the biodiversity in sampled pairs were consistently higher in soil samples, both in the number of discovered species (median 2.7x higher) and the evenness (median 2.66x higher of Faith's phylogenetic diversity). Decreased diversity could be due to the specific environment in the roots of the orchids.    The pH value is a key factor affecting the plant, fungal and bacterial communities on the mining dumps. Soil fungi can grow over a broad range of pH [47], however, soil bacteria are more sensitive to low pH [48], so fungi can dominate this pH spectrum. Values of pH varied from acidic (4.98) to slightly alkaline (7.81).
A high number (57) of fungal species, found in the surrounding soil, tolerate acidic pH of 4.98. Regarding roots, it was 37. Similar to our study, fungi like Cladophialophora sp., Cladosporium sp., Exophiala sp., Mortierella alpina, Mortierella sp., Mucor sp., Mucor hiemalis, Penicillium sp., Umbelopsis sp. provided a great tolerance to a higher concentration of some heavy metals in high acidic to neutral pH of the soils [49]. In regard to to pH, we did not observe anything significant or different in comparison to other studies [50][51][52]. Organisms from extreme habitats have developed strategies for survival and growth in harsh conditions in the production of organic molecules [53]. They have the ability of adaptation to high salinity, radiation, limited nutrients, extreme temperatures and pressure, and variable values of the acidity [54]. In these contaminated areas, metal-resistant fungi occur which have potential in the process of bioremediation [55].   The benefits of mycorrhizas to both plants and fungi is an uptake of nutrients. The fungi obtain carbon from the plants, and in return, enhance a plant's ability to take up minerals or water, subsequently leading to improvement of plant growth and reproduction [56]. Many species of fungi possess the ability to act as an effective biosorbent of the toxic metal. However, these elements may inhibit their growth and affect their reproduction [57].
The composition of fungi in orchid roots is usually much more uniform for orchid species growing in a forest community in the presence of more species of higher plants and trees than for orchids that form plant monocultures on the stony substrate of mining heaps. This difference is not so significant for the fungi present in the soil.
At the same time, the study of fungi in the roots of different species of orchids from different types of habitats and the surrounding technogenic substrate showed that the only species of fungus that occurred in an increased quantity of all the samples was Cortinarius saturatus. In all other fungi, their quantitative composition in the soils and roots of orchids did not correlate. In many cases, significantly present species in the roots were almost non-existent in the soil, and vice versa.
In the study of fungi from the mining sites [52] and recent study of microscopic fungi from five substrates of old environmental loads [50], species, such as Cladosporium sp., Exophiala sp., Mortierella alpina, Mortierella sp., Mucor hiemalis, Mucor sp., Penicillium spinulosum, Penicillium sp., Phialocephala sp., and Umbelopsis sp. agreed with our results.   The function of the genus Mycena, which is one of the representatives to be present in four samples, is to influence the growth of roots [58]. Despite the great biodiversity of fungal genera present in the individual samples, a comparison of the root and soil samples suggests the specificity of both for the host plant. In another study by Cui et al. 2017 [59], Mortierella sp. effectively reduced the heavy metals and significantly shortened the remediation period and improved the poor habitat of mine tailings. Bioaccumulation and biosorption abilities of Penicillium spp. were also studied [60][61][62].

Bacterial Communities
Bacteria have high importance in industrial processes, bioremediation, and heavy metal tolerance [13]. Bioremediation is the process of biological systems cleaning of the polluted environment by the bacteria and fungi, which are important for reclamation, immobilization and detoxification of heavy metals [63].
A total of 30 species of bacteria were detected from five samples of orchids, belonging to eight phyla, Acidobacteria, Actinobacteria, Bacteroidetes, Gemmatimonadetes, Planctomyces, Proteobacteria, Verrucomicrobia, and Tenericutes ( Table 2). The highest biodiversity of bacteria was in the roots and also surrounding soil of E. atrorubens (Jasenie). Additionally, the evenness in sampled pairs was consistently higher in soil samples (median 1.2x higher of Faith's phylogenetic diversity). However, that was not the cause in the number of discovered species as it was in analysis of fungal communities.  Our results show (Figures 4 and 5) that Candidatus Udaeobacter was the dominant bacterium at the Sirk deposit in the roots of two different orchids as well as in technogenic substrate and a high dominance of bacteria from the family Pyrinomonadaceae in a stony substrate on the mining heap in Dobšiná and the related very dominant abundance of the fungus Trichophaea pseudogregaria in the roots of E. atrorubens. The most abundant species in the roots of orchids was Candidatus udaeobacter, Gemmata sp., Singulisphaera sp., Chthoniobacter sp., Pseudomonas sp., Luteolibacter sp., Nocardioides sp., Pirellula sp. (Figure 4). In the surrounding soil, it was Candidatus udaeobacter, Chthoniobacter sp., Pyrinomonadaceae; RB41, Gemmata sp., and Pirellula sp. (Figure 5). Moreover, Candidatus udaeobacter, Candidatus solibacter, and Chthoniobacter sp. were present in four or more soil samples. Furthermore, 11 species tolerated pH of 4.98.
Bacteria from the genus Pseudomonas induce auxin production by plant growth-promoting bacteria [71]. They are also known as very metabolically active bacteria able to utilize many agrochemicals [72][73][74]. Together with Luteolibacter sp., Vicinamibacter sp., and Sphingomonas sp. they were identified by many other studies as an oil-degrading bacteria which is important for bioremediation [75][76][77]. Acidibacter sp. and Candidatus solibacter were suitable to be grown in oil-contaminated soils [78].
The genera representing the heterotrophic strain Planctomycetes have the ability to reduce sulfur and, in particular, play an important role in sugar metabolism [79]. The overlap of the genera present indicates a close connection of the plant with the microbiome, therefore the plant is able to adapt quickly to changing conditions. Nitrosomonadaceae is a strictly aerobic bacteria which has a chemolithoautotrophic metabolism using ammonia as an energy source producing nitrite [80]. Nocardioides spp. are often isolated as plant endophytes and are known to be capable of suppressing crop pathogens [81]. Together with the members of the genus Flavobacterium, they are widely distributed in various environments such as soil, water, and sediment [82].
According to E. atrorubens which was studied at two different localities, we showed that it is not important what species of mycorrhizal fungi and bacteria are associated with orchids, but their presence in the substrate. Therefore, the studied orchids can grow in technogenic or anthropogenic soils and substrates as well as other extreme habitats. The species composition of mycorrhizal fungi in the roots of E. atrorubens is very variable in each locality apart from the Cortinarius saturatus species, which is present in all examined roots of orchids. No other species had a significant quantitative representation in both localities of this orchid. Our research has shown that E. atrorubens can use either one dominant fungus (Dobšiná deposit, up to almost 65% representation of the species Trichophaea pseudogregaria) or even a larger number of species of quantitatively under-represented fungi (Jasenie deposit) for its enormous occurrence in these localities. Species, such as Candidatus solibacter, Bradyrhizobium sp., Burkholderia sp., Gemmatimonas sp., Fimbriiglobus sp., Flavobacterium sp., Pseudomonas sp., Sphingomonas sp., Pedobacter sp., and Verrucomicrobia sp. were also detected in heavy metal contaminated soils [64][65][66][67][68]. A bacterium with the highest relative abundance was Candidatus udaeobacter, which is an abundant soil bacterium that reduces its requirement for soil organic carbon by acquiring costly amino acids and vitamins from the environment [69]. Pirellula sp. found in both roots and soil gains energy from the aerobic oxidation of mono-or disaccharides derived from the cleavage of sulfated polymers produced by algae [70].
Bacteria from the genus Pseudomonas induce auxin production by plant growth-promoting bacteria [71]. They are also known as very metabolically active bacteria able to utilize many agrochemicals [72][73][74]. Together with Luteolibacter sp., Vicinamibacter sp., and Sphingomonas sp. they were identified by many other studies as an oil-degrading bacteria which is important for bioremediation [75][76][77]. Acidibacter sp. and Candidatus solibacter were suitable to be grown in oilcontaminated soils [78].
The genera representing the heterotrophic strain Planctomycetes have the ability to reduce sulfur and, in particular, play an important role in sugar metabolism [79]. The overlap of the genera present indicates a close connection of the plant with the microbiome, therefore the plant is able to adapt quickly to changing conditions. Nitrosomonadaceae is a strictly aerobic bacteria which has a chemolithoautotrophic metabolism using ammonia as an energy source producing nitrite [80]. Nocardioides spp. are often isolated as plant endophytes and are known to be capable of suppressing

Conclusions
Due to its adaptation and survival strategy, the family Orchidaceae has become an interesting target for microbiome identification. In this work, we identified bacterial and fungal communities from the roots and surrounding soil of orchids from five heavy metal contaminated areas of the mining sites Fe deposit Sirk (C. longifolia and E. pontica), Pb-Zn deposit Jasenie (E. atrorubens and P. bifolia), and Ni-Co deposit Dobšiná (E. atrorubens). To sum up, slight deviations in pH of the initial soils do not significantly affect the presence of fungi and bacteria and thus the presence of the studied orchids in these localities. Similarly, the toxic elements (e.g., As, Pb, Cr, Ni, Co, Cu, Fe), in the studied localities, do not affect the occurrence of fungi and bacteria, and also orchids. Furthermore, the study of selected factors influencing the occurrence of orchids showed that the most important factors of the occurrence of orchids in technogenic soils are the presence of a sufficient number of fungi and bacteria, sufficient light and the absence of other plant species in the habitat. Moreover, microbial communities also provide a huge benefit for orchids to be able to grow in these polluted areas. Additionally, some of these microbial communities possess huge biotechnological potential in bioremediation of heavy metal