Introduction

Potentially toxic elements in the environment originate from both natural geological processes and human activities. Natural origins of potentially toxic elements include atmospheric depositions from volcanic sources, dust transport from continental areas or excessive weathering of metal ions, and minerals from rocks (Ernst 1998). However, human routes leading to soil contamination by large amounts of pollutants and chemicals are more aggressive and are nowadays a major problem for agricultural, forest and urban soils. Potentially toxic elements are one of the main causes for concern, since they are persistent in soils and are difficult to remove (Jones et al. 2012). Furthermore, contaminated soil represents a serious threat for the local population inhabiting the area and also negatively affects soil functionality, leading to alterations of several ecosystem processes, including multitrophic interactions. The extent to which the potentially toxic elements are mobilized and become available to exert negative effects depends largely on the soil properties (Andersen et al. 2004).

Multitrophic interactions involving plants, herbivores and parasites have been documented for belowground systems. Plant roots emit various volatile organic compounds, ranging from terpenoids, fatty acid derivatives and sulfur compounds to phenylpropanoids. Emissions are particularly evident in forest soils (Qualley and Dudareva 2008). Volatile organic compounds emissions can be induced by physiological or environmental factors (Rasmann et al. 2005; Laznik and Trdan 2013). Some volatile organic compounds emitted from the plant roots as a response to herbivory, e.g. β-caryophyllene, attract entomopathogenic nematodes (Rasmann et al. 2005; Rasmann and Turlings 2008; Ali et al. 2010). Entomopathogenic nematodes are soft-bodied, non-segmented roundworms that are obligate or sometimes facultative parasites of insects (Kaya and Gaugler 1993). Entomopathogenic nematodes occur naturally in soil; their main orientation system is called chemotaxis. They locate a host in response to carbon dioxide, vibration, volatile organic compounds and other chemical cues (Rasmann et al. 2005; Hallem et al. 2011; Jagodič et al. 2017). Nematodes from two families (Heterorhabditidae and Steinernematidae) have been effectively used as biological insecticides in pest management programmes (Grewal et al. 2005). In addition to plant roots, fungi are also known to be rich producers of volatile organic compounds (Kramer and Abraham 2012). In boreal and temperate forests, the majority of stand-forming tree species live in symbiosis with ectomycorrhizal fungi (Smith and Read 2008). For ectomycorrhizal fungi, volatile organic compounds emissions were detected from mycelium, ectomycorrhizae and fruiting bodies (Splivallo et al. 2011). Volatile organic compounds emitted from ectomycorrhizal fungi affect plant root architecture and morphology (Splivallo et al. 2011; Ditengou et al. 2015), induce plant defence genes, affect bacteria and other fungi and attract insects and mammals (Splivallo et al. 2011). Emission of volatile organic compounds from fungi depends on growth conditions and growth stage (Splivallo et al. 2011; Kramer and Abraham 2012). In environments contaminated with potentially toxic elements, the species composition of ectomycorrhizal fungal communities is changed (Hartley et al. 1997; Colpaert et al. 2011), and ectomycorrhizal colonization of root tips is decreased (Hartley et al. 1997). Therefore, it would be expected that the volatile organic compounds profile and/or concentration emitted from ectomycorrhizal roots will also be affected. This was confirmed in a study by Henke et al. (2015), who reported that aboveground parts of spruce seedlings inoculated with the ectomycorrhizal fungus Tricholoma vaccinum (Schaeff.) P. Kumm. grown in caesium-enriched medium emitted significantly more monoterpenes than those without the fungus or those grown in medium without caesium addition, showing the impact of ectomycorrhizal fungus on plant physiology.

In order to increase our knowledge on the effect of contaminated soil on multitrophic interactions in a terrestrial system, with particular attention to the role of volatile organic compounds, a mesocosm experiment with a multitrophic system composed of (a) European beech (Fagus sylvatica L.) with associated ectomycorrhizal fungi, (b) larvae (grubs) of summer chafer (Amphimallon solstitiale L.), which is a natural root herbivore of different plant species (Laznik and Trdan 2015), and (c) entomopathogenic nematodes (Steinernematidae and Heterorhabditidae) was designed. Beech seedlings were grown in contaminated and non-contaminated soil, where summer chafer larvae were subsequently added. Root-emitted volatile organic compounds were detected using SPME-GC-MS analyses. Volatile organic compounds of interest were tested in a chemotaxis assay with entomopathogenic nematodes. The aims of our investigation were (1) to evaluate growth of seedlings, ectomycorrhizal colonization and ectomycorrhizal communities in different soil types, (2) to assess the differences in volatile organic compounds emissions from beech roots due to soil contamination and root herbivory, and (3) to assess whether the volatile organic compounds from beech roots have any effect on the behaviour of the entomopathogenic nematodes tested.

Materials and methods

Soil collection and chemical analysis

In the mesocosm experiment, two types of soil were used: non-contaminated forest soil (Dystric Cambisol, as classified according to the FAO World Reference Base for Soil Resources) and contaminated garden soil (Urbic Anthrosol). The non-contaminated forest soil was collected from an upper 30-cm layer in urban forest dominated by beech trees on Rožnik Hill near the area of Tivoli Park, Ljubljana, Slovenia, EU (x = 460,055, y = 102,500, Gauss-Kruger coordinate system). Contaminated garden soil was collected from the upper 40 cm of an abandoned garden in the vicinity of a former mine and Pb smelting plant in the city of Žerjav in the Meža Valley, Slovenia, EU (x = 489,300, y = 152,300, Gauss-Kruger coordinate system). The Meža Valley was exposed to more than 300 years of active lead mining and smelting; the activity ceased in 1990 but left behind thousands of hectares of agricultural and forest land polluted with Pb, Zn and Cd (Udovič and Leštan 2009; Voglar and Leštan 2013; Jelušič and Leštan 2015). Potentially toxic elements and pedological analysis of soil was performed at the beginning of the mesocosm experiment. Soil samples were air-dried and sieved to 2 mm (ISO11464 2006). Soil pH was measured in a 1/2.5 (w/v) ratio of soil and 0.01 M CaCl2 suspension (ISO10390 2005). Easily extractable P (P2O5) in the soils was determined spectrophotometrically after calcium-acetate-lactate extraction, as described in ÖNORM L 1087 (1993). In short, plant-available P was extracted by shaking 5 g of dried soil for 2 h at 180 rpm in Ca acetate and Ca lactate solution. The extracts were filtered through pleated filter paper (MN 619 G ¼; Macherey-Nagel, Germany), and 1.0 mL of filtrate was mixed with 16 mL of ammonium heptamolybdate and 2 mL of freshly prepared ascorbic acid. The absorbance at 660 nm on a Cary 50 spectrophotometer (Varian Inc., Palo Alto, CA, USA) was used for quantification. Plant available potassium (K2O) in the soils was measured with flame (acetylene/air) atomic absorption spectrometry (AAS, Varian AA240FS, Varian, Inc., Palo Alto, CA, USA) following the protocol of ÖNORM L1087 (1993). For calculations of easily extractable phosphorus and potassium we used standard calculation factors as described in ÖNORM L 1087 (1993). Soil organic carbon was determined after dry combustion using a CNS elemental analyser at 1350 °C (LECO 2000, LECO Corporation, St. Joseph, MI, USA) according to ISO 10694 (1995). Soil cation exchange capacity (CEC), i.e. the sum of the exchangeable cations of a soil, was determined following the protocol of ÖNORM L 1086-1 (2001).

Planting material and collection of summer chafer larvae

For the experiment, 25 three-year old beech (Fagus sylvatica) seedlings were bought from the certified tree nursery “Omorika d.o.o.” located in north-eastern Slovenia, EU (x = 511,597, y = 162,169, Gauss-Kruger coordinate system), where they were sown and grown outside in a dystric brown soil, cambisol. Five seedlings were immediately destructively analysed for ectomycorrhizal fungi, while 20 seedlings were used for the mesocosm experiment.

In September 2016, 50 summer chafer larvae (third larval stage-L3) were collected from forest soil near the town of Logatec, Slovenia (x = 442,525, y = 80,155, Gauss-Kruger coordinate system). The identification of larvae from the Scarabaeidae family was performed following Laznik and Trdan (2015). Larvae were identified to a species level using the raster pattern on the lower side of their abdomen. All collected larvae were transferred into experimental growing pots 1 day after collection.

Mesocosm experiment

Twenty experimental growing pots, equipped with an automatic irrigation system device made in house (Laboratory for Electronic Devices, Slovenian Forestry Institute, Ljubljana, Slovenia), were filled with homogenized soils (non-contaminated and contaminated) up to 18 L in volume per growing pot. In May 2016, beech seedlings were planted and exposed to environmental conditions in Ljubljana, Slovenia (EU) (x = 459,667, y = 101,406, Gauss-Kruger coordinate system). The automatic irrigation system kept soil moisture above 35% relative humidity. In September 2016, larvae of summer chafer were added into five growing pots with non-contaminated soils and into five growing pots of contaminated soils (5 larvae per pot). The experiment ended in November 2016, when destructive sampling was performed. According to the Slovenian Environment Agency (ARSO 2018) the 10-year average (2007-2017) temperature for the location of the field experiment (Ljubljana, Slovenia), with a continental climate, was 11.8 °C, with average maximum temperature of 16.6 °C and average minimum temperature of −7.5 °C and total annual precipitation of 1415 mm.

Determination of potentially toxic elements in soil

For determination of potentially toxic elements, 2 mm fractions of air-dried soil samples were digested in aqua regia solution, consisting of HCl and HNO3 in a 3:1 ratio (v/v). The samples were then filtered through Whatman No. 4 filter paper and diluted with 0.5 M HNO3 to a total volume of 100 mL. The reference material used in inter-laboratory comparisons was FSCC soil reference Sample B (Soil ringtest 2007, ICP Forests). Pseudo-total potentially toxic elements concentrations were determined by either flame or electrothermal atomic absorption spectrometry after digestion of the samples with aqua regia according to ISO 11466 (1995), using atomic absorption spectrometry (AAS Varian DUO 240 FS/Z + GTA, Varian, Inc., Palo Alto, CA, USA). The limits of quantification were 0.01, 10, 2, 10, 2, 100, 4, 2, 100, 2, 33, 300, 100 and 2 mg per kg of soil for Cd, Pb, Zn, Mn, Cu, Fe, Cr, Ni, Al, Co Mg, Ca, K and Na, respectively. All samples were measured in triplicate (one row, one replicate); reagent blank and analytical duplicates were used to ensure the accuracy and precision of the analysis.

Growth of aboveground parts, ectomycorrhizal communities and lateral root morphology

Before the experiment, root systems of five beech seedlings obtained from the tree nursery were destructively sampled and checked for ectomycorrhizal fungi according to the procedure described below.

At the end of the experiment, seedlings were divided into above- and belowground part by scissors. The height of seedlings above the root collar was measured with a tape measure and the diameter of the stem with a calliper. One lateral root per seedling was removed by scissors and kept refrigerated in a plastic bag. Ectomycorrhizal morphotypes were sorted under an Olympus SZH dissecting microscope (Olympus, Tokyo, Japan) and described according to Agerer (1991) and DEEMY (Agerer and Rambold 2004–2017). From each morphotype, one root tip was removed and put into extraction buffer for molecular identification. Molecular identification of ectomycorrhizae was based on PCR amplification and sequencing of the complete internal transcribed spacer (ITS) regions in nuclear ribosomal DNA (Gardes and Bruns 1993). DNA was extracted with a DNeasy Plant Mini Kit (Qiagen, Hilden, Germany). For amplification of the ITS region, the ITS1f and ITS4 primer pair was used following a modified procedure described in Grebenc and Kraigher (2007). Amplified DNA was separated and excised from the agarose gel, purified with the PCR CleanUp System (Promega Corp., Madison, WI, USA) and sent to a commercial sequencing laboratory (Macrogen Inc., Seoul, South Korea). Obtained sequences were compared to sequences in GenBank (http://www.ncbi.nlm.nih.gov) to obtain species or genus of ectomycorrhizal fungi. Phylogenetic analyses were performed separately for all recorded genera with MEGA7 (Kumar et al. 2016) for detailed phylogenetic positioning and identification of the unknown fungal partner in ectomycorrhizae. Reference sequences for ectomycorrhizal morphotypes were deposited in GenBank under accession numbers MK027144 – MK027209.

Each ectomycorrhizal morphotype and the remaining non-mycorrhizal roots were scanned using an Epson Perfection V700 Photo scanner (Seiko Epson Corp., Suwa, Nagano, Japan) in trays filled with water. Scans were quantified with WinRhizo software (Regent Instruments Inc., Ville de Québec, Canada) to obtain abundances of ectomycorrhizal morphotypes, lateral root length, root tip density and branching density. Species richness (Atlas and Bartha 1981) of ectomycorrhizae was calculated as the number of ectomycorrhizal taxa per each seedling. Relative abundances of ectomycorrhizal taxa were calculated for each treatment (data from five seedlings per treatment were pooled).

Beech roots volatile organic compounds analyses

At destructive sampling, the whole root system of beech was removed from the aboveground part with scissors and quickly washed under tap water to remove adhering soil. Then, roots were frozen in liquid nitrogen and finely ground with a ceramic pestle in a mortar (Laznik et al. 2011). About 0.5 g of ground sample was weighed into a 20 mL headspace vial. The vial was crimped and consecutively heated on a heating block at 55 °C for 45 min. At the same time, the solid-phase microextraction (SPME) sampling fiber (100 μm PDMS, Supelco, Bellefonte, PA, USA) was exposed to the analyte vapours in the headspace of the sample vial for the entire duration of sample heating. For injection, the SPME fiber was manually transferred from the vial into the GC injection port and left inside for about 10 min (also for conditioning it) before sampling the next sample.

SPME-GC-MS analyses were performed on a Trace GC (Thermo Electron Corporation, San Jose, CA, USA) gas chromatograph coupled to a DSQ II single-quadrupole mass spectrometer (Thermo) with an electron ionization (EI) ion-source. The column in use was a ZB-5HT-Inferno column (5% diphenyl–95% dimethylsiloxane; Phenomenex, Torrance, CA, USA) with the dimensions 20 m × 0.18 mm i.d., 0.18 μm film thickness. The oven temperature program was as follows: initial temperature 50 °C (hold time 5 min) with a linear gradient of 12 °C min−1 and a final temperature 250 °C (hold time 2 min). The injector was set in splitless mode at 200 °C, with an initial pressure surge of 200 kPa for 1.5 min, and equipped with a dedicated SPME 1 mm i.d. glass liner (Supelco, Bellefonte, PA, USA). The carrier gas (helium) flow and transfer line temperature were 0.6 mL min−1 and 280 °C, respectively. The mass spectrometer ion source was set at 200 °C and 70 eV ionization energy. The scan range and scan rate were 46-300 amu and 5 scans/s, respectively.

Source and maintenance of entomopathogenic nematodes and synthetic volatile organic compounds

Three commercial entomopathogenic nematode species (Steinernema feltiae [Filipjev], S. carpocapsae [Weiser] and Heterorhabditis bacteriophora Poinar) obtained from Koppert B.V. (Berkel en Rodenrijs, The Netherlands) were used in a chemotaxis assay with selected volatile organic compounds. Final-instar larvae of wax moth (Galleria mellonella L.) were used to rear entomopathogenic nematodes (Laznik and Trdan 2016). Infective juveniles of entomopathogenic nematodes were stored at 4 °C at a density of 2000 infective juveniles mL−1. Only infective juveniles that were less than 2 weeks old were used for testing. We calculated the concentration of the entomopathogenic nematodes suspension using the method developed by Laznik and Trdan (2016). Viability of nematodes was determined before the chemotaxis experiment.

Based on the results obtained from SPME-GC-MS analyses of beech roots from the mesocosm experiment, four synthetically produced volatile organic compounds (Sigma Aldrich) were selected for chemotaxis assay: (1) humulene, (2) β-caryophyllene, (3) borneol and (4) camphor. Synthetic volatile organic compounds were applied at a concentration of 0.03 μg mL−1, which is the average concentration of root volatile organic compounds in the rhizosphere (Weissteiner et al. 2012).

Chemotaxis assay

The chemotaxis assay was based on an experiment by O’Halloran and Burnell (2003) and modified by Laznik and Trdan (2016). For the assay, Petri dishes (ø = 9 cm) containing 25 mL of 1.6% technical agar (Biolife, Milan, Italy), 5 mM potassium phosphate (pH 6.0), 1 mM CaCl2 and 1 mM MgSO4 were used. The experimental assay is shown in Fig. 1. Each treatment had five replicates, and all of the experiments were repeated three times. Petri dishes were kept in a rearing chamber (RK-900 CH, Kambič Laboratory equipment, Semič, Slovenia) at 20 °C and 75% RH. After 24 h, nematodes were immobilized by placing the Petri dishes in a freezer at −20 °C for 3 min. Nematodes were counted under a Nikon C-PS binocular microscope (Nikon Corporation, Tokyo, Japan) at 25 × magnification. The specific chemotaxis index was based on an assay developed by Bargmann and Horvitz (1991) and modified by Laznik and Trdan (2016). The chemotaxis index was calculated as follows:

Fig. 1
figure 1

Three circular marks (ø = 1 cm) were made on the bottom of the plate: first in the centre, then on the right and on the left side of the Petri dish at 1.5 cm from its edge. A 10 μl drop of a tested substance at a concentration of 0.03 μgml−1 was placed with a pipette on the right side of the agar surface (treated area), and 10 μl of distilled water (control area) was placed on the left side of the agar surface (both sides are considered outer segments) (Jagodič et al. 2017). The volatile organic compounds were immediately applied to the agar plates before the application of the nematodes (Laznik and Trdan 2016); a 50 μl drop of 100 infective juveniles was placed in the centre of the agar surface (inner segment). In the control treatment, 10 μl of distilled water was applied to the control and treated area and a 50 μl drop of 100 infective juveniles was placed in the centre of the agar surface

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$$ {\displaystyle \begin{array}{l}\left(\%\mathrm{of}\ \mathrm{infective}\ \mathrm{juvenilesinthetreatmentarea}-\%\mathrm{of}\mathrm{infectivejuvenilesinthecontrolarea}\right)/\\ {}100\%\end{array}} $$

The chemotaxis index varies from 1.0 (perfect attraction) to −1.0 (perfect repulsion), and in the experiments described here, the compounds were classified as follows: ≥ 0.2 as an attractant, from 0.2 to 0.1 as a weak attractant, from 0.1 to −0.1 as having no effect, from −0.1 to −0.2 as a weak repellent and ≤ −0.2 as a repellent to entomopathogenic nematodes (Laznik and Trdan 2016).

Statistical analysis

All the statistical analyses were performed using the Dell Statistica data analysis software system version 13.2 for Microsoft Windows (Dell Inc., Tulsa, OK, USA).

Soil potentially toxic elements determination

To analyse the differences in concentrations between the non-contaminated forest soils and contaminated garden soils with biotic agent and without biotic agent, a Student t-test was conducted. Differences between treatments were considered significant at p < 0.05.

Aboveground growth, ectomycorrhizal communities and lateral root morphology

ANOVA was applied with soil contamination and biotic agent as independent factors and their interaction checked. Data were log transformed where necessary to achieve the criterion of equality of variances. Equality of variances was tested with the Levene test (p < 0.05). As a post-hoc test, the Tukey HSD test was used.

Chemotaxis assay

For all the treatments and controls, preferential movement of nematodes from the inner to the outer segments of the Petri dish (i.e. a directional response) was determined using a paired Student t-test comparing the percentage of infective juveniles in the inner versus the outer segments (differences between treatments were considered significant at p < 0.05). Additionally, to compare the response levels among different species, the average percentage of infective juveniles that moved to the outer segments or stayed in the inner segments was calculated for each dish. The data were compared through an analysis of variance (ANOVA, p < 0.05) (Laznik and Trdan 2016). We tested the significance of individual factors (volatile organic compounds, nematode species, temporal, and spatial replication) and their interactions. According to the ANOVA outputs only the interaction volatile organic compound x nematode species was significant and interpretable.

Additionally, ANOVA was performed on the chemotaxis index values to compare the response among the different entomopathogenic nematode species to the tested volatile organic compounds; the means were separated by Duncan’s multiple range test with a significance level of p < 0.05 (Laznik and Trdan 2016; Jagodič et al. 2017). The same ANOVA analysis (regarding factors and interactions) was used as for a directional response. The data are presented as the mean ± S.E.

Results

Soil properties

Pedological analysis showed that the non-contaminated forest soils had lower pH than contaminated garden soils (Table 1), lower plant-available phosphorus (101 and 573 mg kg−1 P2O5, respectively) and lower potassium (197 and 205 mg kg−1 K2O, respectively). However, the opposite was the case for soil organic matter (SOM) (21% and 6.8%, respectively), while the cation exchange capacity was comparable between both types of soils (29.5 and 28.5 cmolc kg−1, respectively). Analyses of potentially toxic elements (Table 1) revealed high Cd, Pb and Zn concentrations in contaminated garden soil.

Table 1 Chemical properties of non-contaminated forest soil with biotic agent (NC-B) and without biotic agent (NC) and contaminated garden soils with biotic agent (C-B) and without biotic agent (C)
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Growth of aboveground parts, ectomycorrhizal communities and lateral root morphology

Seedling height was significantly affected by soil contamination and by the interaction between soil contamination and biotic agent (summer chafer larvae) (Table 2). Seedlings grown in non-contaminated soil where biotic agent was absent were significantly higher compared to seedlings grown in contaminated soil (regardless of biotic agent presence). Stem diameter was significantly lower for seedlings grown in contaminated soil and no effect of biotic agent was observed (Table 2).

Table 2 Above- and belowground parameters of beech seedlings (mean ± S.E., n = 5) grown in non-contaminated (NC) and contaminated (C) soil in the absence or presence of biotic agent (B), i.e. summer chafer larvae
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Before the onset of the experiment, five ectomycorrhizal taxa were identified on seedling root systems: Hebeloma sacchariolens Quél., Cortinomyces niveus (Vittad.) Bougher & Castellano, Inocybe curvipes P. Karst., Scleroderma areolatum Ehrenb. and Tuber sp. Scleroderma areolatum and Tuber sp. were present in all five seedlings (Fig. 2). According to phylogenetic analyses, Tuber sp. found in our study correspond to Tuber menseri nom. prov. (Bonito et al. 2010; Healy et al. 2016); see Supplementary material (Tab. S1).

Fig. 2
figure 2

Ectomycorrhizal community structure of beech seedlings grown in non-contaminated (NC) and contaminated (C) soil in the absence or presence of biotic agent (summer chafer larvae)

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At the end of the experiment seven taxa were recorded, Cenococcum geophilum Fr. and Peziza sp. in addition to the taxa stated above. Six taxa were present in non-contaminated soil, while only three in contaminated soil (Tuber menseri nom. prov., Scleroderma areolatum and Peziza sp.).

The percentage of mycorrhizal colonization varied from 0.00 to 89.2% for each plant separately, but from 37.2 to 77.4% when data for each treatment were pooled (Table 2). The lowest mycorrhizal colonization was detected in contaminated soil in the absence of summer chafer larvae, but this difference was not statistically significant (Table 2). Species richness was significantly lower in contaminated soil (Table 2), but regardless of treatment, the dominant taxon was Tuber menseri nom. prov. (relative abundance from 72.3 to 91.8%), followed by S. areolatum (from 8.22 to 20.7%).

Soil contamination significantly decreased the length of the lateral roots. However, root tip density was higher in contaminated than in non-contaminated soil. In addition to soil contamination, higher root tip density was also observed due to biotic agent (summer chafer larvae) (Table 2).

Beech roots volatile organic compounds

Analyses of volatile organic compounds in samples by means of solid-phase microextraction (SPME) sampling yielded 14 different compounds from several chemical classes, but mostly terpenes, terpenoids, aldehydes and aromatic compounds (Table 3). In the class of terpenes, levels of humulene and β-caryophyllene were essentially unchanged in dependence on biotic agent (summer chafer larvae). Among the terpenoids, the borneol level was low in contaminated soil without biotic agent, while in the case of biotic agent (in contaminated soil), its level drastically increased; in contrast, in non-contaminated soil, its levels remained comparable between treatments with and without biotic agent. Camphor levels moderately increased in the case of biotic agent in both types of soil. Aldehyde levels, namely 7,10-hexadecadienal and cis-9-hexadecenal, decreased in the presence of biotic agent in both types of soil, while other detected aldehyde levels (e.g. tetradecanal) remained consistent.

Table 3 Results of SPME-GC-MS analysis of volatile organic compounds emitted by beech seedling roots in non-contaminated forest soil with biotic agent (NC-B) and without biotic agent (NC) and contaminated garden soils with biotic agent (C-B) and without biotic agent (C)
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An evident dependence on biotic agent (increase, for both soils) was demonstrated mostly for aromatic compounds (2-tert-butyl-dimethoxy-benzene, 2-isopropyl-4-methyl-anisole, 2-isopropyl-5-methyl-anisole) and cis-p-menth-2,8-dienol. In contrast, m-di-tert-butyl-benzene showed an apparent erratic behaviour (see Table 3). There were also two compounds which were detected only in contaminated soil with biotic agent, cineole and cyclo-octasulfur. The presence of the latter is rather surprising, since it should be of inorganic origin. However, recent advances have indicated that the soil is a huge reservoir and source of biogenic volatile organic compounds, formed from decomposing litter and dead organic material or synthesized by underground living organisms or organs and tissues of plants (Peñuelas et al. 2014). Microorganisms and the plant root system are the major sources for biogenic volatile organic compounds.

Movement of entomopathogenic nematodes and chemotaxis index

Results from the mesocosm confirmed the presence of several volatile organic compounds, including humulene, β-caryophyllene, borneol, and camphor. The directional movement of entomopathogenic nematodes species from inner to outer segments in response to selected volatile organic compounds was significantly influenced by different factors: nematode species, volatile organic compounds, and their interaction (Table 4). A Student t-test showed that the least mobile nematode species was Steinernema feltiae. The average percentage of infective juveniles in the outer segments was 13.5 ± 0.5%. The other tested nematode species were more mobile (S. carpocapsae: 31.0 ± 0.9%; H. bacteriophora: 32.5 ± 1.3%). Volatile organic compounds also significantly influenced the movement of infective juveniles. In humulene treatments on average 28.6 ± 2.2% of infective juveniles moved to outer segments. On the other hand, in camphor treatments on average only 22.7 ± 1.4% of infective juveniles moved from the inner segments.

Table 4 ANOVA results for the directional movement of infective juveniles from the inner to the outer segments of the Petri dish (df for the error term: 224)
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The chemotaxis index values were influenced by different factors: volatile organic compounds and interactions between nematode species and volatile organic compounds (Tables 5 and 6). After 24 h we observed that there were no significant differences in chemotaxis index values among nematode species. Volatile organic compounds influenced the chemotaxis index values. β-caryophyllene proved to be an attractant for S. carpocapsae (chemotaxis index = 0.21 ± 0.02), while humulene was found as a weak attractant for H. bacteriophora (chemotaxis index = 0.17 ± 0.02).

Table 5 ANOVA results for the chemotaxis index values (df for the error term: 224)
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Table 6 Effect of different volatile organic compounds on the chemotactic response of the entomopathogenic nematodes species after 24 h
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Discussion

In our study, we combined the effects of contaminated soil with biotic agent, the larvae of summer chafer, which feed on tree roots (Laznik and Trdan 2015), to observe changes in belowground chemical communication on a multitrophic level. Contaminated garden soils used in our mesocosm experiment were already used in previous studies, e.g. for simulation of the ageing processes, soil recycling and as a substrate for greening with ornamental plants and grasses by Udovič and Leštan (2009); Voglar and Leštan (2013); and Jelušič and Leštan (2015). The aqua regia extractable content of potentially toxic elements in soils indicated high Cd, Pb and Zn pseudo-total concentrations of contaminated garden soils compared to non-contaminated forest soils. The high concentrations of potentially toxic elements have a potential impact on seedling growth, microbial communities (including ectomycorrhizal fungi), underground chemical communication on the multitrophic level and attraction behaviours of entomopathogenic nematodes. Expected significant differences in soils pH were found between the two types of soils, with higher pH of contaminated soil. Although solubility of Cd, Cu, Ni, Pb and Zn is generally lower at higher pH (McBride 1989; Adriano 2001), the pH of rhizosphere soil is acidic (Hinsinger et al. 2003). Cd and Zn cations are potentially toxic elements sensitive to the amount of electrostatic binding sites provided by the organic matter and less susceptible to complexation by dissolved organic carbon (Andersen et al. 2004). On the other hand, Cu, Ni and Pb have a high tendency to form complexes with DOC (Adriano 2001; Andersen et al. 2004). Contrary to other soil parameters, cation exchange capacity was comparable for contaminated and non-contaminated soils. As an important measure of soil fertility, i.e. a large number of plant essential nutrients are taken up as cations and are held on the surfaces of soil minerals, within the crystal framework of some mineral species or as a part of the certain organic compounds (Chapman 1965), it indicated that the potential fertility of both soils was more or less the same.

Tree seedlings planted in contaminated soils are often able to survive for long periods, but their growth is significantly reduced (Dickinson et al. 1991). Although our study was concentrated on belowground parts, aboveground parts were a clear indicator of deteriorated growth conditions, mainly due to changed soil conditions (Table 2). Use of contaminated soil significantly reduced stem height and stem diameter. Similarly to our results, three-year-old seedlings of Fagus sylvatica grown in substrates contaminated with Cd and Pb have shown reduced stem diameter at pH 3, but not at pH 5, and at both pH values when Cd and Pb were combined. On the other hand, no effect was observed on stem elongation (Breckle and Kahle 1992). In the same study, negative effects on elongation of beech primary roots were observed, as well as on root architecture. Roots were more branched and more dense, compact. Even though in our study branching density was not changed, we were able to observe higher root tip density and reduced length of lateral roots. Tree root stunting was also reported as a common effect of potentially toxic elements by Dickinson et al. (1991) and reduced root length was reported for seedlings of Quercus robur L. grown in hydroponic solution containing 0.5 mg Cu L−1 (Wisniewski and Dickinson 2003).

The prevailing ectomycorrhizal taxon of beech seedlings in all conditions, Tuber menseri nom.prov. from /puberulum lineage of the genus Tuber, is a north American native species that has been introduced to Europe and New Zealand and was reported from a broad range of host species (Bonito et al. 2010; Healy et al. 2016). The representatives of the three taxa that persisted in contaminated soil (Tuber sp., Scleroderma sp., and Peziza sp.) have already been reported from brownfield sites contaminated with potentially toxic elements (Jones and Hutchinson 1986; Krpata et al. 2008; Marescotti et al. 2013; Foulon et al. 2016). In addition to potentially toxic elements contamination, the presence of ectomycorrhizal fungal taxa and their species richness could also be affected by soil organic matter content, water retention capacity and soil nutrient status, which is related to soil pH (Erlandson et al. 2016). These are all parameters that significantly differed in our soils.

Commercially important species from the genus Tuber are generally well characterized regarding emissions of volatile organic compounds (Splivallo et al. 2011). For mycelium of Tuber borchii Vittad. from the same (/puberulum) lineage as T. menseri nom. prov., 29 different volatile organic compounds have been reported (Tirillini et al. 2000). Mauriello et al. (2004) have analysed volatile organic compounds from sporocarps of T. borchii and reported large variability among samples. Contrary to findings of Tirillini et al. (2000), they were able to detect tetradecanal, a compound that was a major peak in our samples (16.9 min). β-caryophyllene and δ-cadinene has been reported for mycelium of ectomycorrhizal fungi Laccaria bicolor (Maire) P.D. Orton (Ditengou et al. 2015) and Paxillus involutus (Batsch) Fr. (Müller et al. 2013). As beech seedlings from contaminated and non-contaminated soil in our experiment had the same highly dominant ectomycorrhizal taxon, this could be the reason why volatile organic compounds profiles of both soils without biotic agent were very similar.

Quantity and quality of volatile organic compounds that are released from vegetative plant parts and roots can change dramatically when plants are damaged (Turlings et al. 1995). The effects of summer chafer larvae on plant roots were observed in our case as significantly increased root tip density, which can be explained as compensatory growth following root damage (Johnson et al. 2016). Analyses of volatile organic compounds in samples by means of SPME sampling yielded 14 different compounds from several chemical classes, but mostly terpenes, terpenoids, aldehydes and aromatic compounds. Several compounds (humulene, β- caryophyllene, and tetradecanal) were found in both soil types (contaminated/non-contaminated) in the presence/absence of biotic agent. On the other hand several volatile organic compounds (camphor, 2-tetr-butyl-dimethoxy-benzene, cis-p-menth-2,8-dienol, 2-isopropyl-1-methoxy-4-methylbenzene, and 2-isopropyl-5-methyl-anisole) were represented only when biotic agent (summer chafer larvae) was present. Monoterpene cineole, one of the two compounds which were detected only in contaminated soil with biotic agent, was previously reported to be released from Arabidopsis thaliana (L.) Heynh. roots as a response to interaction with the plant bacterial pathogen Pseudomonas syringae Van Hall and the aphid Diuraphis noxia Kurdjumov (Steeghs et al. 2004). The discovery of cyclo-octasulfur (S8) in our samples from contaminated soil could suggest the presence of sulfur-oxidizing bacteria. Elemental sulfur is the main sulfur compound oxidation product of acid-hydrolysable metal sulfides by sulfur-oxidizing bacteria (Schippers and Sand 1999). Elemental sulfur is stored in bacterial globules as S8 (George et al. 2008; He et al. 2010).

Furthermore, we investigated the influence of synthetic volatile organic compounds excreted by beech seedling roots on the movement of three different entomopathogenic nematode species, S. feltiae, S. carpocapsae and H. bacteriophora. The results of our Petri dish assay showed that the movement and chemosensation of infective juveniles toward and away from volatile organic compounds that were emitted by roots of beech seedlings varied depending on the volatile organic compounds and their interaction. β-caryophyllene proved to be the most attractive compound for S. carpocapsae. Several different studies showed that β-caryophyllene is an attractant for different entomopathogenic nematode species (Rasmann et al. 2005; Laznik and Trdan 2013). Laznik and Trdan (2013) reported that mechanically damaged maize roots release β-caryophyllene. In their investigation β-caryophyllene proved to be an attractant for the entomopathogenic nematodes S. carpocapsae and H. bacteriophora. In a related study, Rasmann et al. (2005) reported that insect-damaged maize roots emit the volatile organic compound β-caryophyllene, which attracts the entomopathogenic nematode species Heterohabditis megidis. Attraction of entomopathogenic nematode species towards humulene was weak in our investigation (weak attraction only to H. bacteriophora), which indicates that this compound could not have an important role on the movement of entomopathogenic nematodes towards the roots of beech seedlings.

The movement of infective juveniles toward different volatile organic compounds was also influenced by the species of entomopathogenic nematodes. S. feltiae proved to be the least mobile species. After 24 h, only 13% of S. feltiae infective juveniles moved to the outer segment. The most mobile species were S. carpocapsae and H. bacteriophora, where approximately 31% of infective juveniles moved to the outer segment. Our results regarding the nematode movement coincide with those in the investigation by Wilson et al. (2012), where they proposed that many species will show different behaviours depending on the substrate in which they forage.

Conclusion

Growth in contaminated soil resulted in decreased height of beech seedlings, reduced stem diameter, decreased length of lateral roots and increased root tip density. Presence of summer chafer larvae increased root tip density as well and negatively affected the height of beech seedlings. Contaminated soil reduced species richness of ectomycorrhizal fungi, but did not change the dominance of Tuber menseri nom. prov. The majority of volatile organic compounds emitted by the beech roots were detected regardless of treatment (e.g. tetradecanal, humulene, β-caryophyllene, etc.). The level of borneol drastically increased in contaminated soil combined with summer chafer larvae. Camphor levels moderately increased in the case of biotic agent in both types of soil. Of four tested volatile organic compounds (humulene, β-caryophyllene, borneol, camphor), β-caryophyllene was an attractant for the entomopathogenic nematode S. carpocapsae and humulene was a weak attractant for H. bacteriophora. As both compounds were present in all treatments, this finding implies root herbivore control in the rhizosphere even in the absence of stress factors, such as direct herbivory or soil contamination.

Industrialization and associated activities such as urbanization are associated with a variety of effects on the soil system, including pollution, conversion of indigenous habitats to various forms of land use, habitat fragmentation and loss and soil community changes (McIntyre 2000). Plant roots emit an incredible variety of compounds known to affect interactions between plants (Erb et al. 2013; Hiltpold et al. 2013) and other organisms (Bonkowski et al. 2009). Hence, volatile organic compounds may be excellent vehicles in the communication between organisms in the rhizosphere. In our study we focused on the volatile organic compounds emitted by beech roots. It is quite possible that other soil-dwelling organisms, i.e. nematodes, also produce volatile organic compounds, but evidence to support this is currently lacking. Understanding more of the complex interactions would not only allow a better understanding of the rhizosphere but could also offer ecologically sound alternatives in pest management in agriculture and forestry (Furlan and Kreutzweiser 2015; Hiltpold et al. 2013; Rasmann et al. 2012). Application near plant roots of capsules containing an attractant (e.g. β-caryophyllene) would help to optimize the use of biological control agents (entomopathogenic nematodes) in plant protection. A further major challenge is to correctly identify the origin of any particular volatile organic compound belowground, especially since many volatile organic compounds are produced only as a result of interactions.