Available online at www.sciencedirect.com Bioresource Technology 99 (2008) 6391–6399 Cultivation of high-biomass crops on coal mine spoil banks: Can microbial inoculation compensate for high doses of organic matter? Milan Gryndler a,1, Radka Sudová b, David Püschel b,*, Jana Rydlová b, Martina Janoušková b, Miroslav Vosátka b b a Institute of Microbiology, Academy of Sciences of the Czech Republic, Vı́deňská 1083, 14200 Prague, Czech Republic Institute of Botany, Academy of Sciences of the Czech Republic, Department of Mycorrhizal Symbioses, 25243 Pruhonice, Czech Republic Received 29 March 2007; received in revised form 21 November 2007; accepted 22 November 2007 Available online 21 February 2008 Abstract Two greenhouse experiments were focused on the application of arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR) in planting of high-biomass crops on reclaimed spoil banks. In the first experiment, we tested the effects of different organic amendments on growth of alfalfa and on the introduced microorganisms. While growth of plants was supported in substrate with compost amendment, mycorrhizal colonization was suppressed. Lignocellulose papermill waste had no negative effects on AMF, but did not positively affect growth of plants. The mixture of these two amendments was found to be optimal in both respects, plant growth and mycorrhizal development. Decreasing doses of this mixture amendment were used in the second experiment, where the effects of microbial inoculation (assumed to compensate for reduced doses of organic matter) on growth of two high-biomass crops, hemp and reed canarygrass, were studied. Plant growth response to microbial inoculation was either positive or negative, depending on the dose of the applied amendment and plant species. Ó 2007 Elsevier Ltd. All rights reserved. Keywords: Compost; Lignocellulose papermill waste; Phalaris arundinacea; Cannabis sativa; Mycorrhizal symbiosis 1. Introduction The extensive surface mining of brown coal in northern Bohemia, Czech Republic, in the second half of the 20th century resulted in the formation of vast areas of spoil banks. They are composed mostly of grey Miocene clays, with characteristics (e.g. low pH and fertility, vulnerability to erosion and low drainage ability) that make these sites unfavorable for plant growth. The application of organic amendments is an effective, yet costly, method for improv- * Corresponding author. Tel.: +420 271015333; fax: +420 271015332. E-mail addresses: gryndler@biomed.cas.cz (M. Gryndler), sudova@ ibot.cas.cz (R. Sudová), puschel@ibot.cas.cz (D. Püschel), rydlova@ ibot.cas.cz (J. Rydlová), janouskova@ibot.cas.cz (M. Janoušková), vosatka@ibot.cas.cz (M. Vosátka). URL: http://www.ibot.cas.cz/mykosym/ (D. Püschel). 1 Tel.: +420 296442382/2652. 0960-8524/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2007.11.059 ing the physical–chemical and microbiological properties of degraded soils. It helps to form functional soil and establish a stable plant cover and enable utilization of spoil banks, e.g. for planting of high-biomass or technical crops. The beneficial effects of organic amendments include decreased soil bulk density and increased water-holding capacity, higher aggregate stability and increased microbial and enzymatic activities (Caravaca et al., 2002, 2003a). The establishment of plant cover in spoil bank substrates can be further facilitated by beneficial soil microorganisms – arbuscular mycorrhizal fungi (AMF) and plant growth promoting rhizobacteria (PGPR). Due to the extensive network of AMF mycelium in the soil, colonized plants are able to effectively exploit nutrients and water from soil (Smith and Read, 1997). AMF can improve soil structure (Wright and Upadhyaya, 1998) and the stability of man-made soils against erosion (Enkhtuya et al., 2005). The ability of AMF to persist in soils with adverse 6392 M. Gryndler et al. / Bioresource Technology 99 (2008) 6391–6399 characteristics as well as their positive effects on plant growth have been repeatedly demonstrated (e.g. Entry et al., 2002; Turnau and Haselwandter, 2002). PGPR can influence plant growth by fixation of aerial nitrogen, increasing nutrient availability in the rhizosphere, affecting root growth and morphology or supporting plant symbiosis with other beneficial microorganisms (Vessey, 2003). The rate of these effects of PGPR can be further modified by AMF; e.g. it was found that the fixation of aerial nitrogen by symbiotic Rhizobium was increased by the presence of AMF (Barea et al., 1989). Positive effects of various organic soil amendments (e.g. farm yard manure or composted papermill sludge) on plant growth and various soil characteristics in degraded and man-made habitats were often reported (e.g. Caravaca et al., 2003b; Juwarkar and Jambhulkar, 2007). The stimulation of AMF development was observed not only in these anthropogenic sites (e.g. Johnson and McGraw, 1988a,b; Noyd et al., 1996), but also in agricultural systems (e.g. Muthukumar and Udaiyan, 2000, 2002; Tanu et al., 2004). The stimulatory effects of organic matter addition on the development of AMF, especially on the growth of extraradical mycelium (Gryndler et al., 2003; Albertsen et al., 2006), could be ascribed to the improvement of physical properties of the growth substrate, especially the porosity of the soil (Joner and Jakobsen, 1995a) or increased CO2 concentration resulting from mineralization of the added organic matter (Bécard and Piché, 1989). In addition, AMF are able to exploit nutrients released by mineralization of organic matter due to the activities of mineralizing microorganisms, as suggested by Joner and Jakobsen (1995b). On the other hand, when applied in high doses, organic amendments (such as manure, sewage sludge or compost) can be harmful to AMF (Thorne et al., 1998; Sáinz et al., 1998), possibly due to toxic components contained in the added organic matter, as suggested by Lambert and Weidensaul (1991). Despite this negative effect, the standard process of spoil banks reclamation encompasses the application of excessive doses of organic amendments (usually at least 500 tons per hectare). This methodology is well justified by good growth of planted crops and by an addi- tional positive aspect – the fact that the physical structure of lignocellulose papermill waste improves the anti-erosion stability of slopes. Alternative technology applying smaller amounts of organic amendments with comparable yields would, however, be useful. Not only it would preserve microbial communities in the soil, it would also significantly reduce costs for the application of organic matter. In our study we examined the hypothesis that the application of microorganisms – AMF and PGPR – to spoil bank clays could increase plant growth and compensate for reduced doses of organic amendments. In the first step, we aimed to determine the optimal type of organic amendment, which would support the growth of plants, but would not be harmful for AMF. In the next experiment the optimized amendment was added to the original spoil bank clay in different doses corresponding to 500, 200, 100 and 0 t/ha in the field. The effect of microbial inoculation on the growth of two high-biomass crops (hemp and reed canarygrass) in these substrates was monitored. 2. Methods Both Experiments I and II were designed as two-factorial with substrate and microbial inoculation as experimental factors. 2.1. Soil and organic substrates Grey Miocene clay was obtained from the spoil bank of a surface mine near Chomutov, northern Bohemia, Czech Republic. Commercial compost used in the study is commonly used for restoration of clay deposits and was prepared by composting urban wastes (Manufacturer: Bioimpro Ltd., Chomutov, Czech Republic). Lignocellulose papermill waste is a technological waste formed mainly by short fibers unusable for paper production (Manufacturer: Mondi Packaging Paper Štětı́ Ltd., Štětı́, Czech Republic). Neither of these organic amendments contained living propagules of AMF. This was proven by a preliminary bioassay test on maize (data not shown). Chemical characteristics of clay and both organic amendments are presented in Table 1. Table 1 Chemical characteristics of the organic amendments and substrates used in cultivation experiments (clay with 4, 10 and 20% of organic amendments) Cox (%) Ntotal (%) C/N P* (mg kg 1) K* (mg kg 1) Na* (mg kg 1) Ca* (mg kg 1) Mg* (mg kg 1) Organic amendments Compost 7.8 Lignocellulose 7.6 16.9 36 1.54 0.31 11 116 15000 430 8800 1400 440 500 3440 57100 1070 2000 Experimental substrates Clay 7.5 Clay 4org 7.6 7.7 Clay 10org Clay 20org 7.7 0.85 1.79 2.34 4.16 0.07 0.09 0.11 0.18 13 21 21 24 9.5 21.6 41.4 125.0 336 361 412 555 70.2 74.4 76.7 88.8 4054 4618 4944 6248 1354 1286 1215 1154 pHH2 O With the exception of compost and lignocellulose, where for nutrients marked with a * total concentrations are given, the data represent available nutrient concentrations (Mehlich, 1984). M. Gryndler et al. / Bioresource Technology 99 (2008) 6391–6399 2.2. Microorganisms AMF inoculum was prepared by mixing the same doses of inocula of Glomus intraradices (BEG140), G. claroideum (BEG96) and G. mosseae (BEG95). These particular isolates originated from the soil in highly disturbed ecosystems (sedimentation ponds and spoil bank) and are, therefore, likely to be adapted to adverse soil conditions. All AMF isolates were cultivated on maize in a zeolite/sand mixture (1:1 v/v) for 4 months prior to the experiment. The resulting inoculum consisted of cultivation substrate containing spores, ERM and colonized maize roots. AMF inoculum was applied under the seedlings at a dose of 15 ml (Experiment I) or 35 ml (Experiment II) per pot. PGPR (from the international collection deposited in the Research Institute of Crop Production, Prague, Czech Republic) were applied as a mixture of equal amounts of cultures of 9 strains of Sinorhizobium (D134, D135, D472, D504, D528, D538, D556, D557, D563) and/or a mixture of 9 strains of Azotobacter (A1, A160, A202, A203, A205, A241, A242, A243 and A244). The bacterial cultures were applied together with planting at the rate of 5  109 cells (suspended in 5 ml of water) per pot. 2.3. Experiment I The first factor, substrate, comprised 4 treatments: (i) pure clay (substrate referred to as clay), (ii) clay mixed with 20% (w/w) of lignocellulose papermill waste (clay + ligno), (iii) clay mixed with 20% of compost (clay + comp) and (iv) clay mixed with 20% of a mixture (1:1, w/w) of lignocellulose papermill waste and compost (clay + ligno + comp). This dose of organic matter approximately corresponded to 500 metric tons of material per hectare, if mixed with the uppermost 25 cm of the clay layer. This represents the lowest actually applied dose of organic materials used in standard agricultural reclamation in the target coal-mining region. Prepared substrates were put into 10  10 cm plastic pots, 0.5 kg of substrate per pot. The second factor, inoculation, encompassed 5 treatments: (i) non-inoculated control, (ii) inoculation with Azotobacter, (iii) inoculation with Sinorhizobium, (iv) inoculation with AMF and (v) inoculation with all the three inoculants together. Each treatment included 10 replicates. Seeds of alfalfa (Medicago sativa L. cv. Vlasta) were surface-sterilized with 10% solution of sodium hypochlorite and sown in each pot. After 2 weeks, plant numbers were adjusted to 5 per pot. Plants were cultivated for 13 weeks (March–June) in a temperated greenhouse (temperature range from 18 to 28 °C) without supplementary lighting. At the harvest, shoot biomass was cut and weighed after drying to constant weight at 70 °C. Root samples were washed and stained with 0.05% Trypan blue in lactoglycerol (Koske and Gemma, 1989). Mycorrhizal colonization of roots was evaluated by the gridline intersect method 6393 (Giovannetti and Mosse, 1980) under a stereomicroscope at 50  magnification. 2.4. Experiment II The first factor, substrate, comprised 4 treatments: (i) pure clay, (ii) clay mixed with 4% (w/w) of organic matter (substrate referred to as clay 4org), (iii) clay mixed with 10% of organic matter (clay 10org) and (iv) clay mixed with 20% of organic matter (clay 20org). Organic material in this experiment consisted of 1:1 w/w mixture of lignocellulose papermill waste and compost. Applied doses corresponded approximately to 0, 100, 200 and 500 metric tons of material per hectare (at 25-cm tillage depth). Prepared substrates were put into 18  18 cm plastic pots (2.5 kg of substrate per pot). The second factor, inoculation, included 3 treatments: (i) non-inoculated control, (ii) inoculation with AMF and (iii) combined inoculation with AMF, Azotobacter and Sinorhizobium. The effects of the above-mentioned factors were tested on two plants: hemp (Cannabis sativa cv. Beniko), and reed canarygrass (Phalaris arundinacea cv. Palaton S.). These species are fast growing and high-biomass producing plants used as energy crops and hemp also for fiber production (Lewandowski et al., 2003; Ranalli and Venturi, 2004). Moreover, they are able to tolerate adverse conditions of spoil bank substrates and are naturally colonized by AMF (Cooke and Lefor, 1998; Fraser and Feinstein, 2005; Citterio et al., 2005). Seeds of hemp or reed canarygrass were surface-sterilized as in Experiment I and were sown in pots filled with the above described substrates. Each treatment included 10 replicates. After 2 weeks, seedlings of hemp and reed canarygrass were thinned to 1 and 5 per pot, respectively. Plants were cultivated for 12 weeks (August–November) in the greenhouse under similar conditions as in Experiment I. At the harvest, the maximum height of plants of both species was measured. Number of tillers per pot was counted for reed canarygrass. Shoot biomass of both species was dried to constant weight at 70 °C and weighed. Samples of dry biomass were ground and digested in HNO3 and H2O2. Concentrations of phosphorus were assessed spectrophotometrically (630 nm, Specol 211) and concentrations of risk elements (Zn, Cu, Mn, Cd and Pb) were determined by ICP–MS (Perkin–Elmer). Root samples were cut into segments and stained as in Experiment I. Root mycorrhizal colonization was quantified visually using modified segment method (Giovannetti and Mosse, 1980) under a compound microscope at 100  magnification. 2.5. Statistical analysis Data were analyzed by one-way or two-way analysis of variance (ANOVA). Prior to ANOVA, the data were checked for normality; if non-normal distribution was 6394 M. Gryndler et al. / Bioresource Technology 99 (2008) 6391–6399 found, the data were subjected either to log transformation (plant parameters and concentrations of risk elements) or were arcsine transformed (mycorrhizal colonization). The data showing non-normal distribution even after the transformation were analysed by the Kruskal–Wallis test (mycorrhizal colonization of alfalfa, Experiment I). Comparisons among means were carried out using Duncan multiple range test at a significance level of P < 0.05 (STATISTICA 5.1’98 Edition). 3. Results 3.1. Experiment I 3.1.1. Growth of plants Growth of alfalfa was significantly affected both by substrate (F = 109.09, P < 0.001) and by inoculation (F = 2.73, P < 0.05). A significant interaction (F = 2.79, P < 0.01) of these two factors was also observed. In pure clay, inoculation with AMF significantly increased growth of alfalfa plants compared to the uninoculated control treatment (Fig. 1). Similar growth stimulation was observed in treatment inoculated with the combined inoculum (AMF + PGPR). However, the application of either of the bacterial cultures alone did not support plants’ growth. The addition of lignocellulose papermill waste caused a perceptible decrease in plant shoot biomass compared to cultivation in pure clay (Fig. 1). In this substrate (clay + ligno), all microbial inoculation treatments (excluding Azotobacter) positively affected plants’ growth. The addition of compost or compost combined with lignocellulose waste caused considerable improvement in the growth of alfalfa. Interestingly, in these two substrates (clay + comp and clay + ligno + comp) the produced biomass Fig. 1. Experiment I – alfalfa: Shoot dry weight (g per pot) of alfalfa in 4 different substrates: pure clay (clay), clay mixed with lignocellulose papermill waste (clay + ligno), clay mixed with compost (clay + comp) and clay mixed with both organic amendments (clay + ligno + comp). Plants were either left non-inoculated (white columns), or were inoculated with AMF (grey columns), Azotobacter (hatched columns), Sinorhizobium (horizontally striped columns) or all microbial inoculants together (black columns). The data are means of ten replicates ± SE. Columns marked by the same letter are not significantly different within a particular substrate; ns – non-significant differences (P < 0.05; Duncan multiple range test). was similar across all inoculation treatments; the addition of AMF or PGPR had no effect on plants’ growth (Fig. 1). 3.1.2. Mycorrhizal colonization Mycorrhizal colonization of alfalfa roots was significantly affected by substrate (v2 = 117.41, P < 0.001) and inoculation (v2 = 14.85, P < 0.05). In pure clay, the roots of alfalfa became highly colonized (almost 50% of root length) by AMF even in treatments receiving no mycorrhizal inoculum (Fig. 2). This indicated the presence of living mycorrhizal fungi in pure clay substrate. However, the addition of mycorrhizal inoculum (either separately or in combination with PGPR) caused significant increase in mycorrhizal colonization. No difference between inoculation treatments in the level of mycorrhizal colonization was observed in clay with the addition of lignocellulose substrate (Fig. 2). In the treatment with added compost, mycorrhizal colonization was almost eliminated throughout all inoculation sub-treatments. Although the inoculation with AMF increased the level of colonization, it was still lower than 10% (Fig. 2). In the substrate where the added compost was combined with lignocellulose waste (clay + ligno + comp), the inhibitory effect of compost on mycorrhizal colonization was considerably decreased and even the uninoculated treatment reached 30% colonization. The application of AMF or AMF + PGPR further increased the extent of root colonization in this substrate (clay + ligno + comp) (Fig. 2). While the inoculation with AMF or with AMF + PGPR had a significant positive effect on mycorrhizal colonization of alfalfa roots in almost all substrates, the addition of PGPR had no such effect across all substrate treatments. Fig. 2. Experiment I – alfalfa: Mycorrhizal colonization of the roots of alfalfa in 4 different substrates: pure clay (clay), clay mixed with lignocellulose papermill waste (clay + ligno), clay mixed with compost (clay + comp) and clay mixed with both organic amendments (clay + ligno + comp). Plants were either left non-inoculated (white columns), or were inoculated with AMF (grey columns), Azotobacter (hatched columns), Sinorhizobium (horizontally striped columns) or all microbial inoculants together (black columns). The data are means of ten replicates ±SE. Columns marked by the same letter are not significantly different within a particular substrate; ns – non-significant differences (P < 0.05; Kruskal–Wallis Multiple Comparison Z-values test). 6395 M. Gryndler et al. / Bioresource Technology 99 (2008) 6391–6399 3.2. Experiment II 3.2.1. Growth of plants Growth of hemp, measured by two parameters (plant height and shoot dry weight), was not affected by inoculation, but was significantly affected by substrate (Table 2). The significant interaction of these two factors indicated that the actual response to applied microbial inoculation differed between substrates. An amendment of clay substrate with organic matter had a significantly positive effect on dry shoot biomass of hemp, with the highest yield observed in the treatment with the highest dose of organic amendment (Table 2). Inoculation with AMF or AMF + PGPR did not influence hemp growth in pure clay. In clay 4org and clay 10org, inoculation with AMF increased plant height. In the highest dose of organic matter, inoculation with AMF decreased shoot biomass and hemp height; this reduction was not so marked if AMF were applied together with PGPR. Growth of reed canarygrass was affected not only by substrate, but also by inoculation (excluding the tiller number parameter) and by the interaction of these two factors (Table 3). As was the case for hemp, the highest biomass production of reed canarygrass was found in uninoculated substrate with the highest dose of organic amendments (Table 3). However, comparable results were also found in the clay 10org treatment inoculated with AMF. The same pattern was observed for tiller production as well. Plant height was significantly increased by the addition of organic matter throughout all treatments (excluding the uninoculated clay 10org treatment). In the substrate with the highest dose of organic matter, the addition of microor- ganisms, either AMF or AMF + PGPR, resulted in a decrease of shoot dry weight and tiller production of reed canarygrass. 3.2.2. Shoot biomass analyses Concentrations of P in shoot biomass of hemp were significantly affected only by substrate (Table 4). In reed canarygrass, concentrations of P in shoot biomass were affected not only by the substrate, but also by inoculation and by the interaction of these two factors (Table 5). No effect of microbial inoculation on P concentration was observed in pure clay, clay 4org and clay 10org. In contrast, in clay 20org the P concentration was significantly higher after both microbial inoculations (AMF or AMF + PGPR) (Table 5). Concentrations of risk elements in both plants species were significantly affected by the substrate, inoculation or interaction of these factors depending on the plant species and element (Tables 4 and 5). In some treatments, bacterial inoculation tended to diminish the effect (either negative or positive) of AMF on element uptake (Cu and Mn for hemp; Cd, Mn and Pb for reed canarygrass). 3.2.3. Mycorrhizal colonization Mycorrhizal colonization of both plant species was significantly affected by substrate and by inoculation (Tables 2 and 3). For reed canarygrass, the interaction of these two factors was also observed; for hemp the interaction was not significant (P = 0.052). A very high level of mycorrhizal colonization of plants cultivated in uninoculated pure clay substrate (75% for Table 2 Experiment II – hemp: effect of substrate (clay with 4, 10 or 20% of organic amendments) and inoculation with soil microorganisms (AMF alone or combined with PGPR) on growth and mycorrhizal parameters of hemp Substrate Inoculation Plant height (cm) Shoot dry weight (g) Clay Uninoculated AMF AMF + PGPR 31.4 ± 9.0 34.1 ± 4.9 38.1 ± 6.0 e de cde 0.63 ± 0.11 0.73 ± 0.09 0.64 ± 0.13 c c c 75 ± 4 84 ± 5 91 ± 3 cd abc a Clay 4org Uninoculated AMF AMF + PGPR 36.9 ± 13.7 50.3 ± 19.2 36.1 ± 15.1 de bcd de 1.04 ± 0.27 1.74 ± 0.45 1.33 ± 0.32 bc bc bc 68 ± 3 91 ± 2 91 ± 1 d ab ab Clay 10org Uninoculated AMF AMF + PGPR 32.1 ± 6.2 53.0 ± 14.8 36.6 ± 10.9 e abc de 0.57 ± 0.10 1.62 ± 0.48 1.24 ± 0.34 c bc bc 67 ± 5 80 ± 3 88 ± 2 d bcd abc Clay 20org Uninoculated AMF AMF + PGPR 69.7 ± 15.2 43.9 ± 12.0 60.9 ± 4.8 a cde ab 4.03 ± 0.76 1.20 ± 0.51 2.47 ± 0.59 a bc ab 40 ± 9 82 ± 4 85 ± 2 e abc abc Factor DF F values/significance (1) Substrate (2) Inoculation (1)  (2) 3 2 6 12.304 0.846 4.375 5.718 0.223 3.686 ** ns ** 4.859 36.279 2.191 ** *** ns (p = 0.052) *** ns ** Mycorrhizal colonization (%) Data are means of ten replicates ± SE. Values within columns marked by the same letter are not significantly different (P < 0.05; Duncan multiple range test). Effects of factors according to twoway ANOVA: ns – non-significant effect, **P < 0.01, ***P < 0.001. DF – degree of freedom. 6396 M. Gryndler et al. / Bioresource Technology 99 (2008) 6391–6399 Table 3 Experiment II – reed canarygrass: effect of substrate (clay with 4, 10 or 20% of organic amendments) and inoculation with soil microorganisms (AMF alone or combined with PGPR) on growth and mycorrhizal parameters of reed canarygrass Substrate Inoculation Plant height (cm) Number of tillers per pot Shoot dry weight (g per pot) Mycorrhizal colonization (%) Clay Uninoculated AMF AMF + PGPR 53.6 ± 2.4 51.8 ± 1.2 51.6 ± 2.4 d d d 17.3 ± 0.9 14.9 ± 0.9 16.3 ± 0.5 ef f ef 1.46 ± 0.15 1.99 ± 0.18 1.86 ± 0.12 g f fg 87 ± 1 86 ± 3 84 ± 3 de de e Clay 4org Uninoculated AMF AMF + PGPR 65.3 ± 2.8 68.8 ± 1.4 61.6 ± 2.8 ab ab bc 25.4 ± 2.0 25.3 ± 1.5 24.5 ± 1.2 d d d 3.48 ± 0.39 4.64 ± 0.60 3.76 ± 0.48 e cde de 88 ± 2 92 ± 1 90 ± 2 de cd de Clay 10org Uninoculated AMF AMF + PGPR 56.6 ± 1.7 70.4 ± 2.0 67.0 ± 2.8 cd a ab 19.9 ± 1.4 37.4 ± 2.0 28.5 ± 2.6 e ab d 2.29 ± 0.30 6.37 ± 0.41 5.02 ± 0.67 f ab bcd 90 ± 1 96 ± 1 96 ± 1 de bc bc Clay 20org Uninoculated AMF AMF + PGPR 66.6 ± 2.1 68.8 ± 2.5 63.9 ± 3.1 ab ab ab 41.5 ± 1.6 31.0 ± 3.4 34.0 ± 1.3 a cd bc 7.62 ± 0.48 4.45 ± 0.28 5.08 ± 0.23 a cde bc 85 ± 3 99 ± 1 97 ± 1 e a ab Factor DF F values/significance (1) Substrate (2) Inoculation (1)  (2) 3 2 6 26.534 4.105 2.9 73.777 0.359 10.556 *** ns *** 70.557 8.457 11.997 *** *** *** 19.265 18.330 5.538 *** *** *** *** * * Data are means of ten replicates ± SE. Values within columns marked by the same letter are not significantly different (P < 0.05; Duncan multiple range test). Effects of factors according to two-way ANOVA: ns – non-significant effect, *P < 0.05, ***P < 0.001. DF – degree of freedom. Table 4 Experiment II – hemp: effect of substrate (clay with 4, 10 or 20% of organic amendments) and inoculation with soil microorganisms (AMF alone or combined with PGPR) on concentrations of phosphorus and risk elements in biomass of hemp Zn (mg kg 1) Cu (mg kg 1) Mn (mg kg 1) Cd (mg kg 1) Pb (mg kg 1) bc de e 0.046 0.029 0.028 abcd d d 0.703 0.602 0.646 abc bcd abcd 119.83 53.43 83.33 b d c 0.047 0.040 0.031 abcd bcd cd 0.808 1.061 0.481 ab a cd a e abc 250.62 48.28 84.57 a d c 0.064 0.050 0.033 a abc cd 0.783 0.896 0.745 abc ab abc 9.28 5.82 7.11 a bc abc 241.68 89.65 100.38 a c bc 0.058 0.036 0.042 ab bcd abcd 0.515 0.462 0.580 bcd d bcd 0.596 15.14 5.267 ns *** *** 39.68 120.5 5.956 *** *** *** 2.258 8.908 0.801 ns *** ns 5.085 0.893 2.681 ** ns * Substrate Inoculation P (%) Clay Uninoculated AMF AMF + PGPR 0.399 0.475 0.475 b ab ab 38.5 50.4 39.4 c abc bc 7.72 7.63 3.91 ab ab e 97.32 40.48 30.93 Clay 4org Uninoculated AMF AMF + PGPR 0.527 0.640 0.665 ab ab a 45.5 36.6 40.9 abc c abc 7.74 4.96 7.78 ab cd ab Clay 10org Uninoculated AMF AMF + PGPR 0.553 0.498 0.665 ab ab a 53.7 45.3 48.6 a abc abc 9.84 3.91 6.79 Clay 20org Uninoculated AMF AMF + PGPR 0.528 0.442 0.510 ab ab ab 44.4 52.9 42.6 abc ab abc Factor DF F values/significance (1) Substrate (2) Inoculation (1)  (2) 3 2 6 2.840 1.216 0.633 ns ns ns * ns ns 2.128 0.387 1.903 Data are means of ten replicates. Values within columns marked by the same letter are not significantly different (P < 0.05; Duncan multiple range test). Effects of factors according to two-way ANOVA: ns – non-significant effect, *P < 0.05, **P < 0.01, ***P < 0.001. DF – degree of freedom. hemp, 87% for reed canarygrass) supported the results of the Experiment I and confirmed a high inoculation potential of AMF in original spoil bank substrate (Tables 2 and 3). However, inoculation with AMF further increased the level of colonization when compared with the control treatment of the relevant substrate. This effect was significant in clay 4org and clay 20org (hemp) and in clay 10org and clay 20org (reed canarygrass). 4. Discussion The addition of compost to spoil bank clay substrate in order to support the growth of planted crops had a strong negative impact on present AMF and almost eliminated mycorrhizal colonization of roots. However, if compost was applied together with lignocellulose papermill waste, its negative effect on AMF was substantially reduced, but 6397 M. Gryndler et al. / Bioresource Technology 99 (2008) 6391–6399 Table 5 Experiment II – reed canarygrass: effect of substrate (clay with 4, 10 or 20% of organic amendments) and inoculation with soil microorganisms (AMF alone or combined with PGPR) on concentrations of phosphorus and risk elements in biomass of reed canarygrass Zn (mg kg 1) P (%) Cu (mg kg 1) Mn (mg kg 1) Cd (mg kg 1) Pb (mg kg 1) Substrate Inoculation Clay Uninoculated AMF AMF + PGPR 0.235 0.197 0.228 ef f ef 44.06 40.22 37.71 cd bcd bcd 5.24 4.69 4.57 cd de de 123.20 80.04 106.38 abcd f de 0.035 0.070 0.042 c a bc 0.894 0.710 0.364 a a d Clay 4org Uninoculated AMF AMF + PGPR 0.269 0.252 0.294 de de cd 35.95 36.57 40.90 e de bcd 4.16 4.69 4.18 e de e 114.32 100.77 112.13 bcde e cde 0.033 0.047 0.021 cd b d 0.478 0.472 0.584 cd ab cd Clay 10org Uninoculated AMF AMF + PGPR 0.390 0.300 0.343 b cd bc 52.48 42.63 52.54 ab bcd ab 5.15 5.55 5.00 cd bc cd 134.91 110.36 118.70 a cde abcde 0.025 0.074 0.024 de a d 0.449 0.702 0.411 cd bcd abc Clay 20org Uninoculated AMF AMF + PGPR 0.264 0.450 0.477 de a a 45.31 59.47 59.47 bc a a 4.93 6.77 6.12 cd a b 126.69 106.35 131.08 abc de ab 0.037 0.086 0.035 c a cd 0.461 0.831 0.369 cd a d Factor DF F values/significance (1) Substrate (2) Inoculation (1)  (2) 3 2 6 59.13 7.559 15.17 *** ns *** 31.62 8.079 6.245 *** ** *** *** *** ns 12.46 99.53 4.016 *** *** ** 1.353 14.30 5.900 ns *** *** *** ** *** 29.49 2.046 5.633 7.486 20.43 1.784 Data are means of ten replicates. Values within columns marked by the same letter are not significantly different (P < 0.05; Duncan multiple range test). Effects of factors according to two-way ANOVA: ns – non-significant effect, **P < 0.01, ***P < 0.001. DF – degree of freedom. the positive effect on plant growth was maintained. Our results suggest that the application of AMF inoculum to the roots of suitable host plants could secure sufficient yield, despite the reduced dose of fertilizing organic amendment. In terms of absolute numbers, both plant species used in Experiment II produced, in general, the highest quantity of shoot biomass when planted in the substrate with the highest dose of organic matter. However, the results of reed canarygrass growth showed that the reduction of organic matter by 50% and the concurrent inoculation with AMF still yielded 83% of biomass (when compared with the standard process in praxis). When the fertilization was further decreased to the lowest dose, the plants yielded 60% of the reference biomass if their growth was supported by AMF inoculation. However, AMF may negatively affect growth of plants if cultivated in certain growth conditions. In general, the negative effect of AMF on plant growth is often observed in nutrient-rich soils or light-deficient conditions, where the plants’ costs of mycorrhiza maintenance exceed the gained benefits (Johnson et al., 1997). This was also documented in our experiment on the height of hemp, tiller production of reed canarygrass and shoot dry biomass of both plant species when planted in the highest dose of organic matter. For the desired positive effect of AMF, the application of excessive doses of amendments should therefore be avoided. A high level of mycorrhizal colonization of roots of both plant species observed in non-inoculated treatments indicated that AMF were abundantly present in the original spoil bank clays. Also Püschel et al. (2008) encountered a very rapid dispersion of AMF propagules at a field experimental site located on a freshly formed spoil bank. Owing to the abundant presence of native AMF in spoil bank clay, the fact that the inoculation with AMF supported growth of reed canarygrass in certain treatments might be surprising. This observation suggests that the added AMF isolates were more efficient in providing the benefits to the plants. This explanation can also correspond with the increase of colonization level that followed the inoculation with AMF. The massive reduction of mycorrhizal colonization of alfalfa roots in the first experiment revealed the strong negative effect of the added compost on AMF. While this is in contradiction with results of Tanu et al. (2004), findings similar to our experiment were reported by Sáinz et al. (1998), who also found significant decrease of mycorrhizal colonization of red clover with increasing proportion of vermicompost from composted urban waste added to the soil. Thorne et al. (1998) observed decrease of mycorrhizal colonization only for introduced but not native AM fungi after the application of composted sewage sludge, which indicated that not all AMF species respond to the addition of compost in the same way. In contrast with Thorne et al. (1998), in our experiment both the introduced and the native AMF were suppressed by compost amendment. The application of lignocellulose papermill waste as the only amendment to the original clay was found to be non-effective for growth stimulation of alfalfa, but it had no negative effect on mycorrhizal colonization of roots. In combination with compost in equal weight ratios, the resultant substrate was found to be the most effective organic amendment. It combined the best of individual amendments and almost eliminated the negative effect of compost on AMF. Positive experience in using papermill waste was reported by Johnson and McGraw (1988b), 6398 M. Gryndler et al. / Bioresource Technology 99 (2008) 6391–6399 who found significantly increased plant biomass and AMF sporulation after application of composted papermill sludge during reclamation of taconite tailings. Results of our study indicate that the simultaneous inoculation with PGPR can, at least for hemp, moderate the above described negative effects of AMF on plants that occurred in substrate with the highest dose of organic amendments. However, apart from this observation, no significant effects of PGPR on plant growth or mycorrhizal colonization appeared throughout our study. This is in contrast with numerous reports that bacteria inhabiting the rhizosphere can considerably modify the growth response of plants to mycorrhizal inoculation (Fitter and Garbaye, 1994; Andrade et al., 1995; Bethlenfalvay et al., 1997). The general lack of plants’ response to PGPR could probably be ascribed to incompatibility between model plants and selected PGPR strains. A wide range of effects of PGPR on AMF, varying from stimulation to reduction of root colonization, were shown e.g. by Germida and Walley (1996) or Requena et al. (1997). This indicates highly specific interactions between rhizosphere bacteria and AMF (Gryndler, 2000). The proper selection of both bacteria and AMF is thus necessary to attain a positive effect on plant growth or root colonization by AMF (Azcón, 1989). Concerning the uptake of risk elements into shoot biomass, the interaction between AMF and PGPR was found. Diminution of the effects of AMF on the uptake of some elements was consistently observed in treatments co-inoculated with PGPR. Analyses of the shoots of high-biomass crops proved that microbial inoculation with AMF and/or PGPR did not increase concentrations of risk elements in the biomass to the level that would exclude their further potential use as a biofuel or source of technical fibers. 5. Conclusions Our study revealed that the addition of compost increases plants’ performance, but at the expense of AMF development and related benefits of mycorrhiza. Considering the beneficial role of mycorrhizal symbiosis for development of plant cover on reclaimed clay substrate, the rarefaction of compost with lignocellulose papermill waste is highly recommended. Although the proposed approach did not yield the biomass at the level of commonly used doses of compost for two model crops, the substantially decreased reclamation costs could offset the modest reduction of yields. 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