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Zn mine tailings
Soil Biology & Biochemistry 38 (2006) 1403–1412 www.elsevier.com/locate/soilbio
Beneficial effects of earthworms and arbuscular mycorrhizal fungi on establishment of leguminous trees on Pb/Zn mine tailings Y. Ma a, N.M. Dickinson b,*, M.H. Wong a a
Department of Biology, Croucher Institute for Environmental Science, Hong Kong Baptist University, Hong Kong SAR, People’s Republic of China b School of Biological and Earth Sciences, Liverpool John Moores University, Byrom Street, Liverpool, Merseyside L3 3AF, UK Received 14 February 2005; received in revised form 2 August 2005; accepted 18 October 2005 Available online 5 January 2006
Abstract Planting trees to stabilize metalliferous mine tailings is a widely used form of land reclamation although substantial soil amendment is invariably required, both to improve the physico-chemical status of the tailings and to ameliorate toxicity prior to planting. Here, we report a glasshouse study of the combined effects of burrowing earthworms (Pheretima guillelmi) and arbuscular mycorrhizal fungi (Glomus spp., AMF) on establishment of a naturally invasive, woody, nitrogen-fixing legume, Leucaena leucocephala, on topsoil-amended Pb/Zn mine tailings. AMF provided the most effective preliminary inoculant, improving N, P and K uptake, but earthworms had more influence improving N nutrition. In most cases, the combined effects of AMF and earthworms were additive and proved to be beneficial to plant growth, plant nutrition and for protection against uptake of toxic metals. AMF influenced metal uptake more than earthworms, but together they reduced mobility of Pb and Zn in soil by as much as 25%. Some minor but significant negative interactions were also evident; for example, earthworms enhanced soil microbial activity but inhibited the beneficial effects of AMF on N2-fixation. We argue that increased attention to ecological interactions in soil could reduce costs and improve the efficacy of restoring a vegetation cover to land impacted by contaminated spoils. q 2005 Elsevier Ltd. All rights reserved. Keywords: Mycorrhizae; Pheretima guillelmi; Leucaena leucocephala; Reclamation; Pb; Zn
1. Introduction The research reported in this paper forms part of a reclamation project on Pb/Zn mine tailings in Guangdong province, south-east China. The mine tailings are largely devoid of vegetation but some species, including Leucaena leucocephala, have started to naturally colonize. L. leucocephala is a fast-growing leguminous shrub from Mexico that is frequently used in agroforestry systems, producing protein-rich foliage for livestock and pods bearing edible seeds. L. leucocephala has also been reported to grow on poor soils and chromite overburdens in India, where it has been found to provide an effective cover to prevent leaching of heavy metals (Rout et al., 1999). However, in Guangdong, as in many places elsewhere, establishment of a vegetation cover relies on firstly converting a hostile and toxic spoil into a functional and sustainable soil, preferably with minimal amendment and associated cost. To do this requires an understanding of * Corresponding author. Tel.: C44 151 231 2190; fax: C44 151 207 3224. E-mail address: [email protected] (N.M. Dickinson).
0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.10.016
the requisite biological components and their role in the conversion process. It is known that arbuscular mycorrhizal fungi (AMF) are ubiquitous and abundant in soils, particularly in more stressed environments, and that they can enhance plant growth and rates of revegetation of mine spoils (Shetty et al., 1995; Jeffries et al., 2003). Fungal hyphae and spores form part of the diet of earthworms, while earthworms have a significant influence on their dispersal, including AMF (Doube et al., 1994a,b). Grazing by earthworms on the soil mycelium of AMF may limit its development or disconnect it from the root, but it may also stimulate its growth (Fitter and Sanders, 1992). Both groups of organisms are likely to play an important role in the creation of a healthy soil from mine spoils. We have found previously that earthworms can survive if mine tailings are amended with at least 20% but preferably 50% of topsoil (Ma et al., 2002, 2003). Earthworm activity increased the yield of L. leucocephala, but also the bioavailability and plant uptake of Pb and Zn. AMF significantly improved growth and establishment, increased Pb and Zn uptake in very dilute tailings, but otherwise decreased uptake (Ma, 2003). However, we have found no record of previous study of the three-way link between
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earthworms, heavy metals and mycorrhizae. In the present work, our aims were to understand the combined effects of both earthworm and AMF inoculation on L. leucocephala and its ability to fix nitrogen. 2. Methods 2.1. Experimental materials Pb/Zn mine tailings were collected from Lechang, Guangdong Province of China. The topsoil mixture was purchased from a commercial supplier (Agribusiness Limited) in Hong Kong. Both the tailings and topsoil mixture were airdried and screened through a 2-mm sieve before use. Seeds of L. leucocephala were collected from the Shaw Campus of Hong Kong Baptist University. Seed germination and the culture of seedlings were the same as described previously (Ma et al., 2003); uniform 6-month-old seedlings were selected for experimental use. An AMF inoculum mixture of Glomus mosseae and Glomus intraradices (Inoculum Endorize-Mix2, Biorize Sarl, France) was used. Earthworms (Pheretima guillelmi) were collected by hand digging from a vegetable farm at Fanling, Hong Kong. All test worms were selected as fully clitellate adults, each approximately 1 g (8 cm). 2.2. Experimental design Experimental treatments consisted of Pb/Zn mine tailings mixed with topsoil in proportions of 0, 25, and 50% (w/w), each inoculated with (i) earthworms (worms), (ii) AMF, (iii) earthwormsCAMF and (iv) a non-inoculated reference (control). Each treatment had four replicates, providing a total of 48 pots. AMF inoculum (200 ml per pot) was mixed well with soil-tailings. Fertilizers (NH4NO3 and K2SO4) were applied (0.4 g N and 0.1 g K kg K1 of air-dried soil-tailings mixtures); no P was added in order to maintain low concentration to encourage more efficient AMF colonization of roots. About 2.5 kg of each soil-tailings mixture was placed into a 3.5 l plastic pot (with mesh cover drainage holes, the mesh aperture was 1 mm!1 mm), and one seedling of 6-month-old L. leucocephala was transplanted into each pot. A dish was placed below each pot and overspill of leachate was carefully avoided during watering. Earthworms were added (10 worms per pot) immediately following planting; to prevent escape of earthworms, inwardly overhanging adhesive tape (0.05 m width) was attached to the rim of each pot. Pots were arranged in a fully randomized design, watered daily and maintained for 3 months under natural light in a temperaturecontrolled greenhouse (22G3 8C).
nodules were also separated; those detached from the root were separated by sieving (200-mm). The nodules were weighed and then immediately incubated in serum vials with purified acetylene (C2H2) at 37 8C for 1 h. Gas from the vials was then removed for ethylene (C2H4) analysis by gas chromatographic fractionation followed by detection using flame ionization. The N2-fixing capacity of nodules was then determined using the Acetylene Reduction Method, which measures the extent of reduction of C2H2 to C2H4 (Sprent, 1969). Randomly selected fine roots (about 100 cm per pot) were preserved in 70% alcohol for assessment of mycorrhizal infection. Root colonization (%) was estimated by microscopy after clearing with 10% KOH and staining with trypan blue (Phillips and Hayman, 1970; McGonigle et al., 1988). Foliage and stems were rinsed with tap water and, with the roots, washed three times with deionized water, oven-dried (65 8C), weighed, ground (100 mm) and digested with a semi-micro Kjeldahl procedure. N determination was carried out using the Berthelot Reaction method and P using the Molybdenum Blue method (Greenberg et al., 1992). Plant components and earthworms were digested in HNO3–HClO4 (4:1 v/v) prior to Pb and Zn determination. Samples of soil-tailings mixture were also air-dried and ground (!74 mm for total metal analyses; !1 mm for other analyses), prior to measurement of pH, EC and DOC (Shimadzu TOC-5000). Organic matter was determined by modified Walkey-Black rapid titration method using H2SO4– K2Cr2O7 (Sparks et al., 1996). Total N and P in soils and tailings were analyzed as described above for plant samples. Soil NHC 4 N was extracted using 2 M KCl for colorimetric determination. P and K were each extracted by 0.5 M NaHCO3 and 1 M HN4OAc, and determined by colorimetry and AAS, respectively (Page et al., 1982; Carter, 1993). Air-dried soiltailings samples were digested with HNO3–HCl–HF (9:3:3 v/v) for total metal determination, and both 1 M NH4OAc 0.005 M DPTA for more mobile and potentially mobile fractions (Ure, 1996; Lindsay and Norvell, 1978). Pb and Zn concentrations in Table 1 Properties of the Pb/Zn mine tailings and topsoil mixture (meanGSD, nZ3) Tailings pH Electrical conductivity (ms cmK1) Organic matter (%)
8.0G0.01 3.7G0.04
7.0G0.01 0.43G0.002
1.0G0.36
5.8G1.45 0.15G0.005
P K
0.78!10 G 0.2!10K4 0.01G0.0036 0.057G0.0076
0.09G0.001 0.26G0.018
Total metal concentrations (mg kgK1)
Pb Zn Cu Cd
4418G372 9827G376 89.9G1.93 32.6G4.52
117G3.66 136G2.71 25.8G0.28 4.85G0.17
DTPA-extractable metal concentrations (mg kgK1)
Pb Zn Cu Cd
1202G254 382.7G77.0 3.48G0.53 n.d.
14.1G0.38 18.4G0.74 1.57G0.18 n.d.
Total nutrients (%)
2.3. Sampling and analysis After 3 months, earthworms were separated from each pot, starved for 3 days to remove gut contents, then washed in deionized water and dried at 65 8C. Tree seedlings heights were measured, prior to separating into foliage, stems and roots. Roots were removed from soil by careful washing and the root
Soil
n.d., not detectable.
N
K3
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Table 2 Soil NHC 4 KN and ‘available’ K as influenced by earthworms and arbuscular mycorrhizal fungi (AMF) after 3 months in different proportions of tailings Proportions of tailings (%)
Inoculation treatment Control
NHC 4 KN
(mg kg
K (mg kgK1)
K1
)
Earthworms a
b
AMF
EarthwormsCAMF c
0 25 50
0.98G0.11 0.81G0.12a 0.64G0.20a
1.98G0.29 1.44G0.26b 0.89G0.25a
1.52G0.27 1.25G0.10bcd 0.87G0.42ad
1.04G0.24ae 1.44G0.31be 1.14G0.11be
0 25 50
9.15G1.66d 6.58G2.49ad 5.35G1.26a
9.73G0.72d 7.38G1.13ad 5.98G0.57a
61.9G7.86b 39.8G9.01c 25.9G5.66d
51.9G5.38c 31.0G4.91b 20.6G1.87e
Control was not inoculated. Different letters of superscript mean a significant difference at P!0.05 in the same row and the same column for each nutrient (one-way ANOVA, Tukey test). Also see Table 7.
all digests and extracts were determined using ICP-AES (Page et al., 1982). Phosphatase activity of soil was measured by colorimetric estimation of the p-nitrophenol released when soil was incubated with buffered (pH 6.5) sodium p-nitrophenyl phosphate solution and toluene, and expressed in micrograms p-nitrophenol per gram of dry soil per hour (Page et al., 1982). Soil dehydrogenase activity was estimated spectrophotometrically (480 nm) to quantify the triphenyl formazan (TPF) production that resulted from an enzymatic reaction when soil samples were incubated in 1% triphenyltetrazolium chloride (TTC) at 37 8C for 24 h (Page et al., 1982).
the N-fixing capacity of root nodules, but earthworm activity played a larger role in reducing N-fixation. With both AMF and earthworms more but less active nodules were formed. Root mycorrhizal colonization increased with increasing proportions of tailings (Fig. 2), and earthworms significantly increased rates of infection. When data for all tailing/soil and inoculation treatments were plotted together (Table 4), root nodulation was positively correlated with soil enzyme activity, but both were negatively correlated with N2-fixing capacity and mycorrhizal colonization. Viewed in these broader terms, with increased AMF infection of roots there appeared to be less
3. Results 3.1. Tailing and topsoil properties The topsoil had a low NPK status but otherwise provided an appropriate ameliorant for the mine tailings (Table 1). The tailings were of high pH, with extremely low nutrients, and high Pb and Zn. There appeared to have been a long enough period of surface exposure of the tailings for a small but significant amount of organic matter to have accumulated. By the end of the experimental period, there had been only negligible effects of earthworms and mycorrhizae on soil pH, EC, DOC and available P; the differences were not statistically significant (data not shown). However, an increase in NHC 4 KN was found with both earthworms and AMF, and available K was substantially increased by AMF inoculation (Table 2). After 3 months, the numbers of earthworms that survived and were active out of the original 10 per pot were: 10 in clean soil, 7–8 in 25% tailings and 2–3 in 50% tailings (P!0.05). 3.2. N-fixation and microbial activity Activity of both microbial enzymes was reduced in proportion to the amount of tailings, much more than due to the effects of earthworms or AMF (Fig. 1). Earthworm inoculation increased enzyme activity, but there was no consistent effect that could be attributed to AMF inoculation. With an increasing proportion of tailings, there was a beneficial effect of both earthworm and AMF inoculation on root nodulation (Table 3). AMF inoculation similarly stimulated
Fig. 1. Soil phosphatase activity (a) and dehydrogenase activity (b) after 3 months’ growth of L. leucocephala in different proportions of tailings. The treatments include inoculation with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control). Different letters indicate a significant difference at P!0.05 when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test). Also see Table 7.
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Table 3 Production and N2-fixing capacity of root nodules of L. leucocephala as influenced by earthworms and arbuscular mycorrhizae in different proportions of tailings Proportions of tailings (%)
Inoculation treatment Control
Fresh weight (g plant ) 0 2.8G0.35a 25 1.8G0.25d 50 0.6G0.17c K1 K1 N2 fixing capacity (mmol C2H4 h g ) 0 146G20.9e 25 181G17.7e 50 293G43.9d
Earthworms
AMF
EarthwormsCAMF
K1
2.3G0.47ad 2.0G0.18d 1.0G0.13b
3.7G0.45c 2.1G0.26ad 1.4G0.48ab
2.5G0.41ad 1.8G0.36d 1.2G0.16b
51.2G11.9a 113G23.7b 221G30.9ed
148G8.63e 276G32.2d 456G99.3c
62.4G16.3a 125G36.0b 155G37.5be
Control was not inoculated. Different letters of superscript mean a significant difference (P!0.05) in the same row and the same column for each parameter. Also see Table 7.
nodulation but more N-fixation (Ma, 2003, data not shown here). 3.3. Plant growth and nutrient uptake After 3 months, there was increased plant growth associated with both AMF and earthworm inoculation only in 50% tailings (Fig. 3), which also suggested a possible additive effect of earthworm and AMF inoculation; combined AMF and earthworm inoculation appeared to increase plant growth equivalent to that in 25% tailings. Foliar nitrogen concentrations were the same whether or not the plants were growing in treatments with tailings, but were enhanced by all ameliorating treatments (Fig. 4). With combined inoculation of AMF and earthworms, foliar P was increased by up to 44.8% in 0% tailings and 26.8% in 50% tailings, whilst foliar K was the same in all tailings dilutions and inoculation treatments (data not shown). Differences were evident in stem concentrations of these nutrients (Fig. 5); in 25% dilutions, K uptake was increased with AMF and AMFC
earthworms treatments, but only by AMF in 50% dilutions. Total uptake of N, P and K by plants was increased by earthworm inoculation both in clean soil and the two tailings treatments (Table 6). 3.4. Pb and Zn bioavailability and uptake There was evidence that soil concentrations of the more mobile fractions of Pb and Zn significantly declined with both earthworm and AMF inoculation (Table 5). The inoculation effect varied with extractant, but amounted to a reduction of soil concentrations of as much as 17% of Pb and 14% of Zn (DTPAextractable) and 25% of Pb and Zn (NH4OAc-extractable). Root and stem concentrations of Pb and Zn reflected the proportion of tailings, but transport to foliage appeared to be highly restricted (Fig. 6). Root concentrations of Pb and Zn were significantly decreased by both types of inoculation, but there was little effect on leaf and stem concentrations. Earthworms had no apparent separate effect on metal concentrations in plant tissues, but had a contributory effect when in combination with AMF. There were significant but much smaller effects of tailings proportions and ameliorating treatments on the mass balance of Pb and Zn, compared to NPK (Table 6). In 25% tailings, earthworms increased the total Pb and Zn uptake by plants, but the only significant effect of AMF and combined inoculation was some reduction on total Pb and Zn uptake in 25% tailings. Amounts of Pb and Zn that accumulated in the earthworm tissues were related to the amount of tailings in the substrata. Highest total body concentrations were 570 mg Pb kgK1 and Table 4 Pearson’s correlation matrix for root nodulation, N2-fixation, mycorrhizal infection, and soil microbial enzyme activity (nZ48) Nodule weight
Fig. 2. Root mycorrhizal colonization of L. leucocephala inoculated with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control), as influenced by the amount of Pb/Zn mine tailings added. Different letters indicate a significant difference at P!0.05 when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test). Also see Table 7.
Nodule weight N2-fixing capacity AMF infection Phosphatase Dehydrogenase
N2-fixing capacity
1.000 K0.450*
1.000
K0.558* 0.736* 0.650*
0.437* K0.723* K0.771*
Levels of significance: *P!0.01.
AMF infection
Phosphatase
Dehydrogenase
1.000 K0.682* K0.562*
1.000 0.847*
1.000
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Fig. 3. Total dry weight yields of L. leucocephala inoculated with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control), as influenced by the amount of Pb/Zn mine tailings added. Different letters indicate a significant difference at P!0.05 when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test).
966 mg Zn kgK1. There were no significant differences between the metal concentrations in earthworms with and without AMF inoculation (Table 7). 4. Discussion 4.1. Effectiveness of inoculation treatments Topsoil amendments to the tailings faciltated survival and growth of plants, AMF and earthworms. Dilution of 50% provided clear evidence of toxicological effects: (i) plant growth
Fig. 5. N, P and K concentrations in stems of L. leucocephala grown in different proportions of Pb/Zn tailings inoculated with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control). Different letters indicate a significant difference (P!0.05) when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test).
Fig. 4. Foliar N concentrations of L. leucocephala grown in different proportions of Pb/Zn tailings inoculated with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control). Different letters indicate a significant difference (P!0.05) when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test).
declined and uptake of Pb and Zn was higher, (ii) only 20–30% earthworm survived after 3 months, (iii) root nodulation was inhibited, and (iv) microbial activity, measured using enzyme assays, was reduced in proportion to the amount of tailings. The microbial response that supports plant growth was evident, even in pots that were not inoculated: both AM colonization of roots and N-fixation were higher in tailings treatments than in clean soil. The results provide a basis to unravel the more subtle effects of AMF communities and burrowing earthworms on the establishment of Leucaena (and associated N-fixation) on the amended mine tailings (Table 8). Beneficial effects to the soils and to plant establishment clearly occurred and these
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Table 5 Mobile fractions of Pb and Zn in different proportions of tailings after 3 months growth of L. leucocephala, measured at the end of the experiment Proportions of tailings (%) DTPA-extractable (mg kg ) Pb 0 25 50 Zn 0 25 50 NH4OAc-extractable (mg kgK1) Pb 0 25 50 Zn 0 25 50
Inoculation treatment Control
Earthworms
AMF
EarthwormsCAMF
13.6G3.12a 232G25.4a 363G16.8a 27.3G6.59a 113G4.23a 133G10.1a
14.0G0.78a 219G4.97a 352G33.4ab 25.5G1.68a 112G1.35a 127G10.7a
13.8G0.61a 199G18.4ba 314G7.56b 26.7G3.01a 96.8G8.17b 127G8.89a
16.0G1.21a 192G5.18b 326G9.45b 28.3G2.41a 101G3.47b 131G3.83a
0.43G0.05a 18.7G2.33a 31.8G2.99a 1.98G0.24a 18.7G1.57a 34.2G0.66a
0.41G0.06a 15.5G0.70b 28.6G3.96ab 1.90G0.26a 15.6G1.34b 30.8G1.52b
0.34G0.07a 15.0G1.41b 23.8G1.82b 1.68G0.17a 14.1G1.04b 30.6G1.86b
0.36G0.01a 14.5G0.62b 25.9G1.28b 1.67G0.15a 14.4G0.99b 29.9G2.14b
K1
Control was not inoculated. Different letters of superscript mean a significant difference (P!0.05) in the same row. Bold text highlights significant treatment effects.
Fig. 6. Lead and Zn concentrations in different parts of L. leucocephala plants grown in different proportions of tailings for 3 months. The treatments include inoculation with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and noninoculated (control). Different letters indicate a significant difference (P!0.05) when comparing the variance within each single tailings treatment according to oneway ANOVA (Tukey test). No significant differences exist between metals in foliage.
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Table 6 Total nutrient and Pb, Zn uptake by L. leucocephala as influenced by earthworms and arbuscular mycorrhizal fungi in different proportions of tailings Proportions of tailings (%)
Total uptake (mg plant K1)
Metals
Control a
Earthworms b
AMF
EarthwormsCAMF
ab
0
N P K Pb Zn
7307 8630a 44,616a 0.123a 1.33a
9191 9684a 49,770ab 0.187b 1.49a
8748 9779ab 53,859b 0.128a 1.42a
9988b 9132b 50,018ab 0.105a 1.35a
25
N P K Pb Zn
6648a 6257a 41,094a 0.962a 2.76a
7988b 7005a 44,636ab 1.26b 3.37c
9004b 7190ab 49,368b 0.859a 2.39ab
9897c 7654b 52,020c 0.655c 2.10b
50
N P K Pb Zn
4676a 4494a 19,151a 1.15a 2.76a
5882b 5707ab 21,606ac 1.21a 2.92a
7469bc 6353bc 27,908bc 1.11a 2.96a
7980c 7267c 29,569b 1.04a 3.02a
Control was not inoculated. Different letters of superscript mean a significant difference (P!0.05) in the same row. Bold text highlights significant treatment effects. Also see Table 7.
effects were generally more pronounced with combined AMF and earthworm inoculation. Inoculation with either earthworms or AMF improved plant nutrition and increased plant growth. Together they reduced heavy metal mobility in soil and plant tissue concentrations, although earthworm activity may increase metal uptake to roots whilst not affecting transport to above-ground plant components. Negative effects of inoculation were relatively minor, concerning the effects of earthworms on reduced rates of N-fixation; their burrowing activities are well know to enhance mineralization of nitrogen from organic matter (Cheng and Wong, 2002; Edwards and Bohlen, 1996), thus reducing the necessity for contributions from root nodules to meet plant growth requirements. Nonetheless, there are obviously quite complex interactions between earthworms, AMF and N-fixation. In overall terms, AMF infection was negatively correlated with N-fixation (Table 4) even though it occurred with both increased nodulation and N-fixation in the presence of tailings (Table 3).
4.2. Effects on soil nutrients and plant growth Earthworms and AMF inoculation stimulated microbial decomposer activity and to mineralization of organic N. Our own previous study on the same mine tailings also found increased NO3–N in Pb/Zn mine tailings after earthworm inoculation (Ma et al., 2003). Earthworms, but not AMF inoculation, significantly stimulated soil dehydrogenase and phosphatase activity, as has been recorded previously (Satchell, 1983). It is known that AMF may directly compete with soil bacteria for carbohydrate substrates (Trevors and van Elsas, 1997). Growth enhancement of L. leucocephala has previously been correlated with AM colonization of roots and uptake of P, Cu and Zn (Manjunath and Habte, 1988). In the present study, improved growth of L. leucocephala with inoculation may have occurred through enhanced uptake of N, P and K. Mobile K in soil was elevated fivefold or more following AMF inoculation, and this was also reflected in stem uptake of this nutrient. AMF hyphae increase the surface area
Table 7 Differences between plant dry weight yields, root mycorrhizal infection, root nodule production, nodule N2-fixing capacity, total nutrient and metal uptake by L. leucocephala, soil phosphatase and dehydrogenase activities, due to (i) different proportions of tailings and (ii) different ameliorating treatments
Dry weight yields Mycorrhizal infection Nodule production N2-fixing capacity Total N uptake Total P uptake Total K uptake Total Pb uptake Total Zn uptake Phosphatase activity Dehydrogenase activity
Tailings
Treatments
Tailings!treatments
26.44*** 232.3*** 120.6*** 85.38*** 20.82*** 30.03*** 118.2*** 180.1*** 92.44*** 226.3*** 357.2***
4.705** 81.89*** 10.17*** 53.54*** 17.55*** 6.193** 8.751*** 6.875** 3.797* 23.54*** 36.14***
1.267 0.363 3.867** 6.015*** 0.705 0.368 0.569 2.266 3.700** 0.424 12.63***
Values are F-values and significance levels from two-way ANOVA. Levels of significance: *P!0.05, **P!0.01, ***P!0.001.
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Table 8 Summary of the beneficial and negative effects of earthworm inoculation and arbuscular mycorrhizal fungi (AMF) inoculation, singly and together, on topsoilamended mine tailings and Leucaena leucocephala Inoculation
Beneficial effects
Negative effects
Earthworms
Increased soil microbial activity Increased AMF infection
Reduced N-fixing capacity of root nodules Possibly contributes to increased plant uptake of Pb and Zn
Increased NHC 4 N in soil Increased foliar N Increased total plant uptake of N Increased root nodulation Increased plant growth AMF
Increased root nodulation Increased N-fixation Increased NHC 4 N in soil Increased foliar N Increased K mobility in soil Increased K uptake by plant Increased plant growth Decreased mobile Pb and Zn in soil Decreased Pb and Zn uptake by roots
Combined earthworms and AMF
Increased N, P, K uptake
Earthworms may inhibit the beneficial effects of AMF on N2-fixation
Improved plant growth Decreased mobile Pb and Zn in soil Possible reduced Pb and Zn uptake Italics indicate the same effects were found separately for both earthworm and AMF inoculation.
for ion absorption from soil (Read, 1984); the link between AM infection and K in plant shoots has been studied previously (Sieverding and Toro, 1988). 4.3. Effects on Pb and Zn bioavailability In earlier work, we studied the separate effects of earthworms and AMF on metal mobility in the same mine tailings. Earthworm inoculation alone increased overall metal uptake, albeit indirectly through improving plant growth rather than increased uptake per se (Ma et al, 2003). The same effect probably occurred in the present study. We found previously that AMF infection (in the absence of earthworms) stimulated uptake of metals by plants when soil metal concentrations were low, but decreased uptake when metal concentration were high (Ma, 2003). In the present experiments, AMF appear to be primarily responsible for protecting plants from excessive metal uptake, whilst combined earthworm and AMF activity reduced likely mobile forms of Pb and Zn in soil by 14–25%. Amelioration of Zn toxicity has been found to occur through the provision of additional adsorptive surfaces on hyphal cell walls and to extrahyphal polysaccharide slime (Hilary et al., 1987; Bradley et al., 1982). Decreased foliar Pb in mycorrhizal plants growing in Pb-polluted soil has been reported previously (Weissenhorn et al., 1995).
(Ydrogo, 1994; Edwards and Bohlen, 1996), as also found in the present study. Earthworm casts may contain up to 10 times as many infective AMF propagules as surrounding soil (Gange, 1993). The relationship between earthworms and N-fixation appears to be more complex; in the present study, earthworms increased root nodulation, but apparently decreased their N2-fixing capacity. However, nodulation and N-fixation is probably a response to nitrogen deficiency, that itself is mitigated by the activity of earthworms. Doube et al. (1994a) showed that earthworms enhanced dispersal of N2-fixing bacteria and their colonization of roots, but that additional nodulation did not significantly affect plant growth. It is also possible to explain why, under higher proportions of tailings, AMF colonization was negatively correlated with root nodule production, but positively correlated with nodule N2-fixing capacity. A high P demand of the N-fixation process can be supplied by AMF, and mycelial growth and mycorrhizal formation are enhanced by Rhizobium; a synergistic process that results in improved rates of P uptake and N-fixation (Azco´n et al., 1991; Xavier and Germida, 2003). High soil N, with or without AMF, leads to a significant decline in nodulation (Jia et al., 2004). 4.5. Conclusion
4.4. Earthworms, AMF and N-fixation Effective mycorrhizal root colonization is constrained by the minimal capacity of AMF for unaided dispersal through soils, but it is known this can be enhanced by earthworms
Reclamation of Pb/Zn mine spoils in Guandong and elsewhere requires techniques that will both ameliorate the constraints of a hostile substrate and enhance the natural attenuation of conditions that are toxic to plant establishment
Y. Ma et al. / Soil Biology & Biochemistry 38 (2006) 1403–1412
and growth. Leucaena is an invasive N-fixing woody plant that is in the early stages of colonization after decades of exposure, or even longer. Arbuscular mycorrhizal fungi clearly provide an important and effective preliminary inoculant; in this experimental work there was as much as 55% root infection in pots that had not been inoculated, and infection levels were highest with increasing proportion of tailings. AMF improve the ability of Leucaena to fix N through its root nodules, improve nutritional status and provide a protective role against toxic metals, but they are naturally slow to disperse. Earthworms aid their dispersal and otherwise improve conditions for plant growth, not least by enhancing soil microbial activity and improve soil nutrient availability. Earthworms are also naturally slow to colonize reclaimed soils, although it has been demonstrated that earthworm inoculation is quite possible if potentially expensive (Butt et al., 1997; Butt, 1999). The combined effects of AMF and earthworms are beneficial in terms of plant growth (O30% yield increase in present experiment), nutrition (improved N, P and K uptake) and protection against toxic metals (up to 25% reduction in uptake), but earthworms may inhibit the beneficial effects of AMF on N2-fixation. The future applications of these ecological studies lies in an opportunity to improve the speed and efficacy of the restoration process through judicious use of topsoil or other amendments combined with the careful encouragement of the soil biota. Acknowledgements The project was supported by the Research Grants Council (HKBU-2049/00M) of the University Grants Committee of Hong Kong. We thank Prof. J. Zhang, Mr. H.T. Poon, Dr. J. Guo and Dr. M. Jiang for their assistance with this work. References Azco´n, R., Rubio, R., Barea, J.M., 1991. Selective interactions between different species of mycorrhizal fungi and Rhizobium meliloti strain, and their effects on growth, N2-fixation (15N) and nutrition of Medicao sativa L. New Phytologist 117, 339–404. Bradley, R., Burt, A.J., Read, D.J., 1982. The biology of mycorrhiza in the Ericaceae. VII. The role of infection in heavy metal resistance. New Phytologist 91, 197–209. Butt, K.R., 1999. Inoculation of earthworms into reclaimed soils: the UK experience. Land Degradation & Development 10, 565–575. Butt, K.R., Frederickson, J., Morris, R.M., 1997. The earthworm inoculation unit technique: an integrated system for cultivation and soil-inoculation of earthworms. Soil Biology & Biochemistry 29, 251–257. Carter, M.R., 1993. Soil Sampling and Methods of Analysis. Lewis Publishers, London. Cheng, J.M., Wong, M.H., 2002. Effects of earthworms on Zn fractionation in soils. Biology and Fertility of Soils 36, 79–86. Doube, B.M., Ryder, M.H., Davoren, C.W., Stephens, P.M., 1994a. Enhanced root nodulation of subterranean clover Trifolium subterraneum by Rhizobium trifolii in the presence of the earthworm Aporrectodea trapezoids. Biology and Fertility of Soils 6, 237–251. Doube, B.M., Stephens, D.M., Davoren, C.W., Ryder, M.H., 1994b. Interactions between earthworms, beneficial soil microorganisms and root pathogens. Applied Soil Ecology 1 (1), 3–10.
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Edwards, C.A., Bohlen, P.J., 1996. Biology and Ecology of Earthworms, third ed. Chapman & Hall, London. Fitter, A.H., Sanders, I.R., 1992. Interactions with the soil fauna. In: Allen, M.F. (Ed.), Mycorrhizal Functioning. Chapman & Hall, New York, pp. 333–354. Gange, A., 1993. Translocation of mycorrhizal fungi by earthworms during early succession. Soil Biology & Biochemistry 25, 1021–1026. Greenberg, A.E., Clesceri, L.S., Eaton, A.D., 1992. Standard Methods for the Examination of Water and Wastewater, 18th ed. American Public Health Association, AWWA, WEF, Washington DC. Hilary, J., Denny, H.J., Wilkins, D.A., 1987. Zinc tolerance in Betula Spp. IV. The mechanism of ectomycorhizal amelioration of zinc toxicity. New Phytologist 106, 545–553. Jeffries, P., Gianinazzi, S., Perotto, S., Turnau, K., Barea, J.-M., 2003. The contribution of arbuscular mycorrhizal fungi in sustainable maintenance of plant health and soil fertility. Biology and Fertility of Soils 37 (1), 1–16. Jia, Y.S., Gray, V.M., Straker, C.J., 2004. The influence of Rhizobium and arbuscular mycorrhizal fungi on nitrogen and phosphorus accumulation by Vicia faba. Annals of Botany 94 (2), 251–258. Lindsay, W.L., Norvell, W.A., 1978. Development of a DTPA soil test for zinc, iron, manganese and copper. Journal of Soil Science Society of America 42, 421–428. Ma, Y., 2003. Earthworms and Mycorrhizae in Phytoremediation of Pb/Zn Mine Tailings: Their Effects on Metal Speciation, Bioavailability and Uptake by Leucaena leucocephala. PhD Thesis, Hong Kong Baptist University. Ma, Y., Dickinson, N.M., Wong, M.H., 2002. Toxicity of Pb/Zn mine tailings to the earthworm Pheretima and the effects of burrowing on metal availability. Biology and Fertility of Soils 36, 79–86. Ma, Y., Dickinson, N.M., Wong, M.H., 2003. Remediation of Pb/Zn mine tailings from Guangdong, China: earthworms (Pheretima guillelmi), trees (Leucaena leucocephala), soil nutrition and metal mobility. Soil Biology & Biochemistry 35 (10), 1369–1379. Manjunath, A., Habte, M., 1988. Development of vesicular-arbuscular mycorrhizal infection and the uptake of immobile nutrients in Leucaena leucocephala. Plant and Soil 106, 97–103. McGonigle, T.P., Fitter, A.H., 1988. Ecological consequences of arthropod grazing on VA mycorrhizal fungi. Proceedings of the Royal Society of Edinburgh 94B, 25–32. Page, A.L., Miller, R.H., Keeney, D.R., 1982. Methods of Soil Analysis— Chemical and Microbiological Properties, second ed. ASA, Inc. and SSSA, Inc., Madison, Wisconsin, USA. Phillips, J.M., Hayman, D.S., 1970. Improved procedures for clearing roots and staining parasitic and vesicular–arbuscular mycorrhizal fungi for rapid assessment of infection. Transaction of British Mycological Society 55, 158–160. Read, C.P.P., 1984. Mycorrhizae: a root–soil interface in plant nutrition. In: Todd, R.L., Giddens, J.E. (Eds.), Microbial–Plant Interactions: ASA Special Publication Number 47. SSSA/ASA/CSSA, Madison, pp. 29–51. Rout, G.R., Samantaray, S., Das, P., 1999. Chromium, nickel and zinc tolerance in Leucaena leucocepahla (K8). Sivae Genetica 48, 3–4. Satchell, J.E., 1983. Earthworm Ecology. Chapman & Hall, London. Shetty, K.G., Hetrick, B.A.D., Schwab, A.P., 1995. Effects of mycorrhizae and fertilizer amendments on Zn tolerance of plants. Environmental Pollution 88 (3), 307–314. Sieverding, E., Toro, S., 1988. Influence of soil water regime on VM mycorrhiza. V. Performance of different VAM fungal species with cassava. Journal of Agronomy and Crop Science 161, 322–332. Sparks, D.L., Oage, A.L., Helmke, P.A., Loeppert, R.H., Soltanpour, P.N., Tabatabai, M.A., Johnston, C.T., Sumner, M.E., 1996. Methods of Soils Analysis, Part 3: Chemical Methods. Soil Science Society of America, Inc., Madison, 1390 pp. Sprent, J.I., 1969. Prolonged reduction of acetylene by detached soybean nodules. Planta 88, 372–375.
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Trevors, J.T., van Elsas, J.D., 1997. Microbial interactions in soil. In: van Elsas, J.D., Trevors, J.T., Wellington, E.M.H. (Eds.), Modern Soil Microbiology. Marcel Dekker, New York, pp. 215–245. Ure, A.M., 1996. Single extraction schemes for soil analysis and related applications. Science of the Total Environment 178, 3–10. Weissenhorn, I., Leyval, C., Belgy, G., Berthelin, J., 1995. Arbuscular mycorrhizal contribution to heavy metal uptake by maize (Zea mays L.) in pot culture with contaminated soil. Mycorrhiza 5, 245–252.
Xavier, L.J.C., Germida, J.J., 2003. Selective interactions between arbuscular mycorrhizal fungi and Rhizobium leguminosarum bv. viceae enhance pea yield and nutrition. Biology and Fertility of Soils 37, 262–267. Ydrogo, H.F.B., 1994. Effecto de las lombrices de tierra (Pontoscolex corethrurus) en las micorrizas vesiculo arbusculares (M.V.A.) en la etapa de crecimento de araza (Eugenia stipitata), achiote (Bixa orellana), pijuayo (Bactris gasipaes), en suelos ultisoles de Yurimaguas. Thesis, University Nac. San Martin, Peru.
Beneficial effects of earthworms and arbuscular mycorrhizal fungi on establishment of leguminous trees on Pb/Zn mine tailings Y. Ma a, N.M. Dickinson b,*, M.H. Wong a a
Department of Biology, Croucher Institute for Environmental Science, Hong Kong Baptist University, Hong Kong SAR, People’s Republic of China b School of Biological and Earth Sciences, Liverpool John Moores University, Byrom Street, Liverpool, Merseyside L3 3AF, UK Received 14 February 2005; received in revised form 2 August 2005; accepted 18 October 2005 Available online 5 January 2006
Abstract Planting trees to stabilize metalliferous mine tailings is a widely used form of land reclamation although substantial soil amendment is invariably required, both to improve the physico-chemical status of the tailings and to ameliorate toxicity prior to planting. Here, we report a glasshouse study of the combined effects of burrowing earthworms (Pheretima guillelmi) and arbuscular mycorrhizal fungi (Glomus spp., AMF) on establishment of a naturally invasive, woody, nitrogen-fixing legume, Leucaena leucocephala, on topsoil-amended Pb/Zn mine tailings. AMF provided the most effective preliminary inoculant, improving N, P and K uptake, but earthworms had more influence improving N nutrition. In most cases, the combined effects of AMF and earthworms were additive and proved to be beneficial to plant growth, plant nutrition and for protection against uptake of toxic metals. AMF influenced metal uptake more than earthworms, but together they reduced mobility of Pb and Zn in soil by as much as 25%. Some minor but significant negative interactions were also evident; for example, earthworms enhanced soil microbial activity but inhibited the beneficial effects of AMF on N2-fixation. We argue that increased attention to ecological interactions in soil could reduce costs and improve the efficacy of restoring a vegetation cover to land impacted by contaminated spoils. q 2005 Elsevier Ltd. All rights reserved. Keywords: Mycorrhizae; Pheretima guillelmi; Leucaena leucocephala; Reclamation; Pb; Zn
1. Introduction The research reported in this paper forms part of a reclamation project on Pb/Zn mine tailings in Guangdong province, south-east China. The mine tailings are largely devoid of vegetation but some species, including Leucaena leucocephala, have started to naturally colonize. L. leucocephala is a fast-growing leguminous shrub from Mexico that is frequently used in agroforestry systems, producing protein-rich foliage for livestock and pods bearing edible seeds. L. leucocephala has also been reported to grow on poor soils and chromite overburdens in India, where it has been found to provide an effective cover to prevent leaching of heavy metals (Rout et al., 1999). However, in Guangdong, as in many places elsewhere, establishment of a vegetation cover relies on firstly converting a hostile and toxic spoil into a functional and sustainable soil, preferably with minimal amendment and associated cost. To do this requires an understanding of * Corresponding author. Tel.: C44 151 231 2190; fax: C44 151 207 3224. E-mail address: [email protected] (N.M. Dickinson).
0038-0717/$ - see front matter q 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.soilbio.2005.10.016
the requisite biological components and their role in the conversion process. It is known that arbuscular mycorrhizal fungi (AMF) are ubiquitous and abundant in soils, particularly in more stressed environments, and that they can enhance plant growth and rates of revegetation of mine spoils (Shetty et al., 1995; Jeffries et al., 2003). Fungal hyphae and spores form part of the diet of earthworms, while earthworms have a significant influence on their dispersal, including AMF (Doube et al., 1994a,b). Grazing by earthworms on the soil mycelium of AMF may limit its development or disconnect it from the root, but it may also stimulate its growth (Fitter and Sanders, 1992). Both groups of organisms are likely to play an important role in the creation of a healthy soil from mine spoils. We have found previously that earthworms can survive if mine tailings are amended with at least 20% but preferably 50% of topsoil (Ma et al., 2002, 2003). Earthworm activity increased the yield of L. leucocephala, but also the bioavailability and plant uptake of Pb and Zn. AMF significantly improved growth and establishment, increased Pb and Zn uptake in very dilute tailings, but otherwise decreased uptake (Ma, 2003). However, we have found no record of previous study of the three-way link between
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earthworms, heavy metals and mycorrhizae. In the present work, our aims were to understand the combined effects of both earthworm and AMF inoculation on L. leucocephala and its ability to fix nitrogen. 2. Methods 2.1. Experimental materials Pb/Zn mine tailings were collected from Lechang, Guangdong Province of China. The topsoil mixture was purchased from a commercial supplier (Agribusiness Limited) in Hong Kong. Both the tailings and topsoil mixture were airdried and screened through a 2-mm sieve before use. Seeds of L. leucocephala were collected from the Shaw Campus of Hong Kong Baptist University. Seed germination and the culture of seedlings were the same as described previously (Ma et al., 2003); uniform 6-month-old seedlings were selected for experimental use. An AMF inoculum mixture of Glomus mosseae and Glomus intraradices (Inoculum Endorize-Mix2, Biorize Sarl, France) was used. Earthworms (Pheretima guillelmi) were collected by hand digging from a vegetable farm at Fanling, Hong Kong. All test worms were selected as fully clitellate adults, each approximately 1 g (8 cm). 2.2. Experimental design Experimental treatments consisted of Pb/Zn mine tailings mixed with topsoil in proportions of 0, 25, and 50% (w/w), each inoculated with (i) earthworms (worms), (ii) AMF, (iii) earthwormsCAMF and (iv) a non-inoculated reference (control). Each treatment had four replicates, providing a total of 48 pots. AMF inoculum (200 ml per pot) was mixed well with soil-tailings. Fertilizers (NH4NO3 and K2SO4) were applied (0.4 g N and 0.1 g K kg K1 of air-dried soil-tailings mixtures); no P was added in order to maintain low concentration to encourage more efficient AMF colonization of roots. About 2.5 kg of each soil-tailings mixture was placed into a 3.5 l plastic pot (with mesh cover drainage holes, the mesh aperture was 1 mm!1 mm), and one seedling of 6-month-old L. leucocephala was transplanted into each pot. A dish was placed below each pot and overspill of leachate was carefully avoided during watering. Earthworms were added (10 worms per pot) immediately following planting; to prevent escape of earthworms, inwardly overhanging adhesive tape (0.05 m width) was attached to the rim of each pot. Pots were arranged in a fully randomized design, watered daily and maintained for 3 months under natural light in a temperaturecontrolled greenhouse (22G3 8C).
nodules were also separated; those detached from the root were separated by sieving (200-mm). The nodules were weighed and then immediately incubated in serum vials with purified acetylene (C2H2) at 37 8C for 1 h. Gas from the vials was then removed for ethylene (C2H4) analysis by gas chromatographic fractionation followed by detection using flame ionization. The N2-fixing capacity of nodules was then determined using the Acetylene Reduction Method, which measures the extent of reduction of C2H2 to C2H4 (Sprent, 1969). Randomly selected fine roots (about 100 cm per pot) were preserved in 70% alcohol for assessment of mycorrhizal infection. Root colonization (%) was estimated by microscopy after clearing with 10% KOH and staining with trypan blue (Phillips and Hayman, 1970; McGonigle et al., 1988). Foliage and stems were rinsed with tap water and, with the roots, washed three times with deionized water, oven-dried (65 8C), weighed, ground (100 mm) and digested with a semi-micro Kjeldahl procedure. N determination was carried out using the Berthelot Reaction method and P using the Molybdenum Blue method (Greenberg et al., 1992). Plant components and earthworms were digested in HNO3–HClO4 (4:1 v/v) prior to Pb and Zn determination. Samples of soil-tailings mixture were also air-dried and ground (!74 mm for total metal analyses; !1 mm for other analyses), prior to measurement of pH, EC and DOC (Shimadzu TOC-5000). Organic matter was determined by modified Walkey-Black rapid titration method using H2SO4– K2Cr2O7 (Sparks et al., 1996). Total N and P in soils and tailings were analyzed as described above for plant samples. Soil NHC 4 N was extracted using 2 M KCl for colorimetric determination. P and K were each extracted by 0.5 M NaHCO3 and 1 M HN4OAc, and determined by colorimetry and AAS, respectively (Page et al., 1982; Carter, 1993). Air-dried soiltailings samples were digested with HNO3–HCl–HF (9:3:3 v/v) for total metal determination, and both 1 M NH4OAc 0.005 M DPTA for more mobile and potentially mobile fractions (Ure, 1996; Lindsay and Norvell, 1978). Pb and Zn concentrations in Table 1 Properties of the Pb/Zn mine tailings and topsoil mixture (meanGSD, nZ3) Tailings pH Electrical conductivity (ms cmK1) Organic matter (%)
8.0G0.01 3.7G0.04
7.0G0.01 0.43G0.002
1.0G0.36
5.8G1.45 0.15G0.005
P K
0.78!10 G 0.2!10K4 0.01G0.0036 0.057G0.0076
0.09G0.001 0.26G0.018
Total metal concentrations (mg kgK1)
Pb Zn Cu Cd
4418G372 9827G376 89.9G1.93 32.6G4.52
117G3.66 136G2.71 25.8G0.28 4.85G0.17
DTPA-extractable metal concentrations (mg kgK1)
Pb Zn Cu Cd
1202G254 382.7G77.0 3.48G0.53 n.d.
14.1G0.38 18.4G0.74 1.57G0.18 n.d.
Total nutrients (%)
2.3. Sampling and analysis After 3 months, earthworms were separated from each pot, starved for 3 days to remove gut contents, then washed in deionized water and dried at 65 8C. Tree seedlings heights were measured, prior to separating into foliage, stems and roots. Roots were removed from soil by careful washing and the root
Soil
n.d., not detectable.
N
K3
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Table 2 Soil NHC 4 KN and ‘available’ K as influenced by earthworms and arbuscular mycorrhizal fungi (AMF) after 3 months in different proportions of tailings Proportions of tailings (%)
Inoculation treatment Control
NHC 4 KN
(mg kg
K (mg kgK1)
K1
)
Earthworms a
b
AMF
EarthwormsCAMF c
0 25 50
0.98G0.11 0.81G0.12a 0.64G0.20a
1.98G0.29 1.44G0.26b 0.89G0.25a
1.52G0.27 1.25G0.10bcd 0.87G0.42ad
1.04G0.24ae 1.44G0.31be 1.14G0.11be
0 25 50
9.15G1.66d 6.58G2.49ad 5.35G1.26a
9.73G0.72d 7.38G1.13ad 5.98G0.57a
61.9G7.86b 39.8G9.01c 25.9G5.66d
51.9G5.38c 31.0G4.91b 20.6G1.87e
Control was not inoculated. Different letters of superscript mean a significant difference at P!0.05 in the same row and the same column for each nutrient (one-way ANOVA, Tukey test). Also see Table 7.
all digests and extracts were determined using ICP-AES (Page et al., 1982). Phosphatase activity of soil was measured by colorimetric estimation of the p-nitrophenol released when soil was incubated with buffered (pH 6.5) sodium p-nitrophenyl phosphate solution and toluene, and expressed in micrograms p-nitrophenol per gram of dry soil per hour (Page et al., 1982). Soil dehydrogenase activity was estimated spectrophotometrically (480 nm) to quantify the triphenyl formazan (TPF) production that resulted from an enzymatic reaction when soil samples were incubated in 1% triphenyltetrazolium chloride (TTC) at 37 8C for 24 h (Page et al., 1982).
the N-fixing capacity of root nodules, but earthworm activity played a larger role in reducing N-fixation. With both AMF and earthworms more but less active nodules were formed. Root mycorrhizal colonization increased with increasing proportions of tailings (Fig. 2), and earthworms significantly increased rates of infection. When data for all tailing/soil and inoculation treatments were plotted together (Table 4), root nodulation was positively correlated with soil enzyme activity, but both were negatively correlated with N2-fixing capacity and mycorrhizal colonization. Viewed in these broader terms, with increased AMF infection of roots there appeared to be less
3. Results 3.1. Tailing and topsoil properties The topsoil had a low NPK status but otherwise provided an appropriate ameliorant for the mine tailings (Table 1). The tailings were of high pH, with extremely low nutrients, and high Pb and Zn. There appeared to have been a long enough period of surface exposure of the tailings for a small but significant amount of organic matter to have accumulated. By the end of the experimental period, there had been only negligible effects of earthworms and mycorrhizae on soil pH, EC, DOC and available P; the differences were not statistically significant (data not shown). However, an increase in NHC 4 KN was found with both earthworms and AMF, and available K was substantially increased by AMF inoculation (Table 2). After 3 months, the numbers of earthworms that survived and were active out of the original 10 per pot were: 10 in clean soil, 7–8 in 25% tailings and 2–3 in 50% tailings (P!0.05). 3.2. N-fixation and microbial activity Activity of both microbial enzymes was reduced in proportion to the amount of tailings, much more than due to the effects of earthworms or AMF (Fig. 1). Earthworm inoculation increased enzyme activity, but there was no consistent effect that could be attributed to AMF inoculation. With an increasing proportion of tailings, there was a beneficial effect of both earthworm and AMF inoculation on root nodulation (Table 3). AMF inoculation similarly stimulated
Fig. 1. Soil phosphatase activity (a) and dehydrogenase activity (b) after 3 months’ growth of L. leucocephala in different proportions of tailings. The treatments include inoculation with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control). Different letters indicate a significant difference at P!0.05 when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test). Also see Table 7.
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Table 3 Production and N2-fixing capacity of root nodules of L. leucocephala as influenced by earthworms and arbuscular mycorrhizae in different proportions of tailings Proportions of tailings (%)
Inoculation treatment Control
Fresh weight (g plant ) 0 2.8G0.35a 25 1.8G0.25d 50 0.6G0.17c K1 K1 N2 fixing capacity (mmol C2H4 h g ) 0 146G20.9e 25 181G17.7e 50 293G43.9d
Earthworms
AMF
EarthwormsCAMF
K1
2.3G0.47ad 2.0G0.18d 1.0G0.13b
3.7G0.45c 2.1G0.26ad 1.4G0.48ab
2.5G0.41ad 1.8G0.36d 1.2G0.16b
51.2G11.9a 113G23.7b 221G30.9ed
148G8.63e 276G32.2d 456G99.3c
62.4G16.3a 125G36.0b 155G37.5be
Control was not inoculated. Different letters of superscript mean a significant difference (P!0.05) in the same row and the same column for each parameter. Also see Table 7.
nodulation but more N-fixation (Ma, 2003, data not shown here). 3.3. Plant growth and nutrient uptake After 3 months, there was increased plant growth associated with both AMF and earthworm inoculation only in 50% tailings (Fig. 3), which also suggested a possible additive effect of earthworm and AMF inoculation; combined AMF and earthworm inoculation appeared to increase plant growth equivalent to that in 25% tailings. Foliar nitrogen concentrations were the same whether or not the plants were growing in treatments with tailings, but were enhanced by all ameliorating treatments (Fig. 4). With combined inoculation of AMF and earthworms, foliar P was increased by up to 44.8% in 0% tailings and 26.8% in 50% tailings, whilst foliar K was the same in all tailings dilutions and inoculation treatments (data not shown). Differences were evident in stem concentrations of these nutrients (Fig. 5); in 25% dilutions, K uptake was increased with AMF and AMFC
earthworms treatments, but only by AMF in 50% dilutions. Total uptake of N, P and K by plants was increased by earthworm inoculation both in clean soil and the two tailings treatments (Table 6). 3.4. Pb and Zn bioavailability and uptake There was evidence that soil concentrations of the more mobile fractions of Pb and Zn significantly declined with both earthworm and AMF inoculation (Table 5). The inoculation effect varied with extractant, but amounted to a reduction of soil concentrations of as much as 17% of Pb and 14% of Zn (DTPAextractable) and 25% of Pb and Zn (NH4OAc-extractable). Root and stem concentrations of Pb and Zn reflected the proportion of tailings, but transport to foliage appeared to be highly restricted (Fig. 6). Root concentrations of Pb and Zn were significantly decreased by both types of inoculation, but there was little effect on leaf and stem concentrations. Earthworms had no apparent separate effect on metal concentrations in plant tissues, but had a contributory effect when in combination with AMF. There were significant but much smaller effects of tailings proportions and ameliorating treatments on the mass balance of Pb and Zn, compared to NPK (Table 6). In 25% tailings, earthworms increased the total Pb and Zn uptake by plants, but the only significant effect of AMF and combined inoculation was some reduction on total Pb and Zn uptake in 25% tailings. Amounts of Pb and Zn that accumulated in the earthworm tissues were related to the amount of tailings in the substrata. Highest total body concentrations were 570 mg Pb kgK1 and Table 4 Pearson’s correlation matrix for root nodulation, N2-fixation, mycorrhizal infection, and soil microbial enzyme activity (nZ48) Nodule weight
Fig. 2. Root mycorrhizal colonization of L. leucocephala inoculated with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control), as influenced by the amount of Pb/Zn mine tailings added. Different letters indicate a significant difference at P!0.05 when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test). Also see Table 7.
Nodule weight N2-fixing capacity AMF infection Phosphatase Dehydrogenase
N2-fixing capacity
1.000 K0.450*
1.000
K0.558* 0.736* 0.650*
0.437* K0.723* K0.771*
Levels of significance: *P!0.01.
AMF infection
Phosphatase
Dehydrogenase
1.000 K0.682* K0.562*
1.000 0.847*
1.000
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Fig. 3. Total dry weight yields of L. leucocephala inoculated with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control), as influenced by the amount of Pb/Zn mine tailings added. Different letters indicate a significant difference at P!0.05 when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test).
966 mg Zn kgK1. There were no significant differences between the metal concentrations in earthworms with and without AMF inoculation (Table 7). 4. Discussion 4.1. Effectiveness of inoculation treatments Topsoil amendments to the tailings faciltated survival and growth of plants, AMF and earthworms. Dilution of 50% provided clear evidence of toxicological effects: (i) plant growth
Fig. 5. N, P and K concentrations in stems of L. leucocephala grown in different proportions of Pb/Zn tailings inoculated with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control). Different letters indicate a significant difference (P!0.05) when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test).
Fig. 4. Foliar N concentrations of L. leucocephala grown in different proportions of Pb/Zn tailings inoculated with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and non-inoculated (control). Different letters indicate a significant difference (P!0.05) when comparing the variance within each single tailings treatment according to one-way ANOVA (Tukey test).
declined and uptake of Pb and Zn was higher, (ii) only 20–30% earthworm survived after 3 months, (iii) root nodulation was inhibited, and (iv) microbial activity, measured using enzyme assays, was reduced in proportion to the amount of tailings. The microbial response that supports plant growth was evident, even in pots that were not inoculated: both AM colonization of roots and N-fixation were higher in tailings treatments than in clean soil. The results provide a basis to unravel the more subtle effects of AMF communities and burrowing earthworms on the establishment of Leucaena (and associated N-fixation) on the amended mine tailings (Table 8). Beneficial effects to the soils and to plant establishment clearly occurred and these
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Table 5 Mobile fractions of Pb and Zn in different proportions of tailings after 3 months growth of L. leucocephala, measured at the end of the experiment Proportions of tailings (%) DTPA-extractable (mg kg ) Pb 0 25 50 Zn 0 25 50 NH4OAc-extractable (mg kgK1) Pb 0 25 50 Zn 0 25 50
Inoculation treatment Control
Earthworms
AMF
EarthwormsCAMF
13.6G3.12a 232G25.4a 363G16.8a 27.3G6.59a 113G4.23a 133G10.1a
14.0G0.78a 219G4.97a 352G33.4ab 25.5G1.68a 112G1.35a 127G10.7a
13.8G0.61a 199G18.4ba 314G7.56b 26.7G3.01a 96.8G8.17b 127G8.89a
16.0G1.21a 192G5.18b 326G9.45b 28.3G2.41a 101G3.47b 131G3.83a
0.43G0.05a 18.7G2.33a 31.8G2.99a 1.98G0.24a 18.7G1.57a 34.2G0.66a
0.41G0.06a 15.5G0.70b 28.6G3.96ab 1.90G0.26a 15.6G1.34b 30.8G1.52b
0.34G0.07a 15.0G1.41b 23.8G1.82b 1.68G0.17a 14.1G1.04b 30.6G1.86b
0.36G0.01a 14.5G0.62b 25.9G1.28b 1.67G0.15a 14.4G0.99b 29.9G2.14b
K1
Control was not inoculated. Different letters of superscript mean a significant difference (P!0.05) in the same row. Bold text highlights significant treatment effects.
Fig. 6. Lead and Zn concentrations in different parts of L. leucocephala plants grown in different proportions of tailings for 3 months. The treatments include inoculation with earthworms (worms), arbuscular mycorrhizal fungi (AMF), both earthworms and arbuscular mycorrhizal fungi (wormsCAMF), and noninoculated (control). Different letters indicate a significant difference (P!0.05) when comparing the variance within each single tailings treatment according to oneway ANOVA (Tukey test). No significant differences exist between metals in foliage.
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Table 6 Total nutrient and Pb, Zn uptake by L. leucocephala as influenced by earthworms and arbuscular mycorrhizal fungi in different proportions of tailings Proportions of tailings (%)
Total uptake (mg plant K1)
Metals
Control a
Earthworms b
AMF
EarthwormsCAMF
ab
0
N P K Pb Zn
7307 8630a 44,616a 0.123a 1.33a
9191 9684a 49,770ab 0.187b 1.49a
8748 9779ab 53,859b 0.128a 1.42a
9988b 9132b 50,018ab 0.105a 1.35a
25
N P K Pb Zn
6648a 6257a 41,094a 0.962a 2.76a
7988b 7005a 44,636ab 1.26b 3.37c
9004b 7190ab 49,368b 0.859a 2.39ab
9897c 7654b 52,020c 0.655c 2.10b
50
N P K Pb Zn
4676a 4494a 19,151a 1.15a 2.76a
5882b 5707ab 21,606ac 1.21a 2.92a
7469bc 6353bc 27,908bc 1.11a 2.96a
7980c 7267c 29,569b 1.04a 3.02a
Control was not inoculated. Different letters of superscript mean a significant difference (P!0.05) in the same row. Bold text highlights significant treatment effects. Also see Table 7.
effects were generally more pronounced with combined AMF and earthworm inoculation. Inoculation with either earthworms or AMF improved plant nutrition and increased plant growth. Together they reduced heavy metal mobility in soil and plant tissue concentrations, although earthworm activity may increase metal uptake to roots whilst not affecting transport to above-ground plant components. Negative effects of inoculation were relatively minor, concerning the effects of earthworms on reduced rates of N-fixation; their burrowing activities are well know to enhance mineralization of nitrogen from organic matter (Cheng and Wong, 2002; Edwards and Bohlen, 1996), thus reducing the necessity for contributions from root nodules to meet plant growth requirements. Nonetheless, there are obviously quite complex interactions between earthworms, AMF and N-fixation. In overall terms, AMF infection was negatively correlated with N-fixation (Table 4) even though it occurred with both increased nodulation and N-fixation in the presence of tailings (Table 3).
4.2. Effects on soil nutrients and plant growth Earthworms and AMF inoculation stimulated microbial decomposer activity and to mineralization of organic N. Our own previous study on the same mine tailings also found increased NO3–N in Pb/Zn mine tailings after earthworm inoculation (Ma et al., 2003). Earthworms, but not AMF inoculation, significantly stimulated soil dehydrogenase and phosphatase activity, as has been recorded previously (Satchell, 1983). It is known that AMF may directly compete with soil bacteria for carbohydrate substrates (Trevors and van Elsas, 1997). Growth enhancement of L. leucocephala has previously been correlated with AM colonization of roots and uptake of P, Cu and Zn (Manjunath and Habte, 1988). In the present study, improved growth of L. leucocephala with inoculation may have occurred through enhanced uptake of N, P and K. Mobile K in soil was elevated fivefold or more following AMF inoculation, and this was also reflected in stem uptake of this nutrient. AMF hyphae increase the surface area
Table 7 Differences between plant dry weight yields, root mycorrhizal infection, root nodule production, nodule N2-fixing capacity, total nutrient and metal uptake by L. leucocephala, soil phosphatase and dehydrogenase activities, due to (i) different proportions of tailings and (ii) different ameliorating treatments
Dry weight yields Mycorrhizal infection Nodule production N2-fixing capacity Total N uptake Total P uptake Total K uptake Total Pb uptake Total Zn uptake Phosphatase activity Dehydrogenase activity
Tailings
Treatments
Tailings!treatments
26.44*** 232.3*** 120.6*** 85.38*** 20.82*** 30.03*** 118.2*** 180.1*** 92.44*** 226.3*** 357.2***
4.705** 81.89*** 10.17*** 53.54*** 17.55*** 6.193** 8.751*** 6.875** 3.797* 23.54*** 36.14***
1.267 0.363 3.867** 6.015*** 0.705 0.368 0.569 2.266 3.700** 0.424 12.63***
Values are F-values and significance levels from two-way ANOVA. Levels of significance: *P!0.05, **P!0.01, ***P!0.001.
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Table 8 Summary of the beneficial and negative effects of earthworm inoculation and arbuscular mycorrhizal fungi (AMF) inoculation, singly and together, on topsoilamended mine tailings and Leucaena leucocephala Inoculation
Beneficial effects
Negative effects
Earthworms
Increased soil microbial activity Increased AMF infection
Reduced N-fixing capacity of root nodules Possibly contributes to increased plant uptake of Pb and Zn
Increased NHC 4 N in soil Increased foliar N Increased total plant uptake of N Increased root nodulation Increased plant growth AMF
Increased root nodulation Increased N-fixation Increased NHC 4 N in soil Increased foliar N Increased K mobility in soil Increased K uptake by plant Increased plant growth Decreased mobile Pb and Zn in soil Decreased Pb and Zn uptake by roots
Combined earthworms and AMF
Increased N, P, K uptake
Earthworms may inhibit the beneficial effects of AMF on N2-fixation
Improved plant growth Decreased mobile Pb and Zn in soil Possible reduced Pb and Zn uptake Italics indicate the same effects were found separately for both earthworm and AMF inoculation.
for ion absorption from soil (Read, 1984); the link between AM infection and K in plant shoots has been studied previously (Sieverding and Toro, 1988). 4.3. Effects on Pb and Zn bioavailability In earlier work, we studied the separate effects of earthworms and AMF on metal mobility in the same mine tailings. Earthworm inoculation alone increased overall metal uptake, albeit indirectly through improving plant growth rather than increased uptake per se (Ma et al, 2003). The same effect probably occurred in the present study. We found previously that AMF infection (in the absence of earthworms) stimulated uptake of metals by plants when soil metal concentrations were low, but decreased uptake when metal concentration were high (Ma, 2003). In the present experiments, AMF appear to be primarily responsible for protecting plants from excessive metal uptake, whilst combined earthworm and AMF activity reduced likely mobile forms of Pb and Zn in soil by 14–25%. Amelioration of Zn toxicity has been found to occur through the provision of additional adsorptive surfaces on hyphal cell walls and to extrahyphal polysaccharide slime (Hilary et al., 1987; Bradley et al., 1982). Decreased foliar Pb in mycorrhizal plants growing in Pb-polluted soil has been reported previously (Weissenhorn et al., 1995).
(Ydrogo, 1994; Edwards and Bohlen, 1996), as also found in the present study. Earthworm casts may contain up to 10 times as many infective AMF propagules as surrounding soil (Gange, 1993). The relationship between earthworms and N-fixation appears to be more complex; in the present study, earthworms increased root nodulation, but apparently decreased their N2-fixing capacity. However, nodulation and N-fixation is probably a response to nitrogen deficiency, that itself is mitigated by the activity of earthworms. Doube et al. (1994a) showed that earthworms enhanced dispersal of N2-fixing bacteria and their colonization of roots, but that additional nodulation did not significantly affect plant growth. It is also possible to explain why, under higher proportions of tailings, AMF colonization was negatively correlated with root nodule production, but positively correlated with nodule N2-fixing capacity. A high P demand of the N-fixation process can be supplied by AMF, and mycelial growth and mycorrhizal formation are enhanced by Rhizobium; a synergistic process that results in improved rates of P uptake and N-fixation (Azco´n et al., 1991; Xavier and Germida, 2003). High soil N, with or without AMF, leads to a significant decline in nodulation (Jia et al., 2004). 4.5. Conclusion
4.4. Earthworms, AMF and N-fixation Effective mycorrhizal root colonization is constrained by the minimal capacity of AMF for unaided dispersal through soils, but it is known this can be enhanced by earthworms
Reclamation of Pb/Zn mine spoils in Guandong and elsewhere requires techniques that will both ameliorate the constraints of a hostile substrate and enhance the natural attenuation of conditions that are toxic to plant establishment
Y. Ma et al. / Soil Biology & Biochemistry 38 (2006) 1403–1412
and growth. Leucaena is an invasive N-fixing woody plant that is in the early stages of colonization after decades of exposure, or even longer. Arbuscular mycorrhizal fungi clearly provide an important and effective preliminary inoculant; in this experimental work there was as much as 55% root infection in pots that had not been inoculated, and infection levels were highest with increasing proportion of tailings. AMF improve the ability of Leucaena to fix N through its root nodules, improve nutritional status and provide a protective role against toxic metals, but they are naturally slow to disperse. Earthworms aid their dispersal and otherwise improve conditions for plant growth, not least by enhancing soil microbial activity and improve soil nutrient availability. Earthworms are also naturally slow to colonize reclaimed soils, although it has been demonstrated that earthworm inoculation is quite possible if potentially expensive (Butt et al., 1997; Butt, 1999). The combined effects of AMF and earthworms are beneficial in terms of plant growth (O30% yield increase in present experiment), nutrition (improved N, P and K uptake) and protection against toxic metals (up to 25% reduction in uptake), but earthworms may inhibit the beneficial effects of AMF on N2-fixation. The future applications of these ecological studies lies in an opportunity to improve the speed and efficacy of the restoration process through judicious use of topsoil or other amendments combined with the careful encouragement of the soil biota. Acknowledgements The project was supported by the Research Grants Council (HKBU-2049/00M) of the University Grants Committee of Hong Kong. We thank Prof. J. Zhang, Mr. H.T. Poon, Dr. J. Guo and Dr. M. Jiang for their assistance with this work. References Azco´n, R., Rubio, R., Barea, J.M., 1991. Selective interactions between different species of mycorrhizal fungi and Rhizobium meliloti strain, and their effects on growth, N2-fixation (15N) and nutrition of Medicao sativa L. New Phytologist 117, 339–404. Bradley, R., Burt, A.J., Read, D.J., 1982. The biology of mycorrhiza in the Ericaceae. VII. The role of infection in heavy metal resistance. New Phytologist 91, 197–209. Butt, K.R., 1999. Inoculation of earthworms into reclaimed soils: the UK experience. Land Degradation & Development 10, 565–575. Butt, K.R., Frederickson, J., Morris, R.M., 1997. The earthworm inoculation unit technique: an integrated system for cultivation and soil-inoculation of earthworms. Soil Biology & Biochemistry 29, 251–257. Carter, M.R., 1993. Soil Sampling and Methods of Analysis. Lewis Publishers, London. Cheng, J.M., Wong, M.H., 2002. Effects of earthworms on Zn fractionation in soils. Biology and Fertility of Soils 36, 79–86. Doube, B.M., Ryder, M.H., Davoren, C.W., Stephens, P.M., 1994a. Enhanced root nodulation of subterranean clover Trifolium subterraneum by Rhizobium trifolii in the presence of the earthworm Aporrectodea trapezoids. Biology and Fertility of Soils 6, 237–251. Doube, B.M., Stephens, D.M., Davoren, C.W., Ryder, M.H., 1994b. Interactions between earthworms, beneficial soil microorganisms and root pathogens. Applied Soil Ecology 1 (1), 3–10.
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