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• Elemental allelopathy suggests that nickel (Ni)-rich leaves shed by hyperaccumulators inhibit the germination and growth of nearby plant species.
• Here, the germination of eight herbaceous species following addition of Alyssum murale biomass or Ni(NO3)2, with the same Ni level added to soil, was assessed. The distribution of Ni in soil was tested by determining Ni phytoavailability and speciation over time.
• Phytoavailable Ni in soil amended with biomass declined rapidly over time due to Ni binding to iron (Fe)/manganese (Mn) oxides in the soil. No significant effects on seed germination were observed. Unlike the Ni complex in Alyssum biomass, more Ni remained soluble and phytoavailable in soil amended with Ni(NO3)2, thus significantly inhibiting seed germination.
• High-Ni leaves shed by hyperaccumulators did not appear to create a ‘toxic zone’ around the plants and inhibit germination or growth of competing plants. The lack of an allelopathic effect was probably related to low Ni availability.
Nickel (Ni) is an essential micronutrient for higher plants (Welch, 1995). Nickel at low levels is important for plant growth, plant senescence, nitrogen metabolism, seed germination and plant disease resistance (Mishra & Kar, 1974; Brown et al., 1987). However, Ni can be phytotoxic when soluble forms of Ni are present in soil in excess. General symptoms of Ni toxicity are interveinal chlorosis of young leaves, reduced growth of roots and shoots, decreased yield and germination inhibition (Brune & Dietz, 1995; Baker & Walker, 1989; Kukier & Chaney, 2004).
Despite the phytotoxic effects of Ni, a number of plant species endemic to Ni-rich serpentine soils have the ability to actively accumulate Ni to concentrations exceeding 0.1% of their shoot dry weight without symptoms of toxicity or yield reduction (Baker et al., 2000). These plant species, called hyperaccumulators, have received increased attention in recent years because of their potential in phytoextraction of metal-contaminated soils (Li et al., 2000).
However, the evolutionary reason for metal hyperaccumulation remains unclear. A number of hypotheses have been proposed to explain the evolution of this phenomenon. Boyd & Martens (1992) listed six principal hypotheses: inadvertent uptake, metal tolerance, disposal from the plant body, drought resistance, pathogen–herbivore defense and interference with neighboring plants. Interference was termed ‘elemental allelopathy’ by Boyd & Martens (1998). Elemental allelopathy refers to the ability of one plant species to affect growth of another, by increasing to toxicity macronutrient or micronutrient contents. To support this hypothesis, Boyd & Jaffré (2001) reported higher Ni levels in the surface soil under the canopy of the New Caledonian Ni hyperaccumulator Sebertia acuminata, compared with the soil under the canopy of nonhyperaccumulator species. However, the hypothesis of elemental allelopathy has not been tested experimentally.
The fallen leaves and stems of Ni hyperaccumulators with high Ni content may increase the total concentration of Ni in surface soil. However, the amount of Ni absorbed by other plants does not fully depend on the net amount of Ni in soil, but is more directly related to the concentration of soluble ions of Ni and the rate of replenishment of this labile pool (Duneman et al., 1991).
Within Ni hyperaccumulators, Ni has been reported to complex with histidine and organic acids such as citrate, malate and malonate (Jaffréet al., 1976; Homer et al., 1991; Krämer et al., 1996), with preferential compartments in vacuoles of epidermal cells or star-like leaf hairs (trichomes) for Ni detoxification (Küpper et al., 2001; Krämer et al., 2000; Broadhurst et al., 2004). The complexing of Ni with organic acids or amino acids can affect chemical solubility and mobility of Ni in the environment. DeKock & Mitchell (1957) found that compared with ionic Ni, Ni chelated with ethylenediaminetetraacetic acid (EDTA), diethylenetriamine-pentaacetic acid (DTPA) or nitrilotriacetic acid (NTA) is absorbed by mustard and tomato at lower concentrations. Studies by Molas & Baran (2004) also showed that barley plants absorb more Ni from its inorganic form (nickel sulfate) than organic complexes (Ni(II)-citrate, Ni(II)-glutamate, or Ni(II)-EDTA) when these chemical forms of Ni were applied to soil at the same Ni concentration. Therefore, chelated Ni was less phytoavailable than free Ni2+ for uptake by plants.
It is also known that bioavailability of heavy metals depends on soil properties such as soil pH, content of organic matter, amorphous Fe, Mn and Al oxides, and soil sorption capacity (Basta et al., 2005). Understanding the speciation of a metal can help assess how strongly the metal is retained in soil and how easily the metal may be released into soil solution. The sequential extraction procedure of Tessier et al. (1979) is commonly applied to evaluate both the actual and potential mobility of metals in the environment (Rudd et al., 1986; Dang et al., 2002). The level of extractable Ni by 0.01 m strontium nitrate was shown to be well correlated with Ni concentrations in crop plant shoots (such as oat, lettuce, etc.); it is an effective measure of phytoavailable Ni in soil (Kukier & Chaney, 2001).
The main objective of this study was to test the hypothesis of elemental allelopathy that when leaves and stems of the Ni hyperaccumulator Alyssum murale are deposited on the soil surface, seed germination of nonhyperaccumulators may be affected. A second focus of this work was to investigate the fate and the distribution of biomass-bound Ni compared with Ni(NO3)2 added in soil by determining Ni phytoavailability and speciation in soil over time.
Materials and Methods
Preparation and analyses of soil and plant biomass
Soil sampling and analyses Two soils were used in this study: a Ni-rich serpentine soil (Brockman variant very gravelly loam; fine, magnesic, mesic Typic Xerochrepts) was collected near Cave Junction, OR, USA; and a low Ni soil (Christiana fine sandy loam; clayey, kaolinitic, mesic Typic Paleudults) was collected at Beltsville, MD, USA. Each soil was mixed in large containers and dried at room temperature. Soils were crushed to pass a 4-mm sieve.
For the analyses of soil properties, dry soil samples were ground and passed through a 2-mm sieve. Five grams of air-dried soil was digested with 10 ml concentrated HNO3 and heated to near dryness on a hotplate, subsequently dissolved in 20 ml 3 m HCl and heated at mild reflux for 2 h. The residue was filtered and diluted to 50 ml with 0.1 m HCl. Total metal – Ni, iron (Fe), manganese (Mn), copper (Cu), cadmium (Cd, and zinc (Zn) – concentrations were determined using atomic absorption spectrophotometry (AAS) (US EPA Method 3050, 1995) with appropriate background correction. Exchangeable Mg and Ca were extracted with Mehlich 3 (Mehlich, 1984), and then measured by inductively coupled plasma atomic emission spectroscopy (ICP-AES). Soil pH was measured in a soil water suspension of 1 g: 1 ml after 1 h (Eckert & Sims, 1995). Organic matter content was determined by loss on ignition (Storer, 1984). The physical and chemical properties of the two soils are listed in Table 1.
Table 1. Physical and chemical characteristics of soils used to assess metal release from Alyssum murale biomass
Organic matter %
Exchangeable ions (mEq 100 g−1) Mg
Ni (mg kg−1)
Cu (mg kg−1)
Zn (mg kg−1)
Cd (mg kg−1)
Total soil metals Mn (g kg−1)
Fe (g kg−1)
Data within the same column are significantly different, except soil pH and Cd (Student's t-test, P < 0.05, n = 4).
Plant biomass collection and analysis Alyssum murale (Waldst. & Kit.), an herbaceous perennial Brassicaceae, has proven to be an effective Ni hyperaccumulator through glasshouse and field trials (Li et al., 2003). Alyssum murale biomass used in this study was collected from plants grown on the Brockman soil near Cave Junction. Shoots were cut 10 cm from the soil surface and placed into paper bags for drying. Biomass was dried for 48 h at 60°C then ground to pass a 1-mm sieve. Although grinding and sieving of biomass does not completely mimic natural leaf fall and decomposition, it was necessary for homogeneity while mixing soils and biomass.
Elemental analysis was performed using AOAC Method 3.014(a) (1984). Two grams of biomass was ashed in a muffle oven at 480°C for 16 h. The ash was digested with 2 ml concentrated HNO3 and heated to near dryness on the hotplate, subsequently dissolved in 10 ml 3 m HCl and refluxed for 2 h on the hotplate. The residue was filtered and diluted to 25 ml with 0.1 m HCl. Nickel concentration was determined by ICP-AES using cobalt as an internal standard. Appropriate standards were also measured to assure quality control.
Plant response to A. murale biomass or inorganic Ni in soil
Alyssum murale biomass was mixed with soils (Christiana soil or Brockman soil): 60 g of biomass was mixed with 2.5 kg of soils (Christiana soil or Brockman soil) in a tray at a rate of 24 g DW biomass kg−1 DW soils. This rate is close to yields (20 t biomass ha−1 soil) we have observed in the field. We selected this rate since it is most likely to produce the hypothesized allopathic effect. Biomass and soil were mixed to homogeneity, and then incubated in a growth chamber for 1 month. A broad, shallow tray was used to mimic biomass addition to soil. The period of 1 month was chosen since Ni was nearly completely released from biomass during this time (Zhang et al., 2005). The growth chamber was set at 24°C and 60% relative humidity, with 14-h photoperiod. The temperature and relative humidity remained constant. The moisture of the mixture of soil and biomass was maintained at near 60% water-holding capacity of soil by regular watering using distilled water. The water-holding capacities of the Oregon and Christiana soils were determined to be 24.4% and 43.7% by the method of Forster (1995).
To simulate how the addition of hyperaccumulator biomass affects the germination and growth of other plants, eight herbaceous species were used in this study: Alyssum montanum L. (mountain gold alyssum), Avena fatua L. (wild oat), Bromus hordeaceus L. (soft brome), Bromus mollis L. (soft chess), Lolium multiflorum Lam. (Italian ryegrass), Lolium perenne L. (perennial ryegrass), Chenopodium album L. (lambsquarter) and Brassica kaber (DC.) L.C. Wheeler (wild mustard). Most species previously have been reported to grow on serpentine soils, although none of the species used were grown on serpentine soils, thus assuring testing of populations that are likely sensitive to toxic concentrations of Ni (Harrison, 1999). Forty-five seeds of each species were sown at intervals of 2–3 cm in each tray. The germination rate of seeds was observed each day for a period of 20 d. Germination was monitored for several additional weeks, but did not change beyond the original 20-d period.
For comparison, the same level of inorganic Ni (nickel nitrate) as applied in A. murale biomass, was added to soil. Nickel nitrate was added by first dissolving in distilled water then adding the water to soil. Nickel was added in an identical manner to the biomass. All other conditions were identical. A control for each soil without Ni addition was assessed. There were four replicates of each treatment combination.
Phytoavailability of nickel in soil
Alyssum murale biomass and 20 g soil (Brockman soil or Christiana soil) were added to cups at the same mass ratio of biomass to soil as described earlier. The biomass and soil were mixed to homogeneity. All cups were placed into a growth chamber for incubation. The conditions of incubation were similar to those already mentioned. The same level of inorganic Ni (Ni(NO3)2) as applied in A. murale biomass was added to soil as a comparison. Soils without Ni addition were also tested as controls. Individual cups were placed into the incubator in a block design to reduce environmental variation.
To determine Ni release from the biomass in soil over time, on days 0, 1, 2, 3, 7, 15, 30 following biomass addition to soils, single cups from each of the four replicates from each treatment were extracted with 40 ml 0.01 m Sr(NO3)2 with continuous shaking for 1 h, then slurries were filtered using an Ahlstrom #513 fluted filter paper (Ahlstrom, Helsinki, Finland) inside a size 40 Whatman (Clifton, NJ, USA) filter paper. Extracted solutions were refrigerated at 4°C until analysed. The Sr(NO3)2-extractable Ni concentrations were determined using AAS.
To assess Ni availability over time, A. murale biomass was added to the Christiana soil because the Christiana soil was able to clearly show the fate of biomass-bound Ni in soil because of to its low total Ni content. For metal speciation, a scheme based on the work of Tessier et al. (1979) was used. Alyssum murale biomass and 1.5 g dry soil at the same mass ratio of biomass to soil as described earlier were mixed in centrifuge tubes (polypropylene, 50 ml), and incubated in the growth chamber. The moisture of the mixture of soil and biomass was maintained at 60% water-holding capacity. On days 0, 0.5, 1, 2, 3, 7, 15, 30, 45, 60, 90, 120, 150 following biomass addition to soils, samples of four replicates were sequentially extracted using the reagents and conditions described in Table 2.
Table 2. Experimental conditions used for sequential extraction of soils
Procedure of extraction
8 ml 1 mol l−1 MgCl2 (pH 7), room temperature, continuous shaking, 1 h
Bound to carbonates
8 ml 1 mol l−1 NaOAc + HOAc (pH 5), room temperature, continuous shaking, 5 h
Bound to iron/manganese oxides
20 ml 0.04 mol l−1 NH2OH.HCl in 25% HOAc, 95 ± 3°C, intermittent shaking, 6 h
Bound to organic matter
3 ml 0.02 mol l−1 HNO3 + 2 ml 30% H2O2 (pH 2), 85 ± 2°C, intermittent shaking, 2 h; 3 ml 30% H2O2 (pH 2), 85 ± 2°C, intermittent shaking, 3 h; 5 ml 3.2 mol l−1 NH4OAc in 20% HNO3 + 7 ml H2O, room temperature, continuous shaking, 30 min
Concentrated HNO3 + 3 m HCl, procedure described as total metal analysis
Following each extraction, mixtures were centrifuged at 2500 r.p.m. for 20 min. The supernatant was filtered using size 40 Whatman filter paper and collected in a vial. Extracted solutions were refrigerated at 4°C until analysed using AAS. Before the next extraction step, the residue was washed with 12 ml distilled water; centrifuged for 20 min and filtered. This second supernatant was collected and analysed to minimize sample dispersion. Ni(NO3)2 with the same quantity of Ni as in Alyssum murale biomass was added to soil as a comparison. The Christiana soil without Ni addition was also tested as a control.
Double rate addition of A. murale biomass
Because the original rate of biomass addition did not produce any apparent effect, and because biomass is added to the soil surface annually with leaf fall, high Ni biomass was added at twice the previous rate. Alyssum murale biomass was first added to soil at a rate of 24 g DW biomass kg−1 DW soil. After incubation of 1 month, more biomass was added to the soil to double the rate. Seed germination and Ni phytoavailability in soil over time were tested as previously described.
All glassware used in all the experiments was soaked in 10% HNO3 (v : v) overnight and then rinsed with distilled water. All reagents used in this study were of analytical grade or better.
Data were analysed using SAS version 8.01 (SAS Institute, Cary, NC, USA). All values reported in the following tables and figures are means based on four replicates. The differences between means for the initial soils analysis were tested using the student's t-test. Treatment effects on seed germination were determined using an anova followed an LSD test for mean separation. The effect of time on Ni concentrations extracted from soil was tested using the one-way anova F-test. A significance level of P < 0.05 was selected for the determination of statistical difference.
Results and Discussion
Response to A. murale biomass or inorganic Ni in soil
Alyssum murale biomass used in this study contained a total Ni concentration of 12.0 g Ni kg−1 DW biomass. The effect of Ni biomass on seed germination of eight herbaceous species in both of the Christiana and Brockman soils is shown in Figs 1 and 2, respectively. Most species achieved a high germination rate in both the control Christiana and the control Brockman soils. The addition of A. murale biomass to either soil had no significant effect on the germination rate of eight species at the rate of 24 g DW biomass kg−1 DW soil (P > 0.05). The roots and shoots of seedlings grew normally without interveinal chlorosis. Further, the double rate addition of A. murale biomass did not significantly reduce the germination of seeds, except for a significant germination reduction by A. montanum and C. album in the Christiana soil and L. perenne in the Brockman soil. It is interesting to note, however, that while generally nonsignificant, greater reduction was observed in the Brockman high Ni soil compared with the Christiana low Ni soil. The sum of Ni added with biomass to an already high Ni soil had greater impact than when the total Ni concentration was lower.
When the same level of inorganic Ni (Ni(NO3)2) as applied with the Alyssum murale biomass was added to soil, each species exhibited symptoms of Ni toxicity, such as interveinal chlorosis, a reduction of length and degree of root system branching, a decrease in leaf dimension, etc. For most of the eight species, the addition of inorganic Ni resulted in a significant decrease in their germination rates (Figs 1 and 2).
Phytoavailability of Ni in soil
The Ni extracted by 0.01 m strontium nitrate in the unamended Christiana soil was close to zero (Fig. 3). The Sr(NO3)2-extractable Ni in the unamended Brockman soil was also low although the total Ni in the Brockman soil was very high (4120 mg Ni kg−1 DW, Table 1). The amount of Sr(NO3)2-extractable Ni in both soils showed no significant change over time (P > 0.05).
Immediately after the addition of A. murale biomass or inorganic Ni to soil, Sr(NO3)2-extractable Ni in soil quickly increased (Fig. 3). Many studies on Ni hyperaccumulators have shown that Ni is accumulated in shoots as Ni complexed with organic and amino acids (Jaffréet al., 1976; Homer et al., 1991; Krämer et al., 1996). The present study indicates that Ni in A. murale biomass was immediately released into soil, and the Ni was highly soluble and phytoavailable. It has also been reported that > 60% of Ni in the stem and leaf tissue is water soluble for the Ni hyperaccumulator Stackhousia tryonii (Bidwell, 2001; Bhatia et al., 2004). A study by Schlegel et al. (1991) suggested contrary results in that high concentrations of Ni-rich soil were found under a Ni hyperaccumulating tree since that resulted in a bacterial population with enhanced Ni tolerance as a result of Ni exposure. We suspect the disparity between the results for our results and Schlegel et al. (1991) may result from differences in spatial distribution of roots and individual bacteria. Bacteria may be fully engulfed in high Ni soil, while plants have the ability to grow away from toxic concentrations of various elements and thus avoid full exposure to high Ni in soil.
The Sr(NO3)2-extractable Ni from the soil amended with A. murale biomass and from the soil amended with inorganic Ni rapidly decreased with the former decreasing faster than the latter over time. Beyond 15 d, Sr(NO3)2-extractable Ni from all amended soils showed few changes. The equilibrium concentration of Sr(NO3)2-extractable Ni in soil amended with biomass was only slightly higher than that of control soil, while the addition of inorganic Ni greatly elevated the equilibrium concentration of extractable Ni in soil (c. 50 mg kg−1 dry soil).
The same trend was shown with the double rate addition of biomass or inorganic Ni. Inorganic Ni greatly increased phytoavailable Ni in soil, while the addition of A. murale biomass only slightly increased phytoavailable Ni in soil. This difference seems to explain the earlier observation that the inorganic Ni added to soil significantly inhibited seed germination of the eight species, while the addition of equal Ni in biomass did not significantly affect seed germination of these species (Figs 1 and 2). The release of Ni from biomass occurred at a very rapid rate, as previously shown, then quickly complexed so as to reduce phytoavailability. Inorganic Ni typically overestimates metal effects. In addition, addition of Ni(NO3)2 can cause a small decline in soil pH which makes Ni more phytoavailable, thus inhibiting the germination and growth of plants (Kukier & Chaney, 2004).
Compared with the Christiana soil, the equilibrium concentration of Sr(NO3)2-extractable Ni in the Brockman soil with the same treatments appears to be relatively low. This demonstrates that the Brockman soil has a stronger ability to bind Ni compared with the Christiana soil. This may result from the fact that the Brockman soil has a slightly higher soil pH level and higher content of organic matter and Fe/Mn oxides (Table 1). The availability of Ni in soil increases as soil pH decreases, and organic matter and Fe/Mn oxides could influence Ni availability (Willaert & Verloo, 1988; Weng et al., 2001).
Nickel redistribution in soil amended with Ni biomass or inorganic Ni
The results of the 5-month sequential extraction test showed the redistribution of Ni and its potential availability in the Christiana soil amended with A. murale biomass with respect to time (Fig. 4a). When A. murale biomass was added to the Christiana soil, the majority of Ni was in the exchangeable fraction. The exchangeable fraction quickly decreased after the addition of biomass and showed few changes beyond 60 d. Nickel bound to Fe/Mn oxides increased rapidly with respect to time, while Ni bound to carbonates reached a maximum concentration at day 15 and then started to decrease. At steady state, the Fe/Mn oxide fraction contained the largest amount of Ni, followed by the carbonate fraction. The concentrations of Ni bound to organic matter and Ni in the residual fraction showed a slight increase with respect to time, but both were only a small proportion (< 3%) of the total Ni in soil.
The phytoavailability of metal in soils decreases following the order of the extraction sequence, from readily available to unavailable (Tessier & Campbell, 1987). The exchangeable fraction may indicate the form of metal that is most available for plant uptake and can be released by merely changing the ionic strength of the medium (Filgueiras et al., 2002). Metals bound to carbonate can easily become available with a reduction of soil pH. The remaining three fractions are generally strongly held by soil constituents and are normally unavailable to plants (Li et al., 1995). From the results of the sequential extraction, with increasing incubation time, the percentage of exchangeable Ni decreased from 78% to 14%, the percentage of Ni bound to carbonate increased from 11% to 18% and the percentage of Ni bound to Fe/Mn oxides increased from 9.5% to 65% (Fig. 4b). Strontium nitrate solution mainly extracted Ni from the exchangeable fraction in soil. This corresponds to the results of strontium nitrate extraction described earlier that showed that the phytoavailable Ni was greatly reduced with respect to time. The Fe/Mn oxides played an important role in immobilization of Ni in the Christiana soil.
In experiments where the same level of inorganic Ni as applied in A. murale biomass was added to the Christiana soil, a similar trend was observed (Fig. 5.). As incubation time increased, the concentration of exchangeable Ni in soil decreased and Ni in other fractions increased. Therefore, phytoavailable Ni in soil gradually decreases with respect to time. However, the percentage of exchangeable Ni in the soil amended with Ni(NO3)2 only decreased from 89% to 56% after a 5-month incubation, and more than half of total Ni in soil was still exchangeable Ni at steady state. A similar result was observed in the studies by Singh & Jeng (1993) where NiCl2 was applied to soil. The percentage of Ni fractions was in the following order: exchangeable Ni > Ni bound to Fe/Mn oxides > Ni bound to carbonates > residual Ni > Ni bound to organic matter. Therefore, compared with the soil amended with biomass, less Ni was bound to Fe/Mn oxides at steady state and more available Ni was present, thus resulting in higher phytotoxicity.
The addition of A. murale biomass or inorganic Ni (Ni(NO3)2) at the same amount as Ni showed that the different forms of Ni greatly influenced the phytoavailability and redistribution of Ni in soil. Biomass-bound Ni was rapidly released and greatly elevated the concentration of phytoavailable Ni in soil for a very short time. However, as incubation time increased, most of soluble Ni was bound to Fe/Mn oxides and silicates in both soils, and phytoavailable Ni in soil amended with Ni biomass quickly decreased with respect to time. The equilibrium concentration of phytoavailable Ni was only slightly higher than the control soil without Ni biomass. When inorganic Ni was added to soil, most Ni was soluble and the equilibrium concentration of phytoavailable Ni was significantly higher than the control soil. As a result of this difference, the addition of inorganic Ni inhibited the germination of eight herbaceous species, while the addition of A. murale biomass (single addition) did not significantly affect germination of seeds.
Based upon these results, the hypothesis that elemental allelopathy can inhibit other less metal-tolerant plants is not supported. Nickel is released from biomass at a rate where it is rapidly bound to soil constituents, rendering Ni unavailable and thus unable to affect seed germination. Most serpentine soils are near neutral pH, and rich in Fe/Mn oxides which strongly adsorb Ni and thus reduce the potential for elemental allelopathy.
We are grateful to Dr C. E. Green for her assistance in AAS and ICP-AES analyses.