Soil Science Society of America Journal 67:895-898 (2003)
© 2003 Soil Science Society of America
DIVISION S-7—FOREST & RANGE SOILS
Aluminum Effects on Picea abies at Low Solution Concentrations
Alexander Heim*,a,
Ivano Brunnera,
Emmanuel Frossardb and
Jörg Lustera
a Swiss Federal Inst. for Forest, Snow, and Landscape Res. WSL, Zürcherstr.111, CH 8903 Birmensdorf, Switzerland
b Swiss Federal Inst. of Technology, Inst. of Plant Sci., Field Station Eschikon, CH 8315 Lindau, Switzerland
* Corresponding author (heim{at}wsl.ch)
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ABSTRACT
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The usual way to evaluate Al toxicity in forest ecosystems is to consider the concentration of Al3+ in soil solution. The objective of this paper is to show that Al may affect tree seedlings even under conditions of very low bulk-soil solution concentrations of Al. Picea abies (L.) H. Karst. seedlings, with or without ectomycorrhizal inoculation [Hebeloma crustuliniforme (Bull.: St. Amans) Quél.], were grown in perlite substrate with addition of 0, 0.1, and 0.5 mM Al. The perlite buffered the pH at values > 5 and no soluble Al was detected in the system. However, plant biomass was significantly reduced when 0.5 mM Al was added without fungal inoculation, and Al uptake was 2 to 3 times higher than in the control treatment. Fungal inoculation compensated the growth reduction, but did not reduce Al uptake. These results indicate that spruce seedlings are able to mobilize and take up Al from solid phases.
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INTRODUCTION
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ALUMINUM HAS LONG BEEN RECOGNIZED as a toxic element for plant growth, and a great number of studies have attempted to determine toxic solution concentrations of Al for different plant species (Cronan and Grigal, 1995). However, the chemistry of Al is complex. There is still discussion on the forms of Al which are available to plants, but it is widely accepted that Al3+ is one of the most toxic species. High concentrations of this ionic species occur in solution only below pH 4.5; therefore, most work has been done under such conditions.
In agricultural systems, plant-available Al is determined by soil extraction procedures to predict the risk of Al toxicity and the need for liming (Sparks, 1996). However, in forest ecosystems, usually solution Al or Ca/Al ratio is regarded as the parameter determining Al effects on tree health (Cronan and Grigal, 1995). Only rarely have efforts been made to link tissue concentrations to extractable Al fractions in the soil (Joslin et al., 1988).
In research on forest decline, several studies have focused on minimum Al solution concentrations limiting conifer growth, and various values have been reported depending on experimental conditions (Schaedle et al., 1989; Hentschel et al., 1993).
In contrast, some authors have reported effects which were not simply caused by Al concentrations in solution. Thus, Arp et al. (1989) found that Al in conifer needles correlated best with dithionite-extractable Al, a solid fraction of Al in the soil. In addition, Godbold et al. (1995) pointed out that toxic effects of Al can appear at rather high pH values of 5, where only small amounts of Al are soluble. The present paper demonstrates significant Al effects on Norway spruce seedlings that occurred under conditions of very low Al solubility at nearly neutral pH.
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MATERIALS AND METHODS
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Culture System
The substrate used for growth was a commercial horticultural perlite with a mean diameter of 5 mm, which was washed thoroughly with sulfuric acid and then rinsed with deionized water until pH was neutral and conductivity below 10 µS cm-1 (J. Colpaert, 1998, personal communication). The perlite contained 9.4 mg g-1 Al, 8.6 mg g-1 Na, 6.1 mg g-1 K, and 9 µg g-1 P as determined by digestion with 1 mL of 40% HNO3 and 40 µL of concentrated HF in a microwave oven at 240°C and 12 MPa (UltraClav, Microwave Laboratory Systems, Leutkirch, Germany) and elemental analysis using ICP-AES (Optima 3000, Perkin-Elmer Corp., Norwalk, CT).
A dry weight of 50 g perlite each was placed into 800-mL polypropylene beakers (13.5-cm height, 9.5-cm diam.), resulting in a filling height of 8 cm. Four holes of 1-mm diam. were drilled into the bottom of the beaker, which allowed water circulation. Two of the beakers were placed into one sterilized, sunlight-transparent plastic bag with a filter allowing gas exchange (Sunbag, Sigma Chemical Co., St. Louis, MO). The unit consisting of bags and beakers filled with perlite was then heated at 105°C for at least 2 d, as autoclaving had proved to be impracticable because it released millimolar concentrations of sodium from the perlite.
The growth solutions were synthetic nutrient solutions which reflected the typical ionic composition of an acidified forest site in Germany, and contained (in µM): 300 NH4NO3, 50 Na2SO4, 100 K2SO4, 30 KH2PO4, 60 MgSO4, 130 CaSO4, 5 MnSO4, 5 FeCl3, 5 H3BO3, 0.1 Na2MoO4, 0.1 ZnSO4, and 0.1 CuSO4 (Jentschke et al., 1991). To the nutrient solutions, 0, 100, or 500 µM AlCl3 were added and the pH was adjusted to 4.0 using HCl. Solutions were 0.2-µm filtered.
Seeds of Norway spruce were surface-sterilized in 30% H2O2 for 40 min and rinsed four times with sterile deionized water. Inocula of H. crustuliniforme were prepared from cultures grown on modified Melin Norkrans agar (Marx, 1969).
Growth solution (250 mL per beaker), seeds (80 per beaker), and inocula (none or 8 per beaker) were added to the beakers in a sterile bench and the bags were closed and transferred to a climate chamber (conditions: 20°C, 50% humidity, 16-h photoperiod, 100 µmol m-2 s-1 photosynthetically active radiation). Nutrient solution was added only once when the experiment was set up to avoid contamination by later additions.
The experiment was set out at three levels of Al addition with or without inoculation with six replicate beakers each, giving a total of 36 beakers or 18 bags. Additionally, control beakers without seeds were prepared in duplicate for all treatments, and 12 control beakers were prepared with 500 µM Al addition and harvested at intervals during the first 2 wk to observe the changes in pH and Al solubility.
Harvest and Analysis
After five months of growth, the bags were opened and plants were harvested. Root and shoot fresh weight were recorded for all beakers. If seeds had germinated, but failed to establish, they were recorded separately and these individuals were not included in the analyses. The plant material was pooled per beaker, frozen in liquid N2 and lyophilized. Shoot and root element concentrations were determined after digestion with 1 mL of 40% HNO3 and 40 µL of concentrated HF in a microwave oven at 240°C and 12 MPa (UltraClav, Microwave Laboratory Systems). Total element concentrations in the digests of roots were measured with ICP-MS (Elan 6000, Perkin Elmer), those of shoots with ICP-AES (Optima 3000, Perkin Elmer).
Two of the 12 control beakers each were harvested at 0, 1, 2, 4, 7, and 14 d. The perlite was crushed with a mortar and pestle to yield the solution in the pores, and centrifuged at 5000 g for 10 min. The supernatant was used for pH and Al measurements. Free Al3+ was determined using capillary electrophoresis (Göttlein, 1998), and total and mononuclear Al were determined by flow injection analysis (Røyset, 1987).
After the five-month experiment, pore solutions were analyzed by the same methods. Additionally, after centrifugation, the perlite was extracted with 20 mL of 0.01 M NaOH for 1 h to give a NaOH-soluble fraction. This latter fraction was passed over a H+–saturated cation exchange resin to remove Na+ ions before analysis. Analysis for organic acids and orthophosphate in pore water and NaOH extract was done by capillary electrophoresis (Heim et al., 2001) with an injection of 55.2 kPa s. Total extracted organic acids and phosphate were calculated as the sum of these two fractions taking into account the carryover of water-soluble ions into the NaOH extract.
Statistical Analysis
A two-way ANOVA was performed on the data with inoculation and Al level as independent factors (Datadesk 6.1.1. for Macintosh). Treatment means were compared using Scheffé's post hoc test at a significance level of 0.05.
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RESULTS AND DISCUSSION
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On addition of the Al solutions to the perlite substrate, buffering reactions increased pH to 5.3 and decreased Al concentrations in solution toward zero, as shown in Fig. 1
. Thus, at the time when the seeds germinated (i.e., about 2–3 wk after the start of the experiments) there were only negligible amounts of Al in solution. At the end of the experiment, pH had risen to 6.4 to 7 and no Al was detected in the pore solutions. Nevertheless, Al-treated plants exhibited decreased biomass production and showed an increased uptake of Al into the shoots of the seedlings compared with pots that had received solutions without Al (Table 1). About 85% (noninoculated treatments) and 158% (inoculated treatments) more Al was taken up into biomass in 500-µM Al treatments compared with controls. Although the additional uptake represents only 3 to 6% of the Al addition, it indicates that the added Al formed a reactive solid Al phase which is available to plant roots to a larger degree than is the Al bound in the mineral structure of perlite. Although the cation exchange capacity of perlite is reported to be very low [
1.5 cmol kg-1 (Robbins, 1999)], the amount of perlite in the pots would have been theoretically sufficient to bind the entire added Al to exchange sites. Another possibility is an amorphous, easily soluble Al-hydroxy-precipitate, which is also likely to form in subsoil horizons during podzolization processes (Gustafsson et al., 2001). It has already been shown that tree roots are able to mobilize solid phase Al in the rhizosphere (Göttlein et al., 1999; Wang et al., 2001). Such mobilization would likely involve localized acidification by protons or organic acids, but these processes are difficult to assess by bulk analytical methods. In the present experiment, some exudation of organic acids, mainly oxalate, occurred in all treatments (Table 2).
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Fig. 1. Time course of pH and Al concentrations in pore solutions of the perlite substrate. For comparison, values of the added experimental solution are given on the left side (TS = treatment solution). Values are means of two replicate determinations.
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Table 1. Norway spruce biomass formed during the experiment, Al concentration of the seedlings, total Al in biomass (sum of shoot and root contents), and final pH of the pore solution. Plants were grown for five months in perlite at three levels of Al addition and inoculated or not with Hebeloma crustuliniforme. Values are means of six replicate beakers per treatment.
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Table 2. Net release of organic acids by Norway spruce seedlings grown for five months in perlite at three levels of Al addition and inoculated or not with Hebeloma crustuliniforme. Values are means of six replicates per treatment.
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Further support for the importance of the plant-available solid Al pool comes from field results presented by Wyttenbach et al. (1991) for a set of 39 forest sites from northern Switzerland. These authors found that a correlation of needle concentration against soil pH, which governs Al solubility, showed substantial residual scatter, while needle concentrations correlated well with EDTA extractable Al in the soil. Similarly, Brunner et al. (1999) found in a pot experiment with 157 soils from Switzerland that shoot Al concentration of Norway spruce varied only slightly although soil pH ranged from 2.95 to 7.41.
A highly rhizotoxic Al species (Comin et al., 1999) that may be formed at pH 5 to 6 is the triskaidekaaluminum [AlO4Al12(OH)24(H2O)127+ or Al-13]. However, the presence of significant amounts of this species in our experiment can be ruled out for two reasons. First, the presence of sulfate concentrations >50 µM strongly interferes with Al-13 formation (Kerven et al., 1995) and second, total Al in the experimental solutions decreased as fast as did Al3+.
Although biomass reduction of noninoculated pots at 500 µM Al addition was compensated in inoculated pots, Al uptake into needles was not reduced by colonization with the ectomycorrhizal fungus H. crustuliniforme (Table 1). Therefore, a direct filtering (Turnau et al., 1996) effect of this fungus is not evident although almost all short roots were covered by a dense fungal mantle. Calculating a P balance showed that inoculated plants had increased their P content by an average of 8% over P content in seeds, using almost 40% of the P added with the nutrient solution. In contrast, noninoculated plants had lost 6% of P from the seed reserve, in particular in the 500-µM Al treatment (Heim, 2000). Similarly, Schier and McQuattie (1995) reported for Pinus strobus L. that amelioration of Al toxicity by mycorrhizal colonization appeared to result from enhanced uptake of nutrients, especially P, rather than from reduced uptake of Al. In contrast, Hentschel et al. (1993) reported that Paxillus involutus (Fries) Fries was able to reduce Al uptake into the needles of Norway spruce grown in a sand culture system. Under field conditions, such varying mycorrhizal effects might complicate any regression model trying to predict needle Al concentration from soil Al fractions.
Our results indicate that uptake and toxicity of Al can occur under conditions when Al concentrations in the soil solution are near zero. Under these conditions, Al is mobilized from plant-available Al phases in the soil. Solution concentrations therefore underestimate Al toxicity hazards on forest soils that are only slightly acidic.
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ACKNOWLEDGMENTS
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We thank the team of our central laboratory for element analyses. Arthur Kölliker is acknowledged for technical assistance. The study was funded by the Swiss National Science Foundation (Grant No. 31-47277.96).
Received for publication September 18, 2002.
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REFERENCES
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ABSTRACT
INTRODUCTION
