Krystyna BOJARCZUK, Jacek OLEKSYN, Piotr KAROLEWSKI, Roma ŻYTKOWIAK
Institute of Dendrology, Polish Academy of Sciences, Parkowa 5, 62-035 Kórnik,
e-mail: bojark@man.poznan.pl
RESPONSE OF SILVER BIRCH (BETULA PENDULA ROTH.) SEEDLINGS TO EXPERIMENTAL VARIATION IN ALUMINUM
CONCENTRATION
Regular research paper
ABSTRACT: The process of soil degradation and destabilization of forest ecosystem by indus- trial pollution is frequently associated with mo- bilization of toxic Al3+ ions. Both these processes exert a negative influence on tree root systems and may even result in the decline of whole for- est stands. One-year-old seedlings of silver birch (Betula pendula Roth.) grown in pots were treat- ed with a range of aluminum sulfate concentra- tions in order to test the effects of Al on growth, root structure, content of phenolic compounds and mineral nutrition of roots and foliage. Plants exposed to Al concentrations exceeding 50 mg Al dm–3 had reduced growth, root structure and nutrient uptake were affected, and a substantial increase of Al concentration occurred in foliage and roots. Concentration of several elements in the foliage and roots declined with increasing Al concentration, including Mg and Ca, and to a lesser extent, P, K, and Na. Most root traits such as root mass or root growth rate were more strongly affected by Al than the foliage. Changes in root Ca, Al and Ca:Al ratio, and root morphol- ogy were detected at the lowest Al concentration (50 mg Al dm–3) indicating usefulness of these traits as early indicators of adverse aluminum ef- fects on plants.
KEY WORDS: Betula pendula, aluminum, Ca:Al ratio, growth, nutrients, root morphology, phenolics
1. INTRODUCTION
Air pollution is a major factor contribut- ing to decline of woody plant growth that can lead to local forest decline in industrialized regions (Drohan et al. 2002). Plants can be affected directly by various toxic gases or in- directly by soil mediated effects (Reich et al.
1994). In many areas of Poland acid precipi- tation is the major reason for considerable acidification of soils. Recent increase in acid- ity of wet deposition in eastern and central European countries is likely a result of a de- crease in alkaline dust components through introduction of more efficient dust removal and insufficient desulfurization in flue gases of power and heating plants (Oleksyn and Reich 1994, Marquardt et al. 2001). Acid- ic deposition can have a noticeable impact on many types of forest soils, due to their low buffering capacity (Marschner 1995, Tao et al. 2002). In acidic soils, indirect negative effects on plants may result from solubili- zation of Al-containing minerals at low pH (Keltjens and van Loenen 1989, Rengel 1992, Reich et al. 1994).
Soil pollution may have a negative effect on the development of root systems, espe- cially in long-lived organisms, such as trees.
The main mechanisms involved in alumi- num toxicity (Boudot et al. 1994) include 1) competition between Al3+, Ca2+ and Mg2+
for the uptake sites of root meristems, lead- ing to decreases in Ca and Mg content, 2) inhibition of meristematic cell division re- sulting in low root growth, 3) alteration of the root membrane structure and function, and 4) decline of nutrient and water uptake by roots. Increase of the concentrations of available Al and decrease in Ca in the soil solution is one of the leading mechanisms by which acidic deposition affects tree vitality (Cronan and Grigal 1995). Consequently, the Ca:Al ratio in plants and soils has been proposed as a useful indicator and predictive tool for estimating tree growth disturbance and potential stress in forest ecosystems (Cronan and Grigal 1995, Göransson and Eldhuset 2001, Wargo et al. 2003, Vanguelova et al. 2005).
Toxic levels of Al3+ frequently affect root structure, function and growth (Vilkka et al.
1990, Boudot et al. 1994, Bojarczuk and Oleksyn 1994, Oleksyn et al. 1996, Silva et al. 2001). As a result decline in uptake of such nutrients as calcium, magnesium, phos- phorus and zinc is observed (Bojarczuk et al. 2002). At the same time, an increase of aluminum and decrease of Ca to Al ratio in roots and above ground plant parts is ob- served (Reich et al. 1994, Oleksyn et al.
1996, Bojarczuk et al. 2002). Excessive ac- cumulation of Al in foliage may cause physi- ological changes. At a heavily polluted site in Poland, we found excessive soil Al and low Mg availability, leading to low needle Mg, high Al, high Al:Ca ratios, reduced photo- synthetic capacity, and increased respiration in Scots pine foliage (Reich et al. 1994).
Among broadleaved trees, birch is re- garded as relatively tolerant to industrial pollution. However, birch decline was often observed in the vicinity of point-source pol- luters (Rachwał and Wit-Rzepka 1989) and occasionally also in regional scale (Mau- er and Palátová 2003).
The objective of this study was to test the effects of a range of Al concentrations in so- lution on growth, root structure, content of phenolic compounds and mineral nutrition of Betula pendula seedlings.
2. MATERIAL AND METHODS Experiments were conducted using one- year-old silver birch (Betula pendula Roth.) seedlings of relatively uniform size (root length 10 cm, shoot length 12 cm, diameter of root collar 3.0–3.5 mm). Seedlings were obtained from the “Zielonka” Arboretum nursery of the Agricultural University in Poznań. In the middle of May the seedlings were planted in pots (17 cm diameter, 13 cm height, 1.4 dm3 volume, one per pot) filled with a 45%:45%:10% (v:v:v) mixture of sand/
perlite/unpolluted forest soil obtained from the birch forest. Seedlings were exposed for five months (149 days) to aluminum sulfate [Al2(SO4)3]in five concentrations 0, 50, 100, 150, and 200 mg Al dm–3. Birch seedlings were treated with Al solutions and fertilized once a week with a nutrient solution (N-NH4 – 0.5%, N-NO3 – 1.6%, K – 2.0%, P – 4.8 g dm–3, S – 2.7 g dm–3, Cu – 5 mg dm–3, Fe – 100 mg dm–3, Mn – 27 mg dm–3, Mo – 5 mg dm–3, B – 26 mg dm–3; adjusted to pH 4.5) at an amount of 0.05 dm3 per pot. The range of aluminum concentrations used in this study was chosen to represent Al levels observed in soils from a variety of polluted environments (Reich et al. 1994, Bojarczuk et al. 2002, Hrdlicka and Kula 2004). The lowest Al level (50 mg dm–3) in the present study was comparable to that found in a soil substrate that originated from an area polluted by a phosphate fertilizer factory which caused slightly reduced growth parameters of B. pendula seedlings (Bojarczuk et al.
2002). In all figures aluminum concentra- tion (0 – 200 mg dm–3) represents a dose of Al2(SO4)3 added to the substrate.
In addition to fertilization, all pots were watered to excess. The experiment was es- tablished in a greenhouse in two randomized complete blocks.
Destructive harvests were conducted at intervals of about seven days. We har- vested plants seventeen times. At each har- vest, we randomly chose four plants from each treatment (i.e. two plants per block) for an analysis of seedling growth dynam- ics. At harvest, the medium was washed from roots with distilled water, diameter at root collar measured and individual plants were separated into leaves, stem, and root.
The projected area of leaves, area and length of roots in three thickness classes (<1 mm, 1–3 mm, and >3 mm), and number of root tips and branching points were measured using image analysis systems WinFolia and WinRhizo (Regent Instruments Inc., Que- bec, Canada). All parts of seedlings were oven-dried (65°C) and dry masses were de- termined.
The content of total soluble phenols (TPh) was determined in 0.1 g of foliage and 0.1 g of roots after double extraction for 15 and 10 min in boiling 95 and 80% ethanol, respectively. The analyses were performed using the spectrophotometric method de- scribed by Johnson and Shaal (1957) and modified by Singleton and Rossi (1965) using the Folin and Ciocalteu’s phenol re- agent. The content of total phenols was ex- pressed in µmol of chlorogenic acid per gram of dry mass. TPh was measured on foliage or roots of three randomly chosen plants per treatment
At the end of the experiment (October) we measured concentrations of total N by the micro-Kjeldahl method and soluble forms of macro- and micronutrients and pollutants in roots and leaves: P, K, Ca, Mg, Fe, Mn, Zn, Cd and Al. Macronutrients were analysed in an extract of 0.03 N acetic acid, and micronu- trients and pollutants in a modified Lindsey extract. Concentrations were determined us- ing the following methods: P colorimetrical- ly with ammoniummolybdate; K, Ca and Na with flame photometry; chlorides and sul- fates nephelometrically; Mg, Fe, Mn, Zn, Cu and Cd with atomic absorption spectroscopy (AAS); and Al colorimetrically with alumi- non. Data for substrate are averaged for each treatment from two combined samples and those for foliage from three randomly chosen plants per treatment.
For all variables, statistical differences among treatments and sampling dates were analyzed using analysis of variance (GLM procedures). Relationships between the sam- pling day and studied traits were made using correlation and regression analyses. For pre- sentation, both correlation and regression are used but we do not assume that direct causal relations are involved. All statistical analyses were conducted with JMP software (version 5.0.1a, SAS Institute, Cary, NC, USA).
3. RESULTS
Results of the final harvest showed that low concentration of aluminum sulfate (50 mg Al dm–3) did not inhibit total seedling or individual organ growth of treated seed- lings (Fig. 1). An Al concentration of 100 mg dm–3 significantly negatively affected shoot (P = 0.05), aboveground (P = 0.02), and seed- ling mass (P = 0.06) and had no effect on leaf and root mass (P = 0.24). A similar pattern of changes was observed in an average growth rate (Fig. 2). For the highest Al concentra- tion (200 mg Al dm–3) most of growth rate parameters were close to zero. In general, leaf mass and growth rates were the least af- fected by elevated levels of Al in the substrate (Figs 1, 2 and 3). An average individual leaf area was relatively unaffected by Al in lower concentrations and declined substantially in the 200 mg Al dm–3 treatment. However, leaf mass was negatively affected at 150 mg Al dm–3. Specific leaf area (SLA, leaf area per unit leaf mass) was the greatest at 0 and 50 mg Al dm–3 (ca. 425 cm2g–1) and lowest at 200 mg Al dm–3 (90 cm2g–1, Fig. 3).
Decline of aboveground and total plant biomass was influenced mainly by Al effects on growth of woody stems. In comparison with the control, woody shoot mass declined by 30, 38 and 88% in the presence of 100, 150 and 200 mg Al dm–3, respectively. In contrast, foliage or root biomass of seedlings grown in the presence of 100 and 150 mg Al dm–3 declined only by 10 to 18% (Fig. 1).
Considerable vulnerability of wood growth to aluminum is also evident from differenc- es in the final root collar diameter (Fig. 4).
B. pendula seedlings grown for 4.5 months in a substrate containing 200 mg Al dm–3 did not show any increase in root collar diameter in comparison with an initial root collar di- ameter at the beginning of the experiment.
At the same time root collar diameter of seedlings grown in the substrates contain- ing 100 and 150 mg Al dm–3 decreased by ca.
25% in comparison with the control or low Al concentration of 50 mg dm–3 (Fig. 4).
Leaf to root area ratio and specific root length (SRL, root length per root mass, m g–1) decline linearly with increase of Al concen- tration in the substrate (Fig. 5). Leaf area to root area ratio changed by 37 fold, from 13.2
Fig. 1. Effect of aluminum sulfate [Al2(SO4)3]in solution on growth parameters (±SE) of Betula pendula seedlings. Measurements were taken at the end of the experiment after 149 days of growth.
to 0.4 between 0 and 200 mg Al dm–3, respec- tively (Fig. 5).
A low level of Al in the substrate (up to 50 mg Al dm–3) did not raise aluminum con- centrations above the level found in other published reports with Betula pendula. Al concentrations exceeding 50 mg Al dm–3 re- sulted in substantial increase of Al concentra- tion in foliage and especially in roots (Fig. 6).
At the same time Ca:Al ratios decreased dra- matically for the 50 mg Al dm–3 substrate concentration (Fig. 6). Such elements as Mg,
Ca and to some extent P, K and Na decreased in foliage and roots with increasing Al con- centration in the substrate (Fig. 7). In gen- eral, increases in substrate Al decreased Ca and Mg concentration more in roots than in foliage. Significant decreases of Ca and Mg in roots was already visible at the lowest Al concentration of 50 mg dm–3 (Fig. 7).
For birch grown in the substrate polluted with Al concentrations of 100 to 150 mg dm–3, the level of total phenols (TPh) was signifi- cantly lower in roots than in foliage. The
Fig. 2. Effect of Al2(SO4)3 concentrations in solution on average (±SE) leaf, shoot, aboveground, root, and total plant growth rate of Betula pendula seedlings.
Fig. 3. Effect of Al2(SO4)3 concentrations in solu- tion on average (±SE) for all harvests: leaf area, leaf mass, and specific leaf area (SLA) of Betula pendula seedlings.
Fig. 4. Final root collar diameter (±SE) of Betula pendula seedlings subjected to five Al2(SO4)3 con- centrations for 149 days.
Fig. 5. Relationships between Al concentration in solution and Betula pendula leaf area to root area ratio (top figure) of treated plants, and between Al concentration and specific root length (m of roots per g of their dry mass, SRL). Average val- ues (±SE) taken at the end of the experiment after 149 days of growth are presented.
Fig. 6. Relationships between Al concentration in solution and Betula pendula leaf and root Al and Ca:Al concentration in treated plants. Average values (±SE) at the end of the experiment after 149 days of growth are presented.
Fig. 7. Relationships between Al concentration in solution and Betula pendula leaf and root nutrient content (%) in treated plants. Average values (±SE) at the end of the experiment after 149 days of growth are shown.
influence of Al substrate pollution on TPh level was statistically significant in roots (P = 0.0045) and leaves (P = 0.02). There were also significant organ (root or foliage) interactions with Al concentration (Fig. 8).
4. DISCUSSION
Elevated aluminum concentrations in the soil substrate treated with Al solution have profound effects on the growth and physiol- ogy of silver birch. Seedlings grown on sub- strates with increased aluminum sulfate were characterized by lower dry mass of above and belowground parts (Fig. 1). Our data in- dicated that growth and development of sil- ver birch, a species that is generally regarded as low in sensitivity to industrial pollution among broad-leaved trees (Boudot et al.
1994, Ernst et al. 1998, Winterhalder et al.
1999, Bojarczuk et al. 2002) can be signifi- cantly limited by substrate contamination with Al. Such contamination is frequently observed in acidic soils in the vicinity of in- dustrial sources of air pollutants (Reich et al. 1994, Drohan et al. 2002).
Low concentrations of aluminum(50 mg Al dm–3) slightly increased leaf, shoot and total aboveground biomass of seedlings and had no effects on root biomass (Fig. 1). This result is in good agreement with data of Clegg and Gobran (1995) who found that growth of Betula pendula seedlings was un- Fig. 8. Average (±SE) total phenols concentra- tion (in µmol of chlorogenic acid per gram of dry mass) in Betula pendula leaves and roots of plants grown at five Al2(SO4)3 concentrations.
affected by up to 81 mg Al dm–3. Positive ef- fects of low Al concentration on growth and biomass of birch seedlings may be explained by Al3+ amelioration of H+ (Kinraide 1993).
Growth enhancement under a condition of slightly elevated Al concentration may also be a result of Al effects on solubility and higher availability of such elements as iron, calcium, phosphorus or magnesium (Keltiens and Loenen 1989, Ericsson et al. 1998).
Aluminum, even in low concentrations, has a major effect on root morphology. Our data indicated a strong negative relationship between plant Al concentration and specific root length (SRL, m g–1). High SRL is con- sidered as one of the best indicators of wa- ter and nutrient acquisition efficiency per unit carbon expenditure (Yanai et al. 1995, Bouma et al. 2000). Reduction or damage of the root system may also lead to higher plant vulnerability to environmental stress- es (Clemensson-Lindell and Persson 1995, Persson and Majdi 1995).
The presence of aluminum in solution re- duced foliage and root concentration of sev- eral mineral elements, such as P, K, Ca, Mg, Na and others (Fig. 7). Generally, the nutrient status of roots was more affected than that of foliage (Figs 6, 7). In all Al treatments, the foliar concentrations of all essential elements were in the range considered sufficient for normal growth of B. pendula (Bergmann 1992, Hrdlicka and Kula 1998, Oleksyn et al. 2000). Natural variation of Ca in B. pen- dula leaves is usually between 0.9 and 1.5%
and depends on foliage age (Oleksyn et al.
2000). Foliar Ca concentrations went below this range for higher Al substrate concen- trations, varying from 0.7 to 0.9, in 100 and 150 mg Al dm–3, respectively (Fig. 7). Decline in root Ca concentrations with increasing substrate Al concentrations were more dra- matic than those for the foliage. At the rela- tively low 50 mg Al dm–3 treatment, root Ca concentration declined by 78% in compari- son with the control.
Reductions in leaf and root tissue nutri- ent contents occurred earlier and in lower concentrations of Al than did reductions in growth. Similarly, in our earlier studies (Bo- jarczuk et al. 2002) we found that nutri- ent disorder preceded any growth decline of plants in pots with soil that originated from
an area polluted by a phosphate fertilizer fac- tory and characterized by a high soil Al level and low Ca:Al ratio.
Foliar Al concentration varied between 112 ppm in the lowest Al treatment (50 mg dm–3) to 1804 ppm in plants exposed to 200 mg Al dm–3 (Fig. 6). On average, in un- polluted sites foliage Al in B. pendula ranges between 50 and 290 ppm (Oleksyn et al.
2000, Bojarczuk et al. 2002, Bojarczuk and Przybył 2005). Aluminum concentra- tion in birch foliage grown in soils obtained from sites subjected to industrial pollu- tion can reach up to 400 ppm (Bojarczuk et al. 2002).
In our study Ca:Al ratio in foliage varied from 70 (control) to 10 (at 150 mg Al dm–3), and in roots between 34 and 0.3 (Fig. 6). The Ca:Al ratio in plants may reflect the com- petition between Ca2+, regarded as the most important base cation, and soluble Al at the root uptake sites (Boudot et al. 1994). Fo- liar Ca:Al ratio (36) at the lowest Al concen- tration (50 mg Al dm–3, Fig. 6), was above the range (15 to 22) found in B. pendula plants grown in soils obtained from the vicinity of a phosphate fertilizer factory that was char- acterized by a high soil Al level and low Ca:
Al ratio (Bojarczuk et al. 2002).
Decrease in root phenols at high alumi- numconcentrations (100–150 mg Al dm–3) of exposure may in part be related to ob- served decreases in root mass and new root growth (low root growth rate, Figs. 1, 2), and also fine root mortality. Furthermore, it is possible that at high Al concentrations, solu- ble phenols formed insoluble polymers with aminoacids and sugars (Howell and Kre- mer 1973), and part of them were leaked into the substrate solution medium, as root cell membranes were destroyed (Zieslin and Abolitz 1994). A similar pattern in foliage and root phenolics (with higher de- crease of these compounds in roots than fo- liage) was also found in an earlier study of one-year-old Scots pine (Pinus sylvestris L.) seedlings grown in a range of nutrient solu- tions containing 0 to 4 mM aluminum nitrate (Oleksyn et al. 1996).
In summary, our results indicate a pos- sible negative impact of Al on a range of cel- lular processes including biomass produc- tion and root structure in Betula pendula
seedlings exposed to various levels of Al in the substrate. Reductions in leaf and root tis- sue nutrient contents occurred earlier and at lower concentrations of Al than did reduc- tions in growth. These results suggest that B. pendula trees may be more susceptible to Al than expected based on previous field ob- servations (Reich et al. 1994).
ACKNOWLEDGEMENTS: This study was supported by The Polish Committee for Scien- tific Research (KBN), grants No 5PO6M00512, 6PO6L04121.
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(Received after revising January 2006)
