EFFECTS OF 03 AND SOIL ACIDIFICATION, ALONE AND IN COMBINATION, ON GROWTH, GAS EXCHANGE RATE AND C H L O R O P H Y L L CONTENT OF RED PINE SEEDLINGS Y U N F E N G S H A N ~, T A K E S H I I Z U T A 2, M A S A T O S H I A O K I 2 and TSUMUGU TOTSUKA 2

t The United Graduate School, Tokyo Universi O, of Agriculture and Technology, Fuchu, Tokyo 183, Japan; 2 Department of Environmental Science and Resources, Facul~. of Agriculture, Tokyo University of Agriculture and Technology, Fuchu, Tokyo 183, Japan (Received: 21 March 1995; accepted in final form 3 June 1996)

Abstract. One-year-old seedlings of red pine (Pinus densiflora Sieb. and Zucc.) were grown in typic red-yellow forest soil (Typic Hapludults) artificially adjusted to pH (H20) 3.15, 3.60 or 3.90 by adding H2S04 solution to the soil (pH 4.60), and exposed to ozone (03) at 1504-10 ppb (nl.L -~) for 8 h a day, 6 days a week, for 16 weeks from June 5 to October 5, 1994. The control seedlings were exposed to charcoal-filtered air (CF) and grown in the soil without the additional supply of H + as H2804 solution during the same period. No significant interactive effects of 03 and soil acidification were observed for the determined seedling parameters in this study. However, 03 caused a reduction in needle dry weight, net photosynthetic rate and chlorophyll contents in the needle tip or on whole-needle weight basis, and stimulated rates of dark respiration and photorespiration. There were no significant effects of 03 on chlorophyll contents in the needle middle and basal parts, transpiration rate or water use efficiency (WUE). On the other hand, the seedlings grown in the soil adjusted to pH 3.60 or 3.90 showed a reduction in needle dry weight, net photosynthetic rate, chlorophyll contents in all the needle parts and WUE, and an increase in the rates of dark respiration and photorespiration. The transpiration rate of the seedlings was not significantly affected by soil acidification. All the seedlings grown in the soil adjusted to pH 3.15 died during the first 4 weeks. Soil and needle analysis suggested that high concentrations of AI and low Ca/AI ratios in the acidified soil were stressors. Key words: chlorophyll content, gas exchange, growth, ozone, red pine, soil acidification

1. Introduction Current ambient 03 levels are well known to damage growth and affect physiological functions of trees (Chappelka and Chevone, 1992). At the same time, acid deposition has become a widespread phenomenon and has been a source of great concern because of its impact on forest ecosystems. Forest declines were reported in Asia, Europe and North America, and gaseous air pollutants such as 03 and acid deposition have been suggested as possible contributors (Totsuka, 1993; Ashmore et al., 1985; Johnson and Siccama, 1983; Woodman, 1987, Krause et al., 1986). Short-term experiments with simulated acid rain (not longer than 14 months) did not provide the experimental evidence for the contribution of acid rain to forest decline (Wood and Bormann, 1977; Reich et al., 1987; Blank et al., 1990; Eamus and Fowler, 1990; Shan and Feng, 1989; Shah et al., 1994 a and b). However, soil acidification caused by long-term acid deposition of anthropogenic air pollutants Water, Air, and Soil Pollution 97: 355-366, 1997. (~) 1997 Kluwer Academic Publishers. Printed in the Netherlands.

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YUNFENGSHANETAL.

was well documented. Falkengren-Grerup (1986, 1987, 1989) reported that the pH (H20) of top soil was decreased by up to 1.5 pH units over a period of 35 years (average decrease 0.8 units) in southern Sweden. Tamm and Hallbacken (1986, 1988) also reported soil acidification in the southwestern Sweden where the subsoil (usually at 70 cm depth) has become 0.5 to 0.7 pH units more acid from 1927 to 1982-84. Matzner et al. (1986) reported that root growth of Norway spruce was significantly inhibited by soil acidification and proposed that soil acidification induced by acid deposition is one of the causes for the disturbance roots in declining forest stands. Furthermore, soil acidification caused a reduction in the dry weight growth of Cryptomeriajaponica D. Don (Izuta et al., 1990; Miva et al., 1994). Many researchers have already investigated the combined effects of 03 and direct acid rain, rather than through soil acidification, on seedlings of forest trees (Chappelka et al., 1986; Edwards et al., 1992; Reich et al., 1987; Shan et al., 1994b). However, there are few studies on the combined effects of 03 and soil acidification caused by acid deposition on seedlings of forest trees. In many areas, 03 and soil acidification caused by acid deposition usually co-occur as major phytotoxic factors. Therefore, it is necessary to investigate their combined effects on growth and physiological functions of trees. The objective of this study was to determine the individual and joint effects of 03 and soil acidification on growth, gas exchange rate and chlorophyll content of red pine seedlings. Red pine was selected as the test species because it is widely distributed in Asian countries such as Japan, Korea and China, and declines of red pine have been reported in many areas in these countries (Totsuka, 1993).

2. Materials and Methods 2.1. SOIL ACIDIFICATION AND GROWTH EXPERIMENT In March, 1994, typic red-yellow soil (Typic Hapludults) from sedimentary rock was collected from soil layer at 0 to 20 cm below a forest floor in Toyohashi, Aichi Prefecture, Japan. The soil was sieved through a 5-mm mesh. The soil pH (H20) was 4.60. Because SO]- is a major component of ambient acid rain in Asian countries such as China and Japan, the pH (H20) of the soil was adjusted by adding 100 mL of H2SO4 solution at 0.2, 0.4 or 0.8 N to 1 L of soil. The amount of H + added to the soil was 2.8, 5.6 or 11.1 g H + 9 m -2 potted soil surface area. Control soil was used without the additional supply of H + and natural pH of this soil is 4.60. The proton loads, pH (H20), pH (KC1) and concentrations of water soluble elements of soil in the four soil treatments are shown in Table I. In May 12, 1994, one-year-old seedlings of red pine (Pinus densiflora Sieb. et Zucc.), supplied from a commercial seedling nursery in Tokyo, Japan, were transplanted into 500 mL plastic pots (one seedling per pot) containing the acidified

357

EFFECTS OF 03 AND SOIL ACIDIFICATION

Table I The proton loads, pH (H20), pH (KCI) and concentrations of water soluble elements of soil just after three soil acidification treatments and control. The element concentrations were expressed on an air-dried soil weight Proton load load (g H + m -2)

pH (H20)

pH (KCI)

Ca

Mg

K AI (#g mg- 1)

0.0 2.8 5.6 11.1

4.60 3.90 3.60 3.15

3.65 3.40 3.24 3.02

21.75 17.59 21.83 22.96

1.74 6.33 12.68 12.95

1 7 . 3 7 n.d. 32.42 2.82 31.68 29.79 29.83 210.95

Mn

Ca/A1 (molar)

0.85 8.03 26.83 35.19

oo 4.21 0.49 0.07

n.d., not detected.

soils or control soil. The 80 seedlings were grown in a greenhouse until June 4, 1994. Then, the seedlings were exposed to 03 at 150 -1- 10 ppb (nL L - 1) or charcoalfiltered air (CF, control) for 8 h per day from 9:00 to 17:00, 6 days a week, for 16 weeks from June 5 to October 5, 1994 in two growth cabinets, respectively. Air temperature, relative humidity and light intensity at the top of the seedlings placed in the growth cabinets were maintained at 30 + 1 ~ 70 + 5% and 410 + 20/zmol m -2 s -I , respectively. The 03 was generated with a silent electrical discharge 03 generator (Nihon Ozone Co., Model O-1-2), and the concentration of 03 in the growth cabinets was continuously monitored with a UV absorption 03 detector (Dashibi Co., Model DY-1500). At the end of the 16 week 03 exposure period, all the seedlings were harvested and dried at 80 ~ for at least one week. The dry weights of leaf, trunk and root were measured. 2.2. MEASUREMENT OF GAS EXCHANGE RATE To determine gas exchange rates of all needles of the seedlings, the aerial parts of a seedling were accommodated into a plant assimilation chamber with an air mixing fan. The measurements were conducted during the final week of the 16 week 03 exposure period as described in the previous section. During the measurement of gas exchanges rates, light intensity at the top of the seedling, air temperature and relative humidity (R.H.) in the plant assimilation chamber were maintained at 700 # mol m -2 S-1 PPFD, 30 + 1 ~ and 70 -I- 5%, respectively. Fresh air was introduced into the chambers at 3 L rain- I. The illumination consisted of two 400 W metal halide lamps (Yoko Lamp, Toshiba Co.) and two 500 W incandescent lamps (Matsushita Co.). The irradiated light was filtered through a heat absorbing water filter and a glass filter which were placed between the illumination system and the plant assimilation chamber, This glass filter is specifically made and can absorb infrared light partially for 750-1000 nm and completely for 1000 nm or above (Koito Industries, Ltd., Japan).

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The rates of net photosynthesis and transpiration were determined by measuring the differences in the CO2 concentration and relative humidity of air between the plant assimilation chamber and the similar blank chamber using an infrared CO2 analyzer (Fuji Electric Co., Model Zap AD 011-55) and a digital humidity analyzer (EG & G Co., Model 911 Dew All), respectively. The dark respiration rate of the seedling was measured in the dark by the same methods. To determine the photorespiration rate of the seedlings, the CO2-free air (CO2 concentration below 2 #L L - l ) was introduced into the plant assimilation chamber and the blank chamber at 3 L m i n - i. The CO2 concentration of air was reduced by passing the air through a plastic pipe containing granular soda lime. The photorespiration rate was determined by the same methods mentioned above. 2.3. MEASUREMENT OF CHLOROPHYLL CONTENT At the end of the 16 week 03 exposure period, mature current needles were sampled in the middle story of seedling crowns, then divided into the tip, middle and basal parts by equal length ratio. The 100 mg fresh needle part samples were used for every measurement. The chlorophyll was extracted from needles with a mixture of acetone, ethanol and deionized water in a ratio of 4.5:4.5:1 for 24 hours in the dark (Chen and Chen, 1984; Shan et al., 1994a). Absorption of the extract was measured at 663 and 645 nm with a spectrophotometer (Shimadzu Co., Model UV-1200), and the concentration of chlorophyll a+b was calculated with formulae proposed by Arnon (1949). 2.4. SOIL AND NEEDLE ANALYSIS The soil was air-dried and sieved through a 5-mm mesh. The mixture of air-dried soil (10 g) and pure water or 1 N KC1 (25 mL) was vigorously stirred, and then was permitted to stand for one hour. After restirring, the pH of the mixture was measured with a pH meter (Horiba Co., M-12). The calibration of the pH meter was performed at the beginning of each 5 measurements. In a constant temperature room at 25 ~ the mixture of air-dried soil (10 g) and pure water (50 mL) was continuously shaken for one hour. Then, the mixture was filtered through a filter paper (Toyo Co., No. 5B). The needle samples were dried in +80 ~ and digested in concentrated H 2 8 0 4 and H 2 0 2 at 440 ~ The concentrations of Ca, Mg, K, A1 and Mn in soil and needle extracts were determined with an atomic absorption spectrophotometer (Shimadzu Co., Model AA-670/GV-6) (Izuta et al., 1994). 2.5.

STATISTICS ANALYSIS

In this present study, two-factor analysis of variance (ANOVA) was performed to test for significance of individual and interactive effects of ozone and soil acidification on parameter of red pine seedlings. The data were pooled in further

359

EFFECTS OF 03 AND SOIL ACIDIFICATION Table II Effects of 03 and soil acidification on dry weights of red pine seedlings a Needle (rag)

Stem (mg)

Root (mg)

Whole-plant (mg)

S o i l p H (H20)

CF

03

CF

03

CF

03

CF

03

pH pH pH pH

608 579 416 death

515 416 400 death

299 295 256 death

258 243 240 death

596 570 488 death

559 468 496 death

1479 1443 1161 death

1333 1127 1135 death

4.60 3.90 3.60 3.15

ANOVA b 03 Soil treatment Linear Quadratic 03 x Soil pH

* * * ns ns

ns ns ns ns ns

ns ns ns ns ns

ns ns ns ns ns

a The seedlings were exposed to 03 at 150 ppb for 16 weeks from June 5 to October 5, 1994 and grown in the soil adjusted to pH 3.90 or 3.60 for 20 weeks from May 12 to October 5, 1994, alone and in combination. The control seedlings were exposed to charcoal-filtered air (CF) and grown in the soil of pH 4.60 during the same period. Each value represents the mean of 10 determinations. b Two-factor ANOVA results: ns = not significant; * p<0.05.

analysis of individual treatment effect and variance for soil treatments (Proton load 0,2,8 or 5,6 g H + m -z) was partitioned into linear and quadratic component using orthogonal polynomial contrasts (Chappelka and Chevone, 1986). The seedlings at pH 3.15 treatment were not included in the statistical analysis because all the seedlings died before 03 treatments.

3. Results

3.1.

V I S I B L E FOLIAR INJURY AND GROWTH RESPONSE

All the seedlings grown in the soil adjusted to pH 3.15 before 03 treatment died during the first 4 weeks, but no visible foliar injury of red pine seedlings was produced by the exposure to 03 at 150 ppb and any of other acidification treatments, alone or in combination. Table II shows the dry weights of parts and whole-plant at the final harvest of red pine seedlings. There were no interactive effects of 03 and soil acidification on the dry weight growth of the seedlings by the statistical analysis of ANOVA. The 03 caused a significant reduction in the needle dry weight of the seedling. The

360

YUNFENG SHAN ET AL. Table III Effects of 03 and soil acidification on net photosynthetic rates on a unit needle dry weight basis (Pn/needle DW) or on a whole plant basis (Pn/plant), photorespiration rate (Rphoto) and dark respiration rate (Rd~k) of red pine seedlings. See legend in Table II Pn/needle DW (mg CO2 g - I DW/h - I )

Pn/plant (mg CO2 plant -I h - I )

Rphoto (mg CO2 g - 1 DWh -1)

Rdark (mg CO2 g - i DWh -1)

Soil pH (H20)

CF

03

CF

O3

CF

03

CF

pH 4.60 pH 3.90 pH 3.60

9.30 8.86 7.79

8.54 7.66 7.85

5.84 5.28 3.31

4.56 3.27 3.27

5. I 1 5.71 7.25

6.64 2.26 6.48 2.89 1 0 . 2 9 4.19

03 3.05 4.45 4.67

ANOVAb 03

*

*

*

*

Soil treatment

*

*

*

*

Linear

*

*

ns

*

Quadratic

ns

ns

ns

ns

03 x Soil pH

ns

ns

ns

ns

soil acidification treatment at soil pH 3.6 was significantly correlated with a linear reduction in needle dry weight, too. 3.2. EFFECTS ON NET PHOTOSYNTHETIC RATES AND RESPIRATION RATES No significant interactive effects of 03 and soil acidification were observed for net photosynthetic rates on a unit needle dry weight basis or on a whole-plant basis of the seedlings, as shown in Table III. However, the exposure to 03 induced significant reductions in the net photosynthetic rates of the seedlings. Also, net photosynthetic rates on the basis of needle dry weight or whole-plant were linearly reduced in the seedlings grown in the soil adjusted to pH 3.60 compared to pH 4.60. The photorespiration rate and dark respiration rate of the seedlings were stimulated by the exposure to 03 or soil acidification, but no significant interactive effects were observed, as shown in Table III. 3.3. EFFECTS ON TRANSPIRATION RATE AND WATER USE EFFICIENCY As shown in Table IV, 03 and soil acidification, alone or in combination, did not alter the transpiration rate of the seedlings. However, there was a significant linear reduction in the water use efficiency (WUE) of the seedling with soil acidification.

EFFECTS OF 03 AND SOIL ACIDIFICATION

361

Table IV Effects of 03 and soil acification on transpiration rates (Tr) and water use efficiency (WUE) on a whole-plant basis of red pine seedlings. See legend in Table II Tr (mg CO2 plant- i h-I)

WUE (rag COz g - 1 H20)

Soil pH (H20)

CF

03

CF

03

pH 4.60 pH 3.90 pH 3.60

0.29 0.27 0.20

0.21 0.19 0.21

20.07 22.00 16.19

22.46 17.76 15.48

ANOVAb 03 Soil treatment Linear Quadratic O3 x Soil pH

3.4.

ns ns ns ns ns

ns * ns ns ns

EFFECTS ON CHLOROPHYLL CONTENT

As shown in Table V, 03 significantly reduced chlorophyll content in the tip part of needles and on a whole-needle fresh weight basis, but did not significantly alter chlorophyll content in the middle or basal parts. As soil pH (H20) value decreased from pH 4.6 to pH 3.6, significant linear reductions in the chlorophyll contents were observed in the top, middle and basal parts of the needles. No significant interactive effects of 03 and soil acidification on the chlorophyll contents of the seedlings were observed.

4. Discussion

4.1.

COMBINED EFFECTS OF 0 3 AND SOIL ACIDIFICATION

Combined effects of 03 and acid substances were emphasized because of their co-occurrence in many of the polluted areas. The 03 in combination with SO2 and NO~ generally results in the greatest suppression of plant growth (Chappelka and Chevone, 1992). Studies of the effects of 03 and direct acid rain or acid mist were reported. Some authors showed a combined effect: white ash seedlings (Chappelka and Chevone, 1986); but other showed no interactive effects: sugar maple and northern red oak (Reich et al., 1986), and loblolly pine (Edwards et al., 1992). In the present study, there were no interactive effects of O3 and soil acidification on growth and physiological functions of red pine seedlings. Therefore, an additive model was

362

YUNFENG SHAN ET AL. Table V Effects of 03 and soil acidification on chlorophyll contents in different parts of needles of red pine seedlings a tip part (rag g - i FW)

middle part (mg g - 1 FW)

basal part (mg g - i FW)

Whole-leaf (mg g - i FW)

Soil pH (HzO)

CF

03

CF

03

CF

O3

CF

03

pH 4.60 pH 3.90 pH 3.60

1.56 1.23 1.15

1.31 0.85 0.84

1.20 0.99 0.85

1.10 0.86 0.79

1.02 0.76 0.62

0.88 0.71 0.59

1.26 1.00 0.87

1.I0 0.81 0.75

ANOVA b 03 Soil treatment Linear Quadratic

** ** *** *

ns ** ** ns

ns ** *** ns

** ** ** ns

03 x Soil pH

ns

ns

ns

ns

a Each value represents the mean of 5 determinations. Two-factor ANOVA results: ns = not significant; * p<0.05; ** p<0.01; *** p<0.001.

adequate to describe the single or combined effects of 03 and soil acidification on red pine seedlings. As shown in Table II-V, the combined treatments resulted in the most serious damage to red pine seedlings. 4.2. 03 EFFECTS Needle dry weight of red pine seedlings was significantly reduced by 0 3 , a s shown in Table II. A non-significant tendency of dry weight reduction in stem, root or whole-plant was seen (Table II). Perhaps the 4-month treatment with 03 at 150 ppb was not sufficient to elicit significant effects on these dry weights. A decrease in the net photosynthetic rates of the O3-exposed seedlings (Table III) agreed well with the results reported by Coyne and Bingham (1982), Wallin et al. (1990), Izuta et al. (1994) and Shan et al. (1994-b). However, dark respiration rates of red pine seedlings were increased by 03 at 150 ppb (Table III). It has been reported that 03 stimulated dark respiration of Picea abies (L.) Karst. (Wallin et al., 1990), and A c e r saccharum March. and hybrid Populus (L.) (Tjoelker et al., 1993). Therefore, the reduction in the net photosynthetic rate and the stimulation in the dark respiration rate probably synergistically enhance the reduction in leaf dry weight of the seedlings exposed to 03. The exposure of wheat plants to 03 reduced the chlorophyll contents in the tip and middle parts of leaves, but did not reduce those in the basal parts (Nie et al., 1993). In our previous study, chlorophyll content in the needle tip part of red pine seedlings exposed to 03 at 300 ppb was significantly decreased, but that in the

EFFECTS OF 03 AND SOIL ACIDIFICATION

363

needle middle part was not altered; by contrast, that in the needle basal part was increased (Shan et al., 1994b). In this study, the exposure to 03 at 150 ppb reduced chlorophyll content in the needle tip part of red pine seedlings, but did not induce a significant alteration of that in the middle or basal part of needle (Table V). These results indicate that the responses of chlorophyll contents to 03 are different among the needle parts of the seedlings. Because of difficulties in making quantitative measurements, there are few studies on the effects of 03 on photorespiration of plants (Miller, 1987; Runeckles and Chevone, 1992). The increase in glycine and serine pool size of O3-exposed plants may be interpreted as a stimulation of photorespiration (Ito et al., 1985). The exposure to 03 at 300 ppb significantly enhanced the photorespiration rate of red pine seedlings (Shan et al., 1994-b). In this study, 150 ppb 03 also enhanced the photorespiration rate of the seedlings (Table III). Since there were no significant effects on chlorophyll content on a whole-needle fresh weight basis, the increase in the photorespiration rate of the seedlings exposed to 03 is considered to be one of the contributing factors for the reduction in the net photosynthetic rate of the seedlings (Table III). 4.3. EFFECTS OF SOIL ACIDIFICATION

Without visible injury, the growth reduction tendency of every plant organ and whole-plant of red pine seedlings was obvious, although only needle dry weight of the seedlings was significantly reduced at p<0.05 with decreasing soil pH (Table II). Also, seedlings did not survive the pH 3.15 treatment. The H + loads to the soils adjusted to pH 3.90, pH 3.60 and pH 3.15 were close to the sum of H + deposition for 32, 64 ot 132 years from wet deposition of pH 4.2 in an area where annual precipitation is 1500 mm, respectively. Similar levels of rainwater pH are found in the most polluted areas in Asia, Europe and North America (Liu et al., 1988; Falkengren-Grerup, 1989; Jagels et al., 1989; cape, 1993). The H + load from wet deposition at pH 4.2 where annual precipitation is 1500 mm was 88.2 mg m -2 year- I. Furthermore, H + load from dry deposition is approximately equal to that from wet deposition (Ulrich et al., 1980; Falkengren-Grerup, 1989). H + loads of three soil acidification treatments in the present study were about the sum of H + deposition from both wet and dry acid depositions for 16, 32 ot 64 years, respectively. Therefore, the results obtained in this study suggest that cumulative H + deposition for 16, 32, or 64 years by wet and dry acid depositions could results in growth reduction or death of red pine seedlings in most areas of red pine forest with comparable rain pH, although the methods of the soil treatment conducted in this study could not be completely realistic. Net photosynthetic rates of red pine seedlings were reduced by soil acidification (Table III). It has been reported that soil acidification also induced a decrease in the net photosynthetic rates of C r y p t o m e r i a j a p o n i c a D. Don. (Ohtagaki et al., 1996). However, the dark respiration rate of red pine seedlings was increased by soil

364

YUNFENG SHAN ET AL. Table VI Element concentrations in needles of red pine seedlings grown for 20 weeks in the acidified soils. Element concentrations are expressed on a needle dry weight basis Soil treatment

Element concentration (rag g - ~) Ca Mg K Mn AI

pH 4.60 pH 3.90 pH 3.60

2.58 2.15 2.84

0.80 0.91 1.09

4.97 5.43 4.54

0.48 0.82 1.26

0.46 0.68 0.97

acidification (Table III). Therefore, it was considered that the reduction in the net photosynthetic rate combined with the stimulation of dark respiration contributed to the reduction in dry weight growth of the seedlings grown in the acidified soils (Table III). As soil acidity increased, chlorophyll content in all parts of the needles was reduced in red pine seedlings (Table V). On the other hand, chlorophyll content of the seedlings was increased by the treatment with simulated acid rain of pH 2.3 or 3.0 (Shan et al., 1994-b). These results show that the short-term effect of simulated acid rain on chlorophyll content of red pine seedlings was completely different from that of soil acidification (Table V). Effects of soil acidification on photorespiration rates of plants have not been reported. In this study, with increasing soil acidity, photorespiration rates of red pine seedlings were significantly increased (Table III). The reduction in the chlorophyll contents and the stimulation of photorespiration might contribute to the decrease of net photosynthetic rates of the seedlings grown in the acidified soils. Water use efficiency (WUE) of red pine seedlings was reduced by soil acidification (Table IV). Because the transpiration rate of the seedlings was not affected (Table IV), the reduction in the WUE is likely attributable to reduction in net photosynthetic rates (Table III). The concentrations of A1 and Mn in the soil and in needles of the seedlings were dramatically increased with increasing soil acidities (Table I, Table VI). The concentrations of K and Mg in the soil and in needles of the seedlings were also dramatically increased. On the other hand, the concentrations of Ca did not change. As a result, Ca/A1 molar ratio in soil was reduced as soil pH reduced. At soil pH 3.60, Ca/A1 molar ratio was 0.49. It was reported that AI concentration was increased by soil acidification and was considered as a cause for the growth reduction of Cryptomeria japonica D. Don (Izuta et al., 1990; Miwa et al., 1994; Totsuka, 1994). Furthermore, A1 toxicity to forest trees will be manifested with Ca/A1 molar ratio <2 (Cronan and Grigal, 1995). Therefore, excess A1 released by soil acidification and low Ca/AI molar ratio in soil may be phytotoxic and could

EFFECTS OF 03 AND SOIL ACIDIFICATION

365

contribute to a reduction in the dry weight growth of red pine seedlings grown in the acidified soil.

Acknowledgements Authors would like to thank Mr. Horie, K. and other staff of The Laboratory of Terrestrial Environment, Tokyo University of Agriculture and Technology, for their helpful discussion, technical support and encouragement. Authors wish to express their appreciation to Japan Society for the Promotion of Science (JSPS) for providing the JSPS Postdoctoral Fellowship to Dr. Shan, Yunfeng.

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