Advanced
Visible injury and growth inhibition of black pine in relation to oxidative stress in industrial areas
Visible injury and growth inhibition of black pine in relation to oxidative stress in industrial areas
Journal of Ecology and Environment. 2010. Dec, 33(4): 333-341
Copyright ©2010, The Ecological Society of Korea
This is an Open Access article distributed under the terms of theCreative Commons Attribution Non-Commercial License(http://creativecommons.org/licenses/by-nc/3.0/)which permits unrestrictednon-commercial use, distribution, and reproduction in any medium,provided the original work is properly cited.
  • Received : July 01, 2010
  • Accepted : July 29, 2010
  • Published : December 01, 2010
Download
PDF
e-PUB
PubReader
PPT
Export by style
Article
Author
Metrics
Cited by
TagCloud
About the Authors
Sim-Hee Han
Department of Forest Resources Development, Korea Forest Research InstituteSuwon 441-350, Korea
simhee02@forest.go.kr
Du-Hyun Kim
Department of Forest Resources Development, Korea Forest Research InstituteSuwon 441-350, Korea
Ja-Jung Ku
Department of Forest Resource Conservation, National ArboretumPocheon 487-821, Korea
Jae Kyung Byun
Department of Forest Conservation, Korea Forest Research InstituteSeoul 130-712, Korea
Jae Cheon Lee
Department of Forest Resources Development, Korea Forest Research InstituteSuwon 441-350, Korea

Abstract
The objective of our study was to investigate the major reasons for the different growth and visible injury on the needles of black pine growing in Ulsan and Yeocheon industrial complex areas, South Korea. After 12 years of growth,we collected climatic and air pollutant data, and analyzed soil properties and the physiological characteristics of black pine needles. Annual and minimum temperatures in Ulsan were higher than those in Yeocheon from 1996 to 2008. Ozone (O 3 ) was the pollutant in greatest concentration in Yeocheon, and whereas the SO 2 concentration in most areas decreased gradually during the whole period of growth, SO 2 concentration in Yeocheon has increased continuously since 1999, where it was the highest out of four areas since 2005. Total nitrogen and cation exchange capacity in Yeocheon soil were significantly lower than those of Ulsan. The average growth of black pine in Yeocheon was significantly smaller than that in Ulsan, and the growth of damaged trees represented a significant difference between the two sites. Photosynthetic pigment and malondialdehyde content and antioxidative enzyme activity in the current needles of black pine in Yeocheon were not significantly different between damaged and healthy trees, but in 1-year-old needles, there were significant differences between damaged and healthy trees. In conclusion, needle damage in Yeocheon black pine can be considered the result of long-term exposure to oxidative stress by such as O 3 or SO 2 , rather than a difference in climatic condition or soil properties, and the additional expense of photosynthate needed to overcome damage or alleviate oxidative stress may cause growth retardation.
Keywords
INTRODUCTION
Since the 1960s, the Korean government has promoted petrochemical and heavy chemical industries in Ulsan,Gyeongsangnam-do, and Yeocheon, Jeollanam-do. Along with national industrial growth, associated environmental problems also arose due to increased contaminants from the exhaust of various factories in the industrial complexes. In fact, harmful substances emitted from chemical fertilizer factories damaged crop and forest production in the 1960s. In addition, industrialization also degraded atmospheric conditions due to increased auto exhausts in large cities and continued increase of ozone concentration as a secondary pollutant. Ozone hazard warnings increased from 1995 to 2008, as the frequency rose from only twice (one day) in 1995; 24 times(12 days) in 1997; 52 times (17 days) in 2000; to 101 times(32 days) in 2008. Also, the ozone hazard warning occurred much earlier (April 25) in 2008 than the previous warning announcement on July 22 in 1995. As of 2008, the highest concentration per hour was 203 ppb in Yeosu, Jeollanam-do (Ministry of Environment 2008).
Interest in pollutants and in their effects on living organisms has increased in the last few decades, as a consequence of the rise in their ground-level concentration and of the widening of their diffusion area. Current levels of the pollutant were high enough to exceed the tolerance threshold of many plants, thus impairing plant growth, reducing crop yields, and altering the composition of plant communities. Pollutants could also induce subtle changes at the physiological and biochemical level, which can lead to different plant responses. Fares et al. (2010) reported that stomatal flux, which is considered responsible for ozone damage, showed a weaker correlation with ozone concentrations than non-stomatal flux during the summer and fall seasons. Therefore, much attention has been given toward understand the mechanisms of plant adaptation to pollutants. Ordinarily, high levels of pollutants can delay growth and development, reduce yield, and, in extreme cases, can inflict lethal injuries on plants. To ensure survival, plants have evolved a range of response strategies to various abiotic stresses; these responses vary with genotypes and among specific stresses (Deepak and Agrawal 2001, Azevedo Neto et al. 2006). At the whole plant level, the effect of stress is usually perceived as a decrease in photosynthesis and growth, alteration in carbon and nitrogen metabolism (Cornic and Massacci 1996, Mwanamwenge et al. 1999, Law and Crafts-Brandner 2001, Han et al. 2007), and reactive oxygen production (Turcsanyi et al. 2000, Schwanz and Polle 2001).
However, there have been few studies on injury to trees by oxidative stress under actual field conditions (Vollenweider and Günthardt-Goerg 2005, Bussotti and Ferretti 2009), especially in terms of physiological damage and defense responses to oxidative stress. In fact, because of the combination of environmental stresses simultaneously occurring in the field, it is difficult to identify the main cause of injuries to tree and natural vegetation under field conditions (Mittler 2006). Since many species exhibit high variability in their symptoms, which could be confused as senescence, such species should not be used in the field as an indicator for ozone injury assessment. Nonetheless, observations of typical symptoms on above-ground plant parts in the field has turned out to be a valuable tool for the assessment of the impact of ambient ozone on sensitive plant species (Skelly et al. 1987, Flagler 1998, Innes et al. 2001). In addition, Bussotti et al. (2003) provided suggestions to improve the reliability of ozone injury assessment on forest plant species by improving the capability of observers to identify and assess ozone symptoms before field surveys are conducted.
Black pine, Pinus thunbergii Parl., is native to coastal areas of Korea and Japan. Because of its resistance to pollution and salt tolerance, it is a popular horticultural tree, and recommended for rehabilitation of reclaimed coastal lands with extreme environmental conditions. Since 1980 in the Yeocheon and Ulsan industrial complexes, however, chlorotic mottle and leaf senescence on black pine needles, which have resulted from long-term exposure to air pollution. Therefore, the breeding of tolerant tree species or varieties is deemed necessary to produce suitable tree species for the restoration of disturbed forest ecosystem in these kinds of environmental conditions. Therefore, in 1996, two experimental black pine forests were established in the Ulsan and Yeocheon industrial complex areas to examine the tolerance of the selected candidate trees. The black pine candidate trees selected from the areas of the Ulsan and Yeocheon industrial complexes were based on established selection standards for visual injury, vitality, and physiological responses for trees.
In this study, our primary objective was to determine the major reasons that cause differential growth and visible needle injury on the needle of black pine growing in the two industrial areas (Yeocheon and Ulsan) in Yeocheon, South Korea. To accomplish this, we collected and analyzed meteorological and air pollution data, analyzed soil properties, and evaluated physiological characteristics such as photosynthetic pigment content, lipid peroxidation, and antioxidative enzyme activity of black pine from the Yeocheon and Ulsan industrial areas, and their possible relationships.
MATERIALS AND METHODS
- Study sites
The study was conducted in two experimental black pine forests, which were established in the vicinities of the Ulsan (San 73 Yongyeon-dong, Nam-gu, Ulsan metropolitan city) and Yeocheon (San 100 Wollae-dong, Yeocheon-si, Jeollanam-do) industrial complexes in 1996. These experimental forests were established to examine the tolerance of black pine as candidate trees against environmental pollution. The selection process was based on established selection standards for visual injury, vitality, and physiological responses of black pine trees in response to two environmental conditions typified in the Ulsan (Dotjilsan) and Yeocheon (Wollae) industrial complex. Prior to the establishment of the experimental forest, seeds from thirty candidate black pine trees were sown in Korea Forest Research Institute (KFRI)’s tree nursery for seedling production. Two-year-old seedlings were transplanted to the Yeocheon and Ulsan sites, which consisted of 10 replicates per family laid in three different blocks. Climatic data were collected from the nearest Suncheon and Ulsan observatories for Yeocheon and Ulsan study sites, respectively, and the annual changes in air temperature and precipitation are shown in Fig. 1 .
Both study sites are close to either a chemical fertilizer factory or petrochemical and heavy chemical industries. Owing to exhaust gas from the industrial complexes, the trees in two experimental forests were exposed to concentrated air pollutants mainly consisting of such as sulfur oxide (SO 2 ) and ozone (O 3 ). Changes in the annual mean concentration of air pollutants from 1996 to 2008 are shown in Fig. 2 .
- Growth and vitality measurement
In 2008, after 12 years of field growth, the diameter of root collar (DRC) of the experimental plants was measured at two sites. To assess the adaptation of trees to pollution, bench marking based on tree phenotypes were done, and vitality and visual symptoms were recorded. Tree vitality was assessed based on leaf tip damage, leaf color, and leaf coloring, and each criterion was graded in four levels. The vitality evaluation method is considered to be a fast decision standard to assess the adaptation of trees in polluted areas. On the basis of growth and vitality, 10 trees representing groups of damaged and healthy trees were chosen from two sites. Fig. 3 showed the average DRC of 10 damaged and healthy trees and overall average DRC of both damaged and healthy trees from the two experimental sites.
- Soil sampling and analysis
To analyze soil characteristics, 18 soil samples (6 samples per block) were collected from each of the two experimental forests. The mineral soil samples at 20-cm depth were collected using a 3.7-cm diameter core, after removing the organic layer. Samples were taken at four locations approximately 1.5 m from the stem to avoid the impact of stem flow. The samples were immediately processed in the laboratory; roots were removed and passed through a 2-mm sieve. The processed soil samples were refrigerated at 4 o C until analysis. Similarly, some subsamples (100 g) were air-dried at room temperature and used for the determination of organic matter content, total nitrogen, available phosphorus, cation exchange capacity (CEC) and exchangeable cations (K + , Na + , Ca 2+ , and Mg 2+ ). Soil pH (soil:water) was determined from refrigerated samples.
- Needle sampling and analysis
Needles from the black pines were collected from branch samples located at a height of 2.5-3.0 m from the sun-exposed crowns for both damaged and healthy trees at the Yeocheon sites in August 2008. Since visible injury was not observed in the black needles from the Ulsan site, needle samples were not collected from there.
A total of 10 trees for each group were included in the sampling. Needles from each branch were segregated into current (new leaf) and 1-year-old needles, and refrigerated at 4 o C until analysis. The total chlorophyll, chlorophyll a, chlorophyll b, and carotenoid content in the needles were determined based on the procedure described by Lichtenthaler (1987).
Lipid peroxidation was determined by measuring the amount of malondialdehyde (MDA) produced by thiobarbituric acid reaction as described by Heath and Packer (1968). To assess resistance to oxidative stress, the activity of superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione reductase (GR), and catalase (CAT) on fresh needles was assayed. SOD was assayed on the basis of the inhibition of reduction of nitro-blue tetrazolium in the presence of xanthine at 530 nm according to the method of Beauchamp and Fridovich (1971). An APX activity assay was carried out in a reaction mixture containing 50 mM phosphate buffer (pH 7.0), 0.5 mM AsA, 0.1 mM EDTA, 0.1 mM H 2 O 2 , and 0.1 mL enzyme extract based on the method of Nakano and Asada (1981). The change in A 290 was recorded for 1 min after the addition of H 2 O 2 . The GR activity assay was carried out in a reaction mixture containing 50 mM phosphate buffer (pH 7.8), 0.1 mM NADPH, 0.5 mM GSSH, and 0.1 mL enzyme extract as described by Carlberg and Mannervik (1985). The change in A 340 was recorded for 5 min after the addition of enzyme extract. The CAT activity was determined by following a two-step procedure based on the relationships of dismutation of H 2 O 2 to H 2 O and molecular oxygen, which is proportional to the concentration of CAT (Fossati et al. 1980). All enzyme activities were measured using a UV-120 (Shimadzu, Kyoto, Japan).
- Statistical analysis
Lager Image
Annual mean maximum and minimum air temperature (a) and total precipitation (b) from 1996 to 2008 at the Suncheon and Ulsan Observatories.
Lager Image
Changes in the annual mean concentration of air pollutants at the National Seoul (5 metro cities) Yeocheon (Wollae) and Ulsan stations
The data were statistically analyzed using Analysis of Variance of a Completely Randomized Design. Means were compared using the Duncan multiple range test at the 5% level. Statistical analyses were done using the SAS System for Windows ver. 8.01 (SAS Institute, Cary, NC, USA).
RESULTS AND DISCUSSION
- Growth response under two different environments
The two study sites had different growth environments for black pine. As shown in Fig. 1 , annual and minimum temperatures in Ulsan were higher than those in Suncheon (Yeocheon site) from 1996 to 2008, whereas the maximum temperature was the same at the two sites. Similarly, the atmospheric concentration of ozone, nitrous oxide, sulfur dioxide, and carbon monoxide, presented in Fig. 2 , revealed values of ozone and sulfur dioxide at the Yeocheon site that were much higher than the national average, indicating the occurrence of air pollution in this area. In addition, the total nitrogen, CEC, and K content in the soil of Yeocheon were significantly lower than those of Ulsan ( Table 1 ).
Differences in environmental conditions could lead to differences in black pine growth between the two areas. Based on the DRC of 14-year-old black pines, transplanted at Yeocheon and Ulsan study area in 1996, the average DRC of black pine in Yeocheon was significantly smaller than that in Ulsan ( Fig. 3 ). It is well known that the per-
Soil characteristics of black pine experimental forests in the Yeocheon and Ulsan study sitesAll values are means of eighteen replicates ± standard deviation. Means with different letters are significantly different at a 5% level using Duncan’s multiple range test.O.M., organic matter; T.N., total nitrogen; Av., available; CEC, cation exchange capacity.
Lager Image
Soil characteristics of black pine experimental forests in the Yeocheon and Ulsan study sites All values are means of eighteen replicates ± standard deviation. Means with different letters are significantly different at a 5% level using Duncan’s multiple range test.O.M., organic matter; T.N., total nitrogen; Av., available; CEC, cation exchange capacity.
Lager Image
Average diameter of root collar (DRC) of damaged and healthy black pine in Yeocheon and Ulsan complex area. All values are means of ten replicates ± standard deviation. Means with different letters are significantly different at a 5% level using Duncan’s multiple range test.
formance of genotypes relative to each other can vary according to the environment, so that genotypes that are superior in one environment may not be correspondingly superior elsewhere. This phenomenon has been formulated from the concept of genotype-environment interaction (Burdon 1977). In addition, Scholz (1989) described that a particular genotype behaves differently in different environments, and different genotypes vary in their reaction to different environments. In our study, the growth retardation of black pine in Yeocheon might be a result of the mixture of environmental factors, such as lower annual temperature and poor soil conditions, as evident in the lower total nitrogen and CEC of soil as compared with that of Ulsan.
However, chlorotic mottle and tip necrosis, the two most common needle symptoms of conifer species exposed to air pollutants such as O 3 or SO 2 , were observed on the needles of black pines in Yeocheon, but not in Ulsan (data not shown). In general, mottle on young and older needles are induced by low-level exposure to O 3 , while tip necrosis in young pines is induced by high-level exposures. This means that the lower annual temperature and soil quality may not only be the factor contributing to the growth retardation of black pine at Yeocheon. Our hypothesis is that the growth retardation of black pine at the Yeocheon site could also be due to the reduction of photosynthetic activity due to the presence of necrotic and chlorotic needles. This could be supported by the fact that chlorotic mottle and tip necrosis on black pine needles are induced by oxidative stresses due to pollutants such as O 3 and SO 2 that are prevalent in the Yeocheon area ( Fig. 2 ). As compared to the other sites, the O 3 concentration in Yeocheon was highest during the whole period of growth. Likewise, the SO 2 concentration in most areas decreased gradually from 1996 to 2008, whereas the SO 2 concentration in Yeocheon has increased continuously since 1999, and it was the highest among the four areas since 2005. Therefore, it may be that long-term exposure to higher O 3 and SO 2 concentration have contributed to needle damage and growth reduction of black pine in Yeocheon. Some research has established that ozone, after diffusion through the stomata, causes short-term oxidative stress that can lead to visible injury (Becker et al. 1989, Chappelka and Samuelson 1998, Bungener et al. 1999). Longer-term exposure could eventually reduce shoot and root biomass (Davison and Barnes 1998) or loss of reproductive output (Black et al. 2000). In addition, Hirano and Morimoto (1999) reported that a large reduction in the series of tree-ring index (TRI) in a stand close to an industrial complex appeared from the 1960s to 1970s, and a significant negative correlation between TRI and sulfur dioxide (SO 2 ) concentration was found in the polluted area. Their results indicated that past reduction in the growth of black pine trees in an industrial area was mainly caused by SO 2.
- Individual differences under identical environmental conditions
In general, individual differences in plant growth parameters could be observed in the same environmental conditions. As shown in Fig. 3 , in the two study sites there were significant differences in the average DRC of damaged and healthy black pine, as rated according to their viability in terms of such as growth and damage level (data not shown). Interestingly, there was not a significant difference in the DRCs of healthy trees between the two sites, whereas the DRCs of damaged trees showed a significant difference between two sites. This observation is parallel to the findings of Scholz (1989) that different genotypes of the same species differ in sensitivity, and the same dose can lead to different symptom intensities in different individuals of the same species. Comparing
Average photosynthetic pigment content in the current and 1-year-old needles of damaged and healthy black pine at the Yeocheon study siteAll values are means of ten replicates ± standard deviation. Means with different letters are significantly different at a 5% level using Duncan’s multiple range test.Chl, chlorophyll.
Lager Image
Average photosynthetic pigment content in the current and 1-year-old needles of damaged and healthy black pine at the Yeocheon study site All values are means of ten replicates ± standard deviation. Means with different letters are significantly different at a 5% level using Duncan’s multiple range test.Chl, chlorophyll.
Lager Image
Average malondialdehyde (MDA) content in the current and 1-year-old needles of damaged and healthy black pine at the Yeocheon study site. All values are means of ten replicates ± standard deviation. Means with different letters are significantly different at a 5% level using Duncan’s multiple range test.
the two genotypes, it shows that the genotypic variation of sensitivity may be great, and identical genotypes react similarity to a given pollution regime. This means that the phenotypic variation of damage in such experiments was governed to a great extent by genetic factors.
Consequently, in both the Yeocheon and Ulsan experimental sites, the growth and vitality of black pine in differed noticeably enough that we have grouped them into damaged and healthy trees for the purposes of genotypic comparison within the same environmental conditions. However, the difference between the damage and the healthy trees was more obvious at the Yeocheon site, which could be ascribed to the overall environmental conditions characterized by lower total nitrogen and CEC in the soil, lower annual temperature, and higher air pollutants. These poor environmental conditions could have hastened such genotypic differences between the two sites. Some black pines in Yeocheon have even showed chlorotic mottle and tip necrosis on the needles.
Table 2 shows the photosynthetic pigment content in the needles of damaged and healthy trees in Yeocheon. Chlorophyll content in the current needle did not significantly between damaged and healthy trees, but a significant difference was observed in 1-year-old needle. The reduction in chlorophyll content could be the result of deficiency in nutrients such as nitrogen (Oren et al. 1993, Tausz et al. 1996) and a decrease in chlorophyll biosynthesis or chlorophyll destruction by oxidative stress (Shimazaki et al. 1980, Heath 1989, Aarti et al. 2006). This is associated with visible symptoms such as chlorotic mottle or tip necrosis on the needle. Typically, reduction in chlorophyll content due to nutrient deficiency occurs easily even in young needles or leaves (Chandler and Dale 1995, Mehne-Jakobs 1996). However, in our study, the difference in chlorophyll content between damaged and healthy trees could not be attributed solely to nutrient deficiency, because we found no significant differences in chlorophyll content between the damaged and healthy needles of the current needles and visible injury symptoms were not observed in the current needles in both groups. On the contrary, the difference in chlorophyll content between the two groups was observed in 1-year-old needles, and visible injury was also observed on 1-year-old needles of damaged trees. We proposed that the chlorophyll reduction observed in this study could be the result of long-term exposure of various oxidative stress rather than nutrient deficiency in the soil as induced by high O 3 and SO 2 concentrations in Yeocheon compared with other areas.
As with the results on chlorophyll content, MDA content, which represents the state of membrane lipid peroxidation (Ranieri et al. 1996), in 1-year-old needles of damaged trees was significantly higher than that of healthy trees, but MDA content in current needles was not significantly different between damaged and healthy trees ( Fig. 4 ). The increase of MDA content in 1-year-old needles of damaged trees was considered to be the effects of long-term exposure to oxidative stress during the growth period, and an increase in lipid peroxidation indicates that damage was occurring at the membrane levels (Calatayud et al. 2006, Han et al. 2009). In a study on age-related peroxidation in annual beech ( Fagus silvatica
Average antioxidative enzyme activity in the current and 1-year-old needles of damaged and healthy black pine at the Yeocheon study siteAll values are means of ten replicates ± standard deviation. Means with different letters are significantly different at a 5% level using Duncan’s multiple range test.SOD, superoxide dismutase; GR, glutathione reductase; APX, ascorbate peroxidase; CAT, catalase.
Lager Image
Average antioxidative enzyme activity in the current and 1-year-old needles of damaged and healthy black pine at the Yeocheon study site All values are means of ten replicates ± standard deviation. Means with different letters are significantly different at a 5% level using Duncan’s multiple range test.SOD, superoxide dismutase; GR, glutathione reductase; APX, ascorbate peroxidase; CAT, catalase.
Mill.) leaves and perennial fir (Abies alba Mill.) needles, Kunert and Ederer (1985) reported that older needles are strongly associated with a higher peroxidation of lipids.
In addition, significant differences in the activities of antioxidative enzymes were shown between damaged and healthy trees in 1-year-old needles, and not in current needle ( Table 3 ), which correlates with the results of lipid peroxidation. It has been reported that chlorotic spruce needles contain higher ascorbate and glutathione levels than green needles (Osswald et al. 1987). In chlorotic needles, the level of total ascorbate and the activities of superoxide dismutase, monodehydroascorbate radical reductase, NAD-malate dehydrogenase, and glucose-6-phosphate dehydrogenase were significantly increased (Polle et al. 1992). These results may indicate that the extent of age-related peroxidative damage of cells seems to be controlled by the potency of antioxidative systems (Kunert and Ederer 1985).
In conclusion, the growth reduction and damage observed on black pine in Yeocheon could be the result of oxidative stress, and of the additional expense of photosynthate as a consequent defense mechanism against oxidative stress due to high ozone and SO 2 concentration in the atmosphere.
References
Aarti PD , Tanaka R , Tanaka A 2006 Effects of oxidative stress on chlorophyll biosynthesis in cucumber (Cucumis sativus) cotyledons. Physiol Plant 128 186 - 197    DOI : 10.1111/j.1399-3054.2006.00720.x
Azevedo Neto AD , Prisco JT , Eneas-Filho J , Abreu CEB , Gomes-Filho E 2006 Effect of salt stress on antioxidative enzymes and lipid peroxidation in leaves and roots of salt-tolerant and salt-sensitive maize genotypes Environ Exp Bot 56 87 - 94    DOI : 10.1016/j.envexpbot.2005.01.008
Beauchamp C , Fridovich I 1971 Superoxide dismutase: improved assays and an assay applicable to acrylamide gels. Anal Biochem 44 276 - 287    DOI : 10.1016/0003-2697(71)90370-8
Becker K , Saurer M , Egger A , Fuhrer J 1989 Sensitivity of white clover to ambient ozone in Switzerland New Phytol 112 235 - 243    DOI : 10.1111/j.1469-8137.1989.tb02379.x
Black VJ , Black CR , Roberts JA , Stewart CA 2000 Impact of ozone on the reproductive development of plants. New Phytol 147 421 - 447    DOI : 10.1046/j.1469-8137.2000.00721.x
Bungener P , Balls GR , Nussbaum S , Geissmann M , Grub A , Fuhrer J 1999 Leaf injury characteristics of grassland species exposed to ozone in relation to soil moisture condition and vapour pressure deficit New Phytol 142 271 - 282    DOI : 10.1046/j.1469-8137.1999.00390.x
Burdon RD 1977 Genetic correlation as a concept for studying genotype-environment interaction in forest tree breeding. Silvae Genet 26 168 - 175
Bussotti F , Ferretti M 2009 Visible injury crown condition and growth responses of selected Italian forests in relation to ozone exposure. Environ Pollut 157 1427 - 1437    DOI : 10.1016/j.envpol.2008.09.034
Bussotti F , Schaub M , Cozzi A , Krauchi N , Ferretti M , Novak K , Skelly JM 2003 Assessment of ozone visible symptoms in the field: perspectives of quality control Environ Pollut 125 81 - 89    DOI : 10.1016/S0269-7491(03)00095-2
Calatayud A , Pomares F , Barreno E 2006 Interactions between nitrogen fertilization and ozone in watermelon cultivar Reina de Corazones in open-top chambers: effects on chlorophyll a fluorescence lipid peroxidation and yield. Photosynthetica 44 93 - 101    DOI : 10.1007/s11099-005-0163-2
Carlberg I , Mannervik B 1985 Glutathione reductase Methods Enzymol 113 484 - 490    DOI : 10.1016/S0076-6879(85)13062-4
Chandler JW , Dale JE 1995 Nitrogen deficiency and fertilization effects on needle growth and photosynthesis in Sitka spruce (Picea sitchensis). Tree Physiol 15 813 - 817
Chappelka AH , Samuelson 1998 Ambient ozone effects on forest trees of the eastern United States: a review New Phytol 139 91 - 108    DOI : 10.1046/j.1469-8137.1998.00166.x
Cornic G , Massacci A 1996 Leaf photosynthesis under drought stress. 47-366 Kluwer Academic Publishers Dordrecht In: Photosynthesis and the Environment (Baker NR ed).
Davison AW , Barnes JD 1998 Effects of ozone on wild plants New Phytol 139 135 - 151    DOI : 10.1046/j.1469-8137.1998.00177.x
Deepak SS , Agrawal M 2001 Influence of elevated CO2on the sensitivity of two soybean cultivars to sulphur dioxide Environ Exp Bot 46 81 - 91    DOI : 10.1016/S0098-8472(01)00086-7
Fares S , McKay M , Holzinger R , Goldstein AH 2010 Ozone fluxes in a Pinus ponderosa ecosystem are dominated by non-stomatal processes: evidence from long-term continuous measurements. Agric For Meteorol 150 420 - 431    DOI : 10.1016/j.agrformet.2010.01.007
Flagler RB 1998 Recognition of Air Pollution Injury to Vegetation: A Pictorial Atlas. 2nd ed. Air & Waste Management Association Pittsburgh PA.
Fossati P , Prencipe L , Berti G 1980 Use of 35-dichloro-2-hydroxybenzenesulfonic acid/4-aminophenazone chromogenic system in direct enzymic assay of uric acid in serum and urine Clin Chem 26 227 - 231
Han SH , Kim DH , Lee JC , Kim PG 2009 Effects of fertilization on physiological parameters in American sycamore (Platanus occidentalis) during ozone stress and recovery phase J Ecol Field Biol 32 149 - 158    DOI : 10.5141/JEFB.2009.32.3.149
Han SH , Kim DH , Lee KY , Ku JJ , Kim PG 2007 Physiological damages and biochemical alleviation to ozone toxicity in five species of genus Acer J Korean For Soc 95 551 - 560
Heath RL 1989 Alteration of chlorophyll in plants upon air pollutant exposure. National Academy Press Washington DC. In: Biologic Markers of Air-Pollution Stress and Damage in Forests (Committee on Biologic Markers of Air-Pollution Damage in Trees National Research Council eds).
Heath RL , Packer L 1968 Photoperoxidation in isolated chloroplasts. I. Kinetics and stoichiometry of fatty acid peroxidation Arch Biochem Biophys 125 189 - 198    DOI : 10.1016/0003-9861(68)90654-1
Hirano T , Morimoto K 1999 Growth reduction of the Japanese black pine corresponding to an air pollution episode. Environ Pollut 106 5 - 12    DOI : 10.1016/S0269-7491(99)00063-9
Innes JL , Skelly JM , Schaub M 2001 Ozone and Broadleaved Species: A Guide to the Identification of Ozone-Induced Foliar Injury. Paul Haupt Publishing Bern.
Kunert KJ , Ederer M 1985 Leaf aging and lipid peroxidation: the role of the antioxidants vitamin C and E. Physiol Plant 65 85 - 88    DOI : 10.1111/j.1399-3054.1985.tb02364.x
Law RD , Crafts-Brandner SJ 2001 High temperature stress increases the expression of wheat leaf ribulose-15-bisphosphate carboxylase/oxygenase activase protein. Arch Biochem Biophys 386 261 - 267    DOI : 10.1006/abbi.2000.2225
Lichtenthaler HK 1987 Chlorophylls and carotenoids: pigments of photosynthetic biomembranes Methods Enzymol 148 350 - 382    DOI : 10.1016/0076-6879(87)48036-1
Mehne-Jakobs B 1996 Magnesium deficiency treatment causes reductions in photosynthesis of well-nourished Norway spruce. Trees Struct Funct 10 293 - 300    DOI : 10.1007/BF02340775
Ministry of Environment 2008 Annual Report of Ambient Air Quality in Korea Ministry of Environment Gwacheon
Mittler R 2006 Abiotic stress the field environment and stress combination. Trends Plant Sci 11 15 - 19    DOI : 10.1016/j.tplants.2005.11.002
Mwanamwenge J , Loss SP , Siddique KHM , Cocks PS 1999 Effect of water stress during floral initiation flowering and podding on the growth and yield of faba bean (Vicia faba L.). Eur J Agron 11 1 - 11    DOI : 10.1016/S1161-0301(99)00003-9
Nakano Y , Asada K 1981 Hydrogen peroxide is scavenged by ascorbate-specific peroxidase in spinach chloroplasts Plant Cell Physiol 22 867 - 880
Oren R , Werk KS , Buchmann N , Zimmermann R 1993 Chlorophyll-nutrient relationships identify nutritionally caused decline in Picea abies stands. Can J For Res 23 1187 - 1195    DOI : 10.1139/x93-150
Osswald WF , Senger H , Elstner EF 1987 Ascorbic acid and glutathione contents of spruce needles from different locations in Bavaria. Z Naturforsch 42 879 - 884
Polle A , Chakrabarti K , Chakrabarti S , Seifert F , Schramel P , Rennenberg H 1992 Antioxidants and manganese deficiency in needles of Norway spruce (Picea abies L.) trees. Plant Physiol 99 1084 - 1089    DOI : 10.1104/pp.99.3.1084
Ranieri A , D'Urso G , Nali C , Lorenzini G , Soldatini GF 1996 Ozone stimulates apoplastic antioxidant systems in pumpkin leaves Physiol Plant 97 381 - 387    DOI : 10.1034/j.1399-3054.1996.970224.x
Scholz F 1989 Genetic research in forest decline implications for nongenetic investigations 325-328 Eidgennssische Anstalt fur das forstliche Versuchswesen Birmensdorf In: Air Pollution and Forest Decline (Bucher JB Bucher-Wallin I eds). Proceeding 14th International Meeting for Specialists in Air Pollution Effects on Forest Ecosystems IUFRO P2.05 1988 Oct 2-8 Interlaken.
Schwanz P , Polle A 2001 Growth under elevated CO2ameliorates defenses against photo-oxidative stress in poplar (Populus alba × tremula) Environ Exp Bot 45 43 - 53    DOI : 10.1016/S0098-8472(00)00079-4
Shimazaki K , Sakaki T , Kondo N , Sugahara K 1980 Active oxygen participation in chlorophyll destruction and lipid peroxidation in SO2-fumigated leaves of spinach Plant Cell Physiol 21 1193 - 1204
Skelly JM , Davis DD , Merrill W , Cameron EA , Brown HD , Drummond DB , Dochinger LS 1987 Diagnosing Injury to Eastern Forest Trees: A Manual for Identifying Damage Caused by Air Pollution Pathogens Insects and Abiotic Stresses. Pennsylvania State University College of Agricultural Sciences University Park PA.
Tausz M , Zellnig G , Bermadinger-Stabentheiner E , Grill D , Katzensteiner K , Glatzel G 1996 Physiological structural and nutritional parameters of Norway spruce needles from declining forest stands in Austria Can J For Res 26 1769 - 1780    DOI : 10.1139/x26-201
Turcsanyi E , Lyons T , Plochl M , Barnes J 2000 Does ascorbate in the mesophyll cell walls form the first line of defence against ozone? Testing the concept using broad bean (Vicia faba L.). J Exp Bot 51 901 - 910    DOI : 10.1093/jexbot/51.346.901
Vollenweider P , Gunthardt-Goerg MS 2005 Diagnosis of abiotic and biotic stress factors using the visible symptoms in foliage Environ Pollut 137 455 - 465    DOI : 10.1016/j.envpol.2005.01.032