Characteristics of tree growth and leaf damage under different O3 concentrations

Pinus bungeana, Platycladus orientalis, Koelreuteria paniculata and Ginkgo biloba were taken to research. 3 open-top chambers with O3 concentration gradient were set up to study the characteristics of tree growth and leaf damage under different O3 concentrations. The results showed that with the increase of O3 concentration, the growth of different tree species were inhibited, but the diameter between different tree species was not significantly different. The plant height of different tree species the plant height of different tree species increased from 0.05m to 0.06m under NF O3 concentration, from 0.01m to 0.03m under NF40 O3 concentration, but from 0.01m to 0.02m under NF80 O3 concentration. The diameter of different tree species at 50cm increased from 0.90cm to 1.40cm under NF O3 concentration, from 0.70cm to 1.00cm under NF40 O3 concentration, and from 0.60cm to 0.80cm under NF80 O3 concentration. Under NF O3 concentration, the leaves of different tree species performed well, without any damage symptoms. With the increase of O3 concentration, leaves were withered and died. Leaves wilted under NF40 O3 concentration, but died and fallen under NF80 O3 concentration. Pinus bungeana and Platycladus orientalis were good at resisting O3 pollution, while Koelreuteria paniculata and Ginkgo biloba were worse. The damage of O3 concentration to plants is more sensitive to broad-leaved trees.


Introduction
In recent years, with the rapid development of global economy and industrialization, the amount of nitrogen oxides, carbon monoxide and hydrocarbons discharged into the atmosphere have increased, which leads to the changes of photochemical O3 (O3) concentration in the troposphere (Karnosky et al.,2004). At the same time, the change of land coverage caused by human activities leads to the decrease of O3 absorption performance on the surface, which leads to the increase of O3 concentration near the surface. It was found that with the rapid development of human activities and industrialization, the nearsurface O3 concentration is increasing at the rate of 0.3% to 2% every year, and it was predicted that the 3 related studies have found that the threshold value of O3-induced plant damage in conifers such as pine trees was between 150 ppb to 2 times the environmental concentration, while the threshold value of O3 induced plant damage in broad-leaved trees such as ginkgo, poplar and birch was between 60 ppb to 1.4 and 1.7 times the environmental concentration (Zhang, 2011). The environmental O3 concentration in Beijing and Guangzhou was 60 nmol·mol -1 (Wang, et al., 2016). Meanwhile, this study refers to the experimental settings of Xinyue (2016) and Niu Junfeng (2012), and set three O3 gradients under the atmospheric O3 concentration in Beijing, adding 40 nmol·mol -1 (NF40) to the O3 environmental concentration (NF) After that, it was doubled (NF80), which exceeded the threshold of O3 induced plant damage, but not much. The three O3 concentration gradients gradually increased, and in the controllable range of O3 damage to plants, they also had significant effects on the growth and physiological characteristics of different tree species, so they were set as follows. From August 2016 to October 2017, the experiment was carried out from 9: 00 to 16: 00 every day. O3 fumigation was carried out on NF40 and NF80 OTCs every two days, and the O3 concentration was controlled by adjusting the O3 amount of O3 generator. The O3 monitor was used to measure the real-time O3 concentration, temperature and humidity data in the gas chambers, and the normal watering management was carried out during the whole experiment. Atmospheric O3 concentration data of normal environment (NF) was obtained from the ambient air quality monitoring station set up in Beijing Botanical Garden in Beijing Environmental Protection Monitoring, where is 300m away from the experimental site.
Three potted seedlings of Pinus bungeana, Platycladus orientalis, Koelreuteria paniculata and Ginkgo biloba were placed in each gas chambers, and the seedlings are all 3 years old. The 3-year-old trees are in the early stage of growth and development, which makes the research results more remarkable (Xu et al., 2008;Li et al., 2018). Pinus bungeana, Platycladus orientalis, Koelreuteria paniculata and Ginkgo biloba are common landscaping tree species in Beijing. The area of Platycladus orientalis ranks second among all tree species in Beijing, and Pinus bungeana, as a pine, has strong ability to absorb pollutants and is insensitive to O3 Koelreuteria paniculata and Ginkgo biloba are sensitive to O3. Choosing these tree species can highlight the results of plant growth and physiological characteristics under different O3 concentrations. The flower pot for seedlings was 40cm in diameter and 50cm in height. Seedling size, crown width, basal diameter and plant height of Pinus bungeana, Platycladus orientalis, Koelreuteria paniculata and Ginkgo biloba in different chambers were basically the same. The plant heights of Pinus bungeana, Platycladus orientalis, Koelreuteria paniculata and Ginkgo biloba were about 1.3m, 1.35m and 1.4m, respectively.

Measurement of plant growth status
Potted seedlings of four kinds of trees were distributed and placed in three OTCs, and three trees were placed in each air chamber, 36 test trees in total; There was little difference among different seedlings. The height and diameter at 50cm of each seedling in different OTCs were measured with diameter at breast height ruler at the beginning. After fumigating with O3 for 14 months, the plant height and diameter at 50cm of each seedling in different OTCs were measured again, and the changes in plant height and diameter at 50cm of each seedling under different O3 concentrations were calculated.

Observation of damage degree of leaves
Observe the leaves of plants and record the change degree of leaf color. Grading was carried out according to the size, quantity and greening degree of the leaf damaged spots, which were observed once every two weeks and recorded, and the injury grade was made according to this (Liu et al., 2015).  Figure 1, and different tree species showed the phenomenon of inhibiting tree growth when O3 concentration rises. Under the control of different O3 concentrations NF, NF40 and NF80, at the beginning of the experiment, the plant height of Pinus bungeana trees were 1.30m, 1.33m and 1.31m respectively, and at the end of the experiment, they were 1.36m, 1.34m and 1.32 m respectively. During the whole experiment, the plant height of Pinus bungeana increased by 0.06m under NF O3 concentration, but only increased by 0.01 m under NF40 and NF80 O3 concentration. Under NF O3 concentration, the plant height of Platycladus orientalis increased from 1.36m to 1.41m, which increased by 0.05m, while under NF40 and NF80 O3 concentration, the plant height only increased by 0.03m and 0.01 m; the plant height of Koelreuteria paniculata increased by 0.06m under NF O3 concentration, but only by 0.02 m under NF40 and NF80 O3 concentration. The plant height of Ginkgo biloba increased by 0.06m under NF O3 concentration, but only by 0.03 m and 0.02m under NF40 and NF80 O3 concentration. It can be seen that the plant height of different tree species increased at NF O3 concentration, ranging from 0.05 m to 0.06 m, while that of different tree species also increased at NF40 O3 concentration, but the range of increase was very limited, with the added value ranging from 0.01 m to 0.03 m. Under NF80 O3 concentration, the plant height of different tree species increased even less, only by 0.01 m to 0.02 m; This shows that the increase of O3 concentration is not conducive to the growth of plants, and can inhibit the growth of trees in a certain range. From different tree species, the inhibition of O3 concentration on trees is not obvious, but there are significant differences among tree species (Table 1, P < 0.05).   Table 1 is the variance analysis of plant height of trees with different O3 concentrations. After repeated two-factor analysis, the results showed that the difference of plant height of trees with different O3 concentrations was statistically significant (P < 0.05). The values of F-crit among tree species and O3 concentrations were 3.74 and 2.76 respectively, which were lower than the F values of 4.87 and 39.11 respectively. This showed that the changes of plant height of trees with different O3 concentrations were significant among tree species and O3 concentrations.  Table 3 is the variance analysis of diameter at 50cm of trees with different O3 concentrations. After repeated two-factor analysis, the results show that the diameter at 50cm of trees with different O3 concentrations has no statistical significance among tree species (P=0.08, P > 0.05), but has statistical significance among O3 concentrations (P=0.001, P < 0.05), and the F-crit value is between tree species and O3 concentrations. The F-crit is larger than the f value (4.42) among tree species and smaller than the f value (18.98) among O3 concentrations, which indicates that the diameter change at 50cm of trees with different O3 concentrations is not significant among tree species, but significant among O3 concentrations, which also indicates that the increase of O3 concentration has little effect on the diameter change among different tree species.

Characteristics of leaves damage with different O3 concentrations
The change characteristics of tree leaves with different O3 concentrations are shown in Figure 3. The leaves of different tree species were in good condition under NF O3 concentration, and the leaves did not have any injury symptoms. With the increase of O3 concentration, leaves withered and died, leaves wilted under NF40 O3 concentration, and leaves wilted, died and fallen under NF80 O3 concentration.
The changes of leaf injury characteristics of trees with different O3 concentrations under manual control are shown in Table 3  and death, and the leaves basically grow normally, and the leaf injury rates were equal to level 0 , all of which were in the level of no injury. Under the O3 concentration of NF40, the leaves of different tree species began to turn yellow and wilted, especially in Koelreuteria paniculata and Ginkgo biloba. The injury rates of Pinus bungeana and Platycladus orientalis were 1% and 10% respectively, and the injury grades were all Grade 1. The leaf injury rates of Koelreuteria paniculata and Ginkgo biloba were 78% and 53%, and the injury grades were 5 and 3 respectively. Under NF80 O3 concentration, a large number of tree species died, especially Koelreuteria paniculata, and Pinus bungeana and Platycladus orientalis were relatively light. The leaf injury rates of Pinus bungeana and Platycladus orientalis were 8% and 16% respectively, and the injury grades were grade 1 and 2 respectively. The leaf injury rates of Koelreuteria paniculata and Ginkgo biloba were 92% and 76%, respectively, and the injury grades were 5. This showed that Pinus bungeana and Platycladus orientalis have stronger ability to resist O3 pollution, and Pinus bungeana is stronger than Platycladus orientalis; Koelreuteria paniculata and Ginkgo biloba have weak ability to resist O3 pollution, and Koelreuteria paniculata has the weakest ability to resist O3 pollution.  The leaves of the whole plant wither in a large area, and the degree of etiolation was further serious 76 5

Discussion
High concentration O3 stress inhibited the growth of trees, resulting in the decline of tree biomass and the change of distribution ratio between aboveground and underground parts (Niu, 2012;Wittig et al., 2009). Under the control of atmospheric carbon dioxide, one-year-old hybrid poplar (Populus tremula×P. tremuloides) has been growing for 2 years at 1.5 times of atmospheric O3 concentration. The results show that the height of one-year-old experimental seedlings has decreased by 20% and 17%, respectively, and the stem dry weight has decreased by 5%, and the root dry weight has decreased by 20% (Niu, 2012;Häikiö et al., 2007); The leaf area growth of Betula pendula seedlings decreased by 36%, and the plant height and basal diameter growth decreased by 15% and 20%, respectively, under the treatment of 1.4-1.7 times atmospheric O3 concentration for six consecutive growth seasons (May 1996-October 2001) (Niu, 2012;Oksanen, 2003); The research on 3-year-old Pinus sylvestris seedlings also showed that the basal diameter growth, stem dry weight accumulation and root dry weight accumulation of the experimental seedlings decreased by 9%, 17% and 19%, respectively, under 1.5-1.6 times atmospheric O3 concentration (Niu, 2012;Utriainen and Holopainen, 2001). Meta-analysis results show that the current near-surface O3 (40 ppb) causes the ratio of underground biomass to aboveground biomass of forest tree species to decrease by about 3% in the middle and high latitudes of the northern hemisphere (Wittig et al., 2009). This study also shows that the increase of O3 slows down the growth rate of plant height and diameter of Pinus bungeana and Koelreuteria paniculata, and plants grow faster under normal environmental O3 concentration. There are differences in the growth and slowing down speed of different tree species diameters, which is due to the different sensitivity of different plants to O3, and the sensitivity is influenced by the plant characteristics and environmental factors (Zhang, et al., 2014;Xu, et al., 2007), which is consistent with the research results of Wan et al. (2013).
The increase of O3 concentration can inhibit the growth of trees, and also damage the leaves of trees. The visible injury symptoms of leaves are caused by O3 entering into tissue cells through the open stomata of the lower epidermis of leaves and reacting with substances on the cell wall and plasma membrane (Fu, et al., 2014;Karnosky D F et al.,2007). When plant leaves are damaged by O3, the most typical feature is that some yellow-brown or brown spots appear on local leaves (Donnelly et al, 2001;Fowler et al, 2008), some of these patches are yellow-brown and some are brown, and different tree species show different color patches. With the growth of plants with high O3 concentration, the symptoms of leaf injury will gradually spread to the whole leaf, which will lead to an increase in the number of epidermal necrotic plaques and the area of injured plaques, until the necrotic plaques cover the upper surface of the whole leaf, but the lower surface of the leaves is not affected much, and everything is normal. If the plants are exposed to high O3 concentration for a long time, the leaves of the whole tree will lose water in a large area until they die, eventually causing the leaves to fall off. Liu et al. (2015) set up three gradient O3 observation chambers, and found that under the condition of high concentration O3 (200 ppb-300 ppb), the old leaves were damaged first, then the mature leaves, and the new leaves were less damaged; At the same time, it was found that Pinus tabulaeformis was basically harmless at O3 concentration of 200ppb, but slightly damaged at O3 concentration of 300ppb, which indicated that Pinus tabulaeformis had strong O3 resistance, which was consistent with the result that Pinus bungeana, as the representative of conifers, was lightly damaged by O3 in this study. In European studies, it was found that with the increase of O3 concentration, more than half of the 65 species showed wilting leaves, yellow leaves and withering in severe cases, but conifers had strong tolerance and low sensitivity to injury (Lie et al., 2014;Timonen et al.,2004); In this study, the leaf injury rates of Pinus bungeana were 1% and 8% under the O3 concentration of NF40 and NF80, respectively, both of which were grade 1, which indicated that the leaf injury degree of Pinus bungeana was low and proved the strong O3 tolerance of conifers. The leaf injury rates of Koelreuteria paniculata under the O3 concentrations of NF40 and NF80 are 78% and 92%, respectively, both of which are of Grade 5, indicating that Koelreuteria paniculata is extremely sensitive to O3 and has poor tolerance to O3, which is determined by the characteristics of tree species. As a broad-leaved tree species, Koelreuteria paniculata grows faster and has a higher O3 absorption rate than conifers such as Pinus bungeana (Mannines et al., 2003), so it is sensitive to O3.

Conclusion
Different tree species inhibit tree growth when O3 concentration increases. Plant height and diameter at 50cm increase slowly when O3 concentration increases, and there is no significant difference in diameter between different tree species when O3 concentration increases. Under the concentration of NF O3, the leaves of different tree species all performed well without any injury symptoms. With the increase of O3 concentration, leaves withered and died, leaves wilted under NF40 O3 concentration, and leaves wilted, died and fallen under NF80 O3 concentration. Pinus bungeana and Platycladus orientalis have stronger ability to resist O3 pollution, while Koelreuteria paniculata and Ginkgo biloba have weaker ability to resist O3 pollution. The damage of O3 concentration to plants is more sensitive to broad-leaved trees.