Method for evaluating sensitivity of Chinese herbaceous peony variety to ozone stress

Abstract

The invention discloses a method for evaluating sensitivity of peony varieties to ozone stress. The method comprises the steps of peony planting, ozone stress treatment and index determination. According to the method, responses of key indexes such as leaf morphological structure, photosynthetic capacity, photosynthetic pigment attenuation, membrane lipid peroxidation degree and antioxidant enzyme activity are integrated, a multi-index collaborative evaluation model is established, and data processing and sensitivity evaluation are carried out. In view of the problems of long growth cycle and lagging of ozone sensitive phenotype of paeonia lactiflora, a sensitivity grading standard based on a leaf morphological structure in a leaf expansion period is provided, the adaptability of a variety in an ozone polluted environment can be predicted in the early planting stage, the breeding screening cycle is shortened, and an evaluation result obtained by the method is rapid and stable and has good application prospects. And the method has scientificity and accuracy, provides reference for the breeding direction and planting planning of the Chinese herbaceous peony, reduces the use of chemical protective agents, and promotes the implementation of an environment-friendly cultivation mode.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant identification, and particularly relates to a method for evaluating the sensitivity of peony varieties to ozone stress. Background Art

[0002] Ground-level ozone is mainly formed by the photochemical reaction of primary pollutants such as nitrogen oxides (NO X ) and volatile organic compounds (VOC S ). With the rapid development of industry and the rapid increase in the number of motor vehicles, the concentration of NO X is continuously increasing, which in turn leads to the increasing concentration of ozone in the air. According to environmental monitoring data, the average value of the 8-hour daily ozone concentration in the near-surface layer of many cities in China has exceeded 50 ppb, and the ozone peak is still increasing at a rate of 3 ppb per year, and the maximum hourly average value even exceeds 100 ppb. The continuously increasing ground-level ozone is bound to cause serious harm to human health, agricultural production and biodiversity.

[0003] Peony (Paeonia lactiflora) is a traditional famous flower in China, with ornamental, medicinal and health care values, and has become a new cut flower in the domestic and international markets, with remarkable economic benefits. Regarding the response of peony growth and development to abiotic stress, a large number of previous studies have sorted out the effects of these abiotic stress factors such as heavy metals, salinity, drought, waterlogging, low temperature, high temperature and weak light on the external morphology, internal physiological and biochemical properties and the content of secondary metabolites of peony, and clarified the physiological mechanism of peony to adapt to abiotic stress factors to improve its stress resistance, providing a theoretical reference for the efficient cultivation of peony. However, different from other abiotic stress factors, the seasonal change of ozone pollution makes the growth and development of some plants particularly vulnerable, because high ozone events often occur during their critical growth periods. Unfortunately, the main production areas of peony in China are also areas with frequent high-concentration ozone pollution, especially the key growth and flowering periods of peony (May) are basically synchronized with the peak period of ozone pollution in the main production areas of peony. Regrettably, although a large number of studies have explored the negative effects of ozone on crops or woody plants, there has been no report on the impact of ozone on horticultural plants, especially on peony, and the evaluation of its tolerance.

[0004] Plant leaves are the interface for material and energy exchange between the surface atmosphere and the terrestrial biosphere, and are also the initial sensing organs for the response to ozone stress. Ozone mainly enters the plant body through stomata. Sensitive plants exposed to ozone for a long time will show apparent visible leaf damage symptoms, trigger various defense and protection mechanisms, then the leaf color gradually fades and is accompanied by a decrease in photosynthetic rate, change the absorption of carbon and mineral nutrients in the body and cause their redistribution, resulting in slow growth and premature senescence, and further weaken the stress resistance, competitiveness and adaptability of plants, and ultimately reduce the carbon sequestration potential. A large number of studies have shown that there are significant differences in the sensitivity of different plant species or different varieties of the same plant species to ozone.

[0005] The sensitivity of crops to ozone is often measured by changes in commercial organs (such as yield, biomass) as the core indicator of its sensitivity. However, for woody and horticultural plants, the progress in the breeding of ozone-resistant varieties is relatively slow at present. Therefore, for peonies, it is necessary to explore the effects of ozone stress on peonies and their genotype differences, and clarify the indicator indicators of peony sensitivity to ozone, so as to select suitable peony varieties for popularization and planting to reduce the damage of ozone pollution. Summary of the Invention

[0006] The purpose of the present invention is to propose a method for evaluating the sensitivity of peony varieties to ozone stress, aiming to solve the deficiencies of the existing methods for evaluating the sensitivity of peony varieties to ozone stress, and provide a rapid and standardized evaluation method.

[0007] This method first clarifies the evaluation of the ozone tolerance of peony varieties. By integrating the responses of key indicators such as leaf morphological structure, photosynthetic capacity, photosynthetic pigment attenuation, degree of membrane lipid peroxidation and antioxidant enzyme activity, a multi-index collaborative evaluation model is established, which significantly improves the scientificity and accuracy of the results; the present invention can achieve early and rapid identification. In view of the problems of the long growth cycle of peonies and the lag of ozone-sensitive phenotypes, a sensitivity grading standard based on the leaf morphological structure at the leaf expansion stage is proposed, which can predict the adaptability of varieties in an ozone-polluted environment at the initial stage of planting and shorten the breeding screening cycle; the present invention also provides the breeding of resistant varieties and ecological layout. By accurately distinguishing high, medium and low sensitivity peony varieties, it provides a reference for the breeding direction and planting plan of peonies, and at the same time provides a popularizable method and approach for the study of peony resistance to ozone stress, reduces the use of chemical protectants, and promotes the implementation of an environmentally friendly cultivation mode.

[0008] The purpose of the present invention is achieved in the following way:

[0009] A method for evaluating the sensitivity of peony varieties to ozone stress, the method comprising the following steps:

[0010] Step 1: Select 2 - 3 - year - old peony seedlings of multiple varieties that are strong and disease - free, and plant them in pots. The substrate of the cultivation pots is a mixture of soil, peat, and coarse sand in a ratio of 1:1:1, with 10 - 18 plants of each variety.

[0011] Step 2: For ozone stress treatment, use open - top chambers (OTCs) for ozone fumigation. The daily ozone stress treatment time is 10 h, and the total stress treatment time is 45 - 55 days. Ozone treatment settings: Using the ozone background concentration in natural atmosphere as a control, ozone stress treatment is carried out by adding 60 ppb to the atmospheric background ozone concentration. For each ozone stress treatment, set 3 OTC replicates, and use a 49i ozone analyzer in each OTC to monitor the ozone concentration at the center position of the chamber and the plant canopy height in real - time.

[0012] Step 3: Measure the photosynthetic characteristic indexes of the upper leaves of peonies, then take the upper leaves to analyze the stomatal traits and specific leaf weight of the leaves. Take another part of the upper leaves, wrap them in aluminum foil, and immerse them in liquid nitrogen to measure the antioxidant physiological parameters.

[0013] Step 4: Evaluate the sensitivity of peony varieties to ozone stress through cluster analysis and correlation analysis methods.

[0014] Preferably, in step 2, the environmental factor values in each OTC are close during the experiment.

[0015] Preferably, in step 2, the total ozone stress treatment time is 50 days.

[0016] Preferably, in step 3, the photosynthetic characteristic indexes of the leaves are one or more of specific leaf weight of the leaves, stomatal characteristics of the leaves, and photosynthetic ability of the leaves.

[0017] Preferably, in step 3, the antioxidant physiological parameters are one or more of the photosynthetic pigment content of the leaves, malondialdehyde, total antioxidant reduction ability, and antioxidant enzyme activity.

[0018] Preferably, in step 4, through cluster analysis and correlation analysis methods, it is evaluated that the responses of indexes such as dry weight per unit area of peony leaves, malondialdehyde, total antioxidant reduction ability, catalase, and superoxide dismutase activity to ozone stress are closely related to the change of net photosynthetic rate of the leaves.

[0019] Therefore, the tolerance of different peony varieties to ozone can be evaluated by the change of specific leaf weight in leaf morphological traits, which can directly guide production practice.

[0020] In view of the increasingly serious trend of ozone pollution in the current peony planting areas (such as Shandong, Jiangsu, Sichuan, Beijing - Tianjin - Hebei region, etc.), the present invention provides a method for evaluating the sensitivity of peony varieties to ozone stress.

[0021] The beneficial effects of the method of the present invention compared with the prior art are reflected in: ① High scientificity and accuracy: By quantifying key indicators such as the morphological structure, photosynthetic capacity, photosynthetic pigment content (chlorophyll a, b, and carotenoids), membrane lipid peroxidation product (MDA), and antioxidant enzyme activities (SOD, CAT, APX) of peony leaves under ozone stress, and combining correlation analysis and clustering algorithms, it is found that the change in leaf specific leaf weight can be used to quickly evaluate the tolerance of peony varieties to ozone, significantly improving the objectivity and reliability of the evaluation results; ② Standardized operation and strong applicability: After clearly specifying the ozone stress concentration (60 ppb higher than the ozone background concentration in the natural atmospheric environment) and the treatment duration of 50 days, sampling and analysis are carried out, which is applicable to the horizontal comparison of different peony varieties, better avoiding the problem of incomparable data caused by inconsistent experimental conditions; ③ Value of early warning and variety screening: This method can quickly identify the sensitivity of varieties at the early growth stage of peonies (such as the leaf expansion stage), providing technical support for the breeding of ozone-resistant varieties and the cultivation layout in urban polluted areas, reducing the risk of loss of ornamental and medicinal values caused by ozone pollution; ④ Low cost and easy to promote: The required instruments (such as ovens, balances, scanners, etc.) are conventional laboratory equipment, without the need for complex molecular biology techniques, suitable for large-scale evaluation of variety resources, especially beneficial for grass-roots peony planting enterprises to apply; ⑤ Significant ecological benefits: By accurately screening resistant varieties, the input of pesticides and protective measures in areas with severe ozone pollution can be reduced, promoting the ecological cultivation of peonies, meeting the needs of the development of green agriculture. Brief Description of the Drawings

[0022] Figure 1 It is the effect of ozone stress on the net photosynthetic rate (Pn) of peony leaves with different sensitivity types.

[0023] In the figure, NF is the ozone concentration in the natural environment atmosphere, and NF60 is the ozone concentration in the natural environment atmosphere + 60 ppb; S, MR, and HR in the figure respectively represent highly sensitive varieties ('Rose Red', 'Purple Orchid', 'Big Red Building', and 'Red Building Brocade Chrysanthemum'), moderately sensitive varieties ('Purple Hibiscus', 'Moon Fairy', 'Peach and Plum Glamour', 'Silver Edge Red Pavilion', 'Pink Face Peach Blossom', 'All Red', 'Spring Dawn', and 'Great Wealth'), and low-sensitive varieties ('Coral Multiform', 'Yang Guifei Out of the Bath', and 'Black Sea Waves') of peony varieties to ozone stress; ** and * in the figure respectively indicate that the differences between treatments are significant at the P < 0.01 and P < 0.05 levels.

[0024] Figure 2 It is the effect of ozone stress on the leaf specific leaf weight (LMA) of peony leaves with different sensitivity types.

[0025] In the figure, NF represents the natural environmental atmospheric ozone concentration, and NF60 represents the natural environmental atmospheric ozone concentration + 60 ppb; in the figure, S, MR, and HR respectively represent highly ozone-stress sensitive peony varieties ('Rose Red', 'Purple Orchid', 'Grand Red Building', and 'Red Building Brocade Chrysanthemum'), moderately sensitive varieties ('Purple Hibiscus', 'Moon Fairy', 'Peach and Plum Glamour', 'Silver Edge Red Pavilion', 'Pink Face Peach Blossom', 'All Red', 'Spring Dawn', and 'Great Wealth'), and low-sensitivity varieties ('Coral Graceful', 'Yang Guifei Out of the Bath', and 'Black Sea Waves'); in the figure, **, *, and ns respectively indicate that there are significant differences among treatments at the P<0.01, P<0.05, and P>0.1 levels.

[0026] Figure 3 It is the correlation analysis of the responses of net photosynthetic rate (Pn), specific leaf weight (LMA), malondialdehyde (MDA), total antioxidant reduction capacity (TOC), superoxide dismutase (SOD), and catalase (CAT) in peony leaves to ozone stress (n = 15); in the figure, * indicates that there is a significant linear correlation at the P<0.05 level between the two parameters. Detailed implementation method

[0027] The preferred embodiments of the present invention will be described in more detail below. Although the preferred embodiments of the present invention are described below, it should be understood that it can be implemented in various forms and should not be limited by the embodiments set forth herein. These embodiments are provided to make the present invention more thorough and complete, and to fully convey the scope of the present invention to those skilled in the art.

[0028] Example 1

[0029] Step 1: Preparation of test materials: For the tested peony varieties, select 2-year-old peony seedlings that are robust and free from diseases and pests. The varieties include 'Great Wealth', 'Black Sea Waves', 'Grand Red Building', 'Purple Orchid', 'Rose Red', 'Spring Dawn', 'Silver Edge Red Pavilion', 'Pink Face Peach Blossom', 'Red Building Brocade Chrysanthemum', 'Moon Fairy', 'Purple Hibiscus', 'All Red', 'Peach and Plum Glamour', 'Coral Graceful', and 'Yang Guifei Out of the Bath', with 10 - 18 plants per variety. The test materials are 15 peony varieties, all of which were divided in 2023 and placed in potted plants. The cultivation pots have a bottom diameter of 18 cm, a top diameter of 25 cm, and a height of 27.5 cm. The substrate is mixed in a ratio of soil, peat, and coarse sand of 1:1:1.

[0030] Step 2: Ozone stress treatment: Use an open-top chamber (OTC) for ozone fumigation treatment. The OTC is a regular octagonal column structure with a diameter of 4.8 m and a height of 2.3 m, and there is an opening with a diameter of 3 m at the top. Ozone treatment settings: NF (natural environmental atmospheric ozone concentration) and NF60 (natural environmental atmospheric ozone concentration + 60 ppb), and each ozone treatment has 3 OTC replicates.

[0031] Ozone stress treatment started on April 11, 2024 and ended on May 30. For the NF60 treatment, fumigation was carried out for 10 hours per day (8:00 - 18:00). In each OTC, a 49i ozone analyzer (Thermo Scientific, USA) was used to continuously monitor the ozone concentration at the central position of all OTC chambers and at the plant canopy height. For the OTCs with high-concentration ozone treatment, the ozone concentration in the chamber was controlled by adjusting the oxygen flow rate supplied through a mass flow meter according to the difference between the monitored ozone concentration and the target ozone treatment concentration. During the experiment, the environmental factors (temperature, humidity, etc.) in each OTC were basically the same.

[0032] Step 3: Index determination. ① Specific leaf weight: First, collect the upper 3 leaves from three single stems of each plant. Use ImageJ software to measure the leaf surface area. Then put the leaves together into a paper bag, dry them at 105°C for 30 minutes, and then at 80°C for 72 hours, and weigh them using a thousandth electronic balance. Calculate the dry weight per unit area of the leaf (specific leaf weight) based on the leaf area and dry weight. ② Leaf stomatal characteristics: Gently remove the dust on the leaf surface, and then apply a layer of transparent nail polish to an area of 1 cm × 1 cm on the back of the leaf. After drying, lift the film with transparent tape and stick it on a microscope slide with the impression side facing up. Observe using a Leica DM 2500 microscope, and calculate the stomatal density by counting the number of stomata in 5 randomly selected representative areas. Measure 10 randomly selected stomata for each sample to determine their length and width, and calculate the stomatal size. ③ Leaf photosynthetic capacity: Use a portable photosynthesis system (LI-6800, LI-COR, Inc., Lincoln, NE, USA) to measure gas exchange parameters. Measure the light-saturated net photosynthetic rate, stomatal conductance, and intercellular CO2 concentration of the upper leaves. All measurements were carried out between 8:30 and 11:30 in the morning on sunny days, and the photosynthetically active radiation saturation level was set at 1200 μmol m -2 ·s -1, the CO2 concentration was 400 ppm, the leaf temperature was 28 °C, and the relative humidity was 50 - 60%. ④ Determination of antioxidant physiological parameters: Cut the upper 3 leaves of a single peony stem and immediately wrap them in aluminum foil and immerse them in liquid nitrogen for later use. After grinding into powder with a cryogenic grinder (MM400, Retsch, Arzberg, Germany), measure the photosynthetic pigment content (chlorophyll a, chlorophyll b, chlorophyll a + b, carotenoids), malondialdehyde, total antioxidant reducing capacity, and antioxidant enzyme activities (SOD, APX, CAT) in the leaves. The specific methods for the above measured parameters are detailed in: Shao, Z.S., Zhang, Y.L., Hu, S.W., Jing, L.Q., Wang, Y.X., Wang, Y.L., Yang, L.X. Identification of key responsive leaf traits for ozone tolerance in six modern indica and japonica rice cultivars (Oryza sativa L.) over two years (Eastern China). Agric. Ecosyst. Environ. 2023, 349, 108451.

[0033] The index data measured by the above various methods are as follows:

[0034] Table 1 Photosynthetic rate, stomatal conductance, and intercellular CO2 concentration of leaves of 15 peony cultivars under non-filtered natural air (NF) and elevated ozone concentration of 60 ppb (NF60)

[0035]

[0036]

[0037] Note: In the figure, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, and C15 represent the peony cultivars 'Rose Red', 'Purple Orchid', 'Big Red Building', 'Red Building Brocade Chrysanthemum', 'Purple Hibiscus', 'Moon Fairy', 'Peach and Plum in Splendid Attire', 'Silver Edge Red Pavilion', 'Pink-Faced Peach Blossom', 'All-Red Hall', 'Spring Dawn', 'Great Wealth', 'Coral in Many Postures', 'Yang Guifei Emerging from the Bath', and 'Black Sea Waves' respectively.

[0038] Table 2 Specific leaf weight, stomatal density, and stomatal size of leaves of 15 peony cultivars under non-filtered natural air (NF) and elevated ozone concentration of 60 ppb (NF60)

[0039]

[0040] Note: In the figure, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, and C15 represent the peony cultivars 'Rose Red', 'Purple Orchid', 'Big Red Mansion', 'Red Mansion Brocade Chrysanthemum', 'Purple Hibiscus', 'Moon Fairy', 'Peach and Plum in Splendid Attire', 'Silver-Edged Red Pavilion', 'Pink-Faced Peach Blossom', 'All-Red', 'Dawn Blossom', 'Great Wealth', 'Coral Graceful', 'Yang Guifei Out of the Bath', and 'Black Sea Waves', respectively.

[0041] Table 3 Contents of chlorophyll b, chlorophyll a, and chlorophyll b in leaves of 15 peony cultivars under non-filtered natural air (NF) and elevated ozone concentration of 60 ppb (NF60)

[0042]

[0043]

[0044] Note: In the figure, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, and C15 represent the peony cultivars 'Rose Red', 'Purple Orchid', 'Big Red Mansion', 'Red Mansion Brocade Chrysanthemum', 'Purple Hibiscus', 'Moon Fairy', 'Peach and Plum in Splendid Attire', 'Silver-Edged Red Pavilion', 'Pink-Faced Peach Blossom', 'All-Red', 'Dawn Blossom', 'Great Wealth', 'Coral Graceful', 'Yang Guifei Out of the Bath', and 'Black Sea Waves', respectively.

[0045] Table 4 Contents of malondialdehyde (MDA), total antioxidant reducing capacity (TOC), and carotenoids in leaves of 15 peony cultivars under non-filtered natural air (NF) and elevated ozone concentration of 60 ppb (NF60)

[0046]

[0047] Note: In the figure, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, and C15 represent the peony cultivars 'Rose Red', 'Purple Orchid', 'Big Red Mansion', 'Red Mansion Brocade Chrysanthemum', 'Purple Hibiscus', 'Moon Fairy', 'Peach and Plum in Splendid Attire', 'Silver-Edged Red Pavilion', 'Pink-Faced Peach Blossom', 'All-Red', 'Dawn Blossom', 'Great Wealth', 'Coral Graceful', 'Yang Guifei Out of the Bath', and 'Black Sea Waves', respectively.

[0048] Table 5 Activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in leaves of 15 peony cultivars under non-filtered natural air (NF) and elevated ozone concentration of 60 ppb (NF60)

[0049]

[0050]

[0051] Note: In the figure, C1, C2, C3, C4, C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, and C15 represent the peony cultivars 'Rose Red', 'Purple Orchid', 'Grand Red Mansion', 'Red Mansion Brocade Chrysanthemum', 'Purple Hibiscus', 'Moon Fairy', 'Peach and Plum in Colorful Makeup', 'Silver-edged Red Pavilion', 'Pink-faced Peach Blossom', 'All-round Red', 'Dawn', 'Great Wealth', 'Coral Graceful', 'Yang Guifei Out of the Bath', and 'Black Sea Waves' respectively.

[0052] Step 4: Data processing and sensitivity evaluation. Photosynthesis of plant leaves is an important indicator to measure its growth and development response under stress conditions. In this invention, the dynamic clustering method of the within-group least squares sum is adopted. According to the response of the photosynthetic rate of the leaves of 15 peony cultivars to ozone stress from large to small, they are divided into three categories: highly sensitive type (peony cultivars are: 'Rose Red', 'Purple Orchid', 'Grand Red Mansion', and 'Red Mansion Brocade Chrysanthemum'), moderately sensitive type (peony cultivars are: 'Purple Hibiscus', 'Moon Fairy', 'Peach and Plum in Colorful Makeup', 'Silver-edged Red Pavilion', 'Pink-faced Peach Blossom', 'All-round Red', 'Dawn', and 'Great Wealth'), and low-sensitive type (peony cultivars are: 'Coral Graceful', 'Yang Guifei Out of the Bath', and 'Black Sea Waves'), which are represented by S, MR, and HR respectively. It can be seen that Figure 1 compared with NF, the NF60 treatment reduced the average photosynthetic rate of the leaves of all peony cultivars by 37.2%. Among them, S, MR, and HR decreased by 52.6% ** (the cultivar variation range is 45.9 - 66.4%), 35.3% ** (the cultivar variation range is 25.4 - 42.0%), and 21.5% * (the cultivar variation range is 20.6 - 22.4%), with the maximum difference reaching 3 times. Further correlation analysis found that the responses of the dry weight per unit area of peony leaves (specific leaf weight, LMA), malondialdehyde (MDA), total antioxidant reduction capacity (TOC), peroxidase activity (CAT), and superoxide dismutase activity (SOD) to ozone stress all had significant linear correlation relationships with the changes in the net photosynthetic rate of the leaves, and the correlation coefficients were 0.835 ** 、-0.456 + 、-0.875 ** 、-0.635 * and -0.613 * . The study on the LMA of the leaves found that ( Figure 2 ) the ozone treatment reduced the average LMA of all peony cultivars by 12.6%. Among them, S, MR, and HR decreased by 17.9% **(The varietal variation range is 13.5 - 22.6%) and 12.1% * (The varietal variation range is 9.4 - 13.4%) and 7.0% ns (The varietal variation range is 5.6 - 8.2%), and there is also a close correlation between LMA and the above - mentioned leaf physiology, enzyme activity and other parameters ( Figure 3 ). Based on this, combined with the advantages of simple and rapid analysis method of specific leaf weight, it can accurately obtain the sensitivity of peony varieties to ozone stress by the change of leaf specific leaf weight.

[0053] The above - mentioned examples prove that the tolerance of different peony varieties to ozone can be evaluated by the change of the easily measured index of specific leaf weight, and then directly guide the production practice. The symbols **, *, +, ns in the upper right of the numbers in the text respectively indicate the difference levels at P < 0.01, P < 0.05, P < 0.1, P > 0.1 levels among the treatments.

[0054] The above dynamic clustering method refers to: Gu Shiliang, Mo Huidong. A new method of dynamic clustering - the method of the smallest within - group sum of squares. Journal of Jiangsu Agricultural College, 1989, 10(4): 1 - 8.

Claims

1. A method for evaluating the sensitivity of peony varieties to ozone stress, characterized in that The method comprises the following steps: Step 1: Select several varieties of healthy, pest-free 2-3 year old peony seedlings and plant them in pots. The potting medium is a mixture of soil, peat and coarse sand in a ratio of 1:1:1, with 10-18 plants of each variety. Step 2: Ozone stress treatment uses an open-top air chamber for ozone fumigation treatment. The ozone stress treatment time is 10 hours per day, and the total stress treatment time is 45-55 days. Ozone treatment settings: The ozone background concentration in the natural atmosphere is used as a control, and the atmospheric background ozone concentration + 60ppb is used for ozone stress treatment. Each ozone stress treatment is set up with 3 OTC replicates. In each OTC, a 49i ozone analyzer is used to monitor the ozone concentration at the center of the air chamber and the height of the plant canopy in real time. Step 3: Determine the photosynthetic characteristic index of the upper leaves of peony, then take the upper leaves to analyze the stomatal characteristics and specific leaf weight of the leaves, take another upper leaf and wrap it in aluminum foil and immerse it in liquid nitrogen to determine the antioxidant physiological parameters; Step 4: Evaluate the sensitivity of peony varieties to ozone stress through cluster analysis and correlation analysis methods.

2. The method according to claim 1, characterized in that: In step 2, the internal environmental factors of each OTC are close during the test.

3. The method according to claim 1, characterized in that: In step 2, the total stress treatment time is 50 days.

4. The method according to claim 1, characterized in that: In step 3, the leaf photosynthetic characteristic index is one or more of leaf specific weight, leaf stomatal characteristics, and leaf photosynthetic capacity.

5. The method according to claim 1, characterized in that: In step 3, the antioxidant physiological parameter is one or more of leaf photosynthetic pigment content, malondialdehyde, total antioxidant reduction capacity and antioxidant enzyme activity.

6. The method according to claim 1, characterized in that: In step 4, cluster analysis and correlation analysis are used to evaluate the response of peony leaf dry weight per unit area, malondialdehyde, total antioxidant reduction capacity, catalase and superoxide dismutase activity indicators to ozone stress, which is closely related to the change of leaf net photosynthetic rate.

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