Influence of melatonin on physiological characteristics of citrus seedlings under drought stress and application of melatonin

Abstract

The invention relates to the field of fruit tree cultivation and treatment, in particular to influence of melatonin on physiological characteristics of citrus seedlings under drought stress and application thereof.The citrus seedlings are selected, melatonin spraying liquid is used for spraying the citrus seedlings of an experimental group at dusk every day, spraying is carried out every two days, spraying is carried out five times in total, and drought stress is carried out again after spraying. The soil humidity is controlled to be 40%-50%, then melatonin spraying liquid is sprayed again, leaves are covered with vaporific liquid drops without dripping during spraying, experimental data are highlighted, and a control group and a control group citrus seedling (normal water supply and distilled water spraying on leaf surfaces) are set at the same time; and observing the influence data of the melatonin on the citrus seedlings under drought stress. Experiments prove that the exogenous melatonin significantly reduces the MDA content of citrus leaves, increases the content of penetration regulation and control substances such as proline, soluble sugar and soluble protein, and enhances the activity of antioxidant enzyme, thereby effectively reducing the damage of drought stress to citrus plants.

Description

Technical Field

[0001] The invention relates to the field of citrus cultivation and management, and in particular to the influence and application of melatonin on the physiological characteristics of citrus seedlings under drought stress. Background Art

[0002] Under drought stress, citrus seedlings are in a drought environment, and the soil water supply is insufficient, and the plants cannot obtain enough water from the soil to maintain normal physiological metabolism. The relative water content (RWC) of leaves will gradually decrease as the degree of drought stress deepens. For example, under mild drought stress, the relative water content of leaves may drop from the normal 80%-90% to about 70%; under severe drought stress, it may drop to 50% or even lower. This is because the rate of water loss is greater than the rate of absorption, and the water content in the cells gradually decreases, causing the leaves to wilt, affecting the normal physiological functions of the leaves, such as photosynthesis and respiration. Under drought stress, the water potential of citrus seedlings will drop significantly. Under normal circumstances, the water potential of citrus seedlings may be between -0.2 and -0.5MPa, while under drought stress, the water potential may drop to -1.0MPa or even lower. The decrease in water potential makes it more difficult for plants to absorb water from the soil, because the water potential difference between the soil and the plant decreases, and the driving force of water is weakened.

[0003] Drought stress can also affect the content of photosynthetic pigments (chlorophyll a, chlorophyll b and carotenoids) in citrus seedlings. In mild drought, the chlorophyll content may increase slightly, which may be an adaptive response of plants to drought stress, increasing the chlorophyll content to improve the ability to capture light energy. However, as the degree of drought stress deepens, the chlorophyll content will gradually decrease. For example, under severe drought stress, the content of chlorophyll a and chlorophyll b may drop by 30% to 50% compared to normal conditions. Similar changes will occur in the content of carotenoids. In addition to participating in the capture of light energy, carotenoids also have the function of protecting photosynthetic organs from damage by excess light energy.

[0004] Drought stress can lead to increased production of reactive oxygen species (such as superoxide anion radicals, hydrogen peroxide, etc.) in citrus seedlings. In order to remove these reactive oxygen species, the antioxidant enzyme system in the plant will be activated. For example, the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) will change. In the early stage of drought stress, SOD activity may increase rapidly, converting superoxide anion radicals into hydrogen peroxide, and then POD and CAT activities increase, decomposing hydrogen peroxide into water and oxygen. Under normal circumstances, SOD activity may be 100-200U / g FW, and may increase to 300-500U / g FW during drought stress; POD activity may increase from the normal 50-100U / g FW to 200-300U / g FW; CAT activity may increase from the normal 10-20U / g FW to 30-50U / g FW.

[0005] In addition to antioxidant enzymes, the content of non-enzymatic antioxidants (such as ascorbic acid, glutathione, etc.) in citrus seedlings will also change under drought stress. Ascorbic acid is an important antioxidant, and its content may increase under drought stress. Under normal circumstances, the ascorbic acid content in citrus seedling leaves may be 50-100 mg / kg FW, which may increase to 150-200 mg / kg FW under drought stress.

[0006] Drought stress severely limits the photosynthesis of plants, thereby affecting their growth and development, and reducing their yield and quality. Melatonin is an amine hormone produced by the pineal gland of mammals and humans, which has multiple physiological functions such as promoting sleep, adjusting jet lag, anti-aging, regulating immunity, and anti-tumor. Existing studies have shown that it also has the function of regulating growth in plants, promoting the regeneration of lateral roots and adventitious roots, scavenging free radicals, improving stress resistance, protecting the integrity of cell membranes, and increasing yields. For example, patents (CN201510565734.9; CN201710560170.9; CN201910627655.4) disclose that melatonin has the function of directly inhibiting and killing bacterial blight pathogens, oomycetes, and powdery mildew pathogens to prevent and control rice bacterial blight, potato late blight, tobacco black shank, and wheat powdery mildew. However, the above studies all directly act on melatonin at the location of the strain to directly kill bacteria.

[0007] The present invention uses one-year-old 'Ashimitsu' citrus seedlings as experimental materials to explore the effects of exogenous melatonin (MT) on leaf physiological characteristics under drought stress conditions. The study found that drought stress inhibited the growth of citrus seedlings, and exogenous melatonin (MT) could effectively alleviate the effects of drought stress on plants. Summary of the invention

[0008] In order to solve the above technical problems, the purpose of the present invention is to provide an effect and application of melatonin on the physiological characteristics of citrus seedlings under drought stress. Exogenous melatonin significantly reduces the MDA content in citrus leaves, increases the content of osmotic regulating substances such as proline, soluble sugar and soluble protein, increases the content of total phenols and flavonoids, and enhances the activity of antioxidant enzymes, thereby effectively reducing the damage of drought to plants.

[0009] To achieve the above objectives, the effects of melatonin on the physiological characteristics of citrus seedlings under drought stress were tested in the following ways:

[0010] 1) Determination of materials

[0011] 75 one-year-old citrus seedlings were selected and planted in planting pots with a diameter and height of 20 cm;

[0012] 2) Plant grouping to create drought stress environment

[0013] 75 citrus seedlings were divided into two groups, with the control group accounting for 2 / 5 and the experimental group accounting for 3 / 5. Both groups were subjected to drought stress. In the experimental group, irrigation was stopped 4 days before the test, and the soil moisture in the pot was measured and controlled at 40% to 50%.

[0014] For the control group, irrigation was stopped 4 days before the experiment, and the soil moisture in the pots was measured and controlled at 70% to 80%.

[0015] 3) Two-group experiment

[0016] Melatonin was dissolved in ethanol and then diluted to prepare a melatonin spray solution. The citrus seedlings in the experimental group were sprayed with the melatonin spray solution in the evening every day, once every 2 days, for a total of 5 sprayings. After spraying, drought stress was applied again, and the soil moisture was controlled at 40% to 50%. After that, the melatonin spray solution was sprayed again. The standard for spraying was that the leaves were covered with mist droplets without dripping.

[0017] At the same time, the citrus seedlings in the control group were watered normally and sprayed with distilled water on the leaves.

[0018] 4) Sampling and measurement data

[0019] Samples were collected once in the morning on the 5th, 8th, 11th and 14th days after the last drought stress spraying of melatonin spray solution. Each time sampling was performed, 10 leaves of the same size and located in the middle of each citrus seedling were taken for index determination. The sampled leaves were dried to evaluate the relative water content of the leaves. The chlorophyll content of the other leaves was measured in time after the leaf edges and veins were removed. Finally, the remaining leaf samples were immediately placed in liquid nitrogen for rapid freezing and stored in an ultra-low temperature freezer at -80°C for subsequent physiological index determination.

[0020] Melatonin was dissolved in ethanol and then diluted with distilled water to 50 mmol / L -1 The melatonin spray solution was placed in a dark place and stored at low temperature. When spraying, the required volume was extracted from the mother solution with a pipette and diluted to 50 μmol·L -1 , 100 μmol·L -1 , 200 μmol·L -1 concentration.

[0021] In the experimental group, under the second drought stress condition, three pots of citrus seedlings were sprayed with a dilution concentration of 50 μmol·L -1 Melatonin spray, 100 μmol·L -1 Melatonin spray and 200 μmol·L -1 Melatonin spray solution and take data separately.

[0022] After each experimental sampling, the collected leaves were cleaned with distilled water in time.

[0023] During the drought stress period, the soil moisture content of each pot of citrus seedlings was maintained at the corresponding moisture level by measuring and watering every evening.

[0024] Application of melatonin on the physiological characteristics of citrus seedlings under drought stress. Melatonin was dissolved in ethanol and then diluted with distilled water to 50 mmol·L -1 The melatonin spray solution was diluted and sprayed on the leaves and soil of citrus seedlings under drought stress in several times and at intervals.

[0025] The beneficial effects of the present invention compared with the prior art are as follows: Studies have found that drought stress inhibits the growth of citrus seedlings, and exogenous melatonin can effectively alleviate the effects of drought stress on plants. Exogenous melatonin treatment significantly reduces the decline in relative water content of leaves and delays the decrease in photosynthetic pigment content. At the same time, exogenous melatonin can significantly reduce the content of malondialdehyde (MDA), increase the activity of antioxidant enzymes, promote the synthesis of osmotic regulating substances, and increase the content of total phenols and flavonoids in leaves. Therefore, exogenous melatonin can increase the content of photosynthetic pigments and osmotic regulating substances in leaves, improve the antioxidant capacity of plants, slow down the membrane peroxidation caused by drought stress, and thus improve the resistance of citrus seedlings to drought stress.

[0026] This experiment proves that 100 μmol·L -1 Melatonin treatment had the best effect on alleviating drought stress in 'Ashirimi' seedlings. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the effect of exogenous melatonin on the relative water content of citrus seedling leaves during drought stress.

[0028] Figure 2 This is the effect of exogenous melatonin on the chlorophyll a content, chlorophyll b content and total chlorophyll content of citrus seedlings during drought stress.

[0029] Figure 3 This is the effect of melatonin on the malondialdehyde content in the leaves of 'Ashirimi' citrus seedlings during drought stress.

[0030] Figure 4 This is the effect of exogenous melatonin on the antioxidant enzyme activities in the leaves of 'Ashirimi' citrus seedlings during drought stress.

[0031] Figure 5 This is the effect of exogenous melatonin on the content of osmotic regulating substances in the leaves of 'Ashirimi' citrus seedlings during drought stress.

[0032] Figure 6 This graph shows the effect of exogenous melatonin on the content of total phenols and flavonoids in 'Ashirimi' citrus seedlings during drought stress. DETAILED DESCRIPTION

[0033] In order to make the technical means, creative features, objectives and effects achieved by the present invention easy to understand, the present invention is further described below in conjunction with specific embodiments.

[0034] 1) Determination of materials

[0035] A total of 75 one-year-old potted Citrus aurantium rootstock 'Ashimitsu' seedlings with robust growth, tree vigor and shape, and basically the same management level were selected as test materials and planted in planting pots with a diameter and height of 20 cm.

[0036] 2) Plant grouping to create drought stress environment

[0037] A total of 75 citrus seedlings were evenly divided into 5 groups, 2 of which were control groups and 3 of which were experimental groups. Each group had 15 pots of citrus seedlings. Both groups were subjected to drought stress at the same time. In the experimental group, irrigation was stopped 4 days before the experiment, and the soil moisture in the pots was measured, and the soil moisture was controlled at 40%-50%. In the control group, irrigation was stopped 4 days before the experiment, and the soil moisture in the pots was measured, and the soil moisture was controlled at 70%-80%. During the drought stress period, the soil moisture content of each pot of citrus seedlings was measured and watered at 18:00 every evening, and the soil moisture content of each pot of citrus seedlings was maintained at the corresponding moisture content level.

[0038] 3) Two-group experiment

[0039] Melatonin was dissolved in ethanol and then diluted with distilled water to 50 mmol / L -1 The melatonin spray solution was placed in a dark place and stored at low temperature. When spraying, the required volume was extracted from the mother solution with a pipette and diluted to 50 μmol·L -1, 100 μmol·L -1 , 200 μmol·L -1 The concentration of

[0040] The citrus seedlings in the experimental group were sprayed with melatonin spray solution in the evening every day, once every 2 days, for a total of 5 sprayings. On the 4th day after the last spraying of melatonin spray solution (defined as the 0th day of drought treatment), drought stress was continued, and the soil moisture was controlled at 40% to 50%. After that, melatonin spray solution was sprayed again. The standard for spraying was that the leaves were covered with mist droplets without dripping.

[0041] Meanwhile, the citrus seedlings in the control group were watered normally and sprayed with distilled water on the leaves.

[0042] 4) Sampling and measurement data

[0043] Samples were collected once in the morning on the 5th, 8th, 11th and 14th days after the last drought stress spraying of melatonin spray solution. Each time sampling was performed, 10 leaves of the same size and located in the middle of each citrus seedling were taken for index determination. After each experimental sampling, the collected leaves were cleaned with distilled water in time. The sampled leaves were dried to evaluate the relative water content of the leaves, and the chlorophyll content of the other leaves was determined in time after the leaf edges and veins were removed. Finally, the remaining leaf samples were immediately placed in liquid nitrogen for rapid freezing and stored in an ultra-low temperature freezer at -80°C for subsequent physiological index determination.

[0044] The relative water content of leaves was determined by the drying method, and the chlorophyll content was detected by referring to the method of Li Hesheng. The free proline content was determined by ninhydrin solution colorimetry, the soluble sugar content was determined by anthrone colorimetry, the soluble protein was determined by Coomassie Brilliant Blue G-250, the malondialdehyde (MDA) content was determined by thiobarbituric acid, the antioxidant enzyme activity was evaluated by the NBT method, the POD enzyme activity was determined by guaiacol colorimetry, and the CAT enzyme activity was determined by UV spectroscopy.

[0045] All data obtained during the experiment were processed by Microsoft Excel software. Multifactor repeated measures variance data analysis was performed using SPSS25.0 software, and the difference significance analysis (P<0.05) was performed using Duncan's new multiple range comparison method, and the results were presented using GraphPad Prism9.0 software.

[0046] In further experiments, we can also divide the citrus seedlings of the experimental group into groups. In the experiment, under the second drought stress, three pots of citrus seedlings were sprayed with a dilution concentration of 50 μmol·L -1 Melatonin spray, 100 μmol·L -1Melatonin spray and 200 μmol·L -1 Melatonin spray solution was used and data were collected respectively. The data results were analyzed as follows:

[0047] 1. Relative water content

[0048] Depend on Figure 1 It can be seen that as the drought stress time prolonged, the relative water content of leaves in the four experimental groups showed a significant downward trend compared with the control group (CK). -1 MT, 100 μmol·L -1 MT and 200 μmol·L -1 The relative water content of leaves in the MT experimental group (DS) was significantly higher than that in the control group (50 μmol·L -1 MT and 200 μmol·L -1 There was no significant difference in MT treatment, 100 μmol·L -1 The relative water content of the MT treatment group was just lower than that of the control group. The maximum water retention effect was achieved on the 8th day, and the difference with the experiment reached the maximum.

[0049] 2. Photosynthetic pigment content

[0050] Depend on Figure 2 It can be seen that as the time of drought stress increases, the contents of chlorophyll a, chlorophyll b and chlorophyll a+b in the experimental group (DS) show a significant decrease compared with the control group (CK). The content of photosynthetic pigments in the DS group is significantly lower than that in the CK group, and the downward trend is alleviated by exogenous melatonin (MT) treatment compared with the DS group. There is no significant difference in the chlorophyll a content in the MT treatment with that in the CK group, and when the MT concentration is 100 μmol·L -1 The decreasing trend of Chlb content and total chlorophyll content was significantly alleviated. Within 5d, 8d, 11d and 14d after drought stress, the difference between MT treatment and DS reached the peak on the 14th day.

[0051] 3. Malondialdehyde (MDA) content

[0052] from Figure 3 It can be seen that the MDA content of the DS group and the MT treatment increased with the extension of water stress time. Compared with the CK group, there was a significant upward trend. At 5d, 8d, 11d, and 14d after drought stress, the MT treatment caused the MDA content to gradually increase, reaching the maximum value on the 14th day, 50μmol·L -1 MT, 100 μmol·L -1 MT and 200 μmol·L -1The MDA content in the leaves of the MT treatment group was significantly lower than that of the DS group. -1 The MDA content in leaves of the MT treatment group was reduced by 45.83% compared with that of the DS group, achieving the maximum stress relief effect.

[0053] 4. Antioxidant enzyme activity

[0054] from Figure 4 It can be seen that under drought stress, the antioxidant enzyme activities of the DS group and the CK group showed an upward trend. With the extension of drought time, the antioxidant enzyme activities of the leaves treated with MT showed a significant trend of first increasing and then decreasing. -1 The SOD activity of MT showed no obvious change trend. -1 The SOD activity of the MT treatment group was significantly different from that of the DS group. -1 With 100 μmol·L -1 The activities of POD and CAT in the MT treatment group were significantly higher than those in the DS group. -1 Compared with the DS group, the SOD activity increased by 13.53%, 8.74%, 4.60% and 14.06% at 5d, 8d, 11d and 14d under MT treatment; compared with the DS group, the POD activity increased by 20.69%, 35.85%, 20.17% and 14.63%; compared with the DS group, the CAT enzyme activity increased by 41.2%, 48.81%, 45.61% and 34.81%, respectively.

[0055] 5. Osmotic regulating substances

[0056] like Figure 5 As shown in the figure, as the drought stress time prolonged, the content of osmotic regulating substances in the leaves of 'Ashirimi' seedlings after drought treatment increased significantly. -1 MT, 100 μmol·L -1 , 200 μmol·L -1 Compared with the experimental group (DS) and the CK group, the soluble sugar and soluble protein contents after MT treatment were significantly higher, 50 μmol·L -1 MT, 100 μmol·L - 1 The proline content of MT was significantly higher than that of DS group. -1There was no significant difference in the proline content of leaves after MT treatment and DS group. After 14 days of drought stress treatment, the proline content of MT treatment reached the maximum difference with DS group. The soluble sugar and soluble protein of MT treatment showed a trend of first increasing and then decreasing, with the maximum difference with DS group at 11 days.

[0057] 6. Total phenolic and flavonoid content

[0058] from Figure 6 It can be seen that under drought stress, the total phenolic and total flavonoid contents in the DS group and the experimental group treated with MT showed a trend of first increasing and then decreasing with time. -1 MT, 100 μmol·L -1 The contents of total phenols and total flavonoids in the MT treatment group were significantly higher than those in the DS group (P<0.05). -1 The MT treatment had the most stable effect, 100 μmol·L -1 The flavonoids in the MT group increased by 35.92% compared with those in the DS group. Therefore, spraying melatonin at an appropriate concentration can increase the content of total phenols and flavonoids in the leaves of citrus seedlings.

[0059] From the above experimental data, we know that drought stress severely limits the photosynthesis of plants, thus affecting their growth and development, and reducing their yield and quality. However, melatonin is an important plant hormone that can improve the drought resistance of plants by regulating various physiological and molecular processes. As a strong oxidative scavenger, melatonin can significantly improve the antioxidant capacity of plants. Under drought conditions, the content of antioxidant enzymes and osmotic regulating substances in citrus seedlings is significantly affected.

[0060] Environmental stress can induce plants to produce excessive reactive oxygen species, initiate lipid peroxidation, and cause damage to cell membrane structure. Although reactive oxygen species play an important role in the normal growth and development of plants, under normal circumstances, the level of ROS is maintained in a dynamic balance. When adversity (such as drought) occurs, the ROS homeostasis of the plant will be destroyed, causing the accumulation of free radicals, which in turn triggers membrane peroxidation, thereby increasing the permeability of the cell membrane and destroying the cell structure. Previous studies have shown that exogenous melatonin (MT) can reduce the content of hydrogen peroxide and MDA and alleviate cell membrane peroxidation. Our previous study found that under drought stress conditions, the MDA content of citrus leaves was significantly higher than that of the control, while the MDA content was significantly reduced after MT treatment. Therefore, it is speculated that MT alleviates drought-induced plasma membrane oxidative damage by removing ROS. This is consistent with previous research results on MT on corn and pepper, and further proves the important protective role of MT on cell membrane structure and function under adverse stress.

[0061] In addition, the level of reactive oxygen species (ROS) is closely related to the activity of antioxidant enzymes in plants. In order to resist the oxidative stress caused by environmental stress, plants need to remove excessive reactive oxygen through a series of antioxidant mechanisms when dealing with oxidative stress caused by adverse stress. Many studies have confirmed that exogenous melatonin (MT) can significantly increase the activity of these antioxidant enzymes, thereby enhancing the ability of plants to adapt to adversity. This study revealed that the antioxidant enzyme activity of citrus leaves treated with drought was significantly improved, especially at 50 μmol·L -1 MT and 100 μmol·L -1 After MT treatment, the activity was further enhanced. Therefore, we speculated that MT may effectively remove ROS and alleviate drought-induced plant damage by improving the antioxidant level of citrus leaves.

[0062] Soluble sugar, soluble protein, proline, etc. are important osmotic regulating substances produced by plants in response to environmental stress. Their content can well reflect the adaptability of plants to adversity. -1 MT and 100 μmol·L -1 Under the action of MT, the soluble sugar, soluble protein and proline content of the plants can be significantly increased. This is consistent with the research results of cucumber and kiwi fruit. It is speculated that MT is likely to promote the production of osmotic regulating substances, thereby stabilizing cell permeability and enhancing the drought resistance of plants. This discovery provides new evidence for a deeper understanding of the role of MT in plant stress resistance.

[0063] As a non-enzymatic antioxidant defense system, phenolic and flavonoid compounds are usually regarded as the second line of defense for plants to resist adversity. They play a minor but key role in the ROS scavenging system, protecting plants from damage caused by adversity. They play an important role in the response of plants to adversity. Phenolic compounds can produce reactive oxygen species (ROS) under stress and can effectively resist biotic and abiotic stresses. In previous work, it was found that under drought conditions, the content of total phenolic substances in the leaves of citrus seedlings first increased and then decreased. This response pattern is similar to the performance of other plants under drought and salt stress. These results provide strong evidence that phenolic compounds play an important role in plant resistance. Previous studies have found that MT treatment can significantly increase the content of total phenolic substances and flavonoids in citrus seedlings. Based on this, it is speculated that MT is very likely to improve the antioxidant capacity of plants by increasing the content of total phenols and flavonoids in plants.

[0064] The results showed that under drought conditions, the water content of citrus leaves decreased, the chlorophyll content decreased, the activity of antioxidant enzymes increased, the osmotic regulating substances increased, and the content of flavonoids increased. On this basis, further studies found that exogenous MT significantly reduced the MDA content of citrus leaves, increased the content of osmotic regulating substances such as proline, soluble sugars and soluble proteins, increased the content of total phenols and flavonoids, and enhanced the activity of antioxidant enzymes, thereby effectively reducing the damage of drought to plants. It is speculated that MT induces plant drought resistance by improving plant antioxidant capacity, osmotic regulating substances and phenolic substances. And when the MT concentration is 100μmol·L -1 The best effect was found in alleviating drought stress of citrus seedlings grown on Citrus aurantium rootstock 'Ashirimi'.

[0065] When cultivating citrus seedlings, we can apply the results of the above experiments to the cultivation of actual trees. The application of melatonin to the physiological characteristics of citrus seedlings under drought stress, melatonin was dissolved in ethanol and then diluted with distilled water to 50mmol·L -1 The melatonin spray solution is diluted and sprayed on the leaves and soil of citrus seedlings under drought stress in batches and intervals. Adjusting the use ratio of melatonin according to the number of trees is also within the protection scope of this embodiment.

[0066] The above is a detailed introduction to the method provided by the present invention for determining the effect of melatonin on the physiological characteristics of citrus seedlings under drought stress. The description of the specific embodiment is only used to help understand the method and its core idea of ​​the present invention. It should be pointed out that for ordinary technicians in this technical field, without departing from the principle of the present invention, the present invention can also be improved and modified in several ways, and these improvements and modifications also fall within the scope of protection of the claims of the present invention.

Claims

1. The effect of melatonin on the physiological characteristics of citrus seedlings under drought stress, characterized in that: Tested by: 1) Determination of materials 75 one-year-old citrus seedlings were selected and planted in planting pots with a diameter and height of 20 cm; 2) Plant grouping to create drought stress environment 75 citrus seedlings were divided into two groups, with the control group accounting for 2 / 5 and the experimental group accounting for 3 / 5. Both groups were subjected to drought stress. In the experimental group, irrigation was stopped 4 days before the test, and the soil moisture in the pot was measured and controlled at 40% to 50%. For the control group, irrigation was stopped 4 days before the experiment, and the soil moisture in the pots was measured and controlled at 70% to 80%. 3) Two-group experiment Melatonin was dissolved in ethanol and then diluted to prepare a melatonin spray solution. The citrus seedlings in the experimental group were sprayed with the melatonin spray solution in the evening every day, once every 2 days, for a total of 5 sprayings. After spraying, drought stress was applied again to control the soil moisture at 40% to 50%, and then the exogenous melatonin spray solution was sprayed again. The standard for spraying was that the leaves were covered with mist droplets without dripping. At the same time, the citrus seedlings in the control group were watered normally and sprayed with distilled water on the leaves. 4) Sampling and measurement data Samples were collected once in the morning on the 5th, 8th, 11th and 14th days after the last spraying of melatonin solution under drought stress. During each sampling, 10 leaves of the same size and located in the middle of each citrus seedling were taken for index determination. After sampling, a portion of the leaves will be dried to assess the relative moisture content of the leaves. The chlorophyll content of the other leaves was measured in time after removing the leaf edges and veins. The remaining leaf samples were immediately placed in liquid nitrogen for rapid freezing and stored in an ultra-low temperature freezer at -80°C for subsequent determination of physiological indicators.

2. The effect of melatonin on the physiological characteristics of citrus seedlings under drought stress according to claim 1, characterized in that: Melatonin was dissolved in ethanol and then diluted with distilled water to 50 mmol / L -1 The melatonin spray solution was placed in a dark place and stored at low temperature. When spraying, the required volume was extracted from the mother solution with a pipette and diluted to 50 μmol·L -1 , 100 μmol·L -1 , 200 μmol·L -1 concentration.

3. The effect of melatonin on the physiological characteristics of citrus seedlings under drought stress according to claim 2, characterized in that: In the experimental group, under the second drought stress condition, three pots of citrus seedlings were sprayed with a dilution concentration of 50 μmol·L -1 Melatonin spray, 100 μmol·L -1 Melatonin spray and 200 μmol·L -1 Melatonin spray solution and take data separately.

4. The effect of melatonin on physiological characteristics of citrus seedlings under drought stress according to claim 1, characterized in that: After each experimental sampling, the collected leaves were cleaned with distilled water in time.

5. The effect of melatonin on physiological characteristics of citrus seedlings under drought stress according to claim 1, characterized in that: During the drought stress period, the soil moisture content of each pot of citrus seedlings was maintained at the corresponding moisture level by measuring and watering every evening.

6. An application of melatonin according to claim 1 on the physiological characteristics of citrus seedlings under drought stress, characterized in that: Melatonin was dissolved in ethanol and then diluted with distilled water to 50 mmol / L -1 The melatonin spray solution was diluted and sprayed on the leaves and soil of citrus seedlings under drought stress in several times and at intervals.

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