Composition for relieving flooding stress of rhizoma polygonati in chicken heads and application of composition

Abstract

The invention belongs to the field of plant maintenance, and particularly relates to a composition for relieving water flooding stress of rhizoma polygonati and application of the composition. The composition disclosed by the invention is composed of decomposed cow dung and a spermidine solution, wherein the concentration of the spermidine solution is 0.1-1 mmol / L. According to the method, mature cow dung is applied to a field before polygonatum sibiricum is planted, and when a flood season comes and polygonatum sibiricum plants grow to about 60-80 cm in height, the damage of water flooding stress to polygonatum sibiricum can be continuously relieved through the maintenance method using the composition, the content of proline in polygonatum sibiricum leaves is increased, and the survival rate of the polygonatum sibiricum is increased. The content of malondialdehyde in the polygonatum sibiricum leaves is reduced, and the activity of antioxidant enzyme in the plant leaves is improved to relieve oxidative damage caused by water flooding stress.

Description

Technical Field

[0001] The invention belongs to the field of plant maintenance, and more specifically, relates to a composition for alleviating waterlogging stress of Polygonatum cyrtonema and an application thereof. Background Art

[0002] Polygonatum sibiricum is a shallow-rooted plant with its main root system distributed within 18-20 cm of the soil surface. It grows naturally in an ecological environment with a deep humus layer under the forest and good soil ventilation. However, in artificial cultivation environments, especially in areas with concentrated rainfall during the rainy season, it is prone to flooding. Under flooding stress, Polygonatum sibiricum will show symptoms of damage such as leaf yellowing and root damage, changes in the antioxidant enzyme system, changes in endogenous hormones, damage to the cell membrane system, an increase in osmotic regulating substances, decreased photosynthesis, and abnormal respiration.

[0003] First, under the hypoxic environment of flooding stress, the leaves of Polygonatum cyrtonema will show drooping, chlorosis, yellowing, scorching, wilting, shrinking, and patchy diseases, and the root absorption capacity will decrease or even rot. Secondly, under flooding stress, due to the obstruction of the normal oxidative phosphorylation level and respiration of Polygonatum cyrtonema, the accumulation of reactive oxygen in the plant will induce the increase of the activity of various antioxidant enzymes. The increase in the activity of antioxidant enzymes can more quickly remove the accumulated reactive oxygen free radicals in the plant body and reduce the toxic effects of reactive oxygen.

[0004] Therefore, it is very necessary to develop a product that can increase the antioxidant enzyme activity of Polygonatum cyrtonema to alleviate the physiological damage caused by waterlogging stress to Polygonatum cyrtonema. Summary of the invention

[0005] The invention aims to provide a composition for alleviating waterlogging stress of Polygonatum cyrtonema and application thereof.

[0006] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0007] The invention provides a composition for alleviating waterlogging stress of Polygonatum cyrtonema. The composition consists of cooked cow dung and a spermidine solution. The concentration of the spermidine solution is 0.1-1 mmol / L, and the mass volume ratio of the cooked cow dung to the spermidine solution is 200-300 g:10-50 ml.

[0008] The cooked cow dung in the composition of the present invention can improve the soil structure and make the soil more loose, which is conducive to the plant roots to stretch and breathe as much as possible in the adverse environment of flooding, while spermidine can regulate the physiological process of plants and enhance the antioxidant defense system. By conducting experimental screening on the two and combining them in a scientific ratio, the flooding stress of Polygonatum cyrtonema can be better alleviated.

[0009] The invention also provides application of the composition in alleviating waterlogging stress of Polygonatum cyrtonema.

[0010] Furthermore, the composition is used to increase the chlorophyll content in Polygonatum cyrtonema leaves under waterlogging stress.

[0011] Furthermore, the composition is used to reduce the content of malondialdehyde in leaves of Polygonatum cyrtonema under waterlogging stress.

[0012] Furthermore, the composition is used to increase the content of proline in leaves of Polygonatum cyrtonema under waterlogging stress.

[0013] Furthermore, the composition is used to increase the activity of antioxidant enzymes in plant leaves to alleviate oxidative damage caused by waterlogging stress.

[0014] Furthermore, the antioxidant enzymes include peroxidase, superoxide dismutase and catalase.

[0015] The present invention also provides a method for alleviating waterlogging stress of Polygonatum cyrtonema using the above-mentioned composition, the steps comprising: applying cooked cow dung in the field before planting, transplanting Polygonatum cyrtonema seedlings into the field, and when the Polygonatum cyrtonema plants grow to a height of 60 to 80 cm, evenly spraying spermidine into a 0.1 to 1 mmol / l aqueous solution on the front and back sides of the plant leaves to prevent waterlogging stress.

[0016] Furthermore, the amount of the cooked cow dung applied in the field is 10 to 13 m 3 / mu.

[0017] Furthermore, the spraying frequency is once a day, 2 to 3 ml is sprayed per plant each time, and the total number of spraying times is 6 to 18 times.

[0018] The present invention has the following beneficial effects:

[0019] The invention provides a composition of a decomposed cow dung matrix and a spermidine solution and an application thereof. Before the end of July, that is, in the vigorous growing season before the rainy season, by using the maintenance method of the composition, the harm of waterlogging stress on Polygonatum cyrtonema can be continuously alleviated, the content of proline in Polygonatum cyrtonema leaves can be increased, the content of malondialdehyde in Polygonatum cyrtonema leaves can be reduced, and the activity of antioxidant enzymes in plant leaves can be increased to reduce oxidative damage caused by waterlogging stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing changes in chlorophyll content after flooding for 1 day, where a on the vertical axis represents chlorophyll a, b represents chlorophyll b, and T represents total chlorophyll in the treatment group.

[0021] Figure 2This is a graph showing changes in chlorophyll content after 2 days of flooding, where a on the vertical axis represents chlorophyll a, b represents chlorophyll b, and T represents total chlorophyll in the treatment group.

[0022] Figure 3 This is a graph showing changes in chlorophyll content after flooding for 4 days, where a in the vertical axis represents chlorophyll a, b represents chlorophyll b, and T represents total chlorophyll in the treatment group.

[0023] Figure 4 This is a graph showing changes in chlorophyll content after 8 days of flooding, where a on the vertical axis represents chlorophyll a, b represents chlorophyll b, and T represents total chlorophyll in the treatment group.

[0024] Figure 5 This is a graph showing the changes in chlorophyll content after flooding for 16 days, where a in the vertical axis represents chlorophyll a, b represents chlorophyll b, and T represents total chlorophyll in the treatment group.

[0025] Figure 6 This is a graph showing the changes in malondialdehyde content after urea peroxide treatment under flooding of different durations.

[0026] Figure 7 This is a graph showing the changes in malondialdehyde content after melatonin treatment under flooding of different durations.

[0027] Figure 8 This is a graph showing the changes in malondialdehyde content after salicylic acid treatment under flooding of different durations.

[0028] Fig. 9 This is a graph showing changes in malondialdehyde content in cooked cow dung treated with flooding for different durations.

[0029] Fig.10 This is a graph showing the changes in malondialdehyde content after spermidine treatment under water flooding of different durations.

[0030] Fig.11 This is a graph showing the changes in malondialdehyde content after treatment with cooked cow dung and spermidine under flooding of different durations.

[0031] Fig.12 This is a graph showing the changes in malondialdehyde content after treatment with uniclostridium dithiocarbazide under flooding of different durations.

[0032] Fig.13 This is a graph showing the changes in superoxide dismutase activity in water-flooded Polygonatum cyrtonema treated with oxidized urea.

[0033] Fig.14 This is a graph showing the changes in superoxide dismutase activity in water-soaked Polygonatum cyrtonema treated with melatonin.

[0034] Fig.15 This is a graph showing the changes in superoxide dismutase activity of water-soaked Polygonatum cyrtonema treated with cooked cow dung.

[0035] Fig.16This is a graph showing the changes in superoxide dismutase activity in water-flooded Polygonatum cyrtonema treated with spermidine.

[0036] Fig.17 This is a graph showing the changes in superoxide dismutase activity in water-flooded Polygonatum cyrtonema treated with cooked cow dung and spermidine.

[0037] Fig.18 This is a graph showing the changes in superoxide dismutase activity of water-flooded Polygonatum cyrtonema treated with uniconazole.

[0038] Fig.19 This is a graph showing the changes in peroxidase activity of water-flooded Polygonatum cyrtonema after being treated with oxidized urea.

[0039] Fig. 20 This is a graph showing the changes in peroxidase activity of water-soaked Polygonatum cyrtonema treated with melatonin.

[0040] Fig.21 This is a graph showing the changes in peroxidase activity of water-flooded Polygonatum cyrtonema treated with salicylic acid.

[0041] Fig. 22 This is a graph showing the changes in peroxidase activity of water-flooded Polygonatum cyrtonema treated with cooked cow dung.

[0042] Fig.23 This is a graph showing the changes in peroxidase activity of water-flooded Polygonatum cyrtonema treated with spermidine.

[0043] Fig.24 This is a graph showing the changes in peroxidase activity of water-flooded Polygonatum cyrtonema treated with cooked cow dung and spermidine.

[0044] Fig.25 This is a graph showing the changes in peroxidase activity of water-flooded Polygonatum cyrtonema treated with unicyclic acid.

[0045] Fig.26 This is a graph showing the changes in catalase activity of water-flooded Polygonatum cyrtonema after being treated with urea.

[0046] Fig. 27 This is a graph showing the changes in catalase activity of water-soaked Polygonatum cyrtonema treated with melatonin.

[0047] Fig.28 This is a graph showing the changes in catalase activity of water-flooded Polygonatum cyrtonema treated with salicylic acid.

[0048] Fig.29 This is a graph showing the changes in catalase activity of water-flooded Polygonatum cyrtonema treated with cooked cow dung.

[0049] Fig.30 This is a graph showing the changes in catalase activity of water-flooded Polygonatum cyrtonema treated with spermidine.

[0050] Fig.31This is a graph showing the changes in catalase activity of water-flooded Polygonatum cyrtonema treated with cooked cow dung and spermidine.

[0051] Fig.32 This is a graph showing the changes in catalase activity of water-flooded Polygonatum cyrtonema treated with chloranthazole.

[0052] Fig.33 This is a graph showing the changes in proline content in water-flooded Polygonatum cyrtonema after being treated with oxidized urea.

[0053] Fig.34 This is a graph showing the changes in proline content in water-soaked Polygonatum cyrtonema treated with melatonin.

[0054] Fig.35 This is a graph showing the changes in proline content in water-flooded Polygonatum cyrtonema treated with salicylic acid.

[0055] Fig.36 This is a graph showing the changes in proline content in water-flooded Polygonatum cyrtonema treated with cooked cow dung.

[0056] Fig.37 This is a graph showing the changes in proline content in water-flooded Polygonatum cyrtonema treated with spermidine.

[0057] Fig.38 This is a graph showing the changes in proline content in water-flooded Polygonatum cyrtonema treated with cooked cow dung and spermidine.

[0058] Fig.39 This is a graph showing the changes in proline content in water-flooded Polygonatum cyrtonema treated with uniconazole. DETAILED DESCRIPTION

[0059] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0060] Example 1

[0061] 1. Experimental methods

[0062] 1. The experiment adopted a completely randomized block design, with normal maintenance control, flooding control, flooding urea peroxide treatment, flooding melatonin treatment, flooding salicylic acid treatment, flooding cow dung treatment, flooding spermidine treatment, flooding cow dung + spermidine treatment, and flooding uniconazole treatment. Each treatment had three concentration levels, 10 potted Polygonatum cyrtonema seedlings, repeated three times, a total of 690 plants, and the experiment was carried out under the shade of Populus canadensis with a canopy density of 0.7. The outdoor flooding test lasted for 16 days, and measurements and data statistical analysis were carried out successively, and all experimental work was completed at the end of August.

[0063] 2. First, in the experimental nursery of Beijing Forestry University, 690 ceramic pots with a height of 26 cm and a diameter of 28 cm at the top were prepared, and the water holes at the bottom were covered with tiles. The volume ratio of the matrix was 5:2:2:1 for humus soil: vermiculite: river sand: peat. The cow dung treatment group used decomposed cow dung to replace peat, river sand and vermiculite, and the volume ratio of cow dung to the matrix was 10%, 30% and 50% respectively. Disinfect with 0.5% potassium permanganate solution for 4 hours. On April 3, two-year-old Polygonatum cyrtonema was collected from the Yanshan Mountain area in Huairou. The first stem segment with the terminal bud was selected. The diameter of the thick end of the head was 4.0 cm and the length was 8.0 cm. Two stem segments were planted in each pot. Dip in 5% CE allicin microemulsion and 500 times naphthaleneacetic acid. First fill the soil to about two-thirds of the height of the pot, and place the stem segments with terminal buds in parallel, with the thick end of the terminal bud slightly higher and the thin end of the tail slightly lower. Then fill the stem segment with substrate, add substrate to 4cm from the upper edge of the pot, water thoroughly, and water thoroughly again after 3 days. On May 30, when the test plant of Polygonatum cyrtonema grew to a height of 70cm, a 5cm deep annular ditch was first dug in the pot treated with urea peroxide, and 4, 8, and 12 bags of urea peroxide were evenly buried at the set level. After covering with soil, flooding treatment was started, and tap water that had been left outdoors for 1 day was used to irrigate the pot to 1cm from the upper edge of the pot, and the water was kept 3cm deep. All experiments were completed on August 9. The specific experimental design is shown in Table 1.

[0064] Table 1: Design of the flooding experiment for Polygonatum cyrtonema.

[0065] Experimental treatment (level) Level 1 Level 2 Level 3 Normal maintenance control (CK) Water 0 Water 0 Water 0 Waterlogged control (WL) Water accumulation 3cm Water accumulation 3cm Water accumulation 3cm Flooding Urea Peroxide Treatment (UP) 4g 8g 12g Melatonin treatment in water (MT) 100μmol / L 500μmol / L 1000μmol / L Flooding salicylic acid treatment (SA) 80mg / L 160mg / L 240mg / L Flooded Cow Dung Treatment (CM) 10％ 30％ 50％ Flooding spermidine treatment (SP) 0.5mmol / L 1.0mmol / L 1.5mmol / L Flooded cow dung + spermidine treatment (CS) 0.5mmol / L 1.0mmol / L 1.5mmol / L Flooding treatment with chlorpyrifos (U) 20mg / L 40mg / L 80mg / L

[0066] Note: In the three levels of flooded cow dung + spermidine treatment group, cow dung accounted for 30% of the substrate volume.

[0067] 2. Testing and data statistics.

[0068] 1. Chlorophyll content: Weigh 0.1g of fresh leaves, chop them into pieces and place them in a centrifuge tube. Add 10mL of a 1:1 volume ratio of 95% ethanol and 80% acetone mixture, and keep it away from light for 48 hours until the green leaf tissue becomes colorless. Weigh 0.1g of fresh leaves, chop them into pieces and place them in a centrifuge tube. Add 10mL of a 1:1 volume ratio of 95% ethanol and 80% acetone mixture, and keep it away from light for 48 hours until the green leaf tissue becomes colorless. Use an enzyme marker to measure the absorbance at wavelengths of 470nm, 645nm, and 663nm, respectively, and calculate the chlorophyll a (Chl.a), chlorophyll b (Chl.b), total chlorophyll (Chl) and carotenoid (Carotenoid) contents.

[0069] 2. MDA content: Thiobarbituric acid method: Take fresh plant leaves, add 5 mL of 5% TCA, grind and centrifuge the homogenate for 10 minutes. Take 2 mL of supernatant, add 2 mL of 0.67% TBA, mix and boil in a 100°C water bath for 30 minutes, centrifuge again. Take the supernatant and measure the absorbance at 450 nm, 532 nm and 600 nm respectively and calculate the MDA content (C, μmol / L) according to the following formula:

[0070] C=6.45(A 532 -A 600 )-0.56A 450 .

[0071] Among them, A 532 is the absorbance at 532 nm, A 600 is the absorbance at 600nm, A 450 It is the absorbance at 450nm.

[0072] 3. Determination of superoxide dismutase (SOD) activity: Collect the leaves of the 5th and 6th nodes of the aboveground part of Polygonatum cyrtonema and the underground part, pack them and freeze them in liquid nitrogen. Weigh 0.1g of leaves, add 6mL of extract and a little quartz sand to grind into a homogenate, and centrifuge the homogenate at 12000g for 20min at 4℃. Take 0.1mL of the enzyme solution supernatant and add 3mL of reaction medium (containing 50mmol·L -1 Methionine, 750 μmol·L -1 NBT, 0.1mmol·L -1 EDTA, 20 μmol·L -1 After mixing, illuminate for 20 minutes and keep it in the dark for 10 minutes. The absorbance is measured at 560 nm. The calculation formula of superoxide dismutase activity is as follows:

[0073] SOD=(Ack-As)×V×60(Ack×0.5×W×t).

[0074] In the formula, Ack represents the absorbance of the blank tube; As represents the absorbance of the sample tube; V represents the total volume of the extract; W represents the sample weight; and t represents the illumination time.

[0075] 4. Determination of peroxidase (POD) activity: Collect the leaves of the 5th and 6th nodes of the aboveground part of Polygonatum cyrtonema and the underground part, pack them and freeze them in liquid nitrogen. Weigh 0.1g of leaves, add 6mL of extract and a little quartz sand to grind into a homogenate, centrifuge the homogenate at 12000g for 20min at 4℃. Take 50μL of the enzyme solution supernatant, add 3mL of reaction solution (300mL of phosphate buffer solution and 704μL of H 2 O 2and 168 μL guaiacol), colorimetry was performed at 470 nm, and the peroxidase activity was calculated as follows:

[0076] POD=ΔD470×V1(0.1×W×V2×t).

[0077] In the formula, △D470 represents the change in absorbance during the reaction time; V1 represents the total volume of the enzyme extract; W represents the sample weight; V2 represents the volume of the enzyme solution during the measurement; and t represents the reaction time.

[0078] 5. Determination of catalase (CAT) activity: Collect the leaves of the 5th and 6th nodes of the aboveground part of Polygonatum cyrtonema and the underground part, pack them and freeze them in liquid nitrogen. Weigh 0.1g of leaves, add 6mL of extract and a little quartz sand to grind into a homogenate, centrifuge the homogenate at 12000g for 20min at 4℃. Take 50μL of the enzyme supernatant, add 3mL of reaction solution (200mL of phosphate buffer solution plus 227.2μL of H 2 O 2 ), colorimetry was performed at 240 nm, and the calculation formula for catalase activity was as follows:

[0079] CAT=(Ack-As)×V1(0.1×W×V2×t).

[0080] In the formula, Ack represents the absorbance of the blank tube; As represents the absorbance of the sample tube; V1 represents the total volume of the enzyme extract; W represents the sample weight; V2 represents the volume of the enzyme solution during the measurement; and t represents the reaction time.

[0081] 6. Proline content: The proline (PRO) content is determined by the ninhydrin colorimetric method. Weigh 0.1g of the middle leaf of Polygonatum cyrtonema, add 3% sulfosalicylic acid and quartz sand, grind, centrifuge at 4℃10000g for 20min, and the supernatant is the proline extract. Take 2mL of the extract, add 2mL of glacial acetic acid and 2mL of acidic ninhydrin solution, heat in a boiling water bath for 30min, and the solution turns red. After cooling, add 5mL of toluene, shake for 30s, let stand for a short time, take the upper liquid and add it to a centrifuge tube and centrifuge at 3000r / min for 5min.

[0082] The supernatant was aspirated and colorimetric analysis was performed at a wavelength of 520 nm. The calculation formula for the proline content was as follows:

[0083] Proline = C x V (a x W).

[0084] Wherein, C is obtained from the standard curve; V represents the volume of the extract; a represents the volume absorbed during the measurement; and W represents the sample weight.

[0085] 3. Results and analysis.

[0086] 1. Chlorophyll content.

[0087] from Figure 1 It can be seen that the chlorophyll content in each treatment after flooding for one day did not change much, and only the chlorophyll a and total chlorophyll contents in the leaves of Polygonatum cyrtonema in the flooding control treatment (WL) were significantly reduced.

[0088] from Figure 2 It can be seen that Polygonatum cyrtonema is more sensitive to waterlogging stress, and the root oxygenation effect is better in the early stage of waterlogging; in contrast, the effect of chlorpyrifos is not obvious in the early stage of waterlogging.

[0089] from Figure 3 It can be seen that on the fourth day of flooding, the chlorophyll content dropped sharply; the UP effect of urea peroxide was more prominent, among which 8g of bagged urea peroxide per pot could effectively alleviate the harm of flooding stress; the anti-stress effect of melatonin was also reflected; pheniconazole maintained cell vitality by maintaining low-level metabolic activity, and finally reflected the protection of mesophyll assimilation cells.

[0090] from Figure 4 It can be seen that after 8 days of flooding, the leaves have a lot of burnt edges and obvious dead spots. The overall performance is that the chlorophyll content is already very low. Relatively speaking, cow dung plus spermidine has the best effect, and salicylic acid and clorac also have obvious effects.

[0091] from Figure 5 It can be seen that after 16 days of flooding, a large number of leaves of Polygonatum cyrtonema died. High doses of spermidine

[0092] The chlorophyll content of Polygonatum cyrtonema plants treated with 1.5mmol / L and cow dung and a medium dose of 40mg / L cloconazole remained relatively high.

[0093] 2. Changes in malondialdehyde content.

[0094] Flooding can have many effects on plant physiology and biochemistry, among which the impact on the biomembrane system and its function is an important aspect. Flooding stress can increase the content of malondialdehyde in plant leaves, causing membrane lipid peroxidation and delipidation in the leaves, which in turn leads to serious damage to cell membrane lipids and enzymes.

[0095] Malondialdehyde is the end product of membrane lipid peroxidation under flooding stress conditions. It can reflect the integrity of cell membrane structure to a certain extent and is closely related to plant resistance. Figure 6It can be seen that, compared with the control CK under normal management, the malondialdehyde content of Polygonatum cyrtonema leaves quickly reached a peak of 61.21 nmol / g after 4 days of flooding, and then the malondialdehyde content dropped rapidly after 8 days of flooding. On the 16th day of flooding, the leaves wilted. The application of urea peroxide (UP1, UP2, and UP3 are levels 1, 2, and 3, respectively) can alleviate the root hypoxia problem after flooding. In the first two days of flooding, although the malondialdehyde content increased, it did not change much. In particular, the high concentration of oxygen supply well protected the mesophyll cell membrane system and prevented serious membrane lipid peroxidation due to flooding. After adding urea peroxide, urea peroxide slowly released oxygen in the aqueous solution. It increased the oxygen content in the soil solution, improved the aeration of the matrix, enhanced the stress resistance of the plant, effectively alleviated the damage of flooding stress, and reduced the degree of damage caused by stress.

[0096] from Figure 7 It can be seen that with the increase of flooding time, flooding stress significantly increased the MDA content of Polygonatum cyrtonema. Under flooding stress alone, the MDA content of leaves flooded for 1d, 2d, and 4d increased significantly. Compared with the control, different concentrations of melatonin treatment (MT1, MT2, MT3, level 1, level 2, and level 3, respectively) reduced the MDA content of Polygonatum cyrtonema under flooding stress.

[0097] from Figure 8 It can be seen that the plants treated with salicylic acid showed a slow increase rate and a low peak value. On the 4th day of flooding, the malondialdehyde content was reduced by 40% compared with the flooded control, indicating that salicylic acid can inhibit the increase of malondialdehyde content in the early stage of flooding after water stress, thereby improving the adaptability of Polygonatum cyrtonema to flooding adversity; afterwards, the malondialdehyde content decreased rapidly with the apoptosis of leaves of the flooded control WL, while the malondialdehyde content of plants treated with different concentrations of salicylic acid (SA1, SA2, SA3, level 1, level 2, and level 3, respectively) decreased slowly, showing a strong ability to resist flooding, indicating that Polygonatum cyrtonema treated with salicylic acid has a strong adaptability to membrane system peroxidation after flooding, which makes the cell membrane have a certain stability, thereby reducing the damage to the plasma membrane in water stress.

[0098] from Fig. 9 It can be seen that the substrate mixed with a high proportion of decomposed cow dung (CM1, CM2, CM3, level 1, level 2, and level 3, respectively) maintained the matrix aeration better in the early stage of flooding, which was beneficial to maintaining the normal state of cell metabolism. Therefore, the degree of membrane lipid peroxidation was significantly weaker than that of the WL treatment; and at the peak period of malondialdehyde on the 4th day, the peak value of the cow dung treatment was relatively low, indicating that the mesophyll cells were less damaged.

[0099] Polyamines are a class of low molecular weight aliphatic nitrogenous bases with biological activity. Under abiotic stress, polyamines can remove free radicals in plants and are closely related to the regulation of plant stress tolerance. They have a powerful function in resisting abiotic stress in plants and are widely used in stress resistance research. Fig.10 It can be seen that spermidine (SP1, SP2, SP3, level 1, level 2, level 3, respectively) can reduce the content of malondialdehyde during the flooding process, thereby protecting the cell membrane system from or reducing the damage of membrane lipid peroxidation.

[0100] from Fig.11 It can be seen that in this experiment, 30% decomposed cow dung as potting matrix combined with spermidine (CS1, CS2, CS3, level 1, level 2, level 3, respectively) showed the most ideal protective function of the mesophyll cell membrane system. Not only did it reduce the content of malondialdehyde, but it also showed good stress resistance in terms of peak height and appearance time. When spermidine was applied to potted Polygonatum cyrtonema in cow dung matrix, malondialdehyde slowly increased after waterlogging stress, and the peak appeared on the 8th day of waterlogging, while the waterlogged control WL treatment appeared on the 4th day after waterlogging, and the maximum malondialdehyde content was 37% lower than that of the control WL. This shows that CS treatment can effectively protect Polygonatum cyrtonema. After suffering from waterlogging stress, the CS-treated plants showed lighter symptoms of chlorosis, yellowing, and scorched leaves, and the symptoms appeared later, which is of great significance in actual production.

[0101] Uniconazole, an inhibitor of gibberellins, is a broad-spectrum azole plant growth regulator that has a strong inhibitory effect on herbaceous or woody monocotyledonous or dicotyledonous crops. As commonly used chemical growth regulators for economic crops, both uniconazole and paclobutrazol can control the excessive growth of stems and leaves, while promoting root growth and increasing the root-crown ratio. The medicinal part of Polygonatum cyrtonema is in the rhizome, and there are problems of excessive growth and lodging during cultivation, so proper chemical control and regulation can achieve the effect of killing two birds with one stone.

[0102] The mechanism of action of uniconazole is similar to that of paclobutrazol. It can regulate vegetative growth, shorten internodes, dwarf plants, promote the growth and differentiation of lateral buds and flower buds, and enhance plant resistance. Uniconazole has the advantages of small dosage, strong activity, no plant deformity, long lasting effect, and safety for humans and animals. It has been widely used in crops such as rice, wheat, corn, fruit trees, and flowers.

[0103] from Fig.12It can be seen that although uniconazole (U1, U2, U3, level 1, level 2, level 3, respectively) had a certain link stress effect at the beginning of the flooding test, the effect was not very significant; in the later stage of the flooding test, the stress resistance of uniconazole gradually came into play, not only the peak value of malondialdehyde decreased, but also the peak value was delayed. Therefore, the application of uniconazole significantly inhibited the accumulation of malondialdehyde content in the leaves of Polygonatum cyrtonema under flooding stress. Related studies have also found that uniconazole can increase the content of osmotic regulating substances, reduce the generation and accumulation of malondialdehyde, increase cell membrane permeability, maintain cell membrane stability, and enhance the ability of cell membrane to resist adversity.

[0104] 3. The role of antioxidant enzymes.

[0105] The antioxidant enzyme system of Polygonatum cyrtonema is an important system for Polygonatum cyrtonema plants to adapt to the environment, maintain normal growth and development, and defend against stress. Among them, glutathione peroxidase, catalase and peroxidase are mainly involved in the metabolic activities of Polygonatum cyrtonema leaves.

[0106] 3.1. Superoxide dismutase SOD.

[0107] Superoxide dismutase (SOD) is an antioxidant enzyme that has been widely studied in recent years. SOD is a metal-containing antioxidant enzyme that is ubiquitous in the plant kingdom. It has the function of catalyzing the dismutation of O·2- to form H 2 O 2 and O 2 , H 2 O 2 Then it is converted into H under the action of CAT, POD, etc. 2 O and O 2 , thereby reducing the toxic effects of reactive oxygen. Therefore, SOD is considered to be a key enzyme to prevent oxidative stress. After flooding stress, excessive water will cause poor soil aeration and hypoxia around the root zone of the plant. This adverse rhizosphere environment will affect the root system's absorption and transport of water and minerals, and will produce redundant reactive oxygen, including superoxide anion free radicals (Oˉ2·), hydrogen peroxide (H 2 O 2 ), hydroxyl radical (·OH) and singlet oxygen ( 1 O 2 ), which leads to an imbalance in the redox state of plant cells, is toxic to cells and may cause cell death.

[0108] Fig.13It shows that the application of urea peroxide effectively increased the activity of SOD under flooding stress, especially in the early stage of flooding. The three treatments of UP1, UP2 and UP3 all showed the ability to enhance the activity of SOD, among which the 8g dose of UP2 was the best. On the fourth day of flooding, the SOD activity of Polygonatum cyrtonema leaves treated with this treatment was significantly higher than that of CK and WL treatments, which were 53% and 25% higher than those of the latter two, respectively, thus effectively removing ROS. It can be seen that removing redundant ROS through the antioxidant enzyme system is the main mechanism for Polygonatum cyrtonema to adapt to flooding stress.

[0109] from Fig.14 It can be seen that under flooding stress, the activity of SOD in Polygonatum cyrtonema was significantly increased. 500μmol / L melatonin treatment can increase SOD activity by 55%.

[0110] from Fig.15 It can be seen that the addition of decomposed cow dung can improve the health of leaves subjected to waterlogging stress. Its internal mechanism lies in that the addition of 50% can significantly improve the SOD activity.

[0111] Fig.16 The results showed that exogenous spermidine could increase the SOD activity of Polygonatum cymbidium under waterlogging stress compared with the control throughout the 16-day stress period of this experiment, indicating that spermidine could protect the cell membrane system of Polygonatum cymbidium leaves under waterlogging stress from the attack of free radicals.

[0112] Fig.17 It shows that adding 30% decomposed cow dung to the matrix combined with foliar spraying of more than 1.0 mmol / L spermidine can keep the SOD in the leaves of Polygonatum cyrtonema at a higher activity level during flooding, and its activity is significantly higher than that of the normal management CK control and the flooding control WL.

[0113] Fig.18 It shows that under flooding stress conditions, spraying 20-40 mg / L of clomiphene citrate can promote the increase of antioxidant enzyme SOD, thereby inhibiting the accumulation of free radicals and reducing the degree of membrane lipid peroxidation. In this experiment, clomiphene citrate took effect slowly. From the 8th to the 16th day of flooding stress, the effect of clomiphene citrate on increasing leaf SOD activity was particularly obvious. Therefore, in the summer cultivation of Polygonatum cyrtonema, low doses should be arranged in advance according to the time of the rainy season to prevent waterlogging from harming Polygonatum cyrtonema.

[0114] 3.2. Superoxide dismutase POD.

[0115] from Fig.19It can be seen that urea peroxide can quickly start the stress response in the first 4 days of flooding stress and quickly increase the activity of POD, which is particularly suitable for reducing the damage of short-term waterlogging to Polygonatum cyrtonema. The use of a higher dose of 8-12g bagged controlled release measures can still increase the POD activity by 40% from the 8th to the 16th day of flooding compared with the flooding control WL treatment, that is, from 1067.34U·g -1 ·min -1 Increased to 1494.57 U·g -1 ·min -1 .

[0116] Fig. 20 The results showed that melatonin treatment enabled the leaves of Polygonatum cyrtonema to maintain a high vitality during waterlogging stress. After 2 days of stress, the POD activity of leaves treated with 100 μmol / L, 500 μmol / L and 1000 μmol / L melatonin foliar spraying was significantly higher than that of the control WL. On the 16th day of waterlogging, the POD activity of leaves of Polygonatum cyrtonema treated with 500 μmol / L melatonin was 1.5 times that of the WL treatment.

[0117] Fig.21 The results showed that salicylic acid (SA) or o-hydroxybenzoic acid salicylic acid (SA) is a plant phenolic compound and a signal molecule that plants synthesize automatically under normal conditions or under biotic and abiotic stresses. It plays a prominent role in regulating plant growth and responses to environmental stresses. For Polygonatum cyrtonema under waterlogging stress, applying 160 mg / L of salicylic acid can significantly increase the activity of POD in leaves; when the concentration increases to 240 mg / L, this positive effect will decrease.

[0118] Fig. 22 The results showed that adding 50% decomposed cow dung to the cultivation medium was beneficial to enhancing the POD activity of Polygonatum cyrtonema leaves under waterlogging stress.

[0119] from Fig.23 It can be seen that after 4 days of waterlogging stress, the POD activity of the WL flooded control plants decreased rapidly, while the POD activity of the plants treated with SP2 at a concentration of 1.0 mmol / L reached the highest, 34% higher than that of the WL treated plants; at the end of waterlogging stress, the POD activity of the plants treated with a high concentration of spermidine 1.5 mmol / L, that is, SP3, was significantly higher than that of the other treatments.

[0120] Fig.24 The results showed that the spraying of 30% decomposed cow dung combined with 1.0mmol / L spermidine showed a strong effect in improving POD activity, and the effect was maintained until the end of flooding.

[0121] Fig.25It shows that in the early stage of flooding stress, uniconazole did not significantly increase POD activity. Even on the first day of flooding stress, the POD activity of the WL flooded control plants was the strongest. As the flooding stress continued, the stress resistance of uniconazole gradually came into play. On the eighth day of flooding, the POD activity reached the highest under the 40 mg / L U2 treatment. Until the 16th day, the POD activity of the plants treated with uniconazole was significantly higher than that of the flooded control.

[0122] 3.3. Catalase CAT.

[0123] Catalase (CAT) is an antioxidant enzyme mainly found in plants and microbial cells. It is a traditional Chinese medicine physiological indicator of plant adaptation to adversity and a major component of the antioxidant defense system formed by plants during the long-term evolution process. CAT and POD are both major enzymes for removing reactive oxygen in plants.

[0124] from Fig.26 It can be seen that starting from the second day of stress, the CAT activity of plants treated with urea peroxide was significantly higher than that of plants treated with other treatments.

[0125] Fig. 27 The results showed that a moderate dose of 500 μmol / L melatonin could rapidly increase CAT activity, and as the waterlogging stress persisted, it was significantly higher than the WL control.

[0126] Fig.28 The results showed that the combination of exogenous SA and CAT can enhance the antioxidant capacity of Polygonatum cyrtonema under water stress conditions, while reducing the degree of damage to related photosynthetic pigments, thereby alleviating the degradation of enzymes related to dark reactions in the photosystem, reducing the degree of damage to the photosynthetic system, and thus improving the plant's resistance to water stress. Starting from the fourth day of flooding treatment, the CAT activity of plants treated with 160 mg / L salicylic acid was significantly higher than that of the WL flooded control plants.

[0127] Fig.29 The results showed that in this experiment, the highest mixing ratio of 50% of the decomposed cow dung matrix could effectively increase the CAT activity of Polygonatum cyrtonema leaves under waterlogging stress.

[0128] Fig.30 The results showed that under flooding stress, the CAT activity peak of Polygonatum cyrtonema plants treated with spermidine appeared on the 4th day, and there was no significant difference between the three concentrations, all significantly higher than the flooding control. However, in the late stage of flooding, the high concentration of SP3, i.e. 1.5mmol / L, performed best, and the CAT activity of the flooded plants under this treatment was the highest.

[0129] Fig.31The results showed that the treatment of mixing 30% of decomposed cow dung with foliar spraying of spermidine was the best for Polygonatum cyrtonema under waterlogging stress. During the entire waterlogging stress process, the CAT activity of Polygonatum cyrtonema under this treatment was higher than that of the control, reaching a significant level on the 2nd, 4th, 8th and 16th days; and the peak appeared on the 8th day of waterlogging. From the 4th day to the 8th day, the CAT activity was at a very high level, effectively protecting the cell membrane system of the stressed plants.

[0130] Fig.32 The results showed that in the initial stage of flooding stress, the three concentrations of uniconazole did not show significant differences from the control. After the second day, the CAT activity of the plants treated with uniconazole was significantly higher than that of the control. The differences reached extremely significant levels on days 8 and 16 of flooding. Therefore, uniconazole is an effective cultivation option for Polygonatum cyrtonema to cope with flooding in the rainy season. In this experiment, the 40 mg / L U2 treatment concentration had a very good effect, and there was no significant difference with the 80 mg / L U3 treatment concentration. In addition, it maintained a stable effectiveness in the later stage of stress.

[0131] 4. Proline.

[0132] Proline is an important amino acid that is known to participate in the biosynthesis of primary metabolism during growth and development. In addition, it is an osmotic regulating substance that reduces osmotic damage. Proline is produced in large quantities under adverse stress, and its accumulation is considered to be an osmotic adaptation mechanism. The accumulation of proline can improve waterlogging resistance and eliminate the damage caused by flooding stress.

[0133] Fig.33 It shows that under flooding stress, the proline content in leaves increases rapidly. After urea peroxide treatment, the proline content increases slowly, with a higher peak value, and then decreases in the later stage. The reason is that in the early stage of stress, the oxygen supply to the roots is sufficient due to urea peroxide, and the transmission of adversity signals is not strong, and the accumulation of proline is not quickly stimulated.

[0134] from Fig.34 It can be seen that after melatonin treatment, the proline content increased rapidly and the high content was maintained for a long time to maintain the osmotic balance of cells under stress and protect the normal function of cells.

[0135] In the case of waterlogging stress, the use of salicylic acid to address the adverse effects is very effective. Fig.35 In the plants treated with salicylic acid, proline increased rapidly. After proline in the flooded control plants began to decline rapidly on the 4th day, the salicylic acid treatment still maintained an upward trend and began to decline on the 8th day. However, the proline content was significantly higher than that in the control at this time, providing a protective effect on the stressed plants.

[0136] Fig.36The results showed that the addition of decomposed cow dung into the substrate slowed down the rate of increase in proline content, but maintained a high proline content for a longer period of time, delaying the time when mesophyll cells were damaged.

[0137] Fig.37 The results showed that spermidine can promote the accumulation of proline in leaf flesh under waterlogging stress, not only increasing the absolute content of proline from 784μg / g to 837μg / g, but also maintaining a high proline accumulation state during waterlogging stress. Waterlogging stress causes an increase in the proline content in Polygonatum cyrtonema, which plays an important role in stabilizing cell protein structure, reducing cell acidity, and removing reactive oxygen, reducing the damage caused by adverse stress to leaves, and maintaining normal growth and development of Polygonatum cyrtonema.

[0138] Fig.38 The results showed that in this experiment, Polygonatum cyrtonema accumulated a large amount of proline in its body when it was subjected to waterlogging stress, thereby improving its adaptability to adverse stress. The treatment of the combination of 30% decomposed cow dung and arginine in the substrate effectively increased the proline accumulation rate in the early stage of stress, and was able to continuously maintain a high concentration of proline. The highest value of proline reached 934μg / g, which was 12% higher than that of spermidine alone. In the experiment, the 1.0mmol / L and 1.5mmol / L spermidine doses were significantly higher than the 0.5mmol / L SP1 treatment, so the water stress relief dose can be determined as 1.0mmol / L to cope with the expected damage of waterlogging to Polygonatum cyrtonema.

[0139] Fig.39 The results showed that S3307, referred to as S3307, is a plant growth retardant that has been proven to be quite effective in practice. It is often used to regulate crop growth and development, enhance stress resistance, and improve crop resistance to environmental stresses, such as flooding, weak light, heavy metals, and saline-alkali stress. Flooding stress is one of the constraints on crop growth. Increased water content in the soil will produce hypoxic conditions in a short period of time, and anaerobic respiration and its metabolites will damage cells. S3307 can regulate the osmotic regulation of cells by regulating the content of proline, soluble protein, and soluble sugar, thereby improving the plant's ability to withstand flooding. In this experiment, under the treatment of S3307, proline accumulation increased rapidly, with a peak value of 883 μg / g on the 4th day of flooding. In the subsequent flooding test, on the 8th and 16th days, the S3307 treatment was significantly higher than the flooded control treatment WL. From this experiment, S3307 has the best sustained effect in the late stage of flooding stress.

[0140] It is worth noting that uniconazole can also improve the root-crown ratio of plants, increase the dry weight of the root system, promote the transportation of nutrients from the above-ground part to the underground part, and develop the root system of the plant. For the ecological cultivation of Polygonatum cyrtonema under the forest, there is a phenomenon of excessive stem and leaf growth during the vigorous growth season, and after 10 to 13 nodes, climbing growth occurs, and the tips of the whorled leaves are hooked, which only gathers the fallen and remotely growing branches and leaves together, creating conditions for the occurrence of spider mites, brown spot disease, mildew, etc. Therefore, before the end of July, that is, the vigorous growth season before the rainy season, spraying uniconazole with a concentration of less than 40 mg / L in advance can not only deal with waterlogging in advance, but also improve the health of Polygonatum cyrtonema and increase the economic yield of the rhizomes.

[0141] It should be noted that when the claims of the present invention involve numerical ranges, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0142] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0143] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A composition for alleviating waterlogging stress of Polygonatum cyrtonema, characterized in that: The composition consists of cooked cow dung and spermidine solution. The concentration of the spermidine solution is 0.1-1 mmol / L, and the mass volume ratio of cooked cow dung to spermidine solution is 200-300 g:10-50 ml.

2. Use of the composition described in claim 1 in alleviating waterlogging stress of Polygonatum cyrtonema.

3. Use of the composition according to claim 2 in alleviating waterlogging stress of Polygonatum cyrtonema, characterized in that: The composition is used for increasing the chlorophyll content in leaves of Polygonatum cyrtonema under waterlogging stress.

4. Use of the composition according to claim 2 in alleviating waterlogging stress of Polygonatum cyrtonema, characterized in that: The composition is used for reducing the content of malondialdehyde in leaves of Polygonatum cyrtonema under waterlogging stress.

5. Use of the composition according to claim 2 in alleviating waterlogging stress of Polygonatum cyrtonema, characterized in that: The composition is used for increasing the content of proline in leaves of Polygonatum cyrtonema under waterlogging stress.

6. Use of the composition according to claim 2 in alleviating waterlogging stress of Polygonatum cyrtonema, characterized in that: The composition is used for improving the activity of antioxidant enzymes in plant leaves to alleviate the oxidative damage caused by waterlogging stress.

7. Use of the composition according to claim 6 in alleviating waterlogging stress of Polygonatum cyrtonema, characterized in that: The antioxidant enzymes include peroxidase, superoxide dismutase and catalase.

8. A method for alleviating waterlogging stress of Polygonatum cyrtonema using the composition of claim 1, characterized in that the steps include: Before planting, apply cooked cow dung in the field, and then transplant the Polygonatum cyrtonema seedlings into the field. When the Polygonatum cyrtonema plants grow to a height of 60 to 80 cm, prepare spermidine into a 0.1 to 1 mmol / l aqueous solution and spray it evenly on the front and back sides of the plant leaves to prevent waterlogging stress.

9. The method for alleviating flooding stress of Polygonatum cyrtonema by using the composition according to claim 8, characterized in that: The amount of cooked cow dung applied in the field is 10-13 m 3 / mu.

10. The method for alleviating waterlogging stress of Polygonatum cyrtonema by using the composition according to claim 8, characterized in that: The spraying frequency is once a day, 2 to 3 ml per plant each time, and the total number of sprayings is 6 to 18 times.

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