Nitrogen fertilizer application method for promoting accumulation of gingko flavonoids, total phenols and antioxidant enzymes

Abstract

The invention discloses a nitrogen fertilizer application method for promoting accumulation of ginkgo flavonoids, total phenols and antioxidant enzymes, and relates to the technical field of plant growth promotion. The nitrogen fertilizer applying method comprises the following steps: performing trunk cutting treatment on three-year-old gingko seedlings 40 cm above the ground, performing different nitrogen form treatment, and applying a nitrification inhibitor and potassium chloride at the same time; the content of flavonoid in leaves is increased, the content of total phenols is increased, the content of antioxidant enzymes CAT / SOD / POD is increased, and the DPPH free radical scavenging capacity is improved. The result of the embodiment shows that the nitrogen fertilizer with the high nitrate-ammonium ratio promotes accumulation of gingko flavonoids and total phenols; the CAT enzyme activity, the DPPH free radical scavenging capacity and the SOD enzyme activity of the ginkgo biloba are remarkably improved by applying a nitrogen fertilizer with the ratio of nitrate to ammonium being 25: 75 and a pure ammonium nitrogen fertilizer, and all nitrogen treatment combinations have a promoting effect on the increase of the POD enzyme activity of the ginkgo biloba leaves.

Description

Technical Field

[0001] The invention belongs to the technical field of plant growth promotion, and more specifically, relates to a nitrogen fertilizer application method for promoting the accumulation of ginkgo flavonoids, total phenols and antioxidant enzymes. Background Art

[0002] Ginkgo biloba L. is the oldest relict plant among the existing gymnosperms and is known as a "living fossil". Ginkgo biloba leaves are rich in flavonoids and terpene lactone compounds and have important medicinal value. EGb761 (Ginkgo biloba extract 761) is a standardized ginkgo leaf extract, and its preparations are widely used in the fields of medicine, health products and food additives. Flavonoids are the most critical active ingredients (accounting for about 24% of its components), mainly composed of quercetin, isorhamnetin and kaempferol. [2] . Flavonoids help protect the plant from biotic and abiotic stresses through their anti-pathogen, anti-oxidant, and reactive oxygen scavenging functions, and act as regulatory and signaling compounds. They also play an important role in treating age-related cognitive decline, tinnitus, vertigo, and Alzheimer's disease in humans. Ginkgo flavonoids are difficult to chemically synthesize, and are currently mainly extracted from ginkgo leaves. Therefore, increasing the content of flavonoids is beneficial to the survival of ginkgo itself and the development of the ginkgo leaf extract pharmaceutical industry, and has great practical value and broad application prospects.

[0003] Fertilization is the most widely used agricultural technical measure, especially nitrogen fertilizer (Nitrogen), because nitrogen is an important component of proteins, nucleic acids and many secondary metabolites, and is essential for the primary and secondary metabolism of plants. The inorganic nitrogen sources that plants can directly absorb and utilize from the soil are mainly nitrate nitrogen and ammonium nitrogen, and a small amount of amide nitrogen can also be absorbed and utilized by plants. Due to the difference in morphology, nitrate nitrogen and ammonium nitrogen cause plants to have different responses to their absorption, transportation and assimilation, thereby affecting plant growth and development, biomass accumulation, secondary metabolism content and quality. A large number of studies have found that nitrogen forms and ratios are beneficial to the growth and development of most plants, and plants have a higher nitrogen utilization rate, but the optimal ratio of the two nitrogen forms varies depending on the plant species, growth environment and growth and development period. In ginkgo, studies have found that nitrogen form and ratio can significantly promote the accumulation of carbon and nitrogen compounds, increase the activity of related enzymes, and benefit the carbon and nitrogen metabolism of ginkgo [Huang Dongjing, Cao Fuliang, Wang Guibin. Effects of inorganic nitrogen forms on carbon and nitrogen metabolism in ginkgo seedlings [J]. Journal of Nanjing Forestry University (Natural Science Edition), 2019, 43(02): 197-202.] [Zheng Jun, Cao Fuliang, Yu Wanwen. Effects of different nitrogen forms and ratios on the quality of ginkgo leaves in the late growth period [J]. Journal of Zhejiang Forestry College, 2007, (05): 564-568.]. In production practice, excessive use of nitrogen fertilizers can lead to a series of environmental problems such as soil acidification, reduced biodiversity and eutrophication of water bodies. Therefore, it is of great significance to optimize the nitrogen fertilizer supply level and form to improve the nitrogen use efficiency (NUE) of ginkgo and obtain a stable yield of flavonoid compounds. Ammonium nitrogen (NH4 + ) and nitrate nitrogen (NO3 - ) is a good source of inorganic nitrogen that can be absorbed and utilized by most plants. In order to avoid the problems of single application toxicity, ion balance destruction, environmental pollution, etc. while improving nitrogen utilization efficiency, the fertilization strategy must be changed, among which the combined application of ammonium nitrate is an effective way.

[0004] Plants' adaptation to the nitrogen nutrition conditions of the long-term growth environment will affect their ability to utilize different forms of nitrogen, and then evolve preferences for different nitrogen sources, forming different nitrogen utilization strategies. In theory, when the inorganic nitrogen form supplied by the soil is consistent with the plant's nitrogen form preference, the optimal nitrogen utilization state of plants can be achieved. For example, under the same dosage conditions, supplying nitrate nitrogen to nitrate-loving crops can achieve better yield increases than ammonium nitrogen, and supplying ammonium nitrogen to ammonium-loving plants can also achieve higher nitrogen utilization efficiency. Previous studies have shown that plants prefer inorganic nitrogen sources rather than organic forms, and the nitrogen preference of plants may also be related to the need for growing plants to maintain their anion or cation balance. For example, many coniferous species such as Scots pine and European larch show strong NH4 + Preference, mainly due to NO3- The absorption of NH4 is regulated by systemic feedback caused by the increase in the concentration of its downstream metabolic product (glutamine), as well as the high energy consumption of the absorption process. Similarly, 21 dominant tree species in temperate and tropical forest ecosystems showed strong NH4 + Preference, but NH4 + The high absorption rate of NO3 will hinder the absorption of cations and cause rhizosphere acidification. Therefore, the combined application of ammonium and nitrate may be more beneficial to the growth and development of plants, mainly by promoting leaf growth, maintaining the stability of rhizosphere pH and increasing cation absorption. - The addition of can increase the synthesis and transport of cytokinins to reduce the NH4 + Toxicity, some studies have also pointed out that the combined application of ammonium nitrate increases the content of auxin rather than cytokinin.

[0005] Nitrogen metabolism provides precursors for flavonoid synthesis in the form of phenylalanine, which is also closely related to amino acid metabolism. Previous studies have shown that the nitrogen supply level and form of plants have a great influence on flavonoid synthesis, but its regulatory role has not been deeply understood. According to the growth / differentiation balance theory, plants always take into account the special needs of each developmental stage and optimize the available resources in primary or secondary metabolism.

[17] . The shikimate pathway connects plant flavonoid biosynthesis and nitrogen metabolism, catalyzing the synthesis of aromatic amino acids (phenylalanine, tyrosine and tryptophan) from carbohydrates in glycolysis and the pentose phosphate pathway. The synthesized phenylalanine enters the main synthesis pathway of flavonoids and phenolic compounds. Phenylalanine ammonia lyase (PAL) is a key enzyme connecting primary metabolism and secondary metabolism. It is very important for the metabolism of flavonoids and is also related to nitrogen nutrition because the phenylalanine pathway catalyzed by PAL releases NH4 + , thereby redistributing nitrogen. Therefore, higher NH4 + The content may reduce NH4 in plants by inhibiting the expression level of PAL + The effect of nitrogen supply form on the accumulation of flavonoids can also be explained by the carbon-nitrogen balance hypothesis, that is, the carbon-nitrogen balance in plants controls the distribution of carbon in the biosynthesis pathway of flavonoids, thereby affecting the accumulation of flavonoids.

[0006] So far, existing studies have mainly focused on the effects of nitrogen on the later growth and development of ginkgo, while the metabolic mechanisms of total phenols and flavonoids in different ammonium nitrate ratios during the vigorous growth period of ginkgo have not been studied in depth. In addition, single nitrogen fertilizer is commonly used in production practice, and there is an overdose phenomenon, ignoring the effects of nitrogen form and ratio on ginkgo growth and leaf quality. Therefore, screening out the optimal ammonium nitrate ratio suitable for high-yield and high-quality leaf ginkgo is particularly important for further exploring the metabolic mechanisms of total phenols and flavonoids in ginkgo leaves. Summary of the invention

[0007] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a method for applying nitrogen fertilizer to promote the accumulation of ginkgo flavonoids, which is used to promote the accumulation of ginkgo flavonoids. Another technical problem to be solved by the present invention is to provide a method for applying nitrogen fertilizer to promote the accumulation of ginkgo total phenols, which is used to promote the accumulation of ginkgo total phenols. Another technical problem to be solved by the present invention is to provide a method for applying nitrogen fertilizer to promote the improvement of the antioxidant capacity of ginkgo, which is used to promote the improvement of the antioxidant capacity of ginkgo.

[0008] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0009] A method for applying nitrogen fertilizer to promote the accumulation of ginkgo flavonoids, comprising:

[0010] 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground;

[0011] 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time;

[0012] 3) finally obtaining ginkgo seedlings with increased flavonoid content in leaves;

[0013] The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen;

[0014] Among them, ammonium nitrogen: nitrate nitrogen = 0-25: 75-100.

[0015] The ratio of ammonium nitrogen to nitrate nitrogen is 25:75 or 0:100; and the organic nitrogen is glycine.

[0016] A method for applying nitrogen fertilizer to promote the accumulation of total phenols in Ginkgo biloba, comprising:

[0017] 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground;

[0018] 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time;

[0019] 3) finally obtaining ginkgo seedlings with increased total phenol content in leaves;

[0020] The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen;

[0021] The ammonium nitrogen: nitrate nitrogen = 75-100: 0-25.

[0022] The ratio of ammonium nitrogen to nitrate nitrogen is 100:0 or 75:25.

[0023] A method for applying nitrogen fertilizer to promote the accumulation of ginkgo antioxidant enzyme CAT, comprising:

[0024] 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground;

[0025] 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time;

[0026] 3) finally obtaining ginkgo seedlings with increased content of antioxidant enzyme CAT;

[0027] The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen;

[0028] The ammonium nitrogen: nitrate nitrogen = 75-100: 0-25.

[0029] The ratio of ammonium nitrogen to nitrate nitrogen is 75:25 or 100:0; and the organic nitrogen is glycine.

[0030] A method for applying nitrogen fertilizer to promote the accumulation of ginkgo antioxidant enzyme SOD, comprising:

[0031] 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground;

[0032] 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time;

[0033] 3) finally obtaining ginkgo seedlings with increased content of the antioxidant enzyme SOD;

[0034] The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen;

[0035] The ammonium nitrogen: nitrate nitrogen = 100:0 or 75:25 or 50:50;

[0036] The organic nitrogen is glycine or urea.

[0037] A method for applying nitrogen fertilizer to promote the accumulation of ginkgo antioxidant enzyme POD, comprising:

[0038] 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground;

[0039] 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time;

[0040] 3) finally obtaining ginkgo seedlings with increased content of antioxidant enzyme POD;

[0041] The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen;

[0042] The ammonium nitrogen: nitrate nitrogen = 0-100: 0-100;

[0043] The organic nitrogen is glycine or urea.

[0044] The ratio of ammonium nitrogen to nitrate nitrogen is 100:0 or 75:25 or 50:50 or 20:75 or 0:100; and the organic nitrogen is glycine or urea.

[0045] A method for applying nitrogen fertilizer to promote the improvement of DPPH free radical scavenging ability of ginkgo, comprising:

[0046] 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground;

[0047] 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time;

[0048] 3) ultimately obtaining ginkgo seedlings with improved DPPH free radical scavenging ability;

[0049] The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen;

[0050] The ammonium nitrogen: nitrate nitrogen = 100:0 or 75:25 or 0:100;

[0051] The organic nitrogen is glycine or urea.

[0052] Compared with the prior art, the present invention has the following beneficial effects:

[0053] The present invention cuts the stem of 3-year-old ginkgo seedlings at 40 cm above the ground, then applies fertilizer, and simultaneously applies nitrification inhibitor and potassium chloride; finally, ginkgo seedlings with increased flavonoid content, increased total phenol content, increased antioxidant enzyme CAT / SOD / POD content, and increased DPPH free radical scavenging ability in leaves are obtained. The results of the embodiment show that low ammonium nitrate ratio nitrogen fertilizer promotes the accumulation of ginkgo flavonoids and total phenols; applying nitrogen fertilizer with an ammonium nitrate ratio of 25:75 and pure ammonium nitrogen fertilizer significantly improves ginkgo CAT enzyme activity, DPPH free radical scavenging ability and SOD enzyme activity, and all nitrogen treatment combinations have a promoting effect on the increase of ginkgo leaf POD enzyme activity. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The activity diagram of superoxide dismutase (SOD) of Ginkgo biloba under different nitrogen forms (different lowercase letters represent significant differences at the (P<0.05) level; A is 100:0, B is 75:25, C is 50:50, D is 25:75, E is 100:0, F is urea, G is glycine, and H is the control group);

[0055] Figure 2 This is a graph showing the DPPH free radical scavenging ability of Ginkgo biloba under different nitrogen forms;

[0056] Figure 3 This is the T-AOC activity diagram of Ginkgo biloba under different nitrogen forms;

[0057] Figure 4 This is a graph showing the effects of different nitrogen forms on the content of ginkgo flavonoids. DETAILED DESCRIPTION

[0058] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art.

[0059] The three-year-old ginkgo seedlings used in this application were purchased from Xuzhou City, Jiangsu Province, China. The plants grew uniformly and were disease-free. They were planted in a 28 cm plastic pot with holes at the bottom (a truncated cone-shaped pot with an upper inner diameter of 26 cm, a lower inner diameter of 21 cm, and a height of 28 cm; the potting medium was a mixture of local yellow loam: nutrient soil = 2:1 (V / V)), with one plant planted in each pot. They were placed in an artificial climate room at Xiashu Forest Farm, Nanjing Forestry University (32°7′N, 119°13′E). After the stem was cut 40 cm above the ground, normal maintenance was performed.

[0060] As shown in Table 1, seven different nitrogen forms were set up (five groups of inorganic nitrogen treatments AE, nitrate nitrogen (NO3 - ) is provided by potassium nitrate, ammonium nitrogen (NH4 + ) provided by ammonium sulfate; 2 groups of organic nitrogen treatments (FG, urea and glycine) and 1 group of blank control (H), a total of 8 treatments, each treatment 15 plants, every 5 plants as a biological replicate. The nitrogen application rate was 5.55g / plant, each treatment was fertilized together, divided into 3 times, and the day of application completion was the start day of the treatment (the 3 times of fertilization were 7.2, 7.12, and 7.22, respectively). At the same time, the nitrification inhibitor dicyandiamide (DCD) was applied (the amount was 7% of the nitrogen application rate, 0.3885g / plant) to ensure K + The supply amount was consistent, and each treatment was made up with KCl.

[0061] Each treatment was placed in the same seedbed and maintained the same management measures. Sampling was performed on the 20th, 40th, 60th and 80th day after the start of the treatment. The samples were quickly frozen in a dry ice sampling box and brought back to the laboratory and stored in a -80℃ ultra-low temperature refrigerator for subsequent physiological index determination.

[0062] Table 1 Experimental design

[0063]

[0064] Note: The table shows the total dosage (g) for each treatment of 15 seedlings.

[0065] The experimental data of this application were calculated and statistically analyzed using Excel software and IBM SPSS Statistics 22.0 software (IBM, NY, USA), and the Duncan multiple comparison method was used to test the significance of the difference. The test data were expressed as mean ± SDs (n = 3) and plotted using Origin 2022 (Origin Lab Inc., USA).

[0066] Example 1 Effect of nitrogen form on antioxidant enzymes in Ginkgo biloba

[0067] 1. Catalase (CAT) activity

[0068] The catalase (CAT) assay kit of Nanjing Jiancheng Bioengineering Institute was used to determine the CAT enzyme activity (ammonium molybdate method). Accurately weigh the tissue weight, add 9 times the volume of physiological saline at a ratio of weight (g): volume (mL) = 1:9, prepare a 10% tissue homogenate under ice water bath conditions, centrifuge at 2500r / min for 10min, and take the supernatant. Mix the tissue homogenate and reagent in the control tube and the test tube according to the operation table, and use a spectrophotometer at a wavelength of 405nm, a light path of 0.5cm, and double distilled water to zero, and measure the absorbance value A of each tube.

[0069]

[0070] Note: 271 is the reciprocal of the slope, a constant, used directly; V 样 : Sampling volume, 0.05mL; V 样总 : total sample volume, mL; T: reaction time, 60 seconds; C Hb : Plant mass concentration: plant mass (g) / total volume of extract (mL); W: tissue fresh weight; △A=A control-A determination.

[0071] The results are shown in Table 2. The single nitrate treatment group A and the group B with an ammonium nitrate ratio of 75:25 showed a downward trend over time, and the overall trend of the other treatment groups was first decreased and then increased. At the 20-day period, the results of variance analysis showed that the CAT activity of each group was significantly different (P<0.01), and the CAT activity of each group decreased with the decrease of the nitrate nitrogen ratio; the CAT activity value of group G was the largest, which increased by 22.50% compared with the control group, significantly higher than the control and other groups, and there was no significant difference in groups D and E and significantly lower than the control and other groups. At the 40-day period, there was no difference in CAT activity among the groups (P>0.05), and fertilization increased the catalase activity of the group, and the CAT activity of each group was greater than that of the control. At the 60-day period, the results of variance analysis showed that the CAT activity of each group was significantly different (P<0.01); the results of multiple analysis showed that the CAT activity of groups A and B was significantly higher than that of the control group, 3.29 times and 3 times that of the control group, respectively; there was no difference between the other groups and the control (P>0.05). At 80 days, the results of variance analysis showed that there were significant differences in CAT activity among the groups (P<0.01). At this time, fertilization had a negative effect on the CAT enzyme activity of Ginkgo biloba leaves, and each group decreased compared with the control group; there was no significant difference between group E and the control (P>0.05), and the other groups were significantly lower than group E (P<0.05).

[0072] Table 2 Effects of different ammonium nitrate ratios on catalase (CAT) in Ginkgo biloba

[0073]

[0074]

[0075] Note: ① Different lowercase letters in the table represent significant differences at the (P<0.05) level; ② A (100:0), B (75:25), C (50:50), D (25:75), E (100:0), F (urea), G (glycine), and H are control groups; ③ 20d, 40d, 60d, and 80d are treatment days, the same below.

[0076] 2. Total superoxide dismutase (SOD) content

[0077] The total superoxide dismutase (SOD) assay kit of Nanjing Jiancheng Bioengineering Research Institute was used to determine the SOD enzyme content in the sample (hydroxylamine method). Accurately weigh the plant tissue at a weight (g): volume (mL) ratio of 1:4, add 4 times the volume of phosphate buffer, prepare a 20% homogenate under ice water bath conditions, 4000 (r / min), centrifuge for 10 minutes, and take the supernatant for determination. According to the operation table, the operation tube and the reagent are fully mixed with a vortex mixer in proportion, placed in a 37°C constant temperature water bath for 40 minutes to mix, and after leaving at room temperature for 10 minutes, use a spectrophotometer at a wavelength of 550nm, use distilled water to zero and perform colorimetry. The conversion formula based on the wet weight of the tissue is:

[0078]

[0079] The results are as follows Figure 1 As shown in the results, except for group F, the effects of different ammonium nitrate ratios on the SOD enzyme activity in ginkgo leaves generally showed a trend of increasing first and then decreasing over time; the SOD enzyme activity in group F showed a trend of gradually increasing over time. At 20 days, the results of variance analysis showed that the SOD enzyme activity of each group was different (P<0.05); the SOD enzyme activity of group A was the highest, increasing by 11.46% compared with the control, and the SOD enzyme activity of group D was the lowest, decreasing by 4.34% compared with the control; the results of multiple comparison analysis showed that the SOD enzyme activity of group A was significantly higher than that of the control, and the SOD enzyme activity of groups D and E was significantly lower than that of the control. At 40 days, the SOD enzyme activity of each group was different (P<0.05), and the SOD activity of group D decreased by 4.84%; the results of multiple comparison analysis showed that the SOD activity of groups A and B was better than that of the control. At 60 days, the results of variance analysis showed that the SOD enzyme activity of each group was significantly different (P<0.05); Group A was the group with the highest SOD enzyme activity, which increased by 19.24% compared with the control group. Multiple analysis showed that Group A was the group with the best SOD enzyme activity. At 80 days, the results of variance analysis showed that the SOD enzyme activity of each group was significantly different (P<0.01); Group E had the lowest SOD enzyme activity, which decreased by 5.04% compared with the control group, and Group A had the highest SOD enzyme activity, which increased by 24.35% compared with the control group; the results of multiple comparison analysis showed that Group A had the best SOD enzyme activity.

[0080] 3. Peroxide reductase (POD) content

[0081] The peroxidase (POD) assay kit of Nanjing Jiancheng Bioengineering Institute was used to determine the POD enzyme content in the sample. Accurately weigh the plant tissue at a weight (g): volume (mL) ratio of 1:9, add 9 times the volume of phosphate buffer, prepare a 10% homogenate under ice water bath conditions, 3500 (r / min), centrifuge for 10 minutes, and take the supernatant for determination. According to the operation table, the operation tube and the reagent are fully mixed with a vortex mixer in proportion, placed in a 37°C constant temperature water bath for 40 minutes to mix, and after leaving at room temperature for 10 minutes, use a spectrophotometer at a wavelength of 420nm, use distilled water to zero and perform colorimetry. The conversion formula based on the wet weight of the tissue is:

[0082]

[0083] Note: V 样 : Sampling volume, 0.05mL; V 样总 : Total sample volume, mL; V 反总 : total reaction volume, mL; T: reaction time, 60 seconds; W: tissue fresh weight.

[0084] The results are shown in Table 3. The POD activity of groups B, C and the control group showed an upward trend over time, while the other groups showed a trend of first increasing and then decreasing over time. The results of variance analysis showed that the data of POD activity in each group at the 20-day stage were significantly different (P<0.01). The POD activity values ​​of the fertilized groups were greater than those of the control group. Among them, the POD activity of group A increased by 1.4 times compared with the control, which was the highest value of POD activity among all groups. The results of multiple comparison analysis showed that the POD enzyme activity of group A was better. The results of variance analysis showed that the data of POD activity in each group at the 40-day stage were significantly different (P<0.01). Fertilization increased the content of ginkgo peroxidase, and each group was higher than the control; among them, group D increased by 71.20%, which was the group with the largest POD activity; the results of multiple comparison analysis showed that groups B and D were significantly better than the control group. The results of variance analysis showed that there was no difference in the data of each group at the 60-day stage. The POD activity of group A was the largest, which increased by 98.08% compared with the control, and the POD activity of group C was the smallest, which decreased by 21.16% compared with the control. The results of variance analysis showed that there were differences in the POD activity data of each group at the 80d stage (P<0.05). The POD activity of the single ammonium nitrogen fertilizer culture group E and the urea group was lower than that of the control group H, which decreased by 3.67% and 7.33% respectively, while the other groups were higher than the control. The group with the highest POD activity was group C, which increased by 57.59% compared with the control. The results of multiple comparison analysis showed that the POD activity value of group C was significantly better than that of the control and other groups.

[0085] Table 3 Effects of different nitrogen forms on peroxidoreductase (POD) in Ginkgo biloba leaves

[0086]

[0087]

[0088] Example 2 Effect of nitrogen form on the total antioxidant capacity of Ginkgo biloba

[0089] 1. DPPH free radical scavenging ability

[0090] The DPPH free radical scavenging ability was determined using the kit of Nanjing Jiancheng Bioengineering Institute (spectrophotometry). Weigh about 0.1g of Ginkgo biloba leaves, cut them into pieces, add 1mL of 80% methanol solution, homogenize them in an ice-water bath, centrifuge for 10min, and refrigerate the supernatant. According to the operation table, mix the sample 80% methanol solution and the working solution, mix them well, centrifuge them at 4000r / min for 5min, and at a wavelength of 517nm, draw 800μL into a cuvette, adjust to zero with 80% methanol, and measure the absorbance value of each tube.

[0091] Sample DPPH free radical scavenging rate (%) = (1-(A measurement-A control) ÷ A blank) × 100%.

[0092] The results are as follows Figure 2 As shown in the figure, except for the F treatment group, the DPPH scavenging ability of ginkgo in the other groups showed a trend of first decreasing and then increasing with the change of treatment time during the whole treatment period, while the F treatment showed an increasing trend with time. During the 20-day period, the DPPH scavenging ability of treatment A was the highest, followed by group B, which increased by 29.10% and 19.94% respectively compared with the control group, and the DPPH scavenging ability of treatment C was the lowest, which decreased by 4.22% compared with the control group; the results of variance analysis showed that there were differences in the DPPH scavenging ability of each group (P<0.05); the results of multiple comparison analysis showed that the DPPH scavenging ability of groups A, B, E, and G was significantly higher than that of the control group, and that of groups C and D was significantly lower than that of the control group. During the 40-day period, the DPPH scavenging ability of treatment B was the highest, which increased by 13.50% compared with the control group, and the DPPH scavenging ability of treatment E was the lowest, which decreased by 13.38% compared with the control; there was no significant difference in the DPPH scavenging ability of each group (P>0.05). At 60 days, the DPPH scavenging ability of treatment B was the highest, which increased by 6.88% compared with the control group, and the DPPH scavenging ability of treatment E was the lowest, which decreased by 16.05% compared with the control group; there was no significant difference in DPPH scavenging ability among the groups (P>0.05). At 80 days, the DPPH scavenging ability of treatment A was the highest, which increased by 5.35% compared with the control group, and the DPPH scavenging ability of treatment E was the lowest, which decreased by 21.37% compared with the control group; there were differences in DPPH scavenging ability among the groups (P<0.05); the results of multiple comparison analysis showed that the DPPH scavenging ability of treatments C, D, E, and G was significantly lower than that of the control, and there was no significant difference between the other treatment groups and the control.

[0093] 2. Total Antioxidant Capacity (T-AOC)

[0094] The total antioxidant capacity (T-AOC) test kit of Nanjing Jiancheng Bioengineering Institute was used for determination (ABTS method). After accurately weighing the tissue, add 9 times the volume of physiological saline at a ratio of weight (g): volume (mL) = 1:9, mechanically homogenize under ice water bath conditions, 12000r / min, centrifuge for 5 minutes, and take the supernatant for determination. The protein concentration was determined using the A045-2 Coomassie Brilliant Blue protein quantitative test kit. According to the operation table, the reagents were mixed evenly and reacted at room temperature for 6 minutes, with a wavelength of 405nm. The OD value of each well was read by the microplate reader. The standard curve was made with the OD value of the standard as the horizontal axis and the concentration of the standard corresponding to each OD value as the vertical axis. The curve formula was obtained using the graphing software, and the OD measured by the sample test tube was substituted to obtain the result.

[0095] The results are as follows Figure 3 As shown in the figure, the effect of different ammonium nitrate ratios on the T-AOC activity of Ginkgo showed an upward trend as time went by, except for the E and G groups. The E and G groups showed a trend of first decreasing and then increasing as time went by. In the 20-day period, the results of variance analysis showed that the T-AOC activity of each group was different (P<0.05); the T-AOC activity of group A was significantly higher than that of other groups, and the T-AOC activity of this group increased by 8.08%, while the T-AOC activity of group D was significantly lower than that of other groups, decreasing by 11.25%; the results of multiple comparison analysis showed that there was no significant difference between group A and the control group (P>0.05), and the other groups were significantly lower than the control group. In the 40-day stage, fertilization treatment reduced the T-AOC activity of Ginkgo, and the T-AOC activity of each group was lower than that of the control group; the results of variance analysis showed that the T-AOC activity of Ginkgo leaves was significantly different among the groups during the 40-day treatment period (P<0.05). At the 60-day stage, the T-AOC activity of ginkgo in all groups was lower than that of the control group, and the T-AOC activity of ginkgo in groups D, E, and G was inhibited; the results of multiple comparison analysis showed that the T-AOC activity of D, E, and G was significantly lower than that of the control group, decreasing by 12.12%, 20.79%, and 22.16%, respectively. At the 80-day stage, the results of variance analysis showed that there was no significant difference among the groups, the T-AOC activity of group B was the highest, increasing by 1.56%, and the T-AOC activity of group E was the lowest, decreasing by 13.96% compared with the control; in general, because groups E and G showed a trend of first decreasing and then increasing with time, excessive organic nitrogen fertilizer had a damaging effect on the T-AOC activity of ginkgo, and the treatment group with a single nitrate nitrogen fertilizer and ammonium nitrate ratio of 75:25 had a negative effect on the T-AOC activity of ginkgo.

[0096] Example 3 Effect of nitrogen form on chlorophyll fluorescence parameters of Ginkgo biloba

[0097] The fluorescence parameters of Ginkgo leaves were measured using an FMS-2 portable pulse-modulated fluorimeter (Hansha Scientific Instruments), including the effective photochemical quantum yield (Fv' / Fm'), the maximum photochemical quenching coefficient (qP), the non-photochemical quenching coefficient (qNP), the non-photochemical quenching (NPQ), the electron transfer efficiency (ETR), the actual photochemical efficiency (ΦPSⅡ) and the maximum chemical efficiency of PSⅡ under dark adaptation (Fv / Fm).

[0098] The results are shown in Table 4. Fv' / Fm' and Fv / Fm reflect the maximum photochemical efficiency of PSⅡ under dark reaction and light reaction, respectively. The results of variance analysis showed that there were significant differences in Fv' / Fm' and Fv / Fm among the groups (P<0.01). Fertilization reduced the maximum photochemical efficiency of Ginkgo biloba leaves under light reaction and dark reaction conditions. The Fv / Fm and Fv' / Fm' of the single ammonium nitrogen fertilizer group E performed better. The results of multiple comparison analysis showed that the Fv / Fm and Fv' / Fm' of the E group were significantly better than those of the other groups, but still less than those of the control group. The results of variance analysis showed that the Fv / Fm and Fv' / Fm' of the E group were significantly better than those of the other groups, but still less than those of the control group. There was a significant difference (P<0.01). The actual photosynthetic efficiency of each group was lower than that of the control group; the results of multiple comparison analysis showed that there was no significant difference between group E and the control group (P>0.05), and the other treatment groups were significantly lower than group E and the control group (P<0.05). qP reflects the ability of plant leaves to convert light energy into chemical energy (i.e., ATP and NADPH used in the dark reaction phase of photosynthesis) under given light conditions. The results of variance analysis showed that qP of each group was significantly different (P<0.01); the results of multiple comparison analysis showed that there was no significant difference between group E and the control group (P>0.05), and the other treatment groups were significantly lower than group E and the control group (P<0.05). The electron transfer rate ETR directly reflects the efficiency of the light reaction of photosynthesis. The higher the ETR, the higher the efficiency of the photosynthetic light reaction, and the plants can use light energy for photosynthesis more effectively. The results showed that fertilization was not conducive to the photosynthetic electron transfer of ginkgo leaves, and the ETR of each group was lower than that of the control. The results of variance analysis showed that the ETR value (representing PSⅡ) had significant differences in the photosynthetic electron transfer rate (P<0.01). The results of multiple comparison analysis showed that there was no significant difference between group E and the control group (P>0.05), and the other treatment groups were significantly lower than group E and the control group (P<0.05).

[0099] Table 4 Chlorophyll fluorescence parameters of Ginkgo biloba leaves under different nitrogen forms

[0100]

[0101] Example 4 Effect of nitrogen form on soluble sugars and total phenols in Ginkgo biloba

[0102] 1. Soluble sugar content

[0103] The soluble sugar content was determined using the kit of Nanjing Jiancheng Bioengineering Research Institute (anthrone colorimetric method). The operation was performed according to the instructions of the kit. In short, 0.2 g of fresh plant leaves were weighed, 2 mL of distilled water was added, and the mixture was ground into a homogenate and poured into a centrifuge tube. The mixture was placed in a boiling water bath for 10 min, cooled and centrifuged at room temperature for 10 min (4000 r / min), and the supernatant was diluted 10 times with distilled water, and the required sample supernatant was prepared after shaking. Then, the sample supernatant, substrate solution and concentrated sulfuric acid were added in sequence according to the operation table. After the reaction, the absorbance of the sample tube, standard tube and blank tube was measured at a wavelength of 620 nm to calculate the soluble sugar content.

[0104]

[0105] Note: ΔA = A test tube - A blank tube; W is the fresh weight of tissue, g; V 提 is the total volume of the extract (distilled water) added, mL; N is the dilution multiple of the sample before testing; C 标准 is the standard sample concentration, 100 μg / mL.

[0106] The results are shown in Table 5. The overall change of soluble sugar with time showed a trend of first rising and then falling. The soluble sugar content of groups D, C and F rebounded at the end of the experiment. At the 20-day period, the results of variance analysis showed that the soluble sugar content of each group was significantly different (P<0.01), and the soluble sugar content of each group was less than that of the control. The negative effects of treatment groups D and E on the soluble sugar content of Ginkgo biloba were relatively small, decreasing by 6.83% and 5.17% respectively. The soluble sugar content of Ginkgo biloba in treatment group C was the smallest, decreasing by 23.5%. There was no difference in the soluble sugar content of each group at the 40-day period (P>0.05), and the soluble sugar content of each group was ranked as: B>G>C>F>E>A. There was no difference in the soluble sugar content of each group at the 60-day period (P>0.05). The soluble sugar content of group E was the highest, increasing by 3.33%, while that of group C decreased by 13.97%, which was the group with the lowest soluble sugar content. There was no difference in soluble sugar content among the groups at 80 days (P>0.05), and the soluble sugar content of each group was lower than that of the control. The soluble sugar content of each group was ranked as follows: F>D>G>E>C>B>A.

[0107] Table 5 Effects of different nitrogen forms on soluble sugars in Ginkgo biloba

[0108]

[0109]

[0110] 2. Total plant phenol content

[0111] The total plant phenols were determined using the Nanjing Jiancheng Bioengineering Research Institute's total plant phenols detection kit (spectrophotometry). Grind fresh ginkgo leaves into powder under liquid nitrogen freezing conditions, weigh about 0.1g of powder, add 2mL of extract, and mix. Extract at 60℃ for 30min, centrifuge at 4000r / min for 10min, and take the supernatant for testing. Add sample supernatant, reagent and distilled water in sequence according to the operation table. Mix the liquid in the tube. Measure the absorbance of the sample tube, standard tube and blank tube at a wavelength of 760nm, and then calculate the total plant phenol content.

[0112] The results are shown in Table 6. The total phenol content of group E showed a trend of increasing first and then decreasing over time, while that of group G showed a trend of decreasing and then increasing. The total phenol content of other treatment groups showed an overall upward trend over time. There was no difference in the total phenol content of each group during the 20-day period (P>0.05), and the total phenol content of each group was less than that of the control. The total phenol content of groups A and G was higher, which was 1.96% lower than that of the control, while the total phenol content of group C was the lowest, which was 17.3% lower than that of the control. There were differences in the total phenol content of each group during the 40-day period (P<0.05). The total phenol content of group G was the highest, which increased by 15.19% compared with the control, followed by the total phenol content of group C, which increased by 12.4% compared with the control, and the total phenol content of group B was the lowest, which decreased by 7.19% compared with the control. There was no difference in the total phenol content of each group during the 60-day period (P>0.05), and the total phenol content of each group was ranked as follows: B>C>F>A>D>E>G. There was no difference in the total phenol content among the groups at 80 days (P>0.05). The total phenol content of group B was the highest, which increased by 1.52% compared with the control. The total phenol content of group E was the lowest, which decreased by 16.92%. The effects of different groups on the total phenol content of ginkgo were different at different times. In general, single nitrate nitrogen culture was not conducive to the formation of total phenolic substances in ginkgo leaves, and fertilizers with low nitrate-ammonium ratio had a positive effect on the total phenol content of ginkgo.

[0113] Table 6 Effects of different nitrogen forms on the total phenol content of Ginkgo biloba

[0114]

[0115]

[0116] Example 5 Effect of Nitrogen Form on Ginkgo Flavonoid Content

[0117] The flavonoid content was determined using a kit from Nanjing Jiancheng Bioengineering Research Institute (colorimetric method). The sample was dried to constant weight and then crushed. About 0.02 g of the sample was weighed, 2 mL of the extract was added, and the sample was shaken and extracted at 60 ° C for 2 hours. After cooling, the sample was centrifuged at room temperature for 10 minutes, and the supernatant was taken. The blank tube, the test tube, and the standard tube were compared with the standard curve using a spectrophotometer at 502 nm to calculate the flavonoid content in the sample.

[0118] Flavonoid content (mg / g) = (ΔA 测定 -0.0141)÷9.0771×total volume of extract (2mL)÷sample mass (g).

[0119] The results are as follows Figure 4 As shown in the figure, the effect of different ammonium nitrate ratios on the content of ginkgo flavonoids showed an upward trend with time, except for the E and G groups, which showed a trend of first decreasing and then increasing with time. There were extremely significant differences among the treatment groups at 20 days (P<0.01); the results of multiple comparison analysis showed that the content of flavonoids in the E group was significantly higher than that in the control group, reaching 3.07 mg·g -1 , an increase of 39.18%, while the content of flavonoids in group C was the least and significantly less than that in the control, a decrease of 12.6% compared with the control. The results of variance analysis showed that there was no significant difference among the groups during the 40-day period, and the content of flavonoids in group E was the highest, reaching 2.85 mg·g -1 , which was 42.22% higher than the control; G treatment had a negative effect on the flavonoid content, which decreased by 9.31% compared with the control group, and was the treatment group with the lowest flavonoid content in the 40d period. The results of variance analysis showed that there was no significant difference in the flavonoid content between the groups and the control group in the 60d period. The flavonoid content of group B was the largest, which increased by 11.1% compared with the control; the flavonoid content of group G was the smallest, which decreased by 13.63% compared with the control group. According to variance analysis, there was no significant difference among the groups in the 80d period (P>0.05). The flavonoid content of group A was the largest, which increased by 20.98% compared with the control, and the flavonoid content of group E was the smallest, which decreased by 15.33% compared with the control.

[0120] In summary, high ammonium nitrate ratio nitrogen fertilizer promoted the accumulation of flavonoids and total phenols in Ginkgo biloba. The application of nitrogen fertilizer with an ammonium nitrate ratio of 25:75 and pure ammonium nitrogen fertilizer significantly increased the CAT enzyme activity, DPPH free radical scavenging ability and SOD enzyme activity of Ginkgo biloba. All nitrogen treatment combinations promoted the increase of POD enzyme activity in Ginkgo biloba leaves. Nitrogen fertilizer application reduced the T-AOC activity of Ginkgo biloba, especially pure nitrate nitrogen fertilizer and the treatment group with an ammonium nitrate ratio of 75:25 had a significant negative effect on the T-AOC activity of Ginkgo biloba. Single ammonium and low ammonium nitrate ratio nitrogen fertilizers were not conducive to the formation of soluble sugars in Ginkgo biloba.

[0121] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A method for applying nitrogen fertilizer to promote the accumulation of ginkgo flavonoids, characterized in that: include: 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground; 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time; 3) finally obtaining ginkgo seedlings with increased flavonoid content in leaves; The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen; Among them, ammonium nitrogen: nitrate nitrogen = 0-25: 75-100.

2. The method for applying nitrogen fertilizer to promote the accumulation of ginkgo flavonoids according to claim 1, characterized in that: The ratio of ammonium nitrogen to nitrate nitrogen is 25:75 or 0:100; and the organic nitrogen is glycine.

3. A method for applying nitrogen fertilizer to promote the accumulation of total phenols in Ginkgo biloba, characterized in that: include: 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground; 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time; 3) finally obtaining ginkgo seedlings with increased total phenol content in leaves; The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen; The ammonium nitrogen: nitrate nitrogen = 75-100: 0-25.

4. The method for applying nitrogen fertilizer to promote the accumulation of total phenols in Ginkgo biloba according to claim 3, characterized in that: The ratio of ammonium nitrogen to nitrate nitrogen is 100:0 or 75:

25.

5. A method for applying nitrogen fertilizer to promote the accumulation of ginkgo antioxidant enzyme CAT, characterized in that: include: 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground; 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time; 3) finally obtaining ginkgo seedlings with increased content of antioxidant enzyme CAT; The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen; The ammonium nitrogen: nitrate nitrogen = 75-100: 0-25.

6. The method for applying nitrogen fertilizer to promote the antioxidant capacity of ginkgo according to claim 5, characterized in that: The ratio of ammonium nitrogen to nitrate nitrogen is 75:25 or 100:0; and the organic nitrogen is glycine.

7. A method for applying nitrogen fertilizer to promote the accumulation of Ginkgo antioxidant enzyme SOD, characterized in that: include: 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground; 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time; 3) finally obtaining ginkgo seedlings with increased content of the antioxidant enzyme SOD; The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen; The ammonium nitrogen: nitrate nitrogen = 100:0 or 75:25 or 50:50; The organic nitrogen is glycine or urea.

8. A method for applying nitrogen fertilizer to promote the accumulation of Ginkgo antioxidant enzyme POD, characterized in that: include: 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground; 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time; 3) finally obtaining ginkgo seedlings with increased content of antioxidant enzyme POD; The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen; The ammonium nitrogen: nitrate nitrogen = 0-100: 0-100; The organic nitrogen is glycine or urea.

9. The method for applying nitrogen fertilizer to promote the antioxidant capacity of ginkgo according to claim 8, characterized in that: The ratio of ammonium nitrogen to nitrate nitrogen is 100:0 or 75:25 or 50:50 or 20:75 or 0:100; and the organic nitrogen is glycine or urea.

10. A method for applying nitrogen fertilizer to promote the improvement of the DPPH free radical scavenging ability of Ginkgo biloba, characterized in that: include: 1) Cut the stems of three-year-old ginkgo seedlings 40 cm above the ground; 2) Applying nitrogen fertilizer to the ginkgo seedlings after the stem cutting treatment, and applying nitrification inhibitors at the same time; 3) ultimately obtaining ginkgo seedlings with improved DPPH free radical scavenging ability; The nitrogen form used in the nitrogen fertilizer is selected from ammonium nitrogen, nitrate nitrogen, and organic nitrogen; The ammonium nitrogen: nitrate nitrogen = 100:0 or 75:25 or 0:100; The organic nitrogen is glycine or urea.

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