Soil conditioner for improving volatile oil content of ligusticum wallichii as well as preparation method and application of soil conditioner
By using soil improvement agents containing organic soil conditioning agents and pathogenic fungus-induced dry powder in Chuanxiong planting, the problem of insufficient volatile oil content of Chuanxiong is solved, and the content and yield of the medicinal materials are significantly improved, and the sustainable development of the Chuanxiong industry has been achieved.
Patent Information
- Application Number
- CN202411717184.3
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2024-11-27
- Publication Date
- 2025-02-25
AI Technical Summary
The prior art is difficult to effectively increase the content of volatile oil of Chuanxiong, which affects the efficacy and yield of medicinal materials.
A soil modification agent is used, including organic soil conditioner, T. acupuncture, Rhizobium induced dried powder, Bacillus licheniformis, Bacillus jelly and Bacillus subtilis. Through the combination of these components, the defense response of plants and the activation of soil microorganisms are stimulated, and the accumulation of volatile oils in Chuanxiong is promoted.
It significantly increased the total volatile oil content of Chuanxiong, especially the content of phenyphthalene components, stabilized the content of ferulic acid, improved the quality and yield of medicinal materials, and ensured the efficacy of Chuanxiong medicinal materials.
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of chuanxiong planting, and in particular to a soil conditioner for increasing the volatile oil content of chuanxiong, and a preparation method and application thereof. Background Art
[0002] Ligusticum chuanxiong Ligusticum chuanxiong The dried rhizome of Hort. As a famous Sichuan native medicinal material, it is currently mainly distributed in Pengzhou, Shifang and Dujiangyan. Chuanxiong is a widely used bulk medicinal material with the effects of promoting blood circulation and qi, dispelling wind and relieving pain, antibacterial and anti-thrombotic. Chuanxiong is rich in volatile oil, which is the main active ingredient. Pharmacological studies have found that Chuanxiong volatile oil has sedative, analgesic, anti-inflammatory, antioxidant, anti-tumor, bone protection and anti-depression effects. It is also a common and excellent Chinese medicine volatile oil penetration enhancer.
[0003] The volatile oil of Chuanxiong mainly includes phthalides, terpenes, amides, aliphatic, aromatic and other types, among which phthalides have the highest content and are the characteristic components of Chuanxiong. The phthalides in Chuanxiong are mainly ligustilide and ligustilide, and the content of ligustilide can account for 50%~80% of the total volatile oil of Chuanxiong. The pharmacological mechanism study shows that the phthalides Z-ligustilide, ligustilide I and ligustilide A in Chuanxiong can pass through the blood-brain barrier, which is the material basis of the efficacy of Chuanxiong volatile oil in treating headaches, and Z-ligustilide plays an important role in protecting nerve cells, bone protection, anti-depression, anti-tumor, anti-inflammatory and other aspects. Phthalide substances are the main indicator components of blood-activating Chinese medicine. By increasing the content of volatile oil, the efficacy of Chuanxiong in promoting blood circulation and removing blood stasis, promoting qi and relieving pain can be enhanced. Therefore, research on improving the volatile oil content of Chuanxiong is of great significance to ensure the clinical efficacy of Chuanxiong.
[0004] The effective ingredients of Chuanxiong are closely related to the ecological environment. As the traditional authentic production area of Chuanxiong, Dujiangyan produces Chuanxiong with stable phthalide content and ratio, and higher ligustilide content than other production areas. This is the chemical characterization of the authenticity of Chuanxiong and an important guarantee of the authenticity of Chuanxiong. The unique soil environment and climatic conditions of the authentic production area have produced high-quality Chuanxiong. However, with the large-scale and continuous cultivation of Chuanxiong, soil problems have also emerged. Soil acidification and imbalance of the microecological environment have affected the production and quality of Chuanxiong. The area of traditional authentic production areas has gradually shrunk. At present, the production of Chuanxiong is mainly concentrated in Pengzhou City and Shifang City. The production of Chuanxiong in this area accounts for more than 90% of the total production of Chuanxiong, and it is currently the main authentic production area of Chuanxiong. Summary of the invention
[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a soil conditioner for increasing the content of volatile oil in Rhizoma Chuanxiong and a preparation method and application thereof.
[0006] The objective of the present invention is achieved through the following technical solutions: A soil conditioner for increasing the volatile oil content of Chuanxiong comprises the following components by weight: 991.5-993.8 parts of an organic soil conditioner, 2-4 parts of 1 billion / g Trichoderma spinulosa, 0.2-1.7 parts of dry powder of Rhizoctonia solani inducer, 0.5-1.5 parts of 10 billion / g Bacillus licheniformis, 0.5-2 parts of 10 billion / g Bacillus gellingii, and 1-2 parts of 10 billion / g Bacillus subtilis.
[0007] The organic soil conditioner comprises the following components in parts by mass: 490-600 parts of rice husk powder, 192-290 parts of humic acid, 41-63 parts of extracellular polysaccharide, 105-139 parts of crude protein, 14-27 parts of crude fat and 1-3 parts of glutamic acid. The raw materials are uniformly mixed to obtain the organic soil conditioner.
[0008] The dry powder of the inducer of Rhizoctonia solani is prepared by the following steps: mycelium of Rhizoctonia solani is inoculated on a PD culture medium with a pH value of 7, and fermented at a temperature of 28-32°C for 6 days, and then the cultured bacteria and the culture medium are separated to obtain a fungal fermentation liquid without bacteria, and the fermentation liquid is inactivated at high temperature, filtered and concentrated, and the concentrated solution is dried to obtain the dry powder of the inducer of Rhizoctonia solani.
[0009] It also includes an early stage Chuanxiong seed dressing agent. Calculated by weight, the raw materials of the Chuanxiong seed dressing agent include the following components: 630-320 parts of dry powder of inducer of Fusarium oxysporum, 276-390 parts of island indole, and 55-290 parts of methyl jasmonate. The raw materials are evenly mixed to obtain the product.
[0010] The dry powder of Fusarium oxysporum elicitor is prepared by the following steps: spores of Fusarium oxysporum are prepared into 2.3*10 7 A single spore suspension of CFU / mL is inoculated onto a PD culture medium at an inoculation rate of 3%, the pH of the culture medium is set between 6.0 and 6.5, and after culturing and fermenting at 25-30°C for 7 days, the cultured bacteria and the culture medium are separated to obtain a fungal fermentation liquid without bacteria, the fermentation liquid is inactivated at high temperature, filtered and concentrated, and the concentrated solution is dried to obtain a dry powder of Rhizoctonia solani inducer.
[0011] The second aspect of the present invention provides a method for preparing a soil conditioner containing volatile oil of Ligusticum chuanxiong, wherein the organic soil conditioner, Trichoderma acanthosporum, dry powder of elicitor of Rhizoctonia solani, Bacillus licheniformis, Bacillus gellinga and Bacillus subtilis are uniformly mixed in proportion to obtain the soil conditioner.
[0012] The third aspect of the present invention provides the use of the above-mentioned soil conditioner in the planting of Chuanxiong. Before sowing Chuanxiong, the soil conditioner is evenly applied to the soil surface, and rotary tilled evenly in the soil layer of 20 cm deep, and fully mixed with the soil surface. The application amount is 200-300 kg / 667 m 2 ; The ratio of Chuanxiong seed dressing agent is 100g to 20kg of Chuanxiong Ling seeds. Dilute the Chuanxiong seed dressing agent 100 times with water before application. Spray the diluted liquid evenly on the surface of Chuanxiong Ling seeds to make it fully contact. Place it in a cool place to dry before sowing.
[0013] In the soil conditioner formula of the present invention, an organic source soil conditioner is specially added. The rich humic acid component in the organic source soil conditioner can adjust the soil pH value, improve and optimize the absorption of nutrients and water by plants, and at the same time, the nutrient elements can quickly enter the plant body, improve the utilization rate of inorganic elements, and promote the synthesis of organic matter in the plant body. In addition, the rich nutrients in the soil conditioner can quickly activate and stimulate the added beneficial microorganisms to reproduce rapidly, supplement the beneficial microorganisms in the soil, and play a role in disease resistance and antibacterial. The organic source soil conditioner contains extracellular polysaccharides, which can activate the plant's own defense mechanism and promote the growth of the plant under different stress conditions.
[0014] In soil conditioners, the dry powder of inducers made from crude toxin of Rhizoctonia solani is used in combination with soil conditioners. On the one hand, it can stimulate the plant's defense response, thereby promoting the accumulation of secondary metabolites; on the other hand, it can stimulate the activation of antagonistic microorganisms of Rhizoctonia solani in the soil, thereby playing a role in disease prevention.
[0015] Fusarium oxysporum is one of the sources of Chuanxiong root rot. It is also an important component of endophytic fungi in Chuanxiong in traditional authentic production areas and is closely related to the synthesis of secondary metabolites of Chuanxiong. Seed dressing with elicitor dry powder made from crude toxin of Fusarium oxysporum can promote the formation of autoimmune antibodies in Chuanxiong seedlings to prevent the occurrence of Chuanxiong root rot, and at the same time regulate metabolic pathways to promote the accumulation of secondary metabolites of Chuanxiong.
[0016] Sea island oligosaccharide is used to dress the seeds of Chuanxiong and Ling. Sea island oligosaccharide is chitosan extracted from the shells of marine crustaceans and prepared through biological enzymatic hydrolysis engineering. It can stimulate the immune-inducing properties of Chuanxiong plants, thereby improving the plant's immunity to the outside world. At the same time, sea island oligosaccharide can regulate the metabolic level in the plant, change the expression of proteins in the body and the accumulation level of secondary metabolites, thereby achieving disease resistance, stress resistance, harm reduction and increased yield of crops.
[0017] Methyl jasmonate is used to dress the seeds of Chuanxiong Ling. Methyl jasmonate plays a vital role in plant growth and development, and in biotic and abiotic stress responses by activating signal transduction molecules in signal transduction pathways in response to different types of stress. As a plant hormone and signal molecule associated with damage resistance, exogenous application of methyl jasmonate can stimulate the expression of plant defense genes. At the same time, methyl jasmonate can promote the growth of plant roots, improve root vitality, and induce the synthesis of secondary metabolites in adventitious roots.
[0018] Chuanxiong phthalide compounds are composed of a six-membered ring and a γ-lactone ring fused together. The difference in their structures mainly lies in the saturation degree of the six-membered ring and the side chain and the configuration of the side chain. Among them, the most representative phthalide materials are Z-ligustilide, ligustilide I and ligustilide A. In different growth stages of Chuanxiong, Z-ligustilide will be converted into ligustilide I and ligustilide A. The structural formulas of the three are shown in Table 1.
[0019] Table 1 Chemical structure Fusarium oxysporum and Rhizoctonia solani are the pathogens of Chuanxiong root rot. After being infected by pathogenic microorganisms, Chuanxiong will produce and accumulate a large amount of secondary metabolites to enhance its own immunity and resistance. After Chuanxiong is infected by Fusarium oxysporum and Rhizoctonia solani and their crude toxins, the metabolic activity of phthalides in Chuanxiong is increased, thereby increasing the content of phthalides in Chuanxiong plants.
[0020] The beneficial effects of the present invention are: The present invention adopts an inducer formed by a crude toxin of a pathogenic fungus, in combination with an organic soil conditioner and a plant growth stimulant, to form a new Chuanxiong soil improver and seed dressing agent. The soil improver and seed dressing agent provided by the present invention can induce the accumulation of secondary metabolites of Chuanxiong, increase the content of total volatile oil of Chuanxiong and phthalide components Z-ligustilide, ligustilide I and ligustilide A in the volatile oil, stabilize the index component ferulic acid of the Chinese Pharmacopoeia, improve the quality of medicinal materials, ensure the efficacy of Chuanxiong, repair the soil ecological environment, ensure the output of Chuanxiong and the development of the industry, realize the sustainable development of the traditional Chinese medicine industry, and have important significance for the development of the Chuanxiong medicinal material industry. DETAILED DESCRIPTION
[0021] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative work are within the scope of protection of the present invention.
[0022] Preparation of Fusarium oxysporum elicitor dry powder: Make spores of Fusarium oxysporum into 2.3*10 7 The single spore suspension of CFU / mL was inoculated into PD medium (pH: 6.0~6.5) at a 3% inoculation rate, and the culture environment temperature was set to between 25~30℃. After 7 days of fermentation, the bacteria and the culture medium were separated by centrifugation to obtain a fungal fermentation liquid without bacteria, and then inactivated at high temperature. After inactivation, the fermentation liquid was filtered to remove large particles of impurities, and the fungal fermentation liquid was concentrated by evaporation or vacuum concentration to increase the concentration. Finally, the concentrated solution was freeze-dried to remove the water in the solution to obtain a dry powder product for use.
[0023] Preparation of dry powder of Rhizoctonia solani elicitor: The mycelium of Rhizoctonia solani was inoculated into PD culture medium (pH=7), and the culture environment temperature was set to between 28-32℃. After 6 days of fermentation, the mycelium and the culture medium were separated by centrifugation to obtain a fungal fermentation liquid without mycelium, and then inactivated at high temperature. After inactivation, the fermentation liquid was filtered to remove large particles of impurities, and the concentration of the fungal fermentation liquid was increased by evaporation or vacuum concentration. Finally, the concentrated solution was freeze-dried to remove the water in the solution to obtain a dry powder product for use.
[0024] Example 1 490 parts of rice husk powder, 290 parts of humic acid, 64 parts of extracellular polysaccharide, 133 parts of crude protein, 21 parts of crude fat and 2 parts of glutamic acid were mixed evenly to obtain an organic source soil conditioner.
[0025] 993.8 parts of organic soil conditioner, 2 parts of Trichoderma spinulosa (1 billion / g), 0.2 parts of Rhizoctonia solani inducer dry powder, 1 part of Bacillus licheniformis (10 billion / g), 1 part of Bacillus gellinga (1 billion / g), and 2 parts of Bacillus subtilis (10 billion / g) were evenly mixed to obtain Chuanxiong soil conditioner.
[0026] 320 parts of Fusarium oxysporum inducer dry powder, 390 parts of island indole and 290 parts of methyl jasmonate were mixed evenly to obtain Chuanxiong seed dressing agent.
[0027] Example 2 560 parts of rice husk powder, 265 parts of humic acid, 53 parts of extracellular polysaccharide, 105 parts of crude protein, 14 parts of crude fat and 3 parts of glutamic acid were mixed evenly to obtain an organic source soil conditioner.
[0028] 991.5 parts of organic soil conditioner, 4 parts of Trichoderma aculeatus (1 billion / g), 1 part of Rhizoctonia solani inducer dry powder, 0.5 parts of Bacillus licheniformis (10 billion / g), 2 parts of Bacillus gellinga (1 billion / g), and 1 part of Bacillus subtilis (10 billion / g) were evenly mixed to obtain Chuanxiong soil conditioner.
[0029] 544 parts of Fusarium oxysporum inducer dry powder, 276 parts of island indole, and 180 parts of methyl jasmonate were mixed evenly to obtain Chuanxiong seed dressing agent.
[0030] Example 3 600 parts of rice husk powder, 192 parts of humic acid, 41 parts of extracellular polysaccharides, 139 parts of crude protein, 27 parts of crude fat and 1 part of glutamic acid were mixed evenly to obtain an organic source soil conditioner.
[0031] 991.5 parts of organic soil conditioner, 3.5 parts of Trichoderma aculeatus (1 billion / g), 1.7 parts of Rhizoctonia solani inducer dry powder, 1.5 parts of Bacillus licheniformis (10 billion / g), 0.5 parts of Bacillus gellinga (1 billion / g), and 1.3 parts of Bacillus subtilis (10 billion / g) were evenly mixed to obtain Chuanxiong soil conditioner.
[0032] 630 parts of Fusarium oxysporum inducer dry powder, 315 parts of island indole and 55 parts of methyl jasmonate were mixed evenly to obtain Chuanxiong seed dressing agent.
[0033] test In late August, the implementation test of the technical solution was carried out in Aoping Town, Pengzhou City, Sichuan Province.
[0034] In late August, about one week before sowing Chuanxiong, the soil conditioners prepared in Examples 1-3 were added at 200-300 kg / 667 m 2 Apply the amount evenly on the soil surface, till it evenly into the 20cm deep soil layer, and mix it thoroughly with the soil.
[0035] Take Chuanxiong Ling seeds, remove surface impurities, mix the Chuanxiong seed dressing agent prepared in Example 1-3 with 100g of Chuanxiong seed dressing and 20kg of Chuanxiong Ling seeds, dilute the Chuanxiong seed dressing agent with water 100 times before application, spray the dilution evenly on the surface of Chuanxiong Ling seeds to make them fully contact, place them in a cool place to dry, and then sow them.
[0036] One week after spreading the soil conditioner, plant the Chuanxiong Ling seeds with a sowing density of 10,000 plants / 667m 2 , equivalent to a density of about 30kg / 667m 2 The field management adopts the conventional management method, and the fertilization refers to the following methods: (1) Fertilizer types Mainly use quick-acting fertilizers.
[0037] (2) Topdressing period The growth period of Chuanxiong is divided into seedling stage, stem and leaf growth period, seedling stage, secondary stem and leaf growth period and rhizome expansion period, and each growth period has obvious overlap. The topdressing period is carried out in the seedling stage, stem growth period and secondary stem and leaf growth period respectively.
[0038] (3) Fertilization method 1) Seedling stage After all seedlings emerge in early to mid-September, 2 Apply 8 kg of potassium sulfate compound fertilizer with N:P2O5:K2O=25:8:10 and total nutrient content of 43%.
[0039] 2) Stem and leaf growth period In mid-October, high-nitrogen compound fertilizer was used as the main fertilizer, with a fertilizer of 1000 kg per 667 m 2 Apply 15 kg of potassium sulfate compound fertilizer with N:P2O5:K2O=25:8:10 and a total nutrient content of 43%; in early December, topdress with balanced compound fertilizer as the main fertilizer, every 667 m 2 Apply 20 kg of potassium sulfate type compound fertilizer with N:P2O5:K2O=15:15:15 and total nutrient content of 45%.
[0040] 3) Secondary stem and leaf growth period In March of the following year, topdressing should be mainly higher potassium fertilizer, with a maximum of 100 kg per 667 m 2 Apply 20 kg of potassium sulfate compound fertilizer with N:P2O5:K2O=15:5:25 and total nutrient content of 45%. The proportion and amount of topdressing throughout the process are detailed in Table 2.
[0041] Table 2 The time and amount of topdressing for Chuanxiong Note: The amount of fertilizer can be increased or decreased according to the condition of the seedlings and applied flexibly.
[0042] A traditional cultivation control was set up, and the soil conditioner and Chuanxiong seed dressing agent provided in the present application were not used for treatment during cultivation.
[0043] Data determination: In late May of the following year, the yield of Chuanxiong was measured in the field and the quality of individual plants of Chuanxiong medicinal materials was determined. The test results are shown in Table 3. At the same time, samples were taken, selected, sliced, dried, crushed, and passed through a No. 2 sieve to make medicinal powder, and the contents of volatile oil and ferulic acid, and the contents of ligustilide I, ligustilide A, and Z-ligustilide were tested.
[0044] (1) Determination of volatile oil content in Rhizoma Chuanxiong: The volatile oil content was determined according to the method for determination of volatile oil in Part IV, 2204 of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 3.
[0045] (2) Determination of ferulic acid content: The ferulic acid content was determined according to the high performance liquid chromatography method (General Chapter 0512) in Part I of the 2020 edition of the Chinese Pharmacopoeia. The results are shown in Table 3.
[0046] (3) Determination of ligustilide I, ligustilide A, and Z-ligustilide: refer to the literature "Determination of the content changes of six main components in the growth process of Ligusticum chuanxiong by UPLC method". The determination results are shown in Table 4.
[0047] Table 3 Data on the production of Ligusticum chuanxiong Table 4 Content of active ingredients in Chuanxiong Table 5 Data on the content of volatile oil in some chuanxiong rhizome It can be seen from the data in the table that the use of the soil improver and Chuanxiong seed dressing provided by the present invention can improve the medicinal ingredients of Chuanxiong, effectively improve the total volatile oil content of Chuanxiong in the producing area, and the increase ratio is 34.38%~46.88%. Among them, Z-ligustilide increases by 28.38%~32.71%, foreign ligustilide I increases by 33.33%~66.67%, and foreign ligustilide A increases by 11.76%~23.53%. At the same time, it can stabilize the pharmacopoeia index component-ferulic acid, and its content is slightly improved, and the ferulic acid content is improved by 0.00%~8.00%, which ensures the quality of Chuanxiong medicinal materials. The use of the soil improver and Chuanxiong seed dressing provided by the present invention can also promote the growth of Chuanxiong, have a certain promoting effect on the yield of Chuanxiong, and the yield is increased by 4.56%~9.14%, which is of great significance to the development of the entire Chuanxiong industry.
[0048] The above is only a preferred embodiment of the present invention. It should be understood that the present invention is not limited to the form disclosed herein, and should not be regarded as excluding other embodiments, but can be used in various other combinations, modifications and environments, and can be modified within the scope of the concept described herein through the above teachings or the technology or knowledge of the relevant field. The changes and modifications made by those skilled in the art shall not deviate from the spirit and scope of the present invention, and shall be within the scope of protection of the claims attached to the present invention.
Claims
1. A soil conditioner for increasing the volatile oil content of Chuanxiong, characterized in that: The invention comprises the following components by weight: 991.5-993.8 parts of organic soil conditioner, 2-4 parts of 1 billion / g Trichoderma spinulosa, 0.2-1.7 parts of dry powder of Rhizoctonia solani inducer, 0.5-1.5 parts of 10 billion / g Bacillus licheniformis, 0.5-2 parts of 10 billion / g Bacillus gellingii, and 1-2 parts of 10 billion / g Bacillus subtilis.
2. The soil conditioner according to claim 1, characterized in that: The organic source soil conditioner comprises the following raw materials in parts by mass: 490-600 parts of rice husk powder, 192-290 parts of humic acid, 41-63 parts of extracellular polysaccharide, 105-139 parts of crude protein, 14-27 parts of crude fat, and 1-3 parts of glutamic acid, and the raw materials are uniformly mixed to obtain the organic source soil conditioner.
3. The soil conditioner according to claim 1, characterized in that: The dry powder of the inducer of Rhizoctonia solani is prepared by the following steps: inoculating the mycelium of Rhizoctonia solani on a PD culture medium with a pH value of 7, fermenting at a temperature of 28-32°C for 6 days, separating the cultured bacteria from the culture medium to obtain a fungal fermentation liquid without bacteria, inactivating the fermentation liquid at high temperature, filtering and concentrating the fermentation liquid, and drying the concentrated solution to obtain the dry powder of the inducer of Rhizoctonia solani.
4. The soil conditioner according to claim 1, characterized in that: It also includes an early stage Chuanxiong seed dressing agent, which includes the following components in parts by mass: 630-320 parts of Fusarium oxysporum inducer dry powder, 276-390 parts of island indole, and 55-290 parts of methyl jasmonate, and the raw materials are evenly mixed to obtain the product.
5. The soil conditioner according to claim 4, characterized in that: The Fusarium oxysporum elicitor dry powder is prepared by the following steps: preparing spores of Fusarium oxysporum into 2.3*10 7 A single spore suspension of CFU / mL is inoculated onto a PD culture medium at an inoculation rate of 3%, the pH of the culture medium is set between 6.0 and 6.5, and after culturing and fermenting at 25-30°C for 7 days, the cultured bacteria and the culture medium are separated to obtain a fungal fermentation liquid without bacteria, the fermentation liquid is inactivated at high temperature, filtered and concentrated, and the concentrated solution is dried to obtain a dry powder of Rhizoctonia solani inducer.
6. A method for preparing a soil conditioner for increasing the volatile oil content of Rhizoma Chuanxiong as claimed in any one of claims 1 to 5, characterized in that: The soil conditioner is obtained by uniformly mixing the organic soil conditioner, Trichoderma acanthosporum, dry powder of Rhizoctonia solani inducer, Bacillus licheniformis, Bacillus gellingii and Bacillus subtilis according to a certain proportion.
7. Use of the soil conditioner for increasing the volatile oil content of Chuanxiong according to any one of claims 1 to 5 in planting Chuanxiong, characterized in that: Before sowing Chuanxiong, apply the soil conditioner evenly on the soil surface, and till it evenly in the 20cm deep soil layer, and mix it thoroughly with the soil surface. The application amount is 200~300kg / 667m 2 .