Method for improving acid aluminum tolerance of sugarcane by efficient combined nitrogen fixation
By controlling the amount of nitrogen fertilizer and selecting appropriate nitrogen-fixing bacteria binding enhancers, the problem of limited sugarcane growth in high-acid-alumina soils was solved, achieving healthy growth of sugarcane and protection against aluminum stress in an efficient combined nitrogen fixation system.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- GUANGXI ZHUANG AUTONOMOUS REGION ACAD OF AGRI SCI
- Filing Date
- 2023-07-21
- Publication Date
- 2026-05-29
AI Technical Summary
Sugarcane growth is limited in high-alumina soils. Existing nitrogen-fixing bacteria species and soil environmental factors lead to unstable nitrogen fixation effects, making it difficult to form an efficient combined nitrogen fixation system.
By controlling the amount of nitrogen fertilizer applied, selecting appropriate nitrogen-fixing bacteria at different growth stages of sugarcane, and using enhancers such as *Bacillus baileyi*, *Gastrointestinal globulus*, and *Azotobacter lipophilus*, combined with indoleacetic acid, eugenol, and vitamin E, a highly efficient combined nitrogen-fixing system is formed.
Sugarcane can grow normally in high-acid aluminum soil, the growth of nitrogen-fixing bacteria is not inhibited, nitrogen fixation capacity is improved, the root system is healthy, and the stunting and yellowing of plants under aluminum stress are prevented. The enhancer works synergistically to improve the nitrogen fixation effect.
Abstract
Description
Technical Field
[0001] This invention relates to the field of sugarcane cultivation technology, and in particular to a method for improving sugarcane's resistance to aluminum acid through efficient combined nitrogen fixation. Background Technology
[0002] Guangxi accounts for over 65% of China's sugarcane cultivation area and sugar production, making it my country's most important sugar-producing region. However, it is also one of the areas in my country with the most severe soil acidification, with approximately 800,000 hectares of sugarcane fields having acidic soil with a pH below 5.0. Aluminum toxicity is the most significant abiotic stress factor limiting crop growth in acidic soils. When soil pH drops below 5.0, complexed and combined aluminum in the soil gradually dissociates, releasing a large amount of active aluminum. 3+ It can poison plants, causing nutrient imbalances, root poisoning, and other serious problems that affect plant growth and development.
[0003] Studies have shown that many nitrogen-fixing bacteria are tolerant to aluminum, and that the nitrogen metabolism and nitrogen-fixing-related physiological activities of these bacteria can affect plant tolerance to aluminum. In the 1980s, a strain of nitrogen-fixing acetic acid bacillus isolated from sugarcane was found to survive long-term and maintain efficient nitrogen fixation at pH ≤ 3.0. Endophytic Hordeum vulgare A1, A23, and X6 in sugarcane have also been shown to be able to fix nitrogen at pH 3.8 and Al. 3+ It grows normally at concentrations as high as 1 mM, and during growth, the pH of the culture medium is increased and Al is removed. 3+ , for Al 3+ The removal rate reached 22.42%. It can be seen that inoculation with nitrogen-fixing bacteria can improve the acid and aluminum tolerance of sugarcane. However, due to the long-term high nitrogen stress, the number of bacteria that can survive at low nitrogen levels in sugarcane cultivars in my country is only equivalent to the lowest level of Brazilian sugarcane varieties (104 CFU / g dry weight), making it difficult to form an efficient nitrogen-fixing system. In addition, the nitrogen-fixing effect is easily affected by the type of nitrogen-fixing bacteria and soil environmental factors, resulting in unstable nitrogen-fixing effect.
[0004] Based on the above problems, it is of great significance to study how to establish an efficient combined nitrogen fixation system so that sugarcane can grow normally in high-alumina soil. Summary of the Invention
[0005] To address the above shortcomings, this invention provides a method for improving sugarcane's tolerance to aluminum acid through efficient synergistic nitrogen fixation. This method establishes an efficient synergistic nitrogen fixation system by controlling nitrogen fertilizer application, selecting suitable nitrogen-fixing bacteria at different growth stages of sugarcane, and employing enhancers, among other measures, enabling sugarcane to grow normally in high-alumina soils. The specific technical solution is as follows:
[0006] A method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation includes:
[0007] Control the amount of nitrogen fertilizer: During the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer, and the fertilizers applied at other times do not contain nitrogen fertilizer.
[0008] Constructing an efficient combined nitrogen fixation system: Applying *Bacillus bailensis* inoculant and enhancer during the initial planting of sugarcane; applying *Gnaphalium erythropoietin* inoculant and enhancer during the sugarcane seedling stage; applying *Gnaphalium lipogenum* inoculant and enhancer during the sugarcane tillering stage; the enhancer comprises the following raw materials in parts by weight: 5-15 parts indoleacetic acid, 1-10 parts eugenol, and 10-20 parts vitamin E.
[0009] Preferably, the nitrogen fertilizer refers to nitrogen-containing inorganic fertilizer, and the amount of nitrogen fertilizer applied in the base fertilizer is 15-25 kg / mu.
[0010] Preferably, the weight ratio of the Bacillus baijerin Kiehl's inoculant to the enhancer is 50-100:1.
[0011] Preferably, the method of using the Bacillus baijerin inoculant and the enhancer is as follows: first, mix the Bacillus baijerin inoculant and the enhancer into a mixture, and then mix the mixture with well-rotted organic fertilizer at a weight ratio of 1:200-300 and apply it as base fertilizer. The amount of base fertilizer used is 1500-2000 kg / mu.
[0012] Preferably, the weight ratio of the *Gnaphalium affine* inoculant to the enhancer is 20-50:1.
[0013] Preferably, the method of using the *Gnaphalium affine* inoculant and the enhancer is as follows: first, mix the *Gnaphalium affine* inoculant and the enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1000-1500 and apply it to the roots, with an application rate of 200-250 mL / hole.
[0014] Preferably, the weight ratio of the lipogenic nitrogen-fixing spirochete agent to the enhancer is 50-80:1.
[0015] Preferably, the method of using the lipogenic nitrogen-fixing spirochete agent and the enhancer is as follows: first, mix the lipogenic nitrogen-fixing spirochete agent and the enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1000-1500 and apply it to the roots, with an application rate of 150-250 mL / hole.
[0016] Preferably, the root application is followed by covering with a layer of biochar with a moisture content of 60-70%.
[0017] Preferably, the root application is done in the evening or morning.
[0018] Compared with the prior art, the beneficial effects of the present invention are:
[0019] 1. The method of the present invention establishes an efficient combined nitrogen fixation system by controlling the amount of nitrogen fertilizer, selecting appropriate nitrogen-fixing bacteria at different growth stages of sugarcane, and using enhancers as an aid, so that sugarcane can grow normally in high-acid aluminum soil.
[0020] 2. In this invention, nitrogen fertilizer is added only when the sugarcane is newly planted throughout its entire growth cycle. Nitrogen fertilizer is not added at other times, which can reduce the inhibitory effect of nitrogen fertilizer on nitrogen-fixing bacteria, promote the growth of nitrogen-fixing bacteria, and improve nitrogen-fixing capacity.
[0021] 3. The *Baiyelinkie* strain of this invention is a rhizosphere nitrogen-fixing bacterium that mainly colonizes the sugarcane rhizosphere. It can synergistically work with indoleacetic acid and eugenol in the enhancer to jointly induce and stimulate the secretion of organic acids from sugarcane roots. These organic acids can react with Al... 3+ A chelation reaction occurs, forming non-toxic compounds that cannot penetrate the roots, thereby reducing active Al in the soil. 3+ The content of [specific ingredient] effectively improved aluminum stress in the field. Simultaneously, the use of *Bacillus baileyi* in conjunction with well-rotted organic fertilizer provided a favorable growth environment for the strain, accelerating its colonization in the rhizosphere and rapidly establishing a dominant population, thus enabling it to fully exert its aluminum removal function.
[0022] 4. *Gastrodia elata* is an obligate endophytic nitrogen-fixing bacterium that is mainly found in sugarcane tissue. When used in the seedling stage, it can infect the epidermal cells of the seedling roots and the cells at the formation site of secondary roots. The repair factors produced after infection work together with the enhancer to quickly repair the damaged sugarcane cells after aluminum stress, enabling the cells to quickly resume normal growth, thereby promoting the normal growth of the seedling roots.
[0023] 5. The *Azotobacter lipophilus* strain used in this study is a facultative endophytic nitrogen-fixing bacterium that can grow in the roots and colonize the root surface and soil. When used during the tillering stage, the growth-promoting factors and enhancers produced by the strain work together to change the root morphology, promote the growth of sugarcane roots, and thus promote the growth of sugarcane, effectively preventing stunting and yellowing of sugarcane plants under aluminum stress.
[0024] 6. The vitamin E in the enhancer of this invention can also prevent eugenol from oxidizing and deteriorating, effectively ensuring that eugenol can perform its function.
[0025] 7. The biochar covering after root application of this invention can provide a good growth environment for nitrogen-fixing bacteria, which is conducive to their growth and colonization. Detailed Implementation
[0026] The specific embodiments of the present invention will be described in detail below, but it should be understood that the scope of protection of the present invention is not limited to the specific embodiments.
[0027] Example 1
[0028] A method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation includes:
[0029] (1) Control the amount of nitrogen fertilizer: During the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer, and the fertilizers applied at other times do not contain nitrogen fertilizer. Nitrogen fertilizer refers to nitrogen-containing inorganic fertilizer. The amount of nitrogen fertilizer applied in the base fertilizer is 15 kg / mu.
[0030] (2) Constructing an efficient combined nitrogen fixation system:
[0031] When planting new sugarcane, apply Bacillus thuringiensis inoculant and enhancer; the weight ratio of Bacillus thuringiensis inoculant and enhancer is 50:1; the application method is as follows: first mix Bacillus thuringiensis inoculant and enhancer into a mixture, then mix the mixture with well-rotted organic fertilizer at a weight ratio of 1:200 and apply it as base fertilizer, with a base fertilizer application rate of 1500 kg / mu.
[0032] Apply red and white spirulina inoculant and enhancer during the sugarcane seedling stage; the weight ratio of red and white spirulina inoculant and enhancer is 20:1. The application method is as follows: first mix red and white spirulina inoculant and enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1000 and apply it to the roots in the evening at a rate of 200 mL / hole. After root application, cover with a 1 cm thick layer of biochar with a moisture content of 60%.
[0033] Apply lipophilic nitrogen-fixing spirochete inoculant and enhancer during the sugarcane tillering stage. The weight ratio of lipophilic nitrogen-fixing spirochete inoculant to enhancer is 50:1. The application method is as follows: first, mix the lipophilic nitrogen-fixing spirochete inoculant and enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1000 and apply it to the roots in the evening. The application rate is 150mL / hole. After root application, cover with a 1cm thick layer of biochar with a moisture content of 60%.
[0034] The above-mentioned reinforcing agent is composed of the following raw materials in parts by weight: 5 parts indoleacetic acid, 1 part eugenol and 10 parts vitamin E.
[0035] Example 2
[0036] A method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation includes:
[0037] (1) Control the amount of nitrogen fertilizer: During the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer, and the fertilizers applied at other times do not contain nitrogen fertilizer. Nitrogen fertilizer refers to nitrogen-containing inorganic fertilizer. The amount of nitrogen fertilizer applied in the base fertilizer is 25 kg / mu.
[0038] (2) Constructing an efficient combined nitrogen fixation system:
[0039] When planting new sugarcane, apply Bacillus thuringiensis inoculant and enhancer; the weight ratio of Bacillus thuringiensis inoculant and enhancer is 100:1; the application method is as follows: first mix Bacillus thuringiensis inoculant and enhancer into a mixture, then mix the mixture with well-rotted organic fertilizer at a weight ratio of 1:300 and apply it as base fertilizer, with a base fertilizer application rate of 2000 kg / mu.
[0040] Apply red and white spirulina inoculant and enhancer during the sugarcane seedling stage. The weight ratio of red and white spirulina inoculant and enhancer is 50:1. The application method is as follows: first, mix red and white spirulina inoculant and enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1500 and apply it to the roots in the morning. The application rate is 250mL / hole. After root application, cover with a 3cm thick layer of biochar with a moisture content of 70%.
[0041] Apply lipophilic nitrogen-fixing spirochete inoculant and enhancer during the sugarcane tillering stage. The weight ratio of lipophilic nitrogen-fixing spirochete inoculant to enhancer is 80:1. The application method is as follows: first, mix the lipophilic nitrogen-fixing spirochete inoculant and enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1500 and apply it to the roots in the morning at a rate of 250 mL / hole. After root application, cover with a 3 cm thick layer of biochar with a moisture content of 70%.
[0042] The above-mentioned reinforcing agent is composed of the following raw materials in parts by weight: 15 parts indoleacetic acid, 10 parts eugenol and 20 parts vitamin E.
[0043] Example 3
[0044] A method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation includes:
[0045] (1) Control the amount of nitrogen fertilizer: During the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer, and the fertilizers applied at other times do not contain nitrogen fertilizer. Nitrogen fertilizer refers to nitrogen-containing inorganic fertilizer. The amount of nitrogen fertilizer applied in the base fertilizer is 20 kg / mu.
[0046] (2) Constructing an efficient combined nitrogen fixation system:
[0047] When planting new sugarcane, apply Bacillus thuringiensis inoculant and enhancer; the weight ratio of Bacillus thuringiensis inoculant and enhancer is 80:1; the application method is as follows: first mix Bacillus thuringiensis inoculant and enhancer into a mixture, then mix the mixture with well-rotted organic fertilizer at a weight ratio of 1:250 and apply it as base fertilizer, with a base fertilizer application rate of 1800 kg / mu.
[0048] Apply red and white spirulina inoculant and enhancer during the sugarcane seedling stage. The weight ratio of red and white spirulina inoculant and enhancer is 45:1. The application method is as follows: first, mix red and white spirulina inoculant and enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1300 and apply it to the roots in the evening. The application rate is 220mL / hole. After root application, cover with a 2cm thick layer of biochar with a moisture content of 65%.
[0049] Apply lipophilic nitrogen-fixing spirochete inoculant and enhancer during the sugarcane tillering stage. The weight ratio of lipophilic nitrogen-fixing spirochete inoculant to enhancer is 70:1. The application method is as follows: first, mix the lipophilic nitrogen-fixing spirochete inoculant and enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1200 and apply it to the roots in the morning at a rate of 200 mL / hole. After root application, cover with a 2 cm thick layer of biochar with a moisture content of 65%.
[0050] The above-mentioned reinforcing agent is composed of the following raw materials in parts by weight: 12 parts indoleacetic acid, 3 parts eugenol and 11 parts vitamin E.
[0051] Comparative Example 1
[0052] Nitrogen fertilizer was applied according to conventional methods, namely, 20 kg / mu as base fertilizer, 15 kg / mu during the tillering stage, 15 kg / mu during the jointing stage, and 15 kg / mu as tail fertilizer. Except for the amount of nitrogen fertilizer, the other steps were the same as in Example 1.
[0053] Comparative Example 2
[0054] In the process of constructing the efficient combined nitrogen fixation system, no reinforcing agent is used, and the other steps are the same as in Example 1.
[0055] Comparative Example 3
[0056] The combination of *Bacillus bailenkrysticus* and its enhancer was used in the seedling stage, the combination of *Bacillus rubrum* and its enhancer was used in the tillering stage, and the combination of *Azotobacter lipogenicus* and its enhancer was used in the new planting stage. Other steps were the same as in Example 1.
[0057] Comparative Example 4
[0058] In the process of constructing the efficient combined nitrogen fixation system, the reinforcing agent used does not contain indoleacetic acid, and the other steps are the same as in Example 1.
[0059] Comparative Example 5
[0060] In the process of constructing the efficient combined nitrogen fixation system, the reinforcing agent used does not contain eugenol, and the other steps are the same as in Example 1.
[0061] In the above embodiments and comparative examples, the *Baiyelinkiella* involved was obtained from the China Agricultural Microbial Culture Collection Center of the Chinese Academy of Agricultural Sciences; the *Rhodospirillum rubrum* and *Rhodospirillum erythrospira* were obtained from the China General Microbial Culture Collection Center of the Institute of Microbiology, Chinese Academy of Sciences; and the *Azospirillum lipogenes* were obtained from the China Center for Type Culture Collection of Wuhan University.
[0062] Effect verification:
[0063] In 2022, the applicant used the method of this invention to plant sugarcane in high-alumina soil to test whether sugarcane could grow normally in high-alumina soil after being planted using the method of this invention. The planting site was located in Dingdang Town, Long'an County, Guangxi Province. The soil type was sandy loam. Before planting, the physical and chemical properties of the sugarcane field soil were tested and found to be: pH < 4.8, organic matter content 19.55 g / kg, total nitrogen content 0.111%, total phosphorus content 0.043%, and total potassium content 0.49%, indicating acidic soil. The sugarcane variety used was Guitang 42. The rows were 10m long with a spacing of 1.5m. Each example and comparative example had 5 rows planted, with 3 replicates. The same field management was applied. A control group was also planted in neutral and aluminum-free soil. The control group received conventional fertilization: 20 kg / mu of nitrogen fertilizer as basal fertilizer, 15 kg / mu during tillering, 15 kg / mu during jointing, and 15 kg / mu as final fertilizer. Twenty days after applying the microbial agent during tillering, 5 plants were randomly selected from each example and comparative example to measure plant height, root weight, and root length, calculating the average values. Leaf tip condition was also observed. The results are shown in Table 1. The methods for observing plant height, root weight, and root length are as follows: When measuring the root system (root length and root weight), first remove the plant from the soil, separate the root system from the plant, wash it, wipe the surface moisture with a cotton cloth, and then measure it; observe the yellowing and wilting of the leaf tips. Yellowing and wilting of more than 80% of the leaves indicates severe yellowing and wilting, while yellowing and wilting of 10-20% of the leaves indicates mild yellowing and wilting (mild yellowing and wilting indicates tolerance to aluminum acid, while severe yellowing and wilting indicates intolerance to aluminum acid); plant height is the height measured from the ground to the first visible thick leaf sheath.
[0064] Table 1. Aluminum resistance of each embodiment and comparative example
[0065] project Plant height (cm) Root weight (g) Root length (cm) plant leaf tips Reference group 58.8 81.0 8.8 No yellowing and wilt Example 1 56.2 78.4 7.9 Mild chlorosis Example 2 53.8 72.1 7.4 Mild chlorosis Example 3 57.6 79.9 8.5 No yellowing and wilt Comparative Example 1 39.5 54.7 5.2 Severe jaundice Comparative Example 2 42.2 58.1 5.9 Severe jaundice Comparative Example 3 44.9 60.1 6.3 Severe jaundice Comparative Example 4 50.5 69.3 6.7 Severe jaundice Comparative Example 5 50.8 69.9 6.8 Severe jaundice
[0066] The test results in the table above show that: (1) When sugarcane is planted in soil with high acidity and aluminum using the method of the present invention, its plant height, root weight, root length and leaf tip are basically the same as those of the control group. The sugarcane does not show stunting, yellowing and other toxic phenomena, and its growth is not hindered. It can be seen that the method of the present invention can enable sugarcane to grow normally in soil with high acidity and aluminum. (2) Controlling the amount of nitrogen fertilizer and applying less nitrogen fertilizer can reduce the inhibitory effect of nitrogen fertilizer on nitrogen-fixing bacteria, which is conducive to the growth of nitrogen-fixing bacteria and improves nitrogen fixation capacity. (3) The application period of Bacillus baileyi, Gracilaria rubra and Azotobacter lipophila will affect the formation of an efficient nitrogen-fixing system. Bacillus baileyi is best used in the new planting period, Gracilaria rubra and Azotobacter rubra is used in the seedling period, and Azotobacter lipophila is used in the tillering period. (4) The synergistic effect of indoleacetic acid and eugenol in the enhancer of the present invention can effectively assist nitrogen-fixing bacteria in forming an efficient nitrogen-fixing system.
[0067] The foregoing description of specific exemplary embodiments of the invention is for illustrative and explanatory purposes. These descriptions are not intended to limit the invention to the precise forms disclosed, and it will be apparent that many changes and variations can be made in accordance with the foregoing teachings. The exemplary embodiments were chosen and described in order to explain the specific principles of the invention and its practical application, thereby enabling those skilled in the art to implement and utilize various different exemplary embodiments of the invention, as well as various different choices and variations. The scope of the invention is intended to be defined by the claims and their equivalents.
Claims
1. A method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation, characterized in that, include: Control the amount of nitrogen fertilizer: During the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer, and the fertilizers applied at other times do not contain nitrogen fertilizer. Constructing an efficient combined nitrogen fixation system: Applying *Bacillus bailensis* inoculant and enhancer during the initial planting of sugarcane; applying *Gnaphalium erythropoietin* inoculant and enhancer during the sugarcane seedling stage; applying *Gnaphalium lipogenum* inoculant and enhancer during the sugarcane tillering stage; the enhancer comprises the following raw materials in parts by weight: 5-15 parts indoleacetic acid, 1-10 parts eugenol, and 10-20 parts vitamin E.
2. The method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 1, characterized in that, The nitrogen fertilizer mentioned refers to nitrogen-containing inorganic fertilizer, and the amount of nitrogen fertilizer applied in the base fertilizer is 15-25 kg / mu.
3. The method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 1, characterized in that, The weight ratio of the Bacillus baijerin inoculant to the enhancer is 50-100:
1.
4. The method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 1, characterized in that, The method of using the Bacillus baijerin inoculant and enhancer is as follows: first, mix the Bacillus baijerin inoculant and enhancer into a mixture, then mix the mixture with well-rotted organic fertilizer at a weight ratio of 1:200-300 and apply it as base fertilizer. The amount of base fertilizer used is 1500-2000 kg / mu.
5. The method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 1, characterized in that, The weight ratio of the *Gnaphalium affine* inoculant to the enhancer is 20-50:
1.
6. The method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 1, characterized in that, The method of using the red and white spirulina inoculant and enhancer is as follows: first, mix the red and white spirulina inoculant and enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1000-1500 and apply it to the roots, with an application rate of 200-250 mL / hole.
7. The method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 1, characterized in that, The weight ratio of the lipogenic nitrogen-fixing spirochete inoculum to the enhancer is 50-80:
1.
8. The method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 1, characterized in that, The method of using the lipogenic nitrogen-fixing spirochete inoculant and the enhancer is as follows: first, mix the lipogenic nitrogen-fixing spirochete inoculant and the enhancer into a mixture, then mix the mixture with water at a weight ratio of 1:1000-1500 and apply it to the roots, with an application rate of 150-250 mL / hole.
9. A method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 6 or 8, characterized in that, After root application, cover with a layer of biochar with a moisture content of 60-70%.
10. A method for improving the aluminum resistance of sugarcane through efficient combined nitrogen fixation according to claim 6 or 8, characterized in that, The root application should be done in the evening or morning.