Method for controlling and inhibiting acidification of sugar cane fields by using nitrogen-fixing bacteria

Abstract

The application discloses a method for controlling and inhibiting sugarcane acidification by using nitrogen-fixing bacteria, and relates to the technical field of controlling and inhibiting sugarcane acidification, and solves the problems that a combined nitrogen-fixing system formed by nitrogen-fixing bacteria and sugarcane is greatly affected by rhizosphere biological adversity, nitrogen-fixing effect is unstable, and nitrogen-fixing efficiency is low. The application reduces the use of nitrogen fertilizer in the whole growth cycle of the sugarcane, and applies a combined nitrogen-fixing bacterial agent and a promoting agent in the sugarcane seedling stage and the elongation stage, respectively. The combined nitrogen-fixing bacterial agent is Raoultella L03, Bacillus azoreus and Rhizosphaera sulcata, and the promoting agent is mannitol, glycerol, succinic acid, sodium molybdate, dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate and glutathione. The application can make the nitrogen-fixing bacteria quickly form a combined nitrogen-fixing system with the sugarcane, so that the purposes of efficient nitrogen fixation, less nitrogen fertilizer application and soil acidification reduction are achieved.

Description

Technical Field

[0001] This invention relates to the field of sugarcane field acidification control technology, and in particular to a method for controlling sugarcane field acidification using nitrogen-fixing bacteria. Background Technology

[0002] Excessive application of nitrogen fertilizer is one of the main causes of soil acidification. In Guangxi, the average annual nitrogen application in sugarcane fields is approximately 550,000 tons, while the nitrogen content in the harvest is only 75,000 tons, resulting in a nitrogen fertilizer utilization efficiency of less than 15%. This severe overuse of nitrogen fertilizer exacerbates soil acidification. Therefore, increasing biological nitrogen fixation and reducing nitrogen fertilizer use are important ways to alleviate soil acidification.

[0003] Sugarcane, as a grass, fixes nitrogen through a synergistic nitrogen-fixing system with nitrogen-fixing bacteria. These bacteria primarily colonize the sugarcane roots, stems, and leaves, forming a loose synergy. Studies have shown that a large number of synergistic nitrogen-fixing bacteria have been isolated from various grasses, exhibiting a rich diversity. For example, Herbaspirillumseropedicae, Gluconacetobacter diazotrophics, Azospirillum spp., Bacillus spp., Enterobacter spp., Pseudomonas spp., Alcaligenes spp., Burkholderia spp., and Azotobacter spp., etc., these co-nitrogen-fixing bacteria can form a co-nitrogen-fixing system with sugarcane, providing nitrogen for sugarcane, thereby reducing the use of nitrogen fertilizer and thus achieving the goal of preventing and controlling soil acidification.

[0004] Reports indicate that some individuals have used associative nitrogen-fixing bacteria (ANOCs) as microbial fertilizers to increase nitrogen fixation, which has helped improve sugarcane yield and product quality. However, the natural associative nitrogen-fixing system has many drawbacks. The formation of an ANOC between ANOCs and sugarcane is greatly affected by rhizosphere biological stress, resulting in unstable nitrogen fixation and low efficiency. Furthermore, research on how to effectively and quickly establish an ANOC between ANOCs and sugarcane to achieve efficient nitrogen fixation is limited. Therefore, how to rapidly establish an ANOC between ANOCs and sugarcane after their application is a problem that needs to be solved. Summary of the Invention

[0005] To address the above shortcomings, this invention provides a method for controlling sugarcane soil acidification using nitrogen-fixing bacteria. This method enables nitrogen-fixing bacteria to rapidly form a combined nitrogen-fixing system with sugarcane, thereby achieving efficient nitrogen fixation, reduced nitrogen fertilizer application, and decreased soil acidification. The specific technical solution is as follows:

[0006] A method for controlling sugarcane acidification using nitrogen-fixing bacteria involves applying nitrogen fertilizer only to the base fertilizer throughout the entire sugarcane growth cycle, while fertilizers applied at other times do not contain nitrogen fertilizer; and applying a combination of nitrogen-fixing bacteria and growth promoters during the sugarcane seedling stage and elongation stage respectively.

[0007] The combined nitrogen-fixing bacterial agent comprises the following bacterial agents in parts by weight: 10-30 parts of nitrogen-fixing bacterium Raoultella L03, 25-45 parts of nitrogen-fixing Bacillus, and 5-15 parts of nitrogen-fixing bacterium Paspalum distichum.

[0008] The accelerator comprises the following raw materials in parts by weight: 40-70 parts mannitol, 30-55 parts glycerol, 35-60 parts succinic acid, 0.001-0.01 parts sodium molybdate, 0.1-0.7 parts dipotassium hydrogen phosphate, 0.1-0.5 parts magnesium sulfate, 0.005-0.01 parts ferrous sulfate, and 0.2-0.7 parts glutathione.

[0009] Preferably, the nitrogen fertilizer refers to nitrogen-containing inorganic fertilizer; the amount of nitrogen fertilizer applied in the base fertilizer is 15-25 kg / mu, and it is applied together with phosphate fertilizer and potassium fertilizer on both sides of the planting ditch.

[0010] Preferably, the combined nitrogen-fixing bacterial agent comprises the following parts by weight: 22 parts of nitrogen-fixing bacterium Raoultella L03, 30 parts of nitrogen-fixing Bacillus, and 12 parts of nitrogen-fixing bacterium Paspalum distichum.

[0011] Preferably, the accelerator comprises the following raw materials in parts by weight: 55 parts mannitol, 50 parts glycerol, 45 parts succinic acid, 0.008 parts sodium molybdate, 0.5 parts dipotassium hydrogen phosphate, 0.3 parts magnesium sulfate, 0.007 parts ferrous sulfate, and 0.4 parts glutathione.

[0012] Preferably, the weight ratio of the combined nitrogen-fixing bacteria agent and the promoter is 5-15:1.

[0013] Preferably, the method of using the combined nitrogen-fixing bacteria agent and the accelerator is as follows: mix the combined nitrogen-fixing bacteria agent and the accelerator according to the weight ratio, add water at 100-200 times the total weight of the combined nitrogen-fixing bacteria agent and the accelerator, mix well to obtain a mixed bacterial solution, and irrigate the sugarcane roots with the mixed bacterial solution.

[0014] Preferably, the amount of the mixed bacterial solution applied is 100-200 mL / strain.

[0015] Preferably, after the mixed bacterial solution is applied, a layer of biochar is applied, with a biochar layer thickness of 1-2 cm.

[0016] Preferably, the mixed bacterial solution is applied in the evening or morning.

[0017] Preferably, the biochar has a moisture content of 60-70%.

[0018] The nitrogen-fixing bacterium Raoultella L03 is a bacterial strain with high nitrogen-fixing activity isolated and screened from the root system of the local sugarcane variety Guitang 28 in Guangxi by the Guangxi Academy of Agricultural Sciences. This strain is Klebsiella plantica and is currently preserved at the Guangxi Academy of Agricultural Sciences.

[0019] Compared with the prior art, the beneficial effects of the present invention are:

[0020] 1. This invention accelerates the colonization of nitrogen-fixing bacteria and improves the nitrogen-fixing capacity of syn-nitrogen-fixing bacteria through a series of technical means, such as controlling the amount of nitrogen fertilizer and providing suitable growth conditions for nitrogen-fixing bacteria, thereby achieving the goals of efficient nitrogen fixation, less nitrogen fertilizer application, and reduced soil acidification.

[0021] 2. In this invention, only the base fertilizer applied during the entire growth cycle of sugarcane contains nitrogen fertilizer, while the fertilizers applied at other times do not contain nitrogen fertilizer, which can reduce the inhibitory effect of nitrogen fertilizer on nitrogen-fixing bacteria.

[0022] 3. In the combined nitrogen-fixing bacteria of this invention, Bacillus azotocinus can stimulate the sugarcane roots to secrete polysaccharides, Azotocinus paspalumana can stimulate the development of sugarcane root hairs, and Azotocinus raoultella L03 can rapidly infect the roots. The combination of the three can quickly attract a large number of nitrogen-fixing bacteria to gather in the roots, forming a population advantage, so that the combined nitrogen-fixing bacteria can quickly form an efficient combined nitrogen-fixing system with sugarcane.

[0023] 4. The mannitol, glycerol, and succinic acid in the promoter of this invention can provide suitable carbon sources for the above three nitrogen-fixing bacteria. Sodium molybdate can promote the growth of nitrogen-fixing bacteria, while dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, and glutathione can promote the secretion of ammonia by nitrogen-fixing Bacillus under acidic soil conditions, increase the environmental pH value, create a good nitrogen-fixing environment for nitrogen-fixing bacteria, and thus improve nitrogen fixation efficiency.

[0024] 5. The biochar covering after root application of the present invention can provide a good growth environment for the combined nitrogen-fixing bacteria, which is conducive to their growth and colonization. Detailed Implementation

[0025] 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.

[0026] The nitrogen-fixing Bacillus and Paspalum distichum of this invention were purchased from the Hebei (Baoding) Agricultural Microbial Strains Industry Technology Research Institute.

[0027] Example 1

[0028] A method for controlling sugarcane acidification using nitrogen-fixing bacteria includes:

[0029] (1) Control the amount of nitrogen fertilizer. During the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer. 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, which is applied together with phosphate fertilizer and potassium fertilizer on both sides of the planting furrow.

[0030] (2) Applying nitrogen-fixing bacteria to construct a combined nitrogen-fixing system. Apply the combined nitrogen-fixing bacteria agent and the promoter during the seedling stage and the elongation stage of sugarcane, respectively. The weight ratio of the combined nitrogen-fixing bacteria agent and the promoter is 5:1. The method of application of the combined nitrogen-fixing bacteria agent and the promoter is as follows: mix the combined nitrogen-fixing bacteria agent and the promoter according to the weight ratio, and add water with a total weight of 100 times the combined nitrogen-fixing bacteria agent and the promoter to make a mixed bacterial solution. In the evening, irrigate the sugarcane roots with the mixed bacterial solution. The application amount of the mixed bacterial solution is 100 mL / plant. After the mixed bacterial solution is applied, cover with a layer of biochar with a moisture content of 60% and a biochar coverage thickness of 1 cm.

[0031] The combined nitrogen-fixing bacterial agent consists of the following parts by weight: 10 parts of nitrogen-fixing bacterium Raoultella L03, 25 parts of nitrogen-fixing Bacillus, and 5 parts of nitrogen-fixing Paspalum distichum.

[0032] The accelerator is composed of the following raw materials in parts by weight: 40 parts mannitol, 30 parts glycerol, 35 parts succinic acid, 0.001 parts sodium molybdate, 0.1 parts dipotassium hydrogen phosphate, 0.1 parts magnesium sulfate, 0.005 parts ferrous sulfate, and 0.2 parts glutathione.

[0033] Example 2

[0034] A method for controlling sugarcane acidification using nitrogen-fixing bacteria includes:

[0035] (1) Control the amount of nitrogen fertilizer. During the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer. 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, which is applied together with phosphorus fertilizer and potassium fertilizer on both sides of the planting furrow.

[0036] (2) Applying nitrogen-fixing bacteria to construct a combined nitrogen-fixing system. Apply the combined nitrogen-fixing bacteria agent and the promoter during the seedling stage and the elongation stage of sugarcane, respectively. The weight ratio of the combined nitrogen-fixing bacteria agent and the promoter is 15:1. The method of application of the combined nitrogen-fixing bacteria agent and the promoter is as follows: mix the combined nitrogen-fixing bacteria agent and the promoter according to the weight ratio, and add water with a total weight of 200 times the combined nitrogen-fixing bacteria agent and the promoter to make a mixed bacterial solution. In the morning, irrigate the sugarcane roots with the mixed bacterial solution. The application amount of the mixed bacterial solution is 200 mL / plant. After the mixed bacterial solution is applied, cover with a layer of biochar with a moisture content of 70% and a biochar covering thickness of 2 cm.

[0037] The combined nitrogen-fixing bacterial agent consists of the following parts by weight: 30 parts of nitrogen-fixing bacterium Raoultella L03, 45 parts of nitrogen-fixing Bacillus, and 15 parts of nitrogen-fixing bacterium Paspalum distichum.

[0038] The accelerator is composed of the following raw materials in parts by weight: 70 parts mannitol, 55 parts glycerol, 60 parts succinic acid, 0.01 parts sodium molybdate, 0.7 parts dipotassium hydrogen phosphate, 0.5 parts magnesium sulfate, 0.01 parts ferrous sulfate, and 0.7 parts glutathione.

[0039] Example 3

[0040] A method for controlling sugarcane acidification using nitrogen-fixing bacteria includes:

[0041] (1) Control the amount of nitrogen fertilizer. During the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer. 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, which is applied together with phosphate fertilizer and potassium fertilizer on both sides of the planting furrow.

[0042] (2) Applying nitrogen-fixing bacteria to construct a combined nitrogen-fixing system. A combination of nitrogen-fixing bacteria and promoter was applied during the seedling and elongation stages of sugarcane, respectively. The weight ratio of the combination of nitrogen-fixing bacteria and promoter was 10:1. The method of application of the combination of nitrogen-fixing bacteria and promoter was as follows: the combination of nitrogen-fixing bacteria and promoter was mixed according to the weight ratio, and water was added to 150 times the total weight of the combination of nitrogen-fixing bacteria and promoter to obtain a mixed bacterial solution. In the evening, the mixed bacterial solution was irrigated on the roots of sugarcane. The application amount of the mixed bacterial solution was 150 mL / plant. After the mixed bacterial solution was applied, a layer of biochar with a moisture content of 65% was covered, and the thickness of the biochar cover was 2 cm.

[0043] The combined nitrogen-fixing bacterial agent consists of the following parts by weight: 22 parts of nitrogen-fixing bacterium Raoultella L03, 30 parts of nitrogen-fixing Bacillus, and 12 parts of nitrogen-fixing bacterium Paspalum distichum.

[0044] The accelerator is composed of the following raw materials in parts by weight: 55 parts mannitol, 50 parts glycerol, 45 parts succinic acid, 0.008 parts sodium molybdate, 0.5 parts dipotassium hydrogen phosphate, 0.3 parts magnesium sulfate, 0.007 parts ferrous sulfate, and 0.4 parts glutathione.

[0045] Comparative Example 1

[0046] Step 1: Controlling Nitrogen Fertilizer Application: Apply inorganic nitrogen fertilizer using conventional methods throughout the sugarcane's growth cycle. Specifically, apply 20 kg / mu of nitrogen fertilizer as base fertilizer, 15 kg / mu as tillering fertilizer, 15 kg / mu as jointing fertilizer, and 15 kg / mu as tailing fertilizer. Except for the nitrogen fertilizer application rate, the other steps are the same as in Example 1.

[0047] Comparative Example 2

[0048] In step 2, the combined nitrogen-fixing bacteria agent does not contain the nitrogen-fixing bacteria Raoultella L03, and the other steps are the same as in Example 1.

[0049] Comparative Example 3

[0050] In step 2, the combined nitrogen-fixing bacterial agent does not contain nitrogen-fixing Bacillus, and the other steps are the same as in Example 1.

[0051] Comparative Example 4

[0052] In step 2, the combined nitrogen-fixing bacteria agent does not contain *Paspalum distichum*, and the other steps are the same as in Example 1.

[0053] Comparative Example 5

[0054] In step 2, glucose is used instead of mannitol, starch is used instead of glycerol, and citric acid is used instead of succinic acid. The other steps are the same as in Example 1.

[0055] Comparative Example 6

[0056] In step 2, the accelerator does not contain dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, or glutathione; the other steps are the same as in Example 1.

[0057] Cultivation experiment:

[0058] The applicant began cultivating sugarcane using the method of this invention in 2020. The cultivation site was located in Dingdang Town, Long'an County, Guangxi Province. The soil type was sandy loam. Before planting, the soil physicochemical properties were tested and found to be: pH 4.5-5.2, organic matter content 22.3 g / kg, total nitrogen content 0.119%, total phosphorus content 0.055%, and total potassium content 0.62%, indicating acidic soil. The cultivated variety was Guitang 42. The sugarcane rows were 10 m long with a row spacing of 1.5 m. Each example and comparative example had 5 rows planted, with 3 replicates, and the same field management was used. Twenty days after applying the combined nitrogen-fixing bacteria and accelerator during the sugarcane elongation period, the nitrogen fixation percentage and nitrogen fixation amount were measured in each example and comparative example. The detection method was to randomly select two whole sugarcane plants from each example, cut them into pieces, mix them thoroughly, and dry them. 1 g of the dried product from each example and comparative example was sent to the Shanghai Chemical Industry Research Institute for determination of total nitrogen content and nitrogen fixation amount. 15 The percentage of nitrogen atoms exceeds the nitrogen fixation percentage (%) = (1- 15 N fx / 15 N nfx )×100%, Nitrogen fixation amount = Nt×nitrogen fixation percentage%, where, 15 N fx For comparison 15 Ni atoms account for over 100% of the total. 15 N nfx For the sample 15The percentage of nitrogen atoms exceeds the standard value, and Nt represents the total nitrogen content. The results are shown in Table 1. After four consecutive years of planting using the method of this invention (planting began in 2020), the soil pH was compared with that before planting, and the results are shown in Table 2.

[0059] Table 1 Comparison of nitrogen fixation performance of each embodiment and comparative example.

[0060] project Nitrogen fixation percentage (%) Nitrogen fixation capacity (mg / g) Example 1 33.2 328.1 Example 2 32.1 317.2 Example 3 33.9 335.0 Comparative Example 1 20.5 202.6 Comparative Example 2 26.6 262.9 Comparative Example 3 28.1 277.7 Comparative Example 4 28.5 281.7 Comparative Example 5 24.9 246.1 Comparative Example 6 25.1 248.1

[0061] Table 2. Soil pH changes after 4 years of continuous planting

[0062] project Soil pH before cultivation Soil pH after 4 years Example 1 4.8 6.9 Example 2 4.5 6.6 Example 3 5.2 7.1 Comparative Example 1 4.9 5.3 Comparative Example 2 4.8 5.5 Comparative Example 3 4.8 5.8 Comparative Example 4 4.9 5.9 Comparative Example 5 4.9 5.6 Comparative Example 6 4.8 5.7

[0063] The above experimental data show that: (1) Reducing the amount of nitrogen fertilizer used during sugarcane growth can reduce the inhibitory effect of nitrogen fertilizer on syn-nitrogen-fixing bacteria, thereby improving nitrogen fixation efficiency. (2) The combined use of Bacillus azotocinus, Paspalum distichum, and Raoultella L03 can quickly attract a large number of nitrogen-fixing bacteria to gather at the roots, forming a population advantage, enabling the syn-nitrogen-fixing bacteria to quickly form an efficient syn-nitrogen-fixing system with sugarcane. (3) Mannitol, glycerol, and succinic acid are the most suitable carbon sources for the syn-nitrogen-fixing bacteria of this invention. (4) The combination of dipotassium hydrogen phosphate, magnesium sulfate, ferrous sulfate, and glutathione provides a good nitrogen-fixing environment for nitrogen-fixing bacteria, ensuring nitrogen fixation efficiency. Therefore, this invention accelerates the colonization of nitrogen-fixing bacteria and improves the nitrogen-fixing capacity of syn-nitrogen-fixing bacteria through a series of technical means such as controlling the amount of nitrogen fertilizer and providing suitable growth conditions for nitrogen-fixing bacteria, thereby achieving the purpose of efficient nitrogen fixation, less nitrogen fertilizer application, and reduced soil acidification.

[0064] 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 controlling sugarcane acidification using nitrogen-fixing bacteria, characterized in that, Throughout the entire growth cycle of sugarcane, only the base fertilizer contains nitrogen fertilizer, while the fertilizers applied at other times do not contain nitrogen fertilizer; and a combination of nitrogen-fixing bacteria and growth promoters are applied during the sugarcane seedling stage and the elongation stage, respectively. The combined nitrogen-fixing bacterial agent comprises the following bacterial agents in parts by weight: 10-30 parts of nitrogen-fixing bacterium Raoultella L03, 25-45 parts of nitrogen-fixing Bacillus, and 5-15 parts of nitrogen-fixing bacterium Paspalum distichum. The accelerator comprises the following raw materials in parts by weight: 40-70 parts mannitol, 30-55 parts glycerol, 35-60 parts succinic acid, 0.001-0.01 parts sodium molybdate, 0.1-0.7 parts dipotassium hydrogen phosphate, 0.1-0.5 parts magnesium sulfate, 0.005-0.01 parts ferrous sulfate, and 0.2-0.7 parts glutathione.

2. The method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 1, characterized in that, The nitrogen fertilizer refers to nitrogen-containing inorganic fertilizer; the amount of nitrogen fertilizer applied in the base fertilizer is 15-25 kg / mu, which is applied together with phosphate fertilizer and potassium fertilizer on both sides of the planting furrow.

3. The method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 1, characterized in that, The combined nitrogen-fixing bacterial agent comprises the following parts by weight: 22 parts of nitrogen-fixing bacterium Raoultella L03, 30 parts of nitrogen-fixing Bacillus, and 12 parts of nitrogen-fixing bacterium Paspalum distichum.

4. The method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 1, characterized in that, The accelerator comprises the following raw materials in parts by weight: 55 parts mannitol, 50 parts glycerol, 45 parts succinic acid, 0.008 parts sodium molybdate, 0.5 parts dipotassium hydrogen phosphate, 0.3 parts magnesium sulfate, 0.007 parts ferrous sulfate, and 0.4 parts glutathione.

5. The method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 1, characterized in that, The weight ratio of the combined nitrogen-fixing bacteria agent and the promoter is 5-15:

1.

6. A method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 1, characterized in that, The method of using the combined nitrogen-fixing bacteria agent and the promoter is as follows: mix the combined nitrogen-fixing bacteria agent and the promoter according to the weight ratio, and add water at 100-200 times the total weight of the combined nitrogen-fixing bacteria agent and the promoter to mix evenly to obtain a mixed bacterial solution, and then irrigate the sugarcane roots with the mixed bacterial solution.

7. A method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 6, characterized in that, The amount of the mixed bacterial solution applied is 100-200 mL per plant.

8. A method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 6, characterized in that, After the mixed bacterial solution is applied, a layer of biochar is placed over it, with a biochar layer thickness of 1-2 cm.

9. A method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 6, characterized in that, The mixed bacterial solution is applied in the evening or morning.

10. A method for controlling sugarcane acidification using nitrogen-fixing bacteria according to claim 8, characterized in that, The biochar has a moisture content of 60-70%.