Bio-organic fertilizer containing Bacillus subtilis HFN04 and its application

Abstract

This invention proposes a bio-organic fertilizer based on Bacillus subtilis HFN04 and its application in controlling cotton wilt disease, relating to the field of microbial application technology. The invention contains Bacillus subtilis (HFN04). Bacillus subtilis HFN04 bio-organic fertilizer has an improving effect on arid and saline-alkali soils in Xinjiang; it contains Bacillus subtilis (… Bacillus subtilis After being applied to the soil, the bio-organic fertilizer of HFN04 forms a dominant microbial community in the plant rhizosphere, which improves the micro-ecological environment of the crop roots, effectively prevents the invasion of pathogens, including cotton wilt, in arid and saline soil environments, and increases cotton yield.

Description

Technical Field

[0001] This invention relates to the field of microbial application technology, and in particular to a bio-organic fertilizer containing Bacillus subtilis HFN04 and its application. Background Technology

[0002] Fusarium wilt, a common soil-borne plant disease in agricultural production, is characterized by its wide spread, short duration, severe damage, and high difficulty in control. The pathogen can survive in the soil environment for many years and exhibits strong resistance. Fusarium wilt has caused serious damage to agriculture worldwide, resulting in enormous economic losses.

[0003] Related studies have shown that Bacillus subtilis has good application effects in controlling cotton wilt. However, Xinjiang, which accounts for more than 90% of the country's total cotton production, is one of the driest regions in my country. Apart from natural factors such as climate, the irrigation areas in Xinjiang generate excessive deep infiltration due to the use of floods and summer high-temperature water-suppressing irrigation and the storage of water in autumn and winter, leading to large-scale secondary soil salinization. The soil in Xinjiang is characterized by chloride or sulfate-chloride accumulation, poor structure of saline-alkali soil, capillary action cavities, and poor permeability. Existing cotton wilt control methods are not effective on cotton in Xinjiang.

[0004] Therefore, there is an urgent need for a method to control cotton wilt disease in arid soil conditions. Summary of the Invention

[0005] In order to solve at least one of the problems existing in the prior art, the present invention provides a bio-organic fertilizer containing Bacillus subtilis HFN04 and its application.

[0006] In a first aspect, the present invention provides a Bacillus subtilis ( Bacillus subtilis HFN04, its accession information is as follows: accession number: CGMCC No.29091; depositary institution: China General Microbiological Culture Collection Center (CGMCC); depositary address: No.3, No.1 Beichen West Road, Chaoyang District, Beijing; deposit date: November 21, 2023.

[0007] This invention collected soil samples from saline-alkali land plots in Linhe County, Inner Mongolia, Huanghua City, Hebei Province, and suburban Beijing. A strain with good soil improvement effects on saline-alkali soil was isolated from these samples. After physicochemical property identification and sequence alignment of its 16S rRNA, gyrA, cheA, and amyA genes, the strain was identified as *Bacillus subtilis*. Bacillus subtilis ), named Bacillus subtilis ( Bacillus subtilis HFN04 has the following physiological characteristics:

[0008] The vegetative cells are rod-shaped, with meso-spores, Gram-positive, and aerobic. They can grow on 20% NaCl nutrient agar. The spores are heat-resistant, produce α-amylase and protease, are catalase-positive, and VP-positive. They hydrolyze starch and do not grow anaerobically.

[0009] The present invention further provides a product containing the aforementioned Bacillus subtilis ( Bacillus subtilis HFN04 bacterial agent.

[0010] The present invention further provides a product containing the aforementioned Bacillus subtilis ( Bacillus subtilis HFN04 is a bio-organic fertilizer. It comprises the following components: ammonium sulfate, urea, monoammonium phosphate, potassium chloride, microbial agent, humic acid, binder, zinc sulfate, borax, and magnesium sulfate; wherein the microbial agent is Bacillus subtilis (…). Bacillus subtilis HFN04, a complex spore suspension of Bacillus amyloliquefaciens, Bacillus gelatinosa, Bacillus laterosporus, Bacillus megaterium and Bacillus licheniformis.

[0011] Preferably, the bio-organic fertilizer comprises, by weight, 100 kg of ammonium sulfate, 230 kg of urea (46% N), 200 kg of monoammonium phosphate (11-49), 240 kg of potassium chloride (62% K2O), 100 kg of microbial agent (2 billion CFU / g, organic matter ≥70%), 130 kg of humic acid (organic matter ≥80%), 50 kg of binder, 2.5 kg of zinc sulfate, 2.5 kg of borax, and 3.0 kg of magnesium sulfate.

[0012] The present invention further provides a product containing the aforementioned Bacillus subtilis ( Bacillus subtilis The role of HFN04 bio-organic fertilizer in the prevention and control of cotton wilt disease.

[0013] The present invention has the following beneficial effects:

[0014] This invention isolates a Bacillus subtilis species from soil. Bacillus subtilis HFN04, this bacterium has an improving effect on arid and saline-alkali soils in Xinjiang. (The text abruptly ends here, likely due to an incomplete sentence or missing information.) Bacillus subtilis When HFN04 is applied during crop growth, it effectively increases crop yield. The Bacillus subtilis (HFN04) provided in this invention... Bacillus subtle HFN04 plays an important role in controlling cotton wilt in saline-alkali soil environments. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in this invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0016] Figure 1 This is a diagram showing the preservation effect of the HFN04 strain under alkaline conditions in Example 2 of the present invention.

[0017] Figure 2 This shows the survival of the HFN04 strain under different temperature conditions in Example 2 of the present invention.

[0018] Figure 3 This is a schematic diagram of the antibacterial experiment results of Bacillus subtilis HFN04 against the growth of Fusarium oxysporum, the causal agent of cotton wilt, provided in Example 2 of the present invention. Detailed Implementation

[0019] It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Where specific techniques or conditions are not specified in the embodiments, they should be performed in accordance with the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified can be purchased from legitimate channels as conventional products.

[0020] Among them, Bacillus licheniformis ( Bacillus licheniformis ), Bacillus amyloliquefaciens ( Bacillus starch liquefier ), Bacillus megaterium (Baeillus megatherium) Both Bacillus jellyoides and Bacillus laterosporus were obtained from Beijing Aerospace Hengfeng Technology Co., Ltd.

[0021] Example 1

[0022] This embodiment provides a method for isolating and purifying Bacillus subtilis (Bt) from soil. Bacillus subtilis The HFN04 process is as follows:

[0023] Twenty-seven soil samples were collected from saline-alkali soils in Linhe, Inner Mongolia; Huanghua, Hebei Province; and the suburbs of Beijing. The samples were air-dried and preserved. Ten grams of soil sample were added to an Erlenmeyer flask containing 90 ml of sterile water and shaken for 30 minutes to prepare a 1 / 10 concentration soil suspension. This suspension was then diluted to a 10⁻³ concentration and incubated on nutrient agar containing 15% NaCl at 30°C for 24-48 hours. Representative bacterial colonies were then selected and preserved.

[0024] Isolation and initial screening of salt-tolerant bacteria: Soil sample dilutions were spread onto nutrient agar containing 15% NaCl and incubated at 30℃ for 24-48 hours. Representative bacterial colonies exhibiting various colony characteristics were selected as initial screening strains. Among these, 21 strains were isolated from soil samples in Inner Mongolia (numbered HFN01, HFN02, HFN03, HFN04, HFN05…HFN21), 16 strains from soil samples in Hebei Province (numbered HFH01, HFH02, HFH03…HFH16), and 17 strains from soil samples in Beijing (numbered HFB01, HFB02, HFB03…HFB17). Secondary screening of salt-tolerant strains: Of the 54 initially screened strains, 43 were identified as Bacillus-like bacteria capable of forming spores on nutrient agar. Spore suspensions of these 43 Bacillus-like bacteria were prepared and treated in a 100℃ water bath for 15 minutes; 7 strains survived. The strains were numbered HFN04, HFN16, HFN20, HFH01, HFB09, HFB10, and HFB17. After morphological observation and nutrient agar culture characteristics, strain HFN04, with its rapid growth rate, was selected for further research and production trials. It was identified and named *Bacillus subtilis* by the Microbial Fertilizer Quality Supervision and Inspection Center of the Ministry of Agriculture and Rural Affairs.

[0025] This embodiment further describes the isolated and purified Bacillus subtilis ( Bacillus subtilis HFN04 was used for identification, such as Figure 1 As shown, Bacillus subtilis ( Bacillus subtilis The physiological characteristics of HFN04 are as follows: vegetative cells are rod-shaped, measuring 0.7-0.8 × 1.6-3.0 μm; mesophytic spores, which are predominantly mesophytic, cylindrical, and sporangia are not enlarged, measuring 0.6-0.7 × 1.0-1.5 μm. The bacteria are motile and Gram-positive. Colony characteristics on solid media are: round, flat, pale yellow, slightly glossy, opaque, with irregular edges; easily diffuses on moist media; older colonies have a rough, wrinkled surface. Physiological and biochemical characteristics: Production temperature is 30-30℃, with an optimal production temperature of 31-33℃; good growth on 15% NaCl nutrient agar; can also grow on 20% NaCl nutrient agar; optimal growth pH is 6.7-7.2; aerobic; spores are heat-resistant; produces α-amylase and protease; positive for catalase and VP reaction; hydrolyzes starch; does not grow anaerobically.

[0026] This embodiment further incorporates Bacillus subtilis (Bacillus subtilis) Bacillus subtilisThe accession number for HFN04 is CGMCC No. 29091; the depositary institution is the China General Microbiological Culture Collection Center (CGMCC); the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; and the deposit date is November 21, 2023.

[0027] Example 2

[0028] This embodiment focuses on Bacillus subtilis (Bacillus subtilis) Bacillus subtilis The resistance to salt, acids and alkalis, and high temperatures of HFN04 was tested using the following methods:

[0029] The tested strain was Bacillus subtilis ( Bacillus subtilis HFN04; the culture medium is beef extract peptone medium, and the culture is carried out at 30~32℃ for 36~52 hours; the chemical reagents used are NaCl, K2HPO4, citric acid, boric acid, etc.

[0030] 1.1 Salt Tolerance Test

[0031] Bacillus subtilis ( Bacillus subtilis HFN04 was inoculated onto nutrient agar containing no NaCl and at concentrations of 0.5%, 5%, 10%, 15%, and 20%, respectively, and incubated at 30°C for 24, 48, and 72 hours. The growth of the strain was observed, and each experiment was repeated three times.

[0032] 1.2 High-salt environment preservation test

[0033] Bacillus subtilis ( Bacillus subtilis The spore suspensions of strain HFN04 were stored at room temperature for 30 days and 60 days in 0%, 6%, 10%, 15%, and 20% NaCl environments, respectively. Viable bacteria were counted in the storage solutions to check the survival ability of the strain in high-salt environments. Each experiment was repeated three times.

[0034] 1.3 Acid and alkali resistance test

[0035] Liquid culture media with different pH values ​​(pH 2.8, 4.4, 5.2, 6.0, 6.8, 7.6, 8.4, 9.2, and 10.0) were prepared by mixing beef extract, peptone liquid medium, and chemical reagents such as K2HPO4, citric acid, NaOH, and boric acid in a certain proportion. The HFN04 strain was inoculated on the above-mentioned mediums with different pH values ​​and cultured at 30°C for 24 hours, 48 ​​hours, and 72 hours. The growth was observed, and each experiment was repeated three times to investigate the growth of the strain in different pH environments, providing a basis for the production of microbial fertilizers.

[0036] 1.4 Preservation test under alkaline conditions

[0037] Bacillus subtilis ( Bacillus subtilisThe spore suspension of strain HFN04 was stored at room temperature in an environment of pH 9.2, with pH 6.8 as a control. Viable cell counts were performed after 2, 4, 6, 8, 10, 12 and 14 months of storage. The experiment was repeated three times to investigate the survival ability of spores in a strongly alkaline environment.

[0038] 1.5 High Temperature Resistance Test

[0039] Bacillus subtilis ( Bacillus subtilis After incubating the spore suspension of strain HFN04 in a water bath at 30℃, 40℃, 50℃, 60℃, 70℃, and 80℃ for 0.5 hours, viable bacteria were counted. Each experiment was repeated three times to determine the high-temperature resistance of the spore suspension.

[0040] 1.6 Low Temperature Resistance Test

[0041] Bacillus subtilis ( Bacillus subtilis The spore suspension of HFN04 was adsorbed with sterilized peat moss at a ratio of 1:6 to prepare a powdered microbial agent, which was then frozen at -18℃ for 380 days. During the storage period, samples were taken regularly to count the viable bacteria and to determine the low-temperature resistance of the strain.

[0042] 1.7 Flat Plate Confrontation Culture Method

[0043] like Figure 1 As shown, the plate confrontation culture method revealed that strain HFN04 had an inhibition rate of 70.69% against the growth of Fusarium oxysporum, the causal agent of cotton wilt.

[0044] The test results for sections 1.1-1.6 are shown below:

[0045] 2.1 Salt Tolerance Test

[0046] Bacillus subtilis ( Bacillus subtilis The HFN04 strain was inoculated onto nutrient agar containing 0-20% NaCl, and the cell growth is shown in Table 1. The results indicate that the HFN04 strain grows best under 0-5% NaCl conditions, followed by 10-15% NaCl conditions, and can still grow under 20% NaCl. This demonstrates that the HFN04 strain can germinate spores, grow and reproduce, and maintain its physiological metabolic activities under high-salt conditions.

[0047] Table 1. Effects of different concentrations of NaCl on the growth of HFNO4 bacteria.

[0048]

[0049] Note: - No growth, + Growth, the number of + indicates the growth status of the bacteria.

[0050] 2.2 High-salt environment preservation test

[0051] Bacillus subtilis ( Bacillus subtilis Table 2 shows the viable cell counts of HFN04 strain spore suspensions after 30 and 60 days of storage at room temperature in different concentrations of NaCl environments.

[0052] Table 2. Survival of HFN04 strain at different salt concentrations (Unit: 10⁻⁶) 8 cuf / ml

[0053]

[0054] As can be seen from the experimental results in Table 2, Bacillus subtilis ( Bacillus subtilis The HFN04 strain can survive normally in high-salt environments. After 60 days of storage at room temperature in 20% NaCl, the survival rate is 88.0%, and after 60 days in a 15% salt solution, the survival rate is 89.0%. After 60 days of storage without NaCl, the bacterial count showed no significant change. These experimental results provide a theoretical basis and scientific evidence for the mixed granulation of the HFN04 strain with high-nutrient fertilizers and organic fertilizers.

[0055] 2.3 Acid and alkali resistance test

[0056] Bacillus subtilis ( Bacillus subtilis Table 3 shows the growth of strain HFN04 on liquid beef extract peptone medium with pH 2.8–10.0. The results indicate that strain HFN04 can grow within the experimental pH range of 4.4–10.0, with optimal growth at pH 6.0–8.4. This demonstrates that the bacterium can grow and reproduce in a strongly alkaline environment, providing a technical basis for its application in improving saline-alkali soils.

[0057] Table 3. Growth of HFN04 strain under different pH conditions

[0058]

[0059] Note: - No growth, + Growth, the number of + indicates the growth status of the bacteria.

[0060] 2.4 Preservation test under alkaline conditions

[0061] Bacillus subtilis ( Bacillus subtilis The spore suspension of strain HFN04 was stored under strongly alkaline conditions for up to 14 months. Viability testing results were as follows: Figure 1 As shown.

[0062] Depend on Figure 1 It can be seen that Bacillus subtilis ( Bacillus subtilisThe HFN04 strain showed no significant change in bacterial count when stored under neutral conditions. After 6 months of storage at pH 9.2, the survival rate was 91.7%, and after 14 months, it was 82.4%. This indicates that the spore-forming properties of this strain are relatively stable under alkaline conditions.

[0063] 2.5 High Temperature Resistance Test

[0064] Bacillus subtilis ( Bacillus subtilis After incubating spore suspensions of strain HFN04 at different temperatures for 0.5 hours, the viable cell count results are as follows: Figure 2 As shown in the results, Bacillus subtilis (…) Bacillus subtle The spores of strain HFN04 can tolerate high temperatures; after being incubated at 80℃ for 0.5 hours, the survival rate reached as high as 93.6%. This is related to the high-temperature treatment at 100℃ used in the secondary screening process of the strain. The ability to tolerate high temperatures is a physiological characteristic of the strain. This characteristic allows for higher-temperature drying treatment in the industrial production of granular microbial fertilizers, laying the foundation for industrial production.

[0065] Bacillus subtilis ( Bacillus subtilis The spore suspension of strain HFN04 was mixed with sterilized peat moss to prepare a peat moss-type bacterial agent with a water content of 25%. The viable cell count results after freezing at -18℃ for 380 days are shown in Table 4.

[0066] Table 4. Low-temperature preservation test of HFN04 bacterial agent

[0067]

[0068] As can be seen from the research results in Table 4, Bacillus subtilis (… Bacillus subtilis The viable cell count of the bacterial agent prepared from strain HFN04 did not change significantly after being stored at -18℃ for 380 days, indicating that the spores of strain HFN04 have strong resistance to low temperatures. After long-term low-temperature storage, the spores can germinate normally upon returning to normal temperature, and their growth characteristics remain unchanged.

[0069] Preparation Example 1

[0070] This embodiment further detects Bacillus subtilis (… Bacillus subtilis The effects of Bacillus subtilis strain HFN04 on improving saline-alkali soil and inhibiting cotton wilt are as follows: Bacillus subtilis (HFN04) was used to treat the soil. Bacillus subtilisA compound bacterial solution was prepared by mixing HFN04 spore suspension, Bacillus amyloliquefaciens, Bacillus mucilaginosus, Bacillus laterosporus, and Bacillus megaterium in a ratio of 6:3:3:1:1. During the preparation of the bacterial agent, 100 kg of microbial agent with an effective viable count ≥2 billion CFU / g (mainly adding microbial metabolites and trace elements in biochelates, while simultaneously treating chloride ions) was added at the initial feeding stage, and steam drum granulation was used. After the granules cooled, 1.5 kg of bacterial powder with an effective viable count ≥200 billion CFU / g was evenly coated onto the surface of the fertilizer granules. The bacterial powder was mixed with an anti-caking agent for even coating; this yielded a bacterial agent for improving saline-alkali soil.

[0071] A bio-organic fertilizer containing Bacillus subtilis HFNO4 is prepared by combining 100 kg of the above-mentioned compound microbial agent (2 billion CFU / g, organic matter ≥70%) with 100 kg of ammonium sulfate, 230 kg of urea (46% N), 200 kg of monoammonium phosphate (11-49), 240 kg of potassium chloride (62% K2O), 130 kg of humic acid (organic matter ≥80%), 50 kg of binder, 2.5 kg of zinc sulfate, 2.5 kg of borax, and 3.0 kg of magnesium sulfate. Specifically, the binder can be one of the following: polyvinylpyrrolidone solution, starch paste, or hydroxypropyl methylcellulose solution; no specific limitation is made here.

[0072] Experimental Example 1

[0073] On April 1, 2022, six core experimental sites were set up in major cotton-growing farms such as Xinhu Farm and Fangcaohu Farm of the Sixth Division to plant cotton. Each experimental site set up a control plot. The experimental plots were treated with the bio-organic fertilizer of this invention at a rate of 100 kg per mu, while the control plots were treated with only local organic fertilizer source sheep manure.

[0074] Example 1

[0075] Effects on soil acidity / alkalinity (pH) and total soil salt content

[0076] pH value is an important basic property of soil and one of the factors affecting soil fertility. It directly affects the state, transformation, and availability of soil nutrients. Measuring soil composition can determine the soil salinity level, and thus whether it affects seed germination and normal crop growth.

[0077] Soil samples were taken from soils treated with bio-organic fertilizer and soils treated with sheep manure alone to determine their pH value and soil composition. The results are as follows:

[0078] Table 5 Soil Composition Table

[0079]

[0080] As shown in Table 5, the average water-soluble salt content at the sampling points where bio-organic fertilizer was applied was 1.63 g / kg, a decrease of 11.72% compared to the control; the average total nitrogen and organic matter content were 1.08 g / kg and 22.31 g / kg, respectively, increases of 18% and 18.39% compared to the control; and the average hydrolyzable nitrogen, available phosphorus, and available potassium content were 52.83 mg / kg, 43.03 mg / kg, and 164.17 mg / kg, respectively, increases of 8.82%, 10.58%, and 7.53% compared to the control. This indicates that applying the bio-organic fertilizer of this invention can significantly improve the physical and chemical properties of cotton soil, reduce the risk of soil salinization, and lay a solid foundation for high cotton yields.

[0081] The cotton yield statistics for the above-mentioned test sites are shown in the following table:

[0082] Table 6 Yield Traits Survey Form (Yield Measurement Statistics)

[0083]

[0084] As can be seen from Table 6, application of products containing Bacillus subtilis (… Bacillus subtilis Compared with the control, the bio-organic fertilizer of HFN04 can effectively increase the seedling survival rate and the number of bolls per plant, without affecting the weight of a single boll, but it can increase the yield of seed cotton per mu by 34.4%.

[0085] The following table shows the yield analysis of cotton lint and seed cotton per mu at the above test sites:

[0086] Table 7 Production Analysis

[0087]

[0088] As can be seen from Table 7: the present invention contains Bacillus subtilis ( Bacillus subtilis The yield of cotton lint and seed cotton per mu under the treatment with HFN04 bio-organic fertilizer increased by 16.81% and 15.24% respectively compared with the control group that did not use the bio-organic fertilizer of this invention.

[0089] The above-ground fresh weight and above-ground dry weight of cotton from the above-mentioned test sites are analyzed in the table below:

[0090] Table 8 Dry Matter Survey Form

[0091]

[0092] As can be seen from Table 8, application of products containing Bacillus subtilis (…) Bacillus subtilis The aboveground fresh weight and aboveground dry weight of cotton grown with HFN04 bio-organic fertilizer were both higher than the control, increasing by 65.83% and 42.25%, respectively. Furthermore, it contains Bacillus subtilis (…). Bacillus subtilisThe fresh weight and dry weight of the underground parts of cotton treated with HFN04 bio-organic fertilizer were significantly higher than those of the control, increasing by 33.02% and 34.33%, respectively. This indicates that microbial agents may mainly promote the accumulation of dry matter in cotton by coordinating the distribution of dry matter, thus laying a solid foundation for high and stable yields.

[0093] The following table summarizes the severity of Fusarium wilt at the aforementioned test sites:

[0094] Table 9. Occurrence of Fusarium wilt in cotton fields (111 Community, 105th Regiment)

[0095]

[0096] As can be seen from Table 9: On July 28, 2022, an investigation of 12 sampling points revealed that the application of products containing Bacillus subtilis (…) Bacillus subtilis The experimental field using HFN04 bio-organic fertilizer showed a reduction in the number of wilt-caused plants compared to the untreated control field, with an 8.8% decrease in disease incidence and a 4.2 decrease in disease index. On August 25, 2022, an investigation of three sampling points revealed that the application of fertilizer containing Bacillus subtilis (…)… Bacillus subtilis The experimental field using HFN04 bio-organic fertilizer showed a reduction in the number of wilt-caused plants compared to the untreated control field, with an 8.6% decrease in disease incidence and a 4.4 decrease in disease index. This indicates that the present invention contains Bacillus subtilis (…). Bacillus subtilis HFN04 bio-organic fertilizer has an inhibitory effect on cotton wilt disease and the disease prevention effect is long-lasting.

[0097] In summary, the Bacillus subtilis (Bacillus subtilis) of the present invention Bacillus subtilis HFN04, this bacterium has an improving effect on arid and saline-alkali soils in Xinjiang; it contains Bacillus subtilis ( Bacillus subtilis After HFN04 bio-organic fertilizer is applied to the soil, beneficial microorganisms form a dominant flora in the plant rhizosphere, improving the micro-ecological environment of crop roots, effectively preventing the invasion of pathogens including wilt, and increasing cotton yield.

[0098] Example 2

[0099] In the Red Globe grape-growing region of the Fifth Division of the Xinjiang Production and Construction Corps along the Tianshan Mountains in northern Xinjiang, grapes were planted on severely saline-alkali land. The land was divided into experimental plots and control plots. The experimental plots were treated with the bio-organic fertilizer of this invention, while the control plots were treated with only local organic fertilizer source sheep manure. Each plot was 0.1 mu (approximately 0.067 hectares) in size, and each group was replicated three times. The local organic fertilizer source sheep manure was mixed with the bio-organic fertilizer obtained in Preparation Example 1, thoroughly stirred, and 10-15% water was added. After fermentation for 10 days, the mixture was applied. A trench was dug 30 cm away from the grape roots, and a 30 cm thick layer of fertilizer was laid, at a rate of 200 kg per mu (approximately 133 kg per hectare), as base fertilizer.

[0100] Topsoil samples (0-20 cm) were taken from plots where the bio-organic fertilizer prepared according to the present invention was applied and from experimental plots where only sheep manure was applied. Soil organic matter content and available nutrient content were measured. The results are as follows:

[0101] Table 10. Effects of the bio-organic fertilizer of the present invention on soil organic matter and available nutrients in the soil.

[0102]

[0103] As can be seen from Table 10, the soil in the vineyard was very barren when only sheep manure was applied, with an organic matter content of only 0.382%. After applying the bio-organic fertilizer of this invention, the soil organic matter content rapidly increased to 1.58%, an increase of 4.14 times.

[0104] The available nitrogen content in the soil of vineyards fertilized only with sheep manure was 13.04 mg / kg. After applying the bio-organic fertilizer of this invention, the available nitrogen content increased to 58.40 mg / kg. This indicates that the soil fertilized only with sheep manure was extremely poor in nutrients and severely deficient in nitrogen. After applying the bio-organic fertilizer, the available nitrogen content increased to a moderate level, nearly 4.5 times higher.

[0105] Grapevines treated with sheep manure alone had low available phosphorus content (1.80 mg / kg), which rapidly increased to 55.45 mg / kg after fertilization, reaching the level of fertile soil, an increase of nearly 31 times. The grapevines treated with this invention's bio-organic fertilizer had previously had 100 kg of superphosphate per acre, resulting in high phosphorus content in the soil.

[0106] The soil available potassium content in the vineyards that only applied sheep manure was higher than that of the soil available nitrogen and available phosphorus, at 81.0 mg / kg, which is considered to be at a medium to low level. After fertilization, the potassium content increased to 145.0 mg / kg, reaching a medium to high potassium level, an increase of nearly 1.8 times.

[0107] The above description shows that after applying the bio-organic fertilizer of this invention, the soil organic matter content increased from less than 0.38% to about 1.58%, an increase of 4.14 times. The soil became more fertile, and the available nutrients in the soil increased accordingly, providing the necessary nutrition for increased crop yield.

[0108] Example 3

[0109] Effects on soil acidity / alkalinity (pH) and total soil salt content

[0110] pH value is an important basic property of soil and one of the factors affecting soil fertility. It directly affects the state, transformation, and availability of soil nutrients. Measuring the total salt content of soil can determine its salinity. A total salt content greater than 0.2% directly affects seed germination and normal crop growth.

[0111] Soil samples were taken from soils treated with bio-organic fertilizer and soils treated with sheep manure alone to determine their pH value and total salt content. The results are as follows:

[0112] Table 11. Effects of the bio-organic fertilizer of the present invention on soil acidity and total soil salinity.

[0113]

[0114] As can be seen from Table 11, the soil pH value of grapes with only sheep manure was 8.64, while the soil pH value was 8.05 after applying the organic fertilizer of this invention. The pH value of grapes decreased and the alkaline soil was improved.

[0115] Total salt content measurements showed that the total salt content of the vineyard soil, when only sheep manure was applied, was 0.264%, classifying it as slightly saline-alkali soil. After fertilization, the total salt content changed, decreasing to 0.103%. This was because the application of bio-organic fertilizer increased the organic matter content of the vineyard soil from 0.382% to 1.58%; the soil structure improved, the non-capillary porosity increased, accelerating soil salt leaching and cutting off soil capillaries, reducing soil moisture evaporation and inhibiting soil salinization. Within a year, the saline-alkali soil was improved, with the surface soil salinity of the vineyard soil decreasing from 0.264% to 0.103%, and the soil desalination rate reaching -60.98%, transforming moderately saline-alkali soil into slightly saline-alkali soil in the same year.

[0116] The experiment employed conventional microbial flora analysis methods (plate viable count) to analyze the species and quantity of viable bacteria in the provided samples. The results are shown in Table 14.

[0117] Table 12. Effects of the bio-organic fertilizer of this invention on the types and quantities of soil microorganisms.

[0118]

[0119] As can be seen from Table 12, there are significant differences in the number of microorganisms in different soil samples. The analysis results also show that the effective microorganisms in Aerospace Hengfeng Bio-fertilizer have grown and multiplied in large numbers in the local soil, stimulating and promoting the growth and reproduction of other indigenous microorganisms. Overall, in all soil samples treated with the bio-organic fertilizer of this invention, the number of microorganisms (bacteria, fungi, and actinomycetes) was higher than that in the plots treated with only sheep manure, with the number varying from several times to tens of times, showing extremely significant differences.

[0120] The analysis results show that there is a certain correlation between the types and quantities of microorganisms in the soil and soil nutrients. Moreover, the soil sample with applied bio-organic fertilizer is darker in color, looser and more elastic than the soil sample with applied sheep manure alone, and there are also certain changes in soil physical properties.

[0121] Example of effect 4

[0122] Impact on crop quality and yield

[0123] In the Red Globe grape-growing region of the Fifth Division of the Xinjiang Production and Construction Corps, severely saline-alkali land was selected; grapes were planted; each plot was 0.1 mu (approximately 0.067 hectares); each group of plots was divided into 4 units, and each unit included 10 plots; units 1-3 were treated with the prepared bio-organic fertilizer at a rate of 200 kg per mu; unit 4 was treated with a fertilizer without mixed microbial agents, namely, a fertilizer prepared from 100 kg ammonium sulfate, 230 kg urea (46% N), 200 kg monoammonium phosphate (11-49%), 240 kg potassium chloride (62% K2O), 130 kg humic acid (organic matter ≥80%), 50 kg binder, 2.5 kg zinc sulfate, 2.5 kg borax, and 3.0 kg magnesium sulfate. Among these, the crop yields in units 1-3 of the three groups all showed a significant increase. The application rate was 200 kg per mu.

[0124] In the first year of grape planting, the yield of grapes with the application of bio-organic fertilizer (average of units 1-3) was 1620 kg / mu, while the grape control (unit 4) yielded 1293 kg / mu.

[0125] In summary, the Bacillus subtilis (Bacillus subtilis) of the present invention Bacillus subtilis HFN04, this bacterium has an improving effect on arid and saline-alkali soils in Xinjiang; it contains Bacillus subtilis ( Bacillus subtilis After HFN04 bio-organic fertilizer is applied to the soil, beneficial microorganisms form a dominant flora in the plant rhizosphere, improving the micro-ecological environment of the crop roots and effectively increasing grape yield.

[0126] Although the present invention has been described in detail above with general descriptions, specific embodiments, and experiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications and improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A substance containing Bacillus subtilis ( Bacillus subtilis The bio-organic fertilizer of HFN04 is characterized by, It includes the following components: ammonium sulfate, urea, monoammonium phosphate, potassium chloride, bacterial agent, humic acid, binder, zinc sulfate, borax and magnesium sulfate; The bacterial agent is Bacillus subtilis (Bacillus subtilis) Bacillus subtilis HFN04, a compound spore suspension of Bacillus amyloliquefaciens, Bacillus mucilaginosus, Bacillus laterosporus, Bacillus megaterium and Bacillus licheniformis; The Bacillus subtilis ( Bacillus subtilis The accession number for HFN04 is CGMCC No. 29091; the depositary institution is the China General Microbiological Culture Collection Center (CGMCC); the depositary address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing; and the deposit date is November 21, 2023.

2. The Bacillus subtilis-containing compound as described in claim 1 (… Bacillus subtilis The bio-organic fertilizer of HFN04 is characterized by, The bio-organic fertilizer comprises, by weight, 100 kg of ammonium sulfate, 230 kg of urea, 200 kg of monoammonium phosphate, 240 kg of potassium chloride, 100 kg of microbial agent, 130 kg of humic acid, 50 kg of binder, 2.5 kg of zinc sulfate, 2.5 kg of borax, and 3.0 kg of magnesium sulfate.

3. A method containing Bacillus subtilis as described in claim 1 (… Bacillus subtilis Application of HFN04 bio-organic fertilizer in the prevention and control of cotton wilt disease.

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