Bacillus tequilensis as well as related products and application thereof

Abstract

The invention discloses bacillus tequilensis as well as a related product and application thereof, and relates to the field of microorganisms, the bacillus tequilensis XH1 provided by the invention is a complete ammonia oxidizing microorganism and can independently oxidize organic nitrogen in soil into NO3 <->-N which can be easily absorbed and utilized by plants so as to realize efficient prevention and control of soil DON leaching loss and promote biological growth; xH1 can be combined with nitrogen-fixing bacteria for use, so that the complete ammonia oxidation capacity can be further improved; the XH1 can also remove ammonia in a water body, and the XH1 has remarkable agricultural application and environmental protection potential.

Description

Technical Field

[0001] The present invention relates to the field of microorganisms, and in particular to Bacillus tequila and related products and applications thereof. Background Art

[0002] Organic fertilizers are widely used in modern agricultural production to restore soil and improve soil fertility, thereby increasing the yield and quality of crops. Organic fertilizers contain rich organic matter and a variety of nutrients, which not only provide plants with essential nutrients, but also promote soil microbial activity, enhance soil water and fertilizer retention capacity, and thus improve crop quality. However, most of the nitrogen in organic fertilizers exists in the form of organic nitrogen. Long-term application will lead to the accumulation of soluble organic nitrogen (Dissolved organic nitrogen, DON) in the soil. Under rainfall or irrigation conditions, DON will be severely lost through leaching. In addition, the soluble organic nitrogen in organic fertilizers is mostly a large molecular structure, which is not conducive to plant absorption and utilization, and the mineralization rate of these macromolecules is slow, and they are converted into nitrate nitrogen (NO3 - -N) is also relatively slow, which is difficult to meet the needs of plant growth. If the application of organic fertilizer is further increased, it will cause more serious DON accumulation and leaching, which will lead to problems such as soil nutrient loss, soil fertility decline, and crop yield reduction. The leached nitrogen will also cause surface water and groundwater pollution. Therefore, seeking an efficient method for mineralizing organic nitrogen is crucial for sustainable agricultural development and environmental protection.

[0003] In order to ensure crop yield and quality, it is very important to increase the mineralization rate of organic nitrogen in organic fertilizers. Microorganisms play an important role in this process. The traditional nitrogen cycle in nature is mainly the process of microorganisms converting NH4 + -N is oxidized to NO2 - -N, and then through nitrification, NO2 - -N is oxidized to NO3 which is easily absorbed by plants - -N. Complete ammonia oxidation is a process of ammonia oxidation performed by special complete ammonia oxidizing microorganisms. Complete ammonia oxidizing microorganisms can achieve the conversion of NH4 + -N to NO3 - -N complete oxidation process can reduce nitrogen loss and facilitate crops to absorb NO3 - -N, thereby promoting crop growth. Since most microorganisms do not have the ability to completely oxidize ammonia, the mineralization of organic nitrogen requires the participation of multiple microorganisms, which is not only inefficient but also prone to produce greenhouse gases such as N2O during the nitrification process.

[0004] In traditional sewage treatment, biological ammonia removal mainly involves two stages: ammonia oxidation and nitrification. The ammonia removal efficiency is low and it is difficult to achieve the goal of complete ammonia removal. Complete ammonia oxidizing microorganisms can be used to remove ammonia from water bodies to reduce the concentration of ammonia nitrogen in the water body, thereby reducing the toxicity of ammonia nitrogen to aquatic organisms and reducing the risk of eutrophication of water bodies. Complete ammonia oxidizing microorganisms used for water body ammonia removal need to have a high affinity for ammonia nitrogen and have a high affinity for NH4 + -N has stronger tolerance and can withstand higher NH4 + -N concentration to complete ammonia oxidation. Currently, there is a lack of safe ammonia oxidizing microorganisms suitable for removing ammonia from water bodies.

[0005] In view of this, the present invention is proposed. Summary of the invention

[0006] The purpose of the present invention is to provide a tequila bacillus and related products and applications thereof.

[0007] The present invention is achieved in that:

[0008] In a first aspect, an embodiment of the present invention provides a Bacillus tequilensis XH1, whose taxonomic name is Bacillus tequilensis and whose deposit number is CGMCC No.29351.

[0009] In a second aspect, an embodiment of the present invention provides a culture obtained by culturing the Bacillus terequina XH1 described in the above embodiment.

[0010] In a third aspect, an embodiment of the present invention provides a biological bacterial agent, which includes: the Bacillus tergilacinus XH1 described in the above embodiment or the culture described in the above embodiment.

[0011] In a fourth aspect, an embodiment of the present invention provides a composition comprising any one of the following: (1) a first microorganism and / or a culture of the first microorganism, and a second microorganism and / or a culture of the second microorganism; (2) a co-culture of the first microorganism and the second microorganism; wherein the first microorganism comprises the Bacillus tergiornosum XH1 described in the above embodiment, and the second microorganism comprises a nitrogen-fixing bacterium.

[0012] In a fifth aspect, an embodiment of the present invention provides a composite bacterial agent, which includes: the microbial composition described in the above embodiment.

[0013] In a sixth aspect, an embodiment of the present invention provides the use of the Bacillus tergilacinus XH1 described in the preceding embodiments, or the culture described in the preceding embodiments, or the biological agent described in the preceding embodiments, or the composition described in the preceding embodiments, or the composite agent described in the preceding embodiments in any of the following: (a) preparing a product with the function of promoting plant growth; (b) promoting plant growth; (c) soil conditioning; (d) preparing a soil conditioner.

[0014] In a seventh aspect, an embodiment of the present invention provides a growth promoter or soil conditioner, the active ingredients of which include: the Bacillus tergilacinus XH1 described in the aforementioned embodiment or the culture described in the aforementioned embodiment or the biological bacterial agent described in the aforementioned embodiment or the composition described in the aforementioned embodiment or the composite bacterial agent described in the aforementioned embodiment.

[0015] In an eighth aspect, an embodiment of the present invention provides the use of the Bacillus tergilacinus XH1 described in the preceding embodiments, or the culture described in the preceding embodiments, or the biological bacterial agent described in the preceding embodiments, or the composition described in the preceding embodiments, or the composite bacterial agent described in the preceding embodiments in wastewater deammonification or in the preparation of wastewater deammonification products.

[0016] In a ninth aspect, an embodiment of the present invention provides a wastewater ammonia removal agent, the active ingredients of which include: the Bacillus tergiornicus XH1 described in the aforementioned embodiment or the culture described in the aforementioned embodiment or the biological bacterial agent described in the aforementioned embodiment or the composition described in the aforementioned embodiment or the composite bacterial agent described in the aforementioned embodiment.

[0017] The present invention has the following beneficial effects:

[0018] (1) Bacillus tergillosis XH1 is a complete ammonia oxidizing microorganism that can independently convert NH4 + -N is oxidized to NO2 - -N, and then through nitrification, NO2 - -N is oxidized to NO3 which is easily absorbed by plants - -N; Applied in agriculture, it can accelerate the mineralization rate of soluble organic nitrogen in organic fertilizers and oxidize it into NO3 which is more easily absorbed by plants - -N, thus promoting plant growth, reducing the application of organic fertilizers and lowering agricultural production costs;

[0019] (2) XH1's complete ammonia oxidation reduces or avoids the accumulation and leaching of DON in the soil, thereby reducing nitrogen pollution to surface water and groundwater, which is beneficial to the protection of aquatic ecosystems;

[0020] (3) XH1 can also be used in combination with nitrogen-fixing bacteria to further improve its complete ammonia oxidation capacity. Nitrogen-fixing bacteria convert atmospheric nitrogen into NH4 + -N, and then oxidized to NO3 by complete ammonia oxidizing bacteria --N, which can not only significantly reduce agricultural production costs and the negative impact of chemical fertilizers and organic fertilizers on the environment, but also effectively reduce nitrogen leaching and greenhouse gas emissions;

[0021] (4) XH1 can also be used to remove ammonia from water bodies, reducing the concentration of ammonia nitrogen in the water, thereby reducing the toxicity of ammonia nitrogen to aquatic organisms and reducing the risk of eutrophication of water bodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings required for use in the embodiments are briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present invention and therefore should not be regarded as limiting the scope. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without creative work.

[0023] Figure 1 (a) shows the morphological characteristics of XH1 on LB solid medium; (b) shows the Gram staining characteristics;

[0024] Figure 2 is the phylogenetic tree of strain XH1;

[0025] Figure 3 (a) is the total leaching loss of soluble organic nitrogen; (b) is the DON concentration of the leaching fluid;

[0026] Figure 4 (a) is the total biomass of dwarf tomatoes; (b) is the yield of dwarf tomatoes; c is the weight of single dwarf tomato fruit;

[0027] Figure 5 (a) is the soluble protein content of dwarf tomatoes; (b) is the lycopene content;

[0028] Figure 6 (a) is the total fresh weight of tobacco; (b) is the height of tobacco plant; (c) is the diameter of tobacco stem; (d) is the maximum leaf area of ​​tobacco;

[0029] Figure 7 (a) is the total biomass of dwarf tomatoes; (b) is the yield of dwarf tomatoes; (c) is the weight of single dwarf tomato fruit;

[0030] Figure 8 (a) is the soluble protein content of dwarf tomatoes; (b) is the lycopene content;

[0031] Fig. 9 (a) is the total fresh weight of tobacco; (b) is the height of tobacco plant; (c) is the diameter of tobacco stem; (d) is the maximum leaf area of ​​tobacco;

[0032] Fig.10The growth characteristics of XH1 in nitrification medium with different NH4Cl concentrations. DETAILED DESCRIPTION

[0033] In order to make the purpose, technical scheme and advantages of the embodiments of the present invention clearer, the technical scheme in the embodiments of the present invention will be described clearly and completely below. If the specific conditions are not specified in the embodiments, they are carried out according to conventional conditions or conditions recommended by the manufacturer. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased commercially.

[0034] In agricultural production, the supply of nitrogen fertilizer is crucial to crop growth. Tomatoes and tobacco are typical nitrate-loving crops, which have a high demand for NO3 - -N have a good response. When the nitrate nitrogen supply is sufficient, it shows better growth effect and higher yield. - -N content can rapidly increase the content of organic acids and mineral cations in tomatoes, which is beneficial to the growth and development of tomatoes, and promotes flower bud differentiation, development and fruit expansion. The nutritional organs of plants will accumulate a large amount of NO3 during the growth period. - -N, but as plants grow, the nitrate nitrogen NO3 in their bodies - -N content gradually decreases, so it is necessary to replenish NO3 in time - -N is the key to ensuring crop yield and quality.

[0035] In addition, NO3 - -N accumulation in plants is a "storage" measure of plants. - -N is also an important osmotic regulating substance in the vacuole. When the carbohydrate synthesis in the plant body decreases and the organic matter content in the vacuole decreases, NO3 - -N can replace and regulate osmotic pressure, and this regulation requires lower energy, so that plants can still grow and develop well under adverse conditions, such as when the soil lacks nutrients.

[0036] Complete ammonia oxidizing microorganisms were only discovered in recent years, and their abundance in soil is extremely low, resulting in few reports on related research and applications. Complete ammonia oxidizing microorganisms have a high affinity for ammonia nitrogen, which can not only improve the ability of microorganisms to bind ammonia nitrogen in organic fertilizers and improve the efficiency of microbial mineralization of organic nitrogen, but also have a more competitive advantage under oligotrophic conditions, even in low NH4 + -N concentration environment, complete ammonia oxidizing microorganisms can also complete NH4 + -N to NO3 - -Oxidation of N.

[0037] The Bacillus tekila XH1 provided in the embodiment of the present invention is a complete ammonia oxidizing microorganism, which has complete ammonia oxidation ability and can independently realize NH4+ -N to NO3 - -N oxidation, and the efficiency is relatively high. It is used in agriculture to accelerate the mineralization of organic nitrogen in organic fertilizers and oxidize it into NO3 which is easily absorbed by plants. - -N, thereby promoting plant growth; at the same time, reducing the accumulation and leaching of DON in the soil, thereby reducing nitrogen pollution to surface water and groundwater, protecting the aquatic ecosystem, and compared with the N2O produced by other microorganisms during nitrification, XH1 releases lower N2O during complete ammonia oxidation, which can reduce the impact on climate change.

[0038] XH1 can be used in combination with nitrogen-fixing bacteria to further increase the production of NO3 by complete ammonia oxidation by XH1. - -N rate, further promoting plant growth, reducing the amount of organic fertilizer applied, and better avoiding DON leaching. Nitrogen-fixing bacteria convert atmospheric nitrogen into NH4 + -N, then oxidized to NO3 by XH1 - -N can not only reduce agricultural production costs and the negative impact of chemical fertilizers and organic fertilizers on the environment, but also effectively reduce nitrogen leaching and greenhouse gas emissions, which is of great significance to the sustainable development of agriculture.

[0039] XH1 can also be used to remove ammonia from water bodies, reducing the concentration of ammonia nitrogen in water bodies, thereby reducing the toxicity of ammonia nitrogen to aquatic organisms and reducing the risk of eutrophication of water bodies. In traditional sewage treatment, biological ammonia removal mainly involves two stages: ammonia oxidation and nitrification. The efficiency of ammonia removal is low, and it is difficult to achieve the goal of complete ammonia removal. XH1 has a high affinity for ammonia nitrogen and is very sensitive to NH4 + -N has stronger tolerance and can withstand higher NH4 + -N concentration to complete ammonia oxidation.

[0040] Specific technical solutions

[0041] On the one hand, an embodiment of the present invention provides a Bacillus tequilensis XH1, whose taxonomic name is Bacillus stequilensis and whose deposit number is CGMCC No.29351.

[0042] Bacillus tequilensis XH1 was registered and deposited with the China General Microbiological Culture Collection on December 18, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 29351. The contact number is: 01064807355.

[0043] On the other hand, an embodiment of the present invention further provides a culture obtained by culturing the Bacillus terequila XH1 described in the above embodiment.

[0044] The present application has no special restrictions on the culture conditions of XH1, and the culture medium and culture conditions currently used for Bacillus tequila can be used.

[0045] On the other hand, an embodiment of the present invention further provides a biological bacterial agent, which includes: the Bacillus tergilacinus XH1 described in the above embodiment or the culture described in any of the above embodiments.

[0046] In some embodiments, the biological agent further comprises a carrier.

[0047] On the other hand, an embodiment of the present invention further provides a composition comprising any of the following:

[0048] (1) a first microorganism and / or a culture of the first microorganism, and a second microorganism and / or a culture of the second microorganism;

[0049] (2) a co-culture of the first microorganism and the second microorganism;

[0050] Wherein, the first microorganism includes the Bacillus tergiraceus XH1 described in the above embodiment, and the second microorganism includes nitrogen-fixing bacteria.

[0051] In some embodiments, the situation of (1) includes: a first microorganism and a second microorganism; or a culture of the first microorganism and a culture of the second microorganism; or a culture of the first microorganism and the second microorganism; or a culture of the first microorganism and the second microorganism.

[0052] On the other hand, an embodiment of the present invention further provides a composite bacterial agent, which includes: the microbial composition described in any of the above embodiments.

[0053] In some embodiments, the composite bacterial agent further comprises a carrier, which can be selected from carriers used in microbial agents in the art.

[0054] On the other hand, the embodiments of the present invention further provide the use of the Bacillus tergilacinus XH1 described in the above embodiments, or the culture described in any of the above embodiments, or the biological bacterial agent described in any of the above embodiments, or the composition described in any of the above embodiments, or the composite bacterial agent described in any of the above embodiments in any of the following:

[0055] (a) preparing products with the function of promoting plant growth; (b) promoting plant growth; (c) soil conditioning; (d) preparing soil conditioners.

[0056] Nitrate nitrogen and ammonium nitrogen are both forms of nitrogen that plants can absorb, but different plants have different preferences for them. Nitrate-loving crops refer to plants that prefer nitrate nitrogen. - - When N supply is sufficient, it shows better growth effect and higher yield.

[0057] In some embodiments, the nitrate-loving plants include any one or more of tomatoes, tobacco, corn, soybeans, rapeseed, spinach, Chinese cabbage, carrots, blueberries, apples, grapes and kiwis.

[0058] In some embodiments, the soil conditioning includes: soil remediation, reducing the accumulation and / or leaching of DON in the soil.

[0059] On the other hand, an embodiment of the present invention further provides a growth promoter or soil conditioner, the active ingredients of which include: the Bacillus tergilacinus XH1 described in the aforementioned embodiment or the culture described in any of the aforementioned embodiments or the biological bacterial agent described in any of the aforementioned embodiments or the composition described in any of the aforementioned embodiments or the composite bacterial agent described in any of the aforementioned embodiments.

[0060] On the other hand, an embodiment of the present invention also provides the use of the Bacillus tergilacinus XH1 described in the preceding embodiment, or the culture described in any preceding embodiment, or the biological bacterial agent described in any preceding embodiment, or the composition described in any preceding embodiment, or the composite bacterial agent described in any preceding embodiment in wastewater deammonification or in the preparation of wastewater deammonification products.

[0061] In addition, an embodiment of the present invention further provides a wastewater ammonia removal agent, the active ingredients of which include: the Bacillus tergilacinus XH1 described in the aforementioned embodiment or the culture described in any of the aforementioned embodiments or the biological bacterial agent described in any of the aforementioned embodiments or the composition described in any of the aforementioned embodiments or the composite bacterial agent described in any of the aforementioned embodiments.

[0062] The features and performance of the present invention are further described in detail below in conjunction with the embodiments.

[0063] Example 1 Screening, identification and preservation of strain XH1

[0064] Strain screening:

[0065] The strains were screened from the red soil of Erwu Mountain, Xichang City, Sichuan Province (27°89'45.04"N, 102°26'44.49"E). The soil physical and chemical properties such as TN, NH4 + -N、NO3 --N, total organic carbon and total organic matter were 0.71±0.06g / kg, 109.75±3.94mg / kg, 192.44±0.75mg / kg, 9.87±0.06g / kg and 67.80±1.31g / kg, respectively.

[0066] Take 1g of soil, add 9ml of sterile water, and prepare a soil suspension by shaking. Take 1mL of soil suspension and add it to LB liquid culture medium for bacterial enrichment, then take 1mL of enriched bacterial liquid and apply it to solid nitrification culture medium for separation and purification. Nitration culture medium: according to NH4Cl 380mg / L, CH3COONa·3H2O 2000mg / L, MgSO4·7H2O 50mg / L, K2HPO4200mg / L, NaCl 120mg / L, MnSO4·4H2O 10mg / L, FeSO4 10mg / L, distilled water 1000mL, adjust pH=7.0 with dilute HCl, then divide and sterilize at 121℃ for 30min. After cooling, the culture medium is obtained; the obtained culture medium can be stored at 4℃ for future use.

[0067] A total of 15 nitrifying bacteria were obtained from the solid nitrification medium and named XH1 to XH15. The nitrification ability of the 15 heterotrophic nitrifying bacteria obtained in the initial screening was studied. After 72 hours of cultivation in the liquid nitrification medium, the nitrification ability of the 15 heterotrophic nitrifying bacteria was studied. - -N production indicates nitrification capacity. NH4 + -N detection adopts the national standard GB / T42485-2023 indigo blue colorimetric method, NO3 - -N detection adopts the national standard DZ / T0064.59-2021 disulfonic acid phenol spectrophotometry. It has strong nitrification ability and can simultaneously detect the ammonia monooxygenase (amoA) gene (which converts NH4 + -N is oxidized to NO2 - -N ammonia oxidation gene) and nitrite oxidase NxrB gene (to convert NO2 - -N is oxidized to NO3 - -N nitrification gene) is XH1. XH1 strain NO3 - The -N production was 25.09 mg / L, significantly higher than that of other strains.

[0068] Strain identification: The selected XH1 was streaked on LB solid medium using the plate streak method. Its morphological characteristics are shown in Figure 1 In (a), the colonies of XH1 are white, round, with irregular edges, a bulge in the middle, and filamentous secretions around them. XH1 was stained by Gram staining, and its Gram staining image is shown in Figure 1In (b), XH1 is purple in Gram stain and belongs to Gram-positive bacteria. The physiological and biochemical characteristics of XH1 are shown in Table 1.

[0069] Table 1 Physiological and biochemical identification

[0070]

[0071]

[0072] 16S rRNA sequencing: DNA sequencing of XH1 was performed using primers 27F and 1492R to obtain the XH1 gene sequence. The XH1 sequence was 1461 bp in total. The phylogenetic tree of the strain was obtained after the XH1 sequence was analyzed using MEGA 5.0 software. The results are as follows: Figure 2 As shown. Through homology comparison, it was found that the sequence similarity between XH1 and Bacillus tequilensis was 98%. The above analysis showed that XH1 belongs to Bacillus tequilensis. (Studies have found that Bacillus tequilensis mainly has functions such as denitrification and biological control, and is mainly used in plant growth promotion).

[0073] The 16S rRNA sequence of Bacillus tequila XH1 is as follows:

[0074]

[0075] Preservation of strains: The screened Bacillus tequilensis XH1 was registered and deposited with the China General Microbiological Culture Collection Center on December 18, 2023. The deposit address is the Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. The deposit number is CGMCC No. 29351. The contact number is: 01064807355.

[0076] The complete ammonia oxidizing Bacillus tequilensis XH1 provided in the embodiment of the present invention has the ability to completely oxidize ammonia, that is, the strain can independently complete the oxidation of NH4 + -N is oxidized to NO2 - -N, then NO2 - -N is oxidized to NO3 - -N ability. When XH1 was cultured in liquid nitrification medium, it was detected that it could convert NH4 + -N is oxidized to NO2 - -N increased the relative expression of ammonia monooxygenase (amoA) gene, and NO2 - -N is oxidized to NO3 -The relative expression of nitrite oxidase (NxrB) gene of -N was increased.

[0077] Example 2: The ability of strain XH1 to control soil DON leaching

[0078] Preparation of bacterial suspension of Bacillus tequila XH1: LB liquid medium was inoculated with 1% XH1 and cultured at 37°C and 180 rpm until OD 600 =1.0, centrifuged at 6000r / m for 10 minutes at 4°C to obtain XH1 bacteria, which were resuspended and washed three times with sterile water. The final XH1 bacterial suspension had an OD of 600 =1.0.

[0079] Leaching experiment: The Erwushan red soil in Xichang City, Sichuan Province (the physical and chemical properties of the soil such as TN, NH4 + -N、NO3 - -N, total organic carbon and total organic matter were 0.71g / kg, 0.11g / kg, 0.192g / kg, 9.87g / kg and 67.80g / kg respectively. Red soil parent material is residual slope deposits of granite weathering, which is very prone to soil erosion, and leaching is easy to cause in the case of poor soil and water conservation measures) as experimental soil, every 5kg soil was divided into pots, and 150g of organic fertilizer with a nitrogen content of 0.4g / kg was added, and the fertilizer was added once and mixed evenly with the soil. The control group did not apply Bacillus tekila XH1, only 100mL of pure water was added, and the treatment group applied 100mL of bacterial suspension of Bacillus tekila XH1 in 5kg soil samples, and the control and treatment groups had 3 parallel samples. 500mL of water was irrigated on the 10th, 20th, 30th, 40th and 60th days, a total of 5 times, and the leaching solution was collected from the bottom tray after 24h of infiltration.

[0080] DON calculation: The TN of the leaching solution adopts the alkaline potassium persulfate colorimetric method of the environmental national standard HJ636-2012. The DON content and concentration of the leaching solution can be obtained by calculation.

[0081] The experimental results are shown in Figure 3 During the whole experimental period, the soil DON leaching loss of the treatment group and the control group was 20.33kg / ha and 30.84kg / ha, respectively, and the soil DON leaching loss of the treatment group was 34.07% lower than that of the control group. At the end of the experiment, the DON content of the leaching solution of the treatment group and the control group was 93.57mg / L and 128.33mg / L, respectively, and the DON content of the leaching solution of the treatment group was 27.08% lower than that of the control group.

[0082] It can be seen that Bacillus tekila XH1 can oxidize DON in the soil into NO3 which is easy for plants to absorb and utilize. - -N, in order to achieve efficient control of soil DON leaching.

[0083] Example 3 Growth-promoting ability of strain XH1 on dwarf tomatoes

[0084] Dwarf tomato growth promotion experiment: Erwushan red soil in Xichang City, Sichuan Province was used as the experimental soil. Every 5kg of soil was divided into pots, and 150g of organic fertilizer with a nitrogen content of 0.4g / kg was added. The fertilizer was added once and mixed evenly with the soil. The treatment group applied 100mL of the bacterial suspension of Bacillus tekila XH1 (the preparation method is described in Example 2) to 5kg of soil samples, and the control group did not apply Bacillus tekila XH1, only 100mL of pure water was added. Both the treatment group and the control group had 3 parallel samples. One dwarf tomato seedling (Solanum abutiloides) was planted in all pots, purchased from Weifang Maitu Agricultural Technology Co., Ltd., China, with a planting period of 65d. All groups were placed outdoors, the lighting conditions were natural sunlight, the temperature range was 23-29°C, and the relative humidity range was 40%-69%.

[0085] Detection of total biomass, fruit yield per plant, single fruit weight, fruit soluble protein content and lycopene content in dwarf tomatoes:

[0086] The total biomass, fruit yield per plant and single fruit weight of dwarf tomatoes were measured by direct weighing method.

[0087] The soluble protein content of the fruit was detected by Coomassie Brilliant Blue staining: 100 mg of Coomassie Brilliant Blue G-250 was weighed, dissolved in 50 mL of 90% ethanol, and then 100 mL of 85% (w / v) phosphoric acid was added. After mixing and dissolving, the volume was made up to 1000 mL with distilled water. A standard curve was prepared using a bovine serum albumin solution of known concentration. 2.5 g of tomato sample was weighed, ground into a homogenate with distilled water, and the supernatant was centrifuged and taken as a soluble protein extract. 1.0 mL of the sample extract was added with Coomassie Brilliant Blue G-250 reagent, and the absorbance was measured at a wavelength of 595 nm after mixing. The protein content was calculated using the standard curve.

[0088] The lycopene content in the fruit was detected by the toluene colorimetric method according to the national standard GB14215-2008.

[0089] At the end of the experiment, the total biomass, fruit yield per plant, single fruit weight, fruit soluble protein content and fruit lycopene content of dwarf tomatoes were significantly different from those of Figure 4 and Figure 5At the end of the experiment, the total biomass of dwarf tomatoes in the treatment group and the control group were 6.12g and 5.79g, respectively, and the total biomass of dwarf tomatoes in the treatment group was 5.70% higher than that in the control group; the yield of dwarf tomatoes in the treatment group and the control group were 138.56g / plant and 89.68g / plant, respectively, and the yield of dwarf tomatoes in the treatment group was 54.51% higher than that in the control group; the single fruit weight of dwarf tomatoes in the treatment group and the control group were 7.54g and 7.24g, respectively, and the single fruit weight of dwarf tomatoes in the treatment group was 4.14% higher than that in the control group; the soluble protein content of dwarf tomatoes in the treatment group and the control group was 1.47mg / g FW and 0.88mg / gFW, respectively, and the soluble protein content of dwarf tomatoes in the treatment group was 67.04% higher than that in the control group; the lycopene content of dwarf tomatoes in the treatment group and the control group was 5.23μg / g FW and 4.28μg / g FW, respectively, and the lycopene content of dwarf tomatoes in the treatment group was 22.19% higher than that in the control group.

[0090] The experimental results showed that Bacillus tequila XH1 significantly promoted the yield and quality of dwarf tomatoes. It can be seen that the complete ammonia oxidation capacity of XH1 can enhance the oxidation of organic nitrogen to NO3 - -N is absorbed by dwarf tomatoes, thereby promoting their growth.

[0091] Example 4 Growth-promoting ability of strain XH1 on tobacco

[0092] Tobacco growth promotion experiment: The red soil of Erwu Mountain in Xichang City, Sichuan Province was used as the experimental soil. Every 5kg of soil was divided into pots, and 150g of organic fertilizer with a nitrogen content of 0.4g / kg was added. The fertilizer was added at one time and mixed evenly with the soil. The treatment group applied 100mL of the bacterial suspension of Bacillus tequila XH1 (the preparation method is described in Example 2) to the 5kg soil sample, and the control group did not apply Bacillus tequila XH1, only 100mL of pure water was added. Both the treatment group and the control group had 3 parallel samples. One tobacco seedling was planted in all pots, and the planting cycle was 65d. All groups were placed outdoors, the lighting conditions were natural sunlight, the temperature range was 23-29°C, and the relative humidity range was 40%-69%.

[0093] The total fresh weight, plant height, stem diameter and maximum leaf area of ​​tobacco were directly measured and calculated.

[0094] At the end of the experiment, the total freshness, plant height, stem diameter and maximum leaf area of ​​tobacco were Figure 6At the end of the experiment, the total fresh weight of tobacco in the treatment group and the control group were 101.43g and 82.71g, respectively, and the total fresh weight of tobacco in the treatment group was 22.63% higher than that in the control group; the tobacco plant heights in the treatment group and the control group were 51.59cm and 50.20cm, respectively, and the tobacco plant heights in the treatment group were 2.76% higher than that in the control group; the tobacco stem diameters in the treatment group and the control group were 8.23mm and 7.05mm, respectively, and the tobacco stem diameters in the treatment group were 16.73% higher than that in the control group; the maximum leaf areas of tobacco in the treatment group and the control group were 598.39cm 2 and 464.80cm 2 The maximum leaf area of ​​tobacco in the treatment group was 28.74% higher than that in the control group. The experimental results showed that Bacillus tequila XH1 significantly promoted the growth of tobacco.

[0095] It can be seen that the complete ammonia oxidation ability of Bacillus tequila XH1 can enhance the oxidation of organic nitrogen to NO3 - -N is absorbed by tobacco, thus promoting tobacco growth.

[0096] Example 5 Co-cultivation of strain XH1 with nitrogen-fixing bacteria can further promote the growth of dwarf tomatoes

[0097] (1) Preparation of Bacillus tergillium seed solution: strain XH1 was inoculated into the above-mentioned liquid nitrification medium at 1% of the volume of the medium, and incubated at 37°C and 180 rpm until the OD 600 =1.0.

[0098] (2) Preparation of nitrogen-fixing bacteria seed solution: Inoculate nitrogen-fixing bacteria into liquid nitrogen-free culture medium at 1% of the culture medium volume and culture at 37°C and 180 rpm until OD 600 =1.0.

[0099] (3) Co-cultivation of strain XH1 and nitrogen-fixing bacteria: The two seed solutions prepared in step (1) and step (2) were inoculated into nitrification medium at a volume of 0.5% of the medium, and cultured at 37°C and 180 rpm until OD 600 =1.0.

[0100] Dwarf tomato growth promotion experiment: The red soil of Erwu Mountain in Xichang City, Sichuan Province was used as the experimental soil. Every 5 kg of soil was divided into pots, and 150 g of organic fertilizer with a nitrogen content of 0.4 g / kg was added. The fertilizer was added at one time and mixed evenly with the soil. Treatment 1 applied 100 mL of a bacterial suspension of Bacillus tergilacinus XH1 (the preparation method is described in Example 2) to 5 kg of soil sample. Treatment 2 applied XH1 and nitrogen-fixing bacteria to co-culture until OD 600=1.0 bacterial suspension 100mL in 5kg soil sample, control group does not apply Tejila Bacillus XH1, only add pure water 100mL, treatment 1-2 and control group have 3 parallel samples. One dwarf tomato seedling (Solanum abutiloides) was planted in all pots, purchased from Weifang Maitu Agricultural Technology Co., Ltd., China, and the planting cycle was 65d. All groups were placed outdoors, the lighting conditions were natural sunlight, the temperature range was 23-29°C, and the relative humidity range was 40-69%.

[0101] At the end of the experiment, the total biomass of dwarf tomatoes, fruit yield per plant, single fruit weight, fruit soluble protein content and fruit lycopene content were analyzed. Figure 7 and Figure 8 The total biomass of dwarf tomatoes in treatment 1, treatment 2 and control group were 6.12g, 15.59g and 5.79g, respectively. The total biomass of dwarf tomatoes in treatment 1 was 5.70% higher than that in the control group, and the total biomass of dwarf tomatoes in treatment 2 was 169.25% higher than that in the control group. The yield of dwarf tomatoes in treatment 1, treatment 2 and control group were 138.56g / plant, 162.06g / plant and 89.68g / plant, respectively. The yield of dwarf tomatoes in treatment 1 was 54.51% higher than that in the control group, and the yield of dwarf tomatoes in treatment 2 was 80.71% higher than that in the control group. The single fruit weight of dwarf tomatoes in treatment 1, treatment 2 and control group were 7.54g, 9.02g and 7.24g, respectively. The single fruit weight of dwarf tomatoes in treatment 1 was 4.14% higher than that in the control group, and the single fruit weight of dwarf tomatoes in treatment 2 was 24.58% higher than that in the control group. The soluble protein content of dwarf tomato fruits in treatment 1, treatment 2 and control group were 1.47mg / g FW, 2.11mg / g FW and 0.88 mg / g FW, the soluble protein content of dwarf tomato fruits in treatment 1 was 67.04% higher than that in the control group, and the soluble protein content of dwarf tomato fruits in treatment 2 was 139.77% higher than that in the control group; the lycopene contents of dwarf tomato fruits in treatment 1, treatment 2 and the control group were 5.23 μg / g FW, 6.54 μg / g FW and 4.28 μg / gFW, respectively, the lycopene content of dwarf tomato fruits in treatment 1 was 22.19% higher than that in the control group, and the lycopene content of dwarf tomato fruits in treatment 2 was 52.81% higher than that in the control group.

[0102] The addition of nitrogen-fixing bacteria can convert nitrogen in the air into NH4 + -N, then oxidized to NO3 by XH1 - -N. Adding nitrogen-fixing bacteria can provide more NH4 + -N, thereby enhancing the ability of XH1 to promote the growth of dwarf tomatoes.

[0103] Example 6 Co-culture of strain XH1 with nitrogen-fixing bacteria can further promote tobacco growth

[0104] (1) Preparation of Bacillus tergillium seed solution: strain XH1 was inoculated into the above-mentioned liquid nitrification medium at 1% of the volume of the medium, and incubated at 37°C and 180 rpm until the OD 600 =1.0.

[0105] (2) Preparation of nitrogen-fixing bacteria seed solution: Inoculate nitrogen-fixing bacteria into liquid nitrogen-free culture medium at 1% of the culture medium volume and culture at 37°C and 180 rpm until OD 600 =1.0.

[0106] (3) Co-cultivation of strain XH1 and nitrogen-fixing bacteria: The two seed solutions prepared in step (1) and step (2) were inoculated into nitrification medium at a volume of 0.5% of the medium, and cultured at 37°C and 180 rpm until OD 600 =1.0.

[0107] Tobacco growth promotion experiment: The red soil of Erwu Mountain in Xichang City, Sichuan Province was used as the experimental soil. Every 5 kg of soil was divided into pots, and 150 g of organic fertilizer with a nitrogen content of 0.4 g / kg was added. The fertilizer was added at one time and mixed evenly with the soil. In treatment 1, 100 mL of a bacterial suspension of Bacillus tergilacinus XH1 (the preparation method is described in Example 2) was applied to 5 kg of soil sample. In treatment 2, XH1 was co-cultured with nitrogen-fixing bacteria until OD 600 =1.0 bacterial suspension 100mL in 5kg soil sample, the control group did not apply Tejila Bacillus XH1, only added 100mL pure water, treatments 1-2 and the control group had 3 parallel samples. One tobacco seedling was planted in all pots, and the planting cycle was 65d. All groups were placed outdoors, the lighting conditions were natural sunlight, the temperature range was 23-29℃, and the relative humidity range was 40-69%.

[0108] At the end of the experiment, the total freshness, plant height, stem diameter and maximum leaf area of ​​tobacco were Fig. 9 The total fresh weights of tobacco in treatment 1, treatment 2 and control group were 101.43 g, 116.26 g and 82.71 g, respectively. The total fresh weight of tobacco in treatment 1 was 22.63% higher than that in the control group, and the total fresh weight of tobacco in treatment 2 was 40.56% higher than that in the control group. The plant heights of tobacco in treatment 1, treatment 2 and control group were 51.59 cm, 58.20 cm and 50.20 cm, respectively. The plant heights of tobacco in treatment 1 were 2.76% higher than that in the control group, and the plant heights of tobacco in treatment 2 were 15.36% higher than that in the control group. The stem diameters of tobacco in treatment 1, treatment 2 and control group were 8.23 ​​cm, 9.62 cm and 7.05 cm, respectively. The stem diameters of tobacco in treatment 1 were 16.73% higher than that in the control group, and the stem diameters of tobacco in treatment 2 were 36.45% higher than that in the control group. The maximum leaf areas of tobacco in treatment 1, treatment 2 and control group were 580.57 cm 2 、598.39cm2 and 464.80cm 2 The maximum leaf area of ​​tobacco in treatment 1 was 24.91% higher than that in the control group, and the maximum leaf area of ​​tobacco in treatment 2 was 28.74% higher than that in the control group.

[0109] The addition of nitrogen-fixing bacteria can convert nitrogen in the air into NH4 + -N, then oxidized to NO3 by XH1 - -N. Adding nitrogen-fixing bacteria can provide more NH4 + -N, thereby enhancing the ability of XH1 to promote tobacco growth.

[0110] Example 7 Strain XH1 has good ammonia tolerance and nitrate production ability

[0111] A nitrifying medium was prepared according to 380 mg / L NH4Cl, 2000 mg / L CH3COONa·3H2O, 50 mg / L MgSO4·7H2O, 200 mg / L K2HPO4, 120 mg / L NaCl, 10 mg / L MnSO4·4H2O, 10 mg / L FeSO4, and 1000 mL distilled water, and the pH was adjusted to 7.0 with dilute HCl.

[0112] On the basis of the above nitrification medium, only the NH4Cl concentration was increased to 400mg / L, 450mg / L, 500mg / L and 550mg / L, and the concentrations of other components remained unchanged. XH1 was inoculated at 1% of the volume of the medium on the nitrification medium with NH4Cl concentrations of 400mg / L, 450mg / L, 500mg / L and 550mg / L, and the OD was measured every 1 hour 600 .

[0113] Ammonia nitrogen tolerance of strain XH1: It can grow normally in nitrification medium with 400mg / L, 450mg / L, 500mg / L and 550mg / L NH4Cl concentrations. Fig.10 XH1 was cultured in nitrification medium with various concentrations of NH4Cl for 7 hours, and the OD 600 As a complete ammonia oxidizing bacterium, XH1 has excellent ammonia nitrogen tolerance and can + -N environment to survive.

[0114] Nitrate production capacity of strain XH1: The nitrate production capacity of XH1 grown in nitrification medium with 400 mg / L, 450 mg / L, 500 mg / L and 550 mg / L NH4Cl concentrations for 72 hours is shown in Table 1. The NO3 production at the above NH4Cl concentrations - -N were 267.98 mg / L, 293.94 mg / L, 303.81 mg / L and 309.89 mg / L respectively.

[0115] Table 1 Nitrate production capacity of XH1 at different ammonia concentrations

[0116]

[0117] Example 8 Effect of strain XH1 on removing ammonia nitrogen from sewage

[0118] Experiment on removing ammonia nitrogen from sewage: Experimental water samples 1, 2, and 3 were collected from an artificial lake 1, an artificial lake 2, and an artificial river 3 in Chengdu, Sichuan Province, respectively. The initial ammonia nitrogen contents were 63.89 mg / L, 104.42 mg / L, and 221.56 mg / L, respectively.

[0119] Take 50 mL of water samples 1, 2, and 3, and inoculate 0.5 mL of the seed solution of strain XH1. The control group is not inoculated with bacterial solution. The samples are cultured at 37°C and 180 r / min for 48 hours, and the ammonia nitrogen concentration is determined.

[0120] NH4 in water samples + -N concentration was detected by the national standard DZ / T 0064.59-2021 Nessler reagent spectrophotometry method.

[0121] Table 2XH1 Deammoniation Capacity in Water

[0122]

[0123] Strain XH1 deammonification ability: Strain XH1 removes NH4 from water + -N capacity is shown in Table 1. The results show that the use of XH1 removes 69.84% to 80.82% of ammonia within 72 hours, which is 55.42% to 77.21% higher than the control group.

[0124] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A Bacillus tequila XH1, characterized in that Its taxonomic name is Bacillus tequilensis and its preservation number is CGMCC No.29351.

2. A culture, characterized in that The product is obtained by culturing the Bacillus tergillosis XH1 described in claim 1.

3. A biological bacterial agent, characterized in that: It includes: The Bacillus tequila XH1 of claim 1 or the culture of claim 2.

4. A composition, characterized in that This includes any of the following: (1) a first microorganism and / or a culture of the first microorganism, and a second microorganism and / or a culture of the second microorganism; (2) a co-culture of the first microorganism and the second microorganism; Wherein, the first microorganism includes the Bacillus tergiurae XH1 described in claim 1, and the second microorganism includes nitrogen-fixing bacteria.

5. A composite bacterial agent, characterized in that: It includes: The microbial composition according to claim 4.

6. Use of the Bacillus tergilacinus XH1 according to claim 1, the culture according to claim 2, the biological agent according to claim 3, the composition according to claim 4, or the composite agent according to claim 5 in any of the following: (a) preparing products with the function of promoting plant growth; (b) promoting plant growth; (c) soil conditioning; (d) preparing soil conditioners.

7. The use according to claim 6, characterized in that: The plants include: nitrate-loving plants; Optionally, the nitrate-loving plants include any one or more of tomatoes, tobacco, corn, soybeans, rapeseed, spinach, Chinese cabbage, carrots, blueberries, apples, grapes and kiwis.

8. A growth promoter or soil conditioner, characterized in that: The active ingredients include: the Bacillus tergiurae XH1 described in claim 1, or the culture described in claim 2, or the biological bacterial agent described in claim 3, or the composition described in claim 4, or the composite bacterial agent described in claim 5.

9. Use of the Bacillus terequila XH1 according to claim 1, the culture according to claim 2, the biological agent according to claim 3, the composition according to claim 4, or the composite agent according to claim 5 in wastewater deammonification or in the preparation of wastewater deammonification products.

10. A sewage ammonia removal agent, characterized in that: The active ingredients include: the Bacillus tergiurae XH1 described in claim 1, or the culture described in claim 2, or the biological bacterial agent described in claim 3, or the composition described in claim 4, or the composite bacterial agent described in claim 5.

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