A bacillus tequilensis and related products and uses thereof

Abstract

The application discloses a bacillus turicatus and related products and application thereof, relates to the field of microorganisms, and provides the bacillus turicatus XH1 which is a complete ammonia-oxidizing microorganism and can independently oxidize organic nitrogen in soil into NO3 ‑ -N, so as to realize efficient control of soil DON leaching, promote biological growth, and further improve the complete ammonia-oxidizing capacity of the bacillus turicatus XH1 in combination with nitrogen-fixing bacteria; the bacillus turicatus XH1 can also remove ammonia in water, and has significant agricultural application and environmental protection potential.

Description

Technical Field

[0001] This invention relates to the field of microbiology, and more specifically, to a Bacillus tekirae and its related products and applications. Background Technology

[0002] Organic fertilizers are widely used in modern agricultural production for soil remediation and fertility enhancement, improving crop yield and quality. Rich in organic matter and various nutrients, organic fertilizers not only provide essential nutrients for plants but also promote soil microbial activity and enhance soil water and fertilizer retention capacity, thereby improving crop quality. However, most nitrogen in organic fertilizers exists in the form of organic nitrogen, and long-term application leads to the accumulation of dissolved organic nitrogen (DON) in the soil. Under rainfall or irrigation conditions, DON is severely lost through leaching. Furthermore, the dissolved organic nitrogen in organic fertilizers is mostly in the form of large molecules, which is not conducive to plant absorption and utilization, and these large molecules have a slow mineralization rate, converting into nitrate nitrogen (NO3), which is easily absorbed by plants. - The rate of nitrogen (DON) leaching is also relatively slow, making it difficult to meet the needs of plant growth. Further increasing the application of organic fertilizer will lead to more severe DON accumulation and leaching, resulting in soil nutrient loss, soil fertility decline, and reduced crop yields. Furthermore, the leached nitrogen will also cause pollution of surface and groundwater. Therefore, finding an efficient method for mineralizing organic nitrogen is crucial for sustainable agricultural development and environmental protection.

[0003] To ensure crop yield and quality, accelerating the mineralization of organic nitrogen in organic fertilizers is crucial, and microorganisms play a vital role in this process. In nature, the traditional nitrogen cycle primarily involves microorganisms converting NH4+ into nitrogen through ammonia oxidation. + -N is oxidized to NO2 - -N, and then through nitration, NO2 - -N is oxidized into NO3, which is easily absorbed by plants. - -N. Complete ammonia oxidation is an ammonia oxidation process carried out by specialized complete ammonia-oxidizing microorganisms, which can convert NH4+ into nitrogen and nitrogen. + -N to NO3 - The complete oxidation process of -N can reduce nitrogen loss and facilitate crop absorption of NO3. - -N, thus promoting crop growth. Since most microorganisms do not have the ability to fully oxidize ammonia, mineralizing organic nitrogen requires the participation of multiple microorganisms, which is not only inefficient but also prone to producing greenhouse gases such as N2O during nitrification.

[0004] In traditional wastewater treatment, biological ammonia removal mainly involves two stages: ammonia oxidation and nitrification. This method has low ammonia removal efficiency and struggles to achieve complete ammonia removal. Complete ammonia-oxidizing microorganisms can be used for ammonia removal in water bodies to reduce ammonia nitrogen concentration, thereby lowering the toxicity of ammonia nitrogen to aquatic organisms and reducing the risk of eutrophication. Complete ammonia-oxidizing microorganisms used for ammonia removal in water bodies need to have a high affinity for ammonia nitrogen and for NH4+. + -N has stronger tolerance and can withstand higher NH4 levels. + Complete ammonia oxidation can be achieved at -N concentrations. Currently, there is a lack of safe ammonia-oxidizing microorganisms suitable for ammonia removal from water bodies.

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

[0006] The purpose of this invention is to provide Bacillus tekirae and its related products and applications.

[0007] This invention is implemented as follows:

[0008] In a first aspect, embodiments of the present invention provide a Bacillus tequilensis XH1, which is taxonomically named Bacillus tequilensis and has the accession number CGMCC No. 29351.

[0009] Secondly, embodiments of the present invention provide a culture obtained by culturing Bacillus tekirae XH1 as described in the foregoing embodiments.

[0010] Thirdly, embodiments of the present invention provide a biological agent comprising: Bacillus tergentii XH1 as described in the foregoing embodiments or the culture as described in the foregoing embodiments.

[0011] Fourthly, embodiments of the present invention provide 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 Bacillus tekirae XH1 as described in the foregoing embodiments, and the second microorganism comprises a nitrogen-fixing bacterium.

[0012] Fifthly, embodiments of the present invention provide a compound microbial agent comprising the microbial composition described in the foregoing embodiments.

[0013] In a sixth aspect, embodiments of the present invention provide the use of Bacillus tekirae XH1 as described in the foregoing embodiments, or the culture as described in the foregoing embodiments, or the biological agent as described in the foregoing embodiments, or the composition as described in the foregoing embodiments, or the compound agent as described in the foregoing embodiments, in any of the following: (a) preparing products that promote plant growth; (b) promoting plant growth; (c) soil conditioning; (d) preparing soil conditioners.

[0014] In a seventh aspect, embodiments of the present invention provide a growth promoter or soil conditioner, the active ingredients of which include: Bacillus terrestris XH1 as described in the foregoing embodiments, or the culture as described in the foregoing embodiments, or the biological agent as described in the foregoing embodiments, or the composition as described in the foregoing embodiments, or the compound agent as described in the foregoing embodiments.

[0015] Eighthly, embodiments of the present invention provide the application of Bacillus terrestris XH1 as described in the foregoing embodiments, or the culture as described in the foregoing embodiments, or the biological agent as described in the foregoing embodiments, or the composition as described in the foregoing embodiments, or the compound agent as described in the foregoing embodiments, in the deammoniation of wastewater or the preparation of products deammoniation of wastewater.

[0016] Ninthly, embodiments of the present invention provide a wastewater ammonia removal agent, the active ingredients of which include: Bacillus terrestris XH1 as described in the foregoing embodiments, or the culture as described in the foregoing embodiments, or the biological agent as described in the foregoing embodiments, or the composition as described in the foregoing embodiments, or the compound agent as described in the foregoing embodiments.

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

[0018] (1) Bacillus tekirae XH1 is a complete ammonia oxidizing microorganism, capable of independently oxidizing NH4+. + -N is oxidized to NO2 - -N, and then through nitration, NO2 - -N is oxidized into NO3, which is easily absorbed by plants. - -N; In agriculture, it can accelerate the mineralization of soluble organic nitrogen in organic fertilizers, oxidizing it into NO3, which is more easily absorbed by plants. - -N, thereby promoting plant growth, reducing the application of organic fertilizer, and lowering agricultural production costs;

[0019] (2) The complete ammonia oxidation of XH1 reduces or avoids the accumulation and leaching of DON in the soil, thereby reducing nitrogen pollution of surface water and groundwater and helping to protect aquatic ecosystems.

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

[0021] (4) XH1 can also be used to remove ammonia from water bodies, reduce 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. Attached Figure Description

[0022] To more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings used in the embodiments will be briefly introduced below. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as a limitation on the scope. For those skilled in the art, other related drawings can be obtained based on these drawings without creative effort.

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

[0024] Figure 2 Phylogenetic tree of strain XH1;

[0025] Figure 3 In the table, (a) represents the total leaching loss of soluble organic nitrogen; and (b) represents the DON concentration in the leaching solution.

[0026] Figure 4 In the table, (a) represents the total biomass of dwarf tomatoes; (b) represents the yield of dwarf tomatoes; and (c) represents the weight of a single dwarf tomato.

[0027] Figure 5 In the table, (a) represents the soluble protein content of dwarf tomatoes; (b) represents the lycopene content.

[0028] Figure 6 In this context, (a) represents the total fresh weight of tobacco; (b) represents the height of the tobacco plant; (c) represents the diameter of the tobacco stem; and (d) represents the maximum leaf area of ​​the tobacco plant.

[0029] Figure 7 In the table, (a) represents the total biomass of dwarf tomatoes; (b) represents the yield of dwarf tomatoes; and (c) represents the weight of a single dwarf tomato.

[0030] Figure 8 In the table, (a) represents the soluble protein content of dwarf tomatoes; (b) represents the lycopene content.

[0031] Figure 9 In this context, (a) represents the total fresh weight of tobacco; (b) represents the height of the tobacco plant; (c) represents the diameter of the tobacco stem; and (d) represents the maximum leaf area of ​​the tobacco plant.

[0032] Figure 10The growth characteristics of XH1 on nitrified media with different NH4Cl concentrations are shown. Detailed Implementation

[0033] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased commercially.

[0034] In agricultural production, the supply of nitrogen fertilizer is crucial for crop growth. Tomatoes and tobacco, as typical nitrate-loving crops, are particularly susceptible to NO3-. - Both nitrate nitrogen and nitrogen showed good responses. When nitrate nitrogen supply was sufficient, better growth and higher yields were observed. Increasing soil NO3... - NO3- content can rapidly increase the levels of organic acids and mineral cations in tomatoes, which is beneficial to the growth and development of tomatoes, and promotes flower bud differentiation and development as well as fruit enlargement. Plant vegetative organs accumulate large amounts of NO3- during the growing season. - -N, but as plants grow, the nitrate nitrogen NO3 in their bodies - -N content gradually decreases, therefore, timely replenishment of NO3 is necessary. - -N is key to ensuring crop yield and quality.

[0035] In addition, NO3 - -N accumulation in plants is a "reserve" mechanism. - -N is also an important osmotic regulator within vacuoles. When carbohydrate synthesis decreases in plants and the organic matter content within vacuoles declines, NO3-... - -N can replace the regulation of osmotic pressure, and this regulation requires less energy, allowing plants to still grow and develop well under adverse conditions, such as when soil nutrients are insufficient.

[0036] Complete ammonia-oxidizing microorganisms have only been discovered in recent years, and their abundance in soil is extremely low, resulting in few reports on related research and applications. These microorganisms have a high affinity for ammonia nitrogen, not only enhancing their ability to bind ammonia nitrogen with organic fertilizer and improving the efficiency of microbial mineralization of organic nitrogen, but also exhibiting a competitive advantage under oligotrophic conditions, even in low NH4 environments. + In an environment with -N concentration, microorganisms that completely oxidize NH4+ can also complete the NH4+ oxidation process. + -N to NO3 - Oxidation of -N.

[0037] The Bacillus tekirae XH1 provided in this embodiment of the invention is a complete ammonia oxidizing microorganism, possessing complete ammonia oxidizing capability and able to independently achieve NH4+ oxidation.+ -N to NO3 - It oxidizes -N with high efficiency, and when applied in agriculture, it can accelerate the mineralization of organic nitrogen in organic fertilizers, oxidizing it into NO3, which is easily absorbed by plants. - XH1 promotes plant growth by reducing the accumulation and leaching of DON in the soil, thereby reducing nitrogen pollution of surface and groundwater and protecting aquatic ecosystems. Compared with the N2O produced by other microorganisms during nitrification, XH1 releases less 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 improve the NO3 production from complete ammonia oxidation. - The rate of nitrogen-fixing bacteria (N-N) further promotes plant growth, reduces the amount of organic fertilizer applied, and better prevents DON leaching. Nitrogen-fixing bacteria convert atmospheric nitrogen into NH4. + -N, then oxidized to NO3 by XH1. - -N not only reduces agricultural production costs and the negative environmental impacts of chemical and organic fertilizers, but also effectively reduces nitrogen leaching and greenhouse gas emissions, which is of great significance to sustainable agricultural development.

[0039] XH1 can also be used for ammonia removal in water bodies, reducing the concentration of ammonia nitrogen and thus lowering its toxicity to aquatic organisms and the risk of eutrophication. Traditional wastewater treatment methods for ammonia removal mainly involve two stages: ammonia oxidation and nitrification. These methods have low efficiency and are difficult to completely remove ammonia. XH1, however, has a high affinity for ammonia nitrogen and for NH4+. + -N has stronger tolerance and can withstand higher NH4 levels. + Complete ammonia oxidation is achieved at a -N concentration.

[0040] Specific technical solutions

[0041] On one hand, the present invention provides a Bacillus equilensis XH1, which is taxonomically named Bacillus equilensis and has the accession number CGMCC No. 29351.

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

[0043] On the other hand, this embodiment of the invention also provides a culture obtained by culturing Bacillus tekirae XH1 as described in the foregoing embodiments.

[0044] This application does not impose any special restrictions on the culture conditions of XH1, and existing culture media and culture conditions used for Bacillus tekiria can be used.

[0045] On the other hand, embodiments of the present invention also provide a biological agent, which includes: Bacillus tekirae XH1 as described in the foregoing embodiments or the culture described in any of the foregoing embodiments.

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

[0047] On the other hand, embodiments of the present invention also provide a composition comprising any one 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] The first microorganism includes Bacillus tekirae XH1 as described in the foregoing embodiments, and the second microorganism includes nitrogen-fixing bacteria.

[0051] In some embodiments, the situation described in (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 a second microorganism; or a culture of the first microorganism and a second microorganism.

[0052] On the other hand, embodiments of the present invention also provide a compound microbial agent, which includes: the microbial composition described in any of the foregoing embodiments.

[0053] In some embodiments, the compound microbial agent further includes a carrier. The carrier may be selected from carriers used in microbial agents in the art.

[0054] On the other hand, embodiments of the present invention also provide the application of Bacillus tekirulae XH1 as described in the foregoing embodiments, or the culture as described in any of the foregoing embodiments, or the biological agent as described in any of the foregoing embodiments, or the composition as described in any of the foregoing embodiments, or the compound agent as described in any of the foregoing embodiments, in any of the following:

[0055] (a) Prepare products that promote plant growth; (b) Promote plant growth; (c) Soil conditioning; (d) Prepare 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. Nitrate-loving crops are more sensitive to NO3-. - When -N supply is sufficient, it exhibits better growth performance and higher yield.

[0057] In some embodiments, the nitrifying plants include any one or more of the following: tomatoes, tobacco, corn, soybeans, rapeseed, spinach, bok choy, carrots, blueberries, apples, grapes, and kiwifruit.

[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, embodiments of the present invention also provide a growth promoter or soil conditioner, the active ingredients of which include: Bacillus tekirae XH1 as described in the foregoing embodiments, or the culture as described in any of the foregoing embodiments, or the biological agent as described in any of the foregoing embodiments, or the composition as described in any of the foregoing embodiments, or the compound agent as described in any of the foregoing embodiments.

[0060] On the other hand, embodiments of the present invention also provide the application of Bacillus tekirulae XH1 as described in the foregoing embodiments, or the culture described in any of the foregoing embodiments, or the biological agent described in any of the foregoing embodiments, or the composition described in any of the foregoing embodiments, or the compound agent described in any of the foregoing embodiments, in wastewater ammonia removal or in the preparation of wastewater ammonia removal products.

[0061] In addition, this invention also provides a wastewater ammonia removal agent, the active ingredients of which include: Bacillus terrestris XH1 as described in the foregoing embodiments, or the culture as described in any of the foregoing embodiments, or the biological agent as described in any of the foregoing embodiments, or the composition as described in any of the foregoing embodiments, or the compound agent as described in any of the foregoing embodiments.

[0062] The features and performance of the present invention will be further described in detail below with reference to embodiments.

[0063] Example 1: Screening, Identification and Preservation of Strains XH1

[0064] Strain screening:

[0065] The microbial strain was selected from Erwu Mountain red soil in Xichang City, Sichuan Province (27°89'45.04”N, 102°26'44.49”E). The soil's physicochemical properties, such as total nitrogen (TN) and NH4+, were analyzed. + -N, NO3 --N, total organic carbon, and total organic matter were 0.71±0.06 g / kg, 109.75±3.94 mg / kg, 192.44±0.75 mg / kg, 9.87±0.06 g / kg, and 67.80±1.31 g / kg, respectively.

[0066] Take 1g of soil and add 9ml of sterile water, then prepare a soil suspension by shaking. Add 1mL of the soil suspension to LB liquid medium for bacterial enrichment, then spread 1mL of the enriched bacterial solution onto solid nitrification medium for separation and purification. Nitrification medium: NH4Cl 380mg / L, CH3COONa·3H2O 2000mg / L, MgSO4·7H2O 50mg / L, K2HPO4 200mg / L, NaCl 120mg / L, MnSO4·4H2O 10mg / L, FeSO4 10mg / L, distilled water 1000mL. Adjust the pH to 7.0 with dilute HCl, then dispense into containers and sterilize at 121℃ for 30min. After cooling, the culture medium is obtained; the obtained culture medium can be stored at 4℃ for later use.

[0067] Fifteen nitrifying bacteria strains were obtained from solid nitrification medium and named XH1 to XH15. The nitrification capacity of these 15 heterotrophic nitrifying bacteria strains was studied. After culturing in liquid nitrification medium for 72 hours, they showed increased nitrification capacity with NO3-. - -N formation indicates nitrification capacity. NH4 + -N detection adopts the national standard GB / T42485-2023 indophenol blue colorimetric method, NO3 - -N detection was performed using the national standard DZ / T0064.59-2021, the disulfonic acid phenol spectrophotometric method. This method has strong nitrification ability and can simultaneously detect the ammonia monooxygenase (amoA) gene (which converts NH4+ into nitrogen). + -N is oxidized to NO2 - The -N ammonia oxidation gene) and the nitrite oxidase NxrB gene (which converts NO2) - -N is oxidized to NO3 - The strain with the -N nitrification gene is XH1. The NO3- of strain XH1... - The -N production was 25.09 mg / L, which was significantly higher than that of the other strains.

[0068] Strain identification: The selected XH1 strain was streaked onto LB agar using the streak plate method. Its morphological characteristics are shown in [reference needed]. Figure 1 In (a), XH1 colonies are white, round, with irregular edges, a raised center, and filamentous secretions around the periphery. XH1 was stained using Gram staining; the Gram staining diagram is shown below. Figure 1In (b), XH1 stains purple with Gram stain, indicating it is a Gram-positive bacterium. 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, which is 1461 bp in length. The phylogenetic tree of the strain was obtained after analyzing the XH1 sequence using MEGA 5.0 software. The results are as follows: Figure 2 As shown. Homology comparison revealed that XH1 shares 98% sequence similarity with Bacillus tequilensis. Based on the above analysis, XH1 belongs to Bacillus tequilensis. (Studies have found that Bacillus tequilensis mainly functions in denitrification and biocontrol, and is primarily used for plant growth promotion).

[0073] The 16S rRNA sequencing of Bacillus tektina XH1 is as follows:

[0074]

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

[0076] The *Bacillus tequilensis* XH1 strain provided in this invention possesses complete ammonia oxidation capability, meaning that this strain can independently complete NH4+ oxidation. + -N is oxidized to NO2 - -N, then NO2 - -N is oxidized to NO3 - The ability of XH1 to generate NH4+ was detected when it was cultured in liquid nitrification medium. + -N is oxidized to NO2 - The relative expression of the -N ammonia monooxygenase (amoA) gene was increased, and NO2 was reduced. - -N is oxidized to NO3 -The relative expression of the -N nitrite oxidase (NxrB) gene is increased.

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

[0078] Preparation of Bacillus tekiria XH1 bacterial suspension: 1% XH1 was inoculated into LB liquid medium and cultured at 37℃ and 180 r / m until OD. 600 =1.0, centrifuged at 6000 r / m for 10 minutes at 4℃ to obtain XH1 cells, resuspended and washed 3 times with sterile water, and finally the XH1 bacterial suspension, OD 600 =1.0.

[0079] Leaching experiment: Erwu Mountain red soil from Xichang City, Sichuan Province (the physicochemical properties of this soil, such as TN and NH4) was used. + -N, NO3 - -N, total organic carbon, and total organic matter were 0.71 g / kg, 0.11 g / kg, 0.192 g / kg, 9.87 g / kg, and 67.80 g / kg, respectively. The red soil parent material (residual slope deposits from weathered granite, highly susceptible to soil erosion, and prone to leaching under poor soil and water conservation conditions) was used as the experimental soil. 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 all at once and mixed thoroughly with the soil. The control group did not receive Bacillus tekirae XH1, only 100 mL of pure water was added. The treatment group received 100 mL of Bacillus tekirae XH1 suspension in 5 kg soil samples. Both the control and treatment groups had three replicates. 500 mL of water was applied on days 10, 20, 30, 40, and 60, for a total of five times. After 24 hours of infiltration, the leachate was collected from the bottom tray.

[0080] DON Calculation: The TN of the leachate was determined using the alkaline potassium persulfate colorimetric method according to the national environmental standard HJ636-2012. The DON content and concentration of the leachate could be obtained through calculation.

[0081] The experimental results are shown in Figure 3 Throughout the experimental period, the DON leaching rates in the treatment group and the control group were 20.33 kg / ha and 30.84 kg / ha, respectively, with the DON leaching rate in the treatment group being 34.07% lower than that in the control group. At the end of the experiment, the DON content in the leachate of the treatment group and the control group were 93.57 mg / L and 128.33 mg / L, respectively, with the DON content in the leachate of the treatment group being 27.08% lower than that in the control group.

[0082] It is evident that Bacillus tekiria XH1 can oxidize DON in the soil into NO3, which is easily absorbed and utilized by plants. - -N, 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: Red soil from Erwu Mountain in Xichang City, Sichuan Province, was used as the experimental soil. 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 all at once and mixed thoroughly with the soil. The treatment group received 100 mL of a suspension of *Solanum abutiloides* XH1 (preparation method described in Example 2) in 5 kg of soil samples. The control group received no *Solanum abutiloides* XH1, only 100 mL of pure water. Both the treatment and control groups had three replicates. One dwarf tomato seedling (*Solanum abutiloides*), purchased from Weifang Maitu Agricultural Technology Co., Ltd., China, was planted in each pot for 65 days. All groups were placed outdoors under natural sunlight, with a temperature range of 23–29℃ and a relative humidity range of 40%–69%.

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

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

[0087] The soluble protein content of the fruit was determined using the Coomassie Brilliant Blue staining method: 100 mg of Coomassie Brilliant Blue G-250 was weighed and dissolved in 50 mL of 90% ethanol, then 100 mL of 85% (w / v) phosphoric acid was added. After mixing and shaking, the solution was diluted 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, added to distilled water, and homogenized. The supernatant was collected by centrifugation as the soluble protein extract. 1.0 mL of the extract was taken, Coomassie Brilliant Blue G-250 reagent was added, and the absorbance was measured at 595 nm. The protein content was calculated using the standard curve.

[0088] The lycopene content in the fruit was determined using 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, soluble protein content of the fruit, and lycopene content of the fruit of the dwarf tomato were measured as follows: 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 6.12 g and 5.79 g, respectively, with the total biomass of the dwarf tomatoes in the treatment group being 5.70% higher than that in the control group; the yield of dwarf tomatoes in the treatment group and the control group were 138.56 g / plant and 89.68 g / plant, respectively, with the yield of the dwarf tomatoes in the treatment group being 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 7.54 g and 7.24 g, respectively, with the single fruit weight of the dwarf tomatoes in the treatment group being 4.14% higher than that in the control group; the soluble protein content of dwarf tomatoes in the treatment group and the control group were 1.47 mg / g FW and 0.88 mg / g FW, respectively, with the soluble protein content of the dwarf tomatoes in the treatment group being 67.04% higher than that in the control group; and the lycopene content of dwarf tomatoes in the treatment group and the control group were 5.23 μg / g FW and 4.28 μg / g FW, respectively, with the lycopene content of the dwarf tomatoes in the treatment group being 22.19% higher than that in the control group.

[0090] Experimental results showed that *Bacillus tektina* XH1 significantly promoted the yield and quality of dwarf tomatoes. This indicates 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: Red soil from Erwu Mountain in Xichang City, Sichuan Province, was used as the experimental soil. 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 all at once and mixed thoroughly with the soil. The treatment group received 100 mL of a suspension of Bacillus tekirae XH1 (preparation method described in Example 2) in 5 kg of soil samples. The control group received no Bacillus tekirae XH1, only 100 mL of pure water. Both the treatment and control groups had three replicates. One tobacco seedling was planted in each pot for 65 days. All groups were placed outdoors under natural sunlight, with a temperature range of 23–29℃ and a relative humidity range of 40%–69%.

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

[0094] At the end of the experiment, the total fresh tobacco yield, plant height, stem diameter, and maximum leaf area were respectively observed. Figure 6At the end of the experiment, the total fresh weight of tobacco in the treatment group and the control group was 101.43g and 82.71g, respectively, with the treatment group's total fresh weight being 22.63% higher than the control group's. The plant height of tobacco in the treatment group and the control group was 51.59cm and 50.20cm, respectively, with the treatment group's plant height being 2.76% higher than the control group's. The stem diameter of tobacco in the treatment group and the control group was 8.23mm and 7.05mm, respectively, with the treatment group's stem diameter being 16.73% higher than the control group's. The maximum leaf area of ​​tobacco in the treatment group and the control group was 598.39cm². 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 indicate that Bacillus tekirae XH1 significantly promotes tobacco growth.

[0095] It is evident that the complete ammonia oxidation capacity of Bacillus tekirae XH1 can enhance the oxidation of organic nitrogen to NO3. - -N is absorbed by tobacco, thereby promoting tobacco growth.

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

[0097] (1) Preparation of Bacillus tekiria seed culture: Inoculate strain XH1 into the above-mentioned liquid nitrification medium at 1% of the culture medium volume, and incubate at 37℃ and 180r / m until OD. 600 =1.0.

[0098] (2) Preparation of nitrogen-fixing bacteria seed culture: Inoculate nitrogen-fixing bacteria into liquid nitrogen-free medium at 1% of the culture medium volume, and culture at 37℃ and 180r / m until OD. 600 =1.0.

[0099] (3) Co-culture of strain XH1 with nitrogen-fixing bacteria: The two seed cultures prepared in steps (1) and (2) were inoculated into nitrification medium at 0.5% of the medium volume and cultured at 37℃ and 180r / m until OD. 600 =1.0.

[0100] Experiment on promoting dwarf tomato growth: Red soil from Erwu Mountain in Xichang City, Sichuan Province was used as the experimental soil. 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 all at once and mixed thoroughly with the soil. Treatment 1 involved adding 100 mL of a suspension of Bacillus tekirae XH1 (preparation method described in Example 2) to each 5 kg soil sample. Treatment 2 involved co-culturing XH1 with nitrogen-fixing bacteria until OD... 600100 mL of a bacterial suspension with a concentration of 1.0 g / L was added to 5 kg of soil sample. The control group received no *Bacillus tekirae* XH1, only 100 mL of pure water. Treatments 1-2 and the control group each had three replicates. One dwarf tomato seedling (*Solanum abutiloides*), purchased from Weifang Maitu Agricultural Technology Co., Ltd., China, was planted in each pot for 65 days. All groups were placed outdoors under natural sunlight, with temperatures ranging from 23 to 29°C and relative humidity from 40% to 69%.

[0101] At the end of the experiment, the total biomass of dwarf tomatoes, fruit yield per plant, weight of a single fruit, soluble protein content of the fruit, and lycopene content of the fruit were measured. The results are shown in [link to experimental data]. Figure 7 and Figure 8 The total biomass of dwarf tomatoes in treatments 1, 2, and the control group were 6.12 g, 15.59 g, and 5.79 g, respectively. Treatment 1 showed a 5.70% higher total biomass than the control group, and Treatment 2 showed a 169.25% higher total biomass. The yields of dwarf tomatoes in treatments 1, 2, and the control group were 138.56 g / plant, 162.06 g / plant, and 89.68 g / plant, respectively. Treatment 1 showed a 54.51% higher yield than the control group, and Treatment 2 showed an 80.71% higher yield. The average fruit weights of dwarf tomatoes in treatments 1, 2, and the control group were 7.54 g, 9.02 g, and 7.24 g, respectively. Treatment 1 showed a 4.14% higher average fruit weight than the control group, and Treatment 2 showed a 24.58% higher average fruit weight. The soluble protein content of dwarf tomatoes in treatments 1, 2, and the control group were 1.47 mg / g FW, 2.11 mg / g FW, and 2.11 mg / g FW, respectively. The soluble protein content of dwarf tomato fruits treated with 0.88 mg / g FW was 67.04% higher than that of the control group, and the soluble protein content of dwarf tomato fruits treated with 0.88 mg / g FW was 139.77% higher than that of the control group. The lycopene content of dwarf tomato fruits treated with 0.88 mg / g FW was 5.23 μg / g FW, 6.54 μg / g FW, and 4.28 μg / g FW, respectively. The lycopene content of dwarf tomato fruits treated with 0.88 mg / g FW was 22.19% higher than that of the control group, and the lycopene content of dwarf tomato fruits treated with 0.88 mg / g FW was 139.77% higher than that of the control group.

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

[0103] Example 6: Co-culturing strain XH1 with nitrogen-fixing bacteria can further promote tobacco growth.

[0104] (1) Preparation of Bacillus tekiria seed culture: Inoculate strain XH1 into the above-mentioned liquid nitrification medium at 1% of the culture medium volume, and incubate at 37℃ and 180r / m until OD. 600 =1.0.

[0105] (2) Preparation of nitrogen-fixing bacteria seed culture: Inoculate nitrogen-fixing bacteria into liquid nitrogen-free medium at 1% of the culture medium volume, and culture at 37℃ and 180r / m until OD. 600 =1.0.

[0106] (3) Co-culture of strain XH1 with nitrogen-fixing bacteria: The two seed cultures prepared in steps (1) and (2) were inoculated into nitrification medium at 0.5% of the medium volume and cultured at 37℃ and 180r / m until OD. 600 =1.0.

[0107] Tobacco growth promotion experiment: Red soil from Erwu Mountain in Xichang City, Sichuan Province was used as the experimental soil. 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 all at once and mixed thoroughly with the soil. Treatment 1 involved adding 100 mL of a suspension of Bacillus tekirae XH1 (preparation method described in Example 2) to each 5 kg soil sample. Treatment 2 involved co-culturing XH1 with nitrogen-fixing bacteria until OD... 600 100 mL of a bacterial suspension with a concentration of 1.0 g / L was added to 5 kg of soil sample. The control group received no *Bacillus tekirae* XH1, only 100 mL of pure water. Treatments 1-2 and the control group each had three replicates. One tobacco seedling was planted in each pot for 65 days. All groups were placed outdoors under natural sunlight, with temperatures ranging from 23 to 29°C and relative humidity from 40% to 69%.

[0108] At the end of the experiment, the total fresh tobacco yield, plant height, stem diameter, and maximum leaf area were respectively recorded in the following figures: Figure 9 The total fresh weight of tobacco in treatments 1, 2, and the 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 height of tobacco in treatments 1, 2, and the control group were 51.59 cm, 58.20 cm, and 50.20 cm, respectively. The plant height of tobacco in treatment 1 was 2.76% higher than that in the control group, and the plant height of tobacco in treatment 2 was 15.36% higher than that in the control group. The stem diameter of tobacco in treatments 1, 2, and the control group were 8.23 ​​cm, 9.62 cm, and 7.05 cm, respectively. The stem diameter of tobacco in treatment 1 was 16.73% higher than that in the control group, and the stem diameter of tobacco in treatment 2 was 36.45% higher than that in the control group. The maximum leaf area of ​​tobacco in treatments 1, 2, and the control group was 580.57 cm². 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 atmospheric nitrogen into NH4. + -N, then oxidized to NO3 by XH1. - -N. The addition of nitrogen-fixing bacteria can provide more NH4. + -N, thereby enhancing XH1's ability to promote tobacco growth.

[0110] Example 7: Strain XH1 exhibits good ammonia tolerance and nitrate production capacity.

[0111] The nitrification medium was prepared using 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, with the pH adjusted to 7.0 using dilute HCl.

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

[0113] Ammonia nitrogen tolerance of strain XH1: It could grow normally in nitrified media with NH4Cl concentrations of 400 mg / L, 450 mg / L, 500 mg / L, and 550 mg / L. Results are shown below. Figure 10 XH1 was cultured for 7 hours in nitrified media of various concentrations of NH4Cl, and the OD... 600 All values ​​reached above 1.00. This demonstrates that XH1, as a complete ammonia-oxidizing bacterium, possesses excellent ammonia nitrogen tolerance and can withstand high concentrations of NH4+. + Survival in -N environments.

[0114] Nitrate production capacity of strain XH1: The nitrate production capacity of XH1 after 72 hours of growth in nitrification media with NH4Cl concentrations of 400 mg / L, 450 mg / L, 500 mg / L, and 550 mg / L is shown in Table 1. NO3 production was also observed at these 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 the removal of ammonia nitrogen from wastewater

[0118] Wastewater ammonia nitrogen removal experiment: Experimental water samples 1, 2 and 3 were collected from artificial lake 1, artificial lake 2 and artificial river 3 in Chengdu, Sichuan Province, respectively, with initial ammonia nitrogen contents of 63.89 mg / L, 104.42 mg / L and 221.56 mg / L.

[0119] Collect 50 mL of water samples 1, 2, and 3, and inoculate each sample with 0.5 mL of bacterial culture of strain XH1. The control group was not inoculated with bacterial culture. The samples were cultured at 37℃ and 180 r / min for 48 hours, and the ammonia nitrogen concentration was measured.

[0120] NH4 in water sample + -N concentration was determined using the Nessler's reagent spectrophotometric method according to national standard DZ / T 0064.59—2021.

[0121] Table 2. Ammonia removal capacity in XH1 water bodies

[0122]

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

[0124] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of Bacillus tergentii XH1, characterized in that, Its taxonomic name is Bacillus tequilensis The accession number is CGMCC No.29351.

2. A culture, characterized in that, It is obtained by culturing Bacillus tekirae XH1 as described in claim 1.

3. A biological agent, characterized in that, It includes: The Bacillus tekirae XH1 as described in claim 1 or the culture as described in claim 2.

4. The use of Bacillus tekirulatus XH1 as described in claim 1, the culture as described in claim 2, or the biological agent as described in claim 3 in any of the following: (a) Preparation of a product that promotes plant growth; (b) Promotion of plant growth; (c) Soil conditioning; (d) Preparation of a soil conditioner; wherein the plant is a nitrate-loving plant; and the soil conditioning is to control the leaching of DON from the soil.

5. The application according to claim 4, characterized in that, The nitrifying plants include any one or more of the following: tomatoes, tobacco, corn, soybeans, rapeseed, spinach, bok choy, carrots, blueberries, apples, grapes, and kiwifruit.

6. A growth promoter or soil conditioner for promoting the growth of nitrate-loving plants, wherein the soil conditioner is used to control the leaching of DON from the soil, characterized in that, Its active ingredients include: Bacillus tekirae XH1 as described in claim 1, or the culture as described in claim 2, or the biological agent as described in claim 3.

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