Ammonia-utilizing bacterium and use thereof

Abstract

The application provides an ammonia-loving bacterium and application thereof, the ammonia-loving bacterium is preserved in Guangdong Microbial Culture Collection Center on July 24, 2025, and the preservation number is GDMCC No.66752; 16S rDNA of the ammonia-loving bacterium is as shown in SEQ ID NO.1; the ammonia-loving bacterium can be used to prepare microbial preparation, realizes the function of the strain; the ammonia-loving bacterium has good phosphorus dissolving capacity and IAA producing capacity, can enhance the resistance of plants, and promotes the growth of plants.

Description

Technical Field

[0001] This invention relates to an ammonia-loving bacterium and its applications, belonging to the field of microbial technology. Background Technology

[0002] In the process of modern agricultural development, the increasing amount of fertilizer used and improper application methods have led to groundwater pollution, resulting in soil salinization, acidification, compaction, and low agricultural productivity. How to achieve sustainable development in modern agriculture is a topic worthy of discussion. Current improvement solutions include biological methods, such as using microbial fertilizers applied directly to the soil. Organic fertilizers are decomposed and transformed by microorganisms into humus, which can improve the soil's buffering capacity, accelerate nutrient decomposition, and promote the conversion of slow-release nutrients. The industry is still exploring more diverse treatment models and more efficient and significant treatment effects. Summary of the Invention

[0003] In order to overcome the shortcomings of the prior art, the first objective of the present invention is to provide an ammonia-loving bacterium with good phosphorus-dissolving ability and IAA-producing ability, which can enhance plant resistance and promote plant growth.

[0004] The second objective of this invention is to provide an application of the aforementioned ammonia-loving bacteria, which is to use the ammonia-loving bacteria in the preparation of microbial preparations to realize the function of the strain.

[0005] The first objective of this invention can be achieved by adopting the following technical solution: an ammonia-loving bacterium, classified and named Ammoniphilus sp . The ammoniaphile was deposited on July 24, 2025, at the Guangdong Provincial Center for Microbial Culture Collection, located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, with accession number GDMCC No. 66752.

[0006] Furthermore, the 16S rDNA of the ammoniaphilus is shown in SEQ ID NO.1.

[0007] The second objective of this invention can be achieved by adopting the following technical solution: the application of ammonia-loving bacteria, used in the preparation of microbial preparations containing ammonia-loving bacteria.

[0008] Furthermore, the formulation is prepared by the following method: inoculating ammonia-loving bacteria into a fermentation substrate for fermentation, and drying the obtained fermentation broth.

[0009] Furthermore, the concentration of the fermentation broth is 7 × 10⁻⁶. 6 -9×10 6 cfu / mL.

[0010] Furthermore, microbial preparations are preparations that promote plant growth and / or resist disease.

[0011] Furthermore, the plant is at least one of wheat, rapeseed, cotton, potato, tomato, and soybean.

[0012] Furthermore, the pathogens that cause disease in plants are at least one of Fusarium oxysporum and Helicobacter pylori.

[0013] Furthermore, the microbial preparation is a preparation that acts on at least one of the plant seeds, roots, leaves, and fruits.

[0014] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0015] 1. The ammonia-loving bacteria of the present invention can be used as a plant treatment agent. Through the life activities of microorganisms, it increases the supply of plant nutrients, improves plant nutrition, and increases growth efficiency and yield.

[0016] 2. The ammonia-loving bacteria of the present invention have a significant inhibitory effect on pathogenic bacteria of crops, which can enhance plant resistance and promote plant growth;

[0017] 3. The microbial preparation of ammonia-loving bacteria of the present invention has flexible application methods, high bacterial activity, no pollution, and conforms to the sustainable development of the environment. Attached Figure Description

[0018] The ammoniaphiles involved in this invention are classified and named as follows: Ammoniphilus The sp., an ammoniaphil, was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No. 66752.

[0019] Figure 1 The colony morphology of strain HJJHmls-Ba3;

[0020] Figure 2 Microscopic morphology of strain HJJHmls-Ba3;

[0021] Figure 3 The growth curve of HJJHmls-Ba3;

[0022] Figure 4 Photographs of the antibacterial test of Fusarium oxysporum CK;

[0023] Figure 5 Photographs of the antibacterial test of Fusarium oxysporum on day 6;

[0024] Figure 6 Photographs of antibacterial test results for Helicobacter pylori CK;

[0025] Figure 7 Photographs of the antibacterial test of Helicobacter pylori after 6 days;

[0026] Figure 8 The morphology of strain HJJHmls-Ba3 on sodium selenite medium with a concentration of 0 μg / mL;

[0027] Figure 9 The morphology of strain HJJHmls-Ba3 on sodium selenite medium with a concentration of 20 μg / mL;

[0028] Figure 10 The morphology of strain HJJHmls-Ba3 on sodium selenite medium with a concentration of 40 μg / mL;

[0029] Figure 11 The morphology of strain HJJHmls-Ba3 on sodium selenite medium with a concentration of 80 μg / mL;

[0030] Figure 12 The morphology of strain HJJHmls-Ba3 on sodium selenite medium with a concentration of 160 μg / mL;

[0031] Figure 13 The morphology of strain HJJHmls-Ba3 on sodium selenite medium with a concentration of 320 μg / mL is shown. Detailed Implementation

[0032] The present invention will now be further described with reference to the accompanying drawings and specific embodiments:

[0033] Example 1:

[0034] An ammonia-loving bacterium was obtained by the following method:

[0035] 1) Isolation and purification of the strain: After sampling, fermentation broth was prepared. 100 μL of the sample solution was added to 900 μL of sterile water, and then the sample was diluted to 10 μL. -4 10 -5 The concentration was determined by incubating the medium in a 70°C water bath for 20 minutes. 100 μL of the resulting liquid was then spread onto NA plates and incubated at 28°C for 72 hours. Bacterial growth was observed on the plates. Once colonies appeared, typical single colonies were streaked onto NA solid medium for isolation and purification. Single colonies were inoculated into 5 mL of sterilized NB broth and incubated at 28°C for 72 hours. The culture was then stored at -80°C with 50% v / v glycerol for later use.

[0036] The isolated and purified single colonies were identified using the following primers:

[0037] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO. 2)

[0038] 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO. 3);

[0039] The 16S rDNA sequence of the ammoniaphilus HJJHmls-Ba3 was obtained, as shown in SEQ ID NO.1.

[0040] 2) The sequence was compared and analyzed using the EZBioCloud database. The results showed that strain HJJHmls-Ba3 was closely related to known type strains in the database. Ammoniphilus resinae The homology of CC-RT-E is 98.36%, suggesting it may be... Ammoniphilus A new species of the genus Ammoniac.

[0041] 3) Upload the genome of the target strain to the Korean EzBioCloud website. Download the sequence of a type strain with high similarity and use the EzBioCloud online tool ANICalculator to calculate the ANI value. Use 95-96% as the cutoff value to determine whether the strains are different species. Upload the genomes of the target strain and related type strains to the online tool Genome-to-Genome Distance Calculator 3.0 on the German Culture Collection website. Use 70% as the cutoff value and calculate the dDDH value to determine whether the strains are different species.

[0042] Table 1 HJJHmls-Ba3 genome ANI and dDDH calculation

[0043]

[0044] The genome of HJJHmls-Ba3 and closely related type strains Ammoniphilus resinae CC-RT-E T In comparison, the ANI value was 70.39%, less than the threshold of 95%, and the dDDH value was 22.0%, less than the threshold of 70%, indicating that it is a new species of ammoniaophila and has been named *Ammoniaophila*. Ammoniphilus sp.)HJJHmls-Ba3. Figure 1 This refers to the colony morphology of the bacterial strain. Figure 2 It is in microscopic form.

[0045] 4) Growth curve determination of HJJHmls-Ba3: The bacterial culture in the cryopreservation tube was inoculated into NB medium at an inoculation rate of 2% v / v and cultured in a 30℃ incubator for 72 h. After two subcultures, the bacteria were washed twice by centrifugation at 5000 r / min for 5 min with PBS buffer. Then, the OD600nm of the strain was adjusted to 1.0 with PBS buffer. The strain was then inoculated into NB medium at an inoculation rate of 2% v / v and cultured in a growth curve analyzer at 30℃. The growth curve of HJJHmls-Ba3 was measured every 10 minutes to obtain the growth curve (3 replicates).

[0046] like Figure 3 As shown, the time for HJJHmls-Ba3 to grow to OD600nm = 1.0 was 34 hours, and the time to reach the stable phase was 88 hours.

[0047] Detection:

[0048] 1) Detection of the antibacterial effect of strain HJJHmls-Ba3:

[0049] The bacterial culture in the cryopreservation tube was inoculated into NB medium at an inoculation rate of 2% v / v and cultured at 30°C for 72 h. After two subcultures, the bacteria were washed twice by centrifugation at 5000 r / min for 5 min with PBS buffer. Then, the OD600nm of the strain was adjusted to 1.0 with PBS buffer. The strain was then inoculated into NB medium at an inoculation rate of 2% v / v and cultured at 30°C for 88 h. The resulting fermentation broth was then used for further processing.

[0050] Add 100 μL of fermentation broth of strain HJJHmls-Ba3 to each of the four wells. Finally, place a uniformly sized bacterial block in the center of the solidified plate and incubate at 30°C for 6-10 days, observing whether the strain grows.

[0051] Prepare mycelial blocks of pathogenic bacteria Fusarium oxysporum and Helicobacter pylori and repeat the experiment according to the above method.

[0052] Table 2. Antibacterial test results of HJJHmls-Ba3: Antibacterial rate (%)

[0053]

[0054] Figures 4-7 The images show the control (CK) parameters for each pathogenic bacterium and the antibacterial test results for *Fusarium oxysporum* and *Helicobacter pylori* at 6 days. The HJJHmls-Ba3 strain showed a significant antibacterial effect.

[0055] 2) Determination of available phosphorus capacity of strain HJJHmls-Ba3:

[0056] The strain (adjusted to OD=1.0) was inoculated at a rate of 2% v / v into 50 mL centrifuge tubes containing 30 mL of organic and inorganic phosphorus liquid culture medium. A blank control (CK) of uninoculated organic and inorganic phosphorus liquid culture medium was used. The tubes were cultured at 28℃ with shaking at 200 rpm for 5 days. Samples were taken at 24h, 48h, 72h, 96h, and 120h, with 5 mL samples taken each time. The samples were centrifuged at 10000 rpm at 4℃ for 5 min, and the supernatant was stored at 4℃ (3 replicates). The soluble phosphorus content in the supernatant at 24h, 48h, 72h, 96h, and 120h was determined using the molybdenum antimony colorimetric method.

[0057] Table 3. Determination of available phosphorus capacity: soluble phosphorus concentration (μg / mL)

[0058]

[0059] The HJJHmls-Ba3 strain has a good phosphorus conversion capacity. For crops that require high levels of phosphorus during their growth, such as wheat, rapeseed, cotton, potatoes, tomatoes, and soybeans, processing it into a microbial agent and mixing it with fertilizers for application to the soil or plants can effectively promote crop growth.

[0060] 3) Determination of IAA production capacity of strain HJJHmls-Ba3:

[0061] A bacterial suspension with OD600=1 was inoculated into 5 mL of NB liquid medium (containing 100 mg / L L-tryptophan) at an inoculation rate of 2% v / v (using 10 mL tubes) and cultured at 28℃ with shaking at 200 rpm for 5 days (3 replicates). 500 μL of the bacterial suspension at different time points (24 h, 48 h, 72 h, 96 h, and 120 h) were collected in 1.5 mL EP tubes, centrifuged at 12000 rpm for 5 min, and the supernatant was collected. 50 μL of the supernatant was transferred to a 96-well plate, and 50 μL of Salkowski colorimetric solution (a mixture of solution A and solution B) was added simultaneously. After incubating the 96-well plate at room temperature in the dark for 30 min, the absorbance at OD530 nm was measured using a microplate reader.

[0062] Table 4 IAA Concentration (mg / L)

[0063]

[0064] 4) Siderophore production capacity of strain HJJHmls-Ba3:

[0065] Inoculate a bacterial suspension with OD600nm=1 at a 2% v / v rate into 5 mL of NB liquid medium (using a 10 mL tube), and incubate at 28°C and 200 rpm for 5 days (3 replicates). Take 500 μL of the bacterial suspension at different time points: 24 h, 48 h, 72 h, 96 h, and 120 h into 1.5 mL EP tubes, centrifuge at 12000 rpm for 5 min, and collect the supernatant for later use.

[0066] 100 μL of supernatant was added to a 96-well plate, and an equal volume of CAS detection solution was added and mixed thoroughly. After standing for 1 hour, the absorbance (As) at 630 nm was measured using a microplate reader. Separately, an equal volume of uninoculated sterile NB medium was mixed with an equal volume of CAS detection solution, and the absorbance was measured as the reference value (Ar). Finally, the amount of siderophores produced was calculated using the formula (Siderophore yield = (Ar - As) / Ar × 100% of siderophore amount unit. A result less than 10% is considered negative for siderophore secretion). Therefore, the siderophore secretion of strain HJJHmls-Ba3 was positive.

[0067] Table 5 Ferrocarrier Production (%)

[0068]

[0069] 5) New selenium-enriched species (sodium selenite converted to monoselenium, qualitative analysis):

[0070] NB solid culture medium was prepared and autoclaved at 121℃ for 30 min. After cooling to 60-80℃, sodium selenite solution (stock solution concentration of 50 mg / mL) was added to make the sodium selenite concentrations in the culture medium 0 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL, respectively. After the plates were inverted and cooled, HJJHmls-Ba3 strain was inoculated on the culture medium and incubated at 37℃ for 24 h. If the strain could grow, absorb and convert sodium selenite on the sodium selenite plate, coffee red monomeric selenium would be formed on the plate surface and stored in the bacterial cells.

[0071] Figures 8-13 The morphology of strain HJJHmls-Ba3 on sodium selenite plates at concentrations of 0 μg / mL, 20 μg / mL, 40 μg / mL, 80 μg / mL, 160 μg / mL, and 320 μg / mL, respectively.

[0072] Example 2:

[0073] Application of HJJHmls-Ba3 strain preparations:

[0074] The bacterial culture of strain HJJHmls-Ba3 was inoculated into a fermentation substrate for fermentation and cultured at 28-32℃ for 60-84 h. The concentration of the fermentation broth was 7×10⁻⁶. 6 -9×10 6 The concentration of cfu / mL was measured by centrifugation and washing. The fermentation broth was then dried to obtain the microbial preparation, which was stored in the dark at a temperature below 30°C.

[0075] 1) Activity detection of microbial agents in soil:

[0076] The sterilized soil (pH 7.8) was aliquoted and then mixed with the microbial preparation at a mass ratio of 1:60. The mixture was incubated at 28-30℃, with soil without the microbial preparation serving as a control. 10g samples from the 0-10cm soil layer were collected at 0h, 24h, and 48h, diluted with solvent, and the effective viable bacteria count was determined. The results are shown in Table 6.

[0077] Table 6 Effective viable bacteria count (cfu / g, 10⁻⁶) 10 )

[0078]

[0079] As shown in Table 6, microbial agents can effectively increase the number of viable bacteria in the soil and improve the soil environment.

[0080] 2) Detection of the effects of microbial agents on wheat growth:

[0081] Formulation group: After diluting the microbial preparation 20-40 times, wheat seeds of Wankenmai 22 were soaked for 5 hours and then germinated for 1 day. They were then cultured under normal light conditions at 20±1℃, maintaining humidity. Germination rate, root length, and whole plant length were measured at 7 and 14 days of culture. After 14 days, 20 wheat seedlings were selected, uprooted, their roots washed clean of soil, dried, and their fresh weight measured. After measurement, their dry weight was determined by drying at 60℃ for 1.2 hours.

[0082] Control group: The culture was prepared by soaking the seed in water without microbial agents, and the culture and assay methods were the same as those of the preparation group.

[0083] Each group contained 100 seeds, and the root length, whole plant length, fresh weight, and dry weight were analyzed and averaged. The results are shown in Table 7.

[0084] Table 7 Wheat Growth Monitoring

[0085]

[0086] Twenty seedlings were randomly selected and cultured for 14 days until maturity. At wheat maturity, spike length, spikelet number, and thousand-grain weight were measured. The average results are shown in Table 8.

[0087] Table 8. Monitoring of Wheat Growth

[0088]

[0089] Compared with the control group, the preparation group showed a significant increase in spikelet number and thousand-grain weight. Therefore, treatment with microbial preparations can significantly improve wheat plant growth, enhance disease resistance, and increase yield.

[0090] For those skilled in the art, various other corresponding changes and modifications can be made based on the technical solutions and concepts described above, and all such changes and modifications should fall within the protection scope of the claims of this invention.

Claims

1. An ammoniaophilic bacterium, characterized in that, The ammoniaphile was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 24, 2025, with accession number GDMCC No. 66752.

2. The ammoniaphile as described in claim 1, characterized in that, The 16S rDNA of the ammonia-loving bacteria is shown in SEQ ID NO.

1.

3. An application of the ammonia-loving bacteria as described in claim 1, characterized in that, The application is for preparing microbial preparations containing ammonia-loving bacteria.

4. The application as described in claim 3, characterized in that, The preparation is obtained by the following method: inoculating ammonia-loving bacteria into a fermentation substrate for fermentation, and drying the obtained fermentation broth.

5. The application as described in claim 4, characterized in that, The concentration of the fermentation broth is 7×10⁻⁶. 6 -9×10 6 cfu / mL.

6. The application as described in claim 3, characterized in that, The microbial preparation is a plant growth promoter and / or disease resistant agent; the pathogens causing plant diseases are at least one of Fusarium oxysporum and Helicobacter pylori.

7. The application as described in claim 6, characterized in that, The plant is at least one of wheat, rapeseed, cotton, potato, tomato, and soybean.

8. The application as described in claim 3, characterized in that, The microbial preparation is a preparation that acts on at least one of plant seeds, roots, leaves, and fruits.

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