Bacillus subtilis K-1 and its application in preventing and controlling onion soft rot

Abstract

Bacillus subtilis K-1 and its application in preventing and controlling onion soft rot belong to the field of biological control technology. In order to explore biocontrol strains that can prevent and control onion soft rot, the present invention separates rotten onion bulbs showing soft rot, and proves that acid-producing Klebsiella can cause onion soft rot after back-grafting; further, the present invention screens strain K-1 from onion rhizosphere soil, which has good antagonism to acid-producing Klebsiella that can cause onion soft rot, and comprehensively identifies antagonistic strain K-1 as Bacillus subtilis based on morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence identification results. The present invention found through experiments that Bacillus subtilis K-1 has good antagonistic stability and has broad application prospects in the biological control of onion soft rot.

Description

Technical Field

[0001] The invention belongs to the technical field of biological control, and particularly relates to Bacillus subtilis K-1 and application thereof in preventing and controlling onion soft rot. Background Art

[0002] Onion soft rot is caused by bacteria and is one of the common diseases of onions during the growth and storage period. It is mainly spread through soil, seeds and root residues, and develops rapidly under high temperature and high humidity conditions. The pathogenic bacteria of onion soft rot are usually Pectobacterium carotovora sub sp.carotovora (Pcc), but there are other bacteria that can cause onion soft rot through insect transmission, rainwater transmission or wound infection. For example, Klebsiella parasitizes in the respiratory tract or intestines of animals and can cause diseases in animals or humans, but recent studies have shown that Klebsiella oxytoca can also cause plant soft rot (Huang Xu. Isolation and identification of pinellia soft rot bacteria and pathogenicity research [D]. Huazhong Agricultural University, 2012.). Onion soft rot not only leads to a significant reduction in onion yield, but also reduces the quality and market value of onions.

[0003] Traditional chemical control methods have the risk of residual problems and environmental pollution, and physical control methods have large investment and slow results, so biological control is considered to be a feasible alternative. Bacillus subtilis is a bacterium widely present in nature, and has attracted widespread attention from various industries due to its unique biological characteristics. In agriculture, Bacillus subtilis can produce endospores, has strong adaptability to environmental factors such as acidity and temperature, and can promote plant growth through biofilm colonization while being non-pathogenic, and enhances plant absorption of nutrients. Therefore, it is often used as a biological control strain in agriculture, such as Bacillus subtilis can inhibit tea ring spot and tea scorch rot pathogens. However, there is no report on the use of Bacillus subtilis to prevent and control onion soft rot caused by acid-producing Klebsiella.

[0004] Onions are prone to many diseases and there are few disease-resistant varieties. In my country's agricultural production, the prevention and control of soft rot is often carried out from chemical and physical aspects, while there is little research on the biological control of onion soft rot, which restricts the development of onion industrialization in my country and cannot meet the actual needs of green agriculture. Therefore, it is urgent to explore biocontrol strains that can prevent and control onion soft rot. Summary of the invention

[0005] In order to explore biocontrol strains that can prevent and treat onion soft rot, the present invention separated rotten onion bulbs showing soft rot, and proved that Klebsiella oxytoca can cause onion soft rot after back-grafting; further, the present invention screened Bacillus subtilis K-1 from the onion rhizosphere soil, which has a good antagonistic effect on Klebsiella oxytoca that can cause onion soft rot.

[0006] In order to solve the above technical problems and achieve corresponding technical effects, the present invention specifically provides the following technical solutions:

[0007] The first object of the present invention is to provide a strain of Bacillus subtilis K-1, which is deposited in the General Microbiology Center of China Microorganism Culture Collection Administration, with a deposit number of CGMCC No.30922, a deposit date of June 11, 2024, and an address of the depository unit at No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0008] The second object of the present invention is to provide the use of the above-mentioned Bacillus subtilis K-1 in inhibiting the growth of Klebsiella oxytoca.

[0009] The third object of the present invention is to provide the use of the above-mentioned Bacillus subtilis K-1 in preventing and controlling onion soft rot.

[0010] In one embodiment of the present invention, the onion soft rot is caused by Klebsiella oxytoca.

[0011] The fourth object of the present invention is to provide a microbial agent containing the above-mentioned Bacillus subtilis K-1.

[0012] The fifth object of the present invention is to provide the use of the above-mentioned microbial agent in inhibiting the growth of Klebsiella.

[0013] The sixth object of the present invention is to provide the use of the above-mentioned microbial agent in preventing and controlling onion soft rot.

[0014] In one embodiment of the present invention, the onion soft rot is caused by Klebsiella oxytoca.

[0015] The seventh object of the present invention is to provide a biological fertilizer containing the above-mentioned Bacillus subtilis K-1.

[0016] The eighth object of the present invention is to provide the use of the above-mentioned biological fertilizer in inhibiting the growth of Klebsiella.

[0017] The ninth object of the present invention is to provide the application of the above-mentioned biological fertilizer in preventing and treating onion soft rot.

[0018] In one embodiment of the present invention, the onion soft rot is caused by Klebsiella oxytoca.

[0019] Beneficial effects of the present invention:

[0020] The present invention separates the rotten bulbs of onions showing soft rot, and after regrafting, it is proved that Klebsiella oxytoca can cause onion soft rot; further, the present invention screens the strain K-1 with good antagonism to Klebsiella oxytoca that can cause onion soft rot from the rhizosphere soil of onions, and comprehensively identifies the antagonistic strain K-1 as Bacillus subtilis based on the morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence identification results. The antagonistic stability test results of the antagonistic strain K-1 against Klebsiella oxytoca show that the crude extract of the antagonistic strain K-1 has better antibacterial activity after being treated in a 30°C or 50°C water bath; the crude extract of the antagonistic strain K-1 has better antibacterial activity when the environmental pH is 5 or 6; the crude extract of the antagonistic strain K-1 has better antibacterial activity after being irradiated with ultraviolet rays for 30min or 60min; the crude extract of the antagonistic strain K-1 has better antibacterial effect after being treated with anhydrous ethanol. The Bacillus subtilis K-1 obtained by the invention has good antagonistic stability and has broad application prospects in the biological control of onion soft rot. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The pictures show the symptoms of disease after strain A-2 was inoculated into various parts of the isolated onion. Figure 1 A in the figure shows the symptoms of the disease after the bulb was inoculated with sterile water. Figure 1 B in the figure shows the symptoms of the disease after the strain A-2 was grafted back into the bulb. Figure 1 C in the figure shows the symptoms of the disease after sterile water was used to graft the leaf sheath. Figure 1 D in the figure is the symptom picture of strain A-2 after grafting back into leaf sheath. Figure 1 E in the figure shows the symptoms of the disease after the bulb leaves were inoculated with sterile water. Figure 1 F in the figure is the symptom picture of the disease after the strain A-2 was grafted back into the bulb leaves;

[0022] Figure 2 This is the characteristic diagram of colony morphology of pathogen A-2;

[0023] Figure 3 This is the morphological characteristic diagram of pathogen A-2 under electron microscope;

[0024] Figure 4 This is the BLAST comparison result of pathogen A-2;

[0025] Figure 5 This is the phylogenetic tree analysis result of pathogen A-2;

[0026] Figure 6This is a graph showing the test results of the antagonistic activity of antagonistic bacteria K-1 against pathogenic bacteria A-2 that causes onion bacterial soft rot;

[0027] Figure 7 This is the colony morphology characteristic diagram of the antagonistic bacteria K-1;

[0028] Figure 8 This is the morphological characteristic diagram of the antagonistic bacteria K-1 under an electron microscope;

[0029] Fig. 9 This is the BLAST comparison result diagram of antagonistic bacteria K-1;

[0030] Fig.10 This is the phylogenetic tree analysis result of antagonistic bacteria K-1;

[0031] Fig.11 The graph is the result of the antagonistic stability test of the crude extract of antagonistic bacteria K-1 against pathogenic bacteria A-2 after being treated at different temperatures;

[0032] Fig.12 The graph is the result of the antagonistic stability test of the crude extract of antagonistic bacteria K-1 against pathogenic bacteria A-2 at different pH values;

[0033] Fig.13 The graph shows the antagonistic stability test results of the crude extract of antagonistic bacteria K-1 against pathogenic bacteria A-2 after being irradiated with ultraviolet light for different periods of time;

[0034] Fig.14 This is the result of the antagonistic stability test of the crude extract of antagonistic bacteria K-1 against pathogenic bacteria A-2 after being treated with different organic reagents. DETAILED DESCRIPTION

[0035] The present invention is further described in detail below in conjunction with specific examples and drawings. The following examples are convenient for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The drugs, reagents, and materials used in the following examples can be purchased through commercial channels unless otherwise specified.

[0036] Test strains:

[0037] The Bacillus subtilis K-1 provided by the present invention is isolated from onion rhizosphere soil with good growth condition at the Xiangyang base of Northeast Agricultural University, and is deposited in the General Microbiology Center of China Microorganism Culture Collection Committee, with a deposit number of CGMCC No.30922 and a deposit date of June 11, 2024. The address of the deposit unit is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0038] Test pathogens:

[0039] The test pathogen Klebsiella oxytoca A-2 was obtained from rotten onion bulbs in the greenhouse of Northeast Agricultural University. The bacterial suspension was prepared and then cultured and purified.

[0040] The culture medium and its formula involved in the present invention are as follows:

[0041] LB medium: 10 g of peptone, 5 g of yeast extract powder, 10 g of sodium chloride, 15-20 g of agar (liquid without agar), finally distilled water to 1000 mL, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0042] Glucose peptone medium: 5 g peptone, 5 g glucose, 2 g potassium dihydrogen phosphate, finally dilute to 1000 mL with distilled water, pH 7.0-7.2, sterilize at 121°C for 20 min.

[0043] Ammonium ferric citrate semisolid medium: 20 g peptone, 5 g sodium chloride, 0.5 g ammonium ferric citrate, 0.5 g sodium thiosulfate pentahydrate, 5-8 g agar, finally dilute to 1000 mL with distilled water, pH 7.0-7.2, sterilize at 121°C for 20 min.

[0044] Starch culture medium: 5 g beef extract, 10 g peptone, 5 g sodium chloride, 2 g soluble starch, 15-20 g agar, finally dilute to 1000 mL with distilled water, pH 7.0-7.2, sterilize at 121°C for 20 min.

[0045] Nitrate medium: 1 g of peptone, 0.5 g of sodium chloride, 0.2 g of potassium nitrate, and finally distilled water to make up to 1000 mL, pH 7.0-7.2, and sterilize at 121°C for 20 min.

[0046] Sodium citrate medium: 1g of monoammonium dihydrogen phosphate, 1g of dipotassium hydrogen phosphate, 15g of sodium chloride, 0.2g of magnesium sulfate, 2g of sodium citrate, 0.2g of magnesium sulfate, 15-20g of agar, distilled water to 1000mL, finally add 10mL of 1% bromovanillin blue ethanol solution, heat to dissolve the ingredients, pH 6.8, sterilize at 121℃ for 20min.

[0047] Peptone water culture medium: 10 g peptone, 5 g sodium chloride, and finally distilled water to 1000 mL, pH 7.0-7.2, sterilized at 121°C for 20 min.

[0048] Gelatin culture medium: 5 g sodium chloride, 10 g peptone, 3 g beef extract, 120 g gelatin. Heat to dissolve the ingredients, dilute to 1000 mL with distilled water, pH 7.2-7.4, and sterilize at 121°C for 20 min.

[0049] Malonate medium: 3 g sodium malonate, 1 g yeast extract, 2 g sodium chloride, 2 g ammonium sulfate, 0.6 g dipotassium hydrogen phosphate, 0.4 g potassium dihydrogen phosphate, 0.025 g bromovanillin blue, distilled water to 1000 mL, pH 6.8, sterilize at 121°C for 20 min.

[0050] Example 1: Application of Bacillus subtilis K-1 in the prevention and treatment of onion root rot

[0051] 1. Isolation and identification of pathogens

[0052] (1) Isolation and inoculation verification of pathogens

[0053] Rotten onion bulbs were collected from the field greenhouse of Northeast Agricultural University and prepared into bacterial suspension. Strain A-2 was obtained after culture and purification. Then the suspension was inoculated back into the isolated onion bulb leaves, onion leaf sheaths and onion bulb disks respectively. Sterile water was used as the control group. The suspension was cultured at 28℃ for 30 days, and the symptoms were observed and recorded.

[0054] The results showed that strain A-2 could cause onion rot when inoculated into the leaves, sheaths and bulbs of detached onion bulbs, and the symptoms were consistent with those of onion soft rot in the field. Figure 1 The results showed that strain A-2 was the pathogen causing onion soft rot.

[0055] (2) Identification of pathogen A-2

[0056] The pathogen A-2 was identified by morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis.

[0057] The specific method of 16S rDNA sequence analysis and identification is to obtain the genomic DNA of the strain, amplify it with universal primers 27F (SEQ ID NO.1) and 1492R (SEQ ID NO.2), and then use the BLAST program (http: / / www.ncbi.nlm.nih.gov / BLAST / ) for sequence analysis and comparison. After determining the reference strain, the phylogenetic tree was constructed using MEGA4.0 software.

[0058] SEQ ID NO.1: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0059] SEQ ID NO. 2: 5'-TACCTTGTTACGACTT-3'.

[0060] The results of morphological characterization of pathogen A-2 on LB medium showed that the surface of the colony of strain A-2 was smooth and opaque, with regular edges and a milky white or slightly yellow color (see Figure 2 ); the bacteria are rod-shaped, with a size of (0.5-0.8) μm × (1-2) μm, without flagella, and with multiple pili (see Figure 3 ).

[0061] LB medium was used for anaerobic experiment and 37°C growth experiment, glucose peptone water medium was used for methyl red experiment and VP experiment, peptone water medium was used for indole experiment, gelatin medium was used for gelatinization experiment, ammonium ferric citrate semisolid medium was used for H2S experiment, starch medium was used for starch hydrolysis experiment, nitrate medium was used for nitrate reduction experiment, citrate medium was used for citrate utilization experiment, and malonate medium was used for malonate utilization experiment. The results of physiological and biochemical characteristics identification of pathogen A-2 are shown in Table 1.

[0062] Table 1 Physiological and biochemical characteristics of pathogen A-2

[0063]

[0064]

[0065] Note: “+”, positive; “-”, negative

[0066] The genome of pathogen A-2 was extracted, and the genome was used as a template to perform PCR amplification using bacterial 16S rDNA universal primers. The PCR product was sequenced, and the resulting sequence (SEQ ID NO.3) was submitted to Genebank and compared using BLASTN software. The comparison results showed that the pathogen strain belonged to the genus Klebsiella and had a homology of more than 99% with the gene sequence of Klebsiella oxytoca (see Figure 4 ), and in the phylogenetic tree the pathogenic strains clustered together with Klebsiella oxytoca ( Figure 5 ). The pathogen A-2 was identified as Klebsiella oxytoca based on the morphological, physiological and biochemical characteristics.

[0067] SEQ ID NO.3:

[0068]

[0069] (II) Screening of antagonistic strains against pathogen A-2 causing onion bacterial soft rot

[0070] The rhizosphere soil of onions with good growth conditions at the Xiangyang Base of Northeast Agricultural University was taken, and the soil sample was separated, and the obtained strain was subjected to an activity experiment against the soft rot pathogen A-2. The specific experimental method is to prepare an LB solid plate containing pathogen A-2, use a hole puncher to punch holes in the LB culture dish (the diameter of the hole is 7.8mm), inoculate equal amounts of crude extracts of the strain to be tested in the holes, and culture at a constant temperature of 28℃ for 24h, repeat 3 times, observe whether there is an inhibition zone and measure the diameter of the inhibition zone.

[0071] The results of the test strains' antagonistic activity against the soft rot pathogen A-2 showed that the test strain K-1 had a high inhibitory effect on the pathogen A-2 that causes onion bacterial soft rot. Figure 6 The antibacterial rate was 76.67%.

[0072] (III) Identification of antagonistic bacteria K-1

[0073] The present invention conducts morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis and identification on the antagonistic bacteria K-1.

[0074] The specific method of 16S rDNA sequence analysis and identification is to obtain the genomic DNA of the strain, amplify it with universal primers 27F (SEQ ID NO.1) and 1492R (SEQ ID NO.2), and then use the BLAST program (http: / / www.ncbi.nlm.nih.gov / BLAST / ) for sequence analysis and comparison. After determining the reference strain, the phylogenetic tree was constructed using MEGA4.0 software.

[0075] The antagonistic bacteria K-1 was cultured on LB medium. The morphological characteristics of the test strain K-1 showed that the colony surface was rough and opaque, the edges were irregular, and the color was slightly yellow (see Figure 7 ); The bacteria are columnar, (0.5-1.7) μm × (1.6-4.3) μm, with multiple flagella around the periphery (see Figure 8 ).

[0076] LB medium was used for anaerobic experiment and 37°C growth experiment, glucose peptone water medium was used for methyl red experiment and VP experiment, peptone water medium was used for indole experiment, gelatin medium was used for gelatinization experiment, ammonium ferric citrate semisolid medium was used for H2S experiment, starch medium was used for starch hydrolysis experiment, nitrate medium was used for nitrate reduction experiment, citrate medium was used for citrate utilization experiment, and malonate medium was used for malonate utilization experiment. The results of morphological characteristics identification of antagonistic bacteria K-1 are shown in Table 2.

[0077] Table 2 Identification results of morphological characteristics of antagonistic bacteria K-1

[0078]

[0079]

[0080] Note: “+”, positive; “-”, negative

[0081] The genome of the antagonist K-1 was extracted, and the genome was used as a template to perform PCR amplification using bacterial 16S rDNA universal primers. The PCR product was sequenced, and the resulting sequence (SEQ ID NO.4) was submitted to Genebank and compared using BLASTN software. The comparison results showed that the antagonist K-1 belonged to the genus Bacillus and had a 100% homology with the gene sequence of Bacillus subtilis (see Fig. 9 ), and in the phylogenetic tree, the antagonist K-1 is clustered with Bacillus subtilis (see Fig.10 ). The antagonistic bacteria K-1 was identified as Bacillus subtilis based on the morphological, physiological and biochemical characteristics.

[0082] SEQ ID NO.4:

[0083]

[0084] (IV) Performance determination of Bacillus subtilis K-1

[0085] (1) Antagonistic stability test of the crude extract of antagonistic bacteria K-1 against pathogenic bacteria A-2 after treatment at different temperatures

[0086] Activate Bacillus subtilis K-1, centrifuge its bacterial solution at 4°C and 10,000 rpm for 10 minutes, and take the supernatant to obtain the crude extract of strain K-1. Activate the soft rot pathogen A-2 and prepare bacteria-containing plates. Take the crude extract of strain K-1 and treat it in a water bath for 30 minutes, and set up a control group (the crude extract of strain K-1 that has not been treated with temperature, the room temperature is about 23°C). Use a hole puncher to punch 3 holes on the plate containing pathogen A-2, and add the crude extract of strain K-1 under different water bath treatments (30, 50, 70, 90°C) into the holes respectively, and set up 3 replicates for each treatment. Culture at 28°C for 2 to 5 days, observe the inhibition zone and measure the diameter of the inhibition zone.

[0087] The results of antagonistic stability test of the crude extract of antagonistic bacteria K-1 after treatment at different temperatures are as follows Fig.11 As shown in the figure, the antibacterial activity was significantly increased at 30℃ and 50℃ compared with the control, and the antibacterial activity was significantly reduced at 70℃ and 90℃. It can be seen that the thermal stability of Bacillus subtilis K-1 can meet the application temperature environment as a biological agent or biological fertilizer.

[0088] (2) Antagonistic stability test of the crude extract of antagonistic bacteria K-1 against pathogenic bacteria A-2 at different environmental pH

[0089] Activate Bacillus subtilis K-1, centrifuge its bacterial solution at 4℃ and 10000rpm for 10min, and take the supernatant to obtain the crude extract of strain K-1. Activate the soft rot pathogen A-2, and prepare LB solid culture medium containing bacteria plates with pH 4, 5, 6, 7, 8, and 9 respectively. Use a hole puncher to punch 3 holes on the plate containing pathogen A-2, add the crude extract of strain K-1 into the holes respectively, and set 3 replicates for each treatment. Culture at 28℃ for 2-5 days, observe the inhibition zone and measure the diameter of the inhibition zone.

[0090] The results of the antagonistic stability test of the crude extract of antagonistic bacteria K-1 at different pH values ​​are as follows Fig.12As shown, the crude extract of strain K-1 at pH = 5 and pH = 6 has the best antibacterial activity against pathogen A-2, the crude extract of strain K-1 at pH = 4 and pH = 7 has a reduced antibacterial activity against pathogen A-2, and the crude extract of strain K-1 at pH = 8 and pH = 9 has a significantly reduced antibacterial activity against pathogen A-2. However, the antagonistic strain K-1 has an antibacterial effect under the treatment of pH 4.0-9.0, indicating that the antagonistic strain still has good antibacterial activity in an overly acidic or alkaline environment.

[0091] (3) Antagonistic stability test of the crude extract of antagonistic bacteria K-1 against pathogenic bacteria A-2 after being treated with ultraviolet light for different periods of time

[0092] Activate Bacillus subtilis K-1, centrifuge its bacterial solution at 4°C and 10,000 rpm for 10 minutes, and take the supernatant to obtain the crude extract of strain K-1. Activate the soft rot pathogen A-2 and prepare bacteria-containing plates. Take 10 mL of the crude extract of Bacillus subtilis K-1 and place it at a distance of 20 cm from a 30W ultraviolet lamp for different irradiation times, and set up a control group (not treated with ultraviolet light). Use a hole puncher to punch 3 holes on the plate containing pathogen A-2, and add the crude extract of strain K-1 under different ultraviolet irradiation times (30min, 60min, 90min, 120min, 150min) into the holes respectively, and set up 3 replicates for each treatment. Culture at 28°C for 2 to 5 days, observe the inhibition zone and measure the diameter of the inhibition zone.

[0093] The results of the antagonistic stability test of the crude extract of antagonistic bacteria K-1 after being treated with ultraviolet light for different times are as follows Fig.13 As shown, compared with the control group, the antibacterial activity of the crude extract of strain K-1 after ultraviolet treatment for 30min and 60min against pathogen A-2 was relatively stable, and the antibacterial activity of the crude extract of strain K-1 after ultraviolet treatment for 90min, 120min and 150min against pathogen A-2 was significantly reduced. It can be seen that the antagonistic effect of the antagonistic strain K-1 is limited by the ultraviolet irradiation time, but it still has a good antibacterial effect under long-term ultraviolet irradiation.

[0094] (4) Antagonistic stability test of the crude extract of antagonistic bacteria K-1 against pathogenic bacteria A-2 after treatment with different organic reagents

[0095] Activate Bacillus subtilis K-1, centrifuge its bacterial solution at 4°C and 10,000 rpm for 10 minutes, and take the supernatant to obtain the crude extract of strain K-1. Activate the soft rot pathogen A-2 and prepare bacteria-containing plates. Add organic solvents to the crude extract of strain K-1 in a volume ratio of 1:9, set up a control group (add an equal volume of water to the crude extract of strain K-1), and culture at 28°C for 5 hours. Use a hole puncher to punch three holes on the plate containing pathogen A-2, add the crude extract of antagonistic bacteria with different organic solvents (anhydrous ethanol, acetone, ethyl acetate, isopropanol) to the holes, and set up 3 replicates for each treatment. Culture at 28°C for 2 to 5 days, observe the inhibition zone and measure the diameter of the inhibition zone.

[0096] The results of antagonistic stability test of the crude extract of antagonistic bacteria K-1 after treatment with different organic reagents are as follows Fig.14 As shown, compared with the control group, the antibacterial activity of the crude extract of antagonistic bacteria treated with ethyl acetate, acetone and isoamyl alcohol against pathogen A-2 was significantly reduced, and the antibacterial activity of the crude extract of antagonistic bacteria treated with anhydrous ethanol against pathogen A-2 was relatively stable. The antagonistic substance had the best solubility in water and anhydrous ethanol, and was easily soluble in acetone, and slightly soluble in ethyl acetate and isoamyl alcohol. This shows that the antagonistic substance of antagonistic bacteria K-1 has good water solubility and alcohol solubility.

[0097] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the claims of the present invention.

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis ) K-1, characterized in that, The deposit number is CGMCC No.30922.

2. The Bacillus subtilis K-1 of claim 1 is effective in inhibiting Klebsiella oxytoca (which can cause onion soft rot) Klebsiella oxytoca ) applications in growth.

3. Use of the Bacillus subtilis K-1 according to claim 1 in preventing and treating onion soft rot caused by Klebsiella oxytoca.

4. A microbial agent containing the Bacillus subtilis K-1 according to claim 1.

5. Use of the microbial agent according to claim 4 in inhibiting the growth of Klebsiella oxytoca which can cause onion soft rot.

6. Use of the microbial agent according to claim 4 in preventing and treating onion soft rot caused by Klebsiella oxytoca.

7. A biological fertilizer containing the Bacillus subtilis K-1 according to claim 1.

8. Use of the biological fertilizer according to claim 7 in inhibiting the growth of acid-producing Klebsiella that can cause onion soft rot.

9. Use of the biological fertilizer according to claim 7 in preventing and treating onion soft rot caused by Klebsiella oxytoca.

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