Application of Siamese Bacillus PHBM2-Ba5 and microbial composition

Abstract

The present invention relates to the field of microbial engineering technology, and in particular to the application of Siamese Bacillus PHBM2-Ba5 and a microbial composition. Bacillus siamensis ) PHBM2‑Ba5, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 30, 2024, with the deposit number CGMCC NO.32126. The above-mentioned Siamese Bacillus PHBM2‑Ba5 and its fermentation broth are used in the preparation of products that inhibit microorganisms or / and promote plant growth. A microbial composition, comprising: the above-mentioned Siamese Bacillus PHBM2‑Ba5. The above-mentioned Siamese Bacillus PHBM2‑Ba5 and microbial composition provided in the present application can effectively inhibit the growth of plant pathogens, can be used as a broad-spectrum antibacterial biocontrol agent, and can effectively promote plant growth and development, and enhance the plant's stress resistance.

Description

Technical Field

[0001] The invention relates to the technical field of microbial engineering, in particular to application of siam bacillus PHBM2-Ba5 and a microbial composition. Background Art

[0002] Disasters caused by plant pathogens are an important reason for the decline in the yield and quality of agricultural and sideline products. The traditional method of preventing and controlling plant pathogens is mainly to spray chemical pesticides. However, a series of problems caused by the long-term use of chemical pesticides, such as pesticide residues and environmental pollution, are increasingly attracting people's attention. It is particularly urgent for us to find new and environmentally friendly prevention and control methods.

[0003] Currently, the biological pesticides on the market are mainly divided into two categories: microbial pesticides and agricultural antibiotics. Bacillus has gradually come into people's view due to its diversity of biological control. The mechanism of action of Bacillus mainly includes competition, antagonism, lysis, induced resistance, promotion of plant growth, and rhizosphere colonization.

[0004] Bacillus siamensis was first isolated from pickled crabs in Thailand and was reported as a new species in 2010. To date, there are still few reports of strains belonging to this species, and a considerable number of strains have been identified only based on 16S rRNA gene sequence homology analysis and morphological characteristics, so there is great uncertainty in the complex taxonomy of Bacillus. At present, there are few studies on Bacillus siamensis. Summary of the invention

[0005] The purpose of the present invention is to solve the shortcomings of the prior art and to propose the application of Siamese Bacillus PHBM2-Ba5 and a microbial composition.

[0006] Bacillus siamensis ( Bacillus siamensis ) PHBM2-Ba5 was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 30, 2024, with the deposit number CGMCC NO.32126.

[0007] Preferably, the 16S rRNA gene sequence of Bacillus siamese PHBM2-Ba5 is shown as SEQ ID NO.1.

[0008] A siam bacillus PHBM2-Ba5 fermentation liquid is obtained by fermenting the siam bacillus PHBM2-Ba5.

[0009] The preparation method of the fermentation broth of Siamese Bacillus PHBM2-Ba5 comprises the following steps: inoculating Siamese Bacillus PHBM2-Ba5 into NB medium, culturing at 25-28°C for 60-84h, subculturing 1-3 times, centrifuging and washing, and adjusting the system concentration to its OD 600nm to 0.8-1.1, and then inoculated into new NB medium and cultured at 25-28℃ for 60-84h.

[0010] The Siamese Bacillus PHBM2-Ba5 or the Siamese Bacillus PHBM2-Ba5 fermentation liquid is used in preparing products for inhibiting microorganisms and / or promoting plant growth.

[0011] Preferably, the inhibited microorganism is at least one of Fusarium oxysporum, Sclerotium sclerotium, and Helminthosporium.

[0012] Preferably, the function of promoting plant growth is achieved by increasing the content of soluble phosphorus, indoleacetic acid and / or iron carrier in the system.

[0013] A microbial composition comprises: the above-mentioned Bacillus siamese PHBM2-Ba5.

[0014] Preferably, the above-mentioned microbial composition further comprises: Lactobacillus plantarum ( Lactiplantibacillus plantarum )Mle-3, Acetobacter pasteurianus ( Acetobacter pasteurianus ).

[0015] Preferably, in the above microbial composition, the live bacterial concentration of Bacillus siamese PHBM2-Ba5 is 6-8×10 9 cfu / g, and the live bacterial concentration of Lactobacillus plantarum Mle-3 was 1.5-4.5×10 8 cfu / g, the concentration of live bacteria of Acetobacter pasteurianus is 8.5-9.5×10 8 cfu / g.

[0016] A microbial preparation is obtained by anaerobic fermentation of the above microbial composition.

[0017] Preferably, the anaerobic fermentation temperature is 28-37°C, and the anaerobic fermentation time is 72-144h. Beneficial effects: The present invention provides the above-mentioned Bacillus siamensis PHBM2-Ba5, which has a significant inhibitory effect on Fusarium spp. (the main pathogen of tomato wilt), Sclerotium solani (the main pathogen of white rot), and Helminthosporium (the main pathogen of corn leaf spot).

[0018] The inhibition rates of the fermentation broth of Bacillus siame PHBM2-Ba5 against Fusarium oxysporum, Sclerotium sclerotium, and Helminthosporium were 62.75%, 86.42%, and 85.33%, respectively.

[0019] At the same time, as the culture time of Siamese Bacillus PHBM2-Ba5 bacterial suspension increases, it effectively decomposes inorganic phosphorus, promotes the existence of phosphorus in the form of soluble phosphorus, and produces indoleacetic acid and iron carriers, which can enhance the growth-promoting performance of plants.

[0020] The applicant has found that the combination of Siamese Bacillus PHBM2-Ba5 with Lactobacillus plantarum Mle-3 and Acetobacter pasteurianus exhibits good synergistic behavior, and the obtained microbial composition is subjected to anaerobic fermentation, so that the obtained fermentation liquid exhibits a stronger inhibitory effect on tomato Fusarium scaber and Sclerotium sclerotiorum, and the microbial composition has better indoleacetic acid production ability and iron carrier production than the single Siamese Bacillus PHBM2-Ba5.

[0021] The above-mentioned Bacillus siamensis PHBM2-Ba5 and microbial composition provided in the present application can effectively inhibit the growth of plant pathogens, can be used as a broad-spectrum antibacterial biocontrol agent, and can effectively promote plant growth and development and enhance plant resistance. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a photo of the colony morphology of Bacillus siamese PHBM2-Ba5.

[0023] Figure 2 This is a microscopic photograph of Bacillus siamensis PHBM2-Ba5.

[0024] Figure 3 Schematic diagram of the confrontation experiment with siam Bacillus PHBM2-Ba5 fermentation broth.

[0025] Figure 4 These are photos of the antibacterial test of the fermentation broth of Bacillus siamensis PHBM2-Ba5 against Fusarium oxysporum.

[0026] Figure 5 These are photos of the antibacterial test of siam Bacillus PHBM2-Ba5 fermentation broth against Sclerotium sclerotiorum.

[0027] Figure 6 These are photos of the antibacterial test of the fermentation broth of Bacillus siamese PHBM2-Ba5 against Helminthosporium.

[0028] Figure 7 This is a photo of the starch decomposition experiment using Bacillus subtilis PHBM2-Ba5.

[0029] Figure 8These are photos of the cellulose decomposition experiment using Bacillus subtilis PHBM2-Ba5.

[0030] Fig. 9 This is the effective phosphorus capacity curve of Bacillus siamensis PHBM2-Ba5.

[0031] Fig.10 This is the indoleacetic acid production capacity curve of siam Bacillus PHBM2-Ba5.

[0032] Fig.11 This is a curve diagram of the amount of siderophore produced by Bacillus siamese PHBM2-Ba5.

[0033] Fig.12 This is a comparison chart of the inhibition rate and inhibition rate growth rate of the fermentation broth of Bacillus siame PHBM2-Ba5 and the microbial composition against tomato Fusarium oxysporum, Sclerotium sclerotium, and Helminthosporium.

[0034] Fig.13 It is a comparison curve of the indoleacetic acid production ability of Bacillus siamese PHBM2-Ba5 and the microbial composition.

[0035] Fig.14 The figure is a comparison curve of the amount of siderophore produced by Bacillus siamese PHBM2-Ba5 and the microbial composition. DETAILED DESCRIPTION

[0036] The present invention will be further explained below in conjunction with specific embodiments.

[0037] The following Lactobacillus plantarum ( Lactiplantibacillus plantarum ) The deposit number of Mle-3 is CGMCC NO.30556, which is isolated by the applicant (Hangzhou Junwu Xiyan Biotechnology Co., Ltd.).

[0038] The following Acetobacter pasteurianus ( Acetobacter pasteurianus ) with a deposit number of CGMCC NO.1.3001, purchased from the General Microbiology Center of China National Microbiological Culture Collection Administration.

[0039] The NB culture medium used below was purchased from Qingdao Hibo Biotechnology with the brand name HB0108.

[0040] The NA medium used below was obtained by adding 2% agar to the above NB medium.

[0041] The PDA culture medium used below was purchased from Qingdao Hibo Biotechnology Co., Ltd., with the brand number HB0233-12.

[0042] The inorganic phosphorus culture medium used below was purchased from Qingdao Hibo Biotechnology with the brand name HB8670-1.

[0043] The organophosphorus culture medium used below was purchased from Qingdao Hibo Biotechnology with the brand number HB8673-1.

[0044] Example 1: Separation, purification, and identification

[0045] This strain was isolated and collected by Huang Dasheng from homemade fermentation broth in Shangcheng District, Hangzhou City, Zhejiang Province, China in June 2024.

[0046] Isolation and purification of strains: From the homemade fermentation broth sample, 100 μL of the sample was taken into 900 μL of sterile water, and then the sample was diluted to 10 -4 , 10 -5 , place in a 70℃ water bath for 20 min, spread 100 μL of the liquid after the water bath on a NA plate, and place in a 28℃ incubator for 72 h.

[0047] Observe the bacterial growth on the plate. After the colonies grow, pick a typical single colony and streak it on the NA solid medium for strain isolation and purification. Inoculate the single colony into 5 mL of sterilized NB medium, incubate at 28°C for 72 hours, and then store it in a -80°C refrigerator with 50% glycerol for later use.

[0048] The isolated strains were identified by morphology (colony morphology such as Figure 1 As shown, the microscopic morphology Figure 2 as shown).

[0049] The isolated and purified single colonies were sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing identification. The primers are as follows:

[0050] 27F: 5'-AGAGTTTGATCMTGGCTCAG-3';

[0051] 1522R: 5'-AAGGAGGTGATCCAGCCGCA-3'.

[0052] Its 16s rDNA sequence:

[0053]

[0054] The BLAST program in NCBI was used to compare the sequences. The results showed that the PHBM2-Ba5 strain was Bacillus siamensis, and its homology with the known Bacillus in the GeneBank database reached 99.86%. It was named Bacillus siamensis ( Bacillus siamensis )PHBM2-Ba5.

[0055] Bacillus siameensis PHBM2-Ba5 was deposited in the General Microbiology Center of China Culture Collection Administration (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing) on ​​September 30, 2024, with the deposit number CGMCC NO.32126.

[0056] Example 2: Antibacterial effect on tomato wilt

[0057] 2.1 Prepare potato dextrose agar plates (PDA medium) in advance and let them dry.

[0058] 2.2 The siam bacillus PHBM2-Ba5 bacterial solution in the cryopreserved tube was inoculated into NB medium at a 2% inoculation rate and cultured in a 28°C incubator for 72 h. After subculturing twice, the bacteria were washed twice by centrifugation (5000 r / min 5 min) with PBS buffer, and then the strain OD was increased by PBS buffer. 600nm Adjust to 1.0, inoculate the strain into NB medium at a 2% inoculation rate, culture at 28°C for 72h, and the resulting fermentation broth is ready for use.

[0059] 2.3 If Figure 3 As shown, 4 Oxford cups (i.e. Figure 3 Then pour the PDA medium into the culture dish, wait for the medium to solidify, and take out the Oxford cup. Add 100 μL of the above-mentioned Siamese Bacillus PHBM2-Ba5 strain fermentation liquid into each well.

[0060] Finally, a tomato Fusarium wilt block of the same size was placed in the center of the solidified plate (which is the main pathogen of tomato wilt). For the blank control, only a block of Sclerotium rolfsii (i.e. Figure 3 The culture was carried out at 28°C for about 5 days, and the growth of Fusarium spp. was observed.

[0061] Inhibition rate = (natural growth diameter of tomato Fusarium spp. - growth diameter of tomato Fusarium spp. after being inhibited by siam Bacillus PHBM2-Ba5 fermentation liquid) ÷ natural growth diameter of tomato Fusarium spp. × 100%

[0062] The results show (such as Figure 4As shown in the figure), Bacillus siame PHBM2-Ba5 had a significant inhibitory effect on Fusarium oxysporum, and the inhibition rate of its fermentation liquid on Fusarium oxysporum was 62.75%.

[0063] Example 3: Antibacterial effect on white rot

[0064] 3.1 Prepare potato dextrose agar plates (PDA medium) in advance and let them dry.

[0065] 3.2 The siam bacillus PHBM2-Ba5 bacterial solution in the cryopreserved tube was inoculated into NB medium at a 2% inoculation rate and cultured in a 28°C incubator for 72 h. After subculturing twice, the bacteria were washed twice by centrifugation (5000 r / min 5 min) with PBS buffer, and then the strain OD was increased by PBS buffer. 600nm Adjust to 1.0, inoculate the strain into NB medium at a 2% inoculation rate, culture at 28°C for 72h, and the resulting fermentation broth is ready for use.

[0066] 3.3 If Figure 3 As shown, four Oxford cups were placed at the bottom of the culture dish, and then the PDA culture medium was poured into the culture dish. After the culture medium solidified, the Oxford cups were taken out. 100 μL of the fermentation liquid of the Siamese Bacillus PHBM2-Ba5 strain was added to each well.

[0067] Finally, place neat small sclerotium blocks of the same size (which are the main pathogens causing white rot) in the center of the solidified plate. For the blank control, only neat small sclerotium blocks (such as Figure 3 The culture was incubated at 28℃ for about 5 days, and the growth of Sclerotium was observed.

[0068] Inhibition rate = (natural growth diameter of Sclerotium uniformis - growth diameter of Sclerotium uniformis after being inhibited by PHBM2-Ba5 fermentation broth of Bacillus siame) ÷ natural growth diameter of Sclerotium uniformis × 100%

[0069] The results show (such as Figure 5 As shown in the figure), Bacillus siameensis PHBM2-Ba5 had a significant inhibitory effect on Sclerotium oxysporum, and the inhibition rate of its fermentation broth on Sclerotium oxysporum reached 86.42%.

[0070] Example 4: Antibacterial effect on corn leaf spot

[0071] Similarly, referring to the confrontation experimental methods in Examples 5 and 6, an antibacterial experiment was conducted on Helminthosporium (which is the main pathogen causing corn leaf blight).

[0072] The results show (such as Figure 6As shown in the figure), Bacillus siamese PHBM2-Ba5 had a significant inhibitory effect on Helminthosporium, and the inhibition rate of its fermentation broth on Helminthosporium was 85.33%.

[0073] Example 5: Determination of the ability to decompose starch and cellulose

[0074] 5.1 Inoculate siam Bacillus PHBM2-Ba5 on starch screening medium (3 replicates) using the spot inoculation method and culture in a 28°C incubator for 5 days. Then add dilute iodine solution to the medium to observe whether hydrolysis circles are generated.

[0075] The composition of starch screening medium is as follows: soluble starch 10g, peptone 2g, yeast extract 5g, NaCl 5g, K2HPO4 3g, agar 20g, add water to make up to 1000mL.

[0076] like Figure 7 As shown, Bacillus siamensis PHBM2-Ba5 has the ability to decompose starch.

[0077] 5.2 Inoculate siam Bacillus PHBM2-Ba5 on cellulose screening medium (3 replicates) by spot seeding method and culture at 30℃ for 5 days. Pour 1mg / mL Congo red solution into the culture dish for staining for 30 minutes, pour out the Congo red solution, soak in 1mol / L NaCl solution for 15 minutes for decolorization, observe whether there is a hydrolysis circle and measure the diameter of the hydrolysis circle.

[0078] The composition of the cellulose screening medium is as follows: sodium carboxymethyl cellulose 10.0 g, (NH4)2SO4 1.0 g, MgSO4•7H2O0.5 g, KH2PO4 1.0 g, NaCl 0.3 g, agar powder 20.0 g, add water to make up to 1000 mL.

[0079] like Figure 8 As shown, Bacillus siameensis PHBM2-Ba5 has the ability to decompose cellulose, and its hydrolysis zone diameter is 2.9±0.1 cm.

[0080] Bacillus can degrade polysaccharides in fungal cell walls by secreting enzymes (such as cellulase and amylase), destroy the structure of pathogens, degrade fungal hyphae, inhibit the growth of pathogens, and ultimately lead to the death of pathogens.

[0081] Example 6: Determination of Available Phosphorus Capacity

[0082] The OD of the siam Bacillus PHBM2-Ba5 bacterial suspension was 600nmAdjust to 1.0, and then inoculate into 50mL centrifuge tubes containing 30mL of organic phosphorus liquid culture medium and inorganic phosphorus liquid culture medium at an inoculum size of 2%, respectively. The organic phosphorus liquid culture medium and inorganic phosphorus liquid culture medium without inoculation of strains were used as blank controls (CK).

[0083] The cells were cultured at 28°C with shaking at 200 rpm for 5 days, and samples were taken at corresponding time points (24 h, 48 h, 72 h, 96 h, and 120 h). 5 mL of the sample was collected each time and centrifuged at 4°C at 10,000 r / min for 5 min. The supernatant was stored at 4°C (repeated 3 times).

[0084] The molybdenum antimony colorimetric method was used to determine the soluble phosphorus content in the supernatant at 24h, 48h, 72h, 96h and 120h.

[0085] like Fig. 9 As shown, Siamese Bacillus PHBM2-Ba5 effectively decomposes inorganic phosphorus and promotes phosphorus to exist in the form of soluble phosphorus, which is convenient for plant absorption and promotes plant growth.

[0086] Example 7: Determination of the ability to produce indoleacetic acid (IAA)

[0087] The OD of the siam Bacillus PHBM2-Ba5 bacterial suspension was 600nm Adjust to 1.0, then inoculate 2% of the inoculum into a 10mL test tube containing 5mL NB liquid medium (containing 100mg / L L-tryptophan), and place in a shaker at 28℃ 200rpm for 5 days (3 replicates). Take 500μL of the bacterial solution cultured at different times (24h, 48h, 72h, 96h, 120h) into a 1.5mL EP tube, centrifuge at 12000rpm for 5min, and keep the supernatant for later use.

[0088] Take 50 μL of supernatant in a 96-well plate and add 50 μL of Salkowski colorimetric solution (mixture of solution A and solution B). After the 96-well plate is placed in the dark at room temperature for 30 minutes, the OD is measured using an enzyme-labeled instrument. 530nm The IAA concentration was calculated from the absorbance value.

[0089] like Fig.10 As shown, Bacillus siameensis PHBM2-Ba5 produces indoleacetic acid. As time goes by, the concentration of indoleacetic acid shows an overall upward trend, which can promote the growth and development of the plant.

[0090] Example 8: Determination of the amount of siderophore produced

[0091] The OD of the siam Bacillus PHBM2-Ba5 bacterial suspension was 600nmAdjust to 1.0, then inoculate 2% of the inoculum into a 10mL test tube containing 5mL of NB liquid medium, and place it in a shaker at 28℃ and 200rpm for 5 days (3 replicates). Take 500μL of the bacterial solution cultured at different times (24h, 48h, 72h, 96h, 120h) into a 1.5mL EP tube, centrifuge at 12000rpm for 5min, and keep the supernatant for later use.

[0092] Take 100 μL of supernatant in a 96-well plate, add an equal volume of CAS detection solution and mix evenly, let it stand for 1 hour, and then use a microplate reader to measure the absorbance at 630 nm (denoted as As). Take another equal volume of uninoculated sterilized NB culture medium and mix it with an equal volume of CAS detection solution, and measure the absorbance at 630 nm (denoted as Ar) as the reference value; then calculate the amount of iron carrier produced according to the following formula (if the calculated result is less than 10%, it is considered that the iron carrier secretion is negative, otherwise it is considered that the iron carrier secretion is positive).

[0093] Amount of iron carrier produced = (Ar-As) ÷ Ar × 100%

[0094] like Fig.11 As shown in the figure, the amount of siderophore produced by Siamese Bacillus PHBM2-Ba5 is basically greater than 10%. It is believed that the siderophore secretion of Siamese Bacillus PHBM2-Ba5 strain is positive, which can provide the required iron elements for plants, promote chlorophyll synthesis and photosynthesis, and enhance the growth-promoting performance of plants. Similarly, Siamese Bacillus PHBM2-Ba5 can produce siderophores, which will force other strains to be unable to efficiently absorb key nutritional resources, thereby forming a competitive advantage over other strains, resulting in the containment of soil-borne pathogens in the plant growth system.

[0095] Example 9: Microbial composition fermentation broth

[0096] The Siamese Bacillus PHBM2-Ba5 bacterial solution, the plant lactobacillus Mle-3 bacterial solution, and the Acetobacter pasteurianus solution were mixed. The live bacterial concentration of Siamese Bacillus PHBM2-Ba5 in the system was 7×10 9 cfu / g, and the live bacterial concentration of Lactobacillus plantarum Mle-3 was 3×10 8 cfu / g, the concentration of live bacteria of Acetobacter pasteurianus is 9×10 8 cfu / g.

[0097] The mixed bacterial liquid was inoculated into a 5L small anaerobic fermentation tank at a 2% inoculation rate. The temperature in the fermentation tank was adjusted to 37±1°C. The fermentation was cultured for 72 hours. Fresh culture medium was added. Then the temperature in the fermentation tank was adjusted to 28±1°C. The fermentation was cultured for 72 hours. The obtained fermentation liquid was used for later use.

[0098] Example 10: Antibacterial effect of the fermentation liquid of the microbial composition on tomato wilt, white rot and corn leaf spot

[0099] Referring to the confrontation experimental methods in Example 2 and Example 3, antibacterial experiments were carried out on tomato Fusarium scabra, Sclerotium uniformis, and Helminthosporium.

[0100] Antibacterial rate growth rate = (antibacterial rate of microbial composition fermentation liquid - antibacterial rate of Siamese Bacillus PHBM2-Ba5 fermentation liquid) ÷ antibacterial rate of Siamese Bacillus PHBM2-Ba5 fermentation liquid × 100%

[0101] like Fig.12 As shown in the figure, the microbial composition has a significant inhibitory effect on tomato Fusarium scabra, Sclerotium sclerotium, and Helminthosporium. However, compared with Bacillus siamensis PHBM2-Ba5, the microbial composition showed a stronger antibacterial effect on tomato Fusarium scabra and Sclerotium sclerotium, and the growth rates of the antibacterial rates produced by the fermentation broth were 16.67% and 10.99%, respectively.

[0102] Example 11: Determination of the ability of microbial composition to produce indoleacetic acid (IAA)

[0103] The OD of the mixed bacterial solution obtained in Example 9 was 600nm Adjust to 1.0, then inoculate 2% of the inoculum into a 10 mL test tube containing 5 mL of NB liquid medium (containing 100 mg / L of L-tryptophan), and place in a shaker at 28°C and 200 rpm for 5 days (3 replicates). Take 500 μL of the bacterial solution cultured at different times (24h, 48h, 72h, 96h, 120h) into a 1.5 mL EP tube, centrifuge at 12000 rpm for 5 minutes, and keep the supernatant for later use.

[0104] Take 50 μL of supernatant in a 96-well plate and add 50 μL of Salkowski colorimetric solution (mixture of solution A and solution B). After the 96-well plate is placed in the dark at room temperature for 30 minutes, the OD is measured using an enzyme-labeled instrument. 530nm The IAA concentration was calculated from the absorbance value.

[0105] like Fig.13 As shown, the microbial composition can also produce indoleacetic acid, and the ability of the microbial composition to produce indoleacetic acid is better than that of Bacillus siamese PHBM2-Ba5 alone.

[0106] Example 12: Determination of the amount of siderophore produced by a microbial composition

[0107] The OD of the mixed bacterial solution obtained in Example 9 was 600nmAdjust to 1.0, then inoculate 2% of the inoculum into a 10mL test tube containing 5mL of NB liquid medium, and place it in a shaker at 28℃ and 200rpm for 5 days (3 replicates). Take 500μL of the bacterial solution cultured at different times (24h, 48h, 72h, 96h, 120h) into a 1.5mL EP tube, centrifuge at 12000rpm for 5min, and keep the supernatant for later use.

[0108] Take 100 μL of supernatant in a 96-well plate, add an equal volume of CAS detection solution and mix evenly, let it stand for 1 hour, and then use a microplate reader to measure the absorbance at 630 nm (denoted as As). Take another equal volume of uninoculated sterile NB medium and mix it with an equal volume of CAS detection solution, and measure the absorbance at 630 nm (denoted as Ar) as the reference value.

[0109] like Fig.14 As shown, the amount of siderophore produced by the microbial combination was greater than 10%, and the amount of siderophore produced was higher than that of Bacillus siamese PHBM2-Ba5 alone.

[0110] In summary, the microbial composition has significant ability to produce indoleacetic acid and the amount of iron carriers produced, and can effectively promote the growth and development of plants and improve the stress resistance of plants.

[0111] The above description is only a preferred specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Any technician familiar with the technical field can make equivalent replacements or changes according to the technical scheme and inventive concept of the present invention within the technical scope disclosed by the present invention, which should be covered by the protection scope of the present invention.

Claims

1. Bacillus siamensis ( Bacillus siamensis ) PHBM2-Ba5, characterized in that, It was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on September 30, 2024, with the deposit number CGMCC NO.32126.

2. A siam Bacillus PHBM2-Ba5 fermentation broth, characterized in that: Obtained by fermentation of the Siamese Bacillus PHBM2-Ba5 described in claim 1.

3. A method for preparing the fermentation liquid of Bacillus siamensis PHBM2-Ba5 as claimed in claim 2, characterized in that: The method comprises the following steps: inoculating Siamese Bacillus PHBM2-Ba5 in NB medium, culturing at 25-28°C for 60-84 hours, subculturing 1-3 times, washing by centrifugation, and adjusting the concentration of the system to its OD 600nm to 0.8-1.1, and then inoculated into new NB medium and cultured at 25-28℃ for 60-84h.

4. Use of the Siamese Bacillus PHBM2-Ba5 according to claim 1 or the fermentation liquid of Siamese Bacillus PHBM2-Ba5 according to claim 2 in the preparation of products for inhibiting microorganisms or / and promoting plant growth; The microorganism is at least one of tomato Fusarium scaber, Sclerotium sclerotium, and Helminthosporium.

5. The use according to claim 4, characterized in that: The function of promoting plant growth is achieved by increasing the content of soluble phosphorus, indoleacetic acid and / or iron carrier in the system.

6. A microbial composition, characterized in that include: The siam Bacillus PHBM2-Ba5 as claimed in claim 1.

7. A microbial preparation, characterized in that: Obtained by anaerobic fermentation of the microbial composition according to claim 6.

Images