Bacillus velezensis MLY204 and microbial preparation and application thereof

Abstract

The invention discloses bacillus velezensis MLY204 as well as a microbial preparation and application of the bacillus velezensis MLY204. The bacillus velezensis MLY204 is a wild type obtained by soil separation. The bacillus velezensis MLY204 strain has bacteriostatic activity on plant pathogens including ralstonia solanacearum, alternaria alternata and fusarium. The invention further provides a microbial preparation of the bacillus velezensis MLY204 and a preparation method of the microbial preparation. The supernate of the microbial preparation also has a strong bacteriostatic effect on ralstonia solanacearum. The microbial preparation of the bacillus velezensis MLY204 provided by the invention has the advantages of high efficiency, safety and environmental protection, and has a wide application prospect in green agriculture.

Description

Technical Field

[0001] This invention relates to the field of agricultural biotechnology, and in particular to a strain of Bacillus belye MLY204 and its microbial preparations and applications. Background Technology

[0002] Bacillus belesiensis ( Bacillus velezensis First isolated from the Velez River in the province of Malaga, Spain in 1999, *Bacillus velecithus* was not reported and named until 2005. Widely distributed in natural environments such as soil, plant rhizosphere, and oceans, and even found in animal intestines, *Bacillus velecithus* is an aerobic or facultative anaerobic Gram-positive bacterium. It exhibits strong resistance to high temperatures, acid and alkali stresses, and salt stresses, growing within a range of 10-45 °C and pH 5.0-10.0. Furthermore, it can form heat-resistant and stress-resistant spores, facilitating its colonization in complex ecological environments.

[0003] Studies have shown that *Bacillus belye* possesses broad-spectrum antibacterial and growth-promoting abilities, making it a biocontrol microorganism with significant application potential. Reported studies have shown that *Bacillus belye* is effective against *Fusarium oxysporum* and *Lycium barbarum* root rot pathogens. Fusarium solani ), bacterial wilt ( Ralstonia solanacearum Banana anthracnose bacteria ( Colletotrichum musae ), scabies bacteria ( Streptomyces acidiscabies Late blight pathogen ( Phytophthora infestans ), black nevus bacteria ( Phyllachora spp. ), early blight pathogen ( Alternaria alternata Bacillus belyceta has shown good inhibitory effects against pathogens such as rice blast fungus, tomato gray mold fungus, watermelon wilt fungus, and rice bacterial blight fungus. The excellent antibacterial effect of Bacillus belyceta against a variety of pathogens makes it a promising candidate for application in green agriculture.

[0004] Bacillus species are known for producing a variety of metabolites with antibacterial activity. *Bacillus belye*, as a novel strain, has shown great potential. Studies have shown that altering the culture medium (carbon / nitrogen source, trace elements) can activate or enhance different secondary metabolic pathways. The strain described in this invention is a wild-type strain isolated from soil, exhibiting broad-spectrum antibacterial activity and inhibitory effects against a variety of plant pathogens. Summary of the Invention

[0005] Objective: In order to overcome the shortcomings of the existing technology, the present invention provides a strain of Bacillus vesiculus MLY204 and its microbial preparation and application, wherein Bacillus vesiculus MLY204 has antibacterial activity against Ralstonia solanacearum strain, Alternaria alternata and Fusarium wilt.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0007] In a first aspect, the present invention provides a strain of Bacillus belyceae MLY204, wherein the taxonomic name of Bacillus belyceae MLY204 is... Bacillus velezensis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36290.

[0008] The Bacillus berreatus MLY204 strain grows rapidly during fermentation and quickly enters the logarithmic growth phase; however, in poor culture medium, the time to enter the logarithmic growth phase is prolonged.

[0009] Secondly, the present invention provides a microbial preparation of Bacillus vesiculus MLY204, comprising Bacillus vesiculus MLY204 fermentation broth, a stabilizer, and a synergist.

[0010] Furthermore, the preparation method of the Bacillus vesiculus MLY204 fermentation broth includes: picking a single colony of Bacillus vesiculus MLY204 and inoculating it into 10-500 mL of sterile fermentation medium, and culturing it at 20-40 ℃ and 100-250 rpm / min for 1-12 h.

[0011] Furthermore, the stabilizers added to each liter of the Bacillus berberis MLY204 fermentation broth include: 10-130 g of glycerol, 0.1-10 mL of 2-5 M sodium hydroxide solution, 1-10 mL of 1-5 M Tris Base solution, 1-20 g of xanthan gum, 1-100 g of sucrose, and 10-120 g of dipropylene glycol.

[0012] Furthermore, the synergistic agents added to each liter of the Bacillus vesiculosus MLY204 fermentation broth include: 1-100 μg of Ralstonia solanacearum lipopolysaccharide and 1-100 μg of Fusarium vomitoxin.

[0013] In some embodiments, the microbial preparation is an aqueous solution.

[0014] Thirdly, the present invention provides a method for preparing a microbial preparation of Bacillus belyssus MLY204, comprising: Select a single colony of Bacillus belye MLY204 and inoculate it into 10-500 mL of sterile fermentation medium. Incubate at 20-40 ℃ and 100-250 rpm / min for 1-12 h. After expanding the culture by inoculation at a rate of 0.1-10 v / v% for 12-24 h, add a synergist, continue culturing for another 1-12 h, and then add a stabilizer to obtain the final product.

[0015] The microbial preparations prepared by this method are rapidly formed in a single step, making them easy to apply in agricultural fields.

[0016] In some embodiments, each liter of the aseptic fermentation medium comprises: 1-10 g of disodium hydrogen phosphate dodecahydrate, 0.1-10 g of potassium dihydrogen phosphate, 0.1-10 g of sodium chloride, 0.1-10 g of ammonium chloride, 0.1-10 g of magnesium sulfate, 0.1-1 g of L-aspartic acid, 0.1-1 g of L-glutamic acid, 0.1-1 g of L-leucine, 0.1-1 g of L-tyrosine, 0.1-1 g of L-alanine, 0.1-1 g of L-valine, 1-10 g of Tween-80, 1-20 g of glucose monohydrate, 0.1-10 g of calcium chloride, 1-10 g of vermiculite, 0.1-10 g of dodecayl alcohol, 5-200 mg of boric acid, 0.1-15 g of zein, 1-10 g of beef extract, 0.1-15 g of peptone, and 10-100 mg of zinc chloride.

[0017] Fourthly, the application of Bacillus vesicularis MLY204 or its microbial preparations in the preparation of products that inhibit the activity of Ralstonia solanacearum strains, Alternaria alternata, and Fusarium.

[0018] The Bacillus berreatus MLY204 strain exhibits antibacterial activity against Ralstonia solanacearum, Alternaria alternata, and Fusarium moniliforme, with an effective inhibition distance of 0.5-4.5 cm on agar plates.

[0019] In some embodiments, the product further includes a supernatant of a microbial preparation of Bacillus belyssus MLY204.

[0020] Here, "supernatant" refers to the liquid after filtering and removing cells from the fermentation broth of Bacillus belysium MLY204. Its components are metabolic products produced during the fermentation of Bacillus belysium MLY204, including water-soluble and lipid-soluble substances.

[0021] Beneficial Effects: The *Bacillus vesiculus* MLY204 provided by this invention is a slow-growing wild-type strain that can utilize various nutrients. Therefore, a small amount of nutrients is sufficient to maintain its inhibitory effect on plant pathogens for a relatively long period, showing broad application prospects for subsequent formulation development and biocontrol. Furthermore, *Bacillus vesiculus* MLY204 exhibits non-cellular contact antibacterial activity against *Ralstonia solanacearum*, *Alternaria alternata*, and *Fusarium*, demonstrating good application value in plant protection, food preservation, and feed mold prevention. Attached Figure Description

[0022] Figure 1 This is a genomic circle diagram of Bacillus belyssus MLY204 in an embodiment of the present invention.

[0023] Figure 2In this embodiment of the invention, Bacillus belye MLY204 was cultured in a rich culture medium (e.g., Figure 2 (as shown in A and B) and barren culture medium (such as...) Figure 2 The growth curve is shown in Figure C.

[0024] Figure 3 This invention aims to screen Bacillus strains and assess their antagonistic effects against Ralstonia solanacearum on NA plates. Figure 3 In Figure A, the antagonistic relationship between Bacillus strain MLY201 and Ralstonia solanacearum strain MLY102 after 7 days of growth on NA plates is shown. Figure 3 In Figure B, the antagonistic relationship between Bacillus strain MLY202 and Ralstonia solanacearum strain MLY102 after 7 days of growth on NA plates is observed. Figure 3 In the middle C, the antagonistic relationship between Bacillus MLY203 and Ralstonia solanacearum MLY102 strains after 7 days of growth on NA plates is shown. Figure 3 D represents the antagonistic relationship between Bacillus MLY204 and Ralstonia solanacearum MLY102 strains after 7 days of growth on NA plates; Figure 3 E represents the antagonistic relationship between Bacillus strain MLY205 and Ralstonia solanacearum strain MLY102 after 7 days of growth on NA plates.

[0025] Figure 4 This invention illustrates the antagonistic effect of Bacillus belyss MLY204 strain against Alternaria alternata strain in an embodiment of the present invention. Figure 4 In Figure A, the Alternaria species was grown on a PDY plate for 7 days. Figure 4 In Figure B, the antagonistic relationship between Bacillus belyssus MLY204 and Alternaria alternata strain after 7 days of growth on PDY is shown.

[0026] Figure 5 This invention illustrates the antagonistic effect of Bacillus belyssus strain MLY204 against Fusarium in an embodiment of the present invention. Figure 5 A1 is Fusarium oxysporum ( Fusarium oxysporum The growth of strain MLY127 on PDY plates for 7 days; Figure 5 A2 is Fusarium solani, the pathogen of solanaceous diseases ( Fusarium solani The growth of strain MLY128 on PDY plates for 7 days; Figure 5 B1 consists of Bacillus belyssus MLY204 and Fusarium oxysporum ( Fusarium oxysporum Antagonistic activity of strain MLY127 after 7 days of growth on PDY plates; Figure 5 B2 consists of Bacillus belyssus MLY204 and Fusarium solani (… Fusarium solani The growth of strain MLY128 on PDY plates for 7 days.

[0027] Figure 6This invention illustrates the antagonistic effect of the supernatant of the microbial preparation of Bacillus vesicularis MLY204 on Ralstonia solanacearum in this embodiment of the invention. Figure 6 Figure A shows the antagonistic effect of sterile liquid culture medium on Ralstonia solanacearum strain MLY102; Figure 6 B and C represent the antagonistic effect of the supernatant on Ralstonia solanacearum strain MLY102; Figure 6 D represents the antagonistic effect of DMSO on Ralstonia solanacearum strain MLY102; Figure 6 E represents the antagonistic effect of lipid-soluble substances in the supernatant on Ralstonia solanacearum strain MLY102.

[0028] Figure 7 The graph shows the test results of viable bacteria count and spore rate in the fermentation broth of a microbial preparation of Bacillus vesiculosus MLY204. Detailed Implementation

[0029] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. The following description of at least one exemplary embodiment is merely illustrative and is in no way intended to limit the present invention or its application or use.

[0030] Unless otherwise specifically stated, the relative arrangement, numerical expressions, and values ​​of the components and steps set forth in these embodiments do not limit the scope of the invention. It should also be understood that, for ease of description, the dimensions of the various parts shown in the drawings are not drawn to actual scale. Techniques, methods, and devices known to those skilled in the art may not be discussed in detail, but where appropriate, such techniques, methods, and devices should be considered part of the specification. In all examples shown and discussed herein, any specific values ​​should be interpreted as merely exemplary and not as limitations. Therefore, other examples of exemplary embodiments may include different values. It should be noted that similar reference numerals and letters in the following drawings denote similar items; therefore, once an item is defined in one drawing, it need not be further discussed in subsequent drawings.

[0031] The present invention will be further described below with reference to the embodiments.

[0032] The materials used in the following examples were obtained from the following sources: Ralstonia solanacearum strain MLY102 was isolated from diseased tobacco plants in Tianzhu County, Guizhou Province; Fusarium oxysporum strain MLY127 and Fusarium solani strain MLY128 were isolated from diseased tobacco plants in Bijie City, Guizhou Province; Bacillus amyloliquefaciens strains MLY201, MLY202, MLY203, MLY204, and MLY205 were isolated from diseased soil samples from flue-cured tobacco plants in Qianxinan Prefecture; and Fusarium vomitoxin was purchased from MedChemExpress.

[0033] Example 1: Antagonistic effect of Bacillus isolates

[0034] Five wild-type Bacillus strains (MLY201, MLY202, MLY203, MLY204, and MLY205) isolated from diseased soil samples from flue-cured tobacco plantations in Qianxinan Prefecture were cultured on NA agar medium for 48 h. After collecting the bacterial cells, they were evenly dispersed in 0.1% Tween-80 aqueous solution and diluted to OD200 with 0.9% sodium chloride. 600 =0.1. Ralstonia solanacearum strain MLY102 was plated on a new NA plate, and 2.5 μL of Bacillus dilution was added to a sterile filter paper disc. The plate was incubated for 7 days to test the antagonistic effect.

[0035] like Figure 3 As shown, strain MLY204 exhibits the best antagonistic effect.

[0036] 16S rDNA sequencing of strain MLY204 was performed, and the results are shown in SEQ ID NO.1. Figure 1 Sequence comparison revealed that strain MLY204 had a 99.93% similarity to the 16S rDNA sequence of Bacillus belysinus in GenBank. Therefore, this example screened and obtained Bacillus belysinus MLY204, which has a strong antagonistic effect against Ralstonia solanacearum strain MLY102.

[0037] SEQ ID NO.1

[0038] The described Bacillus velezensis MLY204 is deposited at the China General Microbiological Culture Collection Center (CGMCC) under accession number CGMCC No. 36290. Deposit date: October 22, 2025. Classification: Bacillus velezensis.

[0039] In this embodiment, 16S rDNA sequencing was also performed on strains MLY201, MLY202, MLY203, MLY205, and MLY102, and the results are as follows:

[0040] Bacillus amyloliquefaciens strain MLY201, taxonomic name: Bacillus amyloliquefaciens, Latin name: Bacillus amyloliquefaciens MLY201.

[0041] Bacillus amyloliquefaciens strain MLY202, taxonomic name: Bacillus amyloliquefaciens, Latin name: Bacillus amyloliquefaciens MLY202.

[0042] Bacillus amyloliquefaciens strain MLY203, taxonomic name: Bacillus amyloliquefaciens, Latin name: Bacillus amyloliquefaciens MLY203.

[0043] Bacillus belysium strain MLY205, taxonomic name: Bacillus belysium, Latin name: Bacillus velezensis MLY205.

[0044] Example 2: Growth of Bacillus belyss MLY204

[0045] In LB broth medium, under conditions of 30 ℃, 20% loading, and shaking at 200 rpm / min, as follows... Figure 2 As shown in Figure A, the time required for Bacillus belye MLY204 to double in the mid-logarithmic growth phase was measured to be 1 h.

[0046] In PDA broth medium, under conditions of 30 ℃, 20% loading, and shaking at 200 rpm / min, as follows... Figure 2 As shown in Figure B, the time required for Bacillus belyss MLY204 to double in the mid-logarithmic growth phase was 2.2 h.

[0047] In M9 liquid medium, under conditions of 30 ℃, 20% loading, and shaking at 200 rpm / min, as follows: Figure 2As shown in Figure C, the time required for Bacillus belyss MLY204 to double in the mid-logarithmic growth phase was measured to be 11 h.

[0048] This indicates that *Bacillus belyssiensis* MLY204 is a slow-growing wild-type strain, and its time to enter the logarithmic growth phase is prolonged in poor culture media. Therefore, supplying a small amount of nutrients is sufficient to maintain the inhibitory effect of *Bacillus belyssiensis* MLY204 on plant pathogens for a relatively long period, showing broad application prospects for the subsequent development of formulations and biocontrol agents.

[0049] Example 3:

[0050] Wild-type Bacillus berreatus strain MLY204, isolated from diseased soil samples from flue-cured tobacco plantations in Qianxinan Prefecture, was cultured on NA agar medium for 48 h. After collecting the bacterial cells, they were evenly dispersed in 0.1% Tween-80 aqueous solution and diluted to OD200 with 0.9% sodium chloride. 600 =0.1. The diluted bacterial suspension was transferred to a PDY plate inoculated with Alternaria MLY62 strain on one side. 2.5 μL of Bacillus dilution was added to a sterile filter paper disc, and the antagonistic effect was tested for 7 days. (The remaining text appears to be unrelated and possibly machine-translated gibberish.) Figure 4 As shown, the Bacillus belyss MLY204 strain achieved an inhibition rate of 65.67% against Alternaria alternata MLY62 strain.

[0051] Example 4:

[0052] Wild-type Bacillus berreatus strain MLY204, isolated from diseased soil samples from flue-cured tobacco plantations in Qianxinan Prefecture, was cultured on NA agar medium for 48 h. After collecting the bacterial cells, they were evenly dispersed in 0.1% Tween-80 aqueous solution and diluted to OD200 with 0.9% sodium chloride. 600 =0.1. The diluted bacterial suspension was transferred to a PDY plate inoculated on one side with *Fusarium oxysporum* strain MLY127 and *Fusarium solani* strain MLY128. 2.5 μl of Bacillus dilution was added to a sterile filter paper disc, and the antagonistic effect was tested after 7 days. (Example: ...) Figure 5 As shown, when Bacillus belyss MLY204 was cultured on PDY plates for 7 days, it showed an inhibition rate of 32.90% against Fusarium oxysporum strain MLY127 and an inhibition rate of 41.67% against Fusarium solani strain MLY128.

[0053] Example 5:

[0054] This embodiment provides a microbial preparation of Bacillus belyssus MLY204, the preparation method of which includes: Bacillus berberis strain MLY204 was inoculated at an inoculum rate of 1% into sterile fermentation medium. The medium formula (1L) was: 1 g disodium hydrogen phosphate dodecahydrate, 10 g potassium dihydrogen phosphate, 0.1 g sodium chloride, 5 g ammonium chloride, 0.1 g magnesium sulfate, 1 g L-aspartic acid, 1 g L-glutamic acid, 0.1 g L-leucine, 0.1 g L-tyrosine, 0.1 g L-alanine, 0.1 g L-valine, 1 g Tween-80, 20 g glucose monohydrate, 0.1 g calcium chloride, 10 g vermiculite, 0.1 g dodecayl alcohol, 5 mg boric acid, 15 g zein, 10 g beef extract, 15 g peptone, and 10 mg zinc chloride. The culture temperature was 20℃, the loading was 20%, and the shaking was carried out at 250 rpm / min. After 24 h of culture, 1 μg of Ralstonia solanacearum lipopolysaccharide and 1 μg of Fusarium vomitoxin were added. After culturing for another 12 h, add 130 g of glycerol, 10 mL of 2 M sodium hydroxide solution, 10 mL of 1 M Tris Base solution, 20 g of xanthan gum, 1 g of sucrose, and 10 g of dipropylene glycol.

[0055] The preparation method of Ralstonia solanacearum lipopolysaccharide includes: selecting a single colony of freshly grown Ralstonia solanacearum strain MLY102, inoculating it in NB liquid medium, and culturing it at 30 ℃ and 200 rpm / min for 48 h. Centrifuging at 10000 rpm for 10 minutes at room temperature and collecting the precipitate. Adding 5 times the weight of sterile water to the precipitate, repeatedly freezing and thawing 5 times, and then sonicating it; adding an equal volume of 90% phenol, stirring vigorously in a 72 ℃ water bath for 30 min, and then centrifuging at 3000 rpm for 30 min at 4 ℃. Collecting the upper aqueous phase, dialyzing with running water for 48 h, dialyzing with deionized water for 12 h, concentrating with 50% polyethylene glycol 2000, and centrifuging to collect the precipitate to obtain Ralstonia solanacearum lipopolysaccharide.

[0056] At this point, the bacterial count of the Bacillus vesiculosus MLY204 preparation reached 1.1 × 10⁻⁶. 8 cfu / mL, with virtually no bacterial precipitate at the bottom after standing.

[0057] Example 6:

[0058] This embodiment provides a microbial preparation of Bacillus belyssus MLY204, the preparation method of which includes: Bacillus berberis strain MLY204 was inoculated at an inoculum rate of 1% into sterile fermentation medium. The medium formula (1L) was: 10 g disodium hydrogen phosphate dodecahydrate, 0.1 g potassium dihydrogen phosphate, 10 g sodium chloride, 10 g ammonium chloride, 10 g magnesium sulfate, 0.1 g L-aspartic acid, 0.1 g L-glutamic acid, 1 g L-leucine, 1 g L-tyrosine, 1 g L-alanine, 1 g L-valine, 10 g Tween-80, 1 g glucose monohydrate, 10 g calcium chloride, 1 g vermiculite, 10 g dodecayl alcohol, 200 mg boric acid, 0.1 g zein, 1 g beef extract, 0.1 g peptone, and 100 mg zinc chloride. The culture temperature was 35 ℃, the loading was 20%, and the mixture was shaken at 100 rpm / min. After culturing for 12 h, 100 μg of Ralstonia solanacearum lipopolysaccharide and 100 μg of Fusarium vomitoxin were added. The preparation method of Ralstonia solanacearum lipopolysaccharide was the same as in Example 5. After culturing for another 1 h, add 10 g of glycerol, 0.1 mL of 5 M sodium hydroxide solution, 1 mL of 5 M Tris Base solution, 1 g of xanthan gum, 100 g of sucrose, and 120 g of dipropylene glycol.

[0059] At this point, the bacterial count of Bacillus belyssus MLY204 preparation reached 0.6 × 10⁻⁶. 8 The concentration was cfu / mL, and after standing, a large amount of bacterial cells precipitated at the bottom.

[0060] Example 7:

[0061] This embodiment provides a microbial preparation of Bacillus belyssus MLY204, the preparation method of which includes: Bacillus berberis strain MLY204 was inoculated into sterile fermentation medium at an inoculation rate of 10%. The medium formula (1L) was as follows: 10 g disodium hydrogen phosphate dodecahydrate, 0.1 g potassium dihydrogen phosphate, 5 g sodium chloride, 0.1 g ammonium chloride, 5 g magnesium sulfate, 0.1 g L-aspartic acid, 1 g L-glutamic acid, 0.5 g L-leucine, 0.3 g L-tyrosine, 0.5 g L-alanine, 0.2 g L-valine, 5 g Tween-80, 10 g glucose monohydrate, 0.1 g calcium chloride, 3 g vermiculite, 3 g dodecayl alcohol, 50 mg boric acid, 1 g zein, 10 g beef extract, 15 g peptone, and 20 mg zinc chloride. The culture temperature was 40 ℃, the loading was 20%, and the mixture was shaken at 180 rpm / min. After culturing for 18 h, 50 μg of Ralstonia solanacearum lipopolysaccharide and 50 μg of Fusarium vomitoxin were added. The preparation method of Ralstonia solanacearum lipopolysaccharide was the same as in Example 5. After culturing for another 10 h, add 90 g of glycerol, 5 mL of 3 M sodium hydroxide solution, 2 mL of 4 M Tris Base solution, 15 g of xanthan gum, 10 g of sucrose, and 100 g of dipropylene glycol.

[0062] At this point, the bacterial count of Bacillus vesiculosus MLY204 preparation reached 4.1 × 10⁻⁶. 9 The concentration was cfu / mL, and a small amount of bacterial cells precipitated at the bottom after standing.

[0063] Example 8:

[0064] This embodiment provides a microbial preparation of Bacillus belyssus MLY204, the preparation method of which includes: Bacillus berberis strain MLY204 was inoculated into sterile fermentation medium at an inoculum size of 5%. The medium formula (1L) was: 3 g disodium hydrogen phosphate dodecahydrate, 0.8 g potassium dihydrogen phosphate, 0.1 g sodium chloride, 0.2 g ammonium chloride, 0.3 g magnesium sulfate, 0.7 g L-aspartic acid, 0.4 g L-glutamic acid, 0.3 g L-leucine, 0.2 g L-tyrosine, 0.3 g L-alanine, 0.6 g L-valine, 1 g Tween-80, 15 g glucose monohydrate, 0.1 g calcium chloride, 2 g vermiculite, 10 g dodecayl alcohol, 100 mg boric acid, 8 g zein, 10 g beef extract, 10 g peptone, and 50 mg zinc chloride. The culture temperature was 30 ℃, the loading was 20%, and the shaking was carried out at 200 rpm / min. After culturing for 18 h, 60 μg of Ralstonia solanacearum lipopolysaccharide and 40 μg of Fusarium vomitoxin were added. The preparation method of Ralstonia solanacearum lipopolysaccharide was the same as in Example 5. After culturing for another 12 h, add 100 g of glycerol, 6 mL of 2 M sodium hydroxide solution, 5 mL of 3 M Tris Base solution, 10 g of xanthan gum, 15 g of sucrose, and 100 g of dipropylene glycol.

[0065] At this point, the bacterial count of the Bacillus vesiculus MLY204 preparation reached 5.6 × 10⁻⁶. 9 cfu / mL, no sterile precipitate at the bottom after standing.

[0066] Example 9:

[0067] The microbial preparation of Bacillus belyssus MLY204 prepared in Example 7 was centrifuged at 5000 rpm for 30 minutes at 4 °C, and the supernatant was filtered through a 0.22 μm filter membrane.

[0068] Add the supernatant to the NA plate containing Ralstonia solanacearum MLY102, as follows: Figure 6As shown in Figure B, the inhibition zone is 1.0 cm in size.

[0069] Example 10:

[0070] The microbial preparation of Bacillus belyssus MLY204 prepared in Example 8 was centrifuged at 12,000 rpm for 10 minutes at 4 °C, and the supernatant was filtered through a 0.22 μm filter membrane.

[0071] Add the supernatant to the NA plate containing Ralstonia solanacearum MLY102, as follows: Figure 6 As shown in Figure C, the inhibition zone is 1.7 cm in size.

[0072] Comparative Example 1:

[0073] Aseptic fermentation medium was prepared using the method described in Example 8. The prepared medium was filtered through a 0.22 μm filter membrane and then added to an NA plate containing Ralstonia solanacearum MLY102. Figure 6 As shown in Figure A, the inhibition zone is 0 cm in size.

[0074] Example 11:

[0075] The microbial preparation of Bacillus belyssus MLY204 prepared in Example 8 was centrifuged at 12,000 rpm for 10 minutes at 4 °C, and the supernatant was filtered through a 0.22 μm filter membrane.

[0076] Extract the supernatant with an equal volume of ethyl acetate, collect the upper organic phase, and continue extraction with an equal volume of ethyl acetate on the lower aqueous phase. Repeat this process three times, and collect and combine the organic phases. Remove the ethyl acetate from the organic phase by rotary evaporation, redissolve it in DMSO, and add it to an NA plate containing Ralstonia solanacearum MLY102. Figure 6 As shown in Figure E, the inhibition zone is 1.3 cm in size.

[0077] Comparative Example 2:

[0078] After filtering DMSO through a 0.22 μm membrane, it was added to an NA plate containing Ralstonia solanacearum MLY102. Figure 6 As shown in D, the inhibition zone size is 0 cm.

[0079] Example 12:

[0080] A microbial preparation of Bacillus belyssus MLY204 was prepared according to Example 8, stored at 28 °C, and samples were taken every 30 days to determine the viable count and spore rate.

[0081] Among them, the viable cell count was determined by dilution and plating method; the spore rate was determined by heating the fermentation broth in an 85℃ water bath for 10 min, diluting it, and then plating and counting to determine the number of spores. The spore rate = number of spores / number of viable cells × 100%.

[0082] like Figure 7 As shown, although the number of viable bacteria and the spore rate both decreased over time, they remained at a high level.

[0083] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the technical principles of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus belyssus MLY204, characterized in that, The classification name of the Bacillus belysium MLY204 is... Bacillus velezensis It is deposited at the China General Microbiological Culture Collection Center, with accession number CGMCC No. 36290.

2. A microbial preparation of Bacillus belyssus MLY204, characterized in that, It includes Bacillus vesiculosus MLY204 fermentation broth, stabilizers, and synergists.

3. The Bacillus belyceca MLY204 microbial preparation according to claim 2, characterized in that, The preparation method of the Bacillus vesiculus MLY204 fermentation broth includes: picking a single colony of Bacillus vesiculus MLY204 and inoculating it into 10-500 mL of sterile fermentation medium, and culturing it at 20-40 ℃ and 100-250 rpm / min for 1-12 h.

4. The Bacillus belyssus MLY204 microbial preparation according to claim 3, characterized in that, The stabilizers added to each liter of the Bacillus berberis MLY204 fermentation broth include: 10-130 g of glycerol, 0.1-10 mL of 2-5 M sodium hydroxide solution, 1-10 mL of 1-5 M Tris Base solution, 1-20 g of xanthan gum, 1-100 g of sucrose, and 10-120 g of dipropylene glycol.

5. The Bacillus belyssus MLY204 microbial preparation according to claim 3, characterized in that, The synergistic agents added to each liter of the Bacillus vesiculosus MLY204 fermentation broth include: 1-100 μg of Ralstonia solanacearum lipopolysaccharide and 1-100 μg of Fusarium vomitoxin.

6. The Bacillus belyceca MLY204 microbial preparation according to claim 2, characterized in that, The microbial preparation is an aqueous solution.

7. A method for preparing a microbial preparation of Bacillus belye MLY204, characterized in that, include: Select a single colony of Bacillus belye MLY204 and inoculate it into 10-500 mL of sterile fermentation medium. Incubate at 20-40℃ and 100-250 rpm / min for 1-12 h. After expanding the culture by inoculation at a rate of 0.1-10 v / v% for 12-24 h, add a synergist, continue culturing for another 1-12 h, and then add a stabilizer to obtain the final product.

8. The method for preparing the microbial preparation of Bacillus belyceum MLY204 according to claim 2 or the microbial preparation of Bacillus belyceum MLY204 according to claim 7, characterized in that, Each liter of the aseptic fermentation medium comprises: 1-10 g of disodium hydrogen phosphate dodecahydrate, 0.1-10 g of potassium dihydrogen phosphate, 0.1-10 g of sodium chloride, 0.1-10 g of ammonium chloride, 0.1-10 g of magnesium sulfate, 0.1-1 g of L-aspartic acid, 0.1-1 g of L-glutamic acid, 0.1-1 g of L-leucine, 0.1-1 g of L-tyrosine, 0.1-1 g of L-alanine, 0.1-1 g of L-valine, 1-10 g of Tween-80, 1-20 g of glucose monohydrate, 0.1-10 g of calcium chloride, 1-10 g of vermiculite, 0.1-10 g of dodecayl alcohol, 5-200 mg of boric acid, 0.1-15 g of zein, 1-10 g of beef extract, 0.1-15 g of peptone, and 10-100 mg of zinc chloride.

9. Application of Bacillus belyss MLY204 or microbial preparations of Bacillus belyss MLY204 in the preparation of products that inhibit the activity of Ralstonia solanacearum strains, Alternaria alternata and Fusarium wilt.

10. The application according to claim 9, characterized in that, The product also includes the supernatant of a microbial preparation of Bacillus vesiculosus MLY204.

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