Bacillus velezensis as well as preparation method and application thereof

Abstract

The invention provides bacillus velezensis as well as a preparation method and application thereof. The bacillus velezensis and the microbial inoculum or composition containing the bacillus velezensis can effectively inhibit pathogenic microorganisms of plant root rot. The culture medium provided by the invention has the advantages of simple and easily available components, low price, simple and feasible fermentation conditions and short fermentation period. Moreover, the prepared fermentation liquor has higher viable count, spore count and spore conversion rate and lower effective viscosity, almost has no influence on subsequent treatment, and can be simply filtered, concentrated and then spray-dried. The prepared powder product is stable in character, and industrial production and popularization and application of bacillus related products are facilitated.

Description

Technical Field

[0001] The invention belongs to the field of microbial fermentation and culture, and specifically relates to Bacillus velez, a preparation method and application thereof. Background Art

[0002] In recent years, with the general public's concern about the safety of chemical pesticides in food and the environment and the ban on dangerous chemical pesticides, people have been prompted to seek safe and effective ways to control plant diseases, and the use of beneficial microorganisms to control plant diseases has received increasing attention. It is to introduce exogenous antagonistic bacteria that are beneficial to plants into the broken natural ecosystem of plants, or to promote the growth of natural beneficial microbial populations by regulating the environment and showing biocontrol activity, and to achieve the purpose of disease prevention through the interaction between microorganisms.

[0003] As a new type of biocontrol microbial factor, Bacillus Velez has attracted much attention in recent years. Research on it is mainly concentrated in the fields of feed, medicine, textiles, aquatic products, sewage treatment, plant protection, etc. Bacillus Velez has shown great potential in plant disease prevention and control, promoting plant growth, inducing systemic resistance, accumulating defense enzymes and their antibacterial active substances. Bacillus is mostly present in the form of spores in agricultural promotion and application, and the goal of fermentation production is to increase the number of effective spores and the sporulation rate.

[0004] Currently, it is still desirable to provide a Bacillus Velezii strain with a high spore conversion rate. Summary of the invention

[0005] In order to solve at least one of the above problems, the present disclosure provides a Velez subtilis, a preparation method and an application thereof. The Velez subtilis provided by the present disclosure can effectively inhibit the pathogenic microorganisms of plant root rot. The provided fermentation medium and the corresponding fermentation culture method further increase the number of effective spores of the Velez subtilis, reduce the viscosity of the fermentation liquid, significantly improve the dispersion effect of the powder after spray drying, and improve the product properties.

[0006] According to one aspect of the present disclosure, there is provided a Bacillus velezensis, wherein the 16S rDNA of the Bacillus velezensis has the nucleotide sequence as shown in SEQ ID NO: 1, or has at least 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 99. .4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0007] In some embodiments, the gyrB gene of Bacillus velez has the nucleotide sequence of SEQ ID NO:2, or at least 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.1%, 98.2%, 98.3%, 98.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 98.1%, 98.2%, 98.3%, 98.9%, 98. .4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

[0008] In some embodiments, the Bacillus velez is a gram-positive bacterium.

[0009] In some embodiments, the Bacillus Velez is capable of inhibiting pathogenic microorganisms that cause plant root rot.

[0010] In some embodiments, the pathogenic microorganisms include one or more of the following: Fusarium spp., Rhizoctonia sp., Phoma spp., Bacillus spp., Pythum spp., Aphanomyces spp., Erwinia spp., or Athelia rolfsii.

[0011] In some embodiments, the plant comprises one or more of the following: Chenopodiaceae, Leguminosae, Cruciferae, Solanaceae, Cucurbitaceae, Rosaceae.

[0012] In some embodiments, the plant comprises one or more of: sugar beet, soybean, bean, carrot, potato, cucumber, strawberry, or tobacco.

[0013] In some embodiments, the Bacillus velezensis is deposited in the China Center for Type Culture Collection (CCTCC), the name of the deposited strain is Bacillus velezensis KNF-209, and the strain deposit number is CCTCC NO.M2022319.

[0014] According to another aspect of the present disclosure, a fermentation medium for the fermentation culture of the Bacillus Velez subtilis is provided, wherein the fermentation medium comprises: 1% to 5% corn starch, and / or 0.1% to 1% bran, wherein "%" represents g / 100 mL.

[0015] In some embodiments, the fermentation medium comprises: corn starch 1%, 2%, 3%, 4% or 5%, and / or bran 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, wherein "%" represents g / 100mL.

[0016] In some embodiments, the fermentation medium further comprises corn flour, soybean meal, corn steep liquor, peptone, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride, manganese chloride and the balance of water or filler.

[0017] In some embodiments, the fermentation medium also includes 1% to 5% corn flour, 0.1% to 1% soybean meal, 1% to 5% corn steep liquor (solid content 45%), 0.1% to 5% peptone, 0.01% to 1% potassium dihydrogen phosphate, 0.01% to 1% magnesium sulfate, 0.01% to 1% calcium chloride, 0.01% to 1% manganese chloride and the remainder water or filler, where "%" represents g / 100mL.

[0018] In some embodiments, the fermentation medium has a pH of 7.0 to 8.0.

[0019] In some embodiments, the fermentation medium has a pH of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0020] According to another aspect of the present disclosure, a culture of Bacillus Velez subtilis is provided, comprising the Bacillus Velez subtilis and spores produced by the Bacillus Velez subtilis; and optionally, further comprising the culture medium.

[0021] In some embodiments, the viscosity of the culture is <160 cP, <150 cP, <140 cP, <130 cP, <120 cP, <110 cP, <100 cP, <90 cP, <80 cP, <70 cP, <60 cP, or <50 cP.

[0022] According to another aspect of the present disclosure, a method for culturing the Bacillus Velez subtilis is provided, the method comprising a fermentation culturing step, the fermentation culturing step comprising culturing the Bacillus Velez subtilis using a fermentation medium comprising 1% to 5% corn starch and / or 0.1% to 1% bran.

[0023] In some embodiments, the fermentation medium comprises: corn starch 1%, 2%, 3%, 4% or 5%, and / or bran 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1%, wherein "%" represents g / 100mL.

[0024] In some embodiments, the fermentation medium further comprises corn meal, soybean meal, corn steep liquor, peptone, potassium dihydrogen phosphate, magnesium sulfate, calcium chloride and / or manganese chloride.

[0025] In some embodiments, the fermentation medium also includes 1% to 5% corn flour, 0.1% to 1% soybean meal, 1% to 5% corn steep liquor (solid content 45%), 0.1% to 5% peptone, 0.01% to 1% potassium dihydrogen phosphate, 0.01% to 1% magnesium sulfate, 0.01% to 1% calcium chloride, and 0.01% to 1% manganese chloride; the remainder is water or filler, where "%" represents g / 100mL.

[0026] In some embodiments, the fermentation medium has a pH of 7.0 to 8.0.

[0027] In some embodiments, the fermentation medium has a pH of 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9 or 8.0.

[0028] In some embodiments, the fermentation culture includes: inoculating the Bacillus Velez subtilis, setting the temperature to 25-35°C, the initial stirring speed to 100-200 rpm, the stirring speed is controlled in association with 25-35% dissolved oxygen, the ventilation ratio is set to (1-2): (1-2), and the pressure is maintained at 0.05-0.1Mpa for fermentation culture.

[0029] In some embodiments, the fermentation culture further includes the steps of sterilizing and cooling the fermentation medium.

[0030] In some embodiments, the sterilization conditions include 110-130° C., 15-60 min.

[0031] In some embodiments, the cooling temperature comprises 25-35°C.

[0032] In some embodiments, the fermentation culture further comprises the step of feeding betaine.

[0033] In some embodiments, the flow rate of betaine comprises 0.15-0.5 g / L per hour.

[0034] In some embodiments, the flow rate of betaine comprises 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.33, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49, or 0.5 g / L per hour.

[0035] In some embodiments, the fermentation culture further includes the steps of strain activation and seed culture.

[0036] In some embodiments, the culture medium used for activating the strain includes 5-20 g of peptone, 1-10 g of yeast extract powder, 5-20 g of sodium chloride, 5-30 g of agar powder, and the remainder of water and filler.

[0037] In some embodiments, the activation comprises culturing at 25-35° C. for 24-72 hours.

[0038] In some embodiments, the seed culture medium includes 1-20 g of peptone, 1-10 g of yeast extract powder, 1-20 g of sodium chloride, and the remainder of water and filler.

[0039] In some embodiments, the seed culturing comprises culturing at 25-35° C. and at a rotation speed of 150-250 rpm for 5-15 hours.

[0040] According to another aspect of the present disclosure, provided is the use of the culture medium in one or more of the following: increasing the effective bacteria yield in a fermentation product of Bacillus Velez, increasing the spore yield in a fermentation product of Bacillus Velez, increasing the spore conversion rate in a fermentation product of Bacillus Velez, and reducing the viscosity of a fermentation product of Bacillus Velez.

[0041] According to another aspect of the present disclosure, a composition is provided, comprising the Bacillus Velez, or the attenuated Bacillus Velez, the irradiated Bacillus Velez, the culture of the Bacillus Velez, the metabolite of the Bacillus Velez, or the supernatant of the Bacillus Velez.

[0042] In some embodiments, the culture of Bacillus velez is a liquid culture, a fermentation culture, or a supernatant of a fermentation culture.

[0043] In some embodiments, the microbial agent further comprises an additive.

[0044] In some embodiments, the additives include one or more of the following: for example, inert components, dispersants, antifreeze agents, surfactants, binders, thickeners, defoamers, pH adjusters, film formers, wetting agents, anti-caking agents, disintegrants, and fillers or water.

[0045] In some embodiments, the composition comprises one or more of the following dosage forms: powder, wettable powder, water dispersible granules, dispersible granules, granules, dispersible powder for seed treatment, suspension, suspension for seed treatment, liquid preparation, solid preparation, freeze-dried preparation, spray preparation and electrostatic spray preparation.

[0046] In some embodiments, the composition further comprises one or more of the following: plants, other microorganisms or chemical insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators or fertilizers, or a combination thereof, wherein these ingredients are present in an amount suitable for promoting plant growth and / or protecting susceptible plants from pathogenic infection.

[0047] According to another aspect of the present disclosure, a method for preparing a composition is provided, comprising the steps of filtering, concentrating and drying the Bacillus Velez subtilis or the fermentation broth to prepare a bacterial agent; preferably spray drying.

[0048] In some embodiments, the preparation method includes the steps of filtering through a 100-mesh vibrating screen, concentrating the filtered filtrate by 5-10 times by centrifugation, and spray drying to prepare a bacterial agent.

[0049] According to another aspect of the present disclosure, provided is the use of the Bacillus Velez subtilis, the fermentation product of the Bacillus Velez subtilis and / or the composition in preventing and controlling agricultural diseases.

[0050] In some embodiments, the agricultural disease comprises plant root rot.

[0051] In some embodiments, the plant root rot comprises one or more of the following: Fusarium root rot, Rhizoctonia root rot, Ophiocordyceps black rot, Sclerotium siliceous root rot, or bacterial scutellaria root rot.

[0052] In some embodiments, the use includes inhibiting pathogenic microorganisms that cause root rot of the plant.

[0053] In some embodiments, the pathogenic microorganisms include one or more of the following: Fusarium spp., Rhizoctonia sp., Phoma spp., Bacillus spp., Pythum spp., Aphanomyces spp., Erwinia spp., or Athelia rolfsii.

[0054] In some embodiments, the plant includes plants of Chenopodiaceae, Leguminosae, Cruciferae, Solanaceae, Cucurbitaceae, and Rosaceae.

[0055] In some embodiments, the plant comprises sugar beet, soybean, bean, carrot, potato, cucumber, strawberry, or tobacco.

[0056] Beneficial effects:

[0057] The use of the Bacillus Velezii and the bacterial agent or composition containing the same provided by the present disclosure can effectively inhibit the pathogenic microorganisms of plant root rot, has broad application prospects in agricultural disease prevention and control, and is conducive to the sustainable development of agriculture.

[0058] The culture medium and culture method provided by the present disclosure are used to ferment and culture the Bacillus velez. The number of viable bacteria, the number of spores and the spore conversion rate in the fermentation liquid are significantly improved. The culture medium provided by the present invention is simple and easy to obtain, low in price, the fermentation conditions are simple and easy, and the fermentation cycle is short. Moreover, the prepared fermentation liquid has a low viscosity and has almost no effect on subsequent treatment. It can be simply filtered and concentrated and then spray-dried. The powder product is stable in properties, which is conducive to industrial production and the promotion and application of Bacillus-related products. BRIEF DESCRIPTION OF THE DRAWINGS

[0059] Figure 1 The macroscopic colony morphology of the Bacillus Velezii KNF-209 strain of the present invention on TSA medium;

[0060] Figure 2 The microscopic morphology of the Bacillus Velezii KNF-209 strain of the present invention under an optical microscope;

[0061] Figure 3 This is the phylogenetic tree of the rDNA sequence of Bacillus velez KNF-20916S of the present invention;

[0062] Figure 4 The phylogenetic tree of the gyrB gene sequence of Bacillus velez KNF-209 of the present invention. DETAILED DESCRIPTION

[0063] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiments. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. In addition, in the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0064] The applicant screened and obtained a strain of Bacillus velezensis from the dynamic intestinal flora. After laboratory fermentation research and formula optimization, the number of spores in the production and transformation process can reach 17.6 billion cfu / mL. The Bacillus velezensis KNF-209 strain of the present invention was deposited in the China Center for Type Culture Collection (CCTCC) on March 25, 2022, with a deposit number of CCTCC NO.M2022319, and a deposit name of Bacillus velezensis KNF-209. The depository address is Wuhan University, Wuhan, China.

[0065] Moreover, the present invention explored experimental conditions to obtain a fermentation medium and a corresponding fermentation culture method. The medium and culture method provided by the present invention were used to ferment the Velezella spores, and the number of liquid spores could reach 2.43×10 10 cfu / ml, the number of effective spores is at least 30% higher than that of the initial formula and process, the spore conversion rate is over 95%, and the number of effective spores of the powder made by spray drying can reach 1.21×10 12 The high content of cfu / ml further increases the number of effective spores of the Velez Bacillus, reduces the viscosity of the fermentation liquid, and avoids the problem of high viscosity of the fermentation liquid in the tank, poor dispersion effect after powder spraying, and affecting the product properties.

[0066] The present invention verifies through experiments that the Bacillus Velezii and the bacterial agent or composition containing the same can inhibit pathogenic microorganisms of plant root rot, including Fusarium spp., Rhizoctonia sp., Phoma spp., Bacillus spp., Pythum spp., Aphanomyces spp., Erwinia spp. or Athelia rolfsii.

[0067] Therefore, using the Bacillus Velezii and the bacterial agent or composition containing the same provided by the present disclosure to control plant root rot is an ideal biological control method with good economic and ecological benefits.

[0068] definition

[0069] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0070] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0071] The expression "about" as used herein is understood by one of ordinary skill in the art and varies within a certain range depending on the context in which it is used. If one of ordinary skill in the art is not aware of the use of the term based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0072] The term "Oxford cup method" used in this article is also called toxicity glass tube or drug sensitivity test cup. The specific operation is to soak the Oxford cup in a certain concentration of the test solution to allow it to absorb a certain amount of the test substance. Then the bacterial solution is evenly applied to the solid culture medium, and the Oxford cup treated with the test solution is inverted on the culture medium and placed in a constant temperature box for incubation. If the bacteria are not sensitive to the test substance, a clear growth area will be formed around the Oxford cup; if the bacteria are sensitive to the test substance, an obvious antibacterial zone will be formed around the Oxford cup.

[0073] The term "standard strain" used in this article is used as a standard for quality control in daily microbiological testing and scientific research. Strains that have typical characteristics of certain bacteria and serve as quality control standards can be called standard strains.

[0074] The term "gyrB gene" used in this article refers to the B subunit of the DNA helicase gyrase. Changes in the higher-order structure of DNA chains or chromosomes require the participation of topoisomerases, including type I and type II. The DNA helicase gyrase is a type II topoisomerase, which consists of two subunits, A and B. GyrB is the gene encoding the B subunit. Like 16S rRNA, gyrB and ISR are widely present in various bacterial cells and are essential genes for cell life processes, but the evolution rate of gyrB is 3 to 6 times that of 16S rRNA. The evolution rate of ISR is also faster than that of 16SrRNA, and can be used as a model molecule for bacterial molecular evolution and system analysis.

[0075] As used herein, the term "sequence identity" refers to the "percentage of sequence identity" or "percentage of sequence identity" between two polynucleotides, i.e., the number of identical matching positions shared by the sequences within a comparison window, taking into account additions or deletions (i.e., gaps) that must be introduced for optimal alignment of the two sequences. A matching position is any position where the same nucleotide is present in both the target sequence and the reference sequence. Since a gap is not a nucleotide, a gap present in the target sequence is not counted. Likewise, since the target sequence nucleotides are counted and the nucleotides from the reference sequence are not counted, gaps present in the reference sequence are not counted. At least 95% sequence identity includes at least 95%, at least 95.1%, at least 95.2%, at least 95.3%, at least 95.4%, at least 95.5%, at least 95.6%, at least 95.7%, at least 95.8%, at least 95.9%, at least 96%, at least 96.1%, at least 96.2%, at least 96.3%, at least 96.4%, at least 96.5%, at least 96.6%, at least 96.7%, at least 96.8%, at least 96.9%, at least 97%, at least 97.1%, at least 97.2%, at least 97.3%, at least 97.4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.9%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 98.9%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.9% over the entire length of the sequence. .4%, at least 97.5%, at least 97.6%, at least 97.7%, at least 97.8%, at least 97.9%, at least 98%, at least 98.1%, at least 98.2%, at least 98.3%, at least 98.4%, at least 98.5%, at least 98.6%, at least 98.7%, at least 98.8%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9% or 100% sequence identity.

[0076] The sequence identity percentage can be calculated by the following process: determine the number of positions where the same amino acid residue or nucleic acid base occurs in both sequences to obtain the number of matching positions, divide the number of matching positions by the total number of positions in the comparison window, and multiply the result by 100 to obtain the sequence identity percentage. The comparison of the sequence and the determination of the sequence identity percentage between the two sequences can be completed using software that is easy to use and download online. Suitable software programs can be obtained from various sources for the comparison of protein and nucleotide sequences. A suitable program for determining the sequence identity percentage is bl2seq, which is a part of the BLAST program suite available from the BLAST website (blast.ncbi.nlm.nih.gov) of the National Center for Biotechnology Information of the U.S. Government. Bl2seq uses BLASTN or BLASTP algorithms to compare between two sequences. BLASTN is used to compare nucleic acid sequences, and BLASTP is used to compare amino acid sequences. Other suitable programs are, for example, a part of the EMBOSS suite of Needle, Stretcher, Water or Matcher, bioinformatics programs, and can also be obtained at www.ebi.ac.uk / Tools / psa.

[0077] The term "fermentation broth" as used herein refers to a preparation produced by cell fermentation, which undergoes no or minimal recovery and / or purification. The fermentation broth may contain the unfractionated or fractionated contents of the fermentation material derived at the end of the fermentation. Typically, the fermentation broth is unfractionated and comprises spent culture medium and, for example, cell debris present after centrifugal removal of microbial cells (e.g., filamentous fungal cells). In certain embodiments, the fermentation broth contains spent cell culture medium, spores, parasporal crystal proteins, and vigorous and / or inactive microbial cells.

[0078] The term "beet root rot" used in this article refers to the general term for beet root rot caused by infection and parasitism of bacteria and fungi. Due to the increase in the number of years of beet cultivation and changes in the cultivation environment, the harm of beet root rot has gradually increased. It is currently one of the main diseases in beet production and occurs to varying degrees in all beet-growing countries. The dominant pathogens of beet root rot are different in different beet-growing countries. In Europe and the United States, Fusarium spp. and Rhizoctonia sp. are dominant, while in Japan, Rhizoctonia sp. is dominant. In my country, the dominant species of beet root rot is Fusarium spp., in addition to some non-dominant species, such as Phomaspp., Pythum spp., Aphanomyces spp., Bacillus spp. and Erwinia spp. Fusarium beet root rot is mainly caused by Fusarium, which mainly infects the wounds of beet tubers and eventually becomes dry rot. Rhizoctonia beet root rot is mainly caused by Rhizoctonia, which starts to infect the base of the beet petiole. The lesion site is sunken to form deep cracks, and the wound surface has varying degrees of lignification. Sometimes dense brown mycelium can be observed near the wound. Ophiocordyceps beet root rot is mainly caused by fungi of the genus Phyllostomia. The beet roots rot from the inside out. After cracks appear on the epidermis, it can be found that all the tissues in the roots except the vessels turn black. Bacterial beet root rot is caused by bacteria such as Echinobacterium beete. When the soil moisture content near the beet root system is high, the root tail of the beet root becomes waterlogged, and sometimes a sticky rotten liquid flows out, with a sour and smelly smell similar to corruption. Beet root rot caused by Sclerotium is mainly caused by Athena Ralstoniae, which starts to infect the root heads of beet tubers and gradually spreads downwards. The lesions soften and sink inward, and white silky mycelium appears on the outside with brown sclerotia on it. When the environment is humid, the mycelium can expand outward along the surface.

[0079] As used herein, the term "controllingly effective amount" refers to an amount sufficient to control harmful microorganisms on plants without causing any significant damage to the treated plants.

[0080] The term "agriculturally acceptable carrier" as used herein encompasses all adjuvants commonly used in the technology of formulation of pest control agents, such as inert components, dispersants, surfactants, tackifiers, adhesives and the like.

[0081] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with the examples. The specific embodiments described herein are only used to explain the present invention and are not intended to constitute any limitation of the present invention. The actual protection scope of the present invention is set forth in the claims. In the following description, the description of known structures and technologies is omitted to avoid unnecessary confusion of the concepts of the present disclosure. Such structures and technologies are also described in many publications. The equipment, instruments, reagents and / or kits used in the following examples are not mentioned in terms of their sources, and are all conventional products commercially available on the market, or obtained by conventional methods known to those skilled in the art.

[0082] Example

[0083] Example 1

[0084] The Bacillus velezensis KNF-209 strain of the present invention is isolated and screened from the motile intestinal flora. After the strain is cultured on TSA solid medium, the colonies on the medium are light yellow, nearly round, dry, opaque, and have irregular colony edges (see Figure 1 ); Microscopic observation revealed that KNF-209 bacteria were rod-shaped, 0.4-0.6 μm × 1.1-3.4 μm in size, arranged singly or in pairs, Gram-positive, with spores in the shape of columns, proximal, and cysts that did not swell (see Figure 2 ).

[0085] The 16S rDNA gene sequence of the KNF-209 strain was amplified using primers 27F and 1492R, and a 1389bp gene fragment was obtained for sequencing to obtain the sequence information of 16s rDNA (SEQ ID NO: 1). The primer sequences were 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 3); 1492R: 5'-GGTTACCTTGTTACGACTT-3' (SEQ ID NO: 4).

[0086] The 16s rDNA sequence (SEQ ID NO: 1) was compared with the 16s rDNA of Bacillus bacteria in the NCBI database, and the sequence similarity is shown in Table 1.

[0087] Table 1. 16s rDNA sequence similarity between Bacillus velez KNF-209 and Bacillus bacteria

[0088] Gene sequence similarity (%) Bacillus bacteria 99.93 <![CDATA[Bacillus siamensis KCTC13613 T (AJVF01000043)]]> 99.93 <![CDATA[Bacillus velezensis CR-502 T (AY603658)]]> 99.78 <![CDATA[Bacillus Subtilis NCIB3610 T (ABQL01000001)]]> 99.71 <![CDATA[Bacillus amyloliquefaciens DSM 7 T (FN597644)]]> 99.64 <![CDATA[Bacillus nakamurai NRRL B-41091 T (LSAZ01000028)]]> 99.57 <![CDATA[Bacillus cabrialesii TE3 T (MK462260)]]> 99.57 <![CDATA[Bacillus inaquosorum KCTC 13429 T (AMXN01000021)]]> 99.57 <![CDATA[Bacillus stercoris JCM30051 T (MN536904)]]> 99.57 <![CDATA[Bacillus tequilensis KCTC 13622 T (AYTO01000043)]]> 99.50 <![CDATA[Bacillus vallismortis DV1-F-3 T (JH600273)]]>

[0089] The strain KNF-209 has the highest gene sequence similarity with Bacillus Siamensis and Bacillus Velezensis, reaching 99.93%.

[0090] The phylogenetic tree of 16S rDNA sequences of KNF-209 and related species was constructed using MEGA software and the neighbor-joining method. Figure 3 ) were repeated 1000 times. The developmental tree nodes in the figure only show the values ​​with Bootstrap values ​​greater than 50%, and the superscript "T" indicates the model strain.

[0091] The species classification of strain KNF-209 was further identified by analyzing the gyrB gene sequence (SEQ ID NO: 2). The conserved gyrB gene sequence of strain KNF-209 was amplified with primers gyrB-F and gyrB-R to obtain a 1043 bp gene fragment, and sequence information was obtained by sequencing. The primer sequences were gyrB-F: 5'-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGN AARTTYGA-3' (SEQ ID NO: 5); gyrB-R: 5'-AGCAGGATACGGATGTGCGAGCCRTCNA CRTCNGCRTCNGTCAT-3' (SEQID NO: 6). Sequence alignment was performed, and the similarity is shown in Table 2.

[0092] Table 2. Sequence similarity of gyrB gene of Bacillus velez KNF-209 and the following Bacillus bacteria

[0093] Gene sequence similarity (%) Bacillus bacteria 98.4 <h2 style=";text-align:left;direction:ltr"><![CDATA[BacillusvelezensisBCRC17467 <h2 style=";text-align:left;direction:ltr"> T <h2 style=";text-align:left;direction:ltr"> (DQ903176)]]><h2 style=";text-align:left;direction:ltr"> 96.0 <h2 style=";text-align:left;direction:ltr"><![CDATA[BacillussiamensisKCTC13613 <h2 style=";text-align:left;direction:ltr"> T <h2 style=";text-align:left;direction:ltr"> (KC608573)]]><h2 style=";text-align:left;direction:ltr"> 95.7 <h2 style=";text-align:left;direction:ltr"><![CDATA[BacillusamyloliquefaciensBCRC11601 <h2 style=";text-align:left;direction:ltr"> T <h2 style=";text-align:left;direction:ltr"> (DQ309294)]]><h2 style=";text-align:left;direction:ltr"> 89.9 <![CDATA[BacillusnakamuraiNRRLB-41091 T (LSAZ01000005)]]> 81.0 <![CDATA[BacilluscabrialesiiTE3 T (RJVS01000021)]]> 80.6 <![CDATA[BacillusstercorisD7XPN1 T (JHCA01000025)]]> 80.5 <h2 style=";text-align:left;direction:ltr"><![CDATA[BacillusvallismortisBCRC17183 <h2 style=";text-align:left;direction:ltr"> T <h2 style=";text-align:left;direction:ltr"> (DQ309298)]]><h2 style=";text-align:left;direction:ltr"> 80.3 <h2 style=";text-align:left;direction:ltr"><![CDATA[BacillussubtilisBCRC10255 <h2 style=";text-align:left;direction:ltr"> T <h2 style=";text-align:left;direction:ltr"> (DQ309293)]]><h2 style=";text-align:left;direction:ltr"> 80.0 <![CDATA[BacillustequilensisKCTC13622 T (AYTO01000007)]]> 80.0 <![CDATA[BacillusinaquosorumNRRLB-23052 T (HQ605914)]]>

[0094] The strain KNF-209 had the highest similarity with the gyrB gene sequence of Bacillus velezensis, reaching 98.4%.

[0095] The phylogenetic tree of gyrB gene sequences of KNF-209 and related species was constructed using MEGA software and the neighbor-joining method. Figure 4 ) were repeated 1000 times. The developmental tree nodes in the figure only show the values ​​with Bootstrap values ​​greater than 50%, and the superscript "T" indicates the model strain.

[0096] Comprehensive analysis of 16S rDNA and gyrB gene sequences, as well as phylogenetic tree information, ultimately identified the strain as Bacillus velezinsis and named KNF-209.

[0097] The Bacillus velezensis KNF-209 strain of the present invention originates from Wuhan Kono Biotechnology Co., Ltd. and has been deposited by the applicant in the China Center for Type Culture Collection (CCTCC) on March 25, 2022. Its deposit number is CCTCC NO.M2022319, and its deposit name is Bacillus velezensis KNF-209. The address of the depository is Wuhan University, Wuhan, China.

[0098] Example 2

[0099] The physiological and biochemical characteristics of KNF-209 strain were identified using BioMérieux VITEK Bacillus Identification Card (BCL) according to the instructions. The results are shown in Table 3.

[0100] Table 3. Physiological and biochemical characteristics of KNF-209 strain

[0101]

[0102] Example 3

[0103] Antibacterial experiment

[0104] The Oxford cup method was used to test the inhibitory effect of Bacillus Velez KNF-209 on beet root rot pathogens.

[0105] 1 Preparation of Bacillus Velez fermentation broth

[0106] Prepare LB liquid culture medium, scrape an appropriate amount of bacterial lawn from the slant of Bacillus Velez-Zero and inoculate it into the sterilized LB liquid culture medium, place it in a shaking incubator at 30°C and 200 rpm for 20 hours to obtain the Bacillus Velez-Zero fermentation liquid.

[0107] 2. Oxford Cup Confrontation Experiment

[0108] Prepare a PDA plate, inoculate the beet root rot pathogen in the center of the plate, place Oxford cups symmetrically at 2 cm above, below, left and right of the inoculation point, add 100 μL of Bacillus Velez fermentation broth to each of the four Oxford cups, and add 100 μL of sterile LB medium as a control, with 3 parallels for each treatment. Cultivate at a constant temperature in a 28°C incubator to observe the inhibitory effect of the fermentation broth on the pathogen.

[0109] The area of ​​pathogens in the control group and the experimental group was measured. The pathogens on the control plate grew outward from the center point, approximately in a circle, and the diameter of the colony was measured to calculate the area. The pathogens in the treatment group grew into a nearly rectangular shape due to the inhibitory effect, and their length and width were measured to calculate the area.

[0110] The inhibition rate was calculated according to the following formula.

[0111]

[0112] The results are shown in Table 4.

[0113] Table 4. Results of the Oxford Cup Confrontation Experiment

[0114] deal with 1 2 3 <![CDATA[Pathogen area of the control group cm 2 > 63.59 63.59 63.59 <![CDATA[Pathogen area in the experimental group cm 2 > 3.24 3.33 3.34 Antibacterial rate 94.90％ 94.76％ 94.75％

[0115] The Oxford Cup confrontation experiment showed that the strain had a strong inhibitory effect on beet root rot pathogens.

[0116] Example 4

[0117] 1. Bacteria activation and seed culture

[0118] First, the KNF-209 strain is activated and seed cultured according to conventional methods to obtain seed liquid. The specific steps are as follows:

[0119] 1) Activation of bacterial strains: a ring of Bacillus Velez subtilis KNF-209 was selected and spread on the slant culture medium, and the inoculated slant was placed in a water-tight constant temperature incubator at 30°C and cultured for 48 hours.

[0120] Slant culture medium: peptone 10g, yeast extract powder 5g, sodium chloride 10g, agar powder 15g, tap water 1000ml, adjust pH to 7.2-7.4, sterilize at 121℃ for 20min after the above raw materials are prepared. Peptone and yeast extract powder are produced by OXOID, and sodium chloride is produced by Sinopharm Chemical Reagent Co., Ltd.

[0121] 2) Seed culture: The activated Bacillus Velezii KNF-209 was inoculated into a seed culture medium at an inoculation amount of 1%, and then cultured in a reciprocating constant temperature shaker at 30° C. and 180 rpm for 10 hours.

[0122] Seed culture medium: 10g peptone, 5g yeast extract powder, 10g sodium chloride, 1000ml tap water, adjust the pH to 7.2-7.4, and sterilize the above raw materials at 121°C for 20min.

[0123] 2. Based on the applicant's previous fermentation test single factor test results and production formula, the fermentation medium was optimized and 3 test formulas were provided as follows:

[0124] 1# culture medium: corn starch 3%, corn flour 1.5%, soybean meal 1%, corn steep liquor (solid content: 45%) 2%, peptone 0.5%, potassium dihydrogen phosphate 0.1%, bran 0.5%, magnesium sulfate 0.07%, calcium chloride 0.05%, manganese chloride 0.05%, the balance is water, adjust the pH to 7.2-7.6.

[0125] 2# culture medium: corn starch 3%, corn flour 1.5%, soybean meal 1.5%, corn steep liquor (solid content: 45%) 2%, peptone 0.5%, potassium dihydrogen phosphate 0.1%, magnesium sulfate 0.07%, calcium chloride 0.05%, manganese chloride 0.05%, the balance is water, adjust the pH to 7.2-7.6.

[0126] Control culture medium: 2% starch, 2.5% corn flour, 1% soybean meal, 2% corn steep liquor (solid content: 45%), 0.5% peptone, 0.1% potassium dihydrogen phosphate, 0.07% magnesium sulfate, 0.05% calcium chloride, 0.05% manganese chloride, the balance is water, and the pH is adjusted to 7.2-7.6.

[0127] "%" of each component of the medium represents g / 100 mL.

[0128] After dissolving the above culture medium according to the formula, adjust the pH to 7.5 and feed it to three 15L fermentation tanks respectively, sterilize it at 121℃ for 30min, and add the seed liquid at a 5% inoculation amount after the material cools to 30℃. Set the fermentation temperature to 30℃ and the initial stirring speed to 150rpm. The stirring speed during the fermentation process is linked to 30% dissolved oxygen. The ventilation ratio during the fermentation process is set to 1:1.3, and the tank pressure is maintained at 0.05-0.065Mpa for fermentation. After 40h of fermentation, take samples to determine the effective live bacteria and spore counts of the fermentation liquid, and use the dilution plate method to measure the bacteria and spore counts. The method is as follows:

[0129] Effective viable count:

[0130] 1) Use a pipette to take 10 ml of fermentation liquid and add it into a covered glass bottle containing 90 ml of sterile water. Shake vigorously for 1 min and ultrasonicate the sample for 10 min to obtain a basic bacterial suspension.

[0131] 2) Take 1 ml of the basic bacterial suspension and add it to a sterile glass test tube containing 9 ml of sterile water to obtain a dilution of 1:1×10 1 The bacterial suspension was serially diluted at 1:10 to obtain 1:1×10 1 , 1: 1×10 2 , 1: 1×10 3 , 1: 1×10 4 , 1: 1×10 5 , 1: 1×10 6 , 1: 1×10 7 , 1: 1×10 8 dilution of bacterial suspension;

[0132] 3) Accurately pipette 0.1 ml of two serially appropriately diluted bacterial suspensions onto the plate, repeat each dilution three times, and count the cells after culturing at 30°C for 24 hours.

[0133] 4) When counting spores, place the basic bacterial suspension in an 80°C water bath for 20 minutes to kill the bacteria and retain the spores, and then count according to the above counting method.

[0134] The test results are shown in Table 5.

[0135] Table 5. Culture results of different culture media

[0136]

[0137]

[0138] The results showed that the number of spores obtained by fermentation with medium 1# was the highest, reaching 2.16×10 10 cfu / ml, and the spore conversion rate was the highest, reaching 96.0%, and was further optimized based on 1# culture medium in the later stage.

[0139] Example 5

[0140] According to the fermentation results of Example 4, the difference between 1# medium and 2# medium is that 1# adds 0.5% bran, the soybean meal content is reduced by 0.5%, and the number of effective spores and the spore conversion rate are both improved. The effect of adding bran or not adding bran on the fermentation results is not verified further.

[0141] Seed activation and seed culture were performed according to the method of Example 4, and fermentation verification was performed using 1# medium and 3# medium, wherein the formula of 3# medium is as follows:

[0142] 3# culture medium: 3% corn starch, 1.5% corn flour, 1% soybean meal, 2% corn steep liquor, 0.5% peptone, 0.1% potassium dihydrogen phosphate, 0.07% magnesium sulfate, 0.05% calcium chloride, 0.05% manganese chloride, and the balance is water. Adjust the pH to 7.2-7.6.

[0143] "%" of each component of the medium represents g / 100 mL.

[0144] After the above culture medium is dissolved according to the formula, the pH is adjusted to 7.5 and then fed to two 15L fermentation tanks respectively, sterilized at 121°C for 30min, and after the material is cooled to 30°C, the seed liquid is added at a 5% inoculation amount, the fermentation temperature is set to 30°C, the initial stirring speed is 150rpm, the stirring speed during the fermentation process is linked to 30% dissolved oxygen, the ventilation ratio during the fermentation process is set to 1:1.3, and the tank pressure is maintained at 0.05-0.065Mpa for fermentation. After 40h of fermentation, the effective viable count and spore count of the fermentation liquid are measured by sampling, and the counting method is the same as in Example 4. The counting results are shown in Table 6.

[0145] Table 6. Culture results of 1# medium and 3# medium

[0146]

[0147] The results showed that the number of effective spores and the conversion rate of 1# medium with bran added were higher than those without bran, and the viscosity difference was not significant. 1# medium was selected as the preferred medium for further optimization to reduce the viscosity.

[0148] Example 6

[0149] 1. Prepare seed solution in the same manner as in Example 4.

[0150] 2. Fermentation culture

[0151] The preferred fermentation formula was used to test the effect of adding betaine (commercially available, purity 97% or more) in different proportions on the number of fermented spores and the viscosity of the tank, and the fermenter without betaine was used as the control. According to the 1# culture medium ingredients in Example 4, the fermenter culture medium liquid volume was 8L, the initial pH was adjusted to 7.5, the sterilization conditions were 0.1Mpa, 121°C, 30min, the inoculation amount was 5%, the culture temperature was 30°C, the initial speed was controlled to be 150rpm, the speed was set to be associated with 30% dissolved oxygen during the fermentation process, the ventilation ratio was 1:1.3, the tank pressure was maintained at 0.05-0.065Mpa during the process, and the feed was added after 12h of fermentation. Among them, the feed of 3 tanks was betaine, and the feed concentration was set to 42.7g / L, 48.0g / L, and 53.3g / L, respectively. The feed amount was 700ml, and 700ml of sterile water was prepared as the control tank feed. The feeding pump speed was uniformly set to 0.5 ml / min, that is, the betaine flow acceleration rates of the three tanks were 0.16 g / L per hour, 0.18 g / L per hour, and 0.20 g / L per hour, respectively. Feeding was stopped after 20 hours of feeding, and fermentation was continued for 36 hours. Microscopic examination showed that almost all spores were formed in the field of vision, and fermentation was stopped. After the fermentation was completed, the fermentation liquid was acidified to pH 4.5 with hydrochloric acid, transferred to post-treatment for further filtration, filtered through a 100-mesh vibrating screen, and the filtered filtrate was concentrated 5-10 times by centrifugation, spray dried, and the powder was collected.

[0152] The effective viable bacteria count and spore count were performed according to the method of Example 4. The results are shown in Table 7.

[0153] Table 7. Effective viable bacteria and spore counts in fermentation culture with betaine added

[0154]

[0155] Through the fermentation test of three tank batches, it was verified that the spore counts of the three batches reached more than 23 billion cfu / ml, which was more than 30% higher than that of the control culture medium. Among them, the effective spore count using 48g / L betaine supplementation (i.e. 0.18g / L per hour) increased by 40% compared with the initial culture medium, and increased by 13.3% compared with the control spore count without betaine. After using the three feeding processes, the viscosity of the tank was greatly reduced. Considering the spore count content comprehensively, it was finally chosen to supplement betaine at a rate of 0.18g / L per hour.

[0156] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. All technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. A Bacillus vezelensis, characterized in that: The 16S rDNA of the Bacillus velez has the nucleotide sequence as shown in SEQ ID NO: 1, or has at least 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.1%, 98.2%, 98.3%, 98.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 98.1%, 98.2%, 98.3%, 98.9%, 98. .4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity.

2. The Bacillus Velezii according to claim 1, characterized in that The gyrB gene of the Bacillus velez has the nucleotide sequence of SEQ ID NO: 2, or has at least 95%, 95.1%, 95.2%, 95.3%, 95.4%, 95.5%, 95.6%, 95.7%, 95.8%, 95.9%, 96%, 96.1%, 96.2%, 96.3%, 96.4%, 96.5%, 96.6%, 96.7%, 96.8%, 96.9%, 97%, 97.1%, 97.2%, 97.3%, 97.4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98.1%, 98.2%, 98.3%, 98.9%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 98.9%, 98.1%, 98.2%, 98.3%, 98.9%, 98. .4%, 97.5%, 97.6%, 97.7%, 97.8%, 97.9%, 98%, 98.1%, 98.2%, 98.3%, 98.4%, 98.5%, 98.6%, 98.7%, 98.8%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or 100% sequence identity, Preferably, the Bacillus velez is deposited in the China Center for Type Culture Collection (CCTCC), the name of the deposited strain is Bacillus velez KNF-209, and the strain deposit number is CCTCC NO.M2022319.

3. A fermentation medium for fermentation culture of Bacillus Velezii according to claim 1 or 2, characterized in that: The fermentation medium comprises 1% to 5% corn starch and / or 0.1% to 1% bran.

4. The fermentation medium according to claim 3, characterized in that The fermentation medium further comprises 1% to 5% corn flour, 0.1% to 1% soybean meal, 1% to 5% corn steep liquor, 0.1% to 5% peptone, 0.01% to 1% potassium dihydrogen phosphate, 0.01% to 1% magnesium sulfate, 0.01% to 1% calcium chloride and / or 0.01% to 1% manganese chloride, and the balance is water or filler; Preferably, the fermentation medium has a pH of 7-8.

5. A method for culturing Bacillus Velezii according to claim 1 or 2, characterized in that: The method comprises a fermentation culture step, wherein the fermentation culture step comprises culturing the Velez subsporus using a fermentation medium, wherein the fermentation medium comprises 1% to 5% corn starch and / or 0.1% to 1% bran.

6. The culture method according to claim 5, characterized in that: The fermentation medium further comprises 1% to 5% corn flour, 0.1% to 1% soybean meal, 1% to 5% corn steep liquor, 0.1% to 5% peptone, 0.01% to 1% potassium dihydrogen phosphate, 0.01% to 1% magnesium sulfate, 0.01% to 1% calcium chloride and / or 0.01% to 1% manganese chloride, and the balance is water or filler; Preferably, the fermentation medium has a pH of 7 to 8; Preferably, the fermentation culture comprises: inoculating the Bacillus Velezii, setting the temperature to 25-35°C, the initial stirring speed to 100-200 rpm, the stirring speed being controlled in association with 25-35% dissolved oxygen, the ventilation ratio being set to (1-2): (1-2), the pressure being maintained at 0.05-0.1 Mpa, and culturing; Preferably, the fermentation culture further comprises the step of feeding betaine; Preferably, the flow rate of betaine includes 0.15-0.5 g / L per hour; Preferably, the method further comprises the steps of strain activation and seed culture before fermentation culture.

7. A composition comprising the Bacillus Velez according to claim 1 or 2, or an attenuated Bacillus Velez, an irradiated Bacillus Velez, a culture of Bacillus Velez, spores produced by Bacillus Velez, a metabolite of Bacillus Velez, or a supernatant of Bacillus Velez.

8. The composition according to claim 7, characterized in that The culture of Bacillus Velez is a liquid culture, a fermentation culture or a supernatant of a fermentation culture; Preferably, the composition further comprises additives such as inert components, dispersants, antifreeze agents, surfactants, binders, thickeners, defoamers, pH adjusters, film formers, wetting agents, anti-caking agents, disintegrants and fillers or water; Preferably, the composition comprises one or more of the following dosage forms: powder, wettable powder, water dispersible granules, dispersible granules, granules, dispersible powder for seed treatment, suspension, suspension for seed treatment, liquid preparation, solid preparation, freeze-dried preparation, spray preparation and electrostatic spray preparation; Preferably, the composition further comprises one or more of the following: plants, other microorganisms or chemical insecticides, fungicides, nematicides, bactericides, herbicides, plant extracts, plant growth regulators or fertilizers, or a combination thereof.

9. A method for preparing a composition, comprising the steps of filtering, concentrating and drying the Bacillus Velez subtilis according to claim 1 or 2 to prepare a bacterial agent; preferably spray drying.

10. Use of the Bacillus Velezii described in claim 1 or 2, or the composition described in claim 7 or 8 in preventing and controlling agricultural diseases; Preferably, the agricultural diseases include plant root rot; Preferably, the plant root rot includes one or more of the following: Fusarium root rot, Rhizoctonia root rot, Ophiocordyceps black rot, Sclerotium wilt root rot or bacterial scutellaria root rot; Preferably, the use comprises inhibiting pathogenic microorganisms causing root rot of the plant; Preferably, the pathogenic microorganisms include one or more of the following: Fusarium spp., Rhizoctonia sp., Phoma spp., Bacillus spp., Pythum spp., Aphanomyces spp., Erwinia spp. or Athelia rolfsii; Preferably, the plant includes one or more of the following: Chenopodiaceae, Leguminosae, Cruciferae, Solanaceae, Cucurbitaceae, Rosaceae; Preferably, the plants include one or more of the following: beet, soybean, bean, carrot, potato, cucumber, strawberry or tobacco.

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