Bacillus and application thereof in inhibiting phytopathogen

Abstract

The invention provides bacillus and application of the bacillus in inhibition of pathogenic fungi. The invention discloses a novel bacillus (Bacillus) for the first time, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) NO.28299, which is called TG1-2 for short. The bacterial strain provided by the invention is separated from rhizosphere soil of weeds, and is bacillus amyloliquefaciens (Bacillus amyloliquefaciens). Detection shows that the bacterium and fermentation liquor thereof have excellent inhibition effects on various phytopathogens, such as verticillium dahliae, magnaporthe oryzae, fusarium graminearum, botrytis cinerea, sclerotinia sclerotiorum and phytophthora. The bacilli disclosed by the invention have broad-spectrum bacteriostatic action and excellent inhibition effect.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and microorganisms, and more specifically, the invention relates to a bacillus and application thereof in inhibiting plant pathogenic bacteria. Background Art

[0002] Plant diseases and insect pests mainly include fungal diseases, bacterial diseases and viral diseases. Among them, fungal diseases are the most common known diseases, with many types of symptoms and appearing in various parts of the plant. Fungal diseases account for about 70-80% of plant diseases. Sometimes, several or even dozens of fungal diseases can be found on a crop.

[0003] Fungal diseases have distinct geographical distributions due to differences in pathogens and hosts. For example, coal mold, anthracnose, and blight are more common in subtropical and tropical regions and cause serious damage; leaf spot caused by smut, Septoria, and Ascochyta, and sclerotinia caused by Sclerotinia mainly occur in temperate regions. Some fungi are weakly parasitic in the north and do not cause disease, but can cause serious diseases in tropical and subtropical regions.

[0004] Plant diseases caused by plant pathogens seriously harm the yield and quality of crops, causing huge economic losses, and are one of the problems that need to be solved urgently in agricultural production. For example, Magnaporthe oryzae, Botrytis cinerea, Fusarium graminearum, Verticillium dahliae, Phytophthora sojae, Sclerotinia sclerotiorum, etc. are the main common pathogens on crops.

[0005] Many pathogens not only cause crop yields to decline, but also leave various toxins, threatening the health of humans and animals. In actual production processes, chemical control measures are often used. However, the abuse of chemical pesticides not only causes the emergence of drug-resistant pathogens, but also causes ecological pollution, which is not conducive to the sustainable development of agriculture.

[0006] Since fungal diseases are more likely to spread through air and water, chemical agents are generally used for protective control, or by improving cultivation and management measures. However, the long-term use of chemical agents often leads to the occurrence of drug resistance and reduced effectiveness; in addition, the pollution of chemicals to the environment should not be underestimated.

[0007] Therefore, effective and safe fungal control is of dual importance in reducing plant diseases and protecting the environment. Summary of the invention

[0008] The object of the present invention is to provide a bacillus and application thereof in inhibiting plant pathogens, wherein the bacillus has a broad-spectrum antibacterial property.

[0009] In a first aspect of the present invention, an isolated Bacillus is provided. The strain is deposited in China General Microbiological Culture Collection Center (CGMCC) with a deposit number of CGMCC NO.28299.

[0010] In one or more embodiments, the strain is Bacillus amyloliquefaciens.

[0011] In one or more embodiments, a cell culture (including fermentation broth), cell metabolites (including surfactin), cell culture supernatant or cell lysate of the Bacillus is provided, which has a property selected from the following group: inhibiting plant pathogens; producing the metabolite surfactin; preferably, the surfactin is surfactin b or surfactin c.

[0012] In one or more embodiments, the cell culture is a fermentation broth, which is obtained by inoculating Bacillus TG1-2 into a liquid fermentation medium for fermentation culture.

[0013] In another aspect of the present invention, the Bacillus, its cell culture, cell metabolites, cell culture supernatant or cell lysis products are used for inhibiting plant pathogens or preparing a composition for inhibiting plant pathogens.

[0014] In one or more embodiments, the plant pathogens include: pathogenic fungi, oomycetes; preferably, including (but not limited to) selected from: Verticillium dahliae, Magnaporthe oryzae, Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, Phytophthora sojae.

[0015] In one or more embodiments, the Bacillus, its cell culture, cell metabolites, cell culture supernatant or cell lysate are used to prevent and control plant diseases caused by the plant pathogens.

[0016] In one or more embodiments, the plant diseases include (but are not limited to): Verticillium wilt (such as occurring in cotton or lettuce), rice blast (such as occurring in rice), Fusarium graminearum (such as occurring in wheat), gray mold (such as occurring in grapes), sclerotinia (such as occurring in soybeans), and Phytophthora (such as occurring in soybeans).

[0017] In another aspect of the present invention, the use of the Bacillus, its cell culture, cell metabolites, cell culture supernatant or cell lysate is provided for: producing or preparing the metabolite surfactin; or preparing a composition containing the surfactin.

[0018] In one or more embodiments, the surfactin is surfactin b or surfactin c.

[0019] In another aspect of the present invention, a method for preparing the metabolite surfactin is provided, comprising culturing the Bacillus sp. to produce surfactin.

[0020] In one or more embodiments, the method for preparing the metabolite surfactin further comprises isolating and purifying the surfactin from the culture; preferably, the surfactin is surfactin b or surfactin c.

[0021] In another aspect of the present invention, a composition (including a biocontrol agent) for inhibiting plant pathogens is provided, which comprises a component selected from the following group: the Bacillus; or a cell culture, cell metabolite, cell culture supernatant or cell lysate of the Bacillus.

[0022] In one or more embodiments, the composition includes, but is not limited to, pesticide compositions, pharmaceutical compositions, and the like.

[0023] In another aspect of the present invention, a method for inhibiting plant pathogens is provided, comprising: using the Bacillus, or its cell culture (including fermentation broth), cell metabolites, cell culture supernatants or cell lysis products to treat an object to be inhibited or degraded (including plants, environments or places containing plants).

[0024] In another aspect of the present invention, a method for preparing the metabolite surfactin is provided, comprising culturing the Bacillus to express surfactin; preferably, the surfactin is surfactin b or surfactin c.

[0025] In one or more embodiments, the method for preparing the metabolite surfactin further comprises: isolating surfactin from the culture product of the Bacillus.

[0026] In another aspect of the present invention, a kit (including a medicine box) for inhibiting plant pathogens is provided, which comprises: the Bacillus; the cell culture, cell metabolites, cell culture supernatant or cell lysate of the Bacillus; and / or the composition for inhibiting plant pathogens.

[0027] Other aspects of the present invention will be apparent to those skilled in the art in view of the disclosure herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is a phylogenetic tree constructed based on the 16S rDNA sequence of Bacillus TG1-2.

[0029] Figure 2 To test the inhibitory effect of Bacillus TG1-2 on plant pathogens.

[0030] Figure 3 The inhibitory effect of Bacillus TG1-2 fermentation broth on plant pathogens.

[0031] Figure 4 The inhibitory effect of surfactin, the antibacterial substance of Bacillus TG1-2, on plant pathogens. DETAILED DESCRIPTION

[0032] After extensive research and screening, the inventors first revealed a new type of Bacillus, with a deposit number of CGMCC NO.28299, referred to as TG1-2. The strain of the present invention is isolated from the rhizosphere soil of weeds and is Bacillus amyloliquefaciens. Detection found that the bacteria and its fermentation liquid have excellent inhibitory effects on a variety of plant pathogens, including pathogenic fungi, oomycetes (such as Verticillium dahliae, rice blast fungus, Fusarium graminearum, gray mold fungus, sclerotinia, Phytophthora, etc.). The antibacterial effect of the Bacillus of the present invention has a broad spectrum, and the antibacterial effect of the bacteria (including its fermentation liquid) is more significant than that of known biocontrol bacteria.

[0033] the term

[0034] As used herein, the terms "bacteria of the present invention", "Bacillus", "Bacillus", and "Bacillus amyloliquefaciens" are used interchangeably and all refer to the bacterial strain with a deposit number of CGMCC NO.28299.

[0035] As used herein, the term "pathogenic microorganism" refers to harmful microorganisms; in a preferred embodiment of the present invention, the pathogenic microorganism is a fungus or oomycete microorganism, also known as a plant pathogen.

[0036] In the present invention, the term "comprising" means that various components can be used together in the mixture or composition of the present invention. Therefore, the terms "consisting mainly of" and "consisting of" are included in the term "comprising".

[0037] As used herein, "industrially acceptable carrier" or "microbiologically acceptable carrier" is a solvent, suspending agent or excipient that is environmentally friendly or harmless to humans and animals and is used to deliver the Bacillus or cell culture, cell metabolites, cell culture supernatants or cell lysates of the present invention to the object to be treated, and is controllable in terms of toxicity and side effects. The carrier can be liquid or solid, and preferably a carrier that can maintain the activity of Bacillus or cell culture, cell metabolites, cell culture supernatants or cell lysates to a high degree.

[0038] Bacillus and its application

[0039] There are a wide variety of microorganisms colonizing the rhizosphere soil of plants. The roots attract and shape the rhizosphere microbial community by secreting a series of metabolites. In turn, the rhizosphere microorganisms expand the plant's ability to adapt to the environment through chemical exudates. On the one hand, rhizosphere microorganisms can induce the plant's immune system to achieve defense against pathogens. On the other hand, the metabolites produced by rhizosphere microorganisms can inhibit the growth of pathogens, thereby reducing their pathogenicity. However, how to obtain functionally ideal microorganisms from the rhizosphere of plants is still challenging. After extensive screening, the inventors screened and obtained the strain of the present invention from the rhizosphere soil of a weed in Zhangpu Salt Field, Fujian Province.

[0040] The strain of the present invention is a living cell. Once the strain of the present invention is obtained, the strain of the present invention can be obtained in large quantities by means of inoculation, passage, regeneration, etc. This is usually done by inoculating it into a solid plate culture medium or a liquid culture medium to expand the culture of the strain to obtain the living cell of the present invention. The obtained living cell can be further subjected to laboratory domestication, genetic breeding, molecular genetic manipulation, etc. to obtain mutants and transformants. In addition, the strain of the present invention can also be used as a bioengineering host cell for heterologous expression.

[0041] In the present invention, in addition to the natural Bacillus strains isolated and obtained by the inventors, variants, domesticated strains or genetically engineered strains thereof are also included. The variants are variants with the same function / improved function; preferably, the variants with the same function / improved function include those having the function of inhibiting plant pathogens or degrading their toxins.

[0042] The variant may be a variant formed by mutagenesis of the strain. Mutation breeding of strains refers to a breeding method that uses physical and chemical factors to induce mutations in the genetic characteristics of animals and plants, and then selects single strains / individuals that meet certain requirements of people from the mutant population, and then cultivates them into new varieties or germplasms. On the basis of paying attention to the main activity of the strain of the present invention, mutagenesis can be used to screen Bacillus variants that have stronger inhibition of plant pathogens; or, mutagenesis can also be used to screen strains that have the same ability to inhibit plant pathogens, but have better growth performance, more ideal environmental compatibility (such as when some toxic side effects occur, transformation is carried out through mutagenesis), and more significant resistance to adversity.

[0043] Mutagenesis can be carried out by using some mutagenesis methods known to those skilled in the art, for example, including: starting strain culture, mutagenesis treatment, observation of a large number of colonies, comparison of properties of interest, and finally screening strains with improved and modified properties of interest. Such mutagenesis can usually be carried out in a high-throughput manner, and preferably can be repeated multiple times or generations.

[0044] The domestication of strains is different from the variation of strains. It generally refers to a method of selectively breeding microorganisms by gradually adapting microorganisms to certain conditions through artificial measures. It generally includes the following steps: starting strain culture accompanied by changes in various conditions / experimental parameters, observing a large number of colonies, comparing the performance of interest, and finally sorting out strains / colonies with improved and modified performance of interest. Through domestication, strains with higher tolerance and activity can be obtained. The domestication of strains generally requires two or more generations to allow the strains to adapt to certain conditions or to improve the performance of interest.

[0045] The strains that are bioengineered / genetically engineered based on the natural strains of the present invention are also included in the present invention. For example, the introduction of exogenous genes and the deletion / knockout of endogenous genes can be carried out in the strains of the present invention, and the basic condition for the transformation is to make the strains of the present invention retain their ability to inhibit plant pathogens. The purpose of the transformation may include: to improve the colonization ability of the strains of the present invention on the surface of the object that needs to be inhibited, or to make the strains of the present invention further produce at least one other excellent performance on the basis of retaining their ability to inhibit plant pathogens: for example, the growth performance is more excellent, the environmental compatibility is more ideal (such as when some toxic side effects occur, they are eliminated by genetic modification), and the ability to resist adversity is more significant. In some common strain transformation work, one or several metabolic flows / signal pathways can be enhanced / weakened by analyzing the metabolic flow / signal pathways in the strains, so that the strain performance can be further optimized.

[0046] Methods for introducing exogenous genes and deleting / knocking out endogenous genes are known in the art, such as using some viral constructs / vectors or non-viral constructs / vectors to carry expression cassettes of exogenous genes to be introduced (including exogenous genes for overexpression, exogenous genes for gene knockout, and exogenous genes for gene editing), introducing the constructs / vectors into strains, and performing targeted transformation on the strains. The exogenous genes may include, but are not limited to: functional genes, enzyme genes, structural genes, miRNA, siRNA, shRNA, and the like.

[0047] In addition, the strain can also be transformed by directly introducing exogenous proteins into the strain. For example, by using some cell-penetrating peptides known in the art, the exogenous proteins can be directly introduced into the strain.

[0048] Based on the clear explanation of the bacillus, its biological characteristics, its functions and its culture method in the present invention, the method of culturing the bacillus in the present invention in large quantities and obtaining its culture and metabolites is familiar to those skilled in the art. That is, the culture or metabolites of the bacillus obtained by these methods, as well as the compositions containing them or their uses are also included in the present invention.

[0049] The culture medium suitable for culturing the Bacillus of the present invention may be varied. It should be understood that any culture medium that can provide the Bacillus with the nutrients necessary for growth and reproduction (such as carbon source, nitrogen source, etc.) is included in the present invention. The culture medium may be solid or liquid; the culture medium may be commercial or laboratory-prepared, such as LB culture medium. The culture medium used to culture various other bacteria in the prior art may be suitable for culturing the Bacillus of the present invention.

[0050] Cell culture, cell metabolites, cell culture supernatants or cell lysates and their applications

[0051] On the basis of obtaining the Bacillus, the present invention also provides the cell culture, cell metabolites, cell culture supernatant or cell lysis products of the Bacillus, which have characteristics selected from the following group: inhibiting plant pathogens; producing the metabolite surfactin; preferably, the surfactin is surfactin b or surfactinc.

[0052] After obtaining the strain of the present invention, those skilled in the art can easily obtain its culture, for example, by referring to some culture media or culture processes provided in the specific embodiments of the present invention, or by using culture media or culture processes that are appropriately modified from the embodiments of the present invention but can also obtain culture, thereby obtaining cell culture. The cell culture contains active strains or substances secreted by them, thereby having the ability to inhibit plant pathogens.

[0053] The cell metabolites are substances produced or secreted by the strains of the present invention during the culture process, which can be directly secreted into the culture medium by the cells, or separated from the cells after certain treatment. The cell products can be separated, purified or concentrated.

[0054] The cell culture supernatant is the culture solution remaining after removing cells and solid impurities during or after the culture of the strain of the present invention, which may be unconcentrated or concentrated. Generally, cells and solid impurities can be removed by methods such as centrifugation and filtration.

[0055] The cell lysis product is a mixture formed by lysing cells using a cell lysing agent during or after culturing the strain of the present invention. The cell lysis product can be a product after solid impurities are removed after lysis. It can be a purified or concentrated product as needed.

[0056] Application and production process

[0057] The present invention also provides the use of the Bacillus of the present invention for inhibiting plant pathogens, or for preparing the cell culture, cell metabolites, cell culture supernatants or cell lysis products, the latter of which can be used to inhibit plant pathogens, or for preparing a kit / medicine kit having the ability to inhibit plant pathogens.

[0058] The bacillus of the present invention has a broad-spectrum inhibitory effect on plant pathogens. In the embodiments of the present invention, its inhibitory effect on various pathogenic fungi and oomycetes is demonstrated. For example, it has a significant inhibitory effect on Verticillium dahliae, rice blast, Fusarium graminearum, Botrytis cinerea, soybean sclerotinia, and soybean phytophthora, including preventing and controlling diseases caused by Verticillium dahliae, diseases caused by rice blast, diseases caused by Fusarium graminearum, diseases caused by Botrytis cinerea, diseases caused by Sclerotinia sclerotiorum, or diseases caused by Phytophthora. Preferably, the plant diseases caused by Verticillium dahliae are cotton Verticillium wilt and lettuce Verticillium wilt, rice blast caused by the rice blast, Fusarium graminearum caused by the Fusarium graminearum, grape gray mold caused by the Botrytis cinerea, soybean sclerotinia caused by the Sclerotinia sclerotiorum, and soybean phytophthora caused by the Phytophthora.

[0059] The invention also discloses the application of the bacillus or its cell culture, cell metabolites, cell culture supernatants or cell lysis products in the preparation of biocontrol agents.

[0060] The present invention also discloses the use of the bacillus in producing or preparing the metabolite surfactin and in preparing a composition containing the surfactin; preferably, the surfactin is surfactin b or surfactin c. Surfactin is one of the effector molecules that exerts a killing effect.

[0061] According to the results demonstrated in the examples of the present invention, surfactin has excellent killing effects on a variety of pathogenic fungi and oomycetes, such as Verticillium dahliae, the causative agent of cotton and lettuce Verticillium wilt, Rice blast fungus, Fusarium graminearum, Botrytis cinerea, Soybean sclerotinia, and Phytophthora sojae.

[0062] Therefore, the fungus, its cell culture, cell metabolites, cell culture supernatant or cell lysate of the present invention is used to: produce or prepare the metabolite surfactin; or prepare a composition containing the surfactin; preferably, the surfactin is surfactin b or surfactin c.

[0063] In order to better apply the bacillus of the present invention to inhibit or degrade, its culture or production process can also be optimized, and the scope of consideration may include optimization of various culture conditions, optimization of various components of the culture medium, etc. It can be achieved by determining some factors that have a greater impact on the culture and inhibition or degradation of the strain. It should be understood that these improvements should also be included in the present invention.

[0064] The bacillus of the present invention can achieve long-term growth and stable passage. The culture medium and culture method used to cultivate the strain of the present invention are not limited to those disclosed in the present invention, and other culture medium and culture methods conventionally used for cultivating fungi can also be applied to the present invention. It is understood that, based on the embodiments provided by the present invention, various culture medium components may be replaced by other components with similar functions, and other components may be appropriately added or some components thereof may be removed or the content of some components thereof may be changed according to the characteristics of the specific strain in different situations.

[0065] The culture system or fermentation system of the present invention can be scaled up for industrial production. Depending on the size of the system, those skilled in the art can make appropriate adjustments based on their general knowledge to facilitate the growth or production of the strain.

[0066] The present invention also provides a method for inhibiting pathogenic microorganisms, comprising treating an object to be inhibited with the Bacillus sp. or its cell culture, cell metabolites, cell culture supernatants or cell lysates of the present invention.

[0067] In a preferred embodiment of the present invention, the cell culture is a fermentation broth obtained by inoculating Bacillus TG1-2 into a liquid fermentation medium for fermentation culture. More preferably, the main components of the liquid culture medium are: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride per 1L of aqueous solution, and the pH value is 7.0. It should be understood that the content of each component can fluctuate by 40 to 0.5%, such as 30%, 20%, 10%, 5%, 2%, etc.

[0068] When culturing in the above fermentation broth, Bacillus TG1-2 was inoculated into the above liquid culture medium. First, Bacillus TG1-2 was inoculated into 10 ml of liquid fermentation medium and cultured until OD 600 =0.6, then inoculated into new liquid culture medium at 5% inoculum and cultured until OD 600 =2.0. The culture and fermentation temperature was 28°C, 200r / min.

[0069] The bacillus of the invention has a very significant effect of inhibiting pathogenic microorganisms, has a high inhibition rate, and has wide industrial application potential.

[0070] The natural form of the Bacillus of the present invention as a biological material will not cause environmental problems like chemical preparations. The present invention provides a new material for the prevention and control of plant pathogens.

[0071] Composition

[0072] The present invention also provides a composition comprising the Bacillus of the present invention. Alternatively, the composition comprises the cell culture, cell metabolites, cell culture supernatants or cell lysates. The composition includes but is not limited to: pesticide compositions, pharmaceutical compositions, etc.

[0073] In addition to the Bacillus sp. of the present invention or the cell culture, cell metabolites, cell culture supernatants or cell lysates, the composition may further include an industrially acceptable carrier or a microbiologically acceptable carrier.

[0074] Generally, the effective dose of the bacillus of the present invention or the cell culture, cell metabolite, cell culture supernatant or cell lysate may vary depending on the mode of administration or the condition of the subject to be treated. In the present invention, "effective amount" refers to an amount that can produce a function or activity on the subject to be treated and can be accepted by the subject to be treated, generally an amount that is controllable in terms of toxicity and side effects, environmentally friendly or harmless to humans and animals.

[0075] The dosage form of the composition can be varied, including but not limited to: lyophilized agent, wettable powder, emulsifiable concentrate, aqueous solution, emulsion, sprayable solution, oily or aqueous dispersion, suspension, powder, granule, or microcapsule. It should be understood that any dosage form that can deliver the composition of the present invention to the object to be treated while maintaining all or part of its activity is desirable. Preferred dosage forms are those that are easy to deliver, such as solutions, liquid sprays, or sprays.

[0076] In the present invention, the agriculturally acceptable carrier may further include an adjuvant. The adjuvant is an auxiliary component that plays an auxiliary regulating function in the composition, such as a surfactant, an adhesion aid or other types of adjuvants.

[0077] The active ingredient content in the concentrated composition is relatively high, such as 20-90wt%, while the active ingredient content in the diluted composition and the composition actually used is relatively low, usually 0.00005-0.5wt%. In addition, other suitable chemical agents, synergists, trace elements, stabilizers, adhesives, wetting agents, dispersants, emulsifiers, penetrants, solvents, fillers and other common components may also be included. The composition of the present invention may also contain other active ingredients, such as insecticides and bactericides.

[0078] The present invention mainly has the following beneficial effects:

[0079] The Bacillus TG1-2 of the present invention has the effect of inhibiting plant pathogens. It has an inhibitory effect on various pathogenic fungi and oomycetes, such as cotton verticillium wilt and lettuce wilt caused by Verticillium dahliae, rice blast caused by Magnaporthe oryzae, Fusarium graminearum caused by Fusarium graminearum, grape gray mold caused by Botrytis cinerea, soybean sclerotinia caused by Sclerotinia sclerotiorum and soybean phytophthora caused by Phytophthora. The Bacillus TG1-2 of the present invention is derived from the rhizosphere soil of a weed in Zhangpu Salt Field, Fujian Province, has no adverse effects on the environment, can be cultured in vitro, has simple culture conditions, and is easy to obtain fermentation liquid. The fermentation liquid can inhibit a variety of plant pathogens, has a broad-spectrum antibacterial effect, and can play an important role in the biological control of farmland plant diseases.

[0080] The use of rhizosphere biocontrol microbial preparations can effectively reduce the use of chemical pesticides. The secondary metabolites of rhizosphere microorganisms are one of the important sources of fungicides, and have the advantages of being pure natural, pollution-free, safe and efficient.

[0081] The present invention is further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples where specific conditions are not specified are usually carried out according to conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, 3rd Edition, Science Press, or according to the conditions recommended by the manufacturer.

[0082] Example 1. Isolation and identification of strains

[0083] The strain of the present invention is isolated and cultured from the rhizosphere soil of high-salt weeds (isolated from the rhizosphere soil of a weed in Zhangpu Salt Field, Fujian Province). The specific method is: the collected rhizosphere soil suspension is diluted and coated on the LB solid culture medium, and placed in an incubator for overnight culture in the dark. After a single colony grows out, a single colony is picked and transferred to a new culture plate, and stored at 4°C until the colony on the culture plate is single and there are no miscellaneous bacteria around, thereby obtaining a purified product of the bacterium, and the bacterium is named TG1-2. In order to explore the optimal growth environment of the bacterium, a variety of different culture conditions were tried. Through testing, it was found that the optimal growth temperature of the bacterium is 28°C, the optimal salt concentration is 1-2M, and the optimal pH is 7.

[0084] Morphological analysis: TG1-2 was inoculated on LB solid medium and cultured overnight at 28°C. TG1-2 on the LB solid medium had irregular edges, was white and sticky, and had wrinkles on the surface.

[0085] Sequencing analysis: The TG1-2 colony was selected and inoculated into LB liquid medium. The genomic DNA was extracted after culturing at 28°C and 200 rpm / min for 48 hours. The 16S rDNA fragment of TG1-2 was amplified with the bacterial universal primers 27F / 1492R:

[0086] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3';

[0087] 1492R: 5'-GGTTACCTTGTTACGACTT-3';

[0088] Afterwards, the single target band detected by electrophoresis was sequenced. The sequencing results were compared with other known sequences by BLAST in NCBI, and gene sequences with homology above 98% were downloaded, and the phylogenetic tree was constructed using MEGA 11 software.

[0089] The analysis results are as follows Figure 1 As shown, the strain is Bacillus amyloliquefaciens.

[0090] Example 2: Inhibitory effect of Bacillus amyloliquefaciens TG1-2 on plant pathogens

[0091] 1. Experimental Methods

[0092] Seven pathogenic fungi and one oomycete were selected to verify the antibacterial effect of Bacillus TG1-2. The selected pathogens are:

[0093] Verticillium dahliae 592 (Vd592)

[0094] Verticillium dahliae Ls17 (VdLs17)

[0095] Rice blast fungus (Magnaporthe oryzae Guy11, MoGuy11);

[0096] Rice blast fungus (Magnaporthe oryzae Fj86, MoFj86);

[0097] Fusarium graminearum PH1 (FgPH1);

[0098] Grape gray mold (Botrytis cinerea, BC);

[0099] Soybean sclerotinia (Sclerotinia sclerotiorum, Ss);

[0100] Phytophthora sojae 6497 (Ps6497);

[0101] The plant pathogenic fungi were inoculated onto PDA solid culture medium respectively and placed in a 28°C incubator for 3-5 days for activation. After activation, in a sterile clean bench, use a sterilized toothpick to pick up the mycelium on the culture medium and draw a 0.5 cm long cross in the center of a new PDA culture plate (the culture plate has a diameter of 9 cm). The oomycetes were inoculated onto V8 solid culture medium in the same culture method as the pathogenic fungi. The Bacillus TG1-2 of Example 1 was inoculated into LB liquid culture medium and placed in a shaker at 28°C and 200 r / min for about 12 hours of shaking culture until its OD 600 =0.6, then added to 200 ml LB liquid medium at a ratio of 5% and cultured with shaking for 24 h until OD 600 =2.0, after shaking, draw 5 μL TG1-2 and spot it around the plate inoculated with plant pathogens, 2.5 cm away from the pathogens. The control groups were inoculated with pathogenic fungi but not TG1-2, and inoculated with pathogens and Bacillus subtilis GB03 (Bacillus subtitles) known to have biocontrol effects. Set up a blank control CK group. Place in a 28°C incubator and culture for 5 days. Record the diameter of the pathogens and calculate the inhibition rate.

[0102]

[0103] 2. Experimental Results

[0104] The results are as follows Figure 2 As shown, the growth diameters of cotton Verticillium dahliae, lettuce Verticillium dahliae, rice blast fungus, Fusarium graminearum, grape gray mold, soybean sclerotinia, and soybean phytopathogens that were cultured with Bacillus TG1-2 for four weeks were significantly smaller than those of the control group (p<0.05, Table 1). This indicates that Bacillus TG1-2 has a significant inhibitory effect on these plant pathogens, and the inhibition rate is more than 40% (Table 2).

[0105] Table 1. Inhibitory effect of Bacillus TG1-2 on the growth of plant pathogens (unit: cm)

[0106]

[0107] Table 2. Inhibition rate of Bacillus TG1-2 against plant pathogens (%)

[0108]

[0109] The above results show that Bacillus TG1-2 has a good inhibitory effect on plant diseases caused by various pathogens.

[0110] Example 3: Preparation of Bacillus TG1-2 fermentation broth

[0111] Bacillus TG1-2 of Example 1 was cultured by shaking according to the method of Example 2 until OD 600 =2.0.

[0112] The shaken bacteria were centrifuged at 7000g and 4°C for 20 min to remove the bacterial cells.

[0113] Afterwards, extraction was performed using a filter membrane with a pore size of 0.45 μm in a sterile clean bench, and the obtained fermentation liquid was the fermentation liquid of TG1-2.

[0114] Example 4: Inhibitory effect of Bacillus TG1-2 fermentation broth on plant pathogens

[0115] 1. Experimental Methods

[0116] The fermentation liquid of Bacillus TG1-2 prepared in Example 3 was mixed with PDA or V8 solid culture medium in a ratio of V:V=1:4 and poured onto a culture plate (6 cm in diameter) containing the fermentation liquid of TG1-2. In a sterile clean bench, the activated plant pathogens were inoculated in the center of the culture plate containing the TG1-2 fermentation liquid and placed in a 28°C incubator for culture. The control group (PDA culture medium with the same proportion of LB liquid culture medium added) was added with PDA culture medium or V8 culture medium with the same proportion of LB liquid culture medium. Bacillus subtilis GB03, which is known to have a biocontrol effect, was used as a positive control. The method for activating plant pathogens is the same as in Example 2.

[0117] 2. Experimental Results

[0118] The results are as follows Figure 3 As shown in Tables 3 and 4.

[0119] Table 3. Inhibitory effect of Bacillus TG1-2 fermentation broth on the growth of plant pathogens

[0120]

[0121] Table 4. Antibacterial rate of Bacillus TG1-2 fermentation liquid on plant pathogens

[0122]

[0123] The fermentation liquid containing Bacillus TG1-2 had strong growth inhibition activity against Verticillium dahliae, Verticillium dahliae, rice blast, Fusarium graminearum, Botrytis cinerea, Sclerotinia sclerotiorum, and Phytophthora sojae. The inhibition rates were 39.11% for Verticillium dahliae, 61.67% for Verticillium dahliae, 100% for both types of rice blast, 87.99% for Fusarium graminearum, 73.66% for Botrytis cinerea, 63.19% for Sclerotinia sclerotiorum, and 20.19% for Phytophthora sojae.

[0124] Among them, the antibacterial rate of Bacillus TG1-2 fermentation liquid against Verticillium dahliae, Magnaporthe grisea, and Fusarium graminearum was significantly higher than that of Bacillus subtilis GB03.

[0125] Example 5: Isolation and identification of antagonistic substances from Bacillus TG1-2

[0126] 1. Experimental Methods

[0127] The fermentation broth of Bacillus TG1-2 obtained in Example 3 was added to a precooled methanol / acetonitrile / water solution (v / v=2:2:1), vortexed, low-temperature ultrasonicated for 30 min, allowed to stand at -20°C for 10 min, centrifuged at 14000g for 20 min at 4°C, the supernatant was vacuum dried, and 100ul of 50% acetonitrile aqueous solution was added for re-dissolution during mass spectrometry analysis, vortexed, and centrifuged at 14000g for 15 min at 4°C.

[0128] After the supernatant was collected in the previous step, UHPLC was used to perform gradient elution using acetonitrile (95%, 65%, 40%, 95%) through a HILIC column. After the supernatant was separated by UHPLC, a Triple TOF 6600 mass spectrometer was used to collect spectra, and finally the mass spectrometry data was analyzed using XCMS software.

[0129] The experiment set Escherichia coli DH5α as the control group. Screen substances with significant up-regulation compared with Escherichia coli. The variable weight value (VIP) obtained by the OPLS-DA model can be used to measure the influence and explanatory power of the expression pattern of each metabolite on the classification and discrimination of each sample, and to mine differential molecules with biological significance. Usually VIP>1 and p value<0.05 are the screening criteria for significant differential metabolites.

[0130] 2. Experimental Results

[0131] As shown in Table 5, the most significantly upregulated metabolite in Bacillus TG1-2 was finally identified as surfactin (surfactin b and surfactin c are different variants). Compared with E. coli DH5α, surfactin b and surfactin c were significantly upregulated (p<0.05), with surfactin b upregulated 4349.07 times and surfactin c upregulated 4571.92 times.

[0132] Table 5. Metabolites upregulated in Bacillus TG1-2

[0133] name VIP FC p value m / z rt(s) Surfactin b 10.33 4349.07 0.01 1022.67 202.506 Surfactin c 13.5 4571.92 0.011 1036.69 201.143

[0134] In the table, name indicates the name of the metabolite; VIP indicates variable projection importance, the larger the value, the more important it is; FC indicates the difference fold; p value indicates significance, p<0.05 indicates a significant difference; m / z indicates mass-to-charge ratio; rt(s) indicates the retention time of the metabolite on the chromatogram.

[0135] Example 6: Antibacterial effect of Bacillus TG1-2 antagonist

[0136] 1. Experimental Methods

[0137] The surfactin isolated and identified in Example 4 was added to PDA or V8 solid culture medium at a concentration of 200 μM and poured into a plate (6 cm in diameter). The activated plant pathogens were inoculated in the center of the culture plate containing TG1-2 fermentation liquid in a sterile clean bench and placed in a 28°C incubator for culture. PDA or V8 culture medium without surfactin was set as a control. The plant pathogen activation method was the same as in Example 2.

[0138] 2. Experimental Results

[0139] like Figure 4 As shown, the growth colonies of cotton Verticillium dahliae, lettuce Verticillium dahliae, rice blast fungus, Fusarium graminearum, grape gray mold, soybean sclerotinia, and soybean Phytophthora on the culture plate containing surfactin were significantly smaller than those in the control group, indicating that surfactin has a good inhibitory effect on the above-mentioned plant pathogens and is an effective antibacterial substance of Bacillus TG1-2 against plant pathogens.

[0140] Biomaterial Deposit

[0141] The Bacillus amyloliquefaciens isolated and obtained in the present invention, referred to as Bacillus TG1-2, has been deposited in the China General Microbiological Culture Collection Center (CGMCC) (CCTCC), with a deposit number of CGMCC NO.28299 and a deposit date of August 30, 2023.

[0142] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for a person of ordinary skill in the art, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims. At the same time, all the documents mentioned in the present invention are cited as references in this application, just as each document is cited as reference separately.

Claims

1. An isolated bacillus strain, which is deposited in the China General Microbiological Culture Collection Center with a deposit number of CGMCC NO.28299.

2. The cell culture, cell metabolite, cell culture supernatant or cell lysate of the Bacillus according to claim 1, which has a property selected from the group consisting of: inhibiting plant pathogens; producing the metabolite surfactin; preferably, the surfactin is surfactin b or surfactin c.

3. The Bacillus according to claim 1, wherein the cell culture, cell metabolites, cell culture supernatant or cell lysate thereof is used for inhibiting plant pathogens or preparing a composition for inhibiting plant pathogens.

4. The use according to claim 3, characterized in that: The plant pathogens include: pathogenic fungi, oomycetes; preferably, they are selected from: Verticillium dahliae, Magnaporthe oryzae, Fusarium graminearum, Botrytis cinerea, Sclerotiniasclerotiorum, Phytophthora sojae.

5. The use according to claim 4, characterized in that: The Bacillus, its cell culture, cell metabolites, cell culture supernatant or cell lysis products are used to prevent and control plant diseases caused by the plant pathogens; preferably, the plant diseases include: Verticillium wilt, rice blast, Fusarium graminearum, gray mold, sclerotinia disease, and Phytophthora.

6. The Bacillus according to claim 1, wherein the cell culture, cell metabolites, cell culture supernatant or cell lysate thereof is used to: produce or prepare the metabolite surfactin; or prepare a composition containing the surfactin; preferably, the surfactin is surfactin b or surfactin c.

7. A method for preparing the metabolite surfactin, comprising culturing the Bacillus according to claim 1 to produce surfactin; preferably, further comprising isolating and purifying the surfactin from the culture; preferably, the surfactin is surfactin b or surfactin c.

8. A composition for inhibiting plant pathogens, comprising a component selected from the group consisting of: the Bacillus according to claim 1; or a cell culture, cell metabolite, cell culture supernatant or cell lysate of the Bacillus.

9. A method for inhibiting plant pathogens, comprising: The object to be inhibited or degraded is treated using the Bacillus described in claim 1, or its cell culture, cell metabolites, cell culture supernatants or cell lysates.

10. A method for preparing a metabolite surfactin, comprising culturing the Bacillus according to claim 1 to express surfactin; preferably, the surfactin is surfactin b or surfactin c.

11. A kit for inhibiting plant pathogens, comprising: The Bacillus according to claim 1; The cell culture, cell metabolites, cell culture supernatants or cell lysates of the Bacillus according to claim 2; and / or The composition for inhibiting plant pathogens as claimed in claim 7.

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