A biocontrol Bacillus amyloliquefaciens BA200 and its application

Abstract

The present invention belongs to the field of modern agricultural technologies, and particularly relates to a biocontrol Bacillus amyloliquefaciens BA200 and its applications. A new biocontrol bacterium BA200 is isolated and identified in the present invention, and its taxonomic name is Bacillus amyloliquefaciens Bacillus amyloliquefaciens , and this strain has significant inhibitory ability against two pathogenic bacteria, Fusarium oxysporum and Phytophthora vignae, which cause cowpea wilt disease, with an inhibition rate of over 60%; the fermentation broth prepared therefrom can significantly reduce the incidence and disease index of cowpea wilt disease. It is an excellent biocontrol microorganism, has application and development potential and important significance in the prevention and control of cowpea wilt disease, and this biocontrol bacterium, Bacillus amyloliquefaciens BA200, has a broad-spectrum biocontrol effect and can inhibit various plant pathogenic bacteria, providing a valuable biocontrol bacterial resource for the preparation of biological fungicides.

Description

Technical Field

[0001] This invention belongs to the field of modern agricultural technology, specifically relating to a biocontrol strain of Bacillus amyloliquefaciens BA200 and its applications. Background Technology

[0002] Soybeans are a globally important food and oil crop, providing essential nutrients for humankind through their rich plant protein and oil content. However, with the increasing planting area and the expansion of soybean continuous cropping, soybean root rot, caused by Fusarium and Phytophthora soybeanii, has become a devastating disease affecting soybean production, severely impacting its economic benefits. This challenge in controlling soil-borne diseases is common among legumes, with cowpeas, another important economic crop, facing even greater difficulties. As one of the six major edible legumes, cowpeas have high economic value. In cowpea cultivation, once soil-borne diseases occur, they can cause widespread wilting of cowpea plants, sometimes resulting in complete crop failure. Among these, cowpea wilt is the most prevalent, damaging, and difficult to control disease in cowpea cultivation. The pathogens causing cowpea wilt mainly include Fusarium oxysporum and Phytophthora chinensis. Similar to soybean root rot, these soil-borne pathogens infect plants from the roots or stem base under suitable conditions, leading to stem base necrosis, leaf wilting, and ultimately, the death of the entire plant. Because cowpea diseases are rampant during growth, over-reliance on chemical pesticides for wilt control can easily result in excessive pesticide use and excessive pesticide residues. Therefore, developing bio-based fungicides to replace chemical fungicides for the control of cowpea wilt is urgently needed.

[0003] Biological control has become one of the most promising strategies for controlling soil-borne diseases because it can protect the farmland ecosystem, improve human and animal safety, and delay the development and spread of pesticide resistance in pathogens. In cowpea disease control, choosing biological control can effectively reduce the use of chemical pesticides. While controlling diseases, it can also promote plant growth, increase crop yield, and significantly improve ecological and social benefits, making it a crucial measure to ensure high-quality and high-yield cowpea production.

[0004] Rhizosphere-promoting bacteria are a major source of biocontrol agents. Currently, the main biocontrol bacteria used in production include Pseudomonas, Streptomyces, and Bacillus. Their disease-resistance mechanisms include lysis, competition, antagonism, and induction of plant resistance. Besides disease resistance, biocontrol bacteria also significantly promote plant growth, such as nitrogen fixation, improved nutrient utilization, and the release of plant hormones. Among them, Bacillus is an important biocontrol bacterium, easily isolated from soil and plants, and exhibiting good disease control effects. This bacterium is characterized by strong resistance, rapid reproduction, simple nutritional requirements, and easy colonization on plant surfaces, which is beneficial for the production and storage of biocontrol agents. In soil ecological restoration, Bacillus also has the functions of phosphorus solubilization, potassium solubilization, and improving soil fertility. Currently, highly effective biocontrol bacteria for cowpea diseases still need to be developed. Summary of the Invention

[0005] The first aspect of the present invention is to provide a strain of Bacillus amyloliquefaciens.

[0006] A second aspect of the present invention is to provide a culture.

[0007] The third objective of this invention is to provide a method for preparing the culture of the second aspect of this invention.

[0008] The fourth aspect of this invention is to provide a microbial agent.

[0009] The fifth aspect of this invention is to provide a method for preparing the microbial agent of the fourth aspect of this invention.

[0010] The sixth aspect of this invention aims to provide the application of the biocontrol bacteria of the first aspect, the culture of the second aspect, and the bacterial agent of the fourth aspect.

[0011] The seventh aspect of this invention aims to provide a product.

[0012] The object of the eighth aspect of the present invention is to provide a method.

[0013] To achieve the above-mentioned objectives of this invention, the technical solution adopted by this invention is as follows:

[0014] In a first aspect, the present invention provides a Bacillus amyloliquefaciens BA200, which is classified and named Bacillus amyloliquefaciens. Bacillus amyloliquefaciens It was deposited on February 24, 2025, at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No: 33636.

[0015] The colony surface of *Bacillus amyloliquefaciens* BA200 is rough; the colonies are round, white, with irregular edges and raised. When cultured statically in liquid, a biofilm forms. Under a microscope, the bacteria are rod-shaped, show Gram-positive staining, and possess antibacterial extracellular enzyme activities such as protease, cellulase, and β-1,3-glucan, as well as the ability to solubilize phosphorus, potassium, and nitrogen, and to produce siderophores and IAA.

[0016] A second aspect of the present invention provides a culture obtained by culturing Bacillus amyloliquefaciens BA200, which is described in the first aspect of the present invention.

[0017] In some embodiments of the present invention, the culture is prepared by inoculating a single colony of activated Bacillus amyloliquefaciens BA200 into LB liquid medium, culturing at 37°C and 180 r / min for 24 h to obtain a seed culture, and then adding 1% of the seed culture to LB liquid medium for further expansion to OD. 600 =0.6 (concentration is 1×10) 7 The fermentation product was obtained by (CFU / mL).

[0018] A third aspect of the present invention provides a method for preparing a culture of the second aspect of the present invention, wherein the biocontrol bacterium BA200 of the first aspect of the present invention is inoculated into a culture medium and cultured to obtain the culture; preferably, the culture medium includes LB medium.

[0019] In some embodiments of the present invention, the culture temperature is 16-42°C; preferably, the culture temperature is 30-37°C.

[0020] In some embodiments of the present invention, the culture time is 12-96 hours; preferably, the culture time is 18-36 hours.

[0021] In some embodiments of the present invention, the preparation method involves inoculating a single colony of activated Bacillus amyloliquefaciens BA200 into LB liquid medium, culturing at 37°C and 180 r / min for 24 h to obtain a seed culture, and then adding 10% of the seed culture to LB liquid medium to continue culturing until OD. 600 =1.0 bacterial suspension was used as a culture of Bacillus amyloliquefaciens BA200.

[0022] In a fourth aspect, the present invention provides a microbial agent comprising the biocontrol bacterium BA200 of the first aspect of the present invention and / or the culture of the second aspect of the present invention.

[0023] In some embodiments of the present invention, the microbial agent is a solid microbial agent or a liquid microbial agent.

[0024] In some embodiments of the present invention, the solid microbial agent comprises a carrier.

[0025] In some embodiments of the present invention, the carrier comprises at least one of mineral materials and plant materials.

[0026] In some embodiments of the present invention, the mineral material comprises at least one of activated carbon, clay, peat moss, vermiculite, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth.

[0027] In some embodiments of the present invention, the plant material comprises at least one of corn flour, soybean flour, and wheat bran.

[0028] In some embodiments of the present invention, the liquid bacterial agent includes a protectant.

[0029] In some embodiments of the present invention, the protective agent includes one or more of glycerol, benzoate, sorbate, polyethylene glycol, and Tween.

[0030] In some embodiments of the present invention, the dosage form of the microbial agent is at least one of liquid, powder or granules.

[0031] A fifth aspect of the present invention provides a method for preparing the bacterial agent of the fourth aspect of the present invention, wherein the bacterial agent is obtained by using Bacillus amyloliquefaciens BA200 of the first aspect of the present invention and / or the culture of the second aspect of the present invention as active ingredients.

[0032] A sixth aspect of the present invention provides the use of Bacillus amyloliquefaciens BA200 of the first aspect of the present invention, the culture of the second aspect of the present invention, and / or the bacterial agent of the third aspect of the present invention in the preparation of products for the prevention and control of plant pathogens.

[0033] In some embodiments of the present invention, the plant pathogen includes Phytophthora soybeanum (… Phytophthora sojae Fusarium oxysporum ( Fusarium oxysporum Phytophthora indicum ( ) Phytophthora vignae Fusarium solani () Fusarium solani Rhizoctonia solani ( ) Rhizoctonia solani ), Flathead anthrax bacteria ( Colletotrichum truncatum ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides Alternaria species ( Alternaria eichhorniae) Polysporum ( ) Nodulisporium sp.), *Lycium chinense* (sp.), *Lycium chinense* Myrothecium inundatum ) and Phytophthora palmatum ( Phytophthora palmivora ).

[0034] The diseases associated with the aforementioned plant pathogens include, but are not limited to:

[0035] Phytophthora in soybean causes Phytophthora root rot and soybean blight.

[0036] Fusarium oxysporum causes wilt disease and has a wide host range, including but not limited to tomatoes, bananas, watermelons, and cowpeas.

[0037] Phytophthora indicum causes cowpea wilt disease.

[0038] Fusarium solani causes root rot, and its hosts include, but are not limited to, soybeans, peas, and cowpeas.

[0039] Rhizoctonia solani causes damping-off and sheath blight.

[0040] Anthrax is caused by the flat-headed anthrax bacterium.

[0041] Colletotrichum anthracnose causes anthrax.

[0042] Alternaria species cause black spot or leaf spot diseases.

[0043] Polysporum species cause sedosporidiosis.

[0044] Varicella asiatica can cause leaf spot or stem rot.

[0045] Phytophthora palmis causes palm disease.

[0046] A seventh aspect of the present invention provides a product comprising the biocontrol bacterium BA200 of the first aspect of the present invention, the culture of the second aspect of the present invention, and / or the bacterial agent of the third aspect of the present invention.

[0047] In some embodiments of the present invention, the product includes excipients; the excipients include at least one of solvent, colorant, stabilizer, preservative, pH adjuster, buffer, surfactant, foaming agent, defoamer, thickener, and carrier.

[0048] In some embodiments of the present invention, the product may also include other antimicrobial active ingredients, including but not limited to antimicrobial peptides, antibiotics, or other biocontrol bacteria.

[0049] An eighth aspect of the present invention provides a method for controlling plant pathogens, comprising the steps of using Bacillus amyloliquefaciens BA200 of the first aspect of the present invention, a culture of the second aspect of the present invention, and / or a microbial agent of the third aspect of the present invention.

[0050] In some embodiments of the present invention, the method includes the step of inoculating cowpeas with the fermentation broth of Bacillus amyloliquefaciens BA200 by root irrigation for biological control of cowpea wilt disease.

[0051] The beneficial effects of this invention are:

[0052] This invention has isolated and identified a new biological control bacterium, named *Bacillus amyloliquefaciens* (BAM). Bacillus amyloliquefaciensThe biocontrol bacterium Bacillus amyloliquefaciens BA200 was deposited at the China General Microbiological Culture Collection Center on [date missing], with accession number CGMCC No. 33636. This strain exhibits significant inhibitory activity against both Fusarium oxysporum and Phytophthora spp., the two main pathogens causing cowpea wilt. The average inhibition rate of biocontrol bacterium BA200 against Fusarium oxysporum exceeds 60%, and the average inhibition rate against Phytophthora spp. exceeds 75%. Furthermore, biocontrol bacterium BA200 has broad-spectrum biological control effects, showing significant inhibitory effects on the mycelial growth of various plant pathogens such as Fusarium solanaceum, Rhizoctonia solani, Anthracnose spp., Colletotrichum gloeosporioides, Alternaria alternata, Polynosporium spp., Phytophthora spp., Lacquer spp., and Phytophthora palmatum. In addition, biocontrol bacterium BA200 possesses antimicrobial extracellular enzyme activities such as protease, cellulase, and β-1,3-glucanase, as well as growth-promoting traits such as phosphorus solubilization, potassium solubilization, nitrogen fixation, and the production of IAA and siderophores. The use of fermentation broth prepared from biocontrol bacteria BA200 for root irrigation of cowpea plants can significantly reduce the incidence of disease and disease index in seedlings, providing a valuable resource for the preparation of biocontrol agents. Attached Figure Description

[0053] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0054] Figure 1 Morphological observation and Gram staining of biocontrol bacterium BA200; Figure 1 The left image shows the colony morphology of biocontrol bacteria BA200 after 24 h of culture on LB medium, and the right image shows the Gram staining of biocontrol bacteria BA200 under a microscope.

[0055] Figure 2 The figure shows the results of the physiological and biochemical characteristics experiment of biocontrol bacterium BA200, where CK represents the control group and 200 is the number of the biocontrol bacterium BA200 used.

[0056] Figure 3 A phylogenetic tree for the biocontrol bacterium BA200 constructed based on its 16S rDNA sequence.

[0057] Figure 4 The graph shows the results of the extracellular enzyme activity and growth-promoting characteristics of biocontrol bacterium BA200, where CK represents the control group and 200 is the number of the biocontrol bacterium BA200 used.

[0058] Figure 5 The image shows the plate confrontation results of biocontrol bacterium BA200 against Fusarium oxysporum, Phytophthora cowpea, and Phytophthora soybean; where CK represents the control group using sterile water, and 200 is the number of the biocontrol bacterium BA200 used.

[0059] Figure 6The results of plate confrontation between biocontrol agent BA200 and various plant pathogens are shown in the figure.

[0060] Figure 7 This study aimed to evaluate the control efficacy of biocontrol bacterium BA200 fermentation broth against Fusarium oxysporum and Phytophthora chinensis in cowpea wilt. The control group (CK) was treated with 50 mL of sterile water. BA200 was treated with only 50 mL of the biocontrol bacterium. Fo was treated with 50 mL of sterile water followed by inoculation with Fusarium oxysporum 24 h later. BA200+Fo was treated with 50 mL of BA200 fermentation broth followed by inoculation with Fusarium oxysporum 24 h later. Pv was treated with 50 mL of sterile water followed by inoculation with Phytophthora chinensis 24 h later. BA200+Pv was treated with 50 mL of BA200 fermentation broth followed by inoculation with Phytophthora chinensis 24 h later. Detailed Implementation

[0061] The following will describe the concept and technical effects of the present invention clearly and completely with reference to embodiments, so as to fully understand the purpose, features and effects of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are all within the scope of protection of the present invention.

[0062] Example 1: Purification of BA200

[0063] The biocontrol bacterium BA200 in this invention was isolated by the inventors from the rhizosphere soil of cowpeas in Yazhou District, Sanya City, Hainan Province.

[0064] Take a small amount of the preserved biocontrol bacterium BA200, dip it into the bacterial solution, and purify it by streaking it on LB solid medium using the dilution plate method. Incubate at 37°C upside down for 24 h to obtain purified single colonies.

[0065] Example 2 Identification of BA200

[0066] 1. Morphological observation

[0067] like Figure 1 As shown, the biocontrol bacterium BA200 on LB solid medium has a rough surface, with round white colonies that are irregular in edge and raised. When the liquid culture is stationary, a bacterial film forms. Under a microscope, the bacteria are rod-shaped and show positive Gram staining results.

[0068] 2. Identification of physiological and biochemical characteristics

[0069] The biocontrol bacterium BA200 was subjected to Gram staining, catalase assay, oxidase assay, methyl red assay, VP assay, indole assay, citrate utilization assay, starch hydrolysis assay, cellulose decomposition assay, and carbon source utilization assay. The experimental methods were based on the "Handbook of Systematic Identification of Common Bacteria," and the results of its physiological and biochemical characterization are shown in Table 1 and... Figure 2 As shown.

[0070] Table 1. Identification results of physiological and biochemical characteristics of biocontrol bacterium BA200

[0071]

[0072] Note: "+" indicates positive, "-" indicates negative.

[0073] 3. Molecular biological identification

[0074] The biocontrol bacterium BA200 obtained in Experiment 1 was used to extract DNA from the biocontrol bacterium BA200 using a bacterial genomic DNA extraction kit. The genome of the bacteria was amplified using universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 2). The 50 μL PCR system consisted of 25 μL of 2×Taq Master Mix, 2 μL of each primer, 1 μL of DNA template, and 20 μL of dd H2O. The amplification program was: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 1 min 15 s, for a total of 30 cycles, followed by a final extension at 72℃ for 10 min. The amplified products were sequenced by 1.5% agarose gel electrophoresis. The sequences were compared for homology in the GenenBank nucleic acid sequence library in NCBI. Known biocontrol bacterial sequences with high homology to the sequence of the antagonistic strain were downloaded and analyzed using MEGAE. Software version 11.0 uses the neighbor-joining method to construct a phylogenetic tree based on the 16S gene. The constructed phylogenetic tree is as follows: Figure 3 As shown.

[0075] The 16S rDNA sequence of biocontrol bacterium BA200 was BLAST-aligned in GenBank. The alignment results showed that the 16S rDNA sequence of biocontrol bacterium BA200 was consistent with... Bacillus amyloliquefaciens (Accession number: HM055610.1) Belonging to the same branch in the system, and based on colony morphology and physiological and biochemical characteristics, BA200 was identified as... Bacillus amyloliquefaciens .

[0076]

[0077] 4. Determination of antibacterial extracellular enzyme activity and growth-promoting properties of biocontrol bacterium BA200

[0078] The antibacterial extracellular enzyme activity of biocontrol bacterium BA200 was detected using protease, cellulase, and β-1,3-glucanase culture media, and the growth-promoting characteristics of biocontrol bacterium BA200 were detected using phosphorus-solubilizing, potassium-solubilizing, nitrogen-fixing, and ferrophilic detection media.

[0079] The protease detection medium consisted of 3.0 g skim milk powder, 4 g agar, and sterile water to a final volume of 200 mL, with a pH of 7.2. The medium was sterilized at 115°C for 30 min. Single colonies were picked up using a sterile inoculation needle and inoculated onto the protease detection medium plate. The plates were incubated at 30°C for 5 days. The presence of a clear zone around the colony was observed; the presence of a clear zone indicated protease activity.

[0080] The cellulase detection medium consisted of: 2 g sodium carboxymethyl cellulose, 2 g peptone, 2 g yeast extract, 1 g NaCl, 0.2 g KH₂PO₄, 4 g agar, and 200 mL sterile water, with a pH of 7.2. The medium was sterilized at 115°C for 30 min. Single colonies were picked up using a sterile inoculation needle and inoculated onto cellulase detection medium plates. The plates were incubated at 30°C for 5 days. The plates were then completely submerged in 1 mg / mL Congo red solution for 30 min to stain the cells. The staining solution was discarded, and the plates were washed with 1 mol / L NaCl solution. The presence of a clear zone indicated cellulase activity.

[0081] The β-1,3-glucanase detection medium consisted of: 0.4 g β-1,3-glucan, 0.01 g Congo red, 0.4 g NaNO3, 0.2 g KH2PO4, 0.1 g KCl, 0.1 g MgSO4·7H2O, 0.002 g FeSO4, 4 g agar, and sterile water to a final volume of 200 mL. The pH was 7.2, and the medium was sterilized at 115℃ for 30 min. Single colonies were picked up using a sterile inoculation needle and inoculated onto β-1,3-glucanase medium plates. The plates were incubated at 30℃ for 5 days, and the presence of a clear zone was observed. The presence of a clear zone indicated the presence of β-1,3-glucanase activity.

[0082] Inorganic phosphorus medium: (NH4)2SO4 0.1 g, MgSO4·7H2O 0.1 g, NaCl 0.04 g, Ca3(PO4)2 2 g, CaCO3 0.1 g, MnSO4 0.004 g, FeSO4 0.004 g, glucose 0.5 g, agar 4 g, diluted to 200 mL with sterile water, pH 7.2, sterilized at 115℃ for 30 min. Single colonies were picked up with a sterile inoculation needle and inoculated onto inorganic phosphorus medium plates. The plates were incubated at 30℃ for 5 days. The presence of a clear zone indicates the ability to decompose inorganic phosphorus.

[0083] Organophosphorus culture medium: (NH4)2SO4 0.1 g, CaCO3 0.1 g, MgSO4·7H2O 0.1 g, NaCl 0.04 g, MnSO4 0.004 g, FeSO4 0.004 g, lecithin 0.04 g, glucose 0.4 g, agar 4 g, diluted to 200 mL with sterile water, pH 7.2, sterilized at 115℃ for 30 min. Single colonies were picked up with a sterile inoculation needle and inoculated onto organophosphorus culture medium plates. The plates were incubated at 30℃ for 5 days. The presence of a clear zone indicates the ability to decompose organophosphorus compounds.

[0084] Nitrogen-free medium: NaCl 0.024 g, KH2PO4 0.1 g, CaCO3 0.04 g, MgSO4·7H2O 0.04 g, sucrose 2 g, agar 4 g, diluted to 200 mL with sterile water, pH 7.2, sterilized at 115℃ for 30 min. Single colonies were picked up using a sterile inoculation needle and inoculated onto nitrogen-free medium plates. The plates were incubated at 30℃ for 5 days. The nitrogen-fixing ability of the biocontrol bacteria was determined by their growth on the medium and the presence or absence of a clear zone around them. The presence of a clear zone around the biocontrol bacteria indicates nitrogen-fixing capability.

[0085] Potassium feldspar medium: (NH4)2SO4 0.2 g, MgSO4·7H2O 0.1 g, NaCl 0.02 g, Na2HPO4 0.4 g, sucrose 2 g, yeast extract 0.1 g, potassium feldspar powder 2 g, agar 4 g, sterile water to a final volume of 200 mL, pH 7.2, sterilized at 115℃ for 30 min. Single colonies were picked up using a sterile inoculation needle and inoculated onto potassium feldspar medium plates. The plates were incubated at 30℃ for 5 days. The presence of a clear zone around the colony indicated potassium solubilizing ability.

[0086] Ferrophilic assay medium: Use a sterile inoculation needle to pick up a single colony and inoculate it onto a CAS assay medium plate. Incubate at 30℃ for 5 days. Observe the formation of an orange-yellow ring around the colony. The presence of an orange-yellow halo indicates that the colony has the ability to produce siderophores.

[0087] IAA production capacity: Add L-tryptophan to LB liquid medium at a dosage of 100 mg / L, incubate at 37℃ and 180 r / min for 72 h, add a small amount of Salkowsk colorimetric solution and protect from light, observe after 30 min, the presence of red color indicates the production of IAA.

[0088] The results of the determination of extracellular enzyme activity and growth-promoting properties of biocontrol bacterium BA200 showed that ( Figure 4 ): Biocontrol bacterium BA200 has antibacterial extracellular enzyme activities such as protease, cellulase, and β-1,3-glucan, as well as the ability to solubilize phosphorus, potassium, fix nitrogen, and produce siderophores and IAA.

[0089] After completing strain identification, the biocontrol bacterium BA200 was deposited on February 24, 2025, at the China General Microbiological Culture Collection Center (No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences), with accession number CGMCC No: 33636. Its suggested taxonomic name is *Bacillus amyloliquefaciens*. Bacillus amyloliquefaciens.

[0090] Example 3: Inhibitory effect of biocontrol bacterium BA200 on Fusarium wilt pathogen of cowpea

[0091] 1. Experimental Methods

[0092] Preparation of biocontrol bacteria culture:

[0093] Single colonies obtained in Experiment 1 were picked and placed in 4 mL of LB medium and cultured at 37°C with shaking at 180 r / min for 24 h. Then, 10% of the colonies were added to 4 mL of LB medium and cultured at 37°C with shaking at 180 r / min to obtain a culture with OD600=1.0.

[0094] The inhibitory effect of the culture of biocontrol bacterium BA200 on Fusarium oxysporum and Phytophthora cowpeae was tested using the plate confrontation method. The culture of biocontrol bacterium BA200 was dropped in a cross shape around the center of the plate at a distance of 3.5 cm. Then, Fusarium oxysporum, Phytophthora cowpeae and Phytophthora sacchari were cut into 2 mm × 2 mm pieces and inoculated into the center of the plate respectively. The plate inoculated only with pathogens served as a control. The plates were incubated at 25°C in the dark. When the control colonies were almost fully covered by the plate, the colony diameter was measured and the colony inhibition rate was calculated.

[0095] Colony inhibition rate (%) = [(colony diameter of control group - colony diameter of treatment group) / colony diameter of control group] × 100.

[0096] 2. Experimental results

[0097] like Figure 5 As shown, the culture of biocontrol bacterium BA200 has a significant inhibitory effect on Fusarium oxysporum, Phytophthora cowpea, and Phytophthora soybean. Its inhibition rate against Fusarium oxysporum is 63.81%, against Phytophthora cowpea is 75.27%, and against Phytophthora soybean is 61.84%.

[0098] Example 4: Inhibitory effect of biocontrol bacterium BA200 on various plant pathogens

[0099] The inhibitory effect of biocontrol bacterium BA200 on various plant pathogens, including Fusarium solanum, was determined using the method described in Experiment Example 3. Fusarium solani Rhizoctonia solani ( ) Rhizoctonia solani ), Flathead anthrax bacteria ( Colletotrichum truncatum ), Colloidal anthrax bacteria ( Colletotrichum gloeosporioides Alternaria ( Alternaria eichhorniae ), Polysporum ( Nodulisporium sp.), *Lycium chinense* (sp.), *Lycium chinense* Myrothecium inundatum ) and Phytophthora palmatum ( Phytophthora palmivora The inhibitory effects of biocontrol agent BA200 on various plant pathogens are shown in Table 2 and... Figure 6 As shown.

[0100] Table 2. Inhibitory effects of biocontrol agent BA200 on various plant pathogens

[0101]

[0102] From Table 2 and Figure 6 It can be seen that Bacillus amyloliquefaciens BA200 has a good inhibitory effect on all eight plant pathogens tested, with an average inhibition rate of over 50%, indicating that Bacillus amyloliquefaciens BA200 has a broad-spectrum biological control effect.

[0103] Example 5: Pot control efficacy determination of biocontrol bacterium BA200

[0104] A single colony obtained in Experiment 1 was picked and placed in 4 mL of LB medium, and cultured at 37°C with shaking at 180 r / min for 24 h. Then, 200 mL of LB medium was added at a 1% volume ratio, and the culture was continued at 37°C with shaking at 180 r / min, yielding an OD600 of 0.6 (concentration 1×10⁻⁶). 7 Fermentation broth (CFU / mL).

[0105] The concentration of the Fusarium oxysporum inoculation conidial suspension was 5 × 10⁻⁶. 61 spore / mL. Phytophthora cowpea was inoculated using the soil-inoculum method, in which Phytophthora cowpea that had covered a 9 cm culture dish was broken up with a syringe and mixed with soil.

[0106] The cowpea variety chosen is "Nongwang Yousheng 380," sourced from Jiangxi Nongwang High-Tech Co., Ltd.

[0107] Treatments included (1) healthy plants (CK); (2) inoculation with Fusarium oxysporum (Fo); (3) inoculation with Phytophthora vulgaris (Pv); (4) inoculation with biocontrol agent BA200 (BA200); (5) biocontrol agent BA200 + Fusarium oxysporum (BA200 + Fo); and (6) biocontrol agent BA200 + Phytophthora vulgaris (BA200 + Pv).

[0108] After sterilization, cowpea seeds are sown in moist vermiculite. When the cowpea seedlings have two fully unfolded true leaves, they are transplanted into moist nutrient substrate and inoculated with 50 mL of a 1×10⁻⁶ solution. 7 The biocontrol bacteria fermentation broth at CFU / mL was used. The control group was inoculated with sterile water, and the pathogens were inoculated 24 h later. The pathogenicity of Fusarium oxysporum was determined by root immersion inoculation, and the pathogenicity of Phytophthora cowpea was determined by soil inoculation. Six strains were used for each treatment, and the results were repeated three times. The incidence of disease in each treatment was recorded.

[0109] Disease incidence includes incidence rate and disease classification, which are divided into 6 levels:

[0110] Grade 0: Vascular bundles are normal, with no symptoms;

[0111] Grade 1: Discoloration of the vascular bundle stem <1 / 4;

[0112] Grade 2: 1 / 4 of the vascular bundle stem ≤ degree of discoloration < 1 / 2;

[0113] Grade 3: The degree of discoloration of the vascular bundles is less than 3 / 4, and some leaves are yellowing and withering;

[0114] Level 4: Discoloration of the vascular bundles is greater than 3 / 4, with leaves turning yellow, withering, and falling off;

[0115] Level 5: The entire plant is wilted, fallen over, or dead.

[0116] Disease index = [∑(number of cowpea plants at each disease level × number of disease levels) / total number of potted cowpea plants × highest disease level] × 100.

[0117] Prevention and control effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0118] The results are shown in Tables 3 and 4. Figure 7 As shown.

[0119] Table 3. Statistical results of disease incidence and control effects of cowpea wilt (Fusarium oxysporum).

[0120]

[0121] From Table 3 and Figure 7 It is known that Bacillus amyloliquefaciens BA200 of the present invention can significantly reduce the severity of cowpea wilt caused by Fusarium oxysporum, indicating that Bacillus amyloliquefaciens BA200 has a preventive and control effect on cowpea wilt caused by Fusarium oxysporum, with a control effect of 72.06%.

[0122] Table 4. Statistical results of incidence and control effects of cowpea wilt (Phytophthora chinensis).

[0123]

[0124] From Table 4 and Figure 7 It is known that Bacillus amyloliquefaciens BA200 of the present invention can significantly reduce the severity of cowpea wilt caused by Phytophthora indica, indicating that Bacillus amyloliquefaciens BA200 has a preventive and control effect on cowpea wilt caused by Phytophthora indica, with a control effect of 83.10%.

Claims

1. A Bacillus amyloliquefaciens BA200, characterized in that: The taxonomic name of the Bacillus amyloliquefaciens BA200 is Bacillus amyloliquefaciens Bacillus amyloliquefaciens , which was deposited in the China General Microbiological Culture Collection Center on February 24, 2025, with the deposit number CGMCC No: 33636.

2. A culture of the Bacillus amyloliquefaciens BA200 according to claim 1, characterized in that: The preparation method of the culture comprises the following steps: Inoculating the Bacillus amyloliquefaciens BA200 according to claim 1 into a medium, culturing to obtain; The temperature of the culturing is 16 - 42 °C; The time of the culturing is 12 - 96 h; The medium comprises LB medium.

3. A preparation method of a culture, comprising the following steps: Inoculating the Bacillus amyloliquefaciens BA200 according to claim 1 into a medium, culturing to obtain; The temperature of the culturing is 16 - 42 °C; The time of the culturing is 12 - 96 h; The medium comprises LB medium.

4. A bacterial agent, comprising the Bacillus amyloliquefaciens BA200 according to claim 1 or the culture according to claim 2.

5. The bacterial agent according to claim 4, characterized in that: The bacterial agent is a solid bacterial agent or a liquid bacterial agent.

6. A preparation method of the bacterial agent according to claim 4 or 5, using the Bacillus amyloliquefaciens BA200 according to claim 1 or the culture according to claim 2 as an active ingredient to obtain the bacterial agent.

7. Use of the Bacillus amyloliquefaciens BA200 according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 4 or 5 in the preparation of a product for controlling plant pathogenic bacteria; The plant pathogenic bacteria are at least one of Phytophthora sojae, Fusarium oxysporum, Phytophthora vignae, Fusarium solani, Rhizoctonia solani, Colletotrichum truncatum, Colletotrichum gloeosporioides, Alternaria spp., Collectotrichum spp., Myrothecium roridum, and Phytophthora palmivora.

8. A product, comprising one of the Bacillus amyloliquefaciens BA200 according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 4 or 5; The product comprises excipients.

9. A method for controlling plant pathogenic bacteria, comprising the step of treating with the Bacillus amyloliquefaciens BA200 according to claim 1, the culture according to claim 2, or the bacterial agent according to claim 4 or 5; The plant pathogenic bacteria are at least one of Phytophthora sojae, Fusarium oxysporum, Phytophthora vignae, Fusarium solani, Rhizoctonia solani, Colletotrichum truncatum, Colletotrichum gloeosporioides, Alternaria spp., Collectotrichum spp., Myrothecium roridum, and Phytophthora palmivora.

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