Biocontrol bacillus amyloliquefaciens BA200 and application thereof

Abstract

The invention belongs to the technical field of modern agriculture, and particularly relates to a biocontrol bacillus amyloliquefaciens BA200 and application thereof. A new biological control bacterium BA200 is separated and identified, is named as bacillus amyloliquefaciens by taxonomy, and has a remarkable inhibition capability on two pathogenic bacteria, namely fusarium oxysporum and phytophthora vigna, causing the cowpea fusarium wilt, and the inhibition rate is up to 60% or above; fermentation liquor prepared from the bacillus amyloliquefaciens BA200 can remarkably reduce the morbidity and disease index of cowpea fusarium wilt, is a good biocontrol microorganism and has application and development potential and important significance in control of cowpea fusarium wilt, and the biocontrol bacterium bacillus amyloliquefaciens BA200 has a broad-spectrum biological control effect, can inhibit various phytopathogens and has a good application prospect. And valuable biocontrol bacterium resources are provided for preparation of biological bactericides.

Description

Technical Field

[0001] The invention belongs to the technical field of modern agriculture, and particularly relates to a biocontrol Bacillus amyloliquefaciens BA200 and an application thereof. Background Art

[0002] Soybean is an important grain and oil crop in the world. Its rich plant protein and oil provide an important source of nutrition for humans. With the increase in soybean planting area and the expansion of soybean continuous cropping, soybean root rot caused by Fusarium and Phytophthora sojae has become a devastating disease that endangers soybean production, seriously damaging the economic benefits of soybean. This dilemma of soil-borne disease prevention and control is common in leguminous crops, among which cowpea, which is also an important economic crop, faces even more severe challenges. As one of the six major edible legume crops, cowpea has a high economic value. In cowpea cultivation, once cowpea soil-borne diseases occur, it will cause large-scale withering of cowpea plants, and even no grains will be harvested in severe cases. Among them, cowpea wilt is the most common, most harmful, and most difficult disease to prevent and control during cowpea cultivation. The pathogens of cowpea wilt are mainly Fusarium oxysporum and Phytophthora vulgaris. Similar to soybean root rot, these soil-borne pathogens infect plants from the roots or stem base under suitable conditions, causing necrosis at the stem base, leaf wilting, and eventually the whole plant withering or even death. Due to the rampant occurrence of diseases during the growth of cowpeas, over-reliance on chemical agents to prevent and control cowpea wilt can easily lead to excessive use of chemical pesticides and excessive pesticide residues. Therefore, it is urgent to develop biogenic fungicides that can replace chemical fungicides for the prevention and control of cowpea wilt.

[0003] Biological control has become one of the most promising strategies for controlling soil-borne diseases because it can protect the farmland ecological environment, improve the safety of humans and animals, and delay the occurrence and development of pathogen resistance. Choosing biological control in cowpea disease prevention and control can effectively reduce the use of chemical pesticides. While preventing and controlling diseases, it can also promote plant growth, increase crop yields, and significantly improve ecological and social benefits. It is one of the important measures to ensure high-quality and high-yield cowpeas.

[0004] Plant rhizosphere growth-promoting bacteria are the main source of biocontrol agents. Currently, the biocontrol bacteria used in production mainly include Pseudomonas, Streptomyces and Bacillus. Their disease resistance mechanisms include lysis, competition, antagonism and induction of plant resistance. In addition to disease resistance, biocontrol bacteria also have the ability to significantly promote plant growth, such as nitrogen fixation, improving plant utilization of nutrients, and releasing plant hormones. Among them, Bacillus is an important biocontrol bacterium that is easy to separate from soil and plants and shows good disease control effects. This bacterium has the characteristics of strong stress resistance, rapid reproduction, simple nutritional requirements, and easy colonization on the surface of plants, which is conducive to the production and storage of biological control agents. In soil ecological restoration, Bacillus also has the functions of dissolving phosphorus, potassium, and improving soil fertility. At present, efficient biocontrol bacteria for cowpea diseases still need to be developed. Summary of the invention

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

[0006] The second aspect of the present invention aims to provide a culture.

[0007] The third aspect of the present invention aims to provide a method for preparing the culture of the second aspect of the present invention.

[0008] The fourth aspect of the present invention aims to provide a bacterial agent.

[0009] The purpose of the fifth aspect of the present invention is to provide a method for preparing the bacterial agent of the fourth aspect of the present invention.

[0010] The sixth aspect of the present invention aims to provide applications 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 the present invention aims to provide a product.

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

[0013] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is: In a first aspect of the present invention, a Bacillus amyloliquefaciens BA200 is provided. The classification name of the Bacillus amyloliquefaciens BA200 is Bacillus amyloliquefaciens Bacillus amyloliquefaciens , was deposited on February 24, 2025 in the General Microbiology Center of China Microorganism Culture Collection Administration (Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing), with the deposit number CGMCC No: 33636.

[0014] The colony surface of Bacillus amyloliquefaciens BA200 is rough, the colony is round and white, the edges are irregular, and the colony is raised; when the liquid culture is static, a biofilm is formed; under a microscope, the bacteria are rod-shaped, and the Gram staining result is positive. It has the activity of protease, cellulase, β-1,3-glucan antibacterial extracellular enzymes, as well as the ability to solubilize phosphate, potassium, fix nitrogen, produce iron carriers and IAA The second aspect of the present invention provides a culture obtained by culturing the Bacillus amyloliquefaciens BA200 of the first aspect of the present invention.

[0015] In some embodiments of the present invention, the culture is to inoculate a single colony of activated Bacillus amyloliquefaciens BA200 into LB liquid culture medium, culture at 37°C, 180 r / min for 24 h to obtain a seed solution, take 1% of the seed solution and add it to the LB liquid culture medium to continue to expand the culture until the OD 600 =0.6 (concentration is 1×10 7 CFU / mL) of fermentation products.

[0016] The third aspect of the present invention provides a method for preparing the culture of the second aspect of the present invention, wherein the biocontrol bacteria BA200 of the first aspect of the present invention is inoculated into a culture medium and cultured to obtain; preferably, the culture medium comprises LB culture medium.

[0017] In some embodiments of the present invention, the culture temperature is 16-42°C; preferably, the culture temperature is 30-37°C.

[0018] In some embodiments of the present invention, the culturing time is 12 to 96 hours; preferably, the culturing time is 18 to 36 hours.

[0019] In some embodiments of the present invention, the preparation method is to inoculate a single colony of activated Bacillus amyloliquefaciens BA200 into LB liquid culture medium, culture at 37°C, 180 r / min for 24 h to obtain seed solution, take 10% of the seed solution and add it to LB liquid culture medium and continue to culture until OD 600 =1.0 was used as the culture medium of Bacillus amyloliquefaciens BA200.

[0020] The fourth aspect of the present invention provides a bacterial 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.

[0021] In some embodiments of the present invention, the bacterial agent is a solid bacterial agent or a liquid bacterial agent.

[0022] In some embodiments of the present invention, the solid bacterial agent comprises a carrier.

[0023] In some embodiments of the present invention, the carrier comprises at least one of a mineral material and a plant material.

[0024] In some embodiments of the present invention, the mineral material comprises at least one of activated carbon, clay, peat soil, vermiculite, talc, kaolin, montmorillonite, white carbon, zeolite, silica, and diatomaceous earth.

[0025] In some embodiments of the invention, the plant material comprises at least one of corn meal, soybean meal and wheat bran.

[0026] In some embodiments of the present invention, the liquid bacterial agent includes a protective agent.

[0027] In some embodiments of the present invention, the protective agent includes one or more of glycerol, benzoate, sorbate, polyethylene glycol, and Tween.

[0028] In some embodiments of the present invention, the bacterial agent is in the form of at least one of a liquid, a powder or a granule.

[0029] The fifth aspect of the present invention provides a method for preparing the bacterial agent of the fourth aspect of the present invention, using the Bacillus amyloliquefaciens BA200 of the first aspect of the present invention and / or the culture of the second aspect as active ingredients to obtain the bacterial agent.

[0030] The sixth aspect of the present invention provides use of the 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 a product for controlling plant pathogens.

[0031] In some embodiments of the present invention, the plant pathogen includes Phytophthora sojae ( Phytophthora sojae ), Fusarium oxysporum ( Fusarium oxysporum )、Phytophthora vulgaris( Phytophthora vignae ) Fusarium solani )、Rhizoctonia solani( Rhizoctonia solani )、Flat-headed Anthrax ( Colletotrichum truncatum ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides ), Alternaria species ( Alternaria eichhorniae , Polysporon spp. Nodulisporium sp.), Pan-mycophyte ( Myrothecium inundatum ) and Phytophthora palmi ( Phytophthora palmivora ).

[0032] Diseases caused by the above plant pathogens include but are not limited to: Phytophthora sojae causes soybean Phytophthora root rot and soybean blight.

[0033] Fusarium oxysporum causes wilt disease and has a wide host range, including but not limited to tomatoes, bananas, watermelons, cowpeas, etc.

[0034] Phytophthora vulgaris causes cowpea wilt.

[0035] Fusarium solani causes root rot, and its hosts include but are not limited to soybeans, peas, cowpeas, etc.

[0036] Rhizoctonia solani causes damping-off and sheath blight.

[0037] Colletotrichum flatheadensis causes anthrax.

[0038] Colletotrichum gloeosporioides causes anthrax.

[0039] Alternaria species cause black spot or leaf spot.

[0040] The bacteria Polysporus spp. cause Scedosporium.

[0041] Ubiquitous mycorrhiza causes leaf spot or stem rot.

[0042] Phytophthora palmi causes Phytophthora palmitoides disease.

[0043] The seventh aspect of the present invention provides a product, comprising the biocontrol bacteria 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.

[0044] In some embodiments of the present invention, the product includes auxiliary materials; the auxiliary materials include at least one of solvents, colorants, stabilizers, preservatives, pH regulators, buffers, surfactants, foaming agents, defoamers, thickeners, and carriers.

[0045] In some embodiments of the present invention, the product may further include other antimicrobial active ingredients, including but not limited to antimicrobial peptides, antibiotics, or other biological control bacteria.

[0046] An eighth aspect of the present invention provides a method for controlling plant pathogens, comprising the steps of using the 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.

[0047] In some embodiments of the present invention, the method includes the step of inoculating the fermentation liquid of Bacillus amyloliquefaciens BA200 onto cowpea by root irrigation to perform biological control of cowpea wilt.

[0048] The beneficial effects of the present invention are: The present invention isolated and identified a new biological control bacterium, named Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens)BA200. The biocontrol bacterium Bacillus amyloliquefaciens BA200 has been deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on 2024-month-day, with the deposit number CGMCC No33636. This strain has significant antibacterial ability against the two main pathogens of cowpea wilt, Fusarium oxysporum and Phytophthora corytae. The average inhibition rate of biocontrol bacteria BA200 against Fusarium oxysporum is more than 60%, and the average inhibition rate against Phytophthora corytae is more than 75%. In addition, biocontrol bacteria BA200 has a broad-spectrum biological control effect, and has obvious antibacterial effect on the mycelial growth of various plant pathogens such as Fusarium solani, Rhizoctonia solani, Colletotrichum flatheaded, Colletotrichum gloeosporioides, Alternaria, Polysporus, Phytophthora sojae, Pan-mycorrhiza uralensis and Phytophthora palmi. In addition, biocontrol bacteria BA200 has antibacterial extracellular enzyme activities of protease, cellulase, and β-1,3-glucanase, as well as growth-promoting properties of phosphate and potassium solubilization, nitrogen fixation, IAA production and iron carrier production. Root irrigation of cowpea plants with the fermentation liquid prepared from the biocontrol bacteria BA200 can significantly reduce the incidence of seedlings and the disease index, providing a valuable resource for the preparation of biocontrol agents. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein: Figure 1 The morphological observation and Gram staining of the biocontrol bacteria BA200; Figure 1 The middle left picture shows the colony morphology of the biocontrol bacteria BA200 after being cultured on LB medium for 24 hours, and the right picture shows the Gram staining of the biocontrol bacteria BA200 under a microscope.

[0050] Figure 2 This is a diagram of the experimental results of the physiological and biochemical characteristics of the biocontrol bacteria BA200, where CK represents the control group and 200 is the number of the biocontrol bacteria BA200 used.

[0051] Figure 3 Phylogenetic tree of biocontrol bacterium BA200 constructed based on 16S rDNA sequence.

[0052] Figure 4 This is a graph showing the results of the determination of the extracellular enzyme activity and growth-promoting properties of the biocontrol bacterium BA200, where CK represents the control group and 200 is the number of the biocontrol bacterium BA200 used.

[0053] Figure 5 This is a plate confrontation result diagram of the biocontrol bacteria BA200 against Fusarium oxysporum, Phytophthora corytae and Phytophthora sojae; CK represents the control group using sterile water, and 200 is the number of the biocontrol bacteria BA200 used.

[0054] Figure 6 This is a graph showing the results of plate confrontation between the biocontrol bacteria BA200 and various plant pathogens.

[0055] Figure 7 is the control effect of the fermentation liquid of the biocontrol bacteria BA200 on cowpea wilt caused by Fusarium oxysporum and Phytophthora vulgaris; CK represents the control group applied with 50 mL of sterile water; BA200 represents the treatment group applied with 50 mL of the biocontrol bacteria only; Fo represents the treatment group applied with 50 mL of sterile water and inoculated with Fusarium oxysporum 24 hours later; BA200+Fo represents the treatment group applied with 50 mL of the fermentation liquid of the biocontrol bacteria BA200 and inoculated with Fusarium oxysporum 24 hours later; Pv represents the treatment group applied with 50 mL of sterile water and inoculated with Phytophthora vulgaris 24 hours later; BA200+Pv represents the treatment group applied with 50 mL of the fermentation liquid of the biocontrol bacteria BA200 and inoculated with Phytophthora vulgaris 24 hours later. DETAILED DESCRIPTION

[0056] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0057] Example 1 Purification of BA200 The biocontrol bacteria BA200 in the present invention is isolated by the inventor from rhizosphere soil of cowpea in the field in Yazhou District, Sanya City, Hainan Province.

[0058] Take the preserved biocontrol bacteria BA200, pick up a small amount of bacterial solution, use the dilution plate spreading method to streak and purify on LB solid culture medium, and invert and culture at 37℃ for 24 hours to obtain a purified single colony.

[0059] Example 2 Identification of BA200 1. Morphological observation like Figure 1 As shown, the surface of the biocontrol bacteria BA200 on LB solid culture medium is rough, the colonies are round and white, the edges are irregular, and the colonies are raised; when the liquid culture is static, a biofilm is formed; under a microscope, the bacteria are rod-shaped and the Gram staining results are positive.

[0060] 2. Identification of physiological and biochemical characteristics The biocontrol bacteria BA200 was subjected to Gram staining, catalase test, oxidase test, methyl red test, VP test, indole test, citrate utilization test, starch hydrolysis, cellulose decomposition test, and carbon source utilization test. The experimental methods refer to the Manual of Common Bacterial System Identification, and the results of its physiological and biochemical characteristics are shown in Table 1 and Figure 2 shown.

[0061] Table 1. Identification results of physiological and biochemical characteristics of biocontrol bacteria BA200

[0062] Note: “+” indicates positive, and “-” indicates negative.

[0063] 3. Molecular Biology Identification The biocontrol bacteria BA200 obtained in Experimental Example 1 was taken, and the DNA of the biocontrol bacteria BA200 was extracted using a bacterial genomic DNA extraction kit. The genome of the bacterium was amplified using the universal bacterial 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 2 × Taq Master Mix 25 μL, 2 μL of each primer, 1 μL of each DNA template, and 20 μL of dd H2O. The amplification program was 95°C pre-denaturation for 5 min, 95°C denaturation for 30 s, 58°C annealing for 30 s, and 72°C extension for 1 min 15 s, for a total of 30 cycles, and finally 72°C extension for 10 min. The amplified products were sequenced by 1.5% agarose gel electrophoresis. The sequences obtained by sequencing were compared with the GenenBank nucleic acid sequence library in NCBI for homology analysis. The sequences of known biocontrol bacteria with high homology to the antagonistic strain sequences were downloaded and the MEGAE The phylogenetic tree based on 16S gene was constructed using the neighbor-joining method in the 11.0 software. Figure 3 shown.

[0064] The 16S rDNA sequence of the biocontrol bacterium BA200 was compared with that of Bacillus amyloliquefaciens (Accession No.: HM055610.1) belong to the same branch in terms of system, and combined with the colony morphology and physiological and biochemical characteristics, BA200 was identified as Bacillus amyloliquefaciens .

[0065]

[0066] 4. Determination of the antibacterial extracellular enzyme activity and growth-promoting properties of the biocontrol bacterium BA200 The antibacterial extracellular enzyme activity of the biocontrol bacterium BA200 was detected using protease, cellulase and β-1,3-glucanase culture media, and the growth-promoting properties of the biocontrol bacterium BA200 were detected using phosphate, potassium, nitrogen fixation and siderophore detection culture media.

[0067] The components of the protease detection medium are: 3.0 g skim milk powder, 4 g agar, and sterile water to 200 mL, pH 7.2, and sterilized at 115℃ for 30 min. Use a sterilized inoculation needle to pick a single colony and inoculate it on the protease detection medium plate, and culture it at 30℃ for 5 days to observe whether there is a transparent circle around it. The presence of a transparent circle indicates that it has protease activity.

[0068] The components of the cellulase detection medium are: 2 g sodium hydroxymethylcellulose, 2 g peptone, 2 g yeast extract, 1 g NaCl, 0.2 g KH2PO4, 4 g agar, 200 mL sterile water, pH 7.2, and sterilized at 115°C for 30 min. Use a sterilized inoculation needle to pick a single colony and inoculate it on the cellulase detection medium plate. Culture it at 30°C for 5 days, and use 1 mg / mL Congo red solution to completely immerse the entire culture dish for staining for 30 min. Pour off the staining solution and wash it with 1 mol / L NaCl solution. Observe whether there is a transparent circle around it. If there is a transparent circle, it means that it has cellulase activity.

[0069] The components of the β-1,3-glucanase detection medium are: β-1,3-glucan 0.4 g, Congo red 0.01 g, NaNO3 0.4 g, KH2PO4 0.2 g, KCl 0.1 g, MgSO4·7H2O 0.1 g, FeSO4 0.002 g, agar 4 g, sterile water to 200 mL, pH 7.2, 115℃ high temperature sterilization for 30 min. Use a sterile inoculation needle to pick a single colony and inoculate it on the β-1,3-glucanase medium plate, culture at 30℃ for 5 days, and observe whether there is a transparent circle around it. The presence of a transparent circle indicates that it has β-1,3-glucanase activity.

[0070] Inorganic phosphorus culture medium: (NH4)2SO40.1 g, MgSO4·7H2O 0.1 g, NaCl 0.04 g, Ca3(PO4)22 g, CaCO30.1 g, MnSO40.004 g, FeSO40.004 g, glucose 0.5 g, agar 4 g, dilute to 200 mL with sterile water, pH 7.2, sterilize at 115℃ for 30 min. Use a sterile inoculation needle to pick a single colony and inoculate it on the inorganic phosphorus culture medium plate, culture at 30℃ for 5 days, and observe whether there is a transparent circle around it. The presence of a transparent circle indicates that it has the ability to decompose inorganic phosphorus.

[0071] Organophosphorus culture medium: (NH4)2SO40.1 g, CaCO30.1 g, MgSO4·7H2O 0.1 g, NaCl 0.04 g, MnSO40.004 g, FeSO40.004 g, lecithin 0.04 g, glucose 0.4 g, agar 4 g, dilute to 200 mL with sterile water, pH 7.2, sterilize at 115℃ for 30 min. Use a sterile inoculation needle to pick a single colony and inoculate it on an organophosphorus culture medium plate, culture at 30℃ for 5 days, and observe whether there is a transparent circle around it. The presence of a transparent circle indicates that it has the ability to decompose organic phosphorus.

[0072] Nitrogen-free culture medium: NaCl 0.024 g, KH2PO4 0.1 g, CaCO3 0.04 g, MgSO4·7H2O 0.04 g, sucrose 2 g, agar 4 g, sterile water to 200 mL, pH 7.2, 115℃ high temperature sterilization for 30 min. Use a sterile inoculation needle to pick a single colony and inoculate it on a nitrogen-free culture medium plate, and culture it at 30℃ for 5 days. The ability to fix nitrogen is determined by whether the biocontrol bacteria can grow on the culture medium and whether a transparent circle is formed around it. The ability of the biocontrol bacteria to grow on the nitrogen-free culture medium and the formation of a transparent circle around it indicates that it has the ability to fix nitrogen.

[0073] Potassium feldspar culture 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 200 mL, pH 7.2, 115℃ high temperature sterilization for 30 min. Use a sterile inoculation needle to pick a single colony and inoculate it on the potassium feldspar culture medium plate, culture at 30℃ for 5 days, and observe whether there is a transparent circle around it. The presence of a transparent circle indicates that it has the ability to dissolve potassium.

[0074] Siderophore detection medium: Use a sterile inoculation needle to pick up a single colony and spot-inoculate it on the CAS detection medium plate. Culture it at 30℃ for 5 days and observe whether there is an orange-yellow circle around it. The presence of an orange-yellow halo indicates that it has the ability to produce siderophores.

[0075] IAA production capacity: Add L-tryptophan at a dosage of 100 mg / L to LB liquid culture medium, culture at 37°C, 180 r / min for 72 h, add a small amount of Salkowsk colorimetric solution and protect from light, observe after 30 min, and red color change indicates the production of IAA.

[0076] The results of the determination of extracellular enzyme activity and growth-promoting properties of the biocontrol bacterium BA200 showed that ( Figure 4 ): The biocontrol bacterium BA200 has the antibacterial extracellular enzyme activities of protease, cellulase, and β-1,3-glucan, as well as the ability to solubilize phosphate, potassium, fix nitrogen, and produce iron carriers and IAA.

[0077] After the strain identification was completed, the biocontrol bacterium BA200 was deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration (No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences) on February 24, 2025, with the deposit number CGMCC No: 33636, and its recommended taxonomic name is: Bacillus amyloliquefaciens Bacillus amyloliquefaciens.

[0078] Example 3 Inhibitory effect of biocontrol bacteria BA200 on cowpea wilt pathogen 1. Experimental methods Preparation of biocontrol bacteria culture: A single colony obtained in Experimental Example 1 was picked and placed in 4 mL LB medium, and cultured at 37°C, 180 r / min with shaking for 24 h. A 10% volume ratio was placed in 4 mL LB medium, and cultured at 37°C, 180 r / min with shaking to obtain a culture with OD600=1.0.

[0079] The plate confrontation method was used to detect the inhibitory effect of the culture of biocontrol bacteria BA200 on Fusarium oxysporum and Phytophthora vulgaris. The culture of biocontrol bacteria BA200 was dropped in a "cross" shape around 3.5 cm away from the center of the plate. Fusarium oxysporum, Phytophthora vulgaris and Phytophthora sojae were cut into 2 mm × 2 mm blocks and inoculated in the center of the plate respectively. The plate inoculated with pathogens only was used as a control. The plates were cultured in the dark at a constant temperature of 25℃. When the control colonies were about to fill the plate, the colony diameter was measured and the colony inhibition rate was calculated.

[0080] Colony inhibition rate (%) = [(colony diameter of control group - colony diameter of treatment group) / colony diameter of control group] × 100.

[0081] 2. Experimental results like Figure 5 As shown in the data, the culture of the biocontrol bacteria BA200 had a significant inhibitory effect on Fusarium oxysporum, Phytophthora corytae and Phytophthora sojae. The inhibition rates against Fusarium oxysporum were 63.81%, against Phytophthora corytae and against Phytophthora sojae were 75.27% and 61.84% respectively.

[0082] Example 4 Inhibitory effect of biocontrol bacteria BA200 on various plant pathogens The method described in Experimental Example 3 was used to determine the inhibitory ability of the biocontrol bacterium BA200 against a variety of plant pathogens, including Fusarium solani ( Fusarium solani )、Rhizoctonia solani( Rhizoctonia solani )、Flat-headed Anthrax ( Colletotrichum truncatum ), Colletotrichum gloeosporioides ( Colletotrichum gloeosporioides )、Alternaria alternata( Alternaria eichhorniae ), Polysporon spp. ( Nodulisporium sp.), Pan-mycophyte ( Myrothecium inundatum ) and Phytophthora palmi ( Phytophthora palmivora The inhibitory effects of biocontrol bacteria BA200 on various plant pathogens are shown in Table 2 and Figure 6 shown.

[0083] Table 2. Inhibitory effect of biocontrol bacteria BA200 on various plant pathogens

[0084] From Table 2 and Figure 6 It can be seen that Bacillus amyloliquefaciens BA200 has a good inhibitory effect on the 8 tested plant pathogens, and the average inhibition rate is higher than 50%, indicating that Bacillus amyloliquefaciens BA200 has a broad-spectrum biological control effect.

[0085] Example 5 Determination of potted plant control efficacy of biocontrol bacteria BA200 The single colony obtained in Experimental Example 1 was placed in 4 mL LB medium and cultured at 37°C, 180 r / min for 24 h. The colony was then placed in 200 mL LB medium at a volume ratio of 1% and cultured at 37°C, 180 r / min. The OD600 value was 0.6 (concentration was 1×10 7 CFU / mL) of fermentation broth.

[0086] The concentration of the conidia suspension inoculated with Fusarium oxysporum was 5×10 6 The inoculation of cowpea phytophthora was carried out by the fungus-soil method, and the cowpea phytophthora that covered a 9 cm culture dish was crushed with a syringe and mixed with soil.

[0087] The cowpea variety chosen is "Nongwang Yousheng 380", from Jiangxi Nongwang High-tech Co., Ltd.

[0088] Treatments: (1) healthy plants (CK); (2) inoculated with Fusarium oxysporum (Fo); (3) inoculated with Phytophthora vulgaris (Pv); (4) inoculated with the biocontrol agent BA200 (BA200); (5) biocontrol agent BA200 + Fusarium oxysporum (BA200 + Fo); (6) biocontrol agent BA200 + Phytophthora vulgaris (BA200 + Pv).

[0089] After being sterilized, cowpea seeds were sown in moist vermiculite. When the two true leaves of the cowpea seedlings were fully expanded, they were transplanted into moist nutrient medium and inoculated with 50 mL of 1×10 7 CFU / mL of biocontrol bacteria fermentation liquid, the control group was inoculated with sterile water, and the pathogenic bacteria were inoculated 24 hours later. The pathogenicity of Fusarium oxysporum was determined by root immersion inoculation method, and the pathogenicity of Phytophthora cowpea was determined by soil method. Each treatment had 6 plants, repeated 3 times, the disease incidence of each treatment was counted, and the disease index was recorded.

[0090] The disease situation includes the incidence rate and disease classification, which are divided into 6 levels: Grade 0: The endovascular stem is normal and there is no disease; Level 1: Discoloration of the endovascular stem is less than 1 / 4; Level 2: Discoloration degree of 1 / 4 of the intravascular stem ≤ 1 / 2; Level 3: Discoloration of 1 / 2 or less of the endovascular stem and less than 3 / 4, with yellowing and withering of leaves; Level 4: Discoloration of the endovascular stem is greater than 3 / 4, and some leaves turn yellow, wither, or fall off; Level 5: The whole plant wilts, falls over or dies.

[0091] Disease index = [∑(number of cowpea plants at each disease level × number of disease levels) / total number of cowpea potted plants × number of the highest disease level] × 100.

[0092] Prevention and treatment effect = (disease index of control group - disease index of treatment group) / disease index of control group × 100%.

[0093] The results are shown in Tables 3, 4 and Figure 7 shown.

[0094] Table 3. Statistical results of the incidence and control effect of cowpea wilt (Fusarium oxysporum)

[0095] From Table 3 and Figure 7 It can be seen that the Bacillus amyloliquefaciens BA200 of the present invention can significantly reduce the severity of the cowpea wilt disease caused by Fusarium oxysporum, indicating that Bacillus amyloliquefaciens BA200 has a preventive and therapeutic effect on cowpea wilt disease caused by Fusarium oxysporum, and the preventive and therapeutic effect is 72.06%.

[0096] Table 4. Statistical results of the incidence and control effects of cowpea wilt (cowpea wilt)

[0097] From Table 4 and Figure 7 It can be seen that the Bacillus amyloliquefaciens BA200 of the present invention can significantly reduce the severity of the cowpea wilt disease caused by Phytophthora cowpea, indicating that Bacillus amyloliquefaciens BA200 has a preventive and therapeutic effect on cowpea wilt disease caused by Phytophthora cowpea, and the preventive and therapeutic effect is 83.10%.

Claims

1. A Bacillus amyloliquefaciens BA200, characterized in that: The classification of the Bacillus amyloliquefaciens BA200 is named Bacillus amyloliquefaciens Bacillus amyloliquefaciens , and was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on February 24, 2025, with the deposit number CGMCC No: 33636.

2. The culture of Bacillus amyloliquefaciens BA200 according to claim 1.

3. The method for preparing the culture according to claim 2, comprising the following steps: The Bacillus amyloliquefaciens BA200 according to claim 1 is inoculated into a culture medium and cultured to obtain. A bacterial agent comprising the Bacillus amyloliquefaciens BA200 according to claim 1 and / 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 method for preparing the bacterial agent according to claim 4 or 5, wherein the bacterial agent is obtained by using the Bacillus amyloliquefaciens BA200 according to claim 1 and / or the culture according to claim 2 as active ingredients.

7. Use of the Bacillus amyloliquefaciens BA200 according to claim 1, the culture according to claim 2, and / or the bacterial agent according to claim 4 or 5 in the preparation of a product for controlling plant pathogens.

8. The use according to claim 7, characterized in that: The plant pathogens include soybean phytophthora, oxysporum fusarium, cowpea phytophthora, solani fusarium, Rhizoctonia solani, flat-headed anthracnose, gloeosporium anthracnose, alternaria, polysporum, pan-myrocytic spores and palm phytophthora.

9. A product comprising at least one of the Bacillus amyloliquefaciens BA200 according to claim 1, the culture according to claim 2, and / or the bacterial agent according to claim 4 or 5; The product includes auxiliary materials.

10. A method for controlling plant pathogens, comprising the step of treating with the Bacillus amyloliquefaciens BA200 according to claim 1, the culture according to claim 2, and / or the bacterial agent according to claim 4 or 5.

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