CN119899784B - A biocontrol strain of Bacillus amyloliquefaciens BA200 and its application - Google Patents

Abstract

The present invention belongs to the field of modern agricultural technology, and specifically relates to a biocontrol bacillus amyloliquefaciens BA200 and its application. The present invention separates and identifies a new biocontrol bacterium BA200, which is taxonomically named Bacillus amyloliquefaciens . The strain has significant inhibitory ability against two pathogenic bacteria, Fusarium oxysporum and Phytophthora cowpea, which cause cowpea wilt, with an inhibition rate of more than 60%. The fermentation liquid prepared by the strain can significantly reduce the incidence and disease index of cowpea wilt, and is a good biocontrol microorganism. It has application development potential and important significance in the prevention and control of cowpea wilt, and the biocontrol bacterium Bacillus amyloliquefaciens BA200 has a broad-spectrum biocontrol effect, can inhibit a variety of plant pathogens, and provides valuable biocontrol bacteria resources for the preparation of biological fungicides.

Description

Biocontrol bacillus amyloliquefaciens BA200 and application thereof

Technical Field

The invention belongs to the technical field of modern agriculture, and particularly relates to a biocontrol bacillus amyloliquefaciens BA200 and application thereof.

Background

Soybeans are globally important grain and oil crops, rich plant proteins and grease provide important nutrition sources for human beings, and with the increase of the planting area of the soybeans and the expansion of the continuous cropping range of the soybeans, soybean root rot caused by fusarium and phytophthora sojae is a destructive disease which damages the production of the soybeans, and the economic benefit of the soybeans is seriously damaged. This dilemma of soil-borne disease control is common among legumes where cowpea, which is an important commercial crop, faces a more serious challenge. Cowpea is one of six soybean crops, and has high economic value. In cowpea planting, once the cowpea soil-borne diseases occur, cowpea plants can be withered in a large area, and even particles are not collected when serious. Wherein, the cowpea wilt disease is the disease which is most commonly generated in the cowpea planting process, most seriously harmed and most difficult to control. The pathogenic bacteria of the cowpea wilt mainly comprise fusarium oxysporum, phytophthora sojae and the like, and similar to soybean root rot, the soil-borne pathogenic bacteria infects plants from the root or stem base of the plants under proper conditions, so that the stem base necrotic lesions and leaf wilts are caused, and finally the whole plant is wilted and even dead. As diseases are rampant in the cowpea growth process, excessive use of chemical pesticides is easy to cause due to excessive dependence on chemical agents for controlling cowpea wilt, and pesticide residues exceed standards, biological source bactericides capable of replacing chemical bactericides are developed and used for controlling cowpea wilt.

Biological control has become one of the most promising strategies for controlling soil-borne diseases, because it can protect the ecological environment of farmlands, improve the safety of humans and animals, and delay the occurrence and development of drug resistance of pathogenic bacteria. The biological control is selected for the prevention and control of the cowpea diseases, so that the use of chemical pesticides can be effectively reduced, the plant growth can be promoted, the crop yield can be increased, the ecological benefit and the social benefit can be obviously improved while the diseases are controlled, and the cowpea disease prevention and control method is one of important measures for ensuring the high quality and the high yield of cowpea.

The plant rhizosphere growth-promoting bacteria are a main source of the biocontrol bacteria, the biocontrol bacteria currently applied to production mainly comprise pseudomonas, streptomyces, bacillus and the like, and disease-resistant action mechanisms comprise bacteriolysis, competition, antagonism, plant resistance induction and the like, and besides the disease-resistant action, the biocontrol bacteria also have the capability of obviously promoting plant growth, such as nitrogen fixation, improvement of plant utilization of nutrient substances, release of plant hormones and the like. Among them, bacillus is an important biocontrol bacterium, is easily separated from soil and plants, and exhibits a good disease control effect. The bacteria have the characteristics of strong stress resistance, rapid propagation, simple nutrition requirement, easiness in field planting on the surface of plants and the like, and are beneficial to the production and storage of biological control agents. In the ecological restoration of soil, bacillus also has the functions of dissolving phosphorus, dissolving potassium, improving soil fertility and the like. At present, high-efficiency biocontrol bacteria aiming at cowpea diseases still need to be developed.

Disclosure of Invention

The object of the first aspect of the invention is to provide a bacillus amyloliquefaciens strain.

The object of the second aspect of the present invention is to provide a culture.

The object of the third aspect of the present invention is to provide a method for producing a culture according to the second aspect of the present invention.

The fourth aspect of the present invention is directed to a microbial agent.

The fifth aspect of the present invention is directed to a method for producing the microbial inoculum of the fourth aspect of the present invention.

The sixth aspect of the present invention is directed to providing the biocontrol bacterium of the first aspect of the present invention, the culture of the second aspect of the present invention, and the use of the microbial inoculum of the fourth aspect of the present invention.

A seventh aspect of the invention is directed to a product.

An object of an eighth aspect of the invention is to provide a method.

In order to achieve the above purpose of the present invention, the present invention adopts the following technical scheme:

In a first aspect of the present invention, there is provided a Bacillus amyloliquefaciens BA200, which is classified as Bacillus amyloliquefaciens Bacillus amyloliquefaciens, which has been deposited at China general microbiological culture Collection center (institute of microbiology, national academy of sciences of China, north Star, no. 1, no. 3, in the Korean region of Beijing) at 2025, month 2 and 24, and has a deposit number of CGMCC No. 33636.

The bacterial colony of the bacillus amyloliquefaciens BA200 has rough surface, round white bacterial colony, irregular edge and raised bacterial colony, forms bacterial film when liquid culture is static, has rod-shaped bacterial body under microscope and positive gram staining result, and has the functions of inhibiting extracellular enzyme activity of protease, cellulase and beta-1, 3-glucan, and the capabilities of phosphate and potassium dissolving, nitrogen fixing, siderophore and IAA generating

In a second aspect of the present invention, there is provided a culture obtained by culturing the Bacillus amyloliquefaciens BA200 of the first aspect of the present invention.

In some embodiments of the invention, the culture is a fermentation product obtained by inoculating a single colony of bacillus amyloliquefaciens BA200 into LB liquid medium, culturing 24h at 37 ℃ and 180 r/min to obtain seed liquid, adding 1% of the seed liquid into LB liquid medium, and continuously expanding culture until OD ₆₀₀ = 0.6 (with the concentration of 1×10 ⁷ CFU/mL).

In a third aspect of the present invention, there is provided a method for producing a culture according to the second aspect of the present invention, comprising inoculating the biocontrol strain BA200 according to the first aspect of the present invention to a medium, and culturing the inoculated biocontrol strain BA200, wherein the medium preferably comprises LB medium.

In some embodiments of the invention, the temperature of the culture is 16-42 ℃, preferably 30-37 ℃.

In some embodiments of the invention, the time of the culturing is 12-96 hours, preferably 18-36 hours.

In some embodiments of the invention, the preparation method comprises inoculating single colony of activated bacillus amyloliquefaciens BA200 into LB liquid medium, culturing at 37 ℃ and 180 r/min for 24: 24 h to obtain seed liquid, adding 10% of seed liquid into LB liquid medium, and continuing to reach OD ₆₀₀ =1.0 to obtain bacterial liquid as a culture of bacillus amyloliquefaciens BA 200.

In a fourth aspect of the invention, there is provided a microbial agent comprising the biocontrol bacterium BA200 of the first aspect of the invention and/or the culture of the second aspect of the invention.

In some embodiments of the invention, the microbial agent is a solid microbial agent or a liquid microbial agent.

In some embodiments of the invention, the solid microbial agent comprises a carrier.

In some embodiments of the invention, the carrier comprises at least one of mineral material, plant material.

In some embodiments of the invention, the mineral material comprises at least one of activated carbon, clay, turfy soil, vermiculite, talc, kaolin, montmorillonite, white carbon, zeolite, silica, diatomaceous earth.

In some embodiments of the invention, the plant material comprises at least one of corn flour, soy flour, and wheat bran.

In some embodiments of the invention, the liquid microbial agent comprises a protective agent.

In some embodiments of the invention, the protective agent comprises one or more of glycerol, benzoate, sorbate, polyglycol, tween.

In some embodiments of the invention, the dosage form of the microbial inoculum is at least one of a liquid, a powder or a granule.

In a fifth aspect of the present invention, there is provided a method for producing a microbial agent according to the fourth aspect of the present invention, wherein the microbial agent is obtained by using the Bacillus amyloliquefaciens BA200 according to the first aspect of the present invention and/or the culture according to the second aspect of the present invention as an active ingredient.

In a sixth aspect of the invention, there is provided the use of bacillus amyloliquefaciens BA200 of the first aspect of the invention, a culture of the second aspect of the invention, and/or a microbial inoculum of the third aspect of the invention for the preparation of a product for controlling phytopathogens.

In some embodiments of the invention, the plant pathogenic bacteria include phytophthora sojae (Phytophthora sojae), fusarium oxysporum (Fusarium oxysporum), phytophthora vignae (Phytophthora vignae), fusarium solani (Fusarium solani), rhizoctonia solani (Rhizoctonia solani), anthrax grub (Colletotrichum truncatum), colletotrichum glomerata (Colletotrichum gloeosporioides), alternaria species (ALTERNARIA EICHHORNIAE), polyporus (nodulisporium sp.), plaque bacteria (Myrothecium inundatum), and phytophthora palmi (Phytophthora palmivora).

Diseases to which the above plant pathogens are directed include, but are not limited to:

Phytophthora sojae causes Phytophthora sojae root rot and epidemic disease in soybean.

Fusarium oxysporum causes wilt and has a broad host range including, but not limited to, tomatoes, bananas, watermelons, cowpeas, and the like.

Phytophthora vignae causes cowpea wilt.

Fusarium solani causes root rot and hosts include, but are not limited to, soybean, pea, cowpea, and the like.

Rhizoctonia solani causes damping off and banded sclerotial blight.

The colletotrichum glomeratum causes anthracnose.

Colletotrichum gloeosporioides causes anthracnose.

Alternaria bacteria cause melasma or leaf spot.

The genus polyspora causes cercospora disease.

The pantotrichum causes leaf spot or stem rot.

Phytophthora palmae causes Phytophthora palmae.

In a seventh aspect of the invention, there is provided a product comprising the biocontrol bacterium BA200 of the first aspect of the invention, the culture of the second aspect of the invention, and/or the microbial inoculum of the third aspect of the invention.

In some embodiments of the invention, the product comprises an adjunct comprising at least one of a solvent, a colorant, a stabilizer, a preservative, a pH adjustor, a buffering agent, a surfactant, a foaming agent, a defoaming agent, a thickening agent, and a carrier.

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

In an eighth aspect of the invention, there is provided a method for controlling phytopathogenic fungi comprising the step of using the bacillus amyloliquefaciens BA200 of the first aspect of the invention, the culture of the second aspect of the invention, and/or the microbial inoculum of the third aspect of the invention.

In some embodiments of the invention, the method comprises the step of inoculating the fermentation broth of bacillus amyloliquefaciens BA200 onto cowpea by root irrigation for biological control of cowpea wilt.

The beneficial effects of the invention are as follows:

The invention separates and identifies a new biological control bacterium, named as bacillus amyloliquefaciens (Bacillus amyloliquefaciens) BA200, and the biological control bacillus amyloliquefaciens BA200 is preserved in China general microbiological culture collection center (CGMCC) No33636 at the month and day of 2024. The strain has remarkable antibacterial capability on two main pathogenic bacteria of fusarium oxysporum and phytophthora sojae causing cowpea wilt, the average inhibition rate of biocontrol bacteria BA200 on fusarium oxysporum reaches more than 60 percent, the average inhibition rate on phytophthora sojae reaches more than 75 percent, in addition, the biocontrol bacteria BA200 has broad-spectrum biological control effect on various plant pathogenic bacteria such as fusarium solani, rhizoctonia solani, colletotrichum glomerata, alternaria, polyporus, phytophthora sojae, pantotrichum, phytophthora palmis and the like, and the biocontrol bacteria BA200 has protease, cellulase, beta-1, 3-glucanase antibacterial extracellular enzyme activity and promotion characteristics of phosphate dissolving, potassium dissolving, nitrogen fixing and IAA and iron carrier generating. The fermentation liquor prepared by the biocontrol bacterium BA200 is used for root irrigation treatment of cowpea plants, so that the morbidity and disease index of seedlings can be remarkably reduced, and precious resources are provided for preparing the biocontrol bacterium agent.

Drawings

The invention is further described with reference to the accompanying drawings and examples, in which:

FIG. 1 is a morphological observation and gram staining diagram of the biocontrol strain BA200, and FIG. 1 is a left-hand diagram showing colony morphology after the biocontrol strain BA200 is cultured on LB medium for 24 h, and a right-hand diagram showing gram staining diagram of the biocontrol strain BA200 under a microscope.

FIG. 2 is a graph showing the results of physiological and biochemical characteristics of the biocontrol strain BA200, wherein CK represents a control group, and 200 is the number of the biocontrol strain BA200 used.

FIG. 3 is a phylogenetic tree of biocontrol strain BA200 constructed based on the 16S rDNA sequence.

FIG. 4 is a graph showing the results of measurement of extracellular enzyme activity and growth-promoting properties of biocontrol strain BA200, wherein CK represents a control group and 200 is the number of biocontrol strain BA200 used.

FIG. 5 is a graph showing the results of the facing of biocontrol BA200 against plates of Fusarium oxysporum, phytophthora cowpea and Phytophthora sojae, wherein CK represents a control group using sterile water and 200 is the number of biocontrol BA200 used.

FIG. 6 is a graph showing the results of the plate-stand of the biocontrol strain BA200 against a plurality of plant pathogenic bacteria.

FIG. 7 shows the control effect of the fermentation broth of biocontrol bacterium BA200 on fusarium oxysporum and phytophthora vignae, wherein CK represents a control group to which 50mL sterile water is applied, BA200 represents a treatment group to which only 50mL biocontrol bacteria are applied, fo represents a treatment group to which 50mL sterile water is applied and 24h is inoculated with fusarium oxysporum, BA200+ Fo represents a fermentation broth to which 50mL biocontrol bacterium BA200 is applied and 24h is inoculated with fusarium oxysporum, pv represents a treatment group to which 50mL sterile water is applied and 24h is inoculated with phytophthora vignae, and BA200+ Pv represents a treatment group to which 50mL biocontrol bacterium BA200 is applied and 24h is inoculated with phytophthora vignae.

Detailed Description

The conception and the technical effects produced by the present invention will be clearly and completely described in conjunction with the embodiments below to fully understand the objects, features and effects of the present invention. It is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments, and that other embodiments obtained by those skilled in the art without inventive effort are within the scope of the present invention based on the embodiments of the present invention.

EXAMPLE 1 purification of BA200

The biocontrol bacterium BA200 in the present invention is from field cowpea rhizosphere soil isolated by the inventors from the state of the cliff in the three-city, hainan province.

Taking the stored biocontrol bacteria BA200, dipping a small amount of bacterial liquid, streaking and purifying on an LB solid culture medium by adopting a dilution plate coating method, and culturing at 37 ℃ in an inverted way for 24 h to obtain a purified single colony.

EXAMPLE 2 identification of BA200

1. Morphological observation

As shown in FIG. 1, biocontrol strain BA200 has rough surface, round white colony, irregular edge and raised colony on LB solid medium, bacterial film formed when liquid culture is static, rod-shaped thallus under microscope and positive gram staining result.

2. Physiological and biochemical characterization

The biocontrol strain BA200 is subjected to gram staining, a contact enzyme experiment, an oxidase experiment, a methyl red experiment, a V-P experiment, an indole experiment, a citrate utilization experiment, a starch hydrolysis experiment, a cellulose decomposition experiment and a carbon source utilization experiment. The experimental method is referred to a common bacteria System identification Manual, and the physiological and biochemical characteristic identification results are shown in Table 1 and FIG. 2.

TABLE 1 biological control bacteria BA200 physiological and biochemical characteristic identification results

Note that "+" indicates positive and "-" indicates negative.

3. Molecular biological identification

The biocontrol strain BA200 obtained in experimental example 1 was taken and DNA of the biocontrol strain BA200 was extracted using a bacterial genomic DNA extraction kit. The genome was amplified using bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTCAG-3', SEQ ID NO: 1) and 1492R (5'-GGTTACCTTGTTACGACTT-3', SEQ ID NO: 2), 50. Mu.L of a PCR system of 2X TAQ MASTER Mix 25. Mu.L each, 2. Mu.L of primers, 1. Mu.L of DNA templates each, dd H ₂ O20. Mu.L of each, 95℃for 5 min of pre-denaturation, 95℃for 30S of denaturation, 58℃for 30S of annealing, 72℃for 1 min for 15S of extension, and 30 cycles of final 72℃for 10 min, the amplified product was sequenced by 1.5% agarose gel electrophoresis, the sequence obtained by sequencing was analyzed for homology comparison in NCBI GenenBank nucleic acid sequence library, a known biocontrol sequence having high homology to the sequence of the antagonistic strain tested was downloaded, a 16S gene-based phylogenetic tree was constructed using the adjacency method using MEGAE 11.0.0 software, and the phylogenetic tree constructed as shown in FIG. 3.

BLAST alignment of the 16S rDNA sequence of biocontrol strain BA200 in Genbank shows that the 16S rDNA sequence of biocontrol strain BA200 and Bacillus amyloliquefaciens (accession number: HM 055610.1) systematically belong to the same branch, and the colony morphology and physiological and biochemical characteristics are combined to identify BA200 as Bacillus amyloliquefaciens.

The 16S rDNA gene sequence of the biocontrol bacterium BA200 is determined as follows ：CGTACTATACTGCAAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTTAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGGTTGTCTGAACCGCATGGTTCAGACATAAAAGGTGGCTTCGGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCGACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAGCTCTGTTGTTAGGGAAGAACAAGTGCCGTTCAAATAGGGCGGCACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGGGCTCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGAGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGGGGGTTTCCGCCCCTTAGTGCTGCAGCTAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAATCCTAGAGATAGGACGTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGACAGAACAAAGGGCAGCGAAACCGCGAGGTTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTATGGAGCCAGCCGCCGAAGTGATCTGCG （SEQ ID NO：3）.

4. Determination of antibacterial extracellular enzyme activity and growth promotion characteristics of biocontrol bacterium BA200

The antibacterial extracellular enzyme activity of the biocontrol bacterium BA200 is detected by using protease, cellulase and beta-1, 3-glucanase culture mediums, and the growth promoting property of the biocontrol bacterium BA200 is detected by using phosphate-dissolving, potassium-dissolving, nitrogen-fixing and ferrite-philic detection culture mediums.

The protease detection culture medium comprises skimmed milk powder 3.0 g, agar 4g, sterile water to volume of 200mL, and high temperature sterilizing at 7.2,115 deg.C for 30 min. The single bacterial drop point is selected by a sterilization inoculation needle and connected to a protease detection culture medium plate, the culture is carried out at a constant temperature of 30 ℃ for 5 d, the transparent circle generation is observed around, and the transparent circle generation indicates that the protease detection culture medium plate has protease activity.

The cellulase detection culture medium comprises sodium hydroxymethyl cellulose 2 g, peptone 2 g, yeast extract 2 g, naCl 1g, KH ₂PO₄ 0.2 g, agar 4g, sterile water 200 mL, and high temperature sterilization 30min at 7.2,115 ℃. Single bacterial drop points are picked by a sterilization inoculation needle and connected to a cellulase detection culture medium plate, the culture is carried out at a constant temperature of 30 ℃ for 5 d, the whole culture dish is completely immersed and dyed for 30min by using 1 mg/mL Congo red solution, the dye solution is poured out, and the culture dish is washed by using 1mol/L NaCl solution. The presence or absence of transparent rings around the periphery was observed, and the presence of transparent rings indicates that the cellulase has cellulase activity.

The beta-1, 3-glucanase detection culture medium comprises beta-1, 3-glucan 0.4 g, congo red 0.01 g、NaNO₃0.4 g、KH₂PO₄0.2 g、KCl 0.1 g、MgSO₄·7H₂O 0.1 g、FeSO₄0.002 g、 agar 4g and sterile water with constant volume of 200 mL and pH of 7.2,115 ℃ and high-temperature sterilization of 30 min. The single bacterial drop point is selected by a sterilization inoculation needle and connected to a beta-1, 3-glucanase culture medium plate, the culture is carried out at a constant temperature of 30 ℃ for 5 d, whether transparent rings are produced around the culture is observed, and the production of the transparent rings indicates that the culture has the beta-1, 3-glucanase activity.

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 4g, sterile water to 200 mL, pH 7.2,115 ℃ high temperature sterilization 30 min. The single bacterial drop point is selected by a sterilization inoculating needle and is connected to an inorganic phosphorus culture medium flat plate, the culture is carried out at a constant temperature of 30 ℃ for 5d, the transparent rings are observed to generate around, and the transparent rings generate to show that the inorganic phosphorus decomposition capability is realized.

The organophosphorus medium ：(NH₄)₂SO₄0.1 g、CaCO₃0.1g、MgSO₄·7H₂O 0.1 g、NaCl 0.04 g、MnSO₄0.004 g、FeSO₄0.004 g、 is lecithin 0.04 g, glucose 0.4 g and agar 4g, the sterile water is fixed to 200 mL, and the pH is 7.2,115 ℃ and the high temperature sterilization is carried out for 30 min. The single bacterial drop point is selected by a sterilization inoculation needle and connected to an organophosphorus medium flat plate, the culture is carried out at a constant temperature of 30 ℃ for 5 d, the transparent rings are observed to generate around, and the transparent rings generate to indicate the capability of decomposing organophosphorus.

Nitrogen-free culture medium, namely NaCl 0.024 g, KH ₂PO₄0.1 g、CaCO₃0.04 g、MgSO₄·7H₂ O0.04 g, sucrose 2g and agar 4g, and sterile water is used for constant volume to 200 mL, and the pH is 7.2,115 ℃ and the temperature is high for sterilization 30min. And (3) selecting a single fungus drop point by using a sterilization inoculation needle, connecting the single fungus drop point to a nitrogen-free culture medium flat plate, culturing at a constant temperature of 30 ℃ for 5 d, judging whether the biocontrol fungus has nitrogen fixation capacity according to whether the biocontrol fungus can grow on the culture medium and whether transparent rings are produced around the biocontrol fungus, wherein the biocontrol fungus can grow on the nitrogen-free culture medium and the transparent rings are produced around the biocontrol fungus to indicate that the biocontrol fungus has nitrogen fixation capacity.

The culture medium ：(NH₄)₂SO₄0.2 g、MgSO₄·7H₂O 0.1 g、NaCl 0.02 g,Na₂HPO₄0.4 g, of potassium feldspar, sucrose 2 g, yeast extract 0.1 g, potassium feldspar powder 2 g and agar 4 g are sterilized at 7.2,115 ℃ and high temperature of 30min by constant volume of sterile water to 200 mL. And (3) selecting a single bacterial drop point by using a sterilization inoculation needle, inoculating the single bacterial drop point on a potassium feldspar culture medium plate, culturing the single bacterial drop point at a constant temperature of 30 ℃ for 5 d, observing whether transparent rings are generated around the single bacterial drop point, and indicating that the single bacterial drop point has potassium dissolving capability by the transparent rings.

And (3) selecting a single bacterial drop point by using a sterilization inoculation needle, inoculating the single bacterial drop point on a CAS detection medium plate, culturing at a constant temperature of 30 ℃ for 5 d, observing that orange rings are generated around the culture medium, and indicating that the culture medium has the capability of generating siderophores by the orange rings.

IAA production ability, namely adding L-tryptophan into LB liquid culture medium according to the addition amount of 100 mg/L, culturing at 37 ℃ and 180 r/min for 72 h, dripping a small amount of Salkowsk colorimetric solution, treating in a dark place, observing after 30 min, and indicating that IAA is produced when the IAA is changed in red.

The results of the measurement of the extracellular enzyme activity and the growth promotion characteristics of the biocontrol bacterium BA200 show (figure 4) that the biocontrol bacterium BA200 has the capabilities of protease, cellulase, beta-1, 3-glucan for inhibiting the extracellular enzyme activity, phosphate and potassium dissolving, nitrogen fixing and siderophore and IAA generating.

After the strain identification is completed, the biocontrol strain BA200 is preserved in China general microbiological culture Collection center (North Star Xili No. 1, 3, institute of microorganisms, academy of sciences of China) for 24 days of 2 months of 2025, with a preservation number of CGMCC No. 33636, and the proposed taxonomy is named as Bacillus amyloliquefaciens Bacillus amyloliquefaciens.

EXAMPLE 3 inhibition of biocontrol bacterium BA200 against cowpea fusarium wilt

1. Experimental method

Preparation of biocontrol fungus culture:

Single colonies obtained in experimental example 1 were picked and placed in 4 mL LB medium, 37℃and 180 r/min shaking culture 24 h, and 10% by volume of the single colonies were placed in 4 mL LB medium, 37℃and 180 r/min shaking culture to obtain a culture with OD 600=1.0.

Detecting the inhibition effect of the culture of the biocontrol bacterium BA200 on fusarium oxysporum and phytophthora vignae by using a plate facing method, dripping the culture of the biocontrol bacterium BA200 in a cross shape around 3.5 cm parts away from the center of the plate, cutting the fusarium oxysporum, the phytophthora vignae and the phytophthora sojae into 2mm multiplied by 2mm bacterial blocks, respectively inoculating the bacterial blocks in the center of the plate, taking the pathogen plate as a reference, culturing in the dark at a constant temperature of 25 ℃, measuring the diameter of a colony when the colony of the reference is grown on the plate quickly, and calculating the colony inhibition rate.

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

2. Experimental results

As shown in fig. 5, the culture of biocontrol strain BA200 has remarkable inhibitory effect on fusarium oxysporum, phytophthora cowpea and phytophthora sojae, with an inhibition rate of 63.81% on fusarium oxysporum, 75.27% on phytophthora sojae and 61.84% on phytophthora sojae.

EXAMPLE 4 inhibition of biocontrol bacterium BA200 against various plant pathogens

The inhibitory ability of biocontrol strain BA200 against a variety of plant pathogenic bacteria including Fusarium solani (Fusarium solani), rhizoctonia solani (Rhizoctonia solani), anthrax grus (Colletotrichum truncatum), colletotrichum gloeosporioides (Colletotrichum gloeosporioides), alternaria alternata (ALTERNARIA EICHHORNIAE), polyporus (nodulisporium sp.), plaque forming bacteria (Myrothecium inundatum) and phytophthora palmi (Phytophthora palmivora) was measured as described in experimental example 3, and the inhibitory effect of biocontrol strain BA200 against a variety of plant pathogenic bacteria is shown in table 2 and fig. 6.

TABLE 2 inhibition of various plant pathogens by biocontrol bacterium BA200

As can be seen from Table 2 and FIG. 6, the Bacillus amyloliquefaciens BA200 has good inhibition effect on the tested 8 plant pathogenic bacteria, and the average inhibition rate is higher than 50%, which indicates that the Bacillus amyloliquefaciens BA200 has broad-spectrum biological control effect.

Example 5 potted plant control efficacy determination of biocontrol bacterium BA200

Single colonies obtained in experimental example 1 were picked and placed in 4 mL LB medium, 37℃and 180 r/min shaking culture 24 and h, 200 mL LB medium was placed in 1% volume ratio, 37℃and 180 r/min shaking culture, and a fermentation broth with OD 600=0.6 (concentration 1X 10 ⁷ CFU/mL) was obtained.

Fusarium oxysporum inoculated conidium suspension at a concentration of 5X 10 ⁶ spores/mL. The phytophthora vignae inoculation adopts a fungus soil method, and the phytophthora vignae growing on a 9 cm culture dish is smashed and stirred with soil by a syringe.

Cowpea variety selection "grower looking at win 380" is from Jiangxi grower looking at high-tech limited company.

Treating (1) healthy plants (CK), (2) inoculating fusarium oxysporum (Fo), (3) inoculating phytophthora vignae (Pv), (4) inoculating biocontrol strain BA200 (BA 200), (5) biocontrol strain BA200+ fusarium oxysporum (BA 200+ Fo), and (6) biocontrol strain BA200+ phytophthora vignae (BA 200+ Pv).

After being disinfected, cowpea seeds are sowed in wet vermiculite, when two true leaves of cowpea seedlings are completely unfolded, the cowpea seeds are transplanted into a wet nutrition matrix, 50mL of biocontrol bacterium fermentation liquor with the concentration of 1X 10 ⁷ CFU/mL is inoculated, sterile water is inoculated in a control group, pathogenic bacteria are inoculated after 24h, fusarium oxysporum is subjected to pathogenicity measurement by adopting a root soaking inoculation method, phytophthora vignae is subjected to pathogenicity measurement by adopting a fungus soil method, 6 plants are treated each time, 3 times of repetition are carried out, the pathogenicity conditions of each treatment are counted, and the disease index is recorded.

The disease conditions include incidence and disease classification, divided into 6 classes, respectively:

grade 0, normal stem bundle in the vascular, no disease;

Grade 1, wherein the color change degree of the stem bundle in the vascular is less than 1/4;

2, the color change degree of the bundle stem in the vascular is less than or equal to 1/4 and less than 1/2;

Grade 3, wherein the color change degree of the stem bundles in the vascular tube is less than or equal to 1/2 and less than or equal to 3/4, and the leaves are yellow and withered;

4, the color change degree of the stem in the vascular is more than 3/4, and the leaves are yellow, withered and shed;

Grade 5, wilting, lodging or dying of the whole plant.

Disease index = [ Σ (number of cowpea plants per disease grade x number of disease grade)/total number of cowpea potted plants x number of highest disease grade ] ×100.

Control effect= (control disease index-treatment disease index)/control disease index x 100%.

The results are shown in tables 3,4 and FIG. 7.

TABLE 3 statistical results of the disease conditions and control effects of cowpea wilt (Fusarium oxysporum)

From table 3 and fig. 7, the bacillus amyloliquefaciens BA200 can obviously reduce the severity of the cowpea wilt caused by fusarium oxysporum, which indicates that the bacillus amyloliquefaciens BA200 has a control effect on the cowpea wilt caused by fusarium oxysporum, and the control effect is 72.06%.

TABLE 4 statistical results of the disease conditions and control effects of cowpea wilt (phytophthora cowpea)

As can be seen from table 4 and fig. 7, the bacillus amyloliquefaciens BA200 of the present invention can significantly reduce the severity of diseases of cowpea wilt caused by phytophthora sojae, which indicates that the bacillus amyloliquefaciens BA200 has a control effect on cowpea wilt caused by phytophthora sojae, and the control effect is 83.10%.

Claims (9)

1. A bacillus amyloliquefaciens BA200, characterized in that:

The class of the bacillus amyloliquefaciens BA200 is named as bacillus amyloliquefaciens Bacillus amyloliquefaciens, and is preserved in China general microbiological culture Collection center (CGMCC) No. 33636 in 2 months and 24 days of 2025.

2. The culture of bacillus amyloliquefaciens BA200 of claim 1, wherein:

The preparation method of the culture comprises the following steps:

inoculating the bacillus amyloliquefaciens BA200 in claim 1 to a culture medium, and culturing to obtain;

The temperature of the culture is 16-42 ℃;

the culture time is 12-96 h;

The culture medium comprises LB culture medium.

3. A method of preparing a culture comprising the steps of:

inoculating the bacillus amyloliquefaciens BA200 in claim 1 to a culture medium, and culturing to obtain;

The temperature of the culture is 16-42 ℃;

the culture time is 12-96 h;

The culture medium comprises LB culture medium.

4. A microbial agent comprising the bacillus amyloliquefaciens BA200 of claim 1 or the culture of claim 2.

5. The microbial agent of claim 4, wherein:

the microbial inoculum is a solid microbial inoculum or a liquid microbial inoculum.

6. The method for producing a microbial inoculum according to claim 4 or 5, wherein the microbial inoculum is obtained by using the bacillus amyloliquefaciens BA200 according to claim 1 or the culture according to claim 2 as an active ingredient.

7. Use of bacillus amyloliquefaciens BA200 according to claim 1, a culture according to claim 2, or a microbial inoculum according to claim 4 or 5 for the preparation of a product for controlling phytopathogens;

The plant pathogenic bacteria are at least one of phytophthora sojae, fusarium oxysporum, phytophthora vignae, fusarium solani, rhizoctonia solani, colletotrichum glomerata, colletotrichum glomeratum, alternaria, polyrhizium sp and phytophthora palmi.

8. A product comprising one of bacillus amyloliquefaciens BA200 of claim 1, the culture of claim 2, or the microbial inoculum of claim 4 or 5;

The product comprises auxiliary materials.

9. A method for controlling phytopathogens comprising the step of treatment with bacillus amyloliquefaciens BA200 according to claim 1, a culture according to claim 2, or a microbial 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 glomerata, colletotrichum glomeratum, alternaria, polyrhizium sp and phytophthora palmi.