Bacillus velezensis and application of compound preparation thereof

Abstract

A bacillus velezensis strain and a compound preparation thereof and application, belong to the technical field of plant disease control. In order to solve the problems of existing chemical control methods of cowpea wilt, such as easy to produce drug resistance, influence ecological environment, pesticide residue and pesticide overproof, etc., the present application screens a bacillus velezensis T9 which has good inhibitory effect on cowpea wilt pathogen. It is found through experiment that the strain T9 has obvious inhibitory effect on various plant pathogenic fungi, the bacterial liquid can effectively prevent and control cowpea wilt, and can effectively promote the growth of cowpea. In addition, the compound preparation prepared by using prochloraz and bacillus velezensis T9 bacterial liquid is irrigated to cowpea seedlings, it is found that the compound preparation has good control effect on cowpea wilt, and can effectively reduce the use amount of chemical agent, and has certain growth promoting effect on cowpea plants.

Description

Technical Field

[0001] This invention belongs to the field of plant disease control technology, specifically relating to a strain of Bacillus belye and its compound agents and applications. Background Technology

[0002] cowpea( Vigna unguiculata (L.) Walp.), also known as cowpea, long bean, string bean, winged bean, and black navel bean, is a common pod vegetable in China. Its annual cultivation area accounts for more than 10% of the country's total vegetable cultivation area, exceeding 500,000 mu (approximately 33,333 hectares), making it an important vegetable and grain crop in my country.

[0003] Cowpea wilt is caused by Fusarium oxysporum (Fusarium oxysporum) Fusarium oxysporum Fusarium wilt is a serious soil-borne disease caused by wilt, and it is one of the most damaging, difficult to control, and economically detrimental diseases affecting cowpeas in Hainan, severely restricting the development of the cowpea industry. It can occur in almost all tropical to subtropical cowpea growing areas, and in severe cases, it can lead to a yield reduction of 70% or even more. The mycelium or chlamydospores of the pathogen invade the cowpea plant through wounds or intercellular spaces in the rootstock, obtaining nutrients from the host to reproduce. The growing mycelium gradually blocks the plant's vascular bundles and secretes harmful substances such as cellulase and pectinase, leading to cell death. This affects the host plant's ability to obtain water and nutrients from the soil, resulting in symptoms similar to physiological wilting throughout the plant. Therefore, after the onset of cowpea wilt, dissection of the rootstock often reveals brownish discolored necrotic tissue, occasionally with pinkish mold. The leaves gradually turn yellow, discolor, and wither from the bottom up.

[0004] Currently, the control of cowpea wilt mainly relies on chemical control. However, the frequent use of chemical agents not only exacerbates the pathogen's resistance but also severely impacts the ecological environment. Furthermore, some farmers, in an effort to control cowpea diseases, increase the frequency and amount of pesticide use, even illegally using banned agents. Combined with the characteristic of cowpeas being harvested every other day, this leads to significant problems such as banned pesticide residues and excessive pesticide levels in cowpeas. Therefore, there is an urgent need for efficient and environmentally friendly control methods to improve this situation. Summary of the Invention

[0005] To address the problems of existing chemical control methods for cowpea wilt, such as easy development of drug resistance, environmental impact, residues of banned pesticides, and pesticide exceeding standards, this invention screened a strain T9 from the rhizosphere soil of healthy cowpea plants in wilt-affected areas. This strain exhibited good inhibitory activity against the cowpea wilt pathogen. Morphological, physiological, biochemical, and molecular biological identification confirmed that the strain was *Bacillus belyesense*, and the strain was preserved. The taxonomic name of this strain is *Bacillus belyesense*. Bacillus amanuensisThe depositary institution is the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit date is February 21, 2025, and the accession number is CGMCC No. 33611. Experiments have shown that *Bacillus belyi* T9 has a significant inhibitory effect on various plant pathogenic fungi. Its bacterial solution can effectively control cowpea wilt and promote cowpea growth. Furthermore, this invention utilizes a compound agent prepared from prochloraz and *Bacillus belyi* T9 bacterial solution for root irrigation of cowpea seedlings. The compound agent showed good control effect against cowpea wilt, effectively reducing the amount of chemical agents used, and also had a certain growth-promoting effect on cowpea plants.

[0006] To solve the above-mentioned technical problems and achieve the corresponding technical effects, the present invention provides the following technical solution:

[0007] The first objective of this invention is to provide a strain of Bacillus belyssus (B. belyssus) Bacillus velezensis T9, with accession number CGMCC No.33611.

[0008] The second objective of this invention is to provide the application of the aforementioned Bacillus belyi T9 in inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi are Fusarium oxysporum xanthophyte, the pathogen causing cowpea wilt (Fusarium oxysporum var. xanthophyte). Fusarium oxysporum f. sp. tracheiphilum Fusarium graminearum, the pathogen of wheat scab ( Fusarium graminearum Fusarium oxysporum, the pathogen of banana wilt ( Fusarium oxysporum f.sp. cubense ), Fusarium oxysporum, the pathogen of coffee leaf blight ( Fusarium lateritium Mango anthracnose pathogen, *Colletotrichum gloeosporioides* (… Colletotrichum gloeosporioides Rice blast pathogen ( ) Magnaporthe grisea ), Sisal leaf rot pathogen Diplosporum cocovenenans ( Lasiodiplodia theobromae ), coconut gray spot disease pathogen *Pseudomonas spp.* Pestalotiopsis microspora ), the pathogen of maize leaf spot disease, Curvularia zeyla ( Curvularia lunata ), Phytophthora blight pathogen ( Phytophthora capsici ), and the pathogen of leaf spot disease ( ) Diaporthe biconispora ), dragon fruit canker pathogen new dark-colored arthropoda ( Neoscytalidium dimidiatum ) or coffee anthrax pathogen, *Caucasus kaharvatus* ( Colletotrichum kahawae ).

[0009] A third object of the present application is to provide the use of the above-mentioned Bacillus velezensis T9 in the preparation of a biocontrol agent, microbial fertilizer or biopesticide for inhibiting plant pathogenic fungi, wherein the plant pathogenic fungi are Fusarium oxysporum f. sp. vasinfectum, Fusarium graminearum, Fusarium oxysporum, Fusarium coffis, Gloeopaxillus mangiferae, Magnaporthe oryzae, Colletotrichum musae, Pythium aphanidermatum, Phytophthora parasitica, Corynespora cassiicola, and Elsinoe kachawa.

[0010] A fourth object of the present application is to provide a biocontrol agent, microbial fertilizer or biopesticide containing the above-mentioned Bacillus velezensis T9.

[0011] A fifth object of the present application is to provide the use of the above-mentioned biocontrol agent, microbial fertilizer or biopesticide in the inhibition of plant pathogenic fungi, wherein the plant pathogenic fungi are Fusarium oxysporum f. sp. vasinfectum, Fusarium graminearum, Fusarium oxysporum, Fusarium coffis, Gloeopaxillus mangiferae, Magnaporthe oryzae, Colletotrichum musae, Pythium aphanidermatum, Phytophthora parasitica, Corynespora cassiicola, and Elsinoe kachawa.

[0012] A sixth object of the present application is to provide a compound preparation, which is composed of a prohexadione water solution with a concentration of 0.0344 µg / mL and a Bacillus velezensis T9 bacterial solution with a concentration of 0.97×10 8 CFU / mL, wherein the volume ratio of the prohexadione water solution to the Bacillus velezensis T9 bacterial solution is (9:1)~(1:9); and the Bacillus velezensis T9 has a preservation number of CGMCC No. 33611.

[0013] In an embodiment of the present application, the volume ratio of the prohexadione water solution to the Bacillus velezensis T9 bacterial solution is (8:2)~(7:3).

[0014] In an embodiment of the present application, the prohexadione water solution is prepared from 98% prohexadione.

[0015] In an embodiment of the present application, the preparation method of the Bacillus velezensis T9 bacterial solution is to inoculate the strain T9 into LB liquid medium and cultivate at 28℃, 180 rpm in the dark for 2 days. In an embodiment of the present application, the preparation method of the Bacillus velezensis T9 bacterial solution is to inoculate the strain T9 into LB liquid medium and cultivate at 28℃, 180 rpm in the dark for 2 days.

[0016] A seventh object of the present application is to provide the use of the above-mentioned complex agent in the prevention and treatment of fusarium wilt of cowpea.

[0017] An eighth object of the present application is to provide the use of the above-mentioned complex agent in promoting the growth of cowpea.

[0018] In an embodiment of the present application, the method of applying the complex agent is root irrigation.

[0019] Advantages of the present application:

[0020] The present application screens a bacillus velezensis T9 strain with good inhibitory effect on the pathogen of cowpea fusarium wilt from the rhizosphere soil of healthy cowpea plants in the cowpea fusarium wilt-occurring plot. It is found through experiments that the strain has certain degree of nitrogen fixation, phosphorus solubilization, potassium solubilization and iron carrier secretion capacity, and has obvious inhibitory effect on various plant pathogenic fungi. In the plate confrontation experiment of the bacterial liquid, the inhibitory rate of the strain T9 on the pathogen of cowpea fusarium wilt, i.e. fusarium oxysporum f. sp. vasinfectum, is 72.76%, the inhibitory rate on the pathogen of wheat scab, i.e. fusarium graminearum, is 72.70%, the inhibitory rate on the pathogen of banana fusarium wilt, i.e. fusarium oxysporum f. sp. cubense, is 67.58%, the inhibitory rate on the pathogen of coffee leaf blight, i.e. fusarium reticulatum, is 61.09%, the inhibitory rate on the pathogen of mango anthracnose, i.e. gloeoporus discolor, is 73.01%, the inhibitory rate on the pathogen of rice blast, i.e. pyricularia oryzae, is 84.61%, the inhibitory rate on the pathogen of sword palm leaf rot, i.e. cochilobolus hagenis, is 69.51%, the inhibitory rate on the pathogen of coconut gray spot, i.e. pestalodiopsiopsi, is 76.10%, the inhibitory rate on the pathogen of corn leaf spot, i.e. cercospora zea, is 75.56%, the inhibitory rate on the pathogen of pepper blight, i.e. phytophthora infestans, is 74.54%, the inhibitory rate on the pathogen of perithecia leaf spot, i.e. diaporthe phaseolorum, is 69.88%, the inhibitory rate on the pathogen of pitaya ulcer disease, i.e. new dark color columnar segment, is 70.89%, and the inhibitory rate on the pathogen of coffee anthracnose, i.e. phyllachora kahawae, is 75.60%. In addition, the results of pot experiment show that, compared with the disease control group, the disease index of cowpea plants treated with T9 bacterial liquid with a concentration of 1×10 7 CFU / mL, 1×10 8 CFU / mL and 1×10 9 CFU / mL decreases by 27.08%, 31.25% and 33.33% respectively. The strain T9 has strong growth-promoting capacity on cowpea plants. Compared with CK, the plant height, leaf area, fresh weight and root length of cowpea plants treated with T9 bacterial liquid with a concentration of 1×10 8 CFU / mL are greatly improved, which increases by 29.95%, 37.85%, 28.66% and 31.07% respectively, and the plant height, leaf area, fresh weight and root length of cowpea plants treated with T9 bacterial liquid with a concentration of 1×10 9Compared with the control group, cowpea plants treated with T9 bacterial solution at CFU / mL showed increases in plant height, leaf area, fresh weight, and root length of 31.30%, 30.71%, 27.16%, and 40.54%, respectively. Therefore, Bacillus belyi T9, as a natural microbial strain, can effectively control plant diseases caused by fungi such as Fusarium oxysporum and has potential commercial application value.

[0021] This invention uses prochloraz as a chemical agent and Bacillus belyceta var. berberis T9 as a biological agent to formulate a compound agent. It was found that a 0.0344 µg / mL aqueous solution of prochloraz and a 0.97 × 10⁻⁶ µg / mL aqueous solution of prochloraz were combined. 8 A compound pesticide prepared from Bacillus vesicularis T9 bacterial suspension at a volume ratio of 8:2 (CFU / mL) effectively controlled cowpea wilt, reducing the disease index by 54% compared to the positive treatment group, achieving a control effect of 63.41%. Furthermore, this compound pesticide promoted the growth of cowpea plants, increasing plant height by 13.89% and 13.10% respectively, leaf area by 66.04% and 33.52% respectively, fresh weight by 25.42% and 18.97% respectively, and root length by 44.47% and 18.19% respectively compared to the positive control and chemical pesticide treatment groups. The compound pesticide provided by this invention can effectively reduce the use of chemical pesticides, lower the risk of pathogen resistance, and protect the ecological environment, demonstrating good development prospects and application potential. Attached Figure Description

[0022] Figure 1 The image shows the results of the antibacterial activity test of strain T9 against the pathogen causing cowpea wilt; among which, Figure 1 In the diagram, A represents the control group, which was inoculated only with the cowpea wilt pathogen. Figure 1 Figure B in the figure shows the results of detecting the inhibitory ability of strain T9 against the pathogen of cowpea wilt using the cross-cross method.

[0023] Figure 2 This is a colony morphology diagram of strain T9 on LB solid medium; where, Figure 2 In the diagram, A represents the morphology of a single colony of strain T9 on LB solid medium. Figure 2 B in the diagram represents the streak culture of strain T9 on LB solid medium;

[0024] Figure 3 The image shows the physiological and biochemical identification results of strain T9; among them, Figure 3 In the figure, A represents the results of the test for the ability of strain T9 to secrete siderophores. Figure 3 In the figure, B represents the results of the nitrogen fixation capacity determination of strain T9. Figure 3 The figure showing C represents the results of potassium-solubilizing ability determination for strain T9. Figure 3 In the figure, D represents the result of the phosphorus solubilization ability determination of strain T9. Figure 3The figure shows the results of the IAA secretion ability assay for strain T9.

[0025] Figure 4 The diagram shows the phylogenetic tree analysis results of the 16S rDNA of strain T9;

[0026] Figure 5 For strain T9 gyrB Figure showing the results of gene phylogenetic phylogenetic tree analysis;

[0027] Figure 6 The image shows the results of the antibacterial activity assay for Bacillus belyssus T9; among them, Figure 6 Figure A shows the results of the assay for the inhibitory activity of strain T9 against Diplostomum cocovenenans, the pathogen causing sisal leaf rot. Figure 6 Figure B in the graph shows the results of the antibacterial activity test of strain T9 against *Discochaeta spp.*, the pathogen of coconut gray spot disease. Figure 6 The figure shows the results of the assay for the inhibitory effect of strain T9 on Curvularia zebrina, the pathogen of maize leaf spot disease. Figure 6 The figure shows the results of the assay for the inhibitory effect of strain T9 on *Normacea glomerata*, the pathogen causing dragon fruit canker. Figure 6 The figure shows the results of the assay for the inhibitory activity of strain T9 against the coffee anthracnose pathogen, *Colletotrichum cahavarensis*. Figure 6 The figure shows the results of the assay for the inhibitory activity of strain T9 against Fusarium oxysporum, the pathogen causing banana wilt. Figure 6 The figure shows the results of the assay for the inhibitory activity of strain T9 against *Colletotrichum gloeosporioides*, the pathogen causing mango anthracnose. Figure 6 The figure shows the results of the assay for the inhibitory activity of strain T9 against Fusarium graminearum, the pathogen of wheat scab. Figure 6 The figure shows the results of the assay for the inhibitory activity of strain T9 against Phytophthora blight pathogen of pepper. Figure 6 The figure shows the results of the assay for the inhibitory effect of strain T9 on the genus *Metacarpa*, the pathogen causing leaf spot disease. Figure 6 The figure shows the results of the assay for the inhibitory activity of strain T9 against Fusarium oxysporum, the pathogen of coffee leaf blight. Figure 6 The figure shows the results of the assay for the inhibitory effect of strain T9 on rice blast fungus.

[0028] Figure 7 The control effect of Bacillus belyi T9 on cowpea wilt disease in a pot experiment is shown in the figure.

[0029] Figure 8 This image shows the effect of Bacillus belyi T9 on promoting the growth of cowpea plants in a pot experiment.

[0030] Figure 9 The graph shows the inhibitory effects of 10 pesticides on the mycelial growth of the pathogen causing cowpea wilt; among them, Figure 9Figure A shows the inhibitory effect of different concentrations of 98% pyraclostrobin on the mycelial growth of the cowpea wilt pathogen. Figure 9 Figure B in the figure shows the inhibitory effect of different concentrations of 98% mancozeb on the mycelial growth of the cowpea wilt pathogen. Figure 9 In the figure, C represents the inhibitory effect of different concentrations of 97% pyraclostrobin on the mycelial growth of the cowpea wilt pathogen. Figure 9 In the figure, D represents the inhibitory effect of different concentrations of 97% thiophanate-methyl on the mycelial growth of the cowpea wilt pathogen. Figure 9 E in the figure represents the inhibitory effect of different concentrations of 97% chlorothalonil on the mycelial growth of the cowpea wilt pathogen. Figure 9 In the figure, F represents the inhibitory effect of different concentrations of 97% azoxystrobin on the mycelial growth of the cowpea wilt pathogen. Figure 9 In the figure, G represents the inhibitory effect of different concentrations of 98% carbendazim on the mycelial growth of the cowpea wilt pathogen. Figure 9 In the figure, H represents the inhibitory effect of different concentrations of 98% prochloraz on the mycelial growth of the cowpea wilt pathogen. Figure 9 In the figure, I represents the inhibitory effect of different concentrations of 99% hymexazol on the mycelial growth of the cowpea wilt pathogen. Figure 10 J in the figure represents the inhibitory effect of different concentrations of 85% propineb on the mycelial growth of the cowpea wilt pathogen; the concentrations of each agent corresponding to the plates from left to right in the inhibitory effect figures on the mycelial growth of the cowpea wilt pathogen correspond to Table 4 in the instruction manual.

[0031] Figure 10 The graph shows the compatibility test results between Bacillus belyceta var. T9 and imazalil; among them, Figure 10 'a' in the text represents the blank control. Figure 10 Figure b shows the growth of strain T9 on an LB agar plate containing 0.01 µg / mL imazalil. Figure 10 In the figure, c represents the growth of strain T9 on an LB agar plate containing 0.05 µg / mL imazalil. Figure 10 In the figure, d represents the growth of strain T9 on LB agar plates containing 0.1 µg / mL imazalil. Figure 10 The figure 'e' represents the growth of strain T9 on an LB agar plate containing 0.5 µg / mL imazalil. Figure 11 f in the figure represents the growth of strain T9 on an LB agar plate containing 1.0 µg / mL imazalil;

[0032] Figure 11 The graph shows the virulence test results of different concentrations of Bacillus belyi T9 bacterial suspensions against the pathogen causing cowpea wilt; among them, Figure 11 In the figure, 'a' represents the control group, which was inoculated only with the cowpea wilt pathogen. Figure 11 In this context, b represents a concentration of 1×10⁻⁶. 5The results of the virulence determination of T9 bacterial suspension at CFU / mL against the pathogen of cowpea wilt are shown in the figure. Figure 11 In this context, c represents a concentration of 1×10⁻⁶. 6 The results of the virulence determination of T9 bacterial suspension at CFU / mL against the pathogen of cowpea wilt are shown in the figure. Figure 11 In this context, d represents a concentration of 1×10⁻⁶. 7 The results of the virulence determination of T9 bacterial suspension at CFU / mL against the pathogen of cowpea wilt are shown in the figure. Figure 11 In this context, 'e' represents a concentration of 1×10⁻⁶. 8 The results of the virulence determination of T9 bacterial suspension at CFU / mL against the pathogen of cowpea wilt are shown in the figure. Figure 11 f represents a concentration of 1×10⁻⁶. 9 The results of the virulence determination of T9 bacterial suspension at CFU / mL against the pathogen of cowpea wilt are shown in the figure. Figure 12 In this context, g represents a concentration of 1×10⁻⁶. 10 The results of the virulence determination of T9 bacterial suspension at CFU / mL against the pathogen of cowpea wilt are shown in the figure.

[0033] Figure 12 The graph shows the virulence test results of different compound formulations against the pathogen of cowpea wilt; among them, Figure 12 In this context, 'a' represents the control group (CK) without the added compounding agent. Figure 13 The volume ratios of the prochloraz aqueous solution and the bacterial suspension of strain T9 in the compound preparations of the plates corresponding to b, c, d, e, f, g, h, i, j, k and l are 10:0, 9:1, 8:2, 7:3, 6:4, 5:5, 4:6, 3:7, 2:8, 1:9 and 0:10, respectively.

[0034] Figure 13 The efficacy of a compound agent of Bacillus vesicular T9 and prochloraz against cowpea wilt is shown in the figure; among them, Figure 13 In the diagram, A represents the control efficacy of the entire cowpea plant. Figure 13 B in the diagram represents the vascular bundle control effect of cowpea plants; Bacillus velezensis In the diagram, a represents the positive control group, b represents the water treatment group, c represents the LB treatment group, d represents the T9 bacterial solution treatment group, e represents the 98% prochloraz treatment group, and f represents the 98% prochloraz + T9 treatment group. Detailed Implementation

[0035] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to specific embodiments and accompanying drawings. It should be noted that the embodiments mentioned below are only for explaining the invention and are not intended to limit the scope of the invention. The embodiments mentioned below are only some embodiments of the invention, not all embodiments. Those skilled in the art can refer to the content of this document and appropriately improve the process parameters to achieve the desired results. It should be particularly pointed out that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in this invention. The methods and applications of this invention have been described through preferred embodiments, and those skilled in the art can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of this invention to realize and apply the technology of this invention. In the art, any embodiments obtained by other skilled personnel without creative effort are protected by this invention.

[0036] Unless otherwise specified, the experimental methods used in the following embodiments are conventional methods. The materials, reagents, culture media, and instruments used are all conventional materials, reagents, culture media, and instruments in the art, and can be obtained commercially by those skilled in the art. Unless otherwise specified, the molecular biology experimental procedures involved in this invention are conventional experimental procedures in the art or can be performed according to the product instructions of the corresponding reagents.

[0037] The Bacillus belyssus strain screened in this invention ( Bacillus velezensis T9, taxonomically named Bacillus belesii Fusarium oxysporum The specimen is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 33611, on February 21, 2025. The address of the depository is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0038] The plant pathogens used in this invention are from the following sources:

[0039] The pathogen causing cowpea wilt is *Fusarium oxysporum*, a vascular-specific strain. tracheiphilum f.sp. Fusarium graminearum Fusarium graminearum, the pathogen of wheat scab ( Fusarium oxysporum Fusarium oxysporum, the pathogen of banana wilt ( cubense f.sp. Fusarium lateritium ), Fusarium oxysporum, the pathogen of coffee leaf blight ( Colletotrichum Mango anthracnose pathogen, *Colletotrichum gloeosporioides* (… gloeosporioides Magnaporthe grisea Rice blast pathogen ( ) Lasiodiplodia theobromae ), Sisal leaf rot pathogen Diplosporum cocovenenans ( Pestalotiopsis microspora), coconut gray spot disease pathogen *Pseudomonas spp.* Curvularia lunata ), the pathogen of maize leaf spot disease, Curvularia zeyla ( Phytophthora capsici ), Phytophthora blight pathogen ( Diaporthe ), and the pathogen of leaf spot disease ( ) biconispora Neoscytalidium dimidiatum ), dragon fruit canker pathogen new dark-colored arthropoda ( Colletotrichum kahawae ) and coffee anthrax pathogen, Colchicum kaharvae ( Figure 1 It is preserved by the Biosafety Center of the China Academy of Inspection and Quarantine in Sanya.

[0040] The culture medium involved in this invention and its composition are as follows:

[0041] LB medium: peptone 10.0 g / L, yeast extract 5.0 g / L, sodium chloride 8.0 g / L, agar 20 g / L, balance ddH2O; sterilize at 121℃ for 20 min.

[0042] PDA medium: Boil 200 g potatoes in water for 15 min and keep the filtrate. Add glucose 18 g / L, agar 18 g / L, and the remainder is ddH2O. Sterilize at 121℃ for 20 min.

[0043] NBRIP Inorganic Phosphorus Medium: Glucose 10.0 g / L, Ammonium Sulfate 0.1 g / L, Magnesium Sulfate Heptahydrate 0.25 g / L, Potassium Chloride 0.2 g / L, Magnesium Chloride 5.0 g / L, Calcium Phosphate 5.0 g / L, Balance ddH2O; Sterilize at 121℃ for 20 min.

[0044] Alexandrite silicate medium: sucrose 5.0 g / L, disodium hydrogen phosphate 2.0 g / L, magnesium sulfate heptahydrate 0.5 g / L, ferric chloride 0.005 g / L, calcium carbonate 0.1 g / L, potassium feldspar 2.0 g / L, balance ddH2O; sterilize at 115℃ for 20 min.

[0045] Assumption nitrogen-free medium: dipotassium hydrogen phosphate 0.2 g / L, magnesium sulfate heptahydrate 0.2 g / L, calcium sulfate dihydrate 0.2 g / L, sodium chloride 0.2 g / L, calcium carbonate 5.0 g / L, mannitol 10.0 g / L, balance ddH2O; sterilize at 121℃ for 20 min.

[0046] Chromium azurite (CAS) medium: The basal medium consists of 100.0 g glucose, 0.5 g magnesium sulfate heptahydrate, 20.0 g peptone, 0.5 g calcium chloride, and 10.0 g agar, with water added to a final volume of 800 mL; the CAS detection solution consists of 0.06 g chromium azurite, 0.0027 g ferric chloride, 0.073 g hexadecyltrimethylammonium bromide (HDTMA), and 100 mL water; the concentration of PIPES in the 10×PIPES buffer is 1 mol / L, and the pH of the buffer is 7.0; the basal medium is sterilized at 115℃ for 20 min, cooled to 60℃, and 100 mL of 10×PIPES buffer preheated to 60℃ and 100 mL of CAS detection solution are slowly added.

[0047] YMB medium: yeast extract 1.0 g / L, mannitol 10.0 g / L, dipotassium hydrogen phosphate 0.5 g / L, magnesium sulfate heptahydrate 0.2 g / L, sodium chloride 0.1 g / L, calcium carbonate 1.0 g / L, L-tryptophan 2.0 g / L, balance ddH2O; sterilize at 151℃ for 20 min.

[0048] Example 1: Screening and identification of antagonistic bacteria against cowpea wilt pathogen

[0049] 1. Screening of antagonistic bacteria against cowpea wilt pathogen

[0050] Rhizosphere soil samples were collected from healthy cowpea plants in areas affected by cowpea wilt disease. Soil separation was performed using the dilution plating method: 10 g of soil sample was placed in a 250 mL Erlenmeyer flask, 90 mL of ddH2O was added, and the sample was placed on a shaker at 28℃ and 180 rpm for 30 min to mix thoroughly; the sample was then placed in an 80℃ water bath and allowed to stand for 10 min, before being diluted to 10 g / L. -2 10 -3 10 -4 10 -5 Dilute the solution; add 10-15 mL of preheated LB medium to the petri dish, and after it cools, take 0.2 mL of each of the above concentrations of diluted solution and add them to different plates. Spread the solution evenly with a glass spreader until dry, and repeat three times. After sealing, place the plate at 28℃ and incubate. Observe the colonies after 2 days. Select suspected colonies and streak them on LB plates for purification culture. Number the strains and save them.

[0051] Using *Fusarium wilt*, the pathogen of cowpea wilt, as a control strain, the strains selected above were initially screened using the plate confrontation method. A 0.5 cm diameter *Fusarium wilt* mycelium was inoculated in the center of a PDA solid medium plate. Four points 2.5 cm from the center of each medium plate were selected using the cross-symmetry method. The same strain was isolated, purified, and preserved at these four points using toothpicks. Strains with antagonistic effects were screened again using the same method and preserved. The colony diameter was measured using the cross-cross method. Inhibition rate (%) = (colony diameter of control group - colony diameter of treatment group) / colony diameter of control group × 100. The experiment was repeated three times, with three replicates for each treatment. Data were compiled using Excel, and SPSS (25) software was used to analyze the significance of data differences. Tukey's Method was used for multiple comparisons.

[0052] Screening tests for antagonistic bacteria against the pathogen of cowpea wilt showed that strain T9 exhibited the strongest inhibitory effect against *Fusarium oxysporum*, the pathogen of cowpea wilt, with an inhibition rate of 72.76% (see...). Figure 2 Therefore, this invention will conduct further research on strain T9.

[0053] 2. Identification of strain T9

[0054] (1) Morphological identification

[0055] Strain T9 was inoculated onto LB solid medium and cultured for several days before observing colony morphology. Figure 3 As shown, the colonies of strain T9 are white, with obvious wrinkles, an uneven and opaque surface, and indistinct spacing between colonies.

[0056] (2) Physiological and biochemical identification

[0057] Determination of phosphorus and potassium solubilization abilities: After activation, strain T9 was inoculated onto NBRIP inorganic phosphorus medium and Alexandrite silicate medium, respectively, and incubated in a constant temperature incubator at 28℃ for 7 days. The presence of transparent oil droplet-like halos around the colonies was observed. If present, it indicated that the strain had phosphorus / potassium solubilization abilities. The size of the halo was measured.

[0058] Determination of nitrogen fixation function: After activation, strain T9 was inoculated onto Assumption nitrogen-free medium and incubated in a constant temperature incubator at 28℃ for 7 days. Colony growth was observed. The ability to grow on Assumption nitrogen-free medium was considered to have nitrogen fixation function. The ammonia fixation capacity was judged based on the diameter of the colony growth.

[0059] Siderophore production capacity determination: After activation, strain T9 was inoculated onto chromium azure medium and incubated in a constant temperature incubator at 28℃ for 7 days. The presence or absence of a yellow halo around the colony was observed, and the size of the halo was measured.

[0060] Determination of auxin (IAA) secretion capacity: Strain T9 was inoculated into YMB medium and cultured at 28°C for 2 days. After centrifugation at 12000 rpm for 2 min, 2 mL of the supernatant was transferred to a test tube, and an equal volume of Salkowski's reagent was added. The mixture was gently shaken and incubated in the dark for 30 min. A red color indicated the production of IAA. YMB culture medium with Salkowski's reagent served as a blank control.

[0061] The results are as follows gyrB As shown, strain T9 possesses a certain degree of nitrogen fixation, phosphorus solubilization, potassium solubilization, and siderophore secretion capabilities. On CAS medium, a clear yellow halo with a diameter of 23.42 mm was observed, indicating that strain T9 has the ability to secrete siderophores. On Assumption nitrogen-free medium, the secretion ring of strain T9 was smaller, indicating a weaker nitrogen fixation ability. On Alexandrite silicate medium, strain T9 produced cross-shaped colonies with a diameter of 35.66 × 42.12 mm, indicating a strong potassium solubilization ability. On NBRIP inorganic phosphorus medium, the secretion ring of strain T9 had a diameter of 11.13 mm, indicating that strain T9 has the ability to decompose organic phosphorus. The addition of Salkowski's reagent to the fermentation supernatant of strain T9 showed no significant color change, indicating that strain T9 has a weak IAA secretion ability.

[0062] (3) Molecular biological identification

[0063] Strain T9 was inoculated into LB liquid medium and incubated at 180 rpm for 16 h. DNA was extracted from strain T9 using a bacterial DNA extraction kit from Tiangen Biotech (Beijing) Co., Ltd., following the manufacturer's instructions. The obtained bacterial DNA was used as a template for PCR amplification. The target genes selected were the ribosomal 16S rDNA gene and the helicase B subunit gene. gyrB The primers used for amplifying 16S rDNA are universal primers 27F (SEQ ID NO.1) and 1492R (SEQ ID NO.2), used for amplification. gyrB The primers are gyrBF (SEQ ID NO.3) and gyrBR (SEQ ID NO.4).

[0064] SEQ ID NO.1: 5′-AGAGTTTGATCCTGGCTCAG-3′;

[0065] SEQ ID NO.2: 5′-TACGGCTACCTTGTTACGACTT-3′;

[0066] SEQ ID NO.3: 5′-GAAGTCATCATGACCGTTCTGCAYGCNGGNGGNAARTTYGA-3′;

[0067] SEQ ID NO. 4: 5'-AGCAGGGTACGGATGTGCGAGCCRTCNACRTCNGCRTCNGTCAT-3'.

[0068] The PCR reaction system consisted of: 12.5 μL of 2×Tap PCR MasterMix, 9.5 μL of ddH2O, 1 μL each of forward and reverse primers, and 1 μL of template DNA. The 16S rDNA amplification program was as follows: pre-denaturation at 95℃ for 5 min; denaturation at 95℃ for 30 s, annealing at 55℃ for 45 s, extension at 72℃ for 1 min, for 35 cycles; and a final extension at 72℃ for 10 min. gyrB The amplification program was as follows: pre-denaturation at 94℃ for 4 min; denaturation at 94℃ for 1 min, annealing at 57℃ for 1 min, extension at 72℃ for 80 s, 30 cycles; and then extension at 72℃ for 10 min.

[0069] PCR amplification products were detected by 1% agarose gel electrophoresis. After observing and photographing the target fragment bands in a gel imaging system, the PCR amplification products were sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. Sequencing results were compared and analyzed for homology in the NCBI database, and highly similar gene sequences and their type strain sequences were downloaded. A phylogenetic tree was constructed using MEGA 11 software with the maximum likelihood method.

[0070] 16S rDNA of strain T9 and gyrB The gene fragment was amplified by PCR, yielding approximately 1400 bp of 16S rDNA and approximately 1200 bp of [other DNA]. gyrB Gene fragments. The PCR amplification products were sent for sequencing, and the obtained sequence (the 16S rDNA sequence is shown in SEQ ID NO.5) was obtained. B. velezensis The gene sequence (as shown in SEQ ID NO. 6) was sequenced on the NCBI website. The 16S rDNA alignment results showed that strain T9 was similar to *Bacillus belye*. gyrB The similarity of CBMB205 (NR_116240.1) is 99.65%. B. velezensis Comparison results showed that strain T9 was similar to Bacillus belye. Figure 4 The similarity between GH1-13 (KT991749.1) and GH1-13 (KT991749.1) was 100%. Highly similar gene sequences and type strain sequences were downloaded. Phylogenetic trees of strain T9's 16S rDNA and gyrB were constructed using MEGA 11 software and analyzed. The results are as follows: Figure 5 and B. velezensisAs shown, strain T9 and Bacillus belye ( Bacillus velezensis The bacteria clustered in the same branch. Therefore, based on morphological and physiological-biochemical identification results, strain T9 was identified as Bacillus belyesense (B. belyesense). Bacillus velezensis This invention preserves strain T9 at the China General Microbiological Culture Collection Center (CGMCC), and its taxonomical name is *Bacillus belyssae*. Fusarium graminearum The accession number is CGMCC No. 33611, the accession date is February 21, 2025, and the address of the depositary is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences.

[0071] SEQ ID NO.5:

[0072]

[0073] SEQ ID NO.6:

[0074]

[0075] Example 2: Determination of the antibacterial activity of Bacillus belyssus T9

[0076] The tested plant pathogenic fungi included Fusarium graminearum, the pathogen of wheat scab (Fusarium graminearum). ​ Fusarium oxysporum, the pathogen of banana wilt ( Fusarium oxysporum f.sp. cubense ), Fusarium oxysporum, the pathogen of coffee leaf blight ( Fusarium lateritium Mango anthracnose pathogen, *Colletotrichum gloeosporioides* (… Colletotrichum gloeosporioides Rice blast pathogen ( ) Magnaporthe grisea ), Sisal leaf rot pathogen Diplosporum cocovenenans ( Lasiodiplodia theobromae ), coconut gray spot disease pathogen *Pseudomonas spp.* Pestalotiopsis microspora ), the pathogen of maize leaf spot disease, Curvularia zeyla ( Curvularia lunata ), Phytophthora capsici, the pathogen of pepper blight, and *Phytophthora*, the pathogen of leaf spot disease. Diaporthe biconispora ), dragon fruit canker pathogen new dark-colored arthropoda ( Neoscytalidium dimidiatum ), coffee anthrax pathogen, *Colletotrichum kaharava* ( Colletotrichum kahawae ).

[0077] The antibacterial activity of strain T9 was determined using the plate inhibition method. Strain T9 was inoculated into LB liquid medium and cultured at 28℃ and 180 rpm for 16 h to obtain bacterial fermentation broth. Using the "cross-hatching method," sterile 5 mm diameter filter paper discs were affixed to four points 2.5 cm apart on the center of PDA culture plates. The tested pathogenic fungus was used as the target; mycelial discs of uniform age were punched from the edge of activated fungal colonies using a sterile 5 mm punch and inoculated into the center of the PDA plate. 1 μL of bacterial fermentation broth was added to the center of each of the four filter paper discs. Each treatment was repeated in triplicate. The treated PDA plates were incubated at 28℃ for 4–5 days, during which colony growth was observed. Plates with only mycelial discs of the tested pathogen inoculated in the center served as controls. When the tested pathogenic fungal colonies in the controls covered 3 / 4 of the culture plate, the antibacterial activity was measured, recorded, and photographed. The formula for calculating the inhibition rate is as follows:

[0078] Inhibition rate (%) = [(Diameter of pathogen growth in control group - Diameter of pathogen block) - (Diameter of pathogen growth in treatment group - Diameter of pathogen block)] / (Diameter of pathogen growth in control group - Diameter of pathogen block) × 100

[0079] The results are shown in Table 1 and Figure 6As shown, strain T9 exhibits good inhibitory activity against *Fusarium oxysporum*, the pathogen of cowpea wilt, and also against other tested pathogenic fungi. For example, it showed an inhibition rate of 72.70% against *Fusarium graminearum*, the pathogen of wheat scab; 67.58% against *Fusarium oxysporum*, the pathogen of banana wilt; 61.09% against *Fusarium oxysporum*, the pathogen of coffee leaf blight; 73.01% against *Colletotrichum gloeosporioides*, the pathogen of mango anthracnose; and 84.61% against *Blastomyces oryzae*, the pathogen of rice blast. The inhibition rates were as follows: 69.51% against *Diplosporium cocovenenans* (the pathogen causing sisal leaf rot); 76.10% against *Polytrichum simulans* (the pathogen causing coconut gray spot); 75.56% against *Curvularia zeylinum* (the pathogen causing maize leaf spot); 74.54% against *Phytophthora* (the pathogen causing pepper blight); 69.88% against *Cytotrichum* (the pathogen causing heart-leaf spot); 70.89% against *Neopterosporium davidii* (the pathogen causing dragon fruit canker); and 75.60% against *Colletotrichum cahavarensis* (the pathogen causing coffee anthracnose).

[0080] Table 1. Results of antibacterial activity assay of Bacillus belyss T9

[0081]

[0082] Example 3: Application of Bacillus belyi T9 in the control of cowpea wilt disease

[0083] For indoor potted cowpea seedlings at the 2-leaf, 1-heart stage, the pathogen *Fusarium oxysporum* was prepared at a concentration of 1×10⁻⁶. 7 CFU / mL spore suspension. After cleaning the soil from the root surface of seedlings, cut off 5 fibrous roots to injure the roots. Soak the roots in the pathogen spore suspension for 15 min before transplanting into flowerpots. Five treatment groups were set up: H2O (water control group, no root injury treatment), FO (positive control group, root injury and root soaking in Fusarium oxysporum spore suspension), FO+1×10 7 T9 (in addition to the FO group, 1×10) 7 T9 bacterial culture (CFU / mL), FO+1×10 8 T9 (in addition to the FO group, 1×10) 8 T9 bacterial culture (CFU / mL), FO+1×10 9 T9 (in addition to the FO group, 1×10) 9 (T9 bacterial suspension at CFU / mL). The T9 bacterial suspension was obtained by inoculating strain T9 into LB liquid medium and culturing it in the dark at 28°C and 180 rpm for 2 days. The inoculation method for the T9 bacterial suspension was root drenching. The first root drenching was done after transplanting, and thereafter, 15 mL of T9 bacterial suspension was applied every 5 days. After 20 days, the disease index was evaluated and the control effect was measured.

[0084] The disease index evaluation for cowpea wilt is based on the following standards: Grade 0: No discoloration of the root and stem vascular bundles, no yellowing or wilting of leaves; Grade 1: Discoloration of less than 1 / 4 of the root and stem vascular bundles, some leaves discolor, no significant change in plant growth; Grade 2: Discoloration of less than 1 / 2 of the root and stem vascular bundles, leaves discolor and wither, plant growth is inhibited; Grade 3: Discoloration of nearly 3 / 4 of the root and stem vascular bundles, significant inhibition of the entire plant's growth, leaves yellowing, wilting, and falling off from the bottom up; Grade 4: Complete discoloration of the root and stem vascular bundles, the entire plant withers and dies. The disease index evaluation and control effect determination are calculated using the following formula:

[0085] Disease index (%) = [Σ(Number of plants with disease at each disease level × Disease level number) / Total number of potted plants × Highest disease index level] × 100

[0086] Prevention and control efficacy (%) = [Disease index of control group - Disease index of treatment group] / Disease index of control group × 100

[0087] The results of the pot experiment showed that the water control group did not develop the disease, while the positive control group for the pathogen *Fusarium oxysporum* developed the disease, with a disease index of 85.41% (Table 2). Figure 7 The plants in the T9 bacterial solution treatment group did not show obvious disease symptoms on the outside, but some brownish-red areas were still visible when the vascular bundles were cut open. The higher the bacterial solution concentration, the lower the disease index, and the better the control effect. Apply 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 Compared to the Fusarium oxysporum positive control group, the disease index of T9 bacterial suspension with CFU / mL decreased by 27.08%, 31.25%, and 33.33%, respectively.

[0088] Table 2. Results of the control effect of Bacillus belyi T9 on cowpea wilt disease in pot experiments.

[0089]

[0090] Example 4: Application of Bacillus belyi T9 in promoting cowpea growth

[0091] For indoor potted cowpea seedlings at the 2-leaf, 1-heart stage, strain T9 was inoculated into LB liquid medium and cultured at 28℃, 180 rpm in the dark for 2 days to obtain bacterial suspension of strain T9. The bacterial suspension was then diluted with sterile water to prepare a 1×10⁻⁶ solution. 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9Three concentrations of bacterial solution (CFU / mL) were used, with sterile water as the control group (CK). Cowpeas were treated with root irrigation using the three concentrations of bacterial solution and sterile water, once every 5 days, with 15 mL of bacterial solution applied each time. After 20 days, the plant morphology indicators (plant height, leaf area, fresh weight, and root length) of cowpea plants after treatment with different concentrations of bacterial solution were measured to evaluate the growth-promoting effect of biocontrol bacteria T9 on cowpea plants.

[0092] The results are shown in Table 3 and Figure 8 As shown, 1×10 7 CFU / mL, 1×10 8 CFU / mL, 1×10 9 All three concentrations of T9 bacterial suspension (CFU / mL) showed a certain growth-promoting effect on cowpea plants. Compared with the control (CK), the concentration of 1×10⁻⁶ CFU / mL was significantly better. 8 CFU / mL and 1×10 9 Treatment with CFU / mL bacterial solution significantly improved the plant height, fresh weight, and root length of cowpea plants. The differences were statistically significant. Specifically, treatment with a concentration of 1×10⁻⁶ CFU / mL resulted in substantial improvements. 8 Cowpea plants treated with CFU / mL bacterial solution showed increased plant height, leaf area, fresh weight, and root length by 29.95%, 37.85%, 28.66%, and 31.07%, respectively, compared to the control group (CK). Treatment with a concentration of 1×10⁻⁶ CFU / mL also showed significant improvements. 9 Compared to the control group, cowpea plants treated with CFU / mL bacterial solution showed increases of 31.30%, 30.71%, 27.16%, and 40.54% in plant height, leaf area, fresh weight, and root length, respectively. In conclusion, cowpea plants treated with bacterial solution of strain T9 showed significant improvements in all plant morphological indicators compared to the sterile water treatment group.

[0093] Table 3. Results of the determination of the ability of Bacillus belyi T9 to promote the growth of cowpea plants in pot experiments.

[0094]

[0095] Note: Different lowercase letters in the same column indicate significant differences between treatments at the 0.05 level.

[0096] Example 5: Preparation of a compound agent for controlling cowpea wilt disease

[0097] 1. Screening of chemical agents for controlling cowpea wilt disease

[0098] The inhibitory effects of various chemical agents (azoxystrobin, mancozeb, pyraclostrobin, thiophanate-methyl, chlorothalonil, azoxystrobin, carbendazim, prochloraz, hymexazol, and propineb) on the mycelial growth of the tested strains of *Fusarium wilt* pathogen of cowpea were determined using the mycelial growth inhibition rate method. In the preliminary screening, five concentrations of each agent that showed a certain inhibitory effect on pathogen growth were used as the test concentrations for each agent (Table 4). Mycelial cakes with uniform growth were punched using a 5 mm diameter punch and inoculated into the center of different drug-containing plates. After incubation at 28℃ for 7 days, the colony diameter of each treatment was measured using the cross-crossing method, and the growth inhibition rate was calculated. The growth inhibition rate (%) was calculated as: [(control colony diameter - 0.5) - (treatment colony diameter - 0.5)] / (control colony diameter - 0.5) × 100. The growth inhibition rate was converted into an inhibition probability value according to the biostatistical probability conversion table. Using the logarithm of the concentration set in the experiment as the x-axis and the inhibition probability value as the y-axis, the dose-response regression equation for different chemical agents on the pathogen of cowpea wilt was calculated. y =a x+ b, and calculate the median inhibitory concentration (EC50) of each agent against the cowpea wilt pathogen using regression equations. 50 EC 90 and correlation coefficient r The value and the slope a value.

[0099] Table 4 Test reagents and their treatment concentrations

[0100]

[0101] Note: TC is the original drug.

[0102] Table 5. Inhibitory effects of 10 agents on mycelial growth of the pathogen causing cowpea wilt.

[0103]

[0104] The selected 10 agents all showed varying degrees of inhibitory effects on the mycelial growth of the pathogen causing cowpea wilt. Among them, 98% prochloraz showed the strongest inhibitory effect on the pathogenic mycelium, EC 100%. 50 The concentration was 0.0344 µg / mL; secondly, 98% carbendazim and 97% pyraclostrobin showed significant inhibitory effects on the mycelium of the cowpea wilt pathogen, EC 100%. 50 The concentrations were 1.3115 µg / mL and 1.6448 µg / mL, respectively; the least effective inhibitory effect was observed in 98% mancozeb, EC 1.3115 µg / mL. 50 It was 171.5795 µg / mL ( Figure 9 (Table 5).

[0105] 2. Compatibility test between Bacillus vesiculosus T9 and imazalil

[0106] 98% prochloraz was selected as the chemical agent. LB agar plates containing 98% prochloraz were prepared at concentrations of 1.0, 0.5, 0.1, 0.05, and 0.01 μg / mL. 100 μL of activated strain T9 was pipetted evenly onto each LB agar plate, with three replicates per treatment. The plates were incubated at 37°C for 2 days, and the growth of strain T9 was observed and its concentration determined.

[0107] The results showed that on drug-containing media with prochloraz concentrations of 0.1 µg / mL and 1.0 µg / mL, the cell concentrations of strain T9 were 1.03 × 10⁻⁶. 10 CFU / mL and 1.24×10 10 The CFU / mL concentration showed a significant difference compared to other treatment groups, with a slight increase in bacterial concentration. The growth of T9 on the other three drug-containing media showed no significant difference. All concentrations of prochloraz-containing media had no significant inhibitory effect on strain T9, indicating good compatibility between T9 and prochloraz (Table 6). Figure 10 ).

[0108] Table 6. Bacterial concentrations of strain T9 on agar plates containing different concentrations of prochloraz.

[0109]

[0110] 3. Virulence determination of Bacillus belyi T9 against the pathogen of cowpea wilt

[0111] After removing strain T9 from the cryopreservation tube, it was streaked for activation and inoculated into LB liquid medium. The culture was then carried out at 28°C and 180 rpm in the dark for 2 days, and the OD was measured. 600 The bacterial solutions were prepared using sterile physiological saline to achieve the following six bacterial concentrations: 1×10⁻⁶ 5 1×10 6 1×10 7 1×10 8 1×10 9 1×10 10 The inhibition rate was calculated using the filter paper disc method with CFU / mL as the target fungus *Fusarium oxysporum*, the pathogen of cowpea wilt. The inhibition rate was then converted into an inhibition probability value. y Based on the logarithm of the drug concentration x With the probability of inhibition y The toxicity regression equation was obtained. y =a x +b, calculate the median inhibitory concentration (EC50) of strain T9. 50 value.

[0112] The results are as follows Figure 11As shown in Table 7, the regression equation for the virulence of strain T9 against the pathogen causing cowpea wilt is as follows: y =0.3768 x +1.991, correlation coefficient r =0.9841, effective medium concentration EC 50 It is 0.97×10 8 CFU / mL.

[0113] Table 7. Inhibitory effect of different concentrations of T9 on the pathogen of cowpea wilt.

[0114]

[0115] 4. Toxicity determination of the compound preparation of Bacillus vesiculosus T9 and imazalil

[0116] The 98% prochloraz technical solution, which has the best inhibitory effect on Fusarium oxysporum, the pathogen of cowpea wilt, was compounded with the bacterial suspension of strain T9. The concentration of prochloraz in the resulting compound was selected from the effective medium concentration EC obtained in Table 5 of step 1 above. 50 (0.0344 µg / mL), the concentration of strain T9 was selected from the effective medium concentration EC obtained in Table 7 of step 3 above. 50 (0.97×10) 8 The specific preparation method for PDA plates containing compound agents (CFU / mL) is as follows: Prepare aqueous solutions of imazalil with a concentration of 0.344 µg / mL and 0.97 × 10⁻⁶ CFU / mL respectively. 9 T9 bacterial suspension at CFU / mL was prepared by mixing prochloraz aqueous solution with T9 bacterial suspension at volume ratios of 0:10, 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, 9:1, and 10:0, respectively, to obtain compound preparations. 10 mL of the compound preparation was added to 100 mL of PDA medium to obtain drug-containing plates. A treatment with an equal volume of sterile water added to the compound preparation served as a blank control (CK). The colony diameter of *Fusarium oxysporum* on the corresponding plates of each compound preparation was measured using the cross-cross method, and the actual inhibition rate and theoretical inhibition rate were calculated. The Horsfall method was used to calculate the synergistic virulence of the combination. Based on the synergistic ratio, the synergistic effect of different formulations was determined. A synergistic ratio IR > 1 indicates a synergistic effect; IR = 1 indicates an additive effect; and IR < 1 indicates an antagonistic effect. The formula for calculating the synergistic ratio is: IR = Eab / Eth; where Eab is the actual inhibition rate of the compound preparation; and Eth is the theoretical inhibition rate of the compound preparation.

[0117] Eth=T9 bacterial solution EC 50 Dosage-actual inhibition rate × volume percentage of T9 bacterial culture in the compound + prochloraz EC 50 Actual inhibition rate of dose × volume percentage of imazalil aqueous solution in the compound.

[0118] The results showed that the mixture ratio (V:V) of prochloraz aqueous solution and T9 bacterial culture was 1:9, 2:8, 3:7, 4:6, 5:5, 6:4, 7:3, 8:2, and 9:1, all of which had a synergistic effect. Specifically, when the mixture ratio was 8:2 and 7:3, the inhibition rates reached 84.87% and 83.66%, respectively, with synergistic ratios of 1.65 and 1.63. Furthermore, when the mixture ratio was 5:5, the synergistic ratio reached 1.72, but the inhibition rate against the pathogen was low, only 61.99% (Table 8). Figure 12 Therefore, when the volume ratio of prochloraz aqueous solution to bacterial culture of strain T9 is 8:2, the resulting compound has the strongest inhibitory effect on the pathogen of cowpea wilt.

[0119] Table 8. Toxicity test results of the compound preparation of Bacillus vesiculosus T9 and imazalil.

[0120]

[0121] Example 6: Efficacy determination of the compound preparation of Bacillus vesiculosus T9 and prochloraz against cowpea wilt disease.

[0122] Prepare cowpea experimental seedlings and a concentration of 1.0 × 10⁻⁶. 7 A CFU / mL suspension of the cowpea wilt pathogen was prepared. Cowpea seedlings with two leaves and a central bud were removed from their seedling trays. The taproot was gently incised with a sterile scalpel, and the seedlings were then immersed in the pathogen suspension for 30 minutes. After 30 minutes, the seedlings were transplanted into flowerpots. Subsequent experiments were conducted after 5 days of seedling growth. Six treatment groups were set up: a water treatment group (CK) (roots were only injured, no treatment with the cowpea wilt pathogen suspension), a LB treatment group (roots were only injured, no treatment with the cowpea wilt pathogen suspension), and a positive control group (FO) (roots were injured and then inoculated with 1.0 × 10⁶ ppm of the pathogen). 8 CFU / mL Fusarium oxysporum), T9 bacterial suspension treatment group (0.97×10⁻⁶) 8 The treatment groups included: a 98% prochloraz treatment group (prochloraz concentration of 0.0344 µg / mL), and a 98% prochloraz + T9 treatment group (98% prochloraz technical solution and T9 bacterial solution volume ratio of 8:2, prochloraz concentration in the solution of 0.0344 µg / mL, and bacterial concentration of 0.97 × 10⁻⁶ CFU / mL). 8 CFU / mL). Apply the treatment to the roots every 5 days, using 15 mL of the agent each time. After 20 days, evaluate the disease index and determine the control effect.

[0123] The disease index evaluation for cowpea wilt is based on the following standards: Grade 0: No discoloration of the root and stem vascular bundles, no yellowing or wilting of leaves; Grade 1: Discoloration of less than 1 / 4 of the root and stem vascular bundles, some leaves discolor, no significant change in plant growth; Grade 2: Discoloration of less than 1 / 2 of the root and stem vascular bundles, leaves discolor and wither, plant growth is inhibited; Grade 3: Discoloration of nearly 3 / 4 of the root and stem vascular bundles, significant inhibition of the entire plant's growth, leaves yellowing, wilting, and falling off from the bottom up; Grade 4: Complete discoloration of the root and stem vascular bundles, the entire plant withers and dies. The disease index evaluation and control effect determination are calculated using the following formula:

[0124] Disease index (%) = [Σ(Number of plants with disease at each disease level × Disease level number) / Total number of potted plants × Highest disease index level] × 100

[0125] Prevention and control efficacy (%) = [Disease index of control group - Disease index of treatment group] / Disease index of control group × 100

[0126] The results are shown in Tables 9 and 10. Figure 13 As shown, all plants in the positive control group inoculated with pathogen spore suspension developed the disease, with a disease index of 85.41%; none of the blank treatment groups, which were only irrigated with water and LB medium, developed the disease; the T9 bacterial suspension treatment group did not show obvious external disease, but some brownish-red areas were still visible when the vascular bundles were cut open, with a disease index of 54.16% and a control effect of 36.58%; the 98% prochloraz treatment group showed yellowing of the plant exterior, and some plants were stunted and withered, suspected of showing symptoms of phytotoxicity, with a disease index of 22.91% and a control effect of 73.17%; the 98% prochloraz + T9 compound treatment group showed no obvious external disease, and the various physiological indicators of the plants were good, with a plant height of (42.64±2.23) cm, which was significantly different from the FO treatment group; the leaf area was (60.54±3.68) cm². 2 The disease incidence rate was significantly lower than that of the FO, water, and LB treatment groups, with an overall disease index of 31.25% and a control effect of 63.41%. In summary, the compound agent of 98% prochloraz + T9 bacterial solution showed good control effect against cowpea wilt, effectively reduced the amount of chemical agents used, and also had a certain growth-promoting effect on cowpea plants.

[0127] Table 9. Control effects of different treatment groups on cowpea wilt disease.

[0128]

[0129] Table 10 Results of plant morphological index measurements in different treatment groups

[0130]

[0131] Note: Different lowercase letters in the same column indicate significant differences between treatments at the 0.05 level.

[0132] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of Bacillus belye ( Bacillus velezensis T9, characterized in that, Its accession number is CGMCCNo.33611.

2. The application of Bacillus belye T9 as described in claim 1 in inhibiting plant pathogenic fungi, characterized in that, The plant pathogenic fungus is *Fusarium oxysporum*, the vascular-specific strain of cowpea wilt. Fusarium oxysporum f. sp. tracheiphilum Fusarium graminearum, the pathogen of wheat scab ( Fusarium graminearum Fusarium oxysporum, the pathogen of banana wilt ( Fusarium oxysporum f.sp. cubense ), Fusarium oxysporum, the pathogen of coffee leaf blight ( Fusarium lateritium Mango anthracnose pathogen, *Colletotrichum gloeosporioides* (… Colletotrichum gloeosporioides Rice blast pathogen ( ) Magnaporthe grisea ), Sisal leaf rot pathogen Diplosporum cocovenenans ( Lasiodiplodia theobromae ), coconut gray spot disease pathogen *Pseudomonas spp.* Pestalotiopsis microspora ), the pathogen of maize leaf spot disease, Curvularia zeyla ( Curvularia lunata ), Phytophthora blight pathogen ( Phytophthora capsici ), and the pathogen of leaf spot disease ( ) Diaporthe biconispora ), dragon fruit canker pathogen new dark-colored arthropoda ( Neoscytalidium dimidiatum ) or coffee anthrax pathogen, *Caucasus kaharvatus* ( Colletotrichum kahawae ).

3. The use of Bacillus belye T9 as described in claim 1 in the preparation of a biocontrol agent for inhibiting plant pathogenic fungi, characterized in that, The plant pathogenic fungi mentioned are: Fusarium oxysporum (a xylem-specific fungus) which causes cowpea wilt, Fusarium graminearum which causes wheat scab, Fusarium oxysporum (a xylem-specific fungus) which causes banana wilt, Fusarium oxysporum (a xylem-specific fungus) which causes coffee leaf blight, Colletotrichum gloeosporioides (a xylem-specific fungus) which causes mango anthracnose, Blastococcus oryzae (a xylem-specific fungus) which causes rice blast, Diplostomum cocovenenum (a xylem-specific fungus) which causes sisal leaf rot, Polytrichum simulans (a xylem-specific fungus) which causes coconut gray spot disease, Curvularia zeylans (a xylem-specific fungus) which causes maize leaf spot disease, Phytophthora infestans (a xylem-specific fungus) which causes pepper blight, Aegilops genus which causes stachys leaf spot disease, Neodalaria glomeratus (a xylem-specific fungus) which causes dragon fruit canker disease, and Colletotrichum cahavar (a xylem-specific fungus) which causes coffee anthracnose.

4. A biocontrol agent containing Bacillus belye T9 as described in claim 1.

5. The application of the biocontrol agent according to claim 4 in inhibiting plant pathogenic fungi, characterized in that, The plant pathogenic fungi mentioned are: Fusarium oxysporum (a xylem-specific fungus) which causes cowpea wilt, Fusarium graminearum which causes wheat scab, Fusarium oxysporum (a xylem-specific fungus) which causes banana wilt, Fusarium oxysporum (a xylem-specific fungus) which causes coffee leaf blight, Colletotrichum gloeosporioides (a xylem-specific fungus) which causes mango anthracnose, Blastococcus oryzae (a xylem-specific fungus) which causes rice blast, Diplostomum cocovenenum (a xylem-specific fungus) which causes sisal leaf rot, Polytrichum simulans (a xylem-specific fungus) which causes coconut gray spot disease, Curvularia zeylans (a xylem-specific fungus) which causes maize leaf spot disease, Phytophthora infestans (a xylem-specific fungus) which causes pepper blight, Aegilops genus which causes stachys leaf spot disease, Neodalaria glomeratus (a xylem-specific fungus) which causes dragon fruit canker disease, and Colletotrichum cahavar (a xylem-specific fungus) which causes coffee anthracnose.

6. A compound medicine, characterized in that, The compound preparation consists of an aqueous solution of prochloraz at a concentration of 0.0344 µg / mL and a solution of 0.97 × 10⁻⁶ ppm. 8 The bacterial suspension of Bacillus vesiculosus T9 was composed of CFU / mL, with a volume ratio of 98% imazalil aqueous solution to Bacillus vesiculosus T9 bacterial suspension of (9:1) to (1:9); the preservation number of Bacillus vesiculosus T9 was CGMCC No. 33611.

7. The compound preparation according to claim 6, characterized in that, The volume ratio of 98% imazalil aqueous solution to Bacillus vesiculosus T9 bacterial solution is (8:2) to (7:3).

8. The application of the compound agent according to claim 6 or 7 in the prevention and control of cowpea wilt disease.

9. The use of the compound agent according to claim 6 or 7 in promoting cowpea growth.

10. The application according to any one of claims 8 or 9, characterized in that, The compound medicine is applied by root irrigation.

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