A strain of Bacillus subtilis and its microbial inoculant and its application in the control of bacterial angular leaf spot in cucumber.

Abstract

This invention provides a strain of Bacillus subtilis and its microbial inoculant, and its application in the control of bacterial angular leaf spot disease in cucumber. The strain is Bacillus subtilis (… Bacillus subtilis Strain A45, with preservation number CGMCC No. 33289. Microbial agents from this strain can effectively inhibit the pathogenic species *Pseudomonas amygdalina*, which causes bacterial angular leaf spot in cucumbers. Pseudomonas amygdali pv. tearful The strain encodes, in addition to known gene clusters for synthesizing antibacterial substances such as cymoxanil, bacitracin, thiophanate-3-carboxylic acid, catechol-type heptaphosphate, purcheminic acid, subtilosin A, and lysozyme, novel gene clusters for synthesizing surfactant, 1-carbapen-2-em-3-carboxylic acid, and tylosin A, as well as two entirely new terpene compound synthesis gene clusters. The microbial agent of this invention not only prevents cucumber angular leaf spot but also significantly promotes cucumber growth.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for plant diseases, and in particular to a strain of Bacillus subtilis and its microbial inoculant and its application in the control of bacterial angular leaf spot in cucumber. Background Technology

[0002] Cucumber angular leaf spot disease is one of the most prevalent and serious diseases in cucumber production. It can occur from the seedling stage to the mature plant stage, with a short onset period and rapid spread, often causing severe economic losses. With the increasing area of ​​protected cucumber cultivation, coupled with the favorable environmental conditions for bacterial diseases in protected cultivation, angular leaf spot has shown a year-on-year upward trend and is now one of the major bacterial diseases affecting protected cucumber production.

[0003] This disease is mainly caused by *Pseudomonas amygdalipv. lachrymans*, Pal., a pathogen originally named *Pseudomonas syringae* pv. lachrymans (Smith & Bryan) Yong, Dye & Wilkie. The pathogen primarily overwinters on seeds and can survive for more than two years. It can also overwinter in the soil with diseased plant debris. It can also enter through stomata, lenticels, hydathodes, and wounds, spreading through rainwater, irrigation, water droplets from greenhouse roofs, condensation, and leaf exudation. Currently, control mainly relies on chemical pesticides. For prevention, formulations containing kasugamycin, kasugamycin, or quinoline copper can be used. For control, multiple applications are necessary, and pesticides with different mechanisms of action should be used in rotation, such as 40% thiamethoxam zinc suspension, 3% thiamethoxam microemulsion, 5% allicin microemulsion, 77% copper hydroxide wettable powder, 30% copper oxychloride wettable powder, and 20% thiamethoxam copper suspension. Bacillus subtilis, Paenibacillus polymyxa, Bacillus thuringiensis, Bacillus licheniformis, and Bacillus velezensis have been widely used in plant disease control. However, research on biological control of bacterial angular leaf spot in cucumber is still limited. Raupach G S and Kloepper J W (2000) found that rhizosphere growth-promoting bacteria (PGPR) Bacillus pumilus INR7, Curtobacterium flaccumfaciens ME1, and Bacillus subtilis GB03, whether used alone or in combination, could reduce the severity index of cucumber angular leaf spot caused by Pseudomonas syringae pv. lachrymans to varying degrees, with the highest control efficacy being 57.37% (Plant disease, 2000, 84(10): 1073-1075). Li Baoju et al. from the Institute of Vegetables and Flowers, Chinese Academy of Agricultural Sciences, invented and published a method of spraying 1×10⁻⁶ spores of cucumber seeds when the cucumber has two leaves and one bud. 8A 5 mL suspension of Bacillus velezensis ZF145 at CFU / mL showed a 71.10% control effect on bacterial angular leaf spot of cucumber caused by Pseudomonas amygdalipv. lachrymans, Pal (CN112746046B). Summary of the Invention

[0004] The purpose of this invention is to provide a strain of Bacillus subtilis and its microbial agent and its application in the prevention and control of bacterial angular leaf spot disease in cucumber.

[0005] This invention is implemented as follows:

[0006] This invention provides a strain of Bacillus subtilis, A45, with accession number CGMCCNo.33289.

[0007] This invention provides the application of Bacillus subtilis in the prevention and control of plant diseases or in the preparation of microbial agents for the prevention and control of plant diseases, wherein the plant disease is bacterial angular leaf spot of cucumber caused by Pseudomonas amygdalinus.

[0008] The present invention also provides a microbial inoculant containing Bacillus subtilis with accession number CGMCC No. 33289 or containing Bacillus subtilis A45 and its metabolites.

[0009] The microbial inoculant provided by this invention is prepared according to the following method:

[0010] (1) Take fresh and activated A45 slant culture and inoculate it into shake flask seed culture medium. Incubate at 28-32℃ and 180-200rpm for 12-16h.

[0011] (2) Inoculate the cultured shake flask seeds into the seed tank at an inoculation rate of 0.3-0.5%, and culture at 28-32℃ with an aeration rate of 0.5-1.0 vvm for 5-8 hours;

[0012] (3) Then transfer the seeds from the seed tank to the fermentation tank at an inoculation rate of 3.0-6.5%, at 28-32℃, with an aeration rate of 0.6-1.2 vvm, and ferment for 20-30 hours. When the spore formation rate is greater than 95%, it is taken as the standard for leaving the tank. The fermentation liquid is obtained after the fermentation is completed.

[0013] (4) Prepare liquid or solid soluble bacterial agents using fermentation broth;

[0014] The PCB medium used for shaking flask seed culture, seed tank, and fermentation tank consisted of the following (g / L): corn steep liquor 80-100, soybean meal 5-10, starch 40-60, sodium chloride 1.5-3.0, manganese sulfate 0.1-0.2, pH 7.0±0.2, sterilized at 121℃ for 30 min.

[0015] In the microbial agent of the present invention, in step (3), the standard for placing the agent in the container is that the spore formation rate is greater than 95% and the spore shedding rate is greater than 90%.

[0016] In the microbial agent of the present invention, the step of preparing the liquid agent in step (4) is as follows: add xanthan gum with a final concentration of 0.1-0.3%, or gellan gum with a final concentration of 0.02-0.05%, or polyvinylpyrrolidone K90 (PVP K90) to the fermentation broth obtained in step (3), mix evenly, and then aseptically fill to obtain the liquid agent; the step of preparing the solid soluble agent in step (4) is as follows: spray dry the fermentation broth obtained in step (3), and then add soluble carriers such as gluten powder and dextrin to obtain the soluble solid agent.

[0017] The application of Bacillus subtilis in the prevention and control of plant diseases according to the present invention involves dipping the roots in a microbial agent during transplanting. The liquid agent is diluted 100-200 times and the soluble solid agent is diluted 300-400 times.

[0018] The beneficial effects of this invention are:

[0019] 1. The Bacillus subtilis A45 microbial agent provided by this invention can effectively inhibit the growth of Pseudomonas amygdali pv. lachrymans, a pathogen that causes bacterial angular leaf spot in cucumbers.

[0020] 2. The mechanism by which the Bacillus subtilis A45 microbial agent provided by this invention effectively inhibits the growth of Pseudomonas amygdali pv. lachrymans, the pathogen causing bacterial angular leaf spot in cucumber, lies in the fact that this strain encodes not only known gene clusters for synthesizing antibacterial substances such as bacitracin, bacitracin, thiophanate-3-carboxylic acid, catechol-type ferrophosphate, purcheminic acid, subtilosin A, and lysozyme, but also novel gene clusters for synthesizing surfactant, 1-carbapen-2-em-3-carboxylic acid, tylosin A, and two entirely new terpene compound synthesis gene clusters. This provides technical support and theoretical basis for the prevention and control of cucumber angular leaf spot.

[0021] 3. The Bacillus subtilis A45 microbial agent of this invention can not only prevent cucumber angular leaf spot caused by the lesion of Pseudomonas aeruginosa, but also has a significant promoting effect on cucumber growth.

[0022] Biological Preservation Instructions

[0023] Bacillus subtilis A45 was isolated from soil samples collected from a high-incidence area of ​​bacterial angular leaf spot of cucumber in Quzhou County, Hebei Province. It was deposited on January 3, 2025 at the China General Microbiological Culture Collection Center, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, Institute of Biology, Chinese Academy of Sciences, with accession number CGMCC No. 33289. Attached Figure Description

[0024] Figure 1 This is a diagram illustrating the antagonistic effect of the PCB fermentation supernatant of Bacillus isolate A45 on Pseudomonas amygdali pv. lachrymans in Example 1.

[0025] Figure 2 This refers to the spore formation stage during fermentation, where the spore formation rate is >95%.

[0026] Figure 3 This refers to the spore shedding stage during fermentation, where the spore shedding rate is >90%. Detailed Implementation

[0027] 1. Strain isolation, screening and identification

[0028] Strain isolation and screening

[0029] Bacillus spores were isolated from rhizosphere soil samples of cucumbers severely affected by angular leaf spot using the plate dilution method. Further screening was conducted to identify antagonistic bacteria against the pathogenic species *Pseudomonas amygdalipv. lachrymans*. A total of 65 antagonistic bacteria (A1-A65) were obtained. The inhibitory effect of the supernatant of the PCB fermentation broth from each isolate on bacterial angular leaf spot of cucumber was determined using the tube-disc method. A45 showed the largest inhibition zone, reaching 30.0 mm.

[0030] Strain identification

[0031] First, A45 was preliminarily identified based on phylogenetic analysis of the 16S rRNA gene sequence, and then its whole genome was sequenced to determine its species-level taxonomic position based on dDDH and ANI values.

[0032] Example 1: Strain Isolation and Screening

[0033] PCB medium (g / L): peptone 5.0, yeast extract 2.5, glucose 1.0, pH: 7.4±0.2, sterilized at 121℃ for 30 min. PCA medium is prepared by adding 15 g / L agar to PCB medium.

[0034] Soil samples were collected from areas with a high incidence of bacterial angular leaf spot disease in cucumbers in Quzhou County, Hebei Province. 10g of soil sample was added to an Erlenmeyer flask containing 10⁻²⁵ glass beads and 90mL of sterile water, and the sample was shaken to prepare a soil suspension. The soil suspension was then boiled in water for 5 minutes and diluted to three gradients: 10⁻⁴, 10⁻⁵, and 10⁻⁶. 0.2mL of the diluted soil suspension was added to the surface of PCA agar plates, spread evenly, and allowed to stand for 5 minutes. The plates were then incubated upside down at 30℃. The growth of isolated microbial colonies was observed over 24-48 hours. Well-grown single colonies were transferred to PCA slant agar and incubated at 30℃ for 24 hours.

[0035] One loopful of fresh culture of the soil isolate was inoculated into a 500 mL Erlenmeyer flask containing 100 mL of PCB medium. After incubation at 30°C and 180-200 rpm for 24 h, the supernatant was collected by aseptic centrifugation. The antagonistic effect of the fermentation broth supernatant of each strain on *Pseudomonas amygdali* pv. *lachrymans* was determined using the tube-disc method. A total of 65 isolates with inhibition zones were screened, of which 18 had inhibition zones larger than 20 mm (Table 1). A45 had the largest inhibition zone, reaching 30 mm. Figure 1 (As shown).

[0036] Table 1. Size of the inhibition zone in the fermentation supernatant of the screened antagonistic strains.

[0037]

[0038] Example 2: Strain Identification

[0039] Extraction of colony PCR template: Add 30.0 μL of cell lysis buffer to a PCR tube, pick a single colony and lyse it at 100℃ for 10 min. Centrifuge at 10000 rpm for 5 min, and use the supernatant as a template for PCR amplification.

[0040] The composition of the PCR amplification reaction system is shown in Table 2. The PCR amplification program was as follows: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 90 s, 30 cycles, followed by a final extension at 72℃ for 5 min. PCR products were detected by electrophoresis and then sequenced using the Sanger method.

[0041] Table 2 PCR reaction system

[0042]

[0043] Sequencing results were compared with all model strain sequences in the Blast and GenBank nucleic acid databases, and it was found that A45 had the same 16S rRNA gene sequence as the Bacillus subtilis model strains ATCC6051 and str168.

[0044] The A45 16S rRNA gene sequence obtained is shown in SEQ ID No. 1.

[0045] Further, a whole-genome shotgun (WGS) strategy was employed to construct libraries with different insert fragments. Next-generation sequencing (NGS) was used on the Illumina NovaSeq platform, and third-generation single-molecule sequencing was performed on the Oxford Nanopore ONT platform to sequence the entire genome of these libraries. The A45 genome is 4215636 bp in size, with a GC content of 43.51%, a circular chromosome, and no plasmids. Genome alignment of the A45 genome with the Type Strain Genome Server (https: / / tygs.dsmz.de / ) revealed a dDDH value of 100% with Bacillus subtilis ATCC6051T. ANI calculations using https: / / www.ezbiocloud.net / tools / ani also showed a 100% alignment. These results indicate that A45 belongs to the taxonomic class of Bacillus subtilis.

[0046] 2. Analysis of genes encoding secondary metabolite synthesis

[0047] We used antiSMASH (https: / / antismash.secondarymetabolites.org) to search for genes encoding secondary metabolites from the determined A45 genome sequence and analyzed the mechanism by which A45 resists the cucumber angular leaf spot pathogen Pseudomonas amygdali pv.lachrymans.

[0048] Example 3: Known gene clusters encoding the synthesis of known antibacterial active products

[0049] A45 was found to encode multiple antibacterial-related gene clusters, including secondary metabolite encoding genes with the same sequences as known genes: Fengycin, Bacillaene, Sublancin, Bacillibactin, Pulcherriminic acid, subtilosin A, and Bacilysin synthesis gene clusters.

[0050] Example 4: Antibacterial active product encoded by a newly synthesized gene cluster

[0051] The A45 gene cluster encoding the Surfactin synthesis showed the highest similarity (82%) to known genes, the 1-carbapen-2-em-3-carboxylic acid synthesis gene cluster showed the highest similarity (16%), and the thailanstatin A synthesis gene cluster showed the highest similarity (10%). Furthermore, A45 also encodes two novel terpene synthesis gene clusters (see Table 3).

[0052] Table 3. Gene clusters encoding secondary metabolite synthesis in the A45 genome identified using antiSMASH analysis and their sequence similarity to known genes.

[0053]

[0054] 3. Fermentation methods and preparation of inoculants

[0055] Freshly activated A45 slant culture is inoculated into shake flask seed culture medium (PCB medium) and cultured on a shaker at 28-32℃ and 180-200 rpm for 12-16 hours. The cultured shake flask seed culture is then inoculated into a seed tank at an inoculation rate of 0.3-0.5% and cultured at 28-32℃ with an aeration rate of 0.5-1.0 vvm for 5-8 hours. The seed culture from the seed tank is then transferred to a fermenter at an inoculation rate of 3.0-6.5% and fermented at 28-32℃ with an aeration rate of 0.6-1.2 vvm for 20-30 hours. Fermentation is considered complete when the spore formation rate is greater than 95%, with the preferred conditions being a spore formation rate greater than 95% and a spore shedding rate greater than 90%.

[0056] The PCB medium used for shaking flask seed culture, seed tank, and fermentation tank consisted of the following (g / L): corn steep liquor 80-100, soybean meal 5-10, starch 40-60, sodium chloride 1.5-3.0, manganese sulfate 0.1-0.2, pH 7.0±0.2, sterilized at 121℃ for 30 min.

[0057] After fermentation, add xanthan gum to the fermentation broth at a final concentration of 0.1-0.3%, or gellan gum at 0.02-0.05%, or polyvinylpyrrolidone K90 (PVP K90) at 0.3-0.5%. After thorough mixing, aseptically fill the mixture to obtain the liquid inoculum. Alternatively, after spray drying the fermentation broth, add soluble carriers such as gluten powder and dextrin until the desired bacterial count is reached to obtain the soluble solid inoculum (powder).

[0058] Example 5: Preparation of Liquid Microbial Agent

[0059] (1) The preserved A45 strain was inoculated onto PCA slant and activated by incubation at 28℃ for 20 h. Three loops of the activated A45 slant strain were then inoculated into a 3L Erlenmeyer flask containing 500mL of seed culture medium (PCB medium).

[0060] Seed culture was obtained by culturing in a shaker at 28℃ and 200rpm for 12 hours.

[0061] (2) The cultured shake flask seeds were inoculated into a 1.5-ton seed tank containing 0.8 tons of fermentation medium at an inoculation rate of 0.3%, and cultured at 28°C, aeration rate of 0.8 vvm, stirring speed of 200 rpm for 6.5 h to obtain the seed tank seeds.

[0062] (3) The cultured seeds from the seed tank were transferred at an inoculum rate of 5% to a 20-ton fermenter containing 14 tons of fermentation medium. The fermentation was carried out at 28°C with an aeration rate of 0.9 vvm and a stirring speed of 160 rpm for 24 hours to obtain the fermentation broth. At the end of fermentation, the spore formation rate was 100%. Figure 2 As shown), the spore shedding rate was 90%. Figure 3 (As shown).

[0063] (4) Add 0.2% (m / v) xanthan gum to the fermentation broth and stir until completely dissolved to obtain a liquid inoculum. Take a sample, dilute and spread it on PCA plates, incubate at 28℃ for 20 h, and count the viable cells. The effective viable cell count of the liquid inoculum was 2.8 × 10⁻⁶.

[0064] 10 10 CFU / mL.

[0065] The culture medium used in both the seed tank and the fermentation tank was (g / L): corn steep liquor 100, soybean meal 7.5, starch 45, sodium chloride 2.0, manganese sulfate 0.1, pH 7.2, sterilized at 121℃ for 30 min.

[0066] After being filled with liquid bacterial agent and stored at room temperature, the survival rates were measured at 3 months, 6 months, and 12 months, respectively, and were 100%, 98.6%, and 95.8%, respectively.

[0067] Example 6: Preparation of Soluble Solid Microbial Agent

[0068] The fermentation broth obtained in step (3) of Example 5 was spray-dried at an inlet temperature of 180°C and an outlet temperature of 90°C to obtain a solid inoculum powder. The effective bacterial count of the powder was found to be 3.1 × 10⁻⁶ on PCA plates. 11 CFU / g.

[0069] Add 1 part solid microbial agent powder to 2 parts soluble carrier, then mix thoroughly to obtain a soluble solid microbial agent with an effective bacterial content of 100 billion / g.

[0070] 4. Usage and dosage

[0071] A45 microbial inoculant is used to control bacterial angular leaf spot in cucumbers caused by *Pseudomonas amygdalipv. lachrymans*. It can be applied from transplanting to maturity, with optimal results when used before or at the early stage of disease. Liquid inoculants can be diluted 150-250 times, with a 200-fold dilution for spraying being most effective. Solid inoculants can be diluted 600-1000 times, with an 800-fold dilution for spraying being most effective. If dipping roots at transplanting, liquid inoculants should be diluted 100-200 times, and solid inoculants 300-400 times.

[0072] Example 7: Effect of A45 liquid fungicide spray on the control of bacterial angular leaf spot in cucumber.

[0073] When the potted cucumber has grown to two leaves and a central bud, inoculate it with 1×10⁶ plants using the foliar spray method. 8 A suspension of CFU / mL of *Pseudomonas amygdali* pv. *lachrymans*, the pathogen causing bacterial angular leaf spot in cucumbers, was sprayed on the cucumber plants. After 24 hours of spraying and allowing the leaves to dry, a 200-fold dilution of *Bacillus subtilis* A45 liquid inoculant was sprayed onto the cucumber plants. The water control was inoculated with the pathogen but not sprayed with A45. After inoculation, cucumber seedlings were placed in a humidifier at 80±5% relative humidity and 26–28℃ for 24 hours, then transferred to a normal seedling greenhouse. This process was repeated three times. Four days after inoculation, when the water control showed full disease development, the disease index and control effect were calculated.

[0074] Cucumber bacterial angular leaf spot: classified according to the leaf surface area occupied by angular leaf spots.

[0075] Grade 0: No lesions;

[0076] Level 1: 0%-5%;

[0077] Level 3: 5%-25%;

[0078] Level 5: 25%-50%;

[0079] Level 7: 50%-75%;

[0080] Level 9: 75% or higher.

[0081] Disease index = 100 × Σ(number of diseased leaves at each level × disease representative value at each level) / (total number of leaves surveyed × highest disease representative value)

[0082] Prevention and control effect (%) = 100 × (control disease index - treatment disease index) / control disease index.

[0083] Table 4A45 shows the effect of liquid bacterial agent spraying on the control of bacterial angular leaf spot in cucumbers in potted plants.

[0084] deal with Concentration and Usage Method Disease index Prevention and control efficacy (%) A45 Liquid Microbial Agent Dilute 200 times and spray 5.52±0.25 80.74±1.33 Comparison Water spray 28.72±1.33 ---

[0085] Example 8: Effect of A45 solid inoculant root dipping on the control and growth promotion of bacterial angular leaf spot in transplanted cucumbers.

[0086] Cucumber seedlings with 3 true leaves were selected. Before transplanting, the control group received no treatment, while the model group received 1×10⁶ seedlings. 8 Root dips with a CFU / mL suspension of *Pseudomonas amygdali* pv. *lachrymans*, the pathogen causing bacterial angular leaf spot in cucumbers; experimental groups used 1×10... 8 A CFU / mL strain of *Pseudomonas amygdalina*, a pathogen causing bacterial angular leaf spot in cucumbers, was used for root dipping with a 1 / 300 concentration of A45 solid inoculant compound suspension. Twenty days after transplanting, the disease index and control effect were calculated, and vine length and leaf count were measured to observe the growth-promoting effect of A45 on cucumbers.

[0087] The methods for calculating the grading, disease index, and prevention and control effects of keratosis are the same as in Example 7.

[0088] Table 5 shows the effect of A45 solid inoculant root dipping on the control of bacterial angular leaf spot in transplanted cucumbers.

[0089]

[0090] Comparative Example 1: Comparison Experiment with Different Fermentation Media

[0091] (1) Take fresh and activated A45 slant culture and inoculate it into shake flask seed culture medium (PCB medium), and culture it in a shaker at 28℃ and 200rpm for 12h.

[0092] (2) Inoculate the cultured shake flask seeds into the seed tank at an inoculation rate of 0.5%, and culture at 28℃ with an aeration rate of 0.7 vvm for 10 h;

[0093] (3) Then transfer the seeds from the seed tank to the fermentation tank at an inoculation rate of 5.5%, ferment at 28°C and an aeration rate of 0.8 vvm for 30 hours.

[0094] The seed culture medium for the seed tank and the fermentation culture medium for the fermenter each use one of the following three types:

[0095] Culture medium 1 (g / L): corn steep liquor 100, soybean meal 7.5, starch 45, sodium chloride 2.0, manganese sulfate 0.1, pH 7.2, sterilized at 121℃ for 30 min.

[0096] Culture medium 2 (g / L): corn steep liquor 90, soybean meal 10.0, starch 55, sodium chloride 3.0, manganese sulfate 0.2, pH 7.2, sterilized at 121℃ for 30 min.

[0097] Culture medium 3 (g / L): peptone 25.0, soybean meal 20.0, sucrose 50.0, sodium chloride 0.8, manganese sulfate 0.4, pH 7.2, sterilized at 121℃ for 30 min.

[0098] After fermentation, the fermentation broth was centrifuged at 10,000 rpm and 4℃ for 6 min. The supernatant was then filtered through a 0.22 μm microporous membrane. The Oxford cup method was used to determine the antibacterial effect of the filtrate against *Pseudomonas amygdali* pv. *lachrymans* (Pal), the pathogen causing bacterial angular leaf spot of cucumber. The results are shown in Table 6. The larger the diameter of the inhibition zone, the better the antibacterial effect.

[0099] Table 6. Antibacterial effects of fermentation broths obtained from different fermentation media under the same conditions against *Pseudomonas amygdalina* species *Pal*, a pathogen causing lacrimal lacrimation.

[0100] culture medium Diameter of the inhibition zone (mm) 1 38 2 36 3 14

[0101] Comparative Example 2:

[0102] (1) Take fresh, activated A45 slant culture and inoculate it into shake flask seed culture medium (PCB medium), at 30℃.

[0103] Incubate on a shaker at 200 rpm for 12 hours.

[0104] (2) Inoculate the cultured shake flask seeds into the seed tank at an inoculation rate of 0.5%, and culture at 30°C with an aeration rate of 0.8 vvm for 8 hours;

[0105] (3) Then, transfer the seeds from the seed tank to the fermentation tank at an inoculation rate of 5.0%, ferment at 30℃ and an aeration rate of 1.0 vvm for 25-30 hours, and place the seeds in the fermentation tank under the following three different conditions. First condition: 100% spore formation rate and 95% spore shedding rate; Second condition: 96% spore formation rate and no spore shedding; Third condition: 85% spore formation rate and no spore shedding.

[0106] The culture medium for both the seed tank and the fermenter was: corn steep liquor 100g, soybean meal 7.5g, starch 45g, sodium chloride 2.0g, manganese sulfate 0.1g, pH 7.2, sterilized at 121℃ for 30min.

[0107] After fermentation, the counts were taken and then counted again after being stored at room temperature for 6 months. The results are shown in Table 7.

[0108] Table 7. Effects of different fermentation endpoint criteria on the survival rate of A45 fermentation broth stored at room temperature.

[0109] Fermentation endpoint (release from tank) standard Survival rate (%) after 6 months of storage at room temperature Spore formation rate: 100% and spore shedding rate: 95%. 99.6±0.25 96% of spores formed and did not fall off. 89.7±2.28 85% of spores formed and did not fall off. 65.7±3.62

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis A45, with accession number CGMCC No.33289.

2. The application of Bacillus subtilis A45 according to claim 1 in the prevention and control of plant diseases or in the preparation of microbial agents for the prevention and control of plant diseases, wherein the plant disease is bacterial angular leaf spot of cucumber caused by *Pseudomonas amygdalina*; the Bacillus subtilis A45 is first prepared into a microbial agent according to the following method: (1) Take fresh and activated A45 slant culture and inoculate it into shake flask seed culture medium. Incubate at 28-32℃ and 180-200rpm for 12-16h. (2) Inoculate the cultured shake flask seeds into the seed tank at an inoculation rate of 0.3-0.5%, and culture at 28-32℃ with an aeration rate of 0.5-1.0 vvm for 5-8 hours; (3) Then, transfer the seeds from the seed tank to the fermentation tank at an inoculation rate of 3.0-6.5%, at 28-32℃, with an aeration rate of 0.6-1.2 vvm, and ferment for 20-30 hours. The standard for leaving the tank is that the spore formation rate is greater than 95% and the spore shedding rate is greater than 90%. Fermentation is completed to obtain the fermentation liquid. (4) Prepare liquid or solid soluble bacterial agents using fermentation broth; in, The PCB medium used for shake-flask seed culture, and the medium composition used in seed tanks and fermenters are: corn steep liquor 80-100 g / L, soybean meal 5-10 g / L, starch 40-60 g / L, sodium chloride 1.5-3.0 g / L, manganese sulfate 0.1-0.2 g / L, pH 7.0±0.2, sterilized at 121℃ for 30 min.

3. A microbial inoculant, characterized in that, Contains Bacillus subtilis A45 with accession number CGMCC No. 33289 or contains Bacillus subtilis A45 and its metabolites; Prepared according to the following method: (1) Take fresh and activated A45 slant culture and inoculate it into shake flask seed culture medium. Incubate at 28-32℃ and 180-200rpm for 12-16h. (2) Inoculate the cultured shake flask seeds into the seed tank at an inoculation rate of 0.3-0.5%, and culture at 28-32℃ with an aeration rate of 0.5-1.0 vvm for 5-8 hours; (3) Then, transfer the seeds from the seed tank to the fermentation tank at an inoculation rate of 3.0-6.5%, at 28-32℃, with an aeration rate of 0.6-1.2 vvm, and ferment for 20-30 hours. The standard for leaving the tank is that the spore formation rate is greater than 95% and the spore shedding rate is greater than 90%. Fermentation is completed to obtain the fermentation liquid. (4) Prepare liquid or solid soluble bacterial agents using fermentation broth; The PCB medium used for shaking flask seeds, as well as the medium used in seed tanks and fermenters, consist of: corn steep liquor 80-100 g / L, soybean meal 5-10 g / L, starch 40-60 g / L, sodium chloride 1.5-3.0 g / L, manganese sulfate 0.1-0.2 g / L, pH 7.0±0.2, sterilized at 121℃ for 30 min.

4. The microbial agent according to claim 3, characterized in that, In step (4), the steps for preparing liquid bacterial agent are as follows: add xanthan gum with a final concentration of 0.1-0.3%, or gellan gum with a final concentration of 0.02-0.05%, or polyvinylpyrrolidone K90 with a final concentration of 0.3-0.5% to the fermentation broth obtained in step (3), mix evenly, and then aseptically fill the mixture to obtain liquid bacterial agent; in step (4), the steps for preparing solid soluble bacterial agent are as follows: spray dry the fermentation broth obtained in step (3), and then add gluten powder and dextrin soluble carrier to obtain soluble solid bacterial agent.

5. The application of Bacillus subtilis A45 in the prevention and control of plant diseases according to claim 2, characterized in that, When transplanting, dip the roots in the microbial agent described in claim 3. The liquid agent is diluted 100-200 times and the soluble solid agent is diluted 300-400 times.

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