Application of Bacillus subtilis in the control of plant diseases and the preparation of microbial agents

Abstract

This invention provides Bacillus subtilis ( Bacillus subtle The application of strain A45 in the control of plant diseases, its application in the preparation of microbial agents for the control of plant diseases, and the prepared microbial agents. Bacillus subtilis A45 is deposited at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 33289. The liquid microbial agent containing Bacillus subtilis A45 provided by this invention effectively inhibits the cucumber target spot pathogen, *Clostridium multiflorum* (…). Corynespora cassicola The growth mechanism of this strain lies in its encoding a gene cluster for the synthesis of phytosalicylic acid, purchemin, subtilosin A, and surfactant. Among these, the surfactant synthesis gene cluster is unique to this strain, exhibiting a maximum sequence similarity of 82%. Experiments show that the liquid inoculant containing Bacillus subtilis A45 provided by this invention can effectively inhibit the growth of *Clostridium multiflorum*, the pathogen causing target spot disease in cucumbers. Corynespora cashew nut The growth of ) can be controlled by field application with a prevention and control effect of up to 80%.

Description

Technical Field

[0001] This invention relates to the field of biological control technology for plant diseases, and in particular to the application of Bacillus subtilis in the control of plant diseases and the preparation of microbial agents thereon. Background Technology

[0002] Cucumber target spot, also known as Corynespora leaf spot or brown spot, is a fungal disease caused by the fungus *Corynespora cassiicola*, a member of the Deuteromycetes. Its incidence has been gradually increasing in recent years, becoming a new leaf disease affecting cucumbers. The disease is mainly spread through wind, rain, irrigation water, farm tools, and diseased plant debris, and thrives in hot and humid environments. *Corynespora cassiicola* is highly infectious and spreads rapidly; the hot and humid microclimate of protected cultivation is conducive to the growth and development of the pathogen and the formation and germination of spores. *Corynespora cassiicola* primarily appears on the upper surface of leaves, but can also occur on the lower surface. In the early stages, circular or irregular brown spots appear, which later expand to form concentric rings, weakening photosynthesis, leading to poor plant growth, reduced fruit yield, and decreased quality. In severe cases, it causes leaf withering and death, fruit drop, and premature vine death. In recent years, target spot has occurred in most greenhouse cucumber growing areas in my country, becoming one of the major diseases affecting cucumbers and seriously threatening the production safety of my country's greenhouse cucumber industry.

[0003] In production, the occurrence of cucumber target spot disease can be controlled by selecting disease-resistant varieties, crop rotation, reducing pathogen accumulation, and improving ventilation and light conditions. Chemical control is the main means of controlling cucumber target spot disease. Fungicides such as fluopyram, boscalid, azoxystrobin, and difenoconazole can be used in the early stage of the disease. However, due to the easy mutation and strong destructiveness of the pathogen, effective control of the disease is difficult. Multiple studies have reported that the resistance of cucumber target spot pathogen to common antifungal drugs is becoming increasingly serious (Zhu Fadi. Study on resistance of *Corynespora cassiicola* to boscalid and its mechanism. Beijing: Chinese Academy of Agricultural Sciences, 2018; Li Xiuhuan. Monitoring of resistance of cucumber target spot pathogen to pyraclostrobin and study on the molecular mechanism of resistance to two new fungicides. Shandong: Shandong Agricultural University, 2021).

[0004] Bacillus subtilis, Paenibacillus polymyxa, and Bacillus velezensis have been widely used in the control of plant diseases. Chinese patent CN103131657B discloses Bacillus subtilis XLBS-01 (accession number CGMCC No. 6692), which can control cucumber target spot disease, but the control effect is not ideal. Summary of the Invention

[0005] The purpose of this invention is to provide the application of Bacillus subtilis in the prevention and control of plant diseases and the microbial agent prepared therefrom.

[0006] This invention is implemented as follows:

[0007] This invention provides the application of a Bacillus subtilis strain A45 in the prevention and control of plant diseases or in the preparation of microbial agents for the prevention and control of plant diseases. The preservation number of strain A45 is CGMCC No. 33289. The plant disease is cucumber target spot disease caused by Corynespora cassiicola.

[0008] Furthermore, the microbial agent is a liquid agent or a solid soluble agent.

[0009] The present invention also provides a microbial inoculant comprising Bacillus subtilis A45 with accession number CGMCC No. 33289 and prepared by 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 1.0-1.5%, at 28-32℃, with an aeration rate of 0.8-1.2 vvm, and culture for 8-10 hours;

[0012] (3) Then transfer the seeds from the seed tank to the fermentation tank at an inoculation rate of 3.5-4.5%, at 28-32℃, with an aeration rate of 0.8-1.2 vvm, and ferment for 36-48 hours. Fermentation is complete when the spores have completely fallen off.

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

[0014] Furthermore, the microbial agent, the seed culture medium in the shake flask in step (1) is PCB culture medium, the composition of PCB culture medium (g / L) is: peptone 25.0, yeast extract 2.5, glucose 1.0, pH: 7.4±0.2, sterilized at 121℃ for 30min;

[0015] Further, the microbial agent, the culture medium used in the seed tank in step (2) and the fermentation tank in step (3) has the following composition (g / L): peptone 20-25, soybean meal 5-8, sucrose 20-25, starch 25-30, manganese sulfate 0.2-0.3, light calcium carbonate 4.0-6.0, pH 7.0±0.2, sterilized at 121℃ for 30min.

[0016] The application of Bacillus subtilis strain A45 provided by this invention in the control of plant diseases involves applying the liquid inoculant prepared according to claim 3 to cucumbers infected with cucumber target spot disease. The effective bacterial content of the liquid inoculant is 3.30 × 10⁻⁶. 11 CFU / g, diluted 200-300 times for spray application.

[0017] The application of Bacillus subtilis strain A45 provided by this invention in the control of plant diseases involves applying the soluble solid bacterial agent prepared according to claim 3 to cucumbers with cucumber target spot disease. The solid bacterial agent has a content of 100 billion / g and is applied by spraying after being diluted 600 times.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] 1. The liquid bacterial agent containing Bacillus subtilis A45 provided by this invention can effectively inhibit the growth of Corynespora cassiicola, the target spot pathogen of cucumber, and the control effect in field application can reach up to 80%.

[0020] 2. The mechanism by which the liquid bacterial agent containing Bacillus subtilis A45 provided by this invention effectively inhibits the growth of Corynespora cassiicola, the pathogen causing target spot disease in cucumber, lies in the fact that this strain encodes a gene cluster for the synthesis of cytosine, prochlorazine, subtilosin A, and surfactant. Among them, the surfactant synthesis gene cluster is different from the synthesis gene of any known organism, with the highest sequence similarity being 82%, providing technical support and theoretical basis for the prevention and control of target spot disease in cucumber.

[0021] Biological Preservation Instructions

[0022] 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

[0023] Figure 1This is a plate inoculation diagram of Corynespora cassiicola and Bacillus during strain screening in Example 1.

[0024] Figure 2 This represents the state at the end of fermentation in Example 5, where 100% of the spores have completely detached. Detailed Implementation

[0025] 1. Strain screening and identification

[0026] Strain screening: Using the plate confrontation inoculation method, strains with antagonistic activity against Corynespora cassiicola were screened from more than 500 Bacillus strains preserved in the laboratory.

[0027] Strain identification: First, A45 was preliminarily identified based on phylogenetic analysis of the 16S rRNA gene sequence, and then whole-genome sequencing was performed to determine its species-level taxonomic position based on dDDH and ANI values.

[0028] Example 1: Strain screening, reference Figure 1 As shown.

[0029] (1) Incubate *Cyclocarya multiflora* at 25°C for 4 days, and use a 5mm punch to take a fungal cake and inoculate it in the center of a PDA plate.

[0030] (2) The Bacillus species to be screened were cultured in PCB liquid medium at 30℃ with shaking for 10 hours. 2 μL of the culture solution was inoculated around the periphery of a *Corynebacterium multiflorum* mycelium cake as the experimental group; 2 μL of sterile water was placed around the periphery of the *Corynebacterium multiflorum* mycelium cake as the control group; both the experimental and control groups were incubated in a 28℃ incubator. When the *Corynebacterium multiflorum* hyphae in the control group completely covered the culture dish, the colony radius of *Corynebacterium multiflorum* was measured. The colony radius of the experimental group was defined as the growth range of *Corynebacterium multiflorum* colonies from the center of the *Corynebacterium multiflorum* mycelium cake to the center of the Bacillus species colony. The inhibition rate of the antagonistic bacteria was calculated based on the colony radius (mm) of the experimental and control groups.

[0031] Inhibition rate (100%) = (Coronavirus radius of control group - Coronavirus radius of treatment group) / Coronavirus radius of control group × 100%.

[0032] The PCB medium (g / L) consisted of: peptone 5.0, yeast extract 2.5, glucose 1.0, pH 7.4±0.2, sterilized at 121℃ for 30 min. PCA medium was prepared by adding 15 g / L of agar to the PCB medium.

[0033] Ultimately, 12 strains with high antagonistic activity were obtained. The inhibition rates of these 12 strains are shown in Table 1. A45 showed the best antibacterial effect, with an inhibition rate as high as 76.3%.

[0034] Table 1. Antibacterial rate of the antagonistic strains obtained from screening against *Corynebacterium multiflorum*.

[0035]

[0036] Example 2: Strain Identification

[0037] 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.

[0038] 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.

[0039] Table 2 PCR reaction system

[0040]

[0041] 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.

[0042] The sequenced A45 16S rRNA gene is as follows (SEQ ID No. 1):

[0043] 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. Calculation of the ANI values ​​of both genomes using https: / / www.ezbiocloud.net / tools / ani also showed a 100% alignment. These results indicate that A45 is taxonomically classified as *Bacillus subtilis*.

[0044] 2. Analysis of genes encoding secondary metabolite synthesis

[0045] 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 target spot pathogen Corynespora cassiicola.

[0046] Example 3: Known gene clusters encoding the synthesis of known antibacterial active products

[0047] As shown in Table 3, A45 was found to encode multiple antifungal-related active gene clusters. The genes encoding secondary metabolites with the same gene sequences as known genes include: Fengycin, Pulcherriminic acid, and subtilosin A synthesis gene cluster.

[0048] Example 4: Antibacterial active product encoded by a newly synthesized gene cluster

[0049] As shown in Table 3, the Surfactin synthesis gene cluster encoded by A45 showed the highest similarity (82%) to known genes, thus confirming that the A45 Surfactin synthesis gene cluster is different from the synthesis genes of any known organism. Furthermore, A45 also encodes two novel terpene compound synthesis gene clusters (Region 3 and Region 6).

[0050] Table 3. Gene clusters encoding secondary metabolite synthesis in the A45 genome identified using antiSMASH analysis and their sequence similarity to known genes.

[0051]

[0052] 3. Fermentation methods and preparation of inoculants

[0053] 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.

[0054] 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 1.0-1.5%, and cultured at 28-32℃ with an aeration rate of 0.8-1.2 vvm for 8-10 hours. The seed culture from the seed tank is then transferred to a fermenter at an inoculation rate of 3.5-4.5%, and fermented at 28-32℃ with an aeration rate of 0.8-1.2 vvm for 36-48 hours. Fermentation is complete when all spores have fallen off. Complete spore fall-off refers to a spore fall-off rate of 100% (both spore formation rate and fall-off rate are 100%).

[0055] The PCB medium used for shaking flask seed culture, seed tank, and fermentation tank consisted of the following (g / L): peptone 20-25, soybean meal 5-8, sucrose 20-25, starch 25-30, manganese sulfate 0.2-0.3, light calcium carbonate 4.0-6.0, pH 7.0±0.2, sterilized at 121℃ for 30 min.

[0056] 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%, mix well, and then aseptically fill to obtain liquid bacterial agent.

[0057] After spray drying the fermentation broth, soluble carriers such as gluten powder and dextrin are added until the appropriate bacterial count is reached, which is the soluble solid bacterial agent (powder).

[0058] Example 5: Preparation of Liquid Microbial Agent

[0059] (1) The preserved A45 strain was inoculated onto PCA slant and activated by culturing at 28°C for 20 h. Three loops of the activated A45 slant strain were inoculated into a 3L Erlenmeyer flask containing 500mL of seed culture medium (PCB medium) and cultured at 30°C and 200rpm for 14 h to obtain the shake flask seed solution.

[0060] (2) The cultured shake flask seeds were inoculated into a seed tank containing 500L of fermentation medium at an inoculation rate of 1.0%, and cultured at 28℃, aeration rate of 0.9vvm, stirring speed of 200rpm for 9.5h to obtain seed tank seeds.

[0061] (3) The cultured seeds from the seed tank were transferred at an inoculum rate of 4.0% to a 20-ton fermenter containing 12.5 tons of fermentation medium. The fermentation was carried out at 28°C with an aeration rate of 1.0 vvm and a stirring speed of 160 rpm for 40 hours until the spore shedding rate reached 100%. Figure 2 The fermentation broth is obtained as the fermentation endpoint.

[0062] (4) Add 0.2% (m / v) xanthan gum to the fermentation broth, stir until completely dissolved to obtain a liquid inoculum, take a sample, dilute and spread on PCA plates, incubate at 28℃ for 20 h for counting, the effective viable count of the liquid inoculum is 3.15×10⁻⁶. 10 CFU / mL.

[0063] The culture medium used in both the seed tank and the fermentation tank consisted of: 25.0 g / L peptone, 5.0 g / L soybean meal, 22.0 g / L sucrose, 27.5 g / L starch, 0.2 g / L manganese sulfate, and 6.0 g / L light calcium carbonate; pH 7.2, sterilized at 121℃ for 30 min.

[0064] 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%, 98.8%, and 96.2%, respectively.

[0065] Example 6: Effect of Fermentation Endpoint Control on the Stability of Liquid Inoculum

[0066] Using the same method as in Example 5, the difference is that the fermentation endpoints were controlled at 90% spore formation rate (no shedding), 100% spore formation rate (individual shedding), and 100% spore shedding rate (both spore formation rate and shedding rate were 100%), respectively, to prepare the corresponding liquid inoculum. The survival rates of the liquid inoculum prepared in Example 5 at different storage times are shown in Table 4.

[0067] Table 4. Effects of different fermentation endpoints on the stability of liquid inoculum.

[0068]

[0069] Example 7: Preparation of Soluble Solid Microbial Agent

[0070] 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 3.30 × 10⁻⁶ on PCA plates. 11CFU / g.

[0071] 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 approximately 100 billion / g.

[0072] 4. Usage and dosage

[0073] This invention relates to a microbial inoculant for controlling target spot disease in cucumbers caused by *Corynespora cassiicola*. It can be applied from transplanting to maturity, with optimal efficacy when used before and at the early stages of disease. Liquid inoculants can be diluted 200-300 times, with a 200-fold dilution for spraying yielding the best results. Soluble solid inoculants can be diluted 500-800 times, with a 600-fold dilution for spraying yielding the best results.

[0074] Example 8: Field control effect of A45 liquid fungicide spray on cucumber target spot disease

[0075] Cucumber target spot disease: classified according to the leaf surface area occupied by the target spot disease.

[0076] Grade 0: No lesions;

[0077] Level 1: 0%-5%;

[0078] Level 3: 5%-25%;

[0079] Level 5: 25%-50%;

[0080] Level 7: 50%-75%;

[0081] Level 9: 75% or higher.

[0082] 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)

[0083] Prevention and control effect (%) = 100 × (control disease index - treatment disease index) / control disease index.

[0084] Two mu (approximately 0.33 hectares) of cucumbers infected with target spot disease were selected in Guantao County, Handan City, Hebei Province. The experiment included four treatments: 43% fluopyram·azoxystrobin suspension (positive control), A45 liquid inoculant, A45 soluble solid inoculant, and water (blank control). Each treatment had four replicates, resulting in 16 plots, each approximately 50 m². The dosages for each treatment were as follows: positive control: 43% fluopyram·azoxystrobin suspension diluted 1000 times; A45 liquid inoculant diluted 200 times; A45 soluble solid inoculant diluted 600 times; and blank control: water. Treatment began when sporadic lesions first appeared, with applications every 7 days for a total of three applications. The control efficacy is shown in Table 5.

[0085] Table 5 shows the control effect of liquid fungicide spray on cucumber target spot disease.

[0086]

[0087] Comparative Example 1: Comparison Experiment with Different Fermentation Media

[0088] Freshly activated A45 slant culture was inoculated into shake flask seed culture medium (PCB medium) and cultured at 28℃ and 200 rpm for 12 h. The cultured shake flask seed culture was then inoculated into a seed tank at a rate of 1.5%, and cultured at 28℃ with an aeration rate of 1.0 vvm for 10 h. The seed culture from the seed tank was then transferred to a fermenter at a rate of 4.5%, and fermented at 28℃ with an aeration rate of 1.0 vvm for 40 h. The seed culture medium in the seed tank and the fermentation medium in the fermenter were the following three different types. After fermentation, the fermentation broth was centrifuged at 10000 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 *Corynespora cassiicola*, the pathogen causing cucumber target spot disease. The results are shown in Table 6. A larger inhibition zone diameter (mm) indicated a better antibacterial effect.

[0089] Culture medium 1 (g / L): peptone 25.0, soybean meal 5.0, sucrose 22.0, starch 27.5, manganese sulfate 0.2, light calcium carbonate 6.0

[0090] Culture medium 2 (g / L): peptone 22.5, soybean meal 6.0, sucrose 22.0, starch 25.0, manganese sulfate 0.2, light calcium carbonate 5.5

[0091] Culture medium 3 (g / L): peptone 28.0, soybean meal 10.0, sucrose 15.0, starch 30.0, manganese sulfate 0.5, light calcium carbonate 3.5

[0092] Table 6. Antibacterial effects of fermentation broths obtained from different fermentation media under the same conditions against *Corynebacterium multiflorum*.

[0093] culture medium Diameter of the inhibition zone (mm) Culture medium 1 42 Culture medium 2 40 Culture medium 3 16

[0094] Comparative Example 2: Comparison of different fermentation conditions under the same fermentation medium

[0095] Both the seed culture medium in the seed tank and the fermentation medium in the fermenter consisted of 25.0 g / L peptone, 5.0 g / L soybean meal, 22.0 g / L sucrose, 27.5 g / L starch, 0.2 g / L manganese sulfate, and 6.0 g / L light calcium carbonate. Freshly activated A45 slant culture was inoculated into shake flask seed culture medium (PCB medium) and cultured at 28℃ and 200 rpm for 12 h. The cultured shake flask seed culture was then inoculated into the seed tank at a rate of 1.5% and cultured at 28℃ with an aeration rate of 1.0 vvm for 10 h. The seed culture from the seed tank was then transferred to the fermenter at a rate of 4.5% and fermented for 40 h under the following conditions: Condition 1: 28℃, 1.0 vvm aeration; Condition 2: 31℃, 1.1 vvm aeration; and Condition 3: 37℃, 0.7 vvm aeration. 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 *Corynespora cassiicola*, the pathogen causing cucumber target spot disease. The larger the diameter (mm) of the inhibition zone, the better the antibacterial effect. The results are shown in Table 7.

[0096] Table 7. Effects of fermentation conditions on the antibacterial effect of A45 fermentation broth.

[0097] Fermentation conditions Diameter of the inhibition zone (mm) Condition 1 42 Condition 2 41 Condition 3 17

Claims

1. A strain of Bacillus subtilis ( Bacillus subtilis The application of strain A45 in the control of plant diseases or in the preparation of microbial agents for the control of plant diseases, wherein strain A45 has the preservation number CGMCC No. 33289, and the plant disease is caused by *Cyclocarya paliurus* (…). Corynesporacassiicola ) causes target spot disease in cucumbers; characterized in that, The application refers to applying the liquid inoculum prepared by the preparation method to cucumber with target spot of cucumber, the effective bacteria content of the liquid inoculum is 3.15×10 10 CFU / mL, 200-300 times dilution spray is used: (1) Freshly activated A45 slant strain was inoculated into a seed flask, and cultured at 28-32°C and 180-200 rpm for 12-16 hours; (2) The cultured seed flask was inoculated into a seed tank at an inoculation amount of 1.0-1.5%, and cultured at 28-32°C with a ventilation amount of 0.8-1.2 v.v.m for 8-10 hours; (3) The seed tank was inoculated into a fermentation tank at an inoculation amount of 3.5-4.5%, and cultured at 28-32°C with a ventilation amount of 0.8-1.2 v.v.m for 36-48 hours, until the spores were completely shed to obtain the fermentation broth; (4) The fermentation broth was used to prepare a liquid bacterial agent.

2. A strain of Bacillus subtilis ( Bacillus subtilis The application of strain A45 in the control of plant diseases or in the preparation of microbial agents for the control of plant diseases, wherein strain A45 has the preservation number CGMCC No. 33289, and the plant disease is caused by *Cyclocarya paliurus* (…). Corynesporacassiicola ) causes target spot disease in cucumbers; characterized in that, The soluble solid bacterial agent prepared by the following preparation method was applied to cucumbers with target spot disease, and the solid bacterial agent content was 100 billion / g, and the spraying was used at a dilution of 600 times: (1) Freshly activated A45 slant strain was inoculated into a seed flask, and cultured at 28-32°C and 180-200 rpm for 12-16 hours; (2) The cultured seed flask was inoculated into a seed tank at an inoculation amount of 1.0-1.5%, and cultured at 28-32°C with a ventilation amount of 0.8-1.2 v.v.m for 8-10 hours; (3) The seed tank was inoculated into a fermentation tank at an inoculation amount of 3.5-4.5%, and cultured at 28-32°C with a ventilation amount of 0.8-1.2 v.v.m for 36-48 hours, until the spores were completely shed to obtain the fermentation broth; (4) The fermentation broth was used to prepare a solid soluble bacterial agent.

3. A microbial inoculant characterized in that, Bacillus subtilis A45 with the preservation number of CGMCC No. 33289 and prepared by the following preparation method: (1) Freshly activated A45 slant strain was inoculated into a seed flask, and cultured at 28-32°C and 180-200 rpm for 12-16 hours; (2) The cultured seed flask was inoculated into a seed tank at an inoculation amount of 1.0-1.5%, and cultured at 28-32°C with a ventilation amount of 0.8-1.2 v.v.m for 8-10 hours; (3) The seed tank was inoculated into a fermentation tank at an inoculation amount of 3.5-4.5%, and cultured at 28-32°C with a ventilation amount of 0.8-1.2 v.v.m for 36-48 hours, until the spores were completely shed to obtain the fermentation broth; (4) The fermentation broth was used to prepare a liquid bacterial agent or a solid soluble bacterial agent.

4. The microbial inoculant of claim 3, wherein, The seed flask in step (1) was a PCB culture medium, and the PCB culture medium was composed of (g / L): peptone 5.0, yeast extract powder 2.5, glucose 1.0, pH: 7.4±0.2, 121°C sterilization for 30 minutes.

5. The microbial inoculant of claim 3, wherein, The medium used in the seed tank in step (2) and the fermentation tank in step (3) consists of (g / L): peptone 20-25, soybean meal 5-8, sucrose 20-25, starch 25-30, manganese sulfate 0.2-0.3, light calcium carbonate 4.0-6.0, pH 7.0±0.2, sterilized at 121℃ for 30 min.

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