Microbial agent for preventing and treating botrytis cinerea of crops and preparation method and application thereof

Abstract

The present application belongs to the technical field of biological control, and particularly relates to a microbial agent for preventing and treating gray mold of crops, and a preparation method and application thereof. The present application provides a microbial agent for preventing and treating gray mold of crops, which comprises a microbial strain and / or a fermentation broth of the microbial strain; the microbial strain comprises one or more of Bacillus subtilis JD-L9, Pseudomonas lundensis DS-PZ-P18 and Pseudomonas chlororaphis HQ-CZ-P12; wherein the Pseudomonas chlororaphis HQ-CZ-P12, the Pseudomonas lundensis DS-PZ-P18 and the Bacillus subtilis JD-L9 have a good inhibition rate on the pathogenic fungus of gray mold, and after being compounded into a compound microbial agent, the compound microbial agent is sprayed on seedlings of medicinal materials, so that the incidence of gray mold during seedling raising is significantly reduced, pesticide residues of the medicinal materials are reduced, and the safety of the medicinal materials is improved.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology, and in particular relates to a microbial agent for controlling gray mold in crops, its preparation method and application. Background Technology

[0002] Gray mold is caused by *Botrytis*, a fungus belonging to the genus *Botrytis* of the family Sderotiniaceae in the phylum Ascomycota. Botrytis cinerea Botrytis cinerea (Gray Mold) and other pathogens of the genus Botrytis are common fungal diseases affecting the growth of crops in fields, greenhouses, and facility-based seedling cultivation. These fungi are widely distributed globally and seriously affect normal plant growth. The disease is called gray mold because a layer of grayish powdery mold forms on infected parts of the plant in its later stages. Botrytis cinerea overwinters in the soil and on diseased plant debris as sclerotia, conidia, and mycelia. It spreads via airflow, watering, or agricultural operations. Its optimal growth temperature is 18–24℃, and its disease development temperature is 4–32℃. It thrives in humidity levels above 90%, making it a low-temperature, high-humidity disease. Furthermore, it has a wide host range and no significant host specificity, infecting over 1400 important economic crops. It is particularly damaging to senescent or mature plant tissues, causing rot of leaves, stems, and fruits, significantly reducing their economic value and resulting in substantial crop and economic losses.

[0003] Currently, in agricultural production, gray mold disease in cash crops is typically controlled using chemical pesticides. Common chemical fungicides include broad-spectrum fungicides such as carbendazim, chlorothalonil, iprodione, and fludioxonil. However, these have poor inhibitory effects and require large dosages. Furthermore, the excessive use of chemical pesticides not only causes environmental pollution and pesticide residues, seriously harming the environment and human health, but also promotes the emergence of "super gray mold isolates" resistant to chemical fungicides. The environmental problems caused by the frequent use of fungicides have prompted the search for eco-friendly alternatives to control gray mold in order to achieve sustainable agricultural development.

[0004] Biological control is environmentally friendly and represents a future development trend. Biocontrol agents (BCAs), such as antagonistic microorganisms, plant essential oils, and plant defense agents, have been reported for use in treating gray mold in various economic crops. Microbial biocontrol agents possess multiple biocontrol functions, including nutrient competition, niche competition, biological antagonism, plant growth promotion, and induction of plant immune resistance. They mainly include dominant biocontrol bacteria such as Bacillus, Pseudomonas, Trichoderma, and Actinomycetes, and are the main biocontrol products. For example, Streptomyces speciosa screened under publication number CN202410617203.9, Pseudomonas aeruginosa screened under CN202410043245.6, and Bacillus polymyxa screened under CN202110612244.5 all have good inhibitory effects on Botrytis cinerea. Meanwhile, probiotics from the in-situ soil of plant rhizosphere have great potential to antagonize Botrytis cinerea. Gray mold disease is highly prevalent during the seedling stage of Chinese medicinal herbs, and there are few effective bacterial resources for controlling gray mold. Therefore, discovering more in-situ biocontrol strains of Chinese medicinal herbs for the prevention and control of gray mold disease in crop seedlings is a technical problem that urgently needs to be solved to promote the facility-based seedling cultivation of bulk Chinese medicinal herbs. Summary of the Invention

[0005] In view of this, the purpose of this invention is to provide a microbial agent for the prevention and control of gray mold in crops. This agent has a good antagonistic effect against Botrytis cinerea and can be widely used as a biocontrol strain for gray mold, with broad prospects.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a microbial agent for controlling gray mold in crops, the microbial agent comprising microbial strains and / or fermentation broth of microbial strains; The Latin name of Bacillus subtilis JD-L9 is Bacillus subtilis It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35947; The Latin name of the *Pseudomonas rheinrichii* DS-PZ-P18 is... Pseudomonas reinekei It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35948; The Latin name of the *Pseudomonas aeruginosa* HQ-CZ-P12 is... Pseudomonas chlororaphis It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35949.

[0007] This invention also provides a method for preparing a microbial inoculant for controlling gray mold in crops, comprising the following steps: Microbial strains are activated and fermented to obtain microbial agents.

[0008] Preferably, the culture medium for activation culture is LB solid medium or TSA solid medium; the temperature for activation culture is 25~35℃; and the activation culture time is 20~28h.

[0009] Preferably, the fermentation culture medium is LB liquid medium or potato glucose liquid medium; the fermentation culture temperature is 28~30℃; the fermentation culture time is 24~36h; and the culture rotation speed is 180~200rpm.

[0010] The present invention also provides the application of the microbial agent described herein or the microbial agent prepared by the method described herein in the preparation of products for the prevention and control of gray mold in crops.

[0011] Preferably, the crop is a medicinal herb.

[0012] Preferably, the gray mold is caused by Botrytis cinerea.

[0013] The present invention also provides a method for preventing and controlling gray mold in crops, comprising the following steps: Use microbial agents to spray or drench crops on leaves; The microbial agent is the microbial agent described above or the microbial agent prepared by the preparation method described above.

[0014] Preferably, the foliar spraying is performed 2 to 4 times, with an interval of 6 to 8 days between each application, and the irrigation rate for each application is 1 to 3 L / hm². 2 .

[0015] Preferably, the root drenching is performed 1 to 3 times, with an interval of 6 to 8 days between each drenching, and the irrigation volume for each drenching is 8 to 12 L / hm. 2 .

[0016] Beneficial effects: This invention provides a microbial agent for controlling gray mold in crops, comprising a microbial strain and / or a fermentation broth of the microbial strain; the microbial strain includes one or more of Bacillus subtilis JD-L9, Pseudomonas reinhardtii DS-PZ-P18, and Pseudomonas aeruginosa HQ-CZ-P12; wherein Pseudomonas aeruginosa ( Pseudomonas chlororaphis HQ-CZ-P12, Pseudomonas rheumatoides ( Pseudomonas reinekeiDS-PZ-P18, Bacillus subtilis ( Bacillus subtilis JD-L9 exhibits a good inhibitory rate against Botrytis cinerea, the pathogen of gray mold. When formulated into a compound microbial agent and sprayed on seedlings of medicinal herbs, it significantly reduces the incidence of gray mold during the seedling stage, lowers pesticide residues in the herbs, and improves their safety. Therefore, using compound microbial agents for the biological control of gray mold caused by Botrytis cinerea in crops is environmentally friendly, has stable efficacy, and, when combined with other growth-promoting agents, shows broad development and application prospects for the biological control of gray mold in the seedling stage of medicinal herbs. Attached Figure Description

[0017] Figure 1 Colony morphology for HQ-CZ-P12, DS-PZ-P18, and JD-L9; Figure 2 Phylogenetic tree of the isolated strains; Figure 3 The results of the plate antagonism experiment in Example 4; Figure 4 The microbial agent in Example 5 is shown to be effective against Botrytis cinerea. Figure 5 The microbial inoculant in Example 6 is shown to control gray mold during the seedling stage of crop facility cultivation.

[0018] Preservation Instructions

[0019] Bacillus subtilis JD-L9, Latin name is Bacillus subtilis It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35947; Pseudomonas reinhardtii DS-PZ-P18, Latin name is Pseudomonas reinekei It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35948; Pseudomonas aeruginosa HQ-CZ-P12, Latin name is Pseudomonas chlororaphis It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35949. Detailed Implementation

[0020] This invention provides a microbial agent for controlling gray mold in crops, the microbial agent comprising microbial strains and / or fermentation broth of microbial strains; The microbial strains include one or more of Bacillus subtilis JD-L9, Pseudomonas rheinrichii DS-PZ-P18 and Pseudomonas aeruginosa DS-PZ-P18. The Latin name of Bacillus subtilis JD-L9 is Bacillus subtilis It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35947; The Latin name of the *Pseudomonas rheinrichii* DS-PZ-P18 is... Pseudomonas reinekei It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35948; The Latin name of the *Pseudomonas aeruginosa* HQ-CZ-P12 is... Pseudomonas chlororaphis It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35949.

[0021] In this invention, the *Pseudomonas aeruginosa* HQ-CZ-P12 belongs to the genus *Pseudomonas*. *Pseudomonas*, as a large microbial group in plant rhizosphere soil, can provide nutrients to the host, antagonize pathogenic microorganisms, and maintain the rhizosphere microecological balance. Furthermore, *Pseudomonas aeruginosa* (… Pseudomonas chlororaphis Bacillus subtilis is one of the most studied biocontrol bacteria. Four subspecies have been reported, all of which produce colored phenazine antibiotic compounds. Most strains of this species are isolated from the rhizosphere of plants and play a protective role against pathogens and nematodes. Bacillus subtle Bacillus subtilis is also one of the biocontrol strains with application potential among Bacillus species. Its mechanisms of action in controlling plant diseases are diverse, including competitive action, production of antimicrobial substances, promotion of plant growth, and induction of plant resistance. Therefore, the use of Pseudomonas and Bacillus species for biological control of plant diseases is effective. The *Pseudomonas aeruginosa* HQ-CZ-P12, *Pseudomonas rhesus* DS-PZ-P18, and *Bacillus subtilis* JD-L9 described in this invention show good antagonistic effects against *Botrytis cinerea* and can be widely used as biocontrol strains for gray mold, with broad prospects.

[0022] This invention also provides a method for preparing a microbial inoculant for controlling gray mold in crops, comprising the following steps: Microbial strains are activated and fermented to obtain microbial agents.

[0023] In this invention, the culture medium for activation culture is LB solid medium or TSA solid medium, preferably LB solid medium; the temperature for activation culture is 25~35℃, preferably 28~32℃, more preferably 30℃; the activation culture time is 20~28h, preferably 22~26h, more preferably 24h.

[0024] In this invention, the fermentation culture medium is LB liquid medium or potato glucose liquid medium, preferably LB liquid medium; the fermentation culture temperature is 28~30℃, preferably 29℃; the fermentation culture time is 24~36h, preferably 28~32h, more preferably 30h; the culture rotation speed is 180~200rpm, preferably 185~195rpm, more preferably 190rpm.

[0025] In this invention, when the microbial strain includes two of Bacillus subtilis JD-L9, Pseudomonas rheumatoides DS-PZ-P18, and Pseudomonas aeruginosa HQ-CZ-P12, the preferred method for preparing the microbial agent is as follows: after activating and fermenting the microbial strains respectively, a fermentation broth is obtained, and the fermentation broth is mixed at a volume ratio of 1:1 to obtain the microbial agent; When the microbial strains include Bacillus subtilis JD-L9, Pseudomonas reinhardtii DS-PZ-P18, and Pseudomonas aeruginosa HQ-CZ-P12, the preferred method for preparing the microbial agent is as follows: after activating and fermenting the three microbial strains respectively, a fermentation broth is obtained, and the fermentation broth is mixed in a volume ratio of 1:1:1 to obtain the microbial agent.

[0026] The present invention also provides the application of the microbial agent described herein or the microbial agent prepared by the method described herein in the preparation of products for the prevention and control of gray mold in crops.

[0027] In this invention, the crop is a medicinal crop, preferably Codonopsis pilosula, Astragalus membranaceus, Glycyrrhiza uralensis, Angelica sinensis, and Scutellaria baicalensis.

[0028] In this invention, the gray mold is caused by Botrytis cinerea.

[0029] The present invention also provides a method for preventing and controlling gray mold in crops, comprising the following steps: Use microbial agents to spray or drench crops on leaves; The microbial agent is the microbial agent described above or the microbial agent prepared by the preparation method described above.

[0030] In this invention, the foliar spraying is performed 2 to 4 times, preferably 3 times, with an interval of 6 to 8 days, preferably 7 days, and the irrigation amount for each spraying is 1 to 3 L / hm². 2 Preferably 2L / hm 2 .

[0031] In this invention, the number of root drenching sessions is 1 to 3, with an interval of 6 to 8 days between each session, preferably 7 days, and the irrigation amount for each session is 8 to 12 L / hm². 2 Preferably 9~11 L / hm 2 Further preferred is 10 L / hm 2 .

[0032] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0033] Example 1: Isolation of the pathogen causing gray mold in Codonopsis pilosula

[0034] Typical Codonopsis pilosula plants infected with gray mold were selected. Leaves from the diseased areas were cut with a scalpel, sterilized with 75% ethanol for 5 seconds, then sterilized with 1% NaClO solution for 5 seconds, and naturally dried before being placed on PDA medium. After colony growth, the single-spore isolation method was used for purification, repeated four times until a single pathogen was obtained. The pathogen causing gray mold in Codonopsis pilosula was isolated. The hyphae showed clear septa, branching in a dendritic pattern, and contained aerial hyphae. The sporangiophores were erect, forming spherical sporangia (containing numerous spores) at the apex, consistent with the colony morphology of Botrytis cinerea. Therefore, it was identified as Botrytis cinerea (gray mold). Botrytis cinerea ).

[0035] Example 2 Screening of dominant growth-promoting strains

[0036] Healthy Codonopsis pilosula and Astragalus membranaceus plants were selected, and their rhizosphere soil was collected. After sieving, 0.1g of the soil was placed in a 15mL sterile centrifuge tube, and 10mL of sterile water was added. The tube was then shaken on a plate shaker at 180rpm for 30min at room temperature. After shaking, the resulting solution was used to isolate the soil bacteria from the roots of Codonopsis pilosula and Astragalus membranaceus using a serial dilution method. The solutions were diluted to 10, 100, 1000, and 10000 times and spread on LB, PDA, and modified Gao's No. 1 medium. Single bacteria were isolated, purified, and cryopreserved. The colony morphology was observed during colony growth. Three strains were named HQ-CZ-P12, DS-PZ-P18, and JD-L9. The colony morphology of the three strains after 1 day of growth is shown in the figure. Figure 1 As shown; The colonies of strain HQ-CZ-P12 on LB medium are orange-yellow, round, without protrusions, with a moist and smooth surface and neat edges; the colonies of strain DS-PZ-P18 on LB medium are white, round, raised, with a moist and smooth surface and neat edges; the colonies of JD-L9 on LB medium are transparent white, round, raised, with a moist and smooth surface and neat edges.

[0037] Example 3 Molecular identification of isolated strains

[0038] PCR amplification of the 16S rDNA of the isolated strain was performed using universal primers, yielding the target band. Fresh bacterial cultures containing the target band were sent to Qingke Biotechnology Co., Ltd. for testing. The complete 16S rDNA sequence of the strain was obtained by removing the primer sequences from both ends of the sequence. The complete sequence was then BLASTed on NCBI, and sequences with extremely high sequence similarity were selected. A phylogenetic tree was constructed using MEGA 11.0 software, as shown in the figure below. Figure 2 As shown; The universal primers are: upstream primer 27F: 5'-AGAGTTTGATCCTGGCTCAG (SEQ ID NO.1), and downstream primer 1492R: 5'-GGTTACCTTGTTACGACTT (SEQ ID NO.2); PCR reaction system (50µL): 20µL Mix, 2µL upstream primer, 2µL downstream primer, 24µL sterile deionized water, 2µL template DNA; PCR reaction procedure: pre-denaturation at 94℃ for 2 min, followed by 30 amplification cycles, each cycle consisting of denaturation at 94℃ for 30 s, annealing at 60℃ for 40 s, extension at 72℃ for 90 s, and a final extension at 72℃ for 3 min. After the cycle, the system is automatically maintained at 4℃, and the target band is then detected on a 1% agarose gel. according to Figure 2 It can be determined that strain HQ-CZ-P12 is *Pseudomonas aeruginosa*. Pseudomonas chlororaphis ), namely Pseudomonas aeruginosa ( Pseudomonas chlororaphis HQ-CZ-P12 and strain DS-PZ-P18 are Pseudomonas reinhardtii (…). Pseudomonas reinekei ), namely Pseudomonas reinhardtii ( Pseudomonas reinekei DS-PZ-P18, strain JD-L9 is Bacillus subtilis ( Bacillus subtilis ), namely Bacillus subtilis ( Bacillus subtilis JD-L9; The 16S rDNA gene sequences of strains HQ-CZ-P12, DS-PZ-P18, and JD-L9 were identified as follows: HQ-CZ-P12: DS-PZ-P18: JD-L9: Example 4 Plate antagonism test of isolated strains against Botrytis cinerea Botrytis cinerea fungi (take the periphery of the colony and use a sterilized punch to obtain a 0.5 cm diameter mycelial cake) that had been cultured for 3 days at 25℃ were inoculated into the center of a PDA plate. Then, single colonies of strains HQ-CZ-P12, DS-PZ-P18, and JD-L9 were scraped from two opposite locations at equal distances from the inoculation point using a pipette tip. The other two opposite corners were left uninoculated (as a control group). A positive control group was prepared by inoculating 100 mg / ml of fludioxonil. The plate was then inverted and incubated at 25℃ for 3 days. Three parallel experiments were performed simultaneously to observe the Botrytis cinerea colony formation. The results are as follows: Figure 3 The diameter of gray mold colonies was measured, and the inhibition rate was calculated. Inhibition rate % = (Coronary diameter of control group - Colony diameter of treatment group) / Colony diameter of control group 100; The colony diameter of *Botrytis cinerea* in the control group was 83.3 mm. The colony diameters of *Botrytis cinerea* treated with strains HQ-CZ-P12, DS-PZ-P18, and JD-L9 were 13.8 mm, 26.3 mm, and 35.3 mm, respectively, with inhibition rates of 83.43%, 68.43%, and 57.62%. Therefore, the results of the plate antagonism test above show that the three biocontrol bacteria HQ-CZ-P12, DS-PZ-P18, and JD-L9 prepared by the present invention have significant antagonistic activity against Botrytis cinerea.

[0039] Example 5: Verification of the preventive effect of microbial inoculants on Botrytis cinerea infection.

[0040] Microbial inoculants (compound inoculants): (1) Activation of strains: HQ-CZ-P12, DS-PZ-P18 and JD-L9 strains were inoculated and streaked onto sterile LB solid medium and activated at 30°C for 24 h to obtain culture plates; (2) Fermentation culture: Single colonies of each of the three strains prepared in step (1) were picked from the plates and inoculated onto sterile LB liquid medium. The plates were cultured at 30℃ and 200rpm for 36h to obtain the fermentation broth of each strain. The OD was measured using sterile medium as a control. 600 The viable cell count of the three bacterial strains at an absorbance of 1 was obtained, which was 10. 8 CFU / ml; (3) The three fermentation liquids prepared in step (2) are mixed in a volume ratio of 1:1:1 to obtain a microbial agent; Verification experiment (Codonopsis pilosula leaves were selected for verification because they showed the worst resistance to gray mold): Healthy Codonopsis pilosula leaves were collected, sterilized with 75% ethanol for 5 seconds, then sterilized with 1% NaClO solution for 3 seconds, rinsed with sterile water, and allowed to air dry. The leaf surface was then cut open at the center using a scalpel. Infection group: The mycelium was inoculated into the wound, the petiole was wrapped with wet cotton, the whole leaf was placed in a petri dish with water, and incubated in a 25℃ incubator. Positive control group: The mycelium was first inoculated at the wound site, the petiole was wrapped with wet cotton, the whole leaf was placed in a petri dish with water, and incubated in a 25℃ incubator. After 24 hours, 100mg / ml fludioxonil was sprayed on the leaf surface. Compound microbial agent treatment group: The mycelium was inoculated into the wound, the petiole was wrapped with wet cotton, the whole leaf was placed in a petri dish with water, and placed in an incubator at 25℃. After 24 hours, the compound microbial agent was sprayed on the leaf surface. After 7 days of culture in each group, the location of the wound in the center of the Codonopsis pilosula leaves and the extent of infection were observed. The results were as follows: Figure 4 As shown; Experiments showed that the microbial agent described in this invention has strong antagonistic properties against the pathogen of gray mold in Codonopsis pilosula in vitro. Compared with the fludioxonil treatment group, no mycelial growth was observed on the leaves of Codonopsis pilosula in the compound agent treatment group, and the leaves were in good condition. The compound agent showed a high control effect on gray mold in Codonopsis pilosula, with an inhibition rate of 100%.

[0041] Example 6: Verification of the preventive effect of microbial inoculants on gray mold infection during the seedling stage of crops in facility-based cultivation.

[0042] A microbial inoculant (compound inoculant) was obtained by referring to Example 5; Four seedling trays each of Codonopsis pilosula, Glycyrrhiza uralensis, Astragalus membranaceus, and Angelica sinensis were selected from seedlings raised in protected facilities with a gray mold incidence rate exceeding 90%. The seedling period was 120-180 days. The substrate consisted of a mixture of vermiculite and perlite in a specific ratio. The seeding density was 1800 seeds per square meter for Astragalus membranaceus, 16000 seeds per square meter for Codonopsis pilosula, 1000 seeds per square meter for Glycyrrhiza uralensis, and 1200 seeds per square meter for Angelica sinensis. Supplemental lighting was provided using a specific light source for 12 hours daily from 7:00 to 19:00. Microbial inoculants were sprayed onto the leaf surface of the seedlings at a specific ratio of 1:10 with water, for a total of three applications, with a 7-day interval between each application. The application rate of the microbial inoculant was 2 L / hm² each time. 2 After 60 days of growth, observe the leaf infection status for gray mold. The results are as follows: Figure 5 As shown; The results showed that the seedlings of all medicinal materials were in good condition after spraying with the compound microbial agent, and the incidence of gray mold disease decreased significantly.

[0043] As can be seen from the above embodiments, the present invention provides a microbial agent for the prevention and control of gray mold in crops, its preparation method and application. Pseudomonas aeruginosa HQ-CZ-P12, Pseudomonas rhei DS-PZ-P18, and Bacillus subtilis JD-L9 have good inhibitory rates against the pathogen of gray mold. After being compounded into a compound microbial agent, spraying it on seedlings of Chinese medicinal herbs significantly reduces the incidence of gray mold during the seedling stage, reduces pesticide residues in medicinal herbs, and improves the safety of Chinese medicinal herbs.

[0044] 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 microbial inoculant for controlling gray mold in crops, characterized in that, The microbial agent includes microbial strains and / or fermentation broth of microbial strains; The microbial strains include one or more of Bacillus subtilis JD-L9, Pseudomonas rheinrichii DS-PZ-P18 and Pseudomonas aeruginosa HQ-CZ-P12. The Latin name of Bacillus subtilis JD-L9 is Bacillus subtilis It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35947. The Latin name of the *Pseudomonas rheinrichii* DS-PZ-P18 is... Pseudomonas reinekei It is deposited at the China General Microbiological Culture Collection Center, located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO.35948; The Latin name of the *Pseudomonas aeruginosa* HQ-CZ-P12 is... Pseudomonas chlororaphis It is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, on April 2, 2026, with accession number CGMCC NO. 35949.

2. A method for preparing a microbial inoculant for controlling gray mold in crops, characterized in that, Includes the following steps: Microbial strains are activated and fermented to obtain microbial agents.

3. The preparation method according to claim 2, characterized in that, The culture medium for activation culture is LB solid medium or TSA solid medium; the temperature for activation culture is 25~35℃; and the activation culture time is 20~28h.

4. The preparation method according to claim 2, characterized in that, The fermentation culture medium is LB liquid medium or potato glucose liquid medium; the fermentation culture temperature is 28~30℃; the fermentation culture time is 24~36h; and the culture rotation speed is 180~200rpm.

5. The application of the microbial agent according to claim 1 or the microbial agent prepared by any one of claims 2 to 4 in the preparation of products for controlling gray mold in crops.

6. The application according to claim 5, characterized in that, The type of crop mentioned is a medicinal herb.

7. The application according to claim 5, characterized in that, The gray mold disease is caused by Botrytis cinerea.

8. A method for controlling gray mold in crops, characterized in that, Includes the following steps: Use microbial agents to spray or drench crops on leaves; The microbial agent is the microbial agent according to claim 1 or the microbial agent prepared by the preparation method according to any one of claims 2 to 4.

9. The method according to claim 8, characterized in that, The foliar spraying is applied 2-4 times, with an interval of 6-8 days between each application, and the spraying rate is 1-3 L / hm² per application. 2 .

10. The method according to claim 9, characterized in that, The root irrigation is performed 1 to 3 times, with an interval of 6 to 8 days between each irrigation, and the irrigation volume for each irrigation is 8 to 12 L / hm. 2 .

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