Microbial agent and application thereof in preventing and treating persimmon anthracnose

Abstract

The present application belongs to the technical field of microorganisms, and particularly relates to a microbial agent and application thereof in prevention and treatment of persimmon anthracnose. The microbial agent comprises Bacillus amyloliquefaciens QGFCH3-3 and Bacillus atrophaeus HHQGTS13-1. The microbial agent can effectively inhibit the persimmon anthracnose pathogen and has a good prevention and treatment effect on the persimmon anthracnose.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, and in particular to a microbial agent and its application in the prevention and control of anthracnose in persimmons. Background Technology

[0002] Persimmons are sweet and nutritious, possessing significant economic and social value. Guangxi Zhuang Autonomous Region is China's largest persimmon producing area, ranking first in both cultivation area and yield nationwide. The main production areas are Gongcheng County, Pingle County, and Laibin City in Guangxi Zhuang Autonomous Region. Anthracnose is one of the major diseases affecting persimmons, primarily damaging leaves, fruits, and branches, causing severe economic losses.

[0003] Anthracnose in persimmon is caused by a deuteromycete fungus, primarily affecting new shoots and fruits, and sometimes infecting leaves. On new shoots, the initial symptoms are small black dots, which later expand into brown, oval lesions with a slightly sunken center, longitudinal cracks, and the formation of small black dots. The lesions can reach 10-20 mm in length. The wood beneath the lesions rots, making diseased branches or seedlings prone to breakage at the lesion site. Lesions at the base of young branches often encircle the stem, causing death above the lesion. On leaves, the lesions are irregularly arranged, initially turning yellow from the veins and petioles, then black. On fruits, in the early stages of infection, pinhead-sized dark brown to blackish-brown spots appear, gradually expanding to over 5 mm. These spots become slightly sunken, nearly circular, with densely packed, slightly ring-shaped, grayish-brown dots (conidiophores) in the center. In humid conditions, pinkish, sticky conidial masses are secreted from these conidia. When the pathogen infects the skin, it forms hard, black lumps inside the flesh. One or two lesions typically appear on a single fruit, but sometimes more than a dozen. Diseased fruits fall off prematurely. When leaves are infected, the disease primarily affects the petioles and veins, initially appearing yellowish-brown, later turning dark brown to black, and forming long, irregular stripes.

[0004] Currently, the control of persimmon anthracnose still relies primarily on chemical agents, such as spraying with prochloraz, dithiazol, zineb, thiophanate-methyl, thiophanate-methyl, mancozeb, or Bordeaux mixture and organic copper preparations. However, the long-term and extensive use of chemical agents has led to a series of problems: on the one hand, chemical residues remain in the soil, water, and atmosphere; on the other hand, pathogens easily develop resistance to chemical agents, resulting in a gradual decline in control effectiveness, necessitating increased dosages and creating a vicious cycle; furthermore, pesticide residues in agricultural products directly threaten human health, contradicting the development concepts of green agriculture and food safety. Therefore, finding environmentally friendly and sustainable alternatives to chemical agents has become an urgent need to promote the green and efficient development of agriculture. Summary of the Invention

[0005] In view of the above, it is necessary to provide a microbial agent that can effectively inhibit the pathogen of persimmon anthracnose and has a good control effect on persimmon anthracnose.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0007] A microbial inoculant comprises *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophaeus* HHQGTS13-1; wherein, *Bacillus amyloliquefaciens* QGFCH3-3 is classified as *Bacillus amyloliquefaciens*, deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 9, 2025, with accession number CGMCC No. 34814; and *Bacillus atrophaeus* HHQGTS13-1 is classified as *Bacillus atrophaeus*, deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The accession date is January 29, 2024, and the accession number is CGMCC No. 29799.

[0008] Furthermore, in the microbial agent, the viable counts of both *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 are not less than 1 × 10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

[0009] Furthermore, the microbial agent also includes Rhodotorula diobovata HHHCJMDS4-1, which is classified as Rhodotorula diobovata and is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 9, 2025, and the accession number is CGMCC No. 34813.

[0010] Furthermore, in the microbial agent, the viable count of the double obovate red yeast HHHCJMDS4-1 is not less than 1×10⁻⁶. 7 CFU / mL or 1×10 7 CFU / g.

[0011] Furthermore, the microbial agent is in the form of a liquid or a solid.

[0012] The preparation method of the microbial agent is as follows: each strain is fermented and cultured separately and then spray-dried to obtain microbial powder, which is then mixed evenly to obtain the solid form of the microbial agent.

[0013] Specifically, the preparation methods for each type of mycelium powder are as follows:

[0014] Bacillus amyloliquefaciens powder: Bacillus amyloliquefaciens QGFCH3-3 was inoculated into LB liquid medium and cultured for 48-72 h, then spray-dried to obtain Bacillus amyloliquefaciens powder;

[0015] Bacillus atrophicus powder: Bacillus atrophicus HHQGTS13-1 was inoculated into LB liquid medium and cultured for 48-72 h, then spray-dried to obtain Bacillus atrophicus powder;

[0016] Double obovate red yeast powder: The double obovate red yeast HHHCJMDS4-1 was inoculated into YPD liquid medium and cultured for 24-48 hours, then spray-dried to obtain double obovate red yeast powder.

[0017] Furthermore, the preparation method of the microbial agent is as follows: after fermenting and culturing each strain separately, centrifuging is performed to obtain bacterial cells, the bacterial cells are resuspended in sterile water to obtain corresponding bacterial suspensions, and the prepared bacterial suspensions are mixed evenly to obtain the liquid form of the microbial agent.

[0018] Specifically, the preparation methods for each bacterial suspension are as follows:

[0019] Bacillus amyloliquefaciens suspension: Bacillus amyloliquefaciens QGFCH3-3 was inoculated into LB liquid medium and cultured for 48-72 h. After the culture was completed, the bacterial cells were obtained by centrifugation and resuspended in sterile water to obtain a Bacillus amyloliquefaciens suspension.

[0020] Bacillus atrophicus suspension: Bacillus atrophicus HHQGTS13-1 was inoculated into LB liquid medium and cultured for 48-72 h. After the culture was completed, the bacterial cells were obtained by centrifugation and resuspended in sterile water to obtain Bacillus atrophicus suspension.

[0021] Double obovate red yeast suspension: The double obovate red yeast HHHCJMDS4-1 was inoculated into YPD liquid medium and cultured for 24-48h. After the culture was completed, the cells were obtained by centrifugation and resuspended in sterile water to obtain the double obovate red yeast suspension.

[0022] The present invention also provides the application of the above-mentioned microbial agent in the prevention and control of anthracnose in persimmon.

[0023] The present invention also provides the application of the above-mentioned microbial agent in inhibiting pathogens that cause anthracnose in persimmons.

[0024] Furthermore, the pathogens causing persimmon anthracnose include: *C. horii*, *C. fructicola*, *C. gloeosporioides*, and *C. fioriniae*.

[0025] The present invention has the following beneficial effects:

[0026] The *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 screened in this invention both exhibit certain antagonistic effects against the pathogen of persimmon anthracnose. Furthermore, the microbial agent obtained by combining *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 has a synergistic effect, effectively enhancing the control of persimmon anthracnose. While *Rhodotorula bisporus* HHHCJMDS4-1 itself does not have an antagonistic effect against the pathogen of persimmon anthracnose, it can promote the colonization and adaptability of *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 on persimmon trees, and also promote the growth and absorption of persimmon trees, thereby effectively improving the field control effect of persimmon anthracnose. Attached Figure Description

[0027] Figure 1 This is a colony diagram of the Bacillus amyloliquefaciens QGFCH3-3 strain on a plate, representing the bacterial colony of this invention.

[0028] Figure 2 This is a microscopic image of the Bacillus amyloliquefaciens QGFCH3-3 strain of the present invention.

[0029] Figure 3 This is a colony diagram of Rhodotorula diobovata HHHCJMDS4-1, the double obovate red yeast of this invention, on a plate.

[0030] Figure 4 This is a microscopic image of Rhodotorula diobovata HHHCJMDS4-1, the double obovate red yeast of this invention.

[0031] Information on the preservation of biological materials

[0032] The strain information deposited in this application is as follows:

[0033] Bacillus amyloliquefaciens QGFCH3-3, classified as Bacillus amyloliquefaciens, is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 9, 2025, and the accession number is CGMCC No. 34814.

[0034] Bacillus atrophaeus HHQGTS13-1, classified as Bacillus atrophaeus, is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is January 29, 2024, and the accession number is CGMCC No. 29799.

[0035] Rhodotorula diobovata HHHCJMDS4-1, taxonomically named Rhodotorula diobovata, is deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on June 9, 2025, with accession number CGMCC No. 34813. Detailed Implementation

[0036] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.

[0037] All features disclosed in this specification, or all steps in all disclosed methods or processes, may be combined in any way, except for mutually exclusive features and / or steps.

[0038] Unless otherwise stated, each feature disclosed in this specification (including any appended claims and abstract) is merely one example of a series of equivalent or similar features.

[0039] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0040] In the following examples, the culture medium used is as follows:

[0041] LB liquid medium: 10.0 g peptone, 5.0 g yeast extract, 5.0 g sodium chloride, 1000 mL pure water, adjust pH to 7.0 ± 0.1 with sodium hydroxide, sterilize at 121℃ for 20 min.

[0042] LB solid medium: 18g agar, 10.0g peptone, 5.0g yeast extract, 5.0g sodium chloride, 1000mL pure water, pH adjusted to 7.0±0.1 with sodium hydroxide, sterilized at 121℃ for 20min.

[0043] YPD liquid culture medium preparation: yeast extract 10 g / L, peptone 20 g / L, glucose 20 g / L, prepared in proportion, sterilized at 115℃ for 30 min.

[0044] Example 1

[0045] This embodiment provides a Bacillus amyloliquefaciens strain QGFCH3-3, which was isolated from a salt sample of Emerald Lake in Da Qaidam, Qinghai Province. The specific isolation and screening method is as follows:

[0046] (1) Strain isolation and screening

[0047] The salt sample was collected from Emerald Lake in Da Qaidam, Qinghai Province, and brought back to the laboratory in a sterile bag, where it was stored at 4℃. 5g of the salt sample was weighed and added to 45mL of sterile water to prepare a concentration of 10. -1 The soil-sampled bacterial suspension was diluted in 10-fold serial dilutions to achieve a bacterial suspension concentration of 10. -1 ~10 -7 After vortex oscillation and mixing, take 10 -3 10 -4 10 -5 Three gradients of 100 μL bacterial suspension were spread onto LB agar plates, with two replicates for each gradient, and incubated at 35°C for 24 h. Representative strains were selected based on colony color, elevation, edge shape, and size, and streaked onto LB agar plates, then incubated at 35°C. After two subcultures, strains with uniform morphology were Gram-stained and examined microscopically. Strains with uniform microscopic examination were confirmed as purified, numbered QGFCH3-3, and stored at -40°C to -80°C (final concentration 25% glycerol) for later use.

[0048] (2) Physiological and biochemical identification of colonies

[0049] Observe the morphology, color, etc. of the colonies of the strain on the surface of LB medium according to Bergey's Manual of Bacteriological Identification (9th Edition). The colony morphology diagram is shown in the figure below. Figure 1As shown; young cultures were selected, smeared, Gram-stained, and observed under a microscope for bacterial morphology, size, Gram staining reaction, and the presence, morphology, and attachment position of spores, etc. The microscopic images are shown below. Figure 2 As shown.

[0050] (3) Sequence analysis of the strain's 16S rRNA

[0051] The strain QGFCH3-3 was sent to the Sequencing and Identification Department of Shanghai Sangon Biotech for sequencing identification. Its sequence is shown below:

[0052]

[0053] The sequence was compared and analyzed in the NCBI ribosome database. 16S rRNA sequence analysis showed that the strain of this invention shared 100% homology with *Bacillus amyloliquefaciens*, meaning the strain is indeed *Bacillus amyloliquefaciens*.

[0054] The microorganisms in this embodiment were preserved as follows:

[0055] Bacillus amyloliquefaciens QGFCH3-3, classified as Bacillus amyloliquefaciens, is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 9, 2025, and the accession number is CGMCC No. 34814.

[0056] The preparation method of the fermentation product of Bacillus amyloliquefaciens QGFCH3-3 in this embodiment is as follows:

[0057] (1) The Bacillus amyloliquefaciens was inoculated into LB liquid medium at an inoculation rate of 3% and cultured at 36-38℃, pH 6.5, and 190-210 r / min for 48-72 h;

[0058] (2) After the culture is completed, if spray drying is performed, Bacillus amyloliquefaciens powder can be obtained; if centrifugation is performed to obtain bacterial cells, the bacterial cells can be resuspended in sterile water to obtain Bacillus amyloliquefaciens suspension.

[0059] In the above preparation method, the culture temperature can be 36℃, 37℃ or 38℃, etc.; the culture rotation speed can be 190r / min, 200r / min or 210r / min, etc.; the culture time can be 48h, 50h, 60h or 72h, etc.

[0060] Example 2

[0061] This embodiment provides a double obovate red yeast (Rhodotorula diobovata) HHHCJMDS4-1, which was isolated from bryophytes in the primeval forest of Tian'e County, Hechi City. The specific isolation and screening method is as follows:

[0062] (1) Isolation and screening of strains

[0063] Weigh 5g of bryophyte samples collected from the primeval forest of Tian'e County, Hechi City. Rinse the samples thoroughly with clean water and blot dry with filter paper. First, immerse the samples in 70% alcohol for 1 minute, then immerse them in 2% sodium hypochlorite solution for 3 minutes for disinfection. Repeat the sodium hypochlorite solution disinfection once more, then treat with 70% alcohol for 30 seconds. Finally, rinse twice with sterile water. After surface disinfection, under aseptic conditions, place the samples in a sterile mortar and add 5mL of sterile water to thoroughly crush them. After standing for 5 minutes, dilute the crushed liquid in a 10-fold gradient to 10. －3 Take 100 μL of each serially diluted solution and spread it onto YPD liquid medium plates, making two parallel spreads for each gradient. Simultaneously, take a portion of the final rinse solution and spread it onto the plates. If no microorganisms grow after incubation, the surface sterilization is complete. Incubate the plates at 28°C. Select representative strains based on colony color, degree of protrusion, edge shape, and size, and streak them onto selection agar medium, incubating at 28°C. After two subcultures, Gram stain and microscopic examination are performed on strains with uniform morphology observed visually. Strains with uniform microscopic examination are considered purified and designated as HHHCJMDS4-1. These strains are then stored at -40°C to -80°C (final concentration 25% glycerol) for later use.

[0064] (2) Physiological and biochemical identification of colonies

[0065] Observe the morphology and color of the colonies of the strain on the surface of Bengal red agar medium according to Bergey's Manual of Bacteriological Identification (9th Edition). The colony morphology diagram is shown below. Figure 3 As shown; young cultures were selected, smeared, Gram-stained, and observed under a microscope for bacterial morphology, size, Gram staining reaction, and the presence, morphology, and attachment position of spores, etc. The microscopic images are shown below. Figure 4 As shown.

[0066] (3) Sequence analysis of the strain's 16S rRNA

[0067] The strain HHHCJMDS4-1 was sent to the Sequencing and Identification Department of Shanghai Sangon Biotech for sequencing identification. Its sequence is shown below:

[0068] .

[0069] The sequence was compared and analyzed in the NCBI ribosome database. The 16S rRNA sequence analysis results showed that the strain of this invention had 100% homology with Rhodotorula diobovata, that is, the strain is Rhodotorula diobovata.

[0070] The microorganisms in this embodiment were preserved as follows:

[0071] Rhodotorula diobovata HHHCJMDS4-1, classified as Rhodotorula diobovata, is deposited at the China General Microbiological Culture Collection Center (CGMCC) at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing. The deposit date is June 9, 2025, and the accession number is CGMCC No. 34813.

[0072] The preparation method of the fermentation product of the double obovate red yeast HHHCJMDS4-1 in this embodiment is as follows:

[0073] (1) The double obovate red yeast was inoculated into YPD liquid medium at an inoculation rate of 4% and cultured at 26-30℃, pH 7.0, and 130-170 r / min for 24-48 h;

[0074] (2) After the culture is completed, if spray drying is performed, double obovate red yeast powder can be obtained; if centrifugation is performed to obtain cells, the cells are resuspended in sterile water to obtain double obovate red yeast suspension.

[0075] In the above preparation method, the culture temperature can be specifically 26℃, 28℃ or 30℃, etc.; the culture rotation speed can be 130r / min, 150r / min or 170r / min, etc.; the culture time can be 24h, 32h, 40h or 48h, etc.

[0076] Example 3

[0077] This embodiment provides a strain of Bacillus atrophaeus, HHQGTS13-1, classified as Bacillus atrophaeus, deposited at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, on January 29, 2024, with accession number CGMCC No. 29799. Detailed strain information is disclosed in Chinese patent application CN120349913A.

[0078] The preparation method of the fermentation product of Bacillus atrophicus HHQGTS13-1 in this embodiment is as follows:

[0079] (1) The Bacillus atrophus was inoculated into LB liquid medium at an inoculation rate of 2% and cultured at 31-33℃, pH 7.0, and 180-200 r / min for 48-72 h;

[0080] (2) After the culture is completed, if spray drying is performed, Bacillus atrophicus powder can be obtained; if centrifugation is performed to obtain bacterial cells, the bacterial cells can be resuspended in sterile water to obtain Bacillus atrophicus suspension.

[0081] In the above preparation method, the culture temperature can be 31℃, 32℃ or 33℃, etc.; the culture rotation speed can be 180r / min, 190r / min or 200r / min, etc.; the culture time can be 48h, 50h, 60h or 72h, etc.

[0082] Example 4

[0083] This embodiment provides a microbial inoculant comprising Bacillus amyloliquefaciens QGFCH3-3 and Bacillus atrophaeus HHQGTS13-1, wherein the viable count of both Bacillus amyloliquefaciens QGFCH3-3 and Bacillus atrophaeus HHQGTS13-1 is not less than 1 × 10⁻⁶. 7 CFU / mL.

[0084] The preparation method of the microbial inoculant is as follows:

[0085] (1) The Bacillus amyloliquefaciens QGFCH3-3 was inoculated into LB liquid medium at an inoculation rate of 3%, and cultured at 38℃, pH 6.5 and 200 r / min for 55 h. After the culture was completed, the bacterial cells were obtained by centrifugation and the bacterial cells were resuspended in sterile water to obtain a Bacillus amyloliquefaciens bacterial suspension.

[0086] (2) The Bacillus atrophicus HHQGTS13-1 was inoculated into LB liquid medium at an inoculation rate of 2%, and cultured at 32℃, pH 7.0 and 180 r / min for 60 h. After the culture was completed, the bacterial cells were obtained by centrifugation and the bacterial cells were resuspended in sterile water to obtain a Bacillus atrophicus bacterial suspension.

[0087] (3) Mix the above-prepared Bacillus amyloliquefaciens suspension and Bacillus niger suspension in equal volumes to obtain the microbial agent.

[0088] Example 5

[0089] This embodiment provides a microbial inoculant comprising Bacillus amyloliquefaciens QGFCH3-3 and Bacillus atrophaeus HHQGTS13-1, wherein the viable count of both Bacillus amyloliquefaciens QGFCH3-3 and Bacillus atrophaeus HHQGTS13-1 is not less than 1 × 10⁻⁶. 7CFU / mL.

[0090] The preparation method of the microbial inoculant is as follows:

[0091] (1) The Bacillus amyloliquefaciens QGFCH3-3 was inoculated into LB liquid medium at an inoculation rate of 3%, and cultured at 38℃, pH 6.5 and 200 r / min for 55 h. After the culture was completed, it was spray-dried to obtain Bacillus amyloliquefaciens powder.

[0092] (2) The Bacillus atrophicus HHQGTS13-1 was inoculated into LB liquid medium at an inoculation rate of 2%, and cultured at 32℃, pH 7.0 and 180 r / min for 60 h. After the culture was completed, it was spray-dried to obtain Bacillus atrophicus powder.

[0093] (3) Mix the above-prepared Bacillus amyloliquefaciens powder and Bacillus niger powder in equal volumes to obtain the microbial agent.

[0094] Example 6

[0095] This embodiment provides a microbial inoculant comprising Bacillus amyloliquefaciens QGFCH3-3, Bacillus atrophaeus HHQGTS13-1, and Rhodotorula diobovata HHHCJMDS4-1, wherein the viable counts of Bacillus amyloliquefaciens QGFCH3-3, Bacillus atrophaeus HHQGTS13-1, and Rhodotorula diobovata HHHCJMDS4-1 are all not less than 1 × 10⁻⁶. 7 CFU / mL.

[0096] The preparation method of the microbial inoculant is as follows:

[0097] (1) The Bacillus amyloliquefaciens QGFCH3-3 was inoculated into LB liquid medium at an inoculation rate of 3%, and cultured at 38℃, pH 6.5 and 200 r / min for 55 h. After the culture was completed, the bacterial cells were obtained by centrifugation and the bacterial cells were resuspended in sterile water to obtain a Bacillus amyloliquefaciens bacterial suspension.

[0098] (2) The Bacillus atrophicus HHQGTS13-1 was inoculated into LB liquid medium at an inoculation rate of 2%, and cultured at 32℃, pH 7.0 and 180 r / min for 60 h. After the culture was completed, the bacterial cells were obtained by centrifugation and the bacterial cells were resuspended in sterile water to obtain a Bacillus atrophicus bacterial suspension.

[0099] (3) The double obovate red yeast HHHCJMDS4-1 was inoculated into YPD liquid medium at an inoculation rate of 4%, and cultured at 28℃, pH 7.0 and 150r / min for 30h. After the culture was completed, the cells were obtained by centrifugation and the cells were resuspended in sterile water to obtain the double obovate red yeast suspension.

[0100] (4) Mix the above-prepared Bacillus amyloliquefaciens suspension, Bacillus pyriformis suspension and Rhodotorula rubra suspension in equal volumes to obtain the microbial agent.

[0101] The "equal volume mixing" used in this embodiment refers to mixing equal volumes of each strain after fermentation, provided that the effective viable cell counts of their fermentation broths are adjusted to a similar range. This mixing method means that the inoculum amount of each strain in the compound microbial agent is approximately equal, which is simple to operate, has good reproducibility, and has been experimentally proven to be effective. "Equal volume mixing" is not an absolute limitation; the mixing volume ratio of the strains can be adjusted. The core is to ensure that the compound microbial agent contains all three functional strains and that their total viable cell count reaches the effective inoculum amount. Equal volume mixing is a reasonable initial strategy based on the standardization of microbial concentrations and is widely accepted by academia and industry. The mixing ratio can be optimized and adjusted according to the needs of actual application scenarios, which still falls within the protection scope of this invention.

[0102] Example 7

[0103] This embodiment provides a microbial inoculant comprising Bacillus amyloliquefaciens QGFCH3-3, Bacillus atrophaeus HHQGTS13-1, and Rhodotorula diobovata HHHCJMDS4-1, wherein the viable counts of Bacillus amyloliquefaciens QGFCH3-3, Bacillus atrophaeus HHQGTS13-1, and Rhodotorula diobovata HHHCJMDS4-1 are all not less than 1 × 10⁻⁶. 7 CFU / g.

[0104] The preparation method of the microbial inoculant is as follows:

[0105] (1) The Bacillus amyloliquefaciens QGFCH3-3 was inoculated into LB liquid medium at an inoculation rate of 3%, and cultured at 38℃, pH 6.5 and 200 r / min for 55 h. After the culture was completed, it was spray-dried to obtain Bacillus amyloliquefaciens powder.

[0106] (2) The Bacillus atrophicus HHQGTS13-1 was inoculated into LB liquid medium at an inoculation rate of 2%, and cultured at 32℃, pH 7.0 and 180 r / min for 60 h. After the culture was completed, it was spray-dried to obtain Bacillus atrophicus powder.

[0107] (3) The double obovate red yeast HHHCJMDS4-1 was inoculated into YPD liquid medium at an inoculation rate of 4%, and cultured at 28℃, pH 7.0 and 150r / min for 30h. After the culture was completed, it was spray-dried to obtain double obovate red yeast powder.

[0108] (4) Mix the above-prepared Bacillus amyloliquefaciens powder, Bacillus pyriformis powder and Rhodotorula buergerianum powder in equal volumes to obtain the solid microbial agent.

[0109] In Examples 4-7 above, the "equal volume mixing" refers to mixing equal volumes after each strain has undergone fermentation, provided that the effective viable cell counts in its fermentation broth are adjusted to a similar range, or mixing equal volumes after each strain has undergone fermentation and drying, provided that the effective viable cell counts in its bacterial powder are adjusted to a similar range. This mixing method means that the inoculation amount of each strain in the compound bacterial agent is approximately equal, which is simple to operate, has good reproducibility, and has been experimentally proven to be effective. "Equal volume mixing" is not an absolute limitation; the mixing volume ratio of the strains can be adjusted. The core is to ensure that the compound bacterial agent contains all three functional strains and that its total viable cell count reaches the effective inoculation amount. Equal volume mixing is a reasonable initial strategy based on the standardization of bacterial concentration and is widely accepted by the academic and industrial communities. The mixing ratio can be optimized and adjusted according to the needs of actual application scenarios, which still falls within the protection scope of this invention.

[0110] Example 8

[0111] This embodiment tests the indoor antibacterial ability of the aforementioned microbial agent. Details are as follows:

[0112] 1. The tested plant pathogenic fungi included: *C. horii*, *C. fructicola*, *C. gloeosporioides*, and *C. fioriniae*.

[0113] 2. Test Method: A 6mm diameter pathogenic bacterial cake was inoculated into the center of a PDA medium plate. The medium for inoculating the pathogenic bacterial cake was divided into 5 groups: one group was inoculated only with *Bacillus amyloliquefaciens* QGFCH3-3 bacterial suspension (S1 group); one group was inoculated only with *Bacillus atrophicus* HHQGTS13-1 bacterial suspension (S2 group); one group was inoculated only with *Rhodotorula buergerianum* HHHCJMDS4-1 bacterial suspension (S3 group); and one group was inoculated with the microbial agent described in Example 4 (i.e., *Bacillus amyloliquefaciens* QGFCH3-3 bacterial suspension + *Bacillus atrophicus*). Group S4 was inoculated with HHQGTS13-1 bacterial suspension, and another group was inoculated with the microbial agent described in Example 6 (i.e., Bacillus amyloliquefaciens QGFCH3-3 bacterial suspension + Bacillus pyriformis HHQGTS13-1 bacterial suspension + Rhodotorula buergerianum HHHCJMDS4-1 bacterial suspension) (Group S5). Each group was inoculated at 20 mm from the center of the plate, with an inoculation volume of 5 μL. At the same time, PDA medium inoculated only with pathogens was used as a control (CK group). The culture was kept at 28℃ for 5 days to observe the antibacterial effect of the strains and calculate the antibacterial rate.

[0114]

[0115] 3. Measurement Results

[0116] The antibacterial rate is shown in Table 1 below.

[0117] Table 1. Results of indoor antibacterial ability tests for each group of bacterial agents.

[0118]

[0119] The data from groups S1, S2, and S4 in Table 1 show that *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 have a certain inhibitory effect on the four pathogens causing persimmon anthracnose: *C. horii*, *C. fructicola*, *C. gloeosporioides*, and *C. fioriniae*. The microbial agents using *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 showed significantly higher inhibition rates against each pathogen than single-strain agents or combinations of other control strains. This indicates that the combined use of *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 has a synergistic effect in inhibiting the pathogens causing persimmon anthracnose. Data from groups S3 and S5 show that *Rhodotorula diodactylate* HHHCJMDS4-1 itself has no inhibitory effect on the four pathogens causing persimmon anthracnose. Combining it with *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 also did not significantly enhance the inhibitory effect on the four pathogens.

[0120] Example 9

[0121] This embodiment tests the field control efficacy of the aforementioned microbial inoculant against persimmon anthracnose. Details are as follows:

[0122] 1. Experimental Procedure: Young branches of 'Gongcheng Persimmon' were used for the experiment. The spore suspension used for infection was *C. horii*. After 7 days of culture to produce conidia, the spore suspension was placed in CMC medium and the concentration was adjusted to 1×10⁻⁶. 6 Prepare spores / mL for later use. First, perform infection with *Haloxylon ammodendron* spore solution. For spore solution infection, we selected young branches from four different locations on *Persimmon gongchengensis*, using a 1.5 mL... The branches were divided into 5 infection zones with a spacing of cm. The zone was evenly sprayed with a small sprayer, and the branches were bagged. The bags were removed on the second day after inoculation. Spraying was carried out 3 days after infection. The sprayed agents were Bacillus amyloliquefaciens QGFCH3-3 suspension (S1 group), Bacillus atrophicus HHQGTS13-1 suspension (S2 group), Rhodotorula bisporus HHHCJMDS4-1 suspension (S3 group), the microbial agent described in Example 4 (i.e., Bacillus amyloliquefaciens QGFCH3-3 suspension + Bacillus atrophicus HHQGTS13-1 suspension) (S4 group), and the microbial agent described in Example 6 (i.e., Bacillus amyloliquefaciens QGFCH3-3 suspension + Bacillus atrophicus HHQGTS13-1 suspension + Rhodotorula bisporus HHHCJMDS4-1 suspension). Each group consisted of branches from four different locations on a tree, with three replicates per group. This constituted one experimental treatment. During all treatments, a control group was established by applying sterile water to the branches.

[0123] 2. Investigation and Disease Grading Methods: On day 15 after infection, each branch was examined individually. The size of the lesions was measured using calipers, and the severity of the disease was graded and recorded. The grading criteria were as follows: Grade 1 if the lesion diameter accounted for 0-20% of the total branch length; Grade 2 if it accounted for 20%-40%; Grade 3 if it accounted for 40%-60%; Grade 4 if it accounted for 60%-80%; and Grade 5 if it accounted for 80%-100%. Based on the disease grade of different branches, the anthracnose disease disease index was calculated after fungicide application.

[0124] ;

[0125] .

[0126] 3. Measurement Results

[0127] The prevention and control effects are shown in Table 2 below.

[0128] Table 2. Field control efficacy of each group of inoculants against persimmon anthracnose.

[0129]

[0130] The data from groups S1, S2, and S4 in Table 2 show that both Bacillus amyloliquefaciens QGFCH3-3 and Bacillus atrophicus HHQGTS13-1 have certain control effects on anthracnose in field-grown persimmons. Furthermore, the microbial agents using Bacillus amyloliquefaciens QGFCH3-3 and Bacillus atrophicus HHQGTS13-1 have a significantly better control effect on anthracnose in field-grown persimmons than single-strain agents or combinations of other control strains. This indicates that the combination of Bacillus amyloliquefaciens QGFCH3-3 and Bacillus atrophicus HHQGTS13-1 has a synergistic effect in the control of persimmon anthracnose. Combining the data from S3 and S5 in Tables 1 and 2, it can be seen that although *Rhodotorula diodactylate* HHHCJMDS4-1 itself does not have an antagonistic effect on the pathogen of persimmon anthracnose, it can promote the colonization and adaptability of *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 on persimmon trees, and can also promote the growth and absorption of persimmon trees. Therefore, its combination with *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophicus* HHQGTS13-1 can effectively improve the control effect of anthracnose in field persimmons.

Claims

1. A microbial inoculant, characterized in that, The microbial agent includes *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophaeus* HHQGTS13-1; wherein the preservation number of *Bacillus amyloliquefaciens* QGFCH3-3 is CGMCC No. 34814, and the preservation number of *Bacillus atrophaeus* HHQGTS13-1 is CGMCC No. 29799; in the microbial agent, the viable count of both *Bacillus amyloliquefaciens* QGFCH3-3 and *Bacillus atrophaeus* HHQGTS13-1 is not less than 1 × 10⁻⁶. 7 CFU / mL.

2. The microbial inoculant according to claim 1, characterized in that, The microbial inoculant also includes Rhodotorula diobovata HHHCJMDS4-1, whose preservation number is CGMCC NO.34813; the viable count of Rhodotorula diobovata HHHCJMDS4-1 is not less than 1×10⁻⁶. 7 CFU / mL.

3. The microbial inoculant according to any one of claims 1-2, characterized in that, The microbial agent is in liquid form.

4. The method for preparing the microbial inoculant as described in claim 3, characterized in that, The preparation method is as follows: each strain is fermented and cultured separately, centrifuged to obtain bacterial cells, the bacterial cells are resuspended in sterile water to obtain corresponding bacterial suspensions, and the prepared bacterial suspensions are mixed evenly to obtain the liquid form of the microbial agent.

5. The application of the microbial agent as described in any one of claims 1-2 in the prevention and control of persimmon anthracnose, characterized in that, The persimmon anthracnose is caused by *C. horii*, *C. fructicola*, *C. gloeosporioides*, and *C. fioriniae*.

6. The application of the microbial agent according to any one of claims 1-2 in inhibiting the pathogen causing persimmon anthracnose, characterized in that, The pathogens causing persimmon anthracnose are: *C. horii*, *C. fructicola*, *C. gloeosporioides*, and *C. fioriniae*.

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