Bio-control inoculant for preventing and controlling strawberry anthrax stem rot as well as preparation method and application of bio-control inoculant

Abstract

The invention belongs to the field of agricultural microorganisms, and particularly relates to a biocontrol inoculant for preventing and controlling strawberry anthrax stem rot as well as a preparation method and application of the biocontrol inoculant. Comprising streptomyces CMA18 and pseudomonas CMPA12; the streptomycete CMA18 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35370, the preservation date is July 24, 2025, the streptomycete CMA18 is classified and named as streptomycete sp., and the streptomycete CMA18 is preserved in the China General Microbiological Culture Collection Center (CGMCC). The pseudomonas strain CMPA12 is preserved in the China General Microbiological Culture Collection Center (CGMCC), the preservation number is CGMCC No.35863, the preservation date is September 11, 2025, and the pseudomonas strain CMPA12 is classified and named as Pseudomonas sp. The biocontrol inoculant provided by the invention can effectively improve the prevention and control effect on strawberry anthrax stem rot.

Description

Technical Field

[0001] This invention belongs to the field of agricultural microbiology, specifically relating to a biocontrol agent for controlling strawberry anthracnose stem rot, its preparation method, and its application. Background Technology

[0002] Strawberries are an important economic crop, but they are susceptible to anthracnose caused by *Anthracnose sicca* (Siamese anthracnose). Colletotrichum siamense Strawberry anthracnose stem rot caused by ) Colletotrichum crown rot of strawberry CCR (Chemical Resistant Disease) seriously affects the sustainable development of its industry. Currently, production mainly relies on chemical pesticides to control this disease, but long-term use of chemical pesticides can easily lead to problems such as increased pathogen resistance, environmental pollution, and pesticide residues in agricultural products.

[0003] Biological control, as a green and environmentally friendly method of disease control, has received widespread attention. While some biocontrol microorganisms have been found to have some control effect on strawberry anthracnose stem rot, their effectiveness against susceptible strawberry varieties is poor when used alone, and their mechanism of action remains unclear, hindering the research and development and promotion of biocontrol products. Therefore, developing a biological control technology that is effective and environmentally friendly is of great significance. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of existing technologies where single biocontrol microorganisms are ineffective in controlling strawberry anthracnose stem rot, and to provide a biocontrol agent for controlling strawberry anthracnose stem rot based on the synergistic effect of Streptomyces and Pseudomonas. The biocontrol agent provided by this invention can improve the control effect against strawberry anthracnose stem rot and reduce the use of chemical pesticides.

[0005] The present invention also provides a method for preparing the above-mentioned biocontrol agent.

[0006] Another objective of this invention is to provide the application of the above-mentioned biocontrol agent in the control of strawberry anthracnose stem rot.

[0007] To achieve the above objectives, the present invention provides the following technical solution: This invention provides a biocontrol agent for controlling strawberry anthracnose stem rot, comprising Streptomyces CMA18 and Pseudomonas CMPA12; wherein Streptomyces CMA18 is deposited at the China General Microbiological Culture Collection Center, accession number: CGMCC No. 35370, deposit date: July 24, 2025, and is classified as Streptomyces. Streptomyces sp. The *Pseudomonas* CMPA12 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 35863, on September 11, 2025, and is classified as follows: Pseudomonas sp. .

[0008] Preferably, the ratio of viable bacteria of Streptomyces CMA18 to Pseudomonas CMPA12 is 1:1 to 1:5.

[0009] This invention also provides a method for preparing the above-mentioned biocontrol agent, comprising the following steps: (1) Streptomyces CMA18 was cultured in ISP2 medium to obtain CMA18 bacterial suspension; (2) Pseudomonas CMPA12 was cultured in LB medium to obtain CMPA12 bacterial suspension; (3) Mix CMA18 bacterial solution and CMPA12 bacterial solution in proportion to obtain the final product.

[0010] Preferably, in step (1), the mass ratio of Streptomyces CMA18 and ISP2 culture medium is 1:50; the composition of ISP2 culture medium is: 4g yeast extract, 10g malt extract, 4g glucose, 20g agar, 1000mL water, and pH 7.2; the culture is carried out at 28℃ and 180r / min for 72-96h.

[0011] Preferably, in step (2), the mass ratio of the Pseudomonas CMPA12 and LB medium is 1:50; the composition of the LB medium is: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL water, pH 7.0; the culture is carried out at 28℃ and 180r / min for 24-48h.

[0012] Another objective of this invention is to provide the application of the above-mentioned biocontrol agent in the control of strawberry anthracnose stem rot.

[0013] Preferably, the biocontrol agent is applied by root drenching; the application concentration is 10^6 Streptomyces CMA18 and Pseudomonas CMPA12 per strawberry plant. 6 -10 8 CFU.

[0014] The biocontrol agent provided by this invention is applied to the strawberry root system. The first application should be made during the strawberry seedling stage or within one week after transplanting, followed by applications every 15-20 days for 2-3 consecutive applications. This effectively controls the occurrence of strawberry anthracnose stem rot.

[0015] In the biocontrol agent provided by this invention, Streptomyces CMA18 is an endophytic Streptomyces species found in strawberries. Streptomyces sp. The strains screened in this invention can be isolated from strawberry roots, colonize strawberry plants, and regulate strawberry root exudates; Pseudomonas CMPA12 is an Asian Pseudomonas (…). Pseudomonas sp. The strains screened in this invention have a significant inhibitory effect on Siamese anthrax bacteria.

[0016] The biocontrol agent provided by this invention controls the number of viable Streptomyces CMA18 and Pseudomonas CMPA12 within a certain ratio range, and the synergistic effect of the two is better, which can more effectively improve the control effect on strawberry anthracnose stem rot.

[0017] Compared with the prior art, the present invention has the following beneficial effects: (1) The microbial agent composition provided by the present invention utilizes the synergistic effect of Streptomyces CMA18 and Pseudomonas CMPA12. Streptomyces CMA18 can regulate strawberry root exudates and promote the colonization of Pseudomonas CMPA12 in strawberry roots, thereby significantly improving the control effect on strawberry anthracnose stem rot. Experimental data show that the composition can inhibit the incidence index of strawberry anthracnose stem rot by 67.3% under sterile conditions and 70.1% under infective conditions, which is much higher than the effect of using Pseudomonas CMPA12 alone (32.6% under sterile conditions and 30.1% under infective conditions).

[0018] (2) The microbial composition of the present invention uses the synergistic effect between microorganisms to control diseases, reduces the use of chemical pesticides, reduces environmental pollution and pesticide residues in agricultural products, and meets the development needs of green agriculture and eco-friendly agriculture.

[0019] (3) The preparation method of the microbial composition of the present invention is simple, convenient to apply, low in cost, and easy to promote and apply, which is of great significance to promoting the healthy and sustainable development of the strawberry industry.

[0020] Preservation Information 1 Strain name: CMA18 Preservation period: July 24, 2025 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Accession number: CGMCC No. 35370 Address of the depositary: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Postal code: 100101 Classification and nomenclature: Streptomyces Streptomyces sp. .

[0021] Preservation Information 2 Strain name: CMPA12 Preservation period: September 11, 2025 Preservation Institution: China General Microbiological Culture Collection Center, China Microbiological Culture Collection Committee Accession number: CGMCC No. 35863 Address of the depositary: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing Postal code: 100101 Category Naming: Pseudomonas sp. . Attached Figure Description

[0022] Figure 1 These are strawberry root bacteria strains with phosphate-solubilizing activity; Figure 2 A comparison chart of IAA production capacity of the strains; Figure 3 The image shows the antagonistic effect of CMA18 on anthracnose in strawberry seedlings; from left to right: blank control, CMA18 group, and Siamese anthracnose treatment group. Figure 4 This is a culture diagram showing the confrontation between CMPA12 and Siamese anthrax bacteria. Detailed Implementation

[0023] The present invention will be further described in detail below with reference to specific embodiments.

[0024] Example 1 Isolation and identification of CMA18 and CMPA12: Root samples of Akihime strawberry were collected from Lanling County, Linyi City, in June 2022. Strawberry root samples were collected using a random selection method. Three varieties of strawberry plants were dug up from the ground, large clumps of soil accumulated at the roots were shaken off, and the entire strawberry plant was collected into sterile sealed bags labeled with the corresponding information for further processing.

[0025] Cut the roots into small segments using scissors sterilized with alcohol. Place the root segments into centrifuge tubes, add 30 mL of sterile 1×PBS buffer, and place the tubes flat on a shaker. Rinse three times for 20 min each time at 180 rpm and 28°C. After rinsing, wipe the plant roots clean with sterile filter paper and place them into 50 mL centrifuge tubes. Set up 6 replicates for each sample. Cut the strawberry root samples washed with 1×PBS buffer into 2 mm segments using sterilized scissors and mix them evenly. Weigh 0.02 g of strawberry root segments and add them to a sterile centrifuge tube. Add 200 μL of sterilized 10 mM MgCl2 solution to the centrifuge tube. Grind the root segments into a homogenate using a sterile grinder. Then transfer the homogenate to a 50 mL centrifuge tube containing 25 mL of 10 mM MgCl2 solution and mix well. Let it stand at room temperature for 15 min. After mixing the diluent thoroughly in the laminar flow hood, pour it into a square dish. Add 160 μL of the mixed diluent to each well of the cell culture plate, except for well H12, which is used as a negative control and no diluent is added.

[0026] Stack 96-well cell culture plates and incubate at room temperature for two weeks. After two weeks, observe the turbidity of the 96-well cell culture plates, i.e., the degree of colony growth. Select cell culture plates with 30% turbidity for subsequent experiments. Using a multi-channel pipette, transfer 10 μL of liquid from each well of the selected 96-well cell culture plates into a 96-well PCR plate, label the wells, and temporarily store at -20°C for subsequent bacterial identification. Simultaneously, add 140 μL of sterile 80% PCR solution to each well of the culture plate and store the cell culture plates at -8°C for later use.

[0027] Centrifuge the previously prepared 96-well PCR plate containing 10 μL using a microplate centrifuge. After centrifugation, open the sealing film and add 16.6 μL of alkaline lysis buffer 1 (alkaline lysis buffer 1: 0.2 mM Na2-EDTA, 25 mM NaOH, adjust pH to around 12, sterilize at 121℃ for 15 min) to each well using a pipette. Mix thoroughly by pipetting, seal the PCR plate, centrifuge again, and then place it in a PCR instrument for high-temperature lysis in an alkaline environment (lysis at 95℃ for 30 min).

[0028] After cooling, the PCR plate was centrifuged. After centrifugation, 16.6 μL of neutralization buffer 2 (neutralization buffer 2: 25 mM Tris-HCl, adjusted to pH 7.5, sterilized at 121℃ for 15 min) was added to each well of the PCR plate in a clean bench. After mixing by pipetting, the liquid after centrifugation was used as the DNA template for the next round of PCR reaction for subsequent PCR experiments.

[0029] The first round of PCR used unlabeled primers: 799 F (SEQ ID NO.1):AACMGGATTAGATACCCKG; 1193 R (SEQ ID NO. 2): ACGTCATCCCCACCTTCC.

[0030] The 16S sequence was amplified using the primers described above.

[0031] The first round of PCR reaction system was as follows: ; The first round of PCR reaction program was as follows: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for 30 cycles; final extension at 72℃ for 5 min.

[0032] After the first round of PCR reaction, the PCR products are tested to ensure that the amplification is correct.

[0033] The product obtained from the first round of PCR was diluted with nuclease-free water, and the 40-fold dilution was used as the template for the second round of amplification.

[0034] Second round PCR reaction system:

[0035] The second round of PCR reaction procedure was as follows: pre-denaturation at 94℃ for 2 min; denaturation at 94℃ for 30 s, annealing at 55℃ for 30 s, extension at 72℃ for 1 min, for 25 cycles; final extension at 72℃ for 5 min. After amplification, 5 μL of each of the positive and negative controls were mixed with DNA loading buffer. Electrophoresis showed that the positive control produced a band of approximately 500 bp, while the negative control produced no band, indicating that the second round of amplification was successful.

[0036] All PCR products from each PCR plate in the second round were mixed together. 40 μL of the PCR mixture was taken from a centrifuge tube and mixed with 12 μL of DNA loading buffer. A 1.0% agarose gel was prepared, and a 2000 bp DNA marker was electrophoresed with the sample at 90V for 50 min. After electrophoresis, the amplified fragments appearing around 500 bp were excised from the gel and recovered using a gel extraction kit. The accurate concentration of DNA was determined using PicoGreen fluorescent dye. The recovered product was purified using magnetic beads, and the purified product was sent for sequencing using Illumina.

[0037] The sequence (SEQ ID NO.3) of strain CMA18 is as follows: The sequence (SEQ ID NO.4) of strain CMPA12 is as follows: Bioinformatics analysis primarily employed the Culturome bioinformatics analysis workflow developed by Liu Yongxin et al. (http: / / bailab.genetics.ac.cn / culturome) and a written R script. Plate and well labels were removed from the original sequences to obtain amplified purified sequences. VSEARCH was used for repeat removal and USEARCH for noise reduction to identify amplified sequence variants (ASVs), and species annotation was completed. Finally, the sequence and species of bacteria growing in the 96-well plate were determined. The resulting ASV table contains the positions of five optimal candidate wells for a specific species. Based on the strain information provided in the ASV table, the position in the 96-well cell culture plate, and the purity of the wells, 2-3 wells from the desired ASV list were selected using a sterile pipette tip and activated on TSA plates. The plates were incubated upside down in a 28°C incubator for 3-4 days. After single colonies grew, multiple purification processes were performed. Finally, single colonies were transferred to shake tubes containing 3 mL of TSB medium and incubated overnight on a shaker. After the bacterial culture became turbid, 750 μL of the bacterial culture and 750 μL of 50% glycerol were taken and frozen for preservation. Subsequently, the growth-promoting and disease-resistant functions of these strains were identified.

[0038] Example 2 Screening of phosphate-solubilizing strains Select a single colony that has grown and inoculate it into LB liquid medium, then incubate on a shaker until OD500. 600 =0.8, pipette 3 μL of bacterial suspension and spot it onto the center of PVK inorganic phosphate medium. Invert the plate and incubate at 28℃ for 7 days. Observe the changes around the bacteria in the culture dish. The appearance of a clear zone around the bacteria indicates that the strain has phosphate-solubilizing ability. The size of the clear zone can be used as an indicator of the strength of phosphate solubilization; the larger the clear zone, the stronger the phosphate-solubilizing ability of the strain. The strain was tested three times.

[0039] LB liquid medium: 10g peptone, 5g yeast extract, and 5g NaCl are dissolved in 1000 mL of deionized water, and the pH is adjusted to about 7.2. The mixture is then autoclaved at 121℃ for 20 min. LB solid medium: 15g agar powder is added per liter to the LB liquid medium.

[0040] PVK Inorganic Phosphorus Solid Culture Medium: 10g glucose, 0.5g (NH4)2SO4, 0.3g NaCl, 0.3g MgSO4·7H2O, 0.03g MnSO4·H2O, 0.03g FeSO4·7H2O, 0.3g KCl, 5g Ca3(PO4)2, 15g agar, 1000 mL deionized water, pH adjusted to approximately 7.4. Autoclave at 121℃ for 20 min.

[0041] The isolated and purified bacteria were screened using PVK inorganic phosphorus medium. Fourteen strains exhibited a clear zone around their colonies. These strains were numbered D7, P8F9, A13, B20, B19, A3, B12, A18 (CMA18 of this invention), D20, P28H4, A16, B11, A12 (CMPA12 of this invention), and D19. These 14 strains underwent secondary screening, and their phosphorus solubilization performance remained stable. The phosphorus solubilization test results are as follows: Figure 1 As shown, strains A12, B12, and P8F9 exhibit the largest clear zones, indicating their strongest phosphorus-solubilizing ability. Additionally, strains A18, B19, D19, and D7 also show relatively clear phosphorus-solubilizing zones. Therefore, it can be concluded that all 14 bacteria possess phosphorus-solubilizing capabilities, dissolving insoluble phosphorus in the soil and promoting plant absorption and utilization of phosphorus, thereby enhancing plant growth.

[0042] The isolated and purified bacteria were screened using PVK inorganic phosphorus medium. Fourteen strains exhibited a clear zone around their colonies. These strains were numbered D7, P8F9, A13, B20, B19, A3, B12, A18 (CMA18 of this invention), D20, P28H4, A16, B11, A12 (CMPA12 of this invention), and D19. These 14 strains underwent secondary screening, and their phosphorus solubilization performance remained stable. The phosphorus solubilization test results are as follows: Figure 1 As shown, strains A12, B12, and P8F9 exhibit the largest clear zones, indicating their strongest phosphorus-solubilizing ability. Additionally, strains A18, B19, D19, and D7 also show relatively clear phosphorus-solubilizing zones. Therefore, it can be concluded that all 14 bacteria possess phosphorus-solubilizing capabilities, dissolving insoluble phosphorus in the soil and promoting plant absorption and utilization of phosphorus, thereby enhancing plant growth.

[0043] Example 3 Preparation of Salkowski colorimetric solution: Mix 50 mL of 35% HClO4 with 1 mL of 0.5 mol / L FeCl3, store in a brown bottle, and prepare fresh each time.

[0044] The isolated test strain was inoculated into LB liquid medium (same as in Example 2) and cultured overnight on a shaker. 200 μL of the bacterial culture was then inoculated into 4 mL of LB liquid medium (containing 1% L-tryptophan at 200 mg / L). A blank LB medium was used as a control. Each treatment was repeated three times. The culture was carried out at 28°C and 180 rpm for 4 days. After the culture was completed, the bacterial culture was centrifuged, and 1 mL of the supernatant was collected. 1 mL of Salkowski colorimetric reagent was added, and the mixture was allowed to stand in the dark for 30 minutes before observing the color change. If the color turned red, the strain was considered to have the ability to produce indoleacetic acid; the darker the color, the stronger the indoleacetic acid production ability.

[0045] The IAA production capacity of each strain was determined by the Salkowski colorimetric solution reaction. Figure 2 As shown in the figure, the results showed that the supernatant of strain A18 (Streptomyces CMA18 of this invention) turned light pink after being left to stand in the dark for 30 minutes, indicating that the strain has the ability to produce IAA.

[0046] Example 4: Antagonistic effect of CMA18 on CCR and verification of its endogeneity Streptomyces from the Strawberry Root Microbiota Resource Bank were compared with Anthracnose (Sinocyclohexane) separately. Colletotrichum siamense Plate confrontation experiments were conducted, and the disease resistance of each strain to CCR was tested on sterile / infected strawberry seedlings.

[0047] The results are as follows Figure 3 As shown, strain CMA18 did not exhibit antagonistic effects against *Anthracnose sicca* in the plate confrontation experiment, nor did it show resistance when inoculated onto sterile strawberry seedlings. However, it showed strong resistance to CCR caused by *Anthracnose sicca* when inoculated onto infected strawberry seedlings. One week after inoculating sterile strawberry seedlings with CMA18, root, stem, and leaf tissues were collected, disinfected, and endophytic bacteria were isolated and cultured. *Streptomyces* strains were isolated from both root and stem tissues, but not from leaf tissues, indicating that CMA18 is an endophytic fungus of *Streptomyces simonii* that can colonize the roots and stems.

[0048] Example 5: Effects of CMA18 on the root microbial community structure of Strawberry (Strawberry 'Zhangji') Virus-free strawberry seedlings were cultivated in unsterilized seedling substrate, and the changes in root microbial community structure before and after CMA18 inoculation were compared. The results showed that the abundance of *Pseudomonas* spp. in the strawberry root system significantly increased after CMA18 inoculation. *Pseudomonas* spp. were directionally isolated, and the isolated strains were subjected to confrontation culture experiments with *Anthracis sinensis*, resulting in a *Pseudomonas* strain CMPA12 that showed significant antagonism against *Anthracis sinensis*. Figure 4 As can be seen, strain CMPA12 exhibited antagonistic effects against Siamese anthrax bacteria in the plate confrontation experiment.

[0049] Example 6: Effects of CMA18 and CMPA12 on CCR Incidence Index The effects of CMA18 and CMPA12 on the CCR disease index were shown in Table 1, with virus-free strawberry seedlings planted in a sterile cultivation system and an unsterilized seedling substrate, respectively.

[0050] Table 1. Control efficacy of CMA18 and CMPA12 against CCR in strawberry seedlings.

[0051] Example 7: Effect of CMA18 on CMPA12 in strawberry root colonization level Based on a sterile substrate cultivation system using virus-free strawberry seedlings, two treatments were established: one with CMA18 and the other with CMPA12 mixed inoculated into strawberry roots, and the other with CMPA12 inoculated alone. Using qPCR with Pseudomonas spp.-specific primers, the colonization level of CMPA12 in strawberry roots was detected. The results showed that CMA18 increased the colonization level of CMPA12 in strawberry roots by 10%. 2 CFU / Kg.

[0052] Example 8 (1) Culture of Streptomyces CMA18: Streptomyces CMA18 was inoculated into TSA liquid medium and cultured at 28℃ and 180 r / min for 80 h to obtain Streptomyces CMA18 bacterial suspension with a viable count of 10. 8 CFU / mL; (2) Culture of Pseudomonas CMPA12: Pseudomonas CMPA12 was inoculated into LB liquid medium and cultured at 28℃ and 180 r / min for 36 h to obtain Pseudomonas CMPA12 bacterial suspension with a viable count of 10. 8 CFU / mL; (3) Preparation of microbial composition: The above Streptomyces CMA18 bacterial solution and Pseudomonas CMPA12 bacterial solution are mixed at a live bacterial ratio of 1:3 to obtain the biocontrol agent.

[0053] Example 9: Efficacy test of microbial composition in controlling strawberry anthracnose stem rot Strawberry seedlings of uniform growth were selected and divided into 4 groups of 30 seedlings each. The following treatments were then performed on each group: Control group: No microorganisms were applied.

[0054] CMA18 group alone: ​​Streptomyces CMA18 bacterial suspension was applied, with 10 live bacteria per plant. 7 CFU.

[0055] CMPA12 group alone: ​​Pseudomonas CMPA12 bacterial suspension was applied, with 10 live bacteria per strain. 7 CFU.

[0056] Experimental group: The microbial composition prepared in Example 8 was applied, with a total viable count of 10 per strain. 7 CFU.

[0057] All treatments were applied via root drenching, with the first application during the strawberry seedling stage, followed by applications every 18 days for a total of two applications. After inoculation with *Anthracnose sicca*, the incidence of strawberry anthracnose stem rot in each group was observed, and the disease inhibition rate was calculated.

[0058] The results showed that the disease incidence index was higher in the control group; the CMA18 group alone had no significant control effect on strawberry anthracnose stem rot; the disease incidence index inhibition rate of the CMPA12 group alone was 32.5%; and the disease incidence index inhibition rate of the experimental group was 70%, which was significantly higher than that of the CMPA12 group alone, indicating that the microbial composition of the present invention has a good control effect.

[0059] Example 9: Comparison of the control effects of microbial compositions with different proportions Microbial compositions containing Streptomyces CMA18 and Pseudomonas CMPA12 in live bacterial ratios of 1:1, 1:3, and 1:5 were prepared according to the method in Example 8. Strawberry seedlings with uniform growth and infectivity were selected, with 30 seedlings per group. Each seedling was treated with a total live bacterial count of 10^6 bacteria via root irrigation. 7 CFU was administered twice consecutively, and the morbidity index inhibition rate was calculated after inoculation with Siamese anthrax bacteria.

[0060] The results showed that the disease index inhibition rate was 65.2% for the composition with a live bacteria ratio of 1:1, 70.0% for the composition with a live bacteria ratio of 1:3, and 68.3% for the composition with a live bacteria ratio of 1:5. Therefore, the control effect was best when the live bacteria ratio of Streptomyces CMA18 to Pseudomonas CMPA12 was 1:3.

[0061] The above description is merely a preferred embodiment of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A biocontrol agent for controlling strawberry anthracnose stem rot, characterized in that, This includes *Streptomyces* CMA18 and *Pseudomonas* CMPA12; *Streptomyces* CMA18 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 35370, deposited on July 24, 2025, and classified as *Streptomyces*. Streptomyces sp. The *Pseudomonas* CMPA12 strain is deposited at the China General Microbiological Culture Collection Center (CGMCC), accession number CGMCC No. 35863, on September 11, 2025, and is classified as follows: Pseudomonas sp. The ratio of viable Streptomyces CMA18 to Pseudomonas CMPA12 is 1:1 to 1:

5.

2. A method for preparing the biocontrol agent as described in claim 1, characterized in that, Includes the following steps: (1) Streptomyces CMA18 was cultured in ISP2 medium to obtain CMA18 bacterial suspension; (2) Pseudomonas CMPA12 was cultured in LB medium to obtain CMPA12 bacterial suspension; (3) Mix CMA18 bacterial solution and CMPA12 bacterial solution in proportion to obtain the final product.

3. The preparation method according to claim 2, characterized in that, In step (1), the mass ratio of Streptomyces CMA18 and ISP2 culture medium is 1:50; the composition of ISP2 culture medium is: 4g yeast extract, 10g malt extract, 4g glucose, 20g agar, 1000mL water, pH 7.2; the culture is carried out at 28℃ and 180r / min for 72-96h.

4. The preparation method according to claim 2, characterized in that, In step (2), the mass ratio of the Pseudomonas CMPA12 and LB medium is 1:50; the composition of the LB medium is: 10g tryptone, 5g yeast extract, 10g sodium chloride, 1000mL water, pH 7.0; the culture is carried out at 28℃ and 180r / min for 24-48h.

5. The application of the biocontrol agent as described in claim 1 in the control of strawberry anthracnose stem rot.

6. The application according to claim 5, characterized in that, The biocontrol agent was applied by root drenching; the concentration was 10^10 *Streptomyces CMA18* and *Pseudomonas CMPA12* per strawberry plant. 6 -10 8 CFU.

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