A biocontrol agent for preventing and controlling strawberry anthracnose stem and crown blight, a preparation method and application thereof

Abstract

The present application belongs to the field of agricultural microorganisms, and particularly relates to a biocontrol agent for preventing and controlling strawberry anthracnose stem and crown blight, a preparation method and application thereof. The biocontrol agent comprises Streptomyces CMA18 and Pseudomonas CMPA12; the Streptomyces CMA18 is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC No. 35370, a preservation date of July 24, 2025, and a classification name of Streptomyces Streptomyces sp. ; the Pseudomonas CMPA12 is preserved in the China General Microbiological Culture Collection Center, has a preservation number of CGMCC No. 35863, a preservation date of September 11, 2025, and a classification name of Pseudomonas sp. The biocontrol agent can effectively improve the prevention and control effect on strawberry anthracnose stem and crown blight.

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:

[0008] 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..

[0009] Preferably, the ratio of the number of viable Streptomyces CMA18 to Pseudomonas CMPA12 is 1:1-1:5.

[0010] The application further provides a preparation method of the biocontrol agent, comprising the following steps:

[0011] (1) culturing Streptomyces CMA18 by using an ISP2 medium to obtain a CMA18 bacterial solution;

[0012] (2) culturing Pseudomonas CMPA12 by using an LB medium to obtain a CMPA12 bacterial solution;

[0013] (3) mixing the CMA18 bacterial solution and the CMPA12 bacterial solution according to a proportion, and the biocontrol agent is obtained.

[0014] Preferably, in step (1), the mass ratio of the Streptomyces CMA18 to the ISP2 medium is 1:50; the ISP2 medium comprises 4 g of yeast extract, 10 g of malt extract, 4 g of glucose, 20 g of agar, 1000 mL of water, and has a pH of 7.2; and the culturing is performed at 28°C and 180 r / min for 72-96 h.

[0015] Preferably, in step (2), the mass ratio of the Pseudomonas CMPA12 to the LB medium is 1:50; the LB medium comprises 10 g of tryptone, 5 g of yeast extract, 10 g of sodium chloride, 1000 mL of water, and has a pH of 7.0; and the culturing is performed at 28°C and 180 r / min for 24-48 h.

[0016] Another object of the application provides the application of the biocontrol agent in preventing and controlling strawberry anthracnose stem and crown blight.

[0017] Preferably, the application mode of the biocontrol agent is root irrigation; and the application concentration is that the total number of viable Streptomyces CMA18 and Pseudomonas CMPA12 applied to each strawberry is 10 6 -10 8 CFU.

[0018] The specific application method of the biocontrol agent provided by the application is that the agent is applied to the strawberry root system. The first application is performed within 1 week after the strawberry seedling stage or transplanting, and then the application is performed every 15-20 days, and the application is continuously performed for 2-3 times, so that the occurrence of strawberry anthracnose stem and crown blight can be effectively prevented and controlled.

[0019] In the biocontrol agent provided by the application, the Streptomyces CMA18 is a strawberry endophytic Streptomyces (Streptomyces sp.) CMA18. Streptomyces sp.), the strain screened by the application can be separated from the strawberry root system, colonize in the strawberry body, and regulate the secretion of the strawberry root system; the Pseudomonas CMPA12 is Pseudomonas asiatica Pseudomonas sp. ), the strain screened by the application has a significant inhibitory effect on Colletotrichum siamense.

[0020] The biocontrol agent provided by the application controls the viable bacterial quantity of the Streptomyces CMA18 and the Pseudomonas CMPA12 in a certain proportion range, and the synergistic effect of the two is better, so that the prevention and control effect on the strawberry anthracnose stem and base rot can be more effectively improved.

[0021] Compared with the prior art, the application has the following beneficial effects:

[0022] (1) The microbial agent composition provided by the application utilizes the synergistic effect of the Streptomyces CMA18 and the Pseudomonas CMPA12, the Streptomyces CMA18 can regulate the secretion of the strawberry root system and promote the colonization of the Pseudomonas CMPA12 in the strawberry root system, so that the prevention and control effect on the strawberry anthracnose stem and base rot can be significantly improved. Experimental data show that the composition can inhibit the incidence index of the strawberry anthracnose stem and base rot by 67.3% under sterile conditions, and by 70.1% under the condition of having bacteria, which is much higher than the effect of using the Pseudomonas CMPA12 alone (32.6% under sterile conditions and 30.1% under the condition of having bacteria).

[0023] (2) The microbial composition of the application utilizes the synergistic effect between microorganisms to prevent and 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.

[0024] (3) The microbial composition of the application has a simple preparation method, is convenient to use, has low cost, is easy to popularize and apply, and has important significance for promoting the healthy and sustainable development of the strawberry industry.

[0025] Preservation information 1

[0026] Strain name: CMA18,

[0027] Preservation time: July 24, 2025,

[0028] Preservation unit: China General Microbiological Culture Collection Center,

[0029] Preservation number: CGMCC No. 35370,

[0030] Address of the preservation unit: No. 3, Yuanmingyuan West Road, Beijing City, Chaoyang District, China Institute of Microbiology, Chinese Academy of Sciences,

[0031] Postal code: 100101,

[0032] Classification name: Streptomyces sp. Streptomyces sp. .

[0033] Deposit information 2

[0034] Strain name: CMPA12,

[0035] Date of deposit: September 11, 2025,

[0036] Deposit unit: China General Microbiological Culture Collection Center,

[0037] Deposit number: CGMCC No. 35863,

[0038] Address of the deposit unit: No. 3, Institute of Microbiology, Chinese Academy of Sciences, Beijing City, Chaoyang District, Beichen West Road No. 1,

[0039] Postal code: 100101,

[0040] Classification name: Pseudomonas sp. . BRIEF DESCRIPTION OF DRAWINGS

[0041] Figure 1 Strains with phosphorus solubilizing activity in strawberry rhizosphere bacteria;

[0042] Figure 2 Strain IAA production ability comparison chart;

[0043] Figure 3 CMA18 on strawberry seedlings anthracnose antagonistic effect chart; from left to right are: blank control, CMA18 group, Thai anthracnose treatment group;

[0044] Figure 4 CMPA12 and Thai anthracnose confrontation culture chart. DETAILED DESCRIPTION

[0045] The present application will be further described in detail below with specific examples.

[0046] Example 1

[0047] Isolation and identification of CMA18 and CMPA12: Akihime strawberry root samples were taken from Lanling County, Linyi City, and the collection time was June 2022. Random selection was used to collect strawberry root samples, three varieties of strawberry plants were dug from the ground, the large pieces of soil gathered around the strawberry roots were shaken off, and then the whole strawberry was collected into a sterile sealed bag with the corresponding information, and then treated.

[0048] The root system is cut into small pieces with scissors after alcohol disinfection, and the root system is loaded into a centrifuge tube. Add 30 mL of sterilized 1x PBS buffer, place it flat on the shaker, and rinse it 3 times at 180 r / min and 28°C for 20 min each time. Finally, rinse the plant roots with sterile filter paper and place them in a 50 mL centrifuge tube. Set 6 replicates for each sample. Use sterilized scissors to cut the strawberry root sample washed with 1x PBS buffer into 2 mm pieces and mix well. Weigh 0.02 g of strawberry root pieces and add them to a sterile centrifuge tube. Add 200 μL of sterilized 10 mM MgCl2 solution to the centrifuge tube. Grind the root pieces to a homogenate state with a sterile grinding rod. Then transfer the ground 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. Mix the diluent evenly in the clean bench, then pour it into a square dish. Add 160 μL of the mixed diluent to each well of the cell culture plate, and do not add diluent to well H12 as a negative control.

[0049] Stack the 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, and select the cell culture plates with 30% turbidity for subsequent experiments. From the selected 96-well cell culture plates, use a syringe to remove 10 μL of liquid from each well and transfer it to a 96-well PCR plate. Label the serial number and store it in a -20°C freezer for subsequent bacterial identification. At the same time, add 140 μL of sterilized 80% to each well of the cell culture plate. Place the cell culture plate in a -8°C freezer for later use.

[0050] Centrifuge the previous 10 μL 96-well PCR plate 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 about 12, 121°C, 15 min sterilization) to each well using a pipette. Mix well by blowing and seal the PCR plate again. Centrifuge and then place it in a PCR instrument for high-temperature lysis in an alkaline environment (95°C for 30 min).

[0051] After cooling, centrifuge the PCR plate. After centrifugation, add 16.6 μL of neutralization buffer 2 (neutralization buffer 2: 25 mM Tris-HCl, adjust pH to about 7.5, 121°C, 15 min sterilization) to each well of the PCR plate in the clean bench. Mix well by blowing and centrifuge the liquid after mixing to serve as the DNA template for the next PCR reaction for subsequent PCR experiments.

[0052] The first round of PCR uses unlabelled primers:

[0053] 799 F (SEQ ID NO. 1): AACMGGATTAGATACCCKG;

[0054] 1193 R (SEQ ID NO. 2): ACGTCATCCCCACCTTCC.

[0055] The 16S sequence is amplified using the above primers.

[0056] The first round of PCR reaction system is as follows:

[0057] ;

[0058] The first round of PCR reaction program is as follows: reaction program: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 30 cycles; final extension at 72°C for 5 min.

[0059] After the first round of PCR reaction, the PCR product is detected to ensure that the amplification is correct.

[0060] The product obtained in the first round of PCR is diluted with nuclease-free water, and the dilution is 40 times, which is used as the second amplification template.

[0061] The second round of PCR reaction system is as follows:

[0062]

[0063] The second round of PCR reaction program is as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 1 min, 25 cycles; final extension at 72°C for 5 min. After amplification, 5 μL of positive and negative controls are taken and mixed with DNA loading buffer, respectively, and electrophoresis detection shows that the positive control produces a band of about 500 bp, and the negative control does not produce a band, which indicates that the second amplification is qualified.

[0064] All PCR products of each PCR plate in the second round are mixed together, 40 μL of PCR mixed product is taken from the centrifugal tube and mixed with 12 μL of DNA loading buffer, and 1.0% agarose gel is prepared. After electrophoresis of 2000 bp DNA Marker and sample at a voltage of 90V for 50 min, the amplified fragments near 500 bp are cut and treated, and the gel recovery kit is used for recovery. The accurate concentration of DNA is determined by PicoGreen fluorescent dye, and the recovered product is purified by magnetic beads. The purified product is sent for sequencing by Illumina.

[0065] The sequence of CMA18 strain (SEQ ID NO. 3) is:

[0066]

[0067] The sequence of the CMPA12 strain (SEQ ID NO. 4) is:

[0068]

[0069] Bioinformatics analysis was mainly performed using the bioinformatics analysis pipeline Culturome developed by Liu Yongxin et al. (http: / / bailab.genetics.ac.cn / culturome) and the R script written by the present inventors. The added plate and well tags were removed from the original sequence to obtain the pure amplified sequence. VSEARCH was used for de-duplication, USEARCH was used for de-noising to identify the amplified sequence variant (ASV), and species annotation was completed. Finally, the sequence and species of the bacteria grown in the 96-well plate were determined. The obtained ASV table will contain the positions of the five best candidate wells of a certain genus. According to the strain information provided by the ASV table, the positions in the 96-well cell culture plate, and the purity of the wells, the required ASV list was selected by using a sterile gun head to dip the liquid in the wells, and 2-3 wells were activated on TSA plates. The plates were cultured in an inverted 28 incubator for 3-4 days. After single colonies grew, multiple purifications were performed, and finally single colonies were picked and transferred to a shaking tube containing 3 mL of TSB medium for overnight culture in a shaker. After the bacterial solution became turbid, 750 μL of bacterial solution and 750 μL of 50% glycerol were aspirated for freezing preservation. Then, the strains were subjected to functional identification of growth promotion and disease resistance.

[0070] Example 2 Screening of Phosphorus-solubilizing Strains

[0071] The grown single colonies were inoculated into LB liquid medium and cultured in a shaker until OD 600 =0.8. 3 μL of bacterial solution was aspirated by a pipette and spotted in the center of PVK inorganic phosphorus medium, which was then cultured in an inverted 28°C incubator for 7 days. The changes around the bacteria in the culture dish were observed. The appearance of a transparent circle around the bacteria indicated that the strain had phosphorus-solubilizing ability. The size of the transparent circle could be used as an indicator of the strength of phosphorus-solubilizing ability. The larger the transparent circle, the stronger the phosphorus-solubilizing ability of the strain. The strain was repeatedly tested for 3 times.

[0072] LB liquid medium: 10 g of proteose peptone, 5 g of yeast extract, 5 g of NaCl, dissolved in 1000 mL of deionized water, adjusted to a pH of about 7.2, and autoclaved at 121°C for 20 min; LB solid medium: 15 g of agar powder was added per liter of LB liquid medium.

[0073] PVK inorganic phosphorus solid medium: 10 g of glucose, 0.5 g of (NH4)2SO4, 0.3 g of NaCl, 0.3 g of MgSO4·7H2O, 0.03 g of MnSO4·H2O, 0.03 g of FeSO4·7H2O, 0.3 g of KCl, 5 g of Ca3(PO4)2, 15 g of agar, 1000 mL of deionized water, and the pH was adjusted to about 7.4. Autoclaved at 121°C for 20 min.

[0074] The 14 strains with transparent circles around the colonies were screened from the PVK inorganic phosphorus medium, and the strain numbers were D7, P8F9, A13, B20, B19, A3, B12, A18 (CMA18 of the application), D20, P28H4, A16, B11, A12 (CMPA12 of the application), and D19. The 14 strains were re-screened, and the phosphorus solubilizing effect was stable. The phosphorus solubilizing determination results are shown in Table 1. Figure 1 As shown in Table 1, the transparent circles of A12, B12, and P8F9 were the largest, indicating that the phosphorus solubilizing ability of the strains was the strongest. In addition, the phosphorus solubilizing circles of A18, B19, D19, and D7 were also relatively obvious. Therefore, it can be said that the 14 strains all have the ability to solubilize phosphorus in the soil, promote the absorption and utilization of phosphorus elements in the soil by plants, and thus promote the growth of plants.

[0075] The 14 strains with transparent circles around the colonies were screened from the PVK inorganic phosphorus medium, and the strain numbers were D7, P8F9, A13, B20, B19, A3, B12, A18 (CMA18 of the application), D20, P28H4, A16, B11, A12 (CMPA12 of the application), and D19. The 14 strains were re-screened, and the phosphorus solubilizing effect was stable. The phosphorus solubilizing determination results are shown in Table 1. Figure 1 As shown in Table 1, the transparent circles of A12, B12, and P8F9 were the largest, indicating that the phosphorus solubilizing ability of the strains was the strongest. In addition, the phosphorus solubilizing circles of A18, B19, D19, and D7 were also relatively obvious. Therefore, it can be said that the 14 strains all have the ability to solubilize phosphorus in the soil, promote the absorption and utilization of phosphorus elements in the soil by plants, and thus promote the growth of plants.

[0076] Example 3

[0077] Preparation of Salkowski colorimetric solution: 35% concentration of HClO450 mL and 0.5 mol / L of FeCl31 mL were uniformly mixed and prepared, and were placed in a brown bottle and prepared immediately before use.

[0078] The isolated test strains were inoculated in LB liquid medium (same as in Example 2), 200 μL of the bacterial solution was taken after overnight culture on a shaker and inoculated into 4 mL (1%, 200 mg / L of L-tryptophan was added) LB liquid medium, blank LB medium was used as a control, each treatment was repeated 3 times, and the culture was carried out on a shaker at 28°C, 180 rpm for 4 days. After the culture, the bacterial solution was centrifuged, 1 mL of the supernatant was taken, 1 mL of Salkowski colorimetric reagent was added, and the color change was observed after 30 min of dark incubation. If the color changed to red, it was considered that the strain had the ability to produce indole acetic acid, and the darker the color, the stronger the ability of the strain to produce indole acetic acid.

[0079] The ability of each strain to produce IAA was determined by Salkowski colorimetric reaction, and the results are shown in Table 1. Figure 2 As shown in Table 1, the supernatant of strain A18 (Streptomyces CMA18 of the present application) also changed to light pink after 30 min of dark incubation, indicating that the strain had the ability to produce IAA.

[0080] Example 4 Antagonistic effect of CMA18 on CCR and verification of its endophytic nature

[0081] The Streptomyces contained in the strawberry root microbial resource library of Zhangjicai were subjected to plate confrontation experiments with Colletotrichum gloeosporioides (P. hennings) and the disease resistance effect of each strain on CCR was detected on strawberry aseptic / inoculated seedlings. Colletotrichum siamense

[0082] The results are shown in Table 2. Figure 3 As shown in Table 2, strain CMA18 did not show antagonistic effect on C. gloeosporioides in the plate confrontation experiment, and did not show disease resistance effect when inoculated on strawberry aseptic seedlings, but showed strong disease resistance effect on C. gloeosporioides induced CCR when inoculated on strawberry inoculated seedlings. CMA18 was inoculated on aseptic strawberry seedlings, and after one week, strawberry root, stem and leaf tissues were taken, surface sterilized and subjected to endophyte isolation and culture. Streptomyces strains were isolated from the root and stem tissues, but not from the leaf tissue, indicating that CMA18 was an endophyte of Zhangjicai strawberry and could colonize in the root and stem.

[0083] Example 5 Effect of CMA18 on the community structure of the root microbial community of Zhangjicai strawberry

[0084] Strawberry virus-free seedlings were cultivated in non-sterilized seedling substrate, and the change in the community structure of the root microbial community before and after CMA18 inoculation was compared. The results showed that after CMA18 inoculation, the abundance of Pseudomonas in the strawberry root significantly increased. Pseudomonas was subjected to directional isolation, and the isolated strains were subjected to confrontation culture experiment with C. gloeosporioides, and a Pseudomonas strain CMPA12 with significant antagonistic effect on C. gloeosporioides was obtained. Figure 4 ​As can be seen from the above table, the strain CMPA12 showed antagonistic effect on C. siamense in the plate confrontation experiment.

[0085] Example 6 Effect of CMA18 and CMPA12 on CCR disease index

[0086] The strawberry virus-free seedlings were respectively planted in the sterile cultivation system and the non-sterilized seedling substrate, and the effect of CMA18 and CMPA12 on the CCR disease index was shown in Table 1.

[0087] Table 1 Control effect of CMA18 and CMPA12 on strawberry seedling CCR

[0088]

[0089] Example 7 Effect of CMA18 on colonization level of CMPA12 in strawberry root system

[0090] Based on the sterile substrate cultivation system of strawberry virus-free seedlings, CMA18 and CMPA12 were mixed to inoculate the strawberry root system, and CMPA12 was inoculated alone as two treatments. The colonization level of CMPA12 in the strawberry root system was detected by qPCR technology based on Pseudomonas specific primers. The results showed that CMA18 increased the colonization level of CMPA12 in the strawberry root system by 10 2 CFU / Kg.

[0091] Example 8

[0092] (1) Culture of Streptomyces CMA18: Streptomyces CMA18 was inoculated in TSA liquid medium and cultured at 28°C and 180 r / min for 80 h to obtain Streptomyces CMA18 liquid, with a viable cell count of 10 8 CFU / mL;

[0093] (2) Culture of Pseudomonas CMPA12: Pseudomonas CMPA12 was inoculated in LB liquid medium and cultured at 28°C and 180 r / min for 36 h to obtain Pseudomonas CMPA12 liquid, with a viable cell count of 10 8 CFU / mL;

[0094] (3) Preparation of microbial composition: The above Streptomyces CMA18 liquid and Pseudomonas CMPA12 liquid were mixed according to the viable cell count ratio of 1:3 to obtain the biocontrol agent.

[0095] Example 9 Effect test of microbial composition on prevention and control of strawberry anthracnose stem and base rot

[0096] Strawberry sterile seedlings with consistent growth were selected and divided into 4 groups, 30 seedlings in each group, and the following treatments were performed respectively.

[0097] Control group: no microbial is applied.

[0098] CMA18 alone group: Streptomyces CMA18 liquid is applied, and the number of live bacteria applied per plant is 10 7 CFU.

[0099] CMPA12 alone group: Pseudomonas CMPA12 liquid is applied, and the number of live bacteria applied per plant is 10 7 CFU.

[0100] Test group: the microbial composition prepared in Example 8 is applied, and the total number of live bacteria applied per plant is 10 7 CFU.

[0101] All treatments are applied by root irrigation, and the first application is performed at the strawberry seedling stage, and then the application is performed every 18 days, and the application is continuously performed twice. After inoculation of Colletotrichum siamense, the strawberry anthracnose stem base rot disease incidence of each group is observed, and the disease index inhibition rate is calculated.

[0102] The results show that the disease index of the control group is high; the CMA18 alone group has no obvious prevention and control effect on strawberry anthracnose stem base rot; the disease index inhibition rate of the CMPA12 alone group is 32.5%; and the disease index inhibition rate of the test group is 70%, which is significantly higher than that of the CMPA12 alone group, indicating that the microbial composition has good prevention and control effect.

[0103] Example 9: Comparison of prevention and control effects of microbial compositions with different proportions

[0104] The microbial compositions with the live bacteria number ratio of Streptomyces CMA18 to Pseudomonas CMPA12 being 1:1, 1:3 and 1:5 are prepared according to the method of Example 8, consistent strawberry seedlings are selected, 30 plants are selected in each group, the total number of live bacteria applied per plant is 10 7 CFU is applied by root irrigation, and the application is continuously performed twice, and the disease index inhibition rate is calculated after inoculation of Colletotrichum siamense.

[0105] The results show that the disease index inhibition rate of the composition with the live bacteria number ratio of 1:1 is 65.2%; the disease index inhibition rate of the composition with the live bacteria number ratio of 1:3 is 70.0%; and the disease index inhibition rate of the composition with the live bacteria number ratio of 1:5 is 68.3%. Therefore, when the live bacteria number ratio of Streptomyces CMA18 to Pseudomonas CMPA12 is 1:3, the prevention and control effect is best.

[0106] The above only describes the preferred embodiments of the present application, and is not used to limit the protection scope of the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

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), 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. The ratio of viable bacteria of Streptomyces CMA18 to Pseudomonas CMPA12 is 1:1 to 1:

5. The sequence of strain CMA18 is shown in SEQ ID NO.3; The sequence of CMPA12 strain is shown in SEQ ID NO.

4.

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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