Method for targeted screening of biocontrol bacteria and application thereof

Abstract

The application discloses a method for screening biocontrol bacteria, which comprises the following steps: coating a sample suspension suspected to contain biocontrol bacteria on a solid culture medium, inoculating a target pathogenic bacterium for co-culture, and screening biocontrol bacteria which can inhibit the growth of the target pathogenic bacterium. The method overcomes the shortcomings of long experimental period and low efficiency of traditional screening methods, improves the screening speed of agricultural biocontrol bacteria, and can obtain more types of agricultural biocontrol bacteria.

Description

Technical Field

[0001] This invention belongs to the field of biological control of plant diseases, and specifically relates to a method for targeted screening of biocontrol bacteria. Background Technology

[0002] Soil-borne diseases are among the most difficult to control in agricultural production. Cultivated medicinal herbs, due to long planting years, high planting density, and excessive fertilizer application, are prone to frequent soil-borne diseases, leading to significant declines in yield and quality. Soil-borne diseases are particularly severe in major root-type medicinal herbs such as ginseng, American ginseng, Panax notoginseng, Astragalus membranaceus, and Angelica sinensis. These diseases mainly include root rot, rust rot, damping-off, and blight, often involving multiple pathogenic microorganisms. Chemical control is currently the mainstream method, and while it is effective in the short term, long-term application can induce drug resistance in pathogens, resulting in widespread pesticide residue exceeding standards. Biological control techniques can effectively reduce pesticide use, and beneficial microorganisms can improve the soil microecological environment, providing sustained control over diseases. Many fungi, bacteria, and actinomycetes, such as Trichoderma harzianum, Bacillus subtilis, and Bacillus polymyxa, have been registered for use.

[0003] However, the vast majority of discovered biocontrol bacteria remain at the laboratory level, with very few actually put into production application. There are two main reasons for this: First, biocontrol bacteria derived from one crop are not ideally effective against diseases in other crops, especially effective biocontrol bacteria for medicinal herbs are extremely rare. Second, while commonly used soil / plant microbiome methods based on gene sequencing can acquire large amounts of data, obtaining actual biocontrol bacteria still relies on culture medium screening methods, which are too inefficient to obtain a large number of target strains in a short time.

[0004] Traditional screening of agricultural biocontrol bacteria primarily employs the dilution plate method, a process known as the "two-step screening" method. The first step involves evenly spreading a soil suspension of appropriate concentration onto a culture medium plate and incubating it at a specific temperature to obtain single colonies. The second step involves purifying these colonies and then using the plate confrontation method to screen for biocontrol bacteria with antagonistic effects, followed by species identification. While the "two-step screening" method can yield a large number of strains in the first step, it cannot determine their function; the second step is necessary to identify the target bacteria. Therefore, the probability of obtaining biocontrol bacteria using traditional screening methods is very low, and it requires a significant amount of time and resources.

[0005] In recent years, microbiome technology has been widely used in soil microbial community research. Although it can obtain information on antagonistic bacteria, it cannot obtain physical microorganisms. While culturomics-based integrated microbial culture methods can obtain diverse colonies, their functions cannot be determined, and they require specific instruments and equipment, resulting in high costs.

[0006] Therefore, establishing a simple and applicable rapid screening method for biocontrol bacteria suitable for grassroots scientific research and agricultural technology departments can greatly improve screening efficiency and reduce costs. Summary of the Invention

[0007] Based on the shortcomings of existing technologies, this invention provides a targeted and rapid screening method for agricultural biocontrol bacteria. This method can target and screen multiple pathogens of different crops to obtain biocontrol bacteria that have antagonistic effects on the target bacteria. It overcomes the disadvantages of long experimental cycles and low efficiency of traditional screening methods, significantly improves the screening speed, and obtains more types of biocontrol bacteria through the design of different culture media.

[0008] The first aspect of the present invention provides a method for targeted and rapid screening of biocontrol bacteria, the method comprising the following steps: spreading a sample suspension suspected of containing biocontrol bacteria on a solid culture medium and inoculating it with target pathogens for co-culture, thereby targeting and screening for biocontrol bacteria.

[0009] In some embodiments, the target pathogen is a plant pathogenic fungus.

[0010] In some embodiments, the sample is a soil suspension or a plant tissue suspension.

[0011] In some implementations, after spreading the sample suspension, the target pathogen is inoculated into the center of the solid culture medium.

[0012] The incubation time after inoculation with the target pathogen is determined based on the growth rate of the target pathogen. In some embodiments, the co-culture time is 24h-240h. In some specific embodiments, the co-culture time is 24h, 48h, 72h, 96h, 120h, 144h, 168h, 192h, 216h, 240h, or any value between them.

[0013] In some embodiments, the co-cultivation temperature is 20-35°C, preferably 23-28°C. In some specific embodiments, the co-cultivation temperature is 20°C, 23°C, 25°C, 28°C, 30°C, 33°C, 35°C, or any value between them.

[0014] In some embodiments, after the co-culture, if the target pathogen shows a significant inhibition zone or band compared to the control without a coated sample suspension, thereby inhibiting the normal growth of the target pathogen, then candidate biocontrol bacteria that inhibit the growth of the target pathogen are preliminarily screened.

[0015] The targeted screening method employed in this invention shortens the traditional "two-step screening" to "one-step screening." By simultaneously inoculating the target pathogen into the sample suspension during dilution and plating on a solid culture medium, and then observing the co-cultured samples, biocontrol bacteria with antagonistic effects against the target pathogen can be quickly obtained, eliminating the need to culture all microorganisms growing on the plate.

[0016] In some embodiments, the sample suspension is coated onto at least two solid culture media.

[0017] In some embodiments, the at least two solid culture media have different types and / or contents of nutrients.

[0018] In some embodiments, the solid culture medium includes LBE solid culture medium and R2D solid culture medium.

[0019] This invention utilizes a combination of culture media for targeted screening, enabling the acquisition of a relatively diverse range of biocontrol bacteria while saving experimental time. Preferably, this invention employs a combination of LBE solid medium and R2D solid medium, which not only shortens the screening cycle but also allows for the simultaneous acquisition of a wider variety of agricultural biocontrol bacteria.

[0020] In some embodiments, the LBE solid culture medium comprises 5.0-15.0g tryptone, 5.0-10.0g NaCl, 5.0-10.0g yeast extract, 15.0-20.0g agar powder and 1L water. Preferably, the pH of the LBE solid culture medium is 7.0-7.4.

[0021] In some embodiments, the R2D solid culture medium comprises 0.2-0.6 g acid-hydrolyzed casein, 0.3-0.5 g yeast extract, 0.3-0.5 g tryptone, 0.2-0.6 g soluble starch, 0.3-0.5 g K2HPO4, 0.1-0.4 g MgSO4, 0.1-0.4 g sodium pyruvate, 15.0-20.0 g agar powder, and 1 L of water. Preferably, the pH of the R2D solid culture medium is 6.8-7.2.

[0022] In some embodiments, the method further includes purifying the screened candidate biocontrol bacteria.

[0023] In some embodiments, the method further includes verifying the effectiveness of the screened candidate biocontrol bacteria.

[0024] In some implementations, the effect verification is performed using the flat plate confrontation method.

[0025] In some embodiments, the method further includes identifying the genus of the screened biocontrol bacteria.

[0026] In some embodiments, the identification of the fungal genus is performed using a combination of morphological and gene sequencing methods.

[0027] In some embodiments, the purification includes inoculating the biocontrol bacteria into LBE liquid medium for cultivation, and then purifying the bacterial culture obtained by streak plating. In some embodiments, the cultivation temperature is 28-35°C, the time is 6-24 hours, and the rotation speed is 100-300 rpm.

[0028] In some embodiments, the method further includes determining, before inoculating the target pathogen, that the target pathogen is capable of growing in the solid culture medium.

[0029] In some embodiments, the target pathogen includes at least one of Fusarium oxysporum, Fusarium solani, Rhizoctonia solani, Ilyonectria robusta, and Dactyonectria novozelandica.

[0030] In some embodiments, the sample suspension includes soil suspension and / or plant tissue suspension.

[0031] In some embodiments, the sample suspension includes soil suspensions of food crops, cash crops, or traditional Chinese medicinal materials.

[0032] In some embodiments, the medicinal materials are selected from rhizome-type medicinal materials.

[0033] In some embodiments, the Chinese medicinal materials are selected from at least one of ginseng, American ginseng, Panax notoginseng, Astragalus membranaceus, Atractylodes lancea, Angelica sinensis, and Codonopsis pilosula.

[0034] In some embodiments, the food crops include wheat, rice, corn, oats, rye, and barley.

[0035] In some embodiments, the cash crops include vegetable crops (tomatoes, eggplants, peppers, cucumbers, etc.), fiber crops (cotton, hemp, etc.), oil crops (peanuts, rapeseed, sesame, soybeans, etc.), sugar crops (beets, sugarcane, etc.), beverage crops (tea, coffee, etc.), hobby crops (tobacco), medicinal crops (ginseng, Panax notoginseng, Angelica sinensis, etc.), and tropical crops (mangoes, coconuts, bananas, pineapples, etc.).

[0036] In some embodiments, the sample suspension comprises a suspension of ground plant tissue.

[0037] In some embodiments, the sample suspension includes gradient-diluted soil suspensions and / or gradient-diluted plant tissue suspensions.

[0038] A second aspect of the invention provides a biocontrol bacterium obtained according to the method described in the first aspect.

[0039] In some embodiments, the biocontrol bacteria include at least one of fungi, bacteria, and actinomycetes.

[0040] A third aspect of the present invention provides the application of the method described in the first aspect or the biocontrol bacteria described in the second aspect in screening agricultural biocontrol bacteria or controlling soil-borne diseases in agricultural production.

[0041] In some implementations, the soil-borne diseases include root rot, stem base rot, rust rot, damping-off, or blight.

[0042] In some embodiments, the method is used to screen biocontrol bacteria for food crops, cash crops, and traditional Chinese medicinal materials.

[0043] In some embodiments, the biocontrol bacteria include at least one of fungi, bacteria, and actinomycetes.

[0044] This invention replaces the traditional two-step screening method for agricultural biocontrol bacteria with a one-step screening method, and simultaneously obtains more varieties of agricultural biocontrol bacteria by using a combination of culture media. This invention shortens the screening cycle for agricultural biocontrol bacteria, saves time and resources for the screening work, significantly reduces the difficulty of obtaining agricultural biocontrol bacteria, and provides a new method for rapidly obtaining large quantities and diversity of agricultural biocontrol bacteria. Attached Figure Description

[0045] Figure 1 The results of the biocontrol screening for Fusarium oxysporum as the target pathogen in Example 2 are shown. (A) shows the pathogen growth test, (B) shows some of the targeted screening plates, and the yellow circle represents the candidate biocontrol bacteria. (C) shows the rescreening verification of the biocontrol bacteria plate confrontation method.

[0046] Figure 2 The results of the biocontrol screening for Fusarium solanum as the target pathogen in Example 3 are shown. (A) is the pathogen growth test, (B) is a partial target screening plate, and the yellow circle represents the candidate biocontrol bacteria. (C) is the rescreening verification of the biocontrol bacteria plate confrontation method.

[0047] Figure 3 The results of the biocontrol screening for Rhizoctonia solani as the target pathogen in Example 4 are shown. (A) is the pathogen growth test, (B) is a partial target screening plate, and the yellow circle represents the candidate biocontrol bacteria. (C) is the rescreening verification of the biocontrol bacteria plate confrontation method.

[0048] Figure 4 The results of the biocontrol screening for the target pathogen *Cryptospira rubrum* in Example 5 are shown. (A) shows the pathogen growth test, (B) shows some of the targeted screening plates, with candidate biocontrol bacteria represented in the yellow circle, and (C) shows the rescreening verification of the biocontrol bacteria plate confrontation method.

[0049] Figure 5 The results of the biocontrol screening for the target pathogen *Rhizoctonia solani* in Example 6 are shown. (A) shows the pathogen growth test, (B) shows some of the targeted screening plates, with candidate biocontrol bacteria represented in the yellow circle, and (C) shows the rescreening verification of the biocontrol bacteria plate confrontation method.

[0050] Figure 6 The results of targeted screening of endophytic control bacteria in American ginseng root in Example 7 are shown. (A) is the pathogen growth test, (B) is a partial targeted screening plate, and the yellow circle represents the candidate biocontrol bacteria. (C) is the rescreening verification of the biocontrol bacteria plate confrontation method. Detailed Implementation

[0051] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. The specific embodiments described herein are for illustrative purposes only and are not intended to limit the invention in any way. Furthermore, descriptions of well-known structures and techniques are omitted in the following description to avoid unnecessarily obscuring the concepts of this disclosure. Such structures and techniques have also been described in many publications.

[0052] Example 1: Culture medium composition and operation steps for targeted screening of biocontrol bacteria

[0053] The soil samples used in this embodiment were collected from the periroot soil of healthy American ginseng plants in a ginseng base in Wendeng District, Weihai City, Shandong Province, and from the periroot soil of healthy peanut plants in a peanut plantation in Wendeng District, Weihai City, Shandong Province. The target pathogens included *Fusarium solani*, the pathogen causing root rot in American ginseng, and *Rhizoctonia solani*, the pathogen causing root rot in astragalus, both from the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0054] In the initial stage of establishing this method, four culture media—LBE, R2D, TSA, and YEM—were designed, with specific formulations shown in Table 1. Soil suspensions were prepared from the peri-root soil of American ginseng and peanut, respectively, with *Fusarium solani* and *Rhizoctonia solani* as target pathogens. A one-step targeted screening process was used to screen for biocontrol bacteria. The screening results (Table 2) showed that the number of biocontrol bacteria obtained from TSA medium was less than that from LBE medium, and the bacterial species were similar; therefore, TSA medium was discarded. Compared to R2D medium, YEM medium yielded a richer variety of biocontrol bacteria; therefore, YEM medium was also discarded. Finally, the combination of LBE and R2D solid culture media was determined.

[0055] Table 1. Formulation of four culture media

[0056] Agar 18g 20g 15g 20g Yeast extract 5g 0.5g / 0.5g dipotassium hydrogen phosphate / 0.4g / 0.5g Sodium chloride 10g / 5g 0.1g trypsin 15g 0.3g 15g / Acid hydrolyzed casein / 0.4g / / Soluble starch / 0.5g / / Anhydrous magnesium sulfate / 0.1g / / Sodium pyruvate / 0.2g / / Soy papain hydrolysate / / 5g / Mannitol / / / 5g Magnesium sulfate heptahydrate / / / 0.2g

[0057] Table 2. Results of targeted screening of biocontrol bacteria on four solid culture media

[0058]

[0059]

[0060] The "one-step" targeted screening of biocontrol bacteria is carried out according to the following steps:

[0061] (1) Culture test of target bacteria: Before targeted screening, the target pathogens were inoculated into various solid culture media and plates and cultured in the dark at 25°C to observe whether they could grow normally.

[0062] (2) Preparation of soil / plant tissue suspension: Weigh 1.0g of soil sample and add it to 9mL of sterile water. Add 2 sterile glass beads and place in a constant temperature shaking incubator (28℃, 180r / min) for shaking incubation for 30min. Use sterile water to serially dilute the soil / plant tissue suspension to 10. -3 10 -4 10 -5 Three dilution gradients are available.

[0063] (3) Targeted screening of biocontrol bacteria: 50 μL of the above-mentioned soil / plant tissue suspensions at various concentrations were evenly spread onto solid culture medium plates. After spreading, the target pathogen was inoculated in the center of the plate, and the plates were incubated at 25°C for several days. The growth of the target pathogen and other microorganisms in the plates was observed. When the target pathogen is unaffected, it will grow in a circular pattern. This method can quickly eliminate plates that do not contain candidate biocontrol bacteria. When the target pathogen shows irregular growth compared to the control, and then a clear inhibition zone or inhibition band appears, it can be preliminarily determined that there are candidate biocontrol bacteria that inhibit the growth of the target pathogen in this plate.

[0064] (4) Purification of biocontrol bacteria: Select candidate biocontrol bacteria from the targeted screening plate and incubate them in liquid LBE medium (same as LBE solid medium formula, except without agar) for 8 hours (30℃, 200rpm). Purify the obtained candidate biocontrol bacteria using the streak plate method.

[0065] (5) Validation of the efficacy of biocontrol bacteria: The antagonistic effect of candidate biocontrol bacteria obtained from targeted screening was validated using the plate confrontation method. Candidate biocontrol bacteria were incubated in liquid LBE medium with shaking for 8 hours (30℃, 200 rpm), counted, and diluted to 1×10⁻⁶. 7 Prepare a bacterial suspension at CFU / mL. Inoculate the target pathogen in the center of a culture medium plate, add 10 μL of the candidate biocontrol bacterial suspension 2 cm away from the pathogen, and incubate at 25°C to verify its antagonistic effect.

[0066] (6) Identification of biocontrol bacteria: Using the bacterial suspension of biocontrol bacteria as a template, the 16S rRNA gene of the biocontrol bacteria was amplified by PCR using 2×Taq PCR StarMix with Loading Dye (Beijing Kangrun Chengye Biotechnology Co., Ltd.). The universal primers for the 16S rRNA gene (27F: 5'-AGAGTTTGATCMTGGCTCAG-3' (SEQ ID NO:1); 1492R: 5'-ACGGTTACCTTACCTTGTTACGACTT-3' (SEQ ID NO:2)) were synthesized by Beijing Liuhe Huada Gene Technology Co., Ltd. The PCR reaction system was 20 μL: 1.0 μL DNA template, 1.0 μL each of forward and reverse primers (10 μm), 10 μL 2×Taq PCR StarMix, and ddH2O to make up to 20 μL. PCR reaction conditions: 94℃ pre-denaturation for 2 min; 94℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, 32 cycles; final extension at 72℃ for 10 min.

[0067] The PCR products were sequenced, and the sequencing results were spliced ​​together. The obtained sequences were uploaded to the NCBI website for BLAST comparison to find closely related strains. The 16S rRNA sequences of the corresponding strains were downloaded, and a phylogenetic tree was constructed using MEGA 7.0 to obtain the identification results of the strains.

[0068] Example 2: Targeted screening of biocontrol bacteria against Fusarium oxysporum in American ginseng soil

[0069] The soil in this embodiment was collected from healthy American ginseng soil from an American ginseng base in Wendeng District, Weihai City, Shandong Province.

[0070] The target pathogen, *Fusarium oxysporum*, was isolated from a root rot sample of American ginseng by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0071] Biocontrol bacteria were targeted for screening according to the method in Example 1.

[0072] The filtering results are as follows Figure 1 As shown, *Fusarium oxysporum* can grow normally in both LBE and R2D media. Figure 1 A). Targeted screening yielded two candidate biocontrol bacteria ( Figure 1 B), after verification using the plate confrontation method, was determined to have an antagonistic effect. Figure 1 C). Upon identification, J31 was identified as Burkholderia cepacia, and Z47 as Bacillus velezensis (Table 3).

[0073] Table 3. Biocontrol bacteria of Fusarium oxysporum obtained from targeted screening

[0074]

[0075] Example 3: Targeted screening of biocontrol bacteria against Fusarium oxysporum in American ginseng soil

[0076] The soil in this embodiment was collected from healthy American ginseng soil from an American ginseng base in Wendeng District, Weihai City, Shandong Province.

[0077] The target pathogen, *Fusarium solani*, was isolated from a root rot sample of American ginseng by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0078] Biocontrol bacteria were targeted for screening according to the method in Example 1.

[0079] The filtering results are as follows Figure 2 As shown, *Fusarium solani* can grow normally in both LBE and R2D media. Figure 2 A). Targeted screening yielded 4 candidate biocontrol bacteria ( Figure 2 B), after verification using the plate confrontation method, was determined to have an antagonistic effect. Figure 2 C). Upon identification, J41 and J42 were both Bacillus velezensis, and B402 and B504 were Serratia fonticola and Pseudomonas sp., respectively (Table 4).

[0080] Table 4. Biocontrol bacteria of Fusarium solani obtained from targeted screening

[0081]

[0082] Example 4: Targeted screening of biocontrol bacteria against Rhizoctonia solani in peanut soil

[0083] The soil in this embodiment was collected from healthy peanut soil from peanut-growing areas in Wendeng District, Weihai City, Shandong Province.

[0084] The target pathogen, Rhizoctonia solani, was isolated from Astragalus membranaceus root rot samples by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0085] Biocontrol bacteria were targeted for screening according to the method in Example 1.

[0086] Some targeted screening results are as follows Figure 3 As shown, Rhizoctonia solani can grow normally in LBE and R2D media. Figure 3 A). Targeted screening yielded 23 candidate biocontrol bacteria, and after verification using the plate confrontation method, 19 of them were determined to have antagonistic effects (partial results are shown in...). Figure 3 B and Figure 3 C). Upon identification, 11 strains were identified as belonging to the genus Burkholderia, including 4 strains of Burkholderia cepacia, 4 strains of Burkholderia cenocepacia, 1 strain of Burkholderia ambifaria, 1 strain of Burkholderia metallica, and 1 strain of Burkholderia sp.; 8 strains were identified as belonging to the genus Pseudomonas, including 6 strains of Pseudomonas koreensis and 2 strains of Pseudomonas fluorescens (Table 5).

[0087] Table 5. Biocontrol bacteria of Rhizoctonia solani obtained from targeted screening

[0088]

[0089] Example 5: Targeted screening of biocontrol bacteria against *Scutellaria barbata* in American ginseng soil

[0090] The soil in this embodiment was collected from healthy American ginseng soil from an American ginseng base in Wendeng District, Weihai City, Shandong Province.

[0091] The target pathogen, *Ilyonectria robusta*, was isolated from a root rot sample of American ginseng by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0092] Biocontrol bacteria were targeted for screening according to the method in Example 1.

[0093] Some targeted screening results are as follows Figure 4 As shown, *Streptomyces rubrum* can grow normally in both LBE and R2D media. Figure 4 A). Targeted screening yielded 9 candidate biocontrol bacteria, and after verification using the plate confrontation method, 6 of them were determined to have antagonistic effects (partial results are shown in...). Figure 4 B and Figure 4 C). Upon identification, 4 strains were classified as Bacillus, including 2 strains of Bacillus subtilis, 1 strain of Bacillus stercoris, and 1 strain of Bacillus toyonensis; 2 strains were classified as Pseudomonas, including 1 strain of Pseudomonas skribbensis and 1 strain of Pseudomonas rhodesiae (Table 6).

[0094] Table 6. Biocontrol bacteria of robust *Rhizoctonia solani* obtained from targeted screening

[0095]

[0096] Example 6: Targeted screening of biocontrol bacteria against *Rhizoctonia solani* in American ginseng soil

[0097] The soil in this embodiment was collected from healthy American ginseng soil from an American ginseng base in Wendeng District, Weihai City, Shandong Province.

[0098] The target pathogen, *Dactyonectria novozelandica*, was isolated from a root rot sample of American ginseng by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0099] Biocontrol bacteria were targeted for screening according to the method in Example 1.

[0100] Some targeted screening results are as follows Figure 5 As shown, *Flammulina davidii* can grow normally in both LBE and R2D media. Figure 5 A). Targeted screening yielded three candidate biocontrol bacteria, which were confirmed to have antagonistic effects after verification using the plate confrontation method (partial results are shown in...). Figure 5 B and Figure 5 C). Upon identification, one strain was identified as Bacillus subtilis, and two strains were identified as Pseudomonas sp. (Table 7).

[0101] Table 7. Biocontrol bacteria of *Rhizoctonia solani* obtained from targeted screening

[0102]

[0103] Example 7: Targeted screening of biocontrol bacteria against *Scutellaria barbata* from American ginseng roots

[0104] The plant samples in this embodiment were collected from healthy American ginseng roots from an American ginseng base in Wendeng District, Weihai City, Shandong Province.

[0105] The target pathogen, *Ilyonectria robusta*, was isolated from a root rot sample of American ginseng by the Institute of Medicinal Plant Development, Chinese Academy of Medical Sciences.

[0106] Biocontrol bacteria targeted screening was performed according to the method in Example 1, wherein the soil sample in step (2) was replaced with a ground plant sample.

[0107] Some targeted screening results are as follows Figure 6 As shown, *Streptomyces rubrum* can grow normally in both LBE and R2D media. Figure 6 A). Targeted screening yielded three candidate biocontrol bacteria, which were confirmed to have antagonistic effects after verification using the plate confrontation method. Figure 6 B and Figure 6 C). Upon identification, one strain was identified as *Pseudomonas* sp., one strain as *Pseudomonas kribbensis*, and one strain as *Bacillus atrophaeus* (Table 8).

[0108] Table 8. Biocontrol bacteria of *Trichoderma viride* obtained from targeted screening in *Panax quinquefolius* roots

[0109]

[0110] The above embodiments demonstrate that this invention, by simultaneously inoculating the target pathogen onto a culture medium plate after soil dilution, and then observing the co-culture, can rapidly obtain biocontrol bacteria with antagonistic effects against the target pathogen, eliminating the need to culture all microorganisms individually. Furthermore, this invention designs a combination of two culture media for targeted screening, which can save experimental time while obtaining a relatively rich variety of biocontrol bacteria.

[0111] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. A method for targeted screening of biocontrol bacteria, the method comprising: Sample suspensions suspected of containing biocontrol bacteria were spread on at least two solid culture media, and target pathogens were inoculated into the center of the solid culture media for co-culture. Biocontrol bacteria that inhibit the growth of the target pathogens were screened. The solid culture media include LBE solid culture medium and R2D solid culture medium. The LBE solid culture medium consists of 15 g tryptone, 10 g NaCl, 5 g yeast extract, 15.0-20.0 g agar powder, and 1 L water, and the pH of the LBE solid culture medium is 7.0-7.

4. The R2D solid culture medium consists of 0.4 g acid-hydrolyzed casein, 0.5 g yeast extract, 0.3 g tryptone, 0.5 g soluble starch, 0.4 g K2HPO4, 0.1 g MgSO4, 0.2 g sodium pyruvate, 15.0-20.0 g agar powder, and 1 L water, and the pH of the R2D solid culture medium is 6.8-7.

2. The target pathogen was selected from Fusarium oxysporum (Fusarium oxysporum) Fusarium oxysporum Fusarium solani ( ) Fusarium solani Rhizoctonia solani ( ), Rhizoctonia solani Rhizoctonia solani ), and robust red shell fungus ( Ilyonectria robusta ) and New Zealand finger scabies ( Dactyonectria novozelandica At least one of the following; The sample suspension is a soil suspension and / or a plant tissue suspension.

2. The method according to claim 1, characterized in that, The co-culture time is 24h-240h, and / or The co-culture temperature is 20-35℃.

3. The method according to claim 1, characterized in that, After co-culturing, if the target pathogen shows a clear inhibition zone or band compared to the control without sample suspension, thereby inhibiting the normal growth of the target pathogen, then candidate biocontrol bacteria that inhibit the growth of the target pathogen are preliminarily screened.

4. The method according to any one of claims 1-3, characterized in that, The method also includes verifying the effectiveness of the screened biocontrol bacteria.

5. The method according to claim 4, characterized in that, The effect verification was conducted using the flat plate confrontation method.

6. The method according to claim 4, characterized in that, The method also includes purifying and identifying the genus of the biocontrol bacteria obtained through screening.

7. The method according to claim 4, characterized in that, The method also includes purifying or identifying the genus of the biocontrol bacteria obtained through screening.

8. The method according to any one of claims 1-3, characterized in that, The method further includes determining, before inoculating the target pathogen, that the target pathogen is capable of growing in the solid culture medium.

9. The method according to claim 1, characterized in that, The sample suspension includes soil suspensions of food crops or cash crops.

10. The method according to claim 9, characterized in that, The sample suspension includes soil suspensions of Chinese medicinal herbs.

11. The method according to claim 1, characterized in that, The sample suspension includes a suspension of ground plant tissue.

12. The method according to claim 10, characterized in that, The medicinal materials mentioned are selected from root and rhizome medicinal materials.

13. The method according to claim 12, characterized in that, The Chinese medicinal materials are selected from at least one of ginseng, American ginseng, Panax notoginseng, Astragalus membranaceus, Atractylodes lancea, Angelica sinensis, and Codonopsis pilosula.

14. The method according to claim 9, characterized in that, The food crops include at least one of wheat, rice, corn, oats, rye, and barley.

15. The method according to claim 9, characterized in that, The cash crops include at least one of vegetable crops, fiber crops, oil crops, sugar crops, beverage crops, hobby crops, or medicinal crops.

16. The application of the method according to any one of claims 1-15 in the prevention and control of soil-borne diseases in agricultural production, wherein the soil-borne disease is selected from Fusarium oxysporum (…). Fusarium oxysporum Fusarium solani ( ) Fusarium solani Rhizoctonia solani ( ) Rhizoctonia solani ), and robust red shell fungus ( Ilyonectria robusta ) and New Zealand finger scabies ( Dactyonectria novozelandica Soil-borne diseases caused by at least one of the following:

17. The method according to claim 16, characterized in that, The method is used to screen biocontrol bacteria for food crops or cash crops.

18. The method according to claim 16, characterized in that, The method described above is used to screen biocontrol bacteria for traditional Chinese medicinal materials.

19. The method according to claim 18, characterized in that, The biocontrol bacteria include bacteria.

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