A Bacillus thuringiensis strain that is highly effective in killing plant parasitic nematodes and its application

Abstract

The present invention provides a strain of Bacillus thuringiensis that is highly effective in killing plant parasitic nematodes and its application, belonging to the field of microbial technology. The present invention screens and obtains a strain of Bacillus thuringiensis (Bacillus thuringiensis) BMB-0181 that is highly effective in killing plant parasitic nematodes from soil, and the deposit number is CCTCC NO:M 20242644. The fermentation liquid of the strain is used to detect and confirm that it can produce high trans-aconitic acid. It is confirmed by bioassay and potted plant experiments that it has high toxicity to southern root-knot nematodes, can effectively control the colonization of southern root-knot nematodes, and has a certain growth-promoting effect on tomatoes. The strain of the present invention can be developed into a new biological nematode pesticide.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial pesticides, and in particular relates to a Bacillus thuringiensis strain capable of effectively killing plant parasitic nematodes and its application. Background Art

[0002] Plant-parasitic nematodes belong to the phylum Nematoda, class Secernentea. About 200 genera and more than 5,000 species of plant-parasitic nematodes have been discovered. They can infect more than 3,000 plant species, including major food crops, fruits and vegetables, and medicinal plants, causing more than 30% yield reduction or even total crop failure, seriously hindering the safe production of food and cash crops in my country.

[0003] Root-knot nematodes are tiny, about 200 microns in length, making them difficult to detect as they spread through the soil. They invade plant root tips and colonize within them, forming knots that hinder nutrient absorption and transmission, making their damage invisible. The holes they penetrate can also lead to complex infestations with other soil-borne diseases. Because they parasitize within the plant, with only the eggs and second-instar larvae outside the plant, the control window is short, making it difficult for pesticides to reach the lesions, making plant nematode control extremely difficult. Nematicides such as avermectin and thiazothiazolinone have long been the mainstays, but years of heavy use have led to the development of resistance. With the gradual banning of highly toxic nematicides, root-knot nematode control in my country will face significant challenges. Biological control offers the most promising approach to root-knot nematode control, but the overall bottleneck is a shortage of viable products.

[0004] Bacillus thuringiensis (Bt) is a typical rod-shaped, Gram-positive bacterium widely found in soil and other natural environments. It produces vegetative "spores" with a unique structure that are highly resistant to the environment. It produces a variety of insecticidal active substances, including insecticidal crystal proteins, vegetative insecticidal proteins, proteases, and trans-aconitic acid, with high insecticidal activity against over 500 species of insects and nematodes. Bt insecticides account for over 70% of the microbial insecticide market, and the area of ​​Bt-transgenic insect-resistant crops cultivated has exceeded 100 million hectares.

[0005] Trans-aconitic acid (TAA) is a small molecule tricarboxylic acid compound with the chemical formula C₆H₆O₆ and a molecular weight of 174. It is also known as trans-1,2,3-propylenetricarboxylic acid or 3-carboxy-2-pentene-1,5-dioic acid. Trans-aconitic acid can be synthesized and excreted by Bacillus thuringiensis (Bt) through the tbr gene. In vitro bioassays have shown that trans-aconitic acid is highly toxic to various plant-parasitic nematodes, including the southern root-knot nematode (M. incognita) and the soybean cyst nematode (H. glycines). It is a novel nematicidal active substance produced by Bt (ZL 20121039147.7; ZL 201210390568.X). TAA synthesis capacity can be used as a tool to predict the nematicidal activity of Bt. Furthermore, trans-aconitic acid is widely present in crops such as corn and sugarcane, and its safety has been confirmed by the FDA, FAO, and EFSA. Therefore, Bt has great potential to be developed as a highly effective nematicide.

[0006] Bionematicides are environmentally friendly. For many years, China has clearly stated its commitment to accelerating the development of biopesticides. The creation of new, highly effective bionematicides is an urgent need for green plant nematode control in my country. Therefore, identifying new strains of Bacillus thuringiensis that are highly effective in killing plant-parasitic nematodes is particularly important. Summary of the Invention

[0007] In view of the current bottleneck problem of resource scarcity in the field of biological control of plant parasitic nematodes, the first object of the present invention is to provide a Bacillus thuringiensis strain that is highly effective in killing plant parasitic nematodes, which can be used for the efficient control of plant parasitic nematode diseases.

[0008] To achieve the above technical objectives, the inventors have long been committed to the research and development of Bacillus nematicidal resources. Through a large number of experimental studies combined with strain isolation and bioinformatics analysis, they have accurately discovered more than a thousand highly effective nematicidal strains from more than 50,000 Bt strains. Through detailed indoor bioassays and potted plant experiments, a strain of Bacillus thuringiensis with high toxicity to plant parasitic nematodes was screened and identified. The applicant named the strain Bacillus thuringiensis BMB-0181 (Bacillus thuringiensis BMB-0181), which was deposited in the China Center for Type Culture Collection of Wuhan University, Hongshan District, Wuhan City, Hubei Province, China on November 26, 2024, with the deposit number CCTCC NO: M20242644.

[0009] It should be noted that the Bacillus thuringiensis BMB-0181 isolated by the present invention has the following morphological and growth characteristics: the vegetative body of the bacterium is short rod-shaped, with or without peritrichous flagella, and is approximately 1.0-2.0 μm × 3.0-6.0 μm in size. It is Gram-positive, and the spores are oval, approximately 0.8 μm × 2.0 μm. When cultured on LB medium at 28°C, the colonies are milky white, waxy, and approximately round with irregular edges (see Figure 1 The optimum growth temperature is 28℃, and it grows well under pH 6-8, with the optimum pH being 7.0-7.2.

[0010] Secondly, a second object of the present invention is to provide the use of a biocontrol Bacillus strain, namely the aforementioned Bacillus thuringiensis BMB-0181, for controlling agricultural nematode diseases; and the use of this strain in the preparation of biopesticides and / or bio-organic fertilizers for controlling agricultural nematode diseases. Preferably, the agricultural nematodes include root-knot nematodes and cyst nematodes. Further preferably, the root-knot nematode is Meloidogyne incognita, and the cyst nematode is Heterodera glycines.

[0011] In addition, the third object of the present invention is to provide a new use of a wild strain in the biosynthesis of trans-aconitic acid, namely, the use of the above-mentioned Bacillus thuringiensis BMB-0181 as a production strain in the fermentation preparation of trans-aconitic acid.

[0012] Furthermore, a fourth object of the present invention is to provide a biocontrol agent, wherein the active ingredient of the biocontrol agent is prepared from the fermentation broth or the supernatant of the fermentation broth of the above-mentioned Bacillus thuringiensis BMB-0181.

[0013] Finally, a fifth object of the present invention is to provide applications of the above-mentioned biological control agent, wherein the applications are selected from one or both of the following: (1) applications for controlling agricultural nematode diseases; and (2) applications for promoting plant growth. Preferably, the agricultural nematodes include root-knot nematodes and cyst nematodes. More preferably, the root-knot nematodes are southern root-knot nematodes, and the cyst nematodes are soybean cyst nematodes. More preferably, the plant is a tomato.

[0014] Compared with the prior art, the Bacillus thuringiensis BMB-0181 strain provided by the present invention has the following significant advantages and advancements:

[0015] (1) This strain is highly toxic to the southern root-knot nematode, with a 100-fold diluted fermentation liquid having an 80% lethality rate against the nematode. It also has no toxic activity against the non-plant parasitic nematode Caenorhabditis elegans, demonstrating its insecticide specificity and environmental friendliness.

[0016] (2) This strain can produce a new nematicidal active substance, trans-aconitic acid, in high yield under laboratory shake flask conditions, with a yield of 2.5 g / L, which is significantly higher than all existing Bacillus thuringiensis strains that can produce trans-aconitic acid.

[0017] (3) The fermentation liquid of the strain Bacillus thuringiensis BMB-0181 of the present invention was applied to tomato seedlings seriously infested with root-knot nematodes. The results showed that the root-knot nematode prevention effect reached 80% in a pot experiment, which was significant and comparable to that of chemical pesticides.

[0018] (4) The fermentation liquid of the strain Bacillus thuringiensis BMB-0181 of the present invention was applied to tomato seedlings seriously infected with root-knot nematodes. The results showed that the medium and low concentrations of the fermentation liquid had a significant growth-promoting effect on tomatoes in potted experiments, with the average plant height increased by 18%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 : is the colony morphology of Bacillus thuringiensis BMB-0181 of the present invention.

[0020] Figure 2 (A) is the HPLC detection spectrum of trans-aconitic acid produced by Bacillus thuringiensis BMB-0181 of the present invention, wherein the peak at 9.75 min is trans-aconitic acid; (B) is the HPLC detection spectrum of a commercial standard of trans-aconitic acid; (C) is a comparison of trans-aconitic acid production between BMB-0181 and other Bacillus thuringiensis strains.

[0021] Figure 3 : Graph showing the nematicidal activity of the fermentation broth and fermentation supernatant of Bacillus thuringiensis BMB-0181 of the present invention after 24 h of dilution treatment.

[0022] Figure 4 : Effects of the fermentation liquid of Bacillus thuringiensis BMB-0181 of the present invention on the root knot number of potted tomatoes, where ** indicates p value < 0.005 in the t test, and **** indicates p value less than 0.0001.

[0023] Figure 5 :(A) is the root sample of the potted experiment of the blank control group with equal amount of fermentation medium; (B) is the root sample of the potted experiment of the avermectin control group; (C) is the root sample of the potted experiment of the group treated with Bacillus thuringiensis BMB-0181 fermentation liquid.

[0024] Figure 6 : Effects of the fermentation liquid of Bacillus thuringiensis BMB-0181 of the present invention on the plant height of potted tomatoes, where * indicates p value < 0.05 in t test. DETAILED DESCRIPTION

[0025] The following are specific embodiments of the present invention, which describe the technical solutions of the present invention in detail. If no specific technical operations or conditions are specified in the examples, the operations are performed according to conventional techniques or conditions described in the literature in the field. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased normally.

[0026] Example 1: Isolation of strains and detection of TAA production ability

[0027] 1. Isolation of Bacillus thuringiensis

[0028] The strains were isolated from soils of various habitats across the country using acetate selection medium (BPA medium for short), and TAA synthesis gene detection was further performed. Finally, TAA production was verified using high performance liquid chromatography (HPLC) to screen and obtain the strains of the present invention.

[0029] (1) BPA culture medium formula: 5 g beef extract, 10 g peptone, 34 g sodium acetate; add distilled water to 1 L and adjust the pH to 7.2-7.4.

[0030] (2) Separation method

[0031] Weigh 1 g of soil samples from various habitats across the country and place them in BPA culture medium. After thorough shaking, incubate them in a shaker at 30°C for 4 h, remove them and heat-treat them in a 75-80°C water bath for 10-15 min. After a short period of rest, pipette 0.5 mL of the culture medium onto a BPA plate (BPA plates are prepared according to conventional methods and supplemented with 2% agar), spread the medium evenly, and invert the plate in a 30°C incubator for incubation for 24 h. Select 3-5 colonies similar to Bacillus thuringiensis and inoculate them onto a BPA slant (BPA slant is prepared according to conventional methods and supplemented with 2% agar), incubate them at 30°C for more than 72 h, and stain them with carbolic acid fuchsin for microscopic examination. Isolates with parasporal crystals are identified as Bacillus thuringiensis (for methods for isolation, screening, classification and identification of microorganisms, see: Yu Ziniu, Bacillus thuringiensis, Science Press, 1990 edition).

[0032] 2. Detection of TAA synthesis genes in Bacillus thuringiensis

[0033] (1) Extraction of total DNA from Bacillus thuringiensis

[0034] After pure culture of Bt, streak the plate on a new LB solid medium (calculated based on 1 L of medium: peptone: 10 g; yeast powder: 5 g; sodium chloride: 10 g; agar powder: 15 g; distilled water is added to 1 L; pH is adjusted to 7.0). Use an inoculating loop to aseptically inoculate 5 mL of LB liquid medium (1 L: peptone: 10 g; yeast powder: 5 g; sodium chloride: 10 g; distilled water is added to 1 L; pH is adjusted to 7.0) and culture overnight in a shaker at 28°C and 200 rpm. Then, aseptically transfer 50 μL of the culture to 5 mL of LB liquid medium and culture under the same conditions for 3-4 hours. Then, centrifuge at 12000 rpm for 0.5 min to collect the bacteria. ① Use 1 mL of STE [formula: 0.1 mol / L NaCl, 10 mmol / L Tris-HCl (pH 8.0), 1 mmol / L ② Add 100 μL of solution I [formula: 1 mol / L Tris-HCl (pH 8.0), 0.5 mol / L EDTA (pH 8.0), 50 mmol / L glucose] and 10 μL lysozyme (concentration: 50 mg / mL) and incubate at 37°C for more than 30 min; ③ Add 200 μL of 2% sodium dodecyl sulfate (SDS) and incubate at 55°C in a water bath for 30 min; ④ Add 200 μL of 5 mol / L NaCl and mix, then add 500 μL of phenol / chloroform / isoamyl alcohol (volume ratio: 25:24:1), centrifuge at 12000 rpm for 5 min, aspirate the supernatant, and repeat the extraction 1-2 times; ⑤ Transfer the upper DNA solution to a 1.5 ml centrifuge tube, add an equal volume of 95% ethanol, let it stand at room temperature for 5 min, then centrifuge at 12000 rpm for 5 min, and precipitate with 200 μL Wash once with 70% ethanol, freeze-dry, and then dissolve in 50 μL TE solution.

[0035] (2) PCR amplification of Bacillus thuringiensis TAA synthesis gene

[0036] Primers were designed based on the sequence of the TAA synthesis gene tbr gene (Du Cuiying. Research on the biosynthesis pathway of small molecule nematicidal toxin trans-aconitic acid and its application in the control of plant root-knot nematodes [D]. Huazhong Agricultural University, 2017.) and amplified. The primer sequences are as follows:

[0037] tbrF:5`-TTAGCTTCTTGGGGTCTAGAATGAAATACCTTG-3`

[0038] tbrR:5`-CCAAGCTTTTATGAACTTGCTGCCCTTCT-3`

[0039] The PCR reaction system was as follows: 10× buffer 2 μl, 2 mmol / L dNTP 1.5 μl, 10 μmol / L primers 0.4 μl each, Taq enzyme 1 U, total bacterial DNA 1 μl, and sterile deionized H2O was added to 20 μl.

[0040] The PCR amplification reaction procedure was as follows: step 1: pre-denaturation at 94°C for 5 min; step 2: denaturation at 94°C for 1 min, step 3: annealing at 55°C for 1 min, step 4: extension at 72°C for 3 min; step 5: go to step 2 and continue running for 35 repetitions; step 6: extension at 72°C for 5 min.

[0041] (3) Sequence determination and analysis of PCR products

[0042] After agarose gel electrophoresis and agarose gel detection, the PCR products were sent to Beijing Aoke Biotechnology Co., Ltd. for sequencing. The full length of the PCR amplification product of 2097 bp was obtained through two sequencing reactions. The sequencing results were entered into NCBI and submitted to the GenBank database for comparative analysis using the Blastn program to verify the amplification product with the tbr Gene sequence similarity. Bacillus thuringiensis strains with consistent amplification products were identified as strains with potential TAA production, and these potential TAA-producing strains were further tested for TAA production in shake flask fermentation.

[0043] 3. Detection of TAA production capacity of Bacillus thuringiensis

[0044] (1) Laboratory shake flask fermentation of Bacillus thuringiensis

[0045] A single colony of Bacillus thuringiensis BMB-0181 was picked and placed in a PA bottle containing 5 ml of LB liquid medium (ingredients: 10 g of tryptone, 5 g of yeast extract, 10 g of NaCl, and distilled water to 1000 mL; adjust the pH to 7.0-7.2) and cultured at 28°C, 220 rpm, and shaking for 12 h. The inoculum volume was transferred to 50 ml of fresh TAA fermentation medium at a rate of 1 / 100 (v / v) (ingredients: 46.91 g / L nitrogen source No. 2, 30.88 g / L carbon source No. 1, 0.333 g / L inorganic salt No. 1, 2 g carbon source No. 2, 30 g nitrogen source No. 1, 2 g nitrogen source No. 3, 0.3 g inorganic salt No. 2, 0.02 g inorganic salt No. 3, 0.02 g inorganic salt No. 4, supplemented with distilled water to 1000 mL; adjusted to pH 7.0-7.2), and fermented at 30°C and 220 r / min for 48 h.

[0046] (2) Detection of TAA content in fermentation broth

[0047] 1 mL of the fermentation broth was transferred to a 1.5 mL centrifuge tube and centrifuged at 10,000 rpm for 2 min. The supernatant was filtered through a 0.22 μm filter membrane and then subjected to HPLC detection.

[0048] HPLC detection method:

[0049] High-performance liquid chromatograph with variable-wavelength ultraviolet detector; chromatography data processor or chromatography workstation; chromatographic column: 300 mm × 7.8 mm (id) stainless steel column packed with polystyrene divinylbenzene resin, 9 μm particle size; autosampler: 50 μL; mobile phase: 5 mM sulfuric acid, sonicated for 15 min, filtered through a membrane, and degassed; column temperature: 55°C; flow rate: 0.6 mL / min; injection volume: 5 μL; detection wavelength: 210 nm; retention time: approximately 9.4 min.

[0050] The above operating parameters are typical. According to the characteristics of different instruments, the given operating parameters can be appropriately adjusted to obtain the best results.

[0051] By detecting the TAA content in the fermentation broth of Bacillus thuringiensis, the difference in TAA production capacity between different strains was determined, and finally a wild Bacillus thuringiensis strain with high TAA production was obtained and named Bacillus thuringiensis BMB-0181 (see Figure 2 (A) Figure 2 (B) and Figure 2 (C)).

[0052] Example 2: Nematicidal bioactivity assay of Bacillus thuringiensis BMB-0181 fermentation broth

[0053] 1. Cultivation and egg hatching of root-knot nematodes

[0054] (1) Plant Jinpeng No. 3 or Nt wild-type tobacco plants. Seeds can be directly sown into the soil for seedling cultivation. When the plant seedlings are about 15-20 cm tall, transplant them into separate pots and use a pipette to evenly inject nematodes onto the root surface. After about 45-60 days, depending on the degree of root-knot nematode infestation in the plant roots, remove the diseased plants and collect the egg masses.

[0055] (2) Collecting egg masses for hatching J2s root-knot nematodes: ① Collect diseased plant roots, clean the roots of mud and impurities, cut the diseased roots into pieces, and place them in a beaker; ② Add an appropriate amount of 10% sodium hypochlorite aqueous solution to the beaker and stir rapidly for 7 minutes (magnetic stirrer); ③ Prepare sieves with mesh sizes of 60, 100, 200, and 500 from top to bottom, add the stirred sample to the sieves, add water and sieve thoroughly. The eggs will be retained on the 500-mesh sieve; ④ Prepare an incubator (two plastic measuring cups with the bottoms removed, 500-mesh nylon cloth sandwiched between them, and place them in a petri dish); ⑤ Pour the cleaned eggs onto the nylon cloth in the incubator, and add water to the incubator petri dish so that the water just touches the nylon cloth; ⑥ After hatching, the J2 larvae will pass through the nylon cloth and incubate at 25℃ for 3-5 days. Collect the J2 and add water to incubate again.

[0056] 2. Plant-parasitic nematode bioactivity assay

[0057] (1) Treatment of Bacillus thuringiensis BMB-0181 fermentation broth

[0058] The fermentation method for Bacillus thuringiensis BMB-0181 is detailed in Example 1 for laboratory shake flask fermentation of Bacillus thuringiensis. The fermentation broth was centrifuged at 4°C, 10,000 rpm for 10 minutes, and the supernatant was aspirated and filtered through a 0.22 μm pore size filter. The supernatant was diluted 10-fold and 100-fold, respectively. The fermentation broth was diluted 10-fold and 100-fold, and the lethality to nematodes was determined.

[0059] (2) In vitro bioassay of plant parasitic nematodes

[0060] ① Pipette 3-day-old nematode J2s into a 1.5ml centrifuge tube, centrifuge at 5000rpm for 1min, and discard the supernatant; ② Resuspend the nematodes in an appropriate amount of ddH2O and determine the worm density under a microscope. The final nematode density is approximately 30 nematodes / μL; ③ Prepare different concentrations of the fermentation broth supernatant to be tested. Use a pipette to add the fermentation broth and nematodes to the corresponding wells of a 96-well bioassay plate in an ultra-clean workbench (20-40 J2s per well). Sterile ddH2O and ICPM medium serve as blank controls. The total volume per well is 100μL; each treatment is repeated three times; ④ Seal the 96-well bioassay plate with sealing film and place it in a 20℃ incubator. After 24h, count the total number of nematodes and the number of deaths in each well.

[0061] In vitro bioassays on plant parasitic nematodes showed that the fermentation supernatant and fermentation stock solution of Bacillus thuringiensis BMB-0181 had a mortality rate of nearly 100% against southern root-knot nematodes when diluted 10 times. When diluted 100 times, the fermentation solution and supernatant could still kill about 80% of the root-knot nematodes in the sample wells (see Figure 3 ). This is sufficient to confirm that Bacillus thuringiensis BMB-0181 has strong lethal activity against the second-instar larvae of the southern root-knot nematode.

[0062] Example 3: Potted experiment on controlling tomato root-knot nematodes with strain BMB-0181

[0063] ① Select healthy tomato seedlings with uniform growth and size as test seedlings (four-leaf, one-heart stage); ② Inoculate 2,000 second-instar larvae of southern root-knot nematodes at the roots of the tomato seedlings; ③ For the fermentation method of Bacillus thuringiensis BMB-0181, refer to the laboratory shake flask fermentation of Bacillus thuringiensis in Example 1. Dilute the fermentation broth stock solution 10 times, 50 times, and 100 times; ④ Use the fermentation broth dilution as rooting water, add 50 ml of the fermentation broth dilution to each pot of diseased tomatoes, and inoculate the control group with an equal volume of blank fermentation medium and avermectin dilution (the dilution multiple is based on the recommended dosage conversion in the instructions) for a total of five treatments, with five replicates for each treatment. After completion, place the tomatoes in a suitable environment for cultivation, based on the complete life history of the nematodes, and count the number of root knots on the tomatoes after 40 days as the standard for disease infection.

[0064] The tomato root-knot nematode pot experiment showed that the fermentation liquid treatment, especially the fermentation liquid diluted 50 times and 10 times, and the application of 50ml per plant, significantly reduced the number of root knots in the plants, which is consistent with the above laboratory bioassay results. At the same time, under the condition of 50 times dilution of the fermentation liquid, the height of the tomato plant was significantly increased compared with the avermectin treatment and the blank fermentation liquid treatment, indicating that the fermentation liquid of strain BMB-0181 has a certain growth-promoting effect at medium and low concentrations (see Figure 4 、 Figure 5 、 Figure 6 ).

[0065] The present invention screened a strain of Bacillus thuringiensis (BMB-0181) from Bacillus thuringiensis, which is highly effective in killing plant-parasitic nematodes. This strain also produces a high yield of trans-aconitic acid, a novel nematicidal active substance, under shake flask fermentation conditions. Bioassays and potted plant experiments confirmed that this strain has high toxicity against the southern root-knot nematode, effectively controlling its colonization. Its effectiveness is comparable to that of the currently popular nematicidal pesticide, avermectin, suggesting significant application and development potential.

Claims

1. A strain of Bacillus thuringiensis that is highly effective in killing plant parasitic nematodes ( Bacillus thuringiensis )BMB-0181, its deposit number is CCTCC NO: M 20242644.

2. The Bacillus thuringiensis according to claim 1 ( Bacillus thuringiensis ) Application of BMB-0181 in preventing and controlling agricultural nematode diseases, wherein the agricultural nematodes are root-knot nematodes and cyst nematodes; the root-knot nematode is the southern root-knot nematode, and the cyst nematode is the soybean cyst nematode.

3. The Bacillus thuringiensis according to claim 1 ( Bacillus thuringiensis ) Use of BMB-0181 in the preparation of biopesticides and / or bio-organic fertilizers for controlling agricultural nematode diseases, wherein the agricultural nematodes are root-knot nematodes and cyst nematodes; the root-knot nematode is the southern root-knot nematode, and the cyst nematode is the soybean cyst nematode.

4. The Bacillus thuringiensis according to claim 1 ( Bacillus thuringiensis )Application of BMB-0181 in the fermentation preparation of trans-aconitic acid.

5. A biological control agent, characterized in that: The active ingredient of the biological control agent is the Bacillus thuringiensis ( Bacillus thuringiensis ) is prepared from the fermentation broth or fermentation supernatant of BMB-0181.

6. The use of the biological control agent according to claim 5, wherein the use is selected from one or both of the following: (1) application for controlling agricultural nematode diseases; (2) application for promoting tomato growth; The agricultural nematodes are root-knot nematodes and cyst nematodes; the root-knot nematodes are southern root-knot nematodes, and the cyst nematodes are soybean cyst nematodes.

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