Bacillus thuringiensis hp-syj2 and application thereof
By screening and applying Bacillus thuringiensis HP-SYJ2 fermentation broth, the problem of poor control effect of underground pests in existing technologies has been solved, achieving a highly efficient and long-lasting biological control effect, which is suitable for the control of crop pests.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- QINGDAO HELP BIOSCI
- Filing Date
- 2026-02-26
- Publication Date
- 2026-06-02
AI Technical Summary
The existing Bacillus thuringiensis strains have unstable control effects on underground pests, and their strains are not adaptable enough to colonize and maintain their activity in the soil environment, resulting in poor biological control effects.
A Bacillus thuringiensis HP-SYJ2 strain is provided, which can produce highly active parasporal crystal proteins during the spore formation period and prepare insecticides through fermentation broth. It can be effectively planted by drenching the roots and soil to control underground pests.
Bacillus thuringiensis HP-SYJ2 has a control effect of over 80% against root-knot nematodes, root-rot nematodes, and cyst nematodes. It has a long-lasting effect, is not prone to developing resistance, and is simple and low-cost to prepare, making it suitable for biological control of crops.
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Abstract
Description
Technical Field
[0001] This application relates to the field of microbial strain technology, and more specifically, to a Bacillus thuringiensis HP-SYJ2 and its applications. Background Technology
[0002] Plant pests are a key factor affecting food security and restricting the yield and quality of agricultural products. Currently, pest control technologies mainly include chemical pesticide control, physical control, agricultural technology control, and biological control. While each technology has its advantages, they also generally have limitations. Biological control technology, due to its low environmental risk, long-lasting effect, ease of synergistic application with other plant protection measures, and effective energy conservation, is widely recognized as an environmentally friendly and resource-saving approach in integrated pest management systems.
[0003] Among numerous biological control methods, using biocontrol microorganisms to control pests is considered a more eco-friendly approach. Common and widely used biocontrol microorganisms include *Beauveria bassiana*, *Metarhizium anisopliae*, *Verticillium spp.*, and *Bacillus thuringiensis*. *Bacillus thuringiensis* (Bt) is a Gram-positive rod-shaped bacterium widely distributed in nature. Its most significant characteristic is the production of one or more highly insecticidal parasporal crystal proteins (δ-endotoxins) during spore formation. In addition, different Bt strains may produce other insecticidal metabolites, such as thuringin, chitinase, cytolysin, and synergistic proteins. The types and amounts of these active substances vary from strain to strain, determining the highly specific insecticidal spectrum of different Bt strains. Typically, a particular strain only exhibits highly effective insecticidal activity against pests of specific orders or families (such as Lepidoptera, Coleoptera, and Diptera).
[0004] Although Bt formulations and Bt genetically modified crops have been successfully applied globally to control various above-ground pests, existing Bt products still face challenges in controlling underground pests (such as grubs, wireworms, and cutworms), including inconsistent efficacy and insufficient strain adaptability. This is mainly due to the complexity of the soil environment, the differences in the behavioral habits of target pests, and the insufficient colonization ability, persistence, and activity of strains in the soil. Therefore, targeted screening of novel Bt strains with high insecticidal activity, strong environmental resistance (such as drought tolerance, temperature tolerance, and acid / alkali tolerance), and excellent soil colonization ability from the soil habitats of target pests has become a key strategy to overcome the current bottlenecks in the biological control of underground pests and improve overall control effectiveness. Summary of the Invention
[0005] To improve the control of underground pests, this application provides a Bacillus thuringiensis HP-SYJ2 strain and its application.
[0006] In the first aspect, this application provides a Bacillus thuringiensis HP-SYJ2 strain, which adopts the following technical solution: A Bacillus thuringiensis HP-SYJ2 strain is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M2025343, deposit date: March 3, 2025, address: Wuhan University, Wuhan, China.
[0007] By adopting the above technical solution, Bacillus thuringiensis can produce one or more parasporal crystal proteins with high insecticidal activity during the spore formation period, and different Bacillus thuringiensis can produce other metabolites with insecticidal activity, thus determining its highly specific insecticidal spectrum.
[0008] Secondly, this application provides an insecticide, which adopts the following technical solution: An insecticide comprising Bacillus thuringiensis HP-SYJ2 or fermentation broth of Bacillus thuringiensis HP-SYJ2.
[0009] By adopting the above technical solution, the insecticide containing Bacillus thuringiensis HP-SYJ2 can be applied to the roots of plants through root irrigation, thereby effectively establishing itself at the roots and killing pests.
[0010] Preferably, the fermentation broth of Bacillus thuringiensis HP-SYJ2 is obtained by the following method: Bacillus thuringiensis HP-SYJ2 is activated, inoculated into liquid culture medium, fermented to obtain fermentation broth, the fermentation broth is centrifuged, the bacterial cells are collected, and resuspended using sterile PBS buffer to obtain fermentation broth.
[0011] By adopting the above technical solution, Bacillus thuringiensis HP-SYJ2 was activated and cultured to obtain a fermentation broth, which can effectively prevent and control plant pests.
[0012] Preferably, the fermentation culture temperature is 28-30℃ and the culture time is 2-3 days.
[0013] Preferably, the viable cell count in the fermentation broth of Bacillus thuringiensis HP-SYJ2 is 2-8 × 10⁻⁶. 8 cfu / ml.
[0014] By adopting the above technical solutions, Bacillus thuringiensis HP-SYJ2 with the above viable bacterial count can more effectively prevent and control plant pests.
[0015] Preferably, the insecticide is one of a suspension concentrate, powder, or granules.
[0016] Secondly, this application provides an application of Bacillus thuringiensis HP-SYJ2, employing the following technical solution: Application of Bacillus thuringiensis HP-SYJ2 or the aforementioned insecticide in the control of underground pests in crops.
[0017] Preferably, when underground pests feed on plant roots or organic matter in the soil, they ingest preparations containing Bacillus thuringiensis HP-SYJ2 spores or crystal proteins. The crystal proteins dissolve and activate in the alkaline midgut of the pests, destroying the intestinal wall cells and thus causing their death.
[0018] In summary, this application has the following beneficial effects: The Bacillus thuringiensis HP-SYJ2 in this application has a highly effective control effect on underground pests, with an average control effect of over 80% against root-knot nematodes, root-rot nematodes, and cyst nematodes. It is highly targeted, not prone to developing resistance, and has good long-lasting efficacy. Furthermore, the preparation method of insecticides containing Bacillus thuringiensis is simple, low-cost, and easy to use, and it has a very broad application prospect in the biological control of crops. Detailed Implementation
[0019] The present application will be further described in detail below with reference to the embodiments. Example
[0020] Example 1: Isolation and screening of Bacillus thuringiensis HP-SYJ2 (1) Isolation and purification of strains 1.1 Soil source: Soil was collected from the Jiaonan tea plantation area in Laoshan, Qingdao. The collected soil was mixed, crushed, and weeds, tree roots and stones were removed. The soil was placed in an ice bucket and stored at 4℃ for later use. The physicochemical properties of the tested soil are shown in Table 1.
[0021] Table 1
[0022] 1.2 Main culture medium: Beef extract peptone solid culture medium: Dissolve 10g peptone, 3g beef extract, 5g sodium chloride and 15g agar in a small amount of distilled water, then make up to 1L with water, and sterilize at 121℃ for 30min.
[0023] Nutrient agar medium formula: 3g beef extract, 5g peptone, 5g sodium chloride, 18g agar, 1000mL distilled water, pH 7-7.2.
[0024] 1.3 Strains Isolation and Purification: Take 10g of test soil and add it to a 250ml Erlenmeyer flask containing 90mL of sterile water (with glass beads). Shake on a shaker for 30min to fully disperse. After purification for 20-30s, take the supernatant and perform 10-fold serial dilutions. -3 ~10 -5 For dilution, heat-treat the diluent in water at 80℃ for 3 min. Use a pipette to transfer 0.1 ml of each dilution gradient and spread it evenly on beef extract peptone solid medium. Perform three replicates for each gradient and incubate upside down at 28℃ for 7 days. Use an inoculation loop to pick single colonies that look like Bacillus spores, stain with carbofuran, and examine under a microscope for the presence of parasporal crystals. Streak colonies with parasporal crystals onto nutrient agar medium for purification until the colony morphology on the plate is uniform. Store on nutrient agar medium at -80℃.
[0025] The selected strain was named Bacillus thuringiensis HP-SYJ2 and deposited on March 3, 2025, at the China Center for Type Culture Collection (address: Wuhan University, Wuhan, China, postcode: 430072), with accession number CCTCC NO: M2025343, and classified as Bacillus thuringiensis.
[0026] 1.4 Screening of highly active strains Disinfected root-knot nematode J2 larvae were selected and placed into sterilized 24-well containers. 2 mL of treatment solution was added to each well, and the containers were incubated at 25°C for 72 hours. The mortality of the nematodes was determined by the needle touch method. The fermentation broth of the strain isolated and purified in 1.3 was used as the treatment solution, and the fermentation medium was used as the blank control. Each treatment was repeated 3 times. The mortality rate was calculated as (number of dead nematodes / number of test nematodes) × 100. The five strains with the strongest antagonistic activity were isolated and purified. The test results are shown in Table 2.
[0027] Table 2
[0028] (2) Characterization of the screened strain HP-SYJ2: 2.1 Morphological characteristics: After selective culture and activation, the colony morphology of the preserved Bacillus thuringiensis HP-SYJ2 was observed. Then, strains were selected for Gram staining, capsule staining and spore staining and the bacterial morphology was observed under a microscope. Bacillus thuringiensis HP-SYJ2 is rod-shaped, with irregular edges, Gram positive, and has spores. The colonies formed after 24 hours of culture on beef extract peptone medium are round or irregular in shape. After 48 hours, they are round, milky white, with irregular edges, flat and moist.
[0029] 2.2 Physiological and biochemical assays: Catalase positive, VP reaction positive, glucose fermentation produces acid, and hydrolyzes starch and gelatin, all of which can utilize citrate.
[0030] Example 2: An insecticide comprising the fermentation broth of Bacillus thuringiensis HP-SYJ2 obtained in Example 1; the preparation method of the insecticide is as follows: Bacillus thuringiensis HP-SYJ2 is inoculated into a solid culture medium and activated by culturing in a constant temperature incubator at 28℃ and 200r / min for 2 days to obtain the seed culture; the formula of the solid culture medium is: 8g beef extract, 10g yeast extract, 20g glucose, 8g peptone, 0.005g MnSO4·H2O, 0.005g K2PO4, 5g NaCl, and distilled water is added to a final volume of 1000 mL, pH 7.0; The seed culture was inoculated into a liquid culture medium and cultured at 28℃ and 200rpm for 3 days to obtain the fermentation broth. The liquid culture medium formula was: 10g peptone, 5g sodium chloride, 10g yeast extract, and 1000mL water.
[0031] Performance testing
[0032] Detection of the toxic activity of Bacillus thuringiensis HP-SYJ2 fermentation broth against nematodes: (1) Test bacterial culture: The original fermentation broth of Bacillus thuringiensis HP-SYJ2 prepared in Example 2 (with a bacterial count of 5×10⁻⁶) was used. 8 (cfu / ml) is used as reagent A; The original fermentation broth (with a bacterial count of 5 × 10⁶) 8 (cfu / ml) was diluted twice with sterile water to prepare reagent B; The original fermentation broth (with a bacterial count of 5 × 10⁶) 8 (cfu / ml) was diluted 10 times with sterile water to serve as reagent C; The original fermentation broth (with a bacterial count of 5 × 10⁶) 8 (cfu / ml) was diluted 50 times with sterile water as reagent D; The original fermentation broth (with a bacterial count of 5 × 10⁶) 8 (cfu / ml) was diluted 100 times with sterile water as reagent E; The original fermentation broth (with a bacterial count of 5 × 10⁶) 8 (cfu / ml) was diluted 500 times with sterile water as reagent F; The original fermentation broth (with a bacterial count of 5 × 10⁶) 8 The reagent G was prepared by diluting the cfu / ml solution 1000 times with sterile water.
[0033] (2) Test root-knot nematodes: The test root-knot nematodes were preserved in indoor pots. The nematodes were bred from tomatoes. When a large number of egg sacs appeared on the test roots, the roots were removed, rinsed with water, and the egg sacs were removed and disinfected in a 0.5% sodium hypochlorite solution for 3 minutes. They were then rinsed 3 times with sterile water and placed in a petri dish containing sterile water. The nematodes were incubated at a constant temperature of 25°C. After 3 days, the newly hatched second-instar larvae were collected every 24 hours. The isolated second-instar larvae were prepared into a suspension with a concentration of 10 larvae / 10 μL and stored for later use.
[0034] (3) Test root rot nematodes: The test root rot nematodes were bred using potatoes. When a large number of egg sacs appeared on the test potatoes, the egg sac potatoes were collected. The egg sacs were collected by washing and sieving. The egg sacs were disinfected in a 0.5% sodium hypochlorite solution for 3 minutes, then rinsed 3 times with sterile water, and placed in a culture dish containing sterile water. They were cultured at a constant temperature of 25°C. After 7 days, the hatched second-instar larvae were collected. The isolated root rot nematodes were prepared into a suspension with a concentration of 6 nematodes / 10 μL and stored for later use.
[0035] (4) Test method: The nematode-killing activity was tested using 96-well cell culture plates. 10 μL of nematode suspension was taken from each well, and then 90 μL of the treatment reagent was added. Sterile water was used as a control. The test was repeated 6 times and cultured in a constant temperature incubator at 25℃. The mortality rate of nematodes was observed and recorded at 24h, 48h, and 72h. The criterion for judging nematode rearing was that the nematodes were rigid and did not move after being stimulated with bristles. Nematode mortality rate = (number of dead nematodes / number of tested nematodes) × 100, and the corrected cumulative mortality rate = (treatment mortality rate - control mortality rate) / (1 - control mortality rate) × 100%. The test results were recorded in Table 3.
[0036] Table 3. Toxicity of Bacillus thuringiensis HP-SYJ2 fermentation broth against nematodes.
[0037] Note: Different lowercase letters in the same column of the table indicate a significant difference at the 0.05 level (p≤0.05).
[0038] As can be seen from the data in Table 3, Bacillus thuringiensis HP-SYJ2 has strong insecticidal activity against root-knot nematodes and root-rot nematodes. Moreover, the higher the concentration of the fermentation broth, the stronger the insecticidal activity. When diluted more than 100 times, the corrected cumulative mortality rate of both types of nematodes was below 53% after 72 hours, indicating that the insecticidal toxicity was weakened and that auxiliary enhancement was required to achieve the desired control effect.
[0039] The control effect of Bacillus thuringiensis HP-SYJ2 fermentation broth on nematodes and underground pests. Plant preparation: Select plump tomato seeds, disinfect them with 70% alcohol for 1 minute, then disinfect them with 0.5% sodium hypophosphite for 1 minute, rinse them 5 times with sterile water, soak them in sterile water at 40℃ for 2 hours, and germinate them at a constant temperature of 27℃ in the dark. After most of the seeds have germinated, select the seeds with consistent germination and sow them.
[0040] Preparation of nematode suspensions: The isolated second-instar larvae of root-knot nematodes, second-instar larvae of root-rot nematodes, and second-instar larvae of sporangioides nematodes were prepared into suspensions at a concentration of 15 larvae / 10 μL and stored for later use.
[0041] Test reagents: The Bacillus thuringiensis HP-SYJ2 fermentation broth prepared in Example 2 was used as the test group; 5% avermectin emulsifiable concentrate diluted 1500 times was used as the control group; and water was used as the blank control group. Control Experiment: The seedling substrate was sterilized. 30% (by volume) of the test agent, control agent, and water were applied to the sterile substrate and kept at 28℃ with humidity for 3 days. Then, second-instar larvae of root-knot nematodes, second-instar larvae of root-rot nematodes, and second-instar larvae of sporangioides nematodes were distributed using the irrigation inoculation method. 15 mL of nematode suspension was inoculated into each hole, covered with a thin layer of soil, and kept moist for 2 days. Each nematode species was inoculated into 3 holes, marked, and sown. Additionally, two holes for each treatment were inoculated with newly hatched larvae of *Gnaphalium affine*, one larva per hole, marked, and sown. Five days after sowing, the test agent, control agent, and water were applied as a root drench at a rate of 30 mL / plant. A total of three treatments were performed, with the first two treatments spaced 3 days apart and the last treatment 7 days apart. All treatments were cultured at 27±1℃, humidity 75%–80%, and a photoperiod of 9h / 15h. Observe and record the seedling growth every day. After 40 days, investigate the number of diseased plants and the disease incidence rate (or mortality rate) of each treatment, and calculate the control effect (or corrected mortality rate). The disease incidence rate = number of diseased plants / total number of plants investigated × 100; the control effect = (disease incidence rate in the control area - disease incidence rate in the treatment area) / disease incidence rate in the control area × 100%. Record the test results in Table 4.
[0042] Table 4. Field control effect of Bacillus thuringiensis HP-SYJ2 fermentation broth
[0043] Note: Different lowercase letters in the same column of the table indicate a significant difference at the 0.05 level (p≤0.05).
[0044] The data in Table 4 show that the Bacillus thuringiensis HP-SYJ2 fermentation broth has a control effect of over 86% against root-knot nematodes, root-rot nematodes, and cyst nematodes, but its control effect is slightly weaker than that of the control agent (5% abamectin EC diluted 1500 times).
[0045] This specific embodiment is merely an explanation of this application and is not intended to limit it. After reading this specification, those skilled in the art can make modifications to this embodiment without contributing any inventive step, but such modifications are protected by patent law as long as they fall within the scope of the claims of this application.
Claims
1. A Bacillus thuringiensis HP-SYJ2 strain, characterized in that, Bacillus thuringiensis HP-SYJ2 is deposited at the China Center for Type Culture Collection (CCTCC), accession number: CCTCC NO: M2025343, deposit date: March 3, 2025, address: Wuhan University, Wuhan, China.
2. An insecticide, characterized in that, The fermentation broth of Bacillus thuringiensis HP-SYJ2 or Bacillus thuringiensis HP-SYJ2 as described in claim 1.
3. The insecticide according to claim 2, characterized in that: The fermentation broth of Bacillus thuringiensis HP-SYJ2 was obtained by the following method: Bacillus thuringiensis HP-SYJ2 was activated, inoculated into liquid culture medium, and fermented to obtain fermentation broth. The fermentation broth was centrifuged, the bacterial cells were collected, and the cells were resuspended in sterile PBS buffer to obtain fermentation broth.
4. The insecticide according to claim 2, characterized in that: The fermentation culture temperature is 28-30℃, and the culture time is 2-3 days.
5. The insecticide according to claim 2, characterized in that: The viable cell count in the fermentation broth of Bacillus thuringiensis HP-SYJ2 was 2-8 × 10⁻⁸. 8 cfu / ml.
6. The insecticide according to claim 2, characterized in that: The insecticide is one of the following: suspension concentrate, powder, or granules.
7. The application of Bacillus thuringiensis HP-SYJ2 as described in claim 1 or the insecticide as described in any one of claims 2-6 in the control of underground pests in crops.