A Bacillus thuringiensis strain with high insecticidal activity against cotton aphids and two-spotted spider mites and its application

Abstract

The present invention belongs to the field of microbial pesticide technology and discloses a strain of Bacillus thuringiensis ZYLT064 with highly effective insecticidal activity against cotton aphids and two-spotted spider mites, and its application. The strain of the present invention has a deposit number of CCTCC NO: M 20242335 at the China Center for Type Culture Collection. The strain can produce thuringiensis and trans-aconitic acid insecticidal active substances, and its fermentation supernatant exhibits significant insecticidal activity against cotton aphids and two-spotted spider mites. The mortality rate of cotton aphids fed in laboratory tests reached 87%, the mortality rate of two-spotted spider mites reached 80.9%, and the mortality rate of two-spotted spider mites tested in greenhouse pots reached 72.5%. Therefore, the strain of the present invention has the potential to be developed into a multifunctional biological control product and has broad application prospects.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial pesticides, and in particular relates to a Bacillus thuringiensis strain having high insecticidal activity against cotton aphids and two-spotted spider mites and its application. Background Art

[0002] Agricultural pests cause enormous economic losses worldwide each year. These pests are numerous, pose serious risks, and are difficult to control. Among them, cotton aphids, one of the world's top ten plant pests, severely restrict the cultivation and production of cotton and other cash crops in my country due to their rapid reproduction, strong adaptability, and susceptibility to insecticide resistance. Meanwhile, the two-spotted spider mite, a global pest, has a wide host range and can harm fruit trees, vegetables, and grain crops. Currently, control of cotton aphids and two-spotted spider mites primarily relies on chemical pesticides, while biological control utilizes microorganisms such as Beauveria bassiana and Pseudomonas syringae. Existing control methods have numerous shortcomings, including diminishing efficacy, increasing insecticide resistance, and environmental pollution, making them inadequate for sustainable agricultural development. Therefore, there is an urgent need to develop new, environmentally friendly biological control agents to effectively combat the damage caused by agricultural pests.

[0003] Biopesticides made from Bacillus thuringiensis (Bt) are widely used in agriculture. In addition to its insecticidal crystal proteins, Bt produces a number of other bioactive substances at different stages of its growth, which exert their insecticidal activity individually or in synergistic ways. However, there are few reports on the use of B. thuringiensis for the control of cotton aphids and spider mites. Therefore, screening for B. thuringiensis strains with high efficacy against cotton aphids and spider mites has great potential for application. Summary of the Invention

[0004] In view of the technical deficiencies, the first object of the present invention is to provide a Bacillus thuringiensis strain that can produce multiple metabolites such as thuringiensis and trans-aconitic acid, and whose fermentation supernatant exhibits highly effective insecticidal activity against cotton aphids and two-spotted spider mites.

[0005] The laboratory of the inventor has long been committed to the construction of a Bt strain resource library. Relying on this resource library and related information databases, through large-scale genome analysis and biological activity determination, Bt strains with excellent biological activity in controlling cotton aphids and two-spotted spider mites were screened.

[0006] Specifically, the strain was sent to the China Center for Type Culture Collection (CCTCC) for preservation on October 25, 2024, and was classified and named Bacillus thuringiensis ZYLT064, with the preservation number CCTCC M20242335. The preservation address is China Center for Type Culture Collection, Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The Bacillus thuringiensis ZYLT064 strain of the present invention not only significantly improves its ability to produce thuringiensisin but also possesses highly effective insecticidal activity against cotton aphids and two-spotted spider mites. This strain provides a valuable resource for the development of highly effective Bt biological agents for the control of cotton aphids and two-spotted spider mites. It also provides important support for addressing difficult-to-control piercing-sucking pests in my country and has broad application prospects in agricultural and industrial production.

[0008] It should be noted that the Bacillus thuringiensis ZYLT064 screened by the present invention has the following morphological characteristics: after being cultured on LB medium at 28°C for 12 hours, the colonies are milky white in color, round in shape, with a frosted surface and irregular edges ( Figure 1 Microscopic observation showed that the strain was rod-shaped, capable of forming spores and producing rhombus-shaped crystals, and had typical characteristics of Bacillus thuringiensis ( Figure 3A In addition, the present invention constructed a phylogenetic tree based on the ZYLT064 genome ( Figure 2 ).

[0009] A second objective of the present invention is to provide a biological control agent with toxic activity against cotton aphids and two-spotted spider mites. The active ingredient is prepared from the fermentation supernatant of Bacillus thuringiensis ZYLT064 of the present invention. The fermentation supernatant contains trans-aconitic acid and thuringiensis. The biological control agent can be formulated as a suspension concentrate, dust, granule, oil suspension, or wettable powder. This agent is highly effective in killing cotton aphids and is suitable for biological control of cotton aphids. Furthermore, the agent exhibits excellent insecticidal activity against two-spotted spider mites, effectively controlling the damage caused by these pests to various crops.

[0010] The third object of the present invention is to provide the following two applications: (1) application of the Bacillus thuringiensis ZYLT064 in controlling cotton aphids and / or two-spotted spider mites; and (2) application of the biological control agent in controlling cotton aphids and / or two-spotted spider mites.

[0011] Compared with the prior art, the Bacillus thuringiensis ZYLT064 and its application provided by the present invention have the following significant advantages and advancements:

[0012] (1) The fermentation supernatant of the strain ZYLT064 of the present invention exhibited highly effective insecticidal activity against cotton aphids. The fermentation supernatant was diluted 10-fold and used in a bioactivity assay against cotton aphids. The results showed that after feeding the aphids for 72 days, the insecticidal rate reached 87%. Furthermore, high-temperature treatment of the fermentation supernatant did not affect the insecticidal effect, demonstrating that the fermentation liquid has a strong toxic effect against cotton aphids and good thermal stability.

[0013] (2) The fermentation supernatant produced by strain ZYLT064 of the present invention exhibits excellent insecticidal activity against two-spotted spider mites. After feeding the fermentation supernatant to two-spotted spider mites for 72 hours, the mortality rate reached 80.9%. Results from greenhouse potted plants showed a mortality rate of 72.5%, demonstrating that the fermentation supernatant has excellent toxicity against two-spotted spider mites.

[0014] (3) The inventors discovered that the natural secondary metabolites of strain ZYLT064 have highly effective insecticidal activity against cotton aphids and two-spotted spider mites, and prepared them into biological control agents, filling the gap in the use of Bacillus thuringiensis to control cotton aphids. This is also the first discovery of the use of natural secondary metabolites of Bacillus thuringiensis for the control of two-spotted spider mites, enriching the strain resources for the efficient control of two-spotted spider mites.

[0015] (4) The strain ZYLT064 of the present invention has the potential to be developed into a multifunctional biocontrol product and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly describes the drawings involved in the embodiments of the present invention. It should be noted that the drawings listed only present part of the contents of the embodiments of the present invention.

[0017] Figure 1 The figure shows the strain morphology of ZYLT064 strain on LB solid medium.

[0018] Figure 2 This is a phylogenetic tree constructed based on the genome of strain ZYLT064.

[0019] Figure 3 shows the cell morphology of strain ZYLT064 under an optical microscope (eyepiece 10×, objective lens 100×) (A) and the production of thuringiensis and trans-aconitic acid after fermentation in a 250 mL shake flask at 28°C for 48 h (B).

[0020] Figure 4 These are the results of the bioactivity test of the fermentation supernatant of strain ZYLT064 against cotton aphids.

[0021] Figure 5 shows the bioactivity test results of the fermentation supernatant of strain ZYLT064 against two-spotted spider mites (A) and the potted bioassay results (B). DETAILED DESCRIPTION

[0022] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention is further described in detail below with reference to the following examples. Unless otherwise specified, the experimental methods used are all reported conventional microbiological procedures. All consumables and reagents used were obtained from commercial sources.

[0023] Example 1: Cultivation and fermentation of Bacillus thuringiensis ZYLT064

[0024] (1) Activation of the strain: The ZYLT064 strain stored in a -80°C cryotube was transferred to LB solid medium and incubated upside down in a 28°C incubator overnight;

[0025] (2) Seed liquid preparation: Pick a single colony from LB solid medium and transfer it to 10 mL LB liquid medium. Incubate at 28°C, 220 rpm, and shake for 12 h.

[0026] (3) Shake flask fermentation: The strain seed liquid was inoculated into LB medium at a 1% inoculum size and cultured at 30°C, 220 rpm, and shaken for 36 h;

[0027] (4) Fermentation in a 50 L fermenter: The seed culture solution was inoculated at a 2% inoculum into the sterilized fermentation medium in the fermenter. The fermentation temperature was 28-30°C, the fermentation time was 36-48 h, and the pH was controlled at about 7.5. During this period, samples were taken for microscopic examination and the content of thuringiensis was detected.

[0028] Example 2: Quantitative Detection of Thuringin Content in Bacillus thuringiensis Fermentation Supernatant by High Performance Liquid Chromatography

[0029] (1) The fermentation supernatant obtained according to the method of Example 1 was filtered and sterilized using a 0.22 μm pore size water filter membrane;

[0030] (2) Prepare 0.1% formic acid, 30 mM ammonium acetate, and methanol and perform sonication to remove bubbles;

[0031] (3) Liquid phase conditions were: chromatographic column Agent HC-C18 (Analytical 4.6x250 mm 5-Micron), mobile phase A was deionized water with 30 mM ammonium acetate and 0.1% acetic acid to adjust the pH, and phase B was methanol. Gradient elution was performed during analysis: the volume content of methanol was 5% from 0 min to 8 min, 5% to 50% from 8 min to 10 min, and 85% to 5% from 10 min to 17 min. The flow rate of the mobile phase was 0.7 mL / min, and the injection volume was 10 μL. After the ZYLT064 strain was fermented in a 250 ml shake flask at 28°C for 48 hours, the yield of thuringiensis was 1.8 g / L ( Figure 3B ).

[0032] Example 3: Quantification of trans-aconitic acid by liquid chromatography-mass spectrometry

[0033] (1) After fermentation of Bacillus thuringiensis ZYLT064, the fermentation supernatant prepared according to the method in Example 1 was filtered using a 0.22 μm pore size water filter membrane and placed in a sampling bottle;

[0034] (2) Prepare 0.1% acetic acid and acetonitrile solution and sonicate to remove bubbles;

[0035] (3) Programming: A ZORBAX SB-Aq analytical column was used, and the parameters were set as follows: injection volume 1 μL, flow rate 0.3 mL / min, single sample run time 4 min, negative ion mode, nebulizer flow 2 L / min, heater flow 10 L / min, interface temperature 300°C, DL temperature 250°C, heating block temperature 400°C, drying gas flow 10 L / min, detection of precursor 173 m / z, and detection of products 128.85 m / z and 84.9 m / z. After fermentation of strain ZYLT064 in a 250 mL shake flask at 28°C for 48 h, the yield of trans-aconitic acid was 0.5 g / L as determined by liquid chromatography-mass spectrometry.

[0036] Example 4: Feeding test of Bacillus thuringiensis ZYLT064 fermentation supernatant to kill cotton aphids

[0037] The fermentation supernatant obtained in Example 1 was diluted 10 times and used as the test sample. The bioactivity of the sample on cotton aphids was determined using the capsule artificial feed method. The specific method is as follows:

[0038] (1) The cage is a double-tube with two openings (d = 2.5 cm, h = 3 cm) made of organic glass. One end of the double-tube is covered with a layer of parafilm and sterilized under ultraviolet light for 30 min. 300 μL of the sample to be tested (the sample and artificial diet are mixed at a volume ratio of 1:9) is added to the center of the parafilm and covered with another piece of parafilm treated with ultraviolet light to form a small capsule containing the sample to be tested.

[0039] (3) Use a small brush to pick up newly hatched 2-day-old nymphs and transfer them to the paraffin film in the breeding cage. Invert it for 30 seconds to ensure that the aphids are fixed and feeding, and then put the breeding cage upright.

[0040] (4) Each double-pass tube was inoculated with 30 cotton aphid nymphs, and each treatment was replicated at least three times;

[0041] (5) After the cotton aphids were connected, the other end of the double-way tube was sealed with gauze and covered with black breathable gauze;

[0042] (6) The bioassay device was placed in an artificial climate chamber at a temperature of 25°C, a photoperiod of 16:8, and a humidity of 70%;

[0043] (7) The feeding and survival of the test insects were observed and counted every day. The feed was changed every 2 days and the number of aphids was counted every 24 hours. The mortality rate of cotton aphids was observed to determine the insecticidal activity of the substance. Three independent repeated experiments were conducted to verify its insecticidal activity against cotton aphids. The results showed that the 10-fold dilution of the fermentation supernatant fed cotton aphids for 72 hours reached 87% of the insecticidal activity against cotton aphids ( Figure 4 ).

[0044] Example 5: Feeding test of the fermentation supernatant of Bacillus thuringiensis ZYLT064 to kill spider mites

[0045] (1) Cut the double-sided tape into 2 cm long pieces and stick them on one end of the slide. Then select healthy female adult mites and stick their backs on the double-sided tape (be careful not to stick the mite legs, antennae and mouthparts). Place 20-30 mites on each slide and place them in a container padded with a wet sponge at 26°C.

[0046] (2) The slide was immersed in the fermentation supernatant (prepared according to the method of Example 1) and gently shaken for 5-10 seconds before being taken out. The mite body and the surrounding excess residual liquid were absorbed with absorbent paper, and the slide was placed on a white disk padded with a wet sponge (the sponge was replenished with water every day to ensure that it was moist), and the slide was placed in a light incubator at 26°C, 60%-70% humidity, and a light cycle of L:D = 16h:8h for breeding and observation. The mortality was checked every 24 hours, and the total number of insects and the number of dead insects were recorded respectively, and the mortality rate was calculated. Three independent repeated experiments were conducted to verify its activity against two-spotted spider mites. The results showed that the fermentation supernatant had an activity of 80.9% against two-spotted spider mites after 72 hours of treatment ( Figure 5A ).

[0047] Example 6: Potted test of the fermentation supernatant of Bacillus thuringiensis ZYLT064 to kill spider mites

[0048] (1) Plant the germinated white kidney beans in a pot filled with nutrient soil and keep them ready for use after the potted plants grow four true leaves to ensure the healthy growth of the potted white kidney beans.

[0049] (2) Use a No. 0 brush to pick 30 adult female mites and place them in a potted white kidney bean plant that has just grown two leaves and has expanded. Allow the mites to move freely for 4 hours.

[0050] (3) Prepare the handheld sprayer and check whether it is intact and ensure there is no leakage.

[0051] (4) Take the sterile fermentation supernatant of the strain obtained in Example 1 and pour it into a sprayer.

[0052] (5) Use a handheld sprayer to evenly spray the front and back of the white kidney bean leaves. Finish spraying when the leaves are moist and the droplets on the leaves have not dripped off. Pay attention when spraying and aim the nozzle at the leaf surface and back of the white kidney bean to ensure that the liquid is evenly covered. Set the aqueous solution as a blank control and 10ug / ml of avermectin as a positive control. Repeat each treatment three times.

[0053] (6) The treated potted plants were cultured in a greenhouse environment with a temperature of 26±1°C, a humidity of 60%-70%, and a light intensity of L:D=16h:8h.

[0054] (7) After spraying, the growth of white kidney bean plants and the impact of spider mites were observed every day, the number of live insects was recorded, and the results were statistically analyzed to evaluate the effect of the fermentation supernatant of the strain. The efficacy calculation method is: mite population reduction rate (%) = (number of live mites before application - number of live mites after application) / number of live mites before application * 100; control effect (%) = (mite population reduction rate of treatment - mite population reduction rate of blank control) / (1 - mite population reduction rate of blank control) * 100. Three independent repeated experiments were conducted to verify its activity in killing two-spotted spider mites. The potted results showed that the fermentation supernatant had 72.5% activity in killing two-spotted spider mites after 72 hours of spraying ( Figure 5B ).

Claims

1. A Bacillus thuringiensis ( Bacillus thuringiensis ) ZYLT064 strain, whose deposit number in China Center for Type Culture Collection is CCTCC NO: M 20242335.

2. A biological control agent, characterized in that: The active ingredient of the biological control agent is the Bacillus thuringiensis ( Bacillus thuringiensis ) was prepared from the fermentation supernatant of ZYLT064.

3. The biological control agent according to claim 2, characterized in that The fermentation supernatant contains trans-aconitic acid and thuringiensis.

4. The biological control agent according to claim 2, characterized in that The biological control agent is in the form of a suspension, a powder, a granule, an oil suspension or a wettable powder.

5. The Bacillus thuringiensis according to claim 1 ( Bacillus thuringiensis ) Application of ZYLT064 in the control of cotton aphids and two-spotted spider mites.

6. Use of the biological control agent according to claim 2 in controlling cotton aphids and two-spotted spider mites.

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