Bacillus thuringiensis with efficient insecticidal activity on cotton aphid and tetranychus urticae and application of bacillus thuringiensis

Abstract

The invention belongs to the technical field of microbial pesticides, and discloses a bacillus thuringiensis ZYLT064 with efficient insecticidal activity on cotton aphid and tetranychus urticae and application of the bacillus thuringiensis ZYLT064. The preservation number of the strain in the China Center for Type Culture Collection (CCTCC) is CCTCC NO: M 20242335; the strain can generate insecticidal active substances of bacillus thuringiensis and trans-aconitic acid, fermented supernate of the strain shows remarkable insecticidal activity on cotton aphids and tetranychus urticae, the fatality rate of the cotton aphids biologically tested by feeding in a laboratory reaches 87%, the fatality rate of the tetranychus urticae is 80.9%, and the fatality rate of the tetranychus urticae biologically tested by potting in a greenhouse reaches 72.5%. Therefore, the strain disclosed by the invention has the potential of being developed into a multifunctional biological control product and has a wide application prospect.

Description

Technical Field

[0001] The 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 an application thereof. Background Art

[0002] Agricultural pests can cause huge economic losses to the world every year. There are many kinds of pests, which are serious and difficult to control. Among them, cotton aphids, as one of the world's top ten plant pests, have serious restrictions on the planting and production of cotton and other economic crops in my country due to their fast reproduction speed, strong adaptability, and easy to develop resistance to pesticides. At the same time, the two-spotted spider mite is a global pest mite with a wide host range, which can harm fruit trees, vegetables and grain crops. At present, the prevention and control of cotton aphids and two-spotted spider mites mainly relies on chemical pesticides, while biological control uses microorganisms such as Beauveria bassiana and Pseudomonas syringae. There are many shortcomings in the existing prevention and control methods, including the gradual weakening of drug efficacy, the increase of pest resistance and the pollution of the environment, which are difficult to meet the needs of green agricultural development. Therefore, it is urgent to develop new environmentally friendly biological control agents to effectively deal with the damage caused by agricultural pests.

[0003] Biopesticides made from Bacillus thuringiensis (Bt) have been widely used in agriculture. In addition to insecticidal crystal proteins, Bt produces many other biologically active substances at different growth stages, which exert their insecticidal activity alone or in a synergistic manner. However, there are few reports on the use of Bacillus thuringiensis to control cotton aphids and two-spotted spider mites. Therefore, screening Bacillus thuringiensis with high efficiency in killing cotton aphids and two-spotted spider mites has great application value. Summary of the invention

[0004] In view of the technical deficiencies, the first object of the present invention is to provide a strain of Bacillus thuringiensis, which can produce multiple metabolites such as thuringiensis and trans-aconitic acid, and its fermentation supernatant shows efficient 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 preventing and controlling cotton aphids and two-spotted spider mites were screened out.

[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 of Wuhan University, Bayi Road, Wuchang District, Wuhan City, Hubei Province.

[0007] The Bacillus thuringiensis ZYLT064 of the present invention not only has a significantly improved ability to produce thuringiensisin, but also has a highly effective insecticidal activity against cotton aphids and two-spotted spider mites. The strain provides a valuable strain resource for the development of Bt biological agents for the efficient prevention and treatment of cotton aphids and two-spotted spider mites, and also provides important support for solving the difficult-to-prevent piercing-sucking insect pests in my country, and has a wide range of 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, frosted on the surface, and irregular on the 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 constructs a phylogenetic tree based on the ZYLT064 genome ( Figure 2 ).

[0009] The second object of the present invention is to provide a biological control agent with toxic activity against cotton aphids and two-spotted spider mites, wherein 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 thuringiensisin, and the dosage form of the biological control agent can be selected from suspension, powder, granule, oil suspension or wettable powder. The agent has a high efficiency in killing cotton aphids and is suitable for biological control of cotton aphids; at the same time, the agent exhibits an excellent insecticidal effect on two-spotted spider mites and can effectively control the harm of harmful mites 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. (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 exhibits highly effective insecticidal activity against cotton aphids. The fermentation supernatant was diluted 10 times and used for the biological activity assay of cotton aphids. The results showed that after feeding cotton aphids for 72 hours, the insecticidal rate reached 87% and the insecticidal effect was not affected by high temperature treatment of the fermentation supernatant, which proved that the fermentation liquid had a strong toxic effect on cotton aphids and had good thermal stability.

[0013] (2) The fermentation supernatant produced by the strain ZYLT064 of the present invention has good insecticidal activity against two-spotted spider mites. After the fermentation supernatant was fed to two-spotted spider mites for 72 hours, the mortality rate of two-spotted spider mites reached 80.9%, and the results of greenhouse potted plants showed that the mortality rate of two-spotted spider mites reached 72.5%, proving that the fermentation supernatant has an excellent toxic effect on 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 biological control product and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] In order to more clearly illustrate the technical solution of the embodiment of the present invention, the drawings involved in the embodiment of the present invention are briefly described below. It should be noted that the listed drawings only present part of the contents of the embodiment 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 thuringiensisin and trans-aconitic acid by strain ZYLT064 after fermentation in a 250 mL shake flask at 28°C for 48 h (B).

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

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

[0022] In order to make the purpose, technical scheme and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. Unless otherwise specified, the experimental methods used are all reported conventional microbiological operation methods. The consumables and reagents used are all from commercial channels.

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

[0024] (1) Activation of bacterial strains: The ZYLT064 strain stored in a -80°C cryotube was transferred to LB solid medium and inverted in a 28°C incubator for overnight culture;

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

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

[0027] (4) Fermentation in a 50 L fermenter: The seed culture solution was inoculated at a 2% inoculation rate 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 the fermentation, samples were taken for microscopic examination and the content of thuringiensis was detected.

[0028] Example 2: Quantitative detection of thuringiensis 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 water filter membrane with a pore size of 0.22 μm;

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

[0031] (3) Liquid phase conditions: chromatographic column Agent HC-C18 (Analytical 4.6x250 mm 5-Micron), mobile phase A is deionized water with 30mM ammonium acetate and 0.1% acetic acid to adjust the pH, and phase B is methanol. Gradient elution was performed during analysis: the volume content of methanol was: 5% from 0min to 8min, 5% to 50% from 8min to 10min, and 85% to 5% from 10min to 17min. The flow rate of the mobile phase was 0.7mL / min, and the injection volume was 10μL. After the ZYLT064 strain was fermented in a 250ml shake flask at 28°C for 48 hours, the production of thuringiensis was 1.8g / 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 water filter membrane with a pore size of 0.22 μm and placed in a sample injection bottle;

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

[0035] (3) Program setting: A ZORBAX SB-Aq analytical column was selected, 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, detection of products 128.85 m / z and 84.9 m / z. After fermentation of strain ZYLT064 at 28 °C in a 250 ml shake flask 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 the fermentation supernatant of Bacillus thuringiensis ZYLT064 to kill cotton aphids

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

[0038] (1) The breeding cage is a double-channel tube (d = 2.5 cm, h = 3 cm) with two upper and lower openings made of organic glass; one end of the double-channel tube is covered with a layer of parafilm, which is sterilized by irradiation under ultraviolet light for 30 min, and 300 μL of the sample to be tested (the sample and artificial feed are mixed at a volume ratio of 1:9) is dripped into the center of the film, 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 25°C, with 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, the number of aphids was counted every 24 hours, and 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 two-spotted spider mites

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

[0046] (2) Immerse the glass slide in the fermentation supernatant (prepared according to the method of Example 1) and gently shake for 5-10 seconds, then take it out, absorb the mite body and the surrounding excess residual liquid with absorbent paper, place it on a white disk padded with a wet sponge (replenish water to the sponge every day to ensure that it is moist), and place it in a light incubator at 26°C, humidity 60%-70%, and light cycle L:D=16h:8h for breeding and observation. Check the mortality every 24 hours, record the total number of insects and the number of dead insects, and calculate the mortality rate. Perform three independent repeated experiments to verify its activity against two-spotted spider mites. The results show that the fermentation supernatant has 80.9% activity against two-spotted spider mites after 72 hours of treatment ( Figure 5A ).

[0047] Example 6: Pot test of the fermentation supernatant of Bacillus thuringiensis ZYLT064 to kill Tetranychus urticae

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

[0049] (2) Use a No. 0 brush to pick 30 adult female mites from 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 sides of the white kidney bean leaves. When the leaves are moist and no droplets fall off, spray until the leaves are sprayed. Pay attention when spraying and aim the spray 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 avermectin 10ug / ml as a positive control. Repeat each treatment three times.

[0053] (6) The treated potted plants are placed 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 for cultivation.

[0054] (7) After spraying, observe the growth of white bean plants and the impact on spider mites every day, record the number of live insects, and statistically analyze the results to evaluate the effect of the fermented 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 after treatment - mite population reduction rate after blank control) / (1 - mite population reduction rate after blank control) * 100. Three independent repeated experiments were conducted to verify its activity against two-spotted spider mites. The potted plant results showed that the fermented supernatant had an activity of 72.5% against two-spotted spider mites after 72 hours of spraying ( Figure 5B ).

Claims

1. A 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 prepared from the fermentation supernatant of Bacillus thuringiensis ZYLT064 according to claim 1.

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. Use of the Bacillus thuringiensis ZYLT064 according to claim 1 in controlling cotton aphids and / or two-spotted spider mites.

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

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