Application of thuringirin in prevention and treatment of cotton aphid, green peach aphid, armyworm and soybean cyst nematode

Abstract

The invention discloses application of thuringirin in prevention and treatment of cotton aphid, green peach aphid, armyworm and soybean cyst nematode, and belongs to the field of microbial pesticides. The invention discovers that the thurinin has the biological activity of killing cotton aphid, green peach aphid, mythimna separata and soybean cyst nematode for the first time, has a wide application prospect, and can be developed into a novel environment-friendly biological control agent.

Description

Technical Field

[0001] This invention belongs to the field of microbial pesticides, specifically, it relates to the application of a compound produced by Bacillus thuringiensis, Bacillus thuringiensis, in the control of cotton aphids, peach aphids, armyworms and soybean cyst nematodes. Background Technology

[0002] Thuringiensin (Thu) is a secondary metabolite of a nucleic acid analog produced by Bacillus thuringiensis, also known as a β-exotoxin. Its structural formula is shown in formula (I). Thu is composed of adenosine, glucose, phosphate groups, and alloviscosine in a 1:1:1:1 ratio, with the molecular formula C. 22 H 32 O 19 N5P has a molecular weight of 701 Da. Current research has not yet achieved the large-scale, stable preparation of pure Bacillus thuringiensis for target bioactivity assays, nor have there been any reports on using Bacillus thuringiensis with a purity higher than 80% for bioactivity evaluation. Current studies still use fermentation broths or low-purity formulations containing Bacillus thuringiensis as research subjects for bioassays. However, fermentation broths usually contain multiple active or interfering components such as insecticidal crystal proteins and small-molecule organic acids, and low-purity Bacillus thuringiensis formulations also have similar problems, making it difficult to accurately attribute the observed bioactivity to Bacillus thuringiensis itself.

[0003]

[0004] (I)

[0005] The cotton aphid (Aphis gossypii), also known as the melon aphid, belongs to the family Aphididae in the order Hemiptera and is one of the major pests in cotton production. It inhibits plant growth by sucking sap from cotton seedlings and transmits various cotton viral diseases. Its honeydew secretions can also cause "sticky cotton," affecting fiber quality. Currently, the main chemical pesticides used to control cotton aphids are neonicotinoid insecticides. Imidacloprid, as a commonly used and highly effective agent, has been shown to have a low LC50 (lowest concentration) against cotton aphids after testing under the same conditions. 50 It was 67.10 μg / mL.

[0006] The peach aphid (Myzus persicae), belonging to the family Aphididae in the order Hemiptera, is one of the most destructive aphid species in the world, damaging more than 400 plant species, including those in the Brassicaceae, Solanaceae, and Cucurbitaceae families. It has a high reproductive rate and a wide host range, directly sucking sap from leaves and young shoots, causing leaf curling, malformation, and other growth disorders. It also secretes honeydew, inducing sooty mold and transmitting various plant viruses, severely impacting yield and quality. While chemical control of the peach aphid is widely used, it faces challenges such as rapid development of resistance, high toxicity to non-target organisms, and easy residues in crops and soil. Therefore, there is an urgent need to develop new, environmentally friendly biological insecticides for the control of the peach aphid.

[0007] Armyworm (Mythimna separata Walker), belonging to the Noctuidae family of Lepidoptera, is a major outbreak pest of grain crops in my country, exhibiting typical migratory characteristics. Armyworms primarily damage gramineous crops such as wheat, corn, and rice. Before the third instar, larvae mainly feed on leaf tissue; after the third instar, they feed along leaf margins, causing severe notching and sometimes even completely consuming the leaf, leaving only the veins. In high densities, the larvae can migrate en masse, causing rapid damage to large areas of farmland. While chemical control can suppress insect populations in the short term during outbreaks, its application is limited by resistance risks, ecological impacts, and pesticide residue restrictions, necessitating safer and more sustainable control technologies. For example, commonly used chemical insecticides for armyworm control include dinotefuran, chlorpyrifos, and lambda-cyhalothrin. Although these have strong insecticidal activity against armyworms, their high toxicity, rapid action, and tendency to produce high pesticide residues in the field pose significant environmental and food safety risks.

[0008] Soybean cyst nematode (SCN) is one of the main soil-borne pathogens causing soybean yield reduction. Its second-instar larvae can actively infect the roots of host plants in the soil, forming cysts. It has a wide host range and can survive in the soil for extended periods. Existing research shows that different nematicides have significant differences in field control efficacy against soybean cyst nematode. For example, abamectin granules require a dosage of 60 kg / hm², thiazophos requires 33 kg / hm², and tea saponin even requires 100 kg / hm² to achieve moderate control efficacy (Luo Ning et al. Control effects of different nematicides on soybean cyst nematode disease [J]. Shaanxi Agricultural Sciences, 2020, 66(01):10-14.).

[0009] In summary, cotton aphids, peach aphids, armyworms, and soybean cyst nematodes all cause serious damage to agricultural production, while existing chemical control measures have shortcomings in terms of resistance, environmental safety, and application restrictions. Therefore, there is an urgent need to provide a novel, safe, and efficient control method or formulation based on Bacillus thuringiensis for the green control of the aforementioned agricultural pests and pathogenic nematodes. Summary of the Invention

[0010] In view of the problems existing in the prior art, the present invention provides the use of Thuringiensin (Thu), a secondary metabolite produced by Bacillus thuringiensis, in the control of cotton aphids, peach aphids, armyworms, and soybean cyst nematodes.

[0011] To achieve the above objectives, the inventors employed a combination of various separation and purification methods, effectively improving the purification efficiency of Bacillus thuringiensis and successfully obtaining several grams of Bacillus thuringiensis with purities of 80.26% and 90.50%. This provides a reliable basis for evaluating its activity against target organisms such as cotton aphids, peach aphids, armyworms, and soybean cyst nematodes. Based on years of experience in biopesticide research, and through extensive experimental research and relentless exploration, the following technical solution was ultimately obtained:

[0012] (1) Application of Bacillus thuringiensis in controlling cotton aphids or inhibiting cotton aphid oviposition. Currently, there are no literature reports on the bioactivity of Bacillus thuringiensis in killing cotton aphids. The experiments of this invention found that 80.26% pure Bacillus thuringiensis showed a lower LC50 value in the indoor encapsulated artificial feed method. 50 The concentration was 31.94 μg / ml, and at this half-lethal concentration, Bacillus thuringiensis was able to inhibit the oviposition capacity of cotton aphids by 51.87%.

[0013] (2) Application of Bacillus thuringiensis in the control of peach aphids. Currently, there are no research reports on the bioactivity of Bacillus thuringiensis in killing peach aphids. Further, the experiments of this invention found that the bioactivity of 90.50% pure Bacillus thuringiensis against peach aphids was determined by LC50. 50 It was 352.23 μg / ml.

[0014] (3) Application of Bacillus thuringiensis in the control of armyworms. Currently, there are no literature reports on the bioactivity of Bacillus thuringiensis in killing cotton aphids. Further preferably, experiments of this invention found that the bioactivity of 90.50% pure Bacillus thuringiensis against armyworms was measured at LC50. 50 It was 94.31 μg / ml.

[0015] (4) Application of Bacillus thuringiensis in killing soybean cyst nematodes. Currently, there are no literature reports on the bioactivity of Bacillus thuringiensis in killing soybean cyst nematodes. Further preferably, experiments of this invention found that the bioactivity of 90.50% pure Bacillus thuringiensis in the laboratory was measured to be LC. 50 It was 29.36 μg / ml.

[0016] Compared with the prior art, the advantages and advancements of the present invention are as follows:

[0017] (1) Imidacloprid, as a commonly used and highly effective pesticide, was found to have a low LC50 against cotton aphids after testing under the same conditions.50 The concentration was 67.10 μg / mL, while the LC50 of the Bacillus thuringiensis obtained in this invention against cotton aphids was... 50 With a concentration of only 31.94 μg / mL, its toxicity is twice that of imidacloprid, demonstrating significantly higher insecticidal efficiency. Furthermore, chemical control of cotton aphids includes acetamiprid, thiamethoxam, and pymetrozine, but the increasing resistance of cotton aphids to various agents and their toxic effects on non-target organisms limit the use of existing chemical pesticides. In contrast, Bacillus thuringiensis, derived from microorganisms, is more suitable as a green biological control agent to replace existing chemical control methods.

[0018] (2) The Bacillus thuringiensis of the present invention exhibits high insecticidal activity against armyworms, and its origin from microorganisms can significantly reduce environmental safety risks while maintaining the preventive effect.

[0019] (3) When controlling soybean cyst nematodes, abamectin granules require a dosage of 60 kg / hm², thiazophos requires 33 kg / hm², and tea saponin even requires 100 kg / hm² to achieve moderate control efficacy. In contrast, the Bacillus thuringiensis in this invention has strong toxic activity and can effectively inhibit the infection of soybean cyst nematodes. At the same time, Bacillus thuringiensis can compensate for the environmental pressure of chemical pesticides and can be used as a green alternative to chemical nematicides.

[0020] (4) This invention is the first to discover that Bacillus thuringiensis has significant insecticidal activity against a variety of major agricultural pests that are seriously harmful, difficult to control, and prone to developing resistance to chemical pesticides, such as cotton aphid, peach aphid, armyworm and soybean cyst nematode.

[0021] (5) Bacillus thuringiensis, as an active ingredient, can be used in the development of new biological insecticides, which can make up for the shortcomings of traditional chemical pesticides, which are highly toxic and have limited effectiveness in controlling such difficult-to-control and easily resistant pests. It has important promotion value and broad industrialization prospects. Attached Figure Description

[0022] Figure 1 (A) Bioactivity assay curve of 80.26% pure Bacillus thuringiensis against cotton aphid; (B) Bioactivity assay curve of 70% imidacloprid against cotton aphid; (C) Effect of 31.94 μg / ml Bacillus thuringiensis on cotton aphid oviposition.

[0023] Figure 2 Bioactivity curve of 90.50% pure Bacillus thuringiensis against peach aphid.

[0024] Figure 3 Bioactivity curve of 90.50% pure Bacillus thuringiensis against armyworm.

[0025] Figure 4Bioactivity curve of 90.50% pure Bacillus thuringiensis against soybean cyst nematode. Detailed Implementation

[0026] The following are specific embodiments of the present invention, which describe the technical solutions and effects of the present invention in detail. Unless otherwise specified, the technical operations or conditions described in the literature in this field are all performed using conventional techniques or conditions. Unless otherwise specified, the reagents or instruments used are all conventional products that can be purchased normally.

[0027] Example 1: Preparation of Bacillus thuringiensis

[0028] Bacillus thuringiensis strain ZYLT064 (CCTCC NO: M 20242335), which produces Bacillus thuringiensis, was streaked onto LB agar plates for activation. Single colonies were then transferred to PA bottles containing LB medium and incubated overnight at 28°C and 220 rpm. Subsequently, 1% of the culture was inoculated into LB seed medium and incubated at 28°C and 220 rpm for 12 h. Finally, 2% of the culture was inoculated into a 50L fermenter for fermentation at 30°C for 36 h, with a packing factor of 60%. The fermentation medium consisted of: glucose 30 g / L, peptone 10 g / L, magnesium sulfate 0.6 g / L, manganese sulfate 0.14 g / L, calcium chloride 0.2 g / L, and 1 mL / L antifoaming agent. After 36 hours of fermentation, the content of thallium was detected by high performance liquid chromatography (HPLC), and the fermentation broth was used to extract, separate and purify thallium. The obtained samples were used in the experiments of Example 2, Example 3, Example 4 and Example 5.

[0029] The steps for separating and purifying thuringin are as follows:

[0030] (1) Centrifuge the fermentation broth to remove the bacterial cells, and then spray dry the fermentation supernatant to obtain powder.

[0031] (2) Dissolve 50g of spray-dried powder in 1L of ddH2O, and then enrich and purify it using a 717 type strong base anion exchange resin. The specific steps are as follows:

[0032] ① Pack 60 g of 717 type resin into a column. First, treat the column alternately with 1 mol / L hydrochloric acid and 1 mol / L sodium hydroxide, rinsing with plenty of water between the acid and alkali treatments. Repeat this treatment three times, with the amount of acid or alkali used each time being twice the volume of the resin. For the final treatment, the anion exchange resin should be converted to the OH form with alkali, using double the amount of alkali. Then rinse with water until neutral.

[0033] ② After pretreatment and column packing of the 717 resin, 1 L of fermentation broth was added. After adsorption, the column was eluted with deionized water until the pH was neutral, then eluted with 0.01 mol / L HCl, followed by elution with 0.05 mol / L HCl at a flow rate of 4 mL / min. The eluent was collected in 40 mL tubes. The pH was adjusted and the Thu content in each eluent was measured. The eluents with high Thu content were combined. The eluents with high Thu content were freeze-dried to obtain Bacillus thuringiensis with a purity of 80.26%.

[0034] (3) The combined eluent with high Thu content was freeze-dried to obtain a dry powder, which was then dissolved in 10 ml ddH2O. The powder was then further purified by semi-preparative liquid chromatography (SLC). The SLC conditions were as follows: PRC-ODS EE0762 column, flow rate 10 mL / min, UV detection wavelength 254 nm, injection volume 1 mL, mobile phase A 0.1% formic acid-water mixture, and mobile phase B methanol. The collected solution was freeze-dried again to obtain thallium thallium with a purity of 90.50%.

[0035] The above steps are repeated multiple times to obtain sufficient bioassay samples.

[0036] Example 2: Indoor biological activity of Bacillus thuringiensis in killing cotton aphids and inhibiting cotton aphid oviposition.

[0037] (1) Dissolve the 80.26% purity Bacillus thuringiensis sample in ultrapure water, filter and sterilize it, and then mix it with liquid feed according to the required dilution to prepare liquid capsules with final concentrations of 1000, 500, 100, 50, 25 and 12.5 μg / mL.

[0038] (2) Use a small brush to pick up 30 nymphs and transfer them to the rearing cage. After introducing the cotton aphids, seal the other end of the double-ended tube with gauze and cover it with black breathable gauze. Invert the cage for 30 seconds to ensure that the aphids are fixed and feeding. Then, turn the rearing cage upright. Each treatment should have at least three replicates, with an aqueous solution without pesticides as a blank control and the chemical pesticide imidacloprid as a positive control. Place the cage in an artificial climate chamber with a temperature of 25 ℃, a photoperiod of 16:8, and a humidity of 70%. When testing the effect on the cotton aphid oviposition rate, 30 3-4 instar cotton aphids and adult cotton aphids were transferred to the rearing cage and then fed according to the above method.

[0039] (3) Observe and count the feeding and survival of the tested insects every day. Change the feed every 2 days and count the insects every 24 hours. Then, count the mortality rate of cotton aphids in each treatment group. If the mortality rate in the control group is <5%, no correction is needed. If the mortality rate in the control group is between 5% and 20%, the mortality rate of the treatment group should be corrected according to the following formula: Corrected mortality rate = [(treatment mortality rate - control mortality rate) / (1 - control mortality rate) × 100%]. If the control mortality rate is >20%, the experiment needs to be repeated. Oviposition rate = number of offspring produced / initial number of adults.

[0040] The results showed that: ① The LC50 of cotton aphids fed with Bacillus thuringiensis for 5 days was [not specified]. 50 The value was 31.94 μg / mL ( Figure 1 A); ② LC50 of cotton aphids fed with imidacloprid for 5 days 50 The value was 67.10 μg / mL ( Figure 1 B); ③ At a concentration of 31.94 μg / mL, Bacillus thuringiensis was able to inhibit the oviposition ability of cotton aphids ( Figure 1 C).

[0041] Example 3: Determination of the bioactivity of Bacillus thuringiensis against Peach Aphid

[0042] (1) 90.50% thallium thallium was prepared into thallium thallium solutions with final concentrations of 50 μg / mL, 100 μg / mL, 400 μg / mL, 1600 μg / mL and 6400 μg / mL respectively using an aqueous solution containing 0.1% Triton. The 0.1% Triton aqueous solution was used as a negative control.

[0043] (2) Immerse the leaves of tobacco Benedict in test solutions of different concentrations for 30 s, remove them, air dry them, and place them in a petri dish containing 1% agar. Each treatment concentration was repeated three times.

[0044] (3) Ten second-instar peach aphids were inoculated onto each treated tobacco leaf and cultured indoors for 5 days. The survival and growth of the peach aphids were recorded.

[0045] The results showed that LC50 of Bacillus thuringiensis against peach aphids was... 50 352.23 μg / ml ( Figure 2 ).

[0046] Example 4: Determination of the bioactivity of Bacillus thuringiensis against armyworms

[0047] (1) Take 90.50% pure thallium and prepare thallium solutions with final concentrations of 50 μg / mL, 100 μg / mL, 150 μg / mL, 200 μg / mL and 300 μg / mL respectively.

[0048] (2) Weigh 0.8 g of artificial feed for armyworms and place it in a petri dish, spread it evenly on the bottom of the dish, and blow dry the surface moisture.

[0049] (3) Add 200 μL of Bacillus thuringiensis solution of different concentrations to each 0.8 g feed dish, allowing it to be completely absorbed into the feed. Then, use a brush to pick up 25 armyworm larvae and place them on the treated feed, with 3 replicates for each concentration. The negative control group was prepared by adding 200 μL of aqueous solution to the feed.

[0050] (4) Place the petri dish in a 26 ℃ incubator and incubate for 7 days. The mortality of the armyworms will be counted.

[0051] The results showed that the LC50 of thiophanate-methyl against armyworms was... 50 It was 94.31 μg / ml ( Figure 3 ).

[0052] Example 5: The toxic activity of Bacillus thuringiensis against soybean cyst nematodes

[0053] Collect J2 instar nematodes and add 1% Triton X-100 at a ratio of 1 / 100 (to prevent nematode loss). Then dilute and adjust the nematode count to a fixed number of nematodes per μL (e.g., 30-40 nematodes / μL). Serially dilute 90.50% Bacillus thuringiensis to final concentrations of 20 μg / mL, 40 μg / mL, 60 μg / mL, 80 μg / mL, and 100 μg / mL, and perform bioassays according to the following system:

[0054] Table 1 Bioassay system of Bacillus thuringiensis samples against soybean cyst nematodes

[0055]

[0056] Three replicates of samples at different concentrations were prepared, and three blank controls were set up. The above system was added to a 96-well plate and incubated at 25°C. During observation, 1-10 μl of NaOH can be added to rule out the possibility of nematode pseudodeath.

[0057] The results showed that after 72 hours, the LC50 of Bacillus thuringiensis against soybean cyst nematodes was found to be [missing information]. 50 29.36 μg / ml ( Figure 4 ).

Claims

1. Use of a Bacillus thuringiensis toxin in the control of cotton aphids.

2. Use according to claim 1, characterized in that, LC50 of the CrylAb protein against cotton aphids 50 The value was 31.94 μg / mL.

3. Use according to claim 2, characterized in that, In the LC 50 At the concentration, the Bacillus thuringiensis toxin can inhibit the oviposition ability of the cotton aphid.

4. Use of a Bacillus thuringiensis toxin in the control of green peach aphids.

5. Use according to claim 4, characterized in that, LC50 of the Bacillus thuringiensis to Myzus persicae 50 The value is 352.23 μg / ml.

6. Use of a Bacillus thuringiensis toxin in the control of armyworms.

7. Use according to claim 6, characterized in that, LC of Bacillus thuringiensis against the armyworm 50 The value is 94.31 μg / ml.

8. Use of a Bacillus thuringiensis toxin in the control of soybean cyst nematodes.

9. Use according to claim 8, characterized in that, LCso of Bacillus thuringiensis against Heterodera glycines 50 The value is 29.36 μg / ml.

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