Method for synergistically killing agricultural pests by utilizing dsRNA (double-stranded ribonucleic acid) nano preparation of chitinase 2 gene of spodoptera frugiperda and abamectin

Abstract

The invention relates to the technical field of pesticide pest control, in particular to a method for synergistically killing agricultural pests by utilizing a spodoptera frugiperda chitinase 2 gene dsRNA nano preparation and abamectin. Through the synergistic effect of the chemical pesticide abamectin and the grassland spodoptera frugiperda chitinase 2 gene dsRNA nano biological agent, the control effect of agricultural pests is improved, the use amount of the chemical pesticide is reduced, and the drug resistance risk of the pests is reduced. The invention not only can exert the quick-acting characteristic of a chemical insecticide, but also provides an efficient and sustainable solution for comprehensive control of agricultural pests by utilizing the advantages of single action object and difficult generation of resistance of a biological insecticide, and is beneficial to reduction of pesticide residues and reduction of environmental pollution; the invention provides important support for pesticide reduction and synergism and green agriculture development, and has a wide application prospect.

Description

Technical Field

[0001] The invention relates to the technical field of pesticide pest control, and in particular to a method for cooperatively killing agricultural pests by using a fall armyworm chitinase 2 gene dsRNA nano preparation and avermectin. Background Art

[0002] The fall armyworm is an important agricultural pest that can pose a serious threat to gramineous crops such as corn. Its larvae are highly adaptable and have high reproductive capacity, and can rapidly expand their population, causing significant agricultural losses. As the fall armyworm is prone to developing resistance to chemical pesticides, integrated pest management strategies have received attention, and combined with biological control measures, they have become an effective way to control its damage.

[0003] At present, chemical pesticides still occupy an important position in field pest control, but they also face many challenges. For example, excessive use of chemical pesticides causes pests to gradually develop resistance and reduce the efficacy of the pesticides, which requires continuous increase in dosage or replacement of drugs, increasing the cost of prevention and control. At the same time, the problem of chemical pesticide residues has caused concerns about food safety and environmental pollution, affecting human health and ecosystem stability. In addition, the damage of chemical pesticides to non-target organisms such as beneficial insects and natural enemies also has an adverse effect on the ecological balance. Obviously, relying on a single control method of chemical pesticides is no longer sufficient to meet the current needs of pest control, and it is urgent to consider other environmentally friendly and sustainable control methods.

[0004] At present, research on new pesticides mainly focuses on the following aspects: (1) developing new pesticides with high efficiency and low toxicity to reduce the negative impact on human health and the environment; (2) using bio-based pesticides, such as products based on RNAi technology, to reduce harm to non-target organisms and ecosystems; (3) combining new pesticides with other environmental protection measures to achieve sustainable agricultural production. Summary of the invention

[0005] The present invention obtains a dsRNA with a high lethality rate to agricultural pests based on the chitinase 2 gene of the fall armyworm. After being prepared into a nanoformulation and used in combination with avermectin, the mortality rate of agricultural pests can be significantly increased, and the reduction in the amount of chemical pesticides and the increase in efficiency can also be achieved. Based on this, the following invention content is proposed.

[0006] In a first aspect, the present invention provides a pesticide combination formulation, which includes a dsRNA nanoformulation of chitinase 2 gene of fall armyworm and avermectin.

[0007] The dsRNA of the chitinase 2 gene of Spodoptera frugiperda is synthesized according to the nucleotide sequence shown in SEQ ID No.1.

[0008] In some embodiments, the nanoformulation includes dsRNA of the chitinase 2 gene of Spodoptera frugiperda and a nanocarrier.

[0009] In some embodiments, the nanocarrier is chitosan.

[0010] The stability and penetration of the chitinase 2 gene dsRNA of the fall armyworm can be enhanced by encapsulation in nanocarriers.

[0011] In a second aspect, the present invention provides a method for preparing the insecticide combination formula, comprising: mixing chitosan, acetic acid and sodium acetate to prepare a chitosan solution; then mixing the chitosan solution with a dsRNA solution of the chitinase 2 gene of the fall armyworm, and preparing the mixture by standing, incubating, vortexing, standing again and centrifuging to obtain the mixture.

[0012] In some embodiments, the concentration of the chitosan solution is 0.05% to 0.5% (w / v), preferably 0.05% to 0.2%.

[0013] In some embodiments, the concentration of the dsRNA solution is 100-1000 ng / μL, preferably 200-800 ng / μL.

[0014] In some embodiments, the chitosan solution and the dsRNA solution are mixed in a volume ratio of (0.4-4):1.

[0015] In some embodiments, the chitosan solution and the dsRNA solution are mixed at a volume ratio of 1:(1.5-2.5).

[0016] In some embodiments, the standing time is more than 2 minutes.

[0017] In some embodiments, the incubation temperature is 50-60°C, preferably 52-58°C.

[0018] In some embodiments, the incubation time is more than 1 min.

[0019] In some embodiments, the vortex time is 10s to 50s, preferably 25s to 35s.

[0020] In some embodiments, the step of allowing to stand again is allowing to stand at room temperature for more than 1 hour.

[0021] In some embodiments, the centrifugation temperature is 2-6° C. and the rotation speed is above 12000 rpm.

[0022] In some embodiments, the centrifugation time is more than 10 min.

[0023] In a third aspect, the present invention provides the use of the insecticide combination formulation or the preparation method in at least one of the following aspects: (1) Agricultural pest management; (2) Agricultural pest control; (3) Lethal agricultural pests.

[0024] In a fourth aspect, the present invention provides a method for killing agricultural pests, comprising: sequentially subjecting the fall armyworm chitinase 2 gene dsRNA nanoformulation and avermectin to the agricultural pests.

[0025] In some embodiments, the dosage of the nanoformulation is 1-3 μg / head; and / or the effective net content of avermectin is 10% (w / w), and the dosage is 700-1200 mg / L (preferably 900-1100 mg / L).

[0026] In some embodiments, the agricultural pests are first treated with the nanoformulation by dripping on the backboard, and then avermectin is sprayed on the agricultural pests 24 hours later.

[0027] In some embodiments, the agricultural pest is a Lepidoptera pest, preferably Spodoptera frugiperda.

[0028] Compared with the prior art, the present invention has the following beneficial effects: The present invention improves the control effect of agricultural pests through the synergistic effect of chemical pesticide avermectin and fall armyworm chitinase 2 gene dsRNA nanobiological agent, reduces the use of chemical pesticides, and reduces the risk of pest resistance. The present invention can not only give play to the quick-acting characteristics of chemical pesticides, but also take advantage of the single target of biological pesticides and the difficulty in generating resistance, providing an efficient and sustainable solution for the comprehensive control of agricultural pests, which is conducive to reducing pesticide residues, reducing environmental pollution, and providing important support for reducing pesticides and increasing efficiency and developing green agriculture, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 These are the test results of the insecticidal effect of the chemical pesticide avermectin at different concentrations.

[0030] Figure 2 This is the regression curve of the toxicity of the chemical pesticide avermectin to the fall armyworm.

[0031] Figure 3 This is the larval phenotype after synergistic treatment of the chitinase 2 gene dsRNA nanoformulation of the fall armyworm and avermectin.

[0032] Figure 4It is the change in the relative expression level of the chitinase 2 gene in the larvae of the fall armyworm after the synergistic treatment of the chitinase 2 gene dsRNA nanoformulation and avermectin.

[0033] Figure 5 This is the death gradient curve of Spodoptera frugiperda larvae after synergistic treatment with dsRNA nanoformulation of chitinase 2 gene and avermectin. DETAILED DESCRIPTION

[0034] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in conjunction with the drawings of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] The present invention relates to molecular biology experiments. If not otherwise specified, reference can be made to the book Molecular Cloning (J. Sambrook, EF Fritsch, T. Maniatis, Science Press, 1994). This book and its subsequent editions are the most commonly used reference books for those skilled in the art when performing molecular biology-related experimental operations. In addition, according to different experimental purposes, those skilled in the art complete the corresponding experiments under the guidance of the operating manuals attached to various commercial kits or entrust specialized companies to perform the experiments.

[0036] In the examples provided in this specification, if no specific techniques or conditions are specified, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be purchased through regular channels.

[0037] Example 1 Experiment on the killing effect of chemical pesticide avermectin on fall armyworm 1. Pesticide preparation Use 20% (v / v) acetone solution to dilute the avermectin stock solution (the effective net content of avermectin is 10% (w / w)) to obtain working concentrations of 700 mg / L, 900 mg / L, and 1200 mg / L.

[0038] 2. Toxicity test of avermectin against fall armyworm 45 3rd-instar, 2-day-old fall armyworms were randomly selected and divided into 3 groups. A 50mL spray bottle was used to evenly spray the diluted pesticide on the larvae, their food and living environment. At the same time, 30 larvae were selected as the blank control group (CK). The treated larvae were placed in a 28℃ constant temperature biochemical incubator and fed with artificial feed (light: dark time = 16h:8h, temperature 26±2℃, humidity 40%).

[0039] 3. Analysis of the toxicity of avermectin in killing fall armyworm After treatment, the living conditions of the larvae were observed and the mortality was recorded. The results showed that compared with the control group, the death rate and number of larvae treated with 700mg / L, 900mg / L, and 1200mg / L insecticides gradually increased, and no more deaths occurred after the third day ( Figure 1 ). Based on the relationship between pesticide concentration, death number and time, the independent regression equation y=4.9087x-9.7197 (R 2 =0.945)( Figure 2 ).

[0040] Example 2 Preparation of dsRNA Nanoformulation of Chitinase 2 Gene of Spodoptera frugiperda This embodiment provides a dsRNA nano preparation of chitinase 2 gene of fall armyworm, and the preparation method thereof is as follows: 1. Synthesis of chitinase 2 gene dsRNA of fall armyworm: Using the nucleotide sequence shown in SEQ ID No.1 as a template, T7 RiboMAX TM The Express RNAi System (Promega) kit was used to synthesize dsRNA by in vitro transcription. The concentration was determined using NaNoDrop 2000 (Thermo scientific). The chitinase 2 gene dsRNA of Spodoptera frugiperda was stored in a -80°C ultra-low temperature refrigerator for future use.

[0041] 2. Preparation of chitosan / dsRNA nanoformulation: 500 ng / μl of fall armyworm chitinase 2 gene dsRNA and 0.1% chitosan solution (mix 59 ml of 0.1 M acetic acid and 41 ml of 0.1 M sodium acetate, add 0.1 g of chitosan powder, and stir with a magnetic stirrer at 37°C for 10 hours) were uniformly mixed in a volume ratio of 2:1, allowed to stand for 2 minutes, incubated at 55°C for 1 minute, vortexed for 30 seconds, allowed to stand at room temperature for 1 hour, centrifuged at 12000 rpm at 4°C for 10 minutes, and the precipitate was the chitosan / dsRNA nanoformulation.

[0042] Example 3 Experiment on the synergistic effect of chemical pesticides and biological agents on fall armyworm The biological agent used in this example is the fall armyworm chitinase 2 gene dsRNA nano preparation prepared in Example 2.

[0043] 1. Experiment on the back-drip of dsRNA nanoparticle preparation of chitinase 2 gene of fall armyworm 45 3-year-old and 2-day-old fall armyworms were selected, with 15 in each group, and 3 replicates were set for each experiment. At the same time, 45 larvae of the same period were selected as a blank control group, and an internal reference control group was set up at the same time. The eGFP dsRNA of the internal reference control group was a dsRNA synthesized by in vitro transcription using SEQ ID No.2 as a template sequence, and the nanoformulation of the internal reference control group was prepared according to the method of Example 2. The nanoformulation was dripped on the back of the larvae at an amount of 2 μg dsRNA per head. The treated larvae were placed in a 28°C constant temperature biochemical incubator and fed with artificial feed for 24 hours (light: dark time = 16h:8h, temperature 26±2°C, humidity 40%).

[0044] 2. Experiment on synergistic treatment of chemical pesticides and biological agents After 24 hours of the above larval treatment, continue to apply LC 50 A 997.1 mg / L avermectin solution was evenly sprayed on the larvae's body surface, food and living environment. The treated larvae were then placed in a 28°C constant temperature biochemical incubator and fed with artificial feed (light: dark time = 16h:8h, temperature 26±2°C, humidity 40%).

[0045] 3. Phenotypic observation of larvae after synergistic treatment After treatment with the chitinase 2 gene dsRNA nanoparticle preparation of fall armyworm, the control group grew well, while most of the larvae in the experimental group refused to eat and their activity weakened 20 hours after treatment. After spraying with avermectin, the larvae relaxed and stretched, lying flat, and their physiological activity decreased. After 3-5 hours, they began to lose vital signs, and their physiological activity further weakened. After 6 hours, they began to die, and the mortality rate reached 100% after 24 hours ( Figure 3 ).

[0046] 4. Detection of chitinase 2 gene silencing effect of fall armyworm after synergistic treatment 48 hours after treatment, three larvae were collected, total RNA was extracted, reverse transcribed into first-strand cDNA, and the expression of chitinase 2 gene was detected by real-time PCR. β-actin was used as the internal reference gene, the upstream and downstream primers of chitinase 2 gene of Spodoptera frugiperda in real-time PCR were shown as SEQ ID No.3 and SEQ ID No.4, the upstream and downstream primers of the internal reference gene were shown as SEQ ID No.5 and SEQ ID No.6, and the real-time PCR reaction system was as follows: upstream primer 0.3 μL, downstream primer 0.3 μL, 2×SYBR Green Taq HS Premix 5 μL (AG Accurate Biology), DEPC water 3.4 μL, cDNA 1 μL. The reaction program was as follows: 95℃, 10s; 95℃, 15s; 55℃, 30s; 72℃, 30s; 39 cycles.

[0047] The results showed that compared with the control group, the expression of chitinase 2 gene decreased significantly by nearly 10 times after 48 hours of biological agent treatment ( Figure 4 ).

[0048] 5. Death curve of fall armyworm after synergistic treatment The larval death was recorded after treatment. The results showed that compared with the blank control and internal reference control, the death rate and number of larvae in the experimental group after synergistic treatment were significantly increased, and the 24-hour mortality rate reached 100%, with significant insecticidal effect ( Figure 5 ).

[0049] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A pesticide combination formula, characterized in that: The insecticide combination formula comprises a dsRNA nano preparation of chitinase 2 gene of fall armyworm and avermectin.

2. The insecticide combination formulation according to claim 1, characterized in that: The dsRNA of the chitinase 2 gene of Spodoptera frugiperda was synthesized according to the nucleotide sequence shown in SEQ ID No.

1.

3. The insecticide combination formulation according to claim 2, characterized in that: The nano preparation comprises dsRNA of chitinase 2 gene of fall armyworm and a nano carrier.

4. The insecticide combination formulation according to claim 3, characterized in that: The nanocarrier is chitosan.

5. The method for preparing the insecticide combination formulation according to any one of claims 1 to 4, characterized in that: include: Chitosan, acetic acid and sodium acetate are mixed to prepare a chitosan solution; the chitosan solution is then mixed with a chitinase 2 gene dsRNA solution of Spodoptera frugiperda, and the mixture is allowed to stand, incubated, vortexed, allowed to stand again and centrifuged to obtain a solution.

6. Use of the insecticide combination formulation according to any one of claims 1 to 4 or the preparation method according to claim 5 in at least one of the following aspects: (1) Agricultural pest management; (2) Agricultural pest control; (3) Lethal agricultural pests.

7. A method for killing agricultural pests, characterized in that: include: The fall armyworm chitinase 2 gene dsRNA nano preparation and avermectin described in claim 1 are sequentially acted on agricultural pests.

8. The method according to claim 7, characterized in that The dosage of the nano preparation is 1~3μg / head; and / or, the effective net content of avermectin is 10% (w / w), and the dosage is 700~1200 mg / L.

9. The method according to claim 7, characterized in that First, the agricultural pests were treated with nano-preparations by dripping on the back, and then avermectin was sprayed on the agricultural pests 24 hours later.

10. The method according to any one of claims 7 to 9, characterized in that: The agricultural pests are lepidopteran pests, preferably fall armyworm.

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