Insecticide combination formula containing Spodoptera frugiperda chitinase A gene dsRNA nano preparation

Abstract

The invention relates to the technical field of pesticide pest control, in particular to a pesticide combination formula containing a spodoptera frugiperda chitinase A gene dsRNA nano preparation. According to the invention, the Spodoptera frugiperda chitinase A gene dsRNA nano preparation and the abamectin are used in a combined manner, the control effect of agricultural pests is improved through the synergistic effect of a chemical pesticide and a biological preparation, the use amount of the chemical pesticide is reduced, and the drug resistance risk of the pests is reduced. Meanwhile, the combined formula disclosed by the invention can effectively reduce pesticide residues and reduce environmental pollution, provides an efficient and sustainable solution for comprehensive prevention and control of agricultural pests, 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 an insecticide combination formula containing a dsRNA nano preparation of chitinase A gene of fall armyworm. Background Art

[0002] Fall Armyworm Spodoptera frugiperda ) is a pest that seriously threatens global agricultural production. It is widely distributed in the Americas, Africa, Asia and other regions, causing serious economic losses to a variety of crops such as corn, rice, and wheat. Its rapid spread and high reproductive capacity pose a huge challenge to traditional prevention and control methods.

[0003] Traditional control measures mainly rely on chemical pesticides. Although these pesticides can quickly reduce the pest population in the short term, long-term use brings many problems. First, the excessive use of chemical pesticides leads to pest resistance, which gradually weakens the effectiveness of pesticides and requires continuous increase in dosage or frequent replacement of pesticides, further exacerbating the problems of pesticide residues and environmental pollution. Secondly, the damage of chemical pesticides to non-target organisms (such as natural enemies, pollinating insects, etc.) and ecosystems has also aroused widespread concern. Therefore, seeking more sustainable and environmentally friendly pest control strategies has become an inevitable trend.

[0004] As an emerging pest control method, biological agents have received increasing attention. Biological agents mainly include pathogenic microorganisms (such as bacteria, fungi and viruses), plant extracts and RNA pesticides based on RNA interference (RNAi) technology. Among them, RNAi technology induces the degradation or translation inhibition of specific mRNA in cells through double-stranded RNA (dsRNA), thereby inhibiting the expression of target genes, and has become a promising biological control tool. However, the effect of using biological agents alone in field control is slow and farmers are not very accepting. Therefore, how to optimize the application of biological agents and improve their control effect has become an urgent problem to be solved.

[0005] In this context, the coordinated use of chemical pesticides and new biological pesticides has gradually attracted people's attention. By rationally matching chemical pesticides with biological agents, the effect of mutual complementation and assistance can be achieved. On the one hand, chemical pesticides can quickly reduce the number of pest populations and reduce pests' resistance to biological agents. On the other hand, by targeting and inhibiting the expression of key genes in pests through RNA interference technology, efficient and specific prevention and control effects can be achieved. Summary of the invention

[0006] Based on this, the present invention provides a pesticide combination formula with a synergistic effect of killing agricultural pests, including a dsRNA nano preparation of chitinase A gene of fall armyworm and avermectin.

[0007] The present invention can not only significantly improve the mortality of agricultural pests but also achieve the reduction in the amount of chemical pesticides and the increase in their efficacy by jointly applying the fall armyworm chitinase A gene dsRNA nano preparation and avermectin.

[0008] Preferably, the Spodoptera frugiperda chitinase A gene dsRNA is synthesized according to the nucleotide sequence shown in SEQ ID No.1.

[0009] The dsRNA synthesized using the above nucleotide sequence has a high mortality rate against agricultural pests.

[0010] Preferably, the nanoformulation comprises dsRNA of chitinase A gene of Spodoptera frugiperda and a nanocarrier.

[0011] Preferably, the nanocarrier is chitosan.

[0012] Preferably, the preparation method of the fall armyworm chitinase A gene dsRNA nanoformulation comprises: mixing chitosan, acetic acid and sodium acetate to prepare a chitosan solution; then mixing the chitosan solution with a dsRNA solution, and preparing the mixture by standing, incubating, vortexing, standing again and centrifuging to obtain the prepared nanoformulation.

[0013] The nanoformulation prepared by the above method has good dsRNA protection effect, high penetration efficiency, simple operation, and can specifically silence gene expression.

[0014] Preferably, the concentration of the chitosan solution is 0.05% to 0.5% (w / v), more preferably 0.05% to 0.2%.

[0015] Preferably, the concentration of the dsRNA solution is 100-1000 ng / μL, more preferably 200-800 ng / μL.

[0016] Preferably, the chitosan solution and the dsRNA solution are mixed in a volume ratio of (0.4-4):1.

[0017] More preferably, the chitosan solution and the dsRNA solution are mixed in a volume ratio of 1:(1.5-2.5).

[0018] Preferably, the standing time is more than 2 minutes.

[0019] Preferably, the incubation temperature is 50-60°C, preferably 52-58°C.

[0020] Preferably, the incubation time is more than 1 min.

[0021] Preferably, the vortex time is 10s to 50s, more preferably 25s to 35s.

[0022] Preferably, the further standing is standing at room temperature for more than 1 hour.

[0023] Preferably, the centrifugal temperature is 2-6°C and the rotation speed is above 12000 rpm.

[0024] Preferably, the centrifugation time is more than 10 min.

[0025] Furthermore, the present invention provides the use of the combined formulation in agricultural pest management, agricultural pest control or killing agricultural pests.

[0026] Furthermore, the present invention provides a method for killing agricultural pests, comprising: sequentially acting the fall armyworm chitinase A gene dsRNA nano preparation and avermectin on the agricultural pests.

[0027] Preferably, the dosage of the nano preparation is 1-3 μg / head (more preferably 1.5-2.5 μg / head); and / or, the effective net content of avermectin is 10% (w / w), and the dosage is 700-1200 mg / L (more preferably 900-1100 mg / L).

[0028] Preferably, the nano-formulation is firstly applied to agricultural pests by dripping on the backboard, and avermectin is sprayed on the agricultural pests 24 hours later.

[0029] In some embodiments, 48 ​​hours after treatment with the nanoformulation, i.e., 24 hours after treatment with avermectin, the mortality rate of fall armyworm larvae reaches 100%.

[0030] Preferably, the agricultural pest is a Lepidoptera pest, more preferably the fall armyworm.

[0031] Compared with the prior art, the present invention has the following beneficial effects: The present invention uses the fall armyworm chitinase A gene dsRNA nano preparation and avermectin in combination, improves the control effect of agricultural pests through the synergistic effect of chemical pesticides and biological preparations, reduces the use of chemical pesticides, and reduces the risk of pest resistance. At the same time, the combined formula of the present invention can effectively reduce pesticide residues, reduce environmental pollution, and provide an efficient and sustainable solution for the comprehensive control of agricultural pests, which has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 These are the test results of the insecticidal effect of the chemical pesticide avermectin at different concentrations.

[0033] Figure 2 This is the regression curve of the toxicity of the chemical pesticide avermectin to the fall armyworm.

[0034] Figure 3 It is the larval phenotype after synergistic treatment of chitinase A gene dsRNA nanoformulation of Spodoptera frugiperda and avermectin.

[0035] Figure 4 It is the change in the relative expression level of the chitinase A gene in the larvae of the fall armyworm after the synergistic treatment of the chitinase A gene dsRNA nanoformulation and avermectin.

[0036] Figure 5 This is the death gradient curve of Spodoptera frugiperda larvae after synergistic treatment with dsRNA nanoformulation of chitinase A gene and avermectin. DETAILED DESCRIPTION

[0037] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be described clearly and completely below. 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.

[0038] 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.

[0039] 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.

[0040] 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.

[0041] 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%).

[0042] 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 ).

[0043] Example 2 Preparation of dsRNA Nanoformulation of Chitinase A Gene of Spodoptera frugiperda This embodiment provides a dsRNA nano preparation of chitinase A gene of fall armyworm, and the preparation method thereof is as follows: 1. Synthesis of chitinase A gene dsRNA of Spodoptera frugiperda 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 A gene dsRNA of Spodoptera frugiperda was stored in a -80°C ultra-low temperature refrigerator for future use.

[0044] 2. Preparation of chitosan / dsRNA nanoformulations 500 ng / μl of fall armyworm chitinase A gene dsRNA was evenly mixed with 0.1% chitosan solution (59 ml of 0.1 M acetic acid and 41 ml of 0.1 M sodium acetate were mixed evenly, 0.1 g of chitosan powder was added, and the mixture was stirred at 37°C with a magnetic stirrer for 10 hours) 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 4°C 12000 rpm for 10 minutes, and the precipitate was the chitosan / dsRNA nanoformulation.

[0045] 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 A gene dsRNA nano preparation prepared in Example 2.

[0046] 1. Experiment on the back-drip of dsRNA nanoparticle preparation of chitinase A 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%).

[0047] 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%).

[0048] 3. Phenotypic observation of larvae after synergistic treatment After treatment with the chitinase A gene dsRNA nanoparticle preparation of the 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 straightened their bodies, their physiological activity decreased, and they began to lose vital signs 3-5 hours later. The physiological activity of the larvae further weakened, and they began to die after 6 hours. The mortality rate reached 100% after 24 hours ( Figure 3 ).

[0049] 4. Detection of chitinase A 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 A gene was detected by real-time PCR. β-actin was used as the internal reference gene. The upstream and downstream primers of chitinase A gene of Spodoptera frugiperda in real-time PCR are shown in SEQ ID No.3 and SEQ ID No.4, and the upstream and downstream primers of the internal reference gene are shown in SEQ ID No.5 and SEQ ID No.6. The real-time PCR reaction system is 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 is as follows: 95℃, 10s; 95℃, 15s; 55℃, 30s; 72℃, 30s; 39 cycles.

[0050] The results showed that compared with the control group, the expression of chitinase A gene decreased significantly by nearly 10 times after 48 hours of biological agent treatment ( Figure 4 ).

[0051] 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 larval death rate and number after synergistic treatment were significantly increased, with a 24-hour mortality rate of 100%, and a significant insecticidal effect ( Figure 5 ).

[0052] 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 formulation, characterized in that: Including dsRNA nanoformulation of chitinase A gene of fall armyworm and avermectin.

2. The combined formulation according to claim 1, characterized in that: The fall armyworm chitinase A gene dsRNA is synthesized according to the nucleotide sequence shown in SEQ ID No.

1.

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

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

5. The combined formulation according to claim 4, characterized in that: The preparation method of the fall armyworm chitinase A gene dsRNA nano preparation comprises: mixing chitosan, acetic acid and sodium acetate to prepare a chitosan solution; then mixing the chitosan solution with a dsRNA solution, and preparing the preparation through standing, incubating, vortexing, standing again and centrifugal precipitation.

6. Use of the combined formulation according to any one of claims 1 to 5 in agricultural pest management, agricultural pest control or killing agricultural pests.

7. A method for killing agricultural pests, characterized in that: include: The fall armyworm chitinase A 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 usage amount of the nano preparation is 1-3 μg / head; and / or the effective net content of avermectin is 10% (w / w), and the usage amount is 700-1200 mg / L.

9. The method according to claim 7, characterized in that: The nano-preparation is firstly applied to agricultural pests by dripping on the backboard, and then avermectin is 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 pest is a Lepidoptera pest, more preferably the fall armyworm.

Images