Toll gene dsRNA and its application in enhancing the efficacy of avermectin in controlling citrus psyllids

Abstract

This invention discloses Toll Gene dsRNA and its application in enhancing the efficacy of avermectin in controlling citrus psyllids. This invention provides a targeted... DcToll A technical solution for controlling citrus psyllids using a combination of dsRNA and avermectin, specifically silencing the citrus psyllid's dsRNA. DcToll Inhibiting the immune response of citrus psyllids by targeting genes can reduce their potential tolerance to abamectin. The method provided by this invention holds promise for achieving a synergistic effect between immunosuppression and chemical agents, offering a new technical approach to improving the efficacy of abamectin and delaying the development of resistance, thus contributing to achieving reduced pesticide use and increased efficiency.

Description

Technical Field

[0001] This invention belongs to the field of biological control technology for agricultural pests, specifically involving Toll Gene dsRNA and its application in improving the efficacy of avermectin in controlling citrus psyllids. Background Technology

[0002] Avermectin is a broad-spectrum biological pesticide derived from microorganisms. It paralyzes and kills pests by acting on their neuromuscular system, and is characterized by high selective toxicity and relative environmental friendliness. However, with its widespread use, target pests in many areas have developed resistance to it, limiting its sustained effectiveness in pest control.

[0003] RNA interference (RNAi) technology, by introducing specific double-stranded RNA (dsRNA) to silence target genes, demonstrates its advantages in precision and environmental friendliness in pest control. This technology can specifically knock down key genes in pests, interfering with their normal physiological functions, with minimal impact on non-target organisms, providing a new approach for green pest control.

[0004] Toll The innate immune pathway is a core pathway for insects to respond to infections caused by pathogens such as Gram-positive bacteria. Once activated, this pathway initiates a series of intracellular signal transductions, ultimately inducing the expression of effector molecules such as antimicrobial peptides and enhancing the insect's immune defense. It is noteworthy that avermectin is derived from the Gram-positive bacterium *Streptomyces griseus* (…). Streptomyces avermitilis During its action, it may also activate the pest's... Toll It can disrupt the immune pathway, thereby inducing an antibacterial response in insects, potentially weakening the control effect of the agent.

[0005] Citrus psyllid ( Diaphorina citri The insect *Hemiberlesia lataniae* is the obligate vector of *Huanglongbing*, posing a serious threat to the citrus industry. Currently, control relies mainly on chemical pesticides, but long-term use has led to increasing pesticide resistance in pests and growing environmental pollution. Therefore, developing efficient, green, and sustainable control strategies is particularly urgent. Summary of the Invention

[0006] Based on the shortcomings and defects of existing technologies, this invention aims to provide... Toll Gene dsRNA and its application in improving the efficacy of avermectin in controlling citrus psyllids.

[0007] The first objective of this invention is to provide a reduction DcToll The application of gene expression level in improving the control of citrus psyllids with avermectin, as described above DcToll The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Preferably, the reduction DcTollGene expression level formulations are targeted DcToll dsRNA of genes.

[0009] Preferably, the target DcToll The nucleotide sequence of the target region of the gene's dsRNA is shown in SEQ ID NO.10.

[0010] Preferably, the target DcToll The method for preparing dsRNA of a gene includes the following steps:

[0011] S1. RNA was extracted from citrus psyllids and reverse transcribed to obtain cDNA;

[0012] S2. Using the cDNA obtained in step S1 as a template; using the primer with the nucleotide sequence shown in SEQ ID NO.6 as the upstream primer and the primer with the nucleotide sequence shown in SEQ ID NO.7 as the downstream primer, amplify fragment 1; using the primer with the nucleotide sequence shown in SEQ ID NO.8 as the upstream primer and the primer with the nucleotide sequence shown in SEQ ID NO.9 as the downstream primer, amplify fragment 2.

[0013] S3. Clone fragment 1 and fragment 2 sequentially into the pET30a expression vector to construct the recombinant plasmid pET30a. DcToll It was then transformed into Escherichia coli BL21(DE3)RNase III strain to obtain pET30a. DcToll Recombinant bacteria;

[0014] S4. Select pET30a DcToll Recombinant bacterial monoclonal culture to OD 600 Approximately 0.4 mg / L was added with 1 mM IPTG, and expression was induced at 37°C for 4 h. Bacterial cells were collected, lysed in an 80°C water bath for 15 min, and the supernatant was collected after centrifugation. dsDcToll was precipitated with ethanol. Further purification yielded dsDcToll, which is a targeted protein. DcToll dsRNA of genes.

[0015] Preferably, the culture in step S4 is carried out by culturing in LB liquid medium containing 50 mg / L kanamycin at 37°C and 200 rpm with shaking.

[0016] A second objective of this invention is to provide a method for controlling citrus psyllids, comprising the following steps: applying a targeted agent to plant branches infested with citrus psyllids. DcToll After a period of time, avermectin was administered to the dsRNA of the gene. DcToll The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0017] Preferably, the method comprises the following steps: Applying dsRNA targeting the DcToll gene to the plant branches with Diaphorina citri, and then applying abamectin 72 h later.

[0018] Preferably, the Diaphorina citri is the 5th instar nymph of Diaphorina citri.

[0019] Preferably, the nucleotide sequence of the target region of the dsRNA targeting the DcToll gene is as shown in SEQ ID NO.10.

[0020] Preferably, the concentration of the dsRNA targeting the DcToll gene is 500 ng / µL, and the concentration of the abamectin is 1.047 mg / L.

[0021] Advantages of the present invention:

[0022] The present invention silences the DcToll gene of Diaphorina citri through RNAi technology, inhibits its immune response, and reduces the potential tolerance of Diaphorina citri to abamectin. The method provided by the present invention is expected to achieve the synergistic effect of immunosuppression and chemical agents, provide a new technical idea for improving the efficacy of abamectin and delaying the development of resistance, and contribute to the reduction of pesticide dosage and the increase of efficacy. Description of the drawings

[0023] Figure 1 is the effect of abamectin LC 50 treatment on the gene expression level of the 5th instar nymph of Diaphorina citri DcToll ; *** indicates a significant difference at the p <0.001 level.

[0024] Figure 2 is the multiple alignment of the amino acid sequences of Toll proteins of Diaphorina citri and 6 other insects; among them, Dc: Diaphorina citri Diaphorina citri , Cw: Psylla chinensis Cyamophila willieti , Ps: Plautia stali Plautia stali , Ac: Arma chinensis Arma chinensis , Pa: Periplaneta americana Periplaneta americana , Zn: Zootermopsis angusticollis Zootermopsis nevadensis , Ha: Harmonia axyridis Harmonia axyridis .

[0025] Figure 3 is the phylogenetic tree constructed based on the Toll gene sequences of 11 insects; among them, Ac: Arma chinensis Arma chinensis , Hh: Halyomorpha halys Halyomorpha halys , Ps: Eurygaster testudinaria Plautia stali , Sfr: Spodoptera frugiperda Spodoptera frugiperdaSl: Spodoptera litura Spodoptera litura Dc: Citrus psyllid Diaphorina citri Ag: Cotton aphid Aphis gossypii Sfu: White-backed planthopper Sogatella furcifera Nl: Brown planthopper Nilaparvata lugens Bt: Whitefly Bemisia tabaci Ha: Ladybug Harmonia axyridis .

[0026] Figure 4 yes DcToll Gene expression profiles in citrus psyllid adults at different developmental stages and with different body colors; different letters indicate significant differences ( p <0.05).

[0027] Figure 5 yes DcToll Gene expression levels in different tissues of 5th instar nymphs of the citrus psyllid; different letters indicate significant differences (p<0.05).

[0028] Figure 6 Is feeding dsDcToll to citrus psyllids DcToll The effect of gene expression levels; *** indicates that... p The difference at levels <0.001 was significant.

[0029] Figure 7 This represents the effect of different treatment groups on the survival rate of 5th instar nymphs of the citrus psyllid; different letters indicate significant differences ( p <0.05). Detailed Implementation

[0030] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0031] Example 1

[0032] 1. Indoor toxicity test of abamectin against citrus psyllids

[0033] Avermectin technical grade was purchased from Hebei Zhongbaolv Agricultural Technology Co., Ltd. Its generic name is Avermectin (AVM), in powder form, with an active ingredient content of 72%. 0.1389 g of the technical grade was weighed and dissolved in 1 mL of acetone to prepare a stock solution with a concentration of 100,000 mg / L. The stock solution was serially diluted to five concentrations (0.01, 0.1, 1, 10, and 100 mg / L) using an aqueous solution containing 0.1% Tween 80 (v / v). The 0.1% Tween 80 solution was used as a control.

[0034] (1) Determination of toxicity to eggs

[0035] Fifty male and fifty female adults, each 10 days after emergence, were placed on Murraya paniculata plants with more than 10 tender buds, covered by a net, to lay eggs for 24 hours. Branches with similar egg-laying numbers (approximately 40 eggs per branch) were selected as explant materials and immersed in abamectin solutions of various concentrations and a control solution for 30 seconds. The base of the branches was wrapped with sterile, water-moistened absorbent cotton, and each branch was placed individually into a 50 mL centrifuge tube, the tube opening sealed with gauze, forming an independent treatment. Each treatment was replicated six times.

[0036] (2) Toxicity determination against 5th instar nymphs

[0037] Select uniformly growing Murraya paniculata branches, immerse them in abamectin solutions of various concentrations and a control solution for 30 seconds, then remove and air dry indoors under natural conditions. Select healthy 5th instar nymphs of uniform size and gently transfer them to the treated branches using a soft brush. Insert the base of the branch into a PCR tube containing 200 μL of sterile water, seal the tube opening with sealing film, and place the entire setup into a 50 mL centrifuge tube, covering the opening with gauze to form an independent treatment. Each treatment contains 20 nymphs, with 6 replicates per treatment.

[0038] (3) Toxicity determination against male and female adults

[0039] The treatment method for Murraya paniculata branches is the same as (2). After briefly anesthetizing the female and male adults with CO2, they were inoculated into the treated branches. Each branch was inoculated with 20 insects (all of the same sex, i.e., all female adults or all male adults). The branches were covered with fine nets to prevent the adults from escaping. Each net was considered as one treatment. There were 6 replicates for each treatment.

[0040] All treatment devices were placed in a constant temperature incubator under the following conditions: temperature 27 ± 0.5 ℃, photoperiod L:D = 14h:10h, and relative humidity 65 ± 5%. The mortality and egg hatching of the test insects were observed and recorded daily. After 3 days, the total number of insects, cumulative mortality, total number of eggs, and cumulative hatching were tallied. The toxicity regression equation and median lethal concentration (LC50) were calculated using SPSS software. 50 The results showed that avermectin had a LC50 effect on different developmental stages of the citrus psyllid. 50 The order from highest to lowest is: egg > adult male and female > 5th instar nymph (Table 1).

[0041] Table 1. Results of indoor toxicity tests of abamectin against citrus psyllids.

[0042]

[0043] 2. Avermectin against 5th instar nymphs of citrus psyllids DcToll Effects of gene expression levels

[0044] According to citrus psyllidsDcToll The gene (GenBank accession number: XM_026829993.1, its CDS sequence is shown in SEQ ID NO.1) was used to design qPCR primers online at https: / / www.primer3plus.com. Upstream primer... DcToll -qF is: 5'-ATGTGAGAAGACAGCCATTG-3' (SEQ ID NO.2), downstream primer DcToll -qR is: 5'-GGACCTTTTGGTTAGTCTCA-3' (SEQ ID NO.3).

[0045] Select branches of Murraya paniculata with uniform growth and dilute them separately with LC50 solution of abamectin. 50 After immersing in a 1.047 mg / L concentration of the drug solution and a control solution for 30 seconds, the nymphs were removed and allowed to air dry at room temperature. Healthy 5th instar nymphs of uniform size were selected and gently transferred to the treated branches using a soft brush. The base of the branch was inserted into a PCR tube containing 200 μL of sterile water, and the tube opening was sealed with sealing film. The entire apparatus was placed in a 50 mL centrifuge tube, and the tube opening was covered with gauze, constituting one treatment. Twenty nymphs were inoculated on each branch, and each treatment was replicated 9 times. The apparatus was placed in an artificial climate chamber at 27 ± 0.5 ℃, 65 ± 5% RH, and L:D=14:10 h for rearing. After 72 h of rearing, the test insects were collected, and total RNA was extracted from 5th instar nymphs of the citrus psyllid using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Nanjing Novizan Biotechnology Co., Ltd., catalog number RC112). cDNA was synthesized using the HiScript III RTSuperMix for qPCR Reverse Transcription Kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number R433-01). Using the cDNA as a template, real-time quantitative PCR (qPCR) was performed using the TB Green Premix Ex Taq II Kit (TAKARA, catalog number RR820A) on citrus psyllids. DcActin (GenBank accession number: DQ675553.1) is the internal reference gene, and the relative expression level is calculated using the 2-ΔΔCt method. DcActin upstream primer DcActin -qF is 5'-TGTTCCAACCTTCCTTCCTG-3' (SEQ ID NO.4), downstream primer DcActin -qR is 5'-GTGTTGGCGTACAGGTCCTT-3' (SEQ ID NO.5). For example... Figure 1 As shown, avermectin LC 50 After concentration treatment, the 5th instar nymphs of the citrus psyllid... DcTollGene expression levels were significantly upregulated.

[0046] 3. Citrus psyllid DcToll Gene sequence analysis

[0047] Based on citrus psyllids DcToll The gene sequence (GenBank accession number XM_026829993.1) (as shown in SEQ ID NO.1) was obtained using NCBI BLASTP on the NCBI website (https: / / blast.ncbi.nlm.nih.gov / ). DcToll The sequences of homologous proteins from closely related species were obtained, and multiple amino acid sequences were aligned using DNAman 9.0 software. The results are as follows: Figure 2 As shown, the citrus psyllid (Dc) exhibits high similarity to the Chinese scholar tree psyllid (Cw), the small peperomia bug (Ps), the black-bellied bug (Ac), the American cockroach (Pa), and the wet wood termite (Zn). A phylogenetic tree was constructed using MEGA 11.1.8 software and the nearest neighbor join (NJ) method, as shown below. Figure 3 As shown, the citrus psyllid (Dc) clusters with the cotton aphid (Ag) and exhibits a highly conserved evolutionary relationship with other insects such as the takin bug (Ac) and the tea-winged bug (Hh).

[0048] 4. Citrus psyllid DcToll Spatiotemporal expression pattern analysis of genes

[0049] According to citrus psyllids DcToll The CDS sequence of the gene (GenBank accession number: XM_026829993.1) was used to design qPCR primers online using the website https: / / www.primer3plus.com. Upstream primer... DcToll -qF is: 5'-ATGTGAGAAGACAGCCATTG-3' (SEQ ID NO.2), downstream primer DcToll -qR is: 5'-GGACCTTTTGGTTAGTCTCA-3' (SEQ ID NO.3).

[0050] Total RNA was extracted from different developmental stages (eggs, nymphs of all instars, and adults of different ages / body colors) and different tissues (head, legs, wing buds, fat body, midgut, and epidermis) of citrus psyllids using the FastPure Cell / Tissue Total RNA Isolation Kit V2 (Nanjing Novizan Biotechnology Co., Ltd., catalog number RC112). cDNA was synthesized using the HiScript III RT SuperMix for qPCR reverse transcription kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number R433-01). Using the cDNA as a template, real-time quantitative PCR (qPCR) was performed using the TB Green Premix Ex Taq II kit (TAKARA, catalog number RR820A) on citrus psyllids. DcActin (GenBank accession number: DQ675553.1) is the internal reference gene, and the relative expression level is calculated using the 2-ΔΔCt method. DcActin upstream primer DcActin -qF is 5'-TGTTCCAACCTTCCTTCCTG-3' (SEQ ID NO.4), downstream primer DcActin -qR is 5'-GTGTTGGCGTACAGGTCCTT-3' (SEQ ID NO. 5).

[0051] The results showed that DcToll The gene is expressed at all developmental stages of the citrus psyllid, with the highest expression levels in the blue and yellow female adults. Figure 4 The tissue expression profile results are as follows: Figure 5 As shown, the expression was present in different tissues of the 5th instar nymphs, with the highest expression level in the head.

[0052] 5. Large-scale preparation of dsDcToll

[0053] (1) PCR amplification of the insert fragment

[0054] Primers used for cloning into the pET30a vector (containing homologous arms and restriction enzyme sites EcoR1, Xhol): Upstream primer hpDcToll-F1 for insert fragment 1 is 5'-GCTGATATCGGATCCGAATTCTGTATTGAGTCCTCCGTACT-3' (SEQ ID NO. 6); Downstream primer hpDcToll-R1 for insert fragment 1 is 5'-GTGGTGGTGGTGGTGCTCGAGAATATGGCACGTGTGAGATT-3' (SEQ ID NO. 7). Upstream primer hpDcToll-pet-F2 for insert fragment 2 is 5'-TTATCAATCTCACACGTGCCATATTGAGCTCGAACGTCTGCAATTTTCTCA-3' (SEQ ID NO. 8); Downstream primer hpDcToll-pet-R2 for insert fragment 2 is 5'-GTGGTGGTGGTGGTGCTCGAGTGTATTGAGTCCTCCGTACT-3' (SEQ ID NO. 9). Using cDNA from 5th instar nymphs of the citrus psyllid as a template, insert fragments 1 and 2 were amplified using the primers described above.

[0055] (2) Inducible expression of dsDcToll

[0056] Inserts 1 and 2 were sequentially cloned into the pET30a expression vector to construct the recombinant plasmid pET30a. DcToll The virus was transformed into *Escherichia coli* BL21(DE3)RNase III strain. Simultaneously, the empty vector pET30a was transformed as a negative control. Single colonies were picked and inoculated into LB broth containing 50 mg / L kanamycin, and cultured at 37°C with shaking at 200 rpm until OD500. 600 Approximately 0.4. IPTG was added to a final concentration of 1 mM, and expression was induced at 37°C for 4 h. The bacterial cells were collected, lysed in an 80°C water bath for 15 min, and the supernatant was collected after centrifugation. The dsRNA (dsDcToll, whose target region nucleotide sequence is shown in SEQ ID NO.10) was initially purified by ethanol precipitation.

[0057] (3) Purification of dsDcToll

[0058] Add 2 volumes (40 μL) of DEPC-concentrated water to the supernatant (20 μL), followed by 1.5 volumes (30 μL each) of saturated phenol and chloroform. After thorough shaking and mixing, extract the product. Take approximately 60 µL of the upper aqueous phase and add 1 / 10 volume (6 μL) of sodium acetate and 2.5 volumes (150 μL) of anhydrous ethanol. Incubate at -20°C to precipitate nucleic acids. Wash the precipitate with 70% ethanol, air dry, and dissolve in DEPC-concentrated water. Verify the product size and purity using agarose gel electrophoresis and quantify using a nucleic acid concentration analyzer.

[0059] (4) Preparation of dsEGFP

[0060] The nucleotide fragment of EGFP (GenBank accession number LC336961.1) dsRNA is SEQ ID NO.11. Using the T7 High Yield RNA Transcription kit (Nanjing Novizan Biotechnology Co., Ltd., catalog number TR101-01), with EGFP DNA as a template, and EGFP-RNAi-F (SEQ ID NO.12, lowercase T7 sequence): 5'-gatcactaatacgactcactatagggagaCCTGAAGTTCATCTGCACCAC-3' as the upstream primer, and EGFP-RNAi-R (SEQ ID NO.13, lowercase T7 sequence): 5'-gatcactaatacgactcactatagggagaCTCCAGCAGGACCATGTGATC-3' as the downstream primer, the EGFP dsRNA fragment (dsEGFP) was synthesized and finally dissolved in enzyme-free water to obtain an aqueous solution of dsEGFP.

[0061] 6. dsDcToll interference efficiency detection

[0062] Healthy 5th instar nymphs of the citrus psyllid were transferred to tender shoots of Murraya paniculata using a paintbrush. They were fed a Parafilm clamp feeding method, receiving 500 ng / μL of dsDcToll or dsEGFP (control) solution. The feeding device was placed in an artificial climate chamber at 27 ± 0.5 ℃, 65 ± 5% RH, and L:D = 14:10 h. After 72 h of rearing, the insects were collected, total RNA was extracted and reverse transcribed, and detected by qPCR. DcToll Gene expression levels. The results showed ( Figure 6 In the treatment group fed dsDcToll, DcToll The gene expression level was significantly lower than that of the dsEGFP control group, with a silencing efficiency of 50.44%.

[0063] 7. Bioactivity assay of dsDcToll and avermectin in combination

[0064] Healthy 5th instar nymphs of the citrus psyllid were collected and fed with dsEGFP (500 ng / μL) or dsDcToll (500 ng / μL) using the Parafilm membrane clamp nutrient solution method. The feeding device was placed in an artificial climate chamber at 27 ± 0.5 ℃, 65 ± 5% RH, and L:D=14:10 h for 72 h before the insects were collected for subsequent abamectin experiments.

[0065] The following five treatments were set up: ① ddH2O (blank control), ② dsEGFP (blank control), ③ abamectin AVM (LC). 50 Concentration, 1.047 mg / L), ④ dsDcToll, ⑤ dsDcToll + avermectin AVM (LC 50 Concentration, 1.047 mg / L). Nine-mile fragrance branches were soaked in ddH2O and abamectin AVM LC, respectively. 50 After immersing in the concentrated solution for 30 seconds, the insects were removed and allowed to air dry at room temperature. They were then inoculated onto branches, with 20 insects per branch and 6 replicates per treatment. The rearing conditions for the insects were the same as described above. The insects were observed for 3 consecutive days after treatment, and the mortality rate was recorded. Results ( Figure 7 This indicates that dsDcToll is similar to avermectin LC. 50 The nymph mortality rate in the combined concentration treatment group reached 74.51%, which was significantly higher than that in the treatment groups using abamectin alone (50.98%) or dsDcToll (21.67%), demonstrating that the two had a significant synergistic effect.

Claims

1. Reduce DcToll The application of gene expression level in improving the efficacy of avermectin in controlling citrus psyllids is characterized by, The aforementioned DcToll The nucleotide sequence of the gene is as shown in SEQ ID NO.1; the reduction described herein DcToll Gene expression levels are achieved through targeted application. DcToll dsRNA of genes.

2. The application according to claim 1, characterized in that, The targeting DcToll The nucleotide sequence of the target region of the dsRNA of the gene is shown as SEQ ID NO.

10.

3. The application according to claim 2, characterized in that, The targeting DcToll The method of making a dsRNA of a gene comprises the steps of: S1. RNA was extracted from citrus psyllids and reverse transcribed to obtain cDNA; S2. Using the cDNA obtained in step S1 as a template; using the primer with the nucleotide sequence shown in SEQ ID NO.6 as the upstream primer and the primer with the nucleotide sequence shown in SEQ ID NO.7 as the downstream primer, amplify fragment 1; using the primer with the nucleotide sequence shown in SEQ ID NO.8 as the upstream primer and the primer with the nucleotide sequence shown in SEQ ID NO.9 as the downstream primer, amplify fragment 2. S3. Fragments 1 and 2 were cloned into pET30a expression vector in turn to construct recombinant plasmid pET30a DcToll , which was transformed into E. coli BL21(DE3) RNase III strain to obtain recombinant bacteria transformed with pET30a DcToll . S4. Select pET30a DcToll Recombinant bacterial monoclonal culture to OD 600 The concentration was 0.4, 1 mM IPTG was added, and expression was induced at 37℃ for 4 h; the bacterial cells were collected, lysed in an 80℃ water bath for 15 min, centrifuged, and the supernatant was collected. dsDcToll was precipitated by ethanol; dsDcToll was obtained by further purification. The dsDcToll is a targeted protein. DcToll dsRNA of genes.

4. Use according to claim 3, characterized in that, The culture in step S4 is as follows: cultured in LB liquid medium containing 50 mg / L kanamycin at 37°C and 200 rpm with shaking.

5. A method for controlling citrus psyllids, characterized in that, Includes the following steps: Apply targeted treatment to branches of plants infested with citrus psyllids DcToll After a period of time, avermectin was administered to the dsRNA of the gene. DcToll The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The method according to claim 5, characterized in that, Includes the following steps: Apply targeted treatment to branches of plants infested with citrus psyllids DcToll Avermectin was administered 72 h after the gene's dsRNA was detected.

7. The method according to claim 5, characterized in that, The citrus psyllid mentioned is a 5th instar nymph of the citrus psyllid.

8. The method according to claim 5, characterized in that, The target DcToll The nucleotide sequence of the target region of the gene's dsRNA is shown in SEQ ID NO.

10.

9. The method according to claim 5, characterized in that, The target DcToll The concentration of the gene's dsRNA was 500 ng / µL, and the concentration of the avermectin was 1.047 mg / L.

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