Application of dsRNA in the control of insecticide-resistant whiteflies

Abstract

This invention provides the application of dsRNA in the control of insecticide-resistant whiteflies, belonging to the field of pesticide technology, specifically a... BtUGPase The application of dsRNA of the gene in the control of bromocyanamide and fluoxazolamide-resistant whiteflies is discovered and confirmed in this invention. BtUGPase The gene is closely related to UGT-mediated resistance in whiteflies to bromocyanamide and fluoxazolamide. When whiteflies feed on... BtUGPase After dsRNA administration, resistance to bromocyanamide and fluoxazolamide was significantly reduced.

Description

Technical Field

[0001] This invention belongs to the field of pesticide technology, specifically relating to the application of dsRNA in the control of insecticide-resistant whiteflies. Background Technology

[0002] whiteflies Bemisia tabaci (Gennadius)(Hemipetra:Alteyrodidae) belongs to the apocryphae insects, and its developmental stages are divided into three stages: egg, nymph, and adult. Due to its wide distribution and many host plant species, it has become a major agricultural pest in tropical, subtropical, and temperate regions.

[0003] Currently, the main method for controlling whiteflies is chemical control, which involves spraying various insecticides on pests or plant leaves. Chemical control is characterized by its rapid effectiveness and significant insecticidal effect.

[0004] Due to the long-term use of insecticides, whiteflies have inevitably developed varying levels of resistance to many traditional insecticides (such as neonicotinoids and pyrethroids). The rapid development of whitefly resistance has become a major challenge in current pest control. To address this problem, insecticides with novel mechanisms of action and no cross-resistance are needed as alternative agents for resistance management. Against this backdrop, broflanilide (a diamide insecticide) and fluoxazolamide (a novel isoxazoline insecticide) have attracted considerable attention due to their unique mechanisms of action and good control efficacy.

[0005] Bromnipotent, the first diamide insecticide to exhibit good insecticidal activity against piercing-sucking pests, targets ryanodine receptors in insect muscle tissue, inducing sustained muscle contractions in whiteflies, impairing their locomotion and ultimately causing death. Due to its unique mechanism of action, it exhibits no cross-resistance with other traditional insecticides, making it an effective control method for whiteflies in the field. However, due to repeated use, whitefly populations in some parts of China have developed significant resistance to bromnipotent, thus necessitating the search for new methods to delay the development of resistance.

[0006] Fluoxazolamide represents a novel class of isoxazoline neurotoxins that interfere with the insect nervous system through both contact and stomach poisoning. This compound specifically targets γ-aminobutyric acid (GABA) receptors, disrupting nerve conduction in whiteflies, leading to hyperexcitability, convulsions, and even death. In recent years, due to the increasing resistance of whiteflies to traditional insecticides, fluoxazolamide has received widespread attention as an alternative agent. Through screening insecticides for over 20 generations, whitefly populations exhibiting resistance to fluoxazolamide exceeding 100 times were obtained, indicating a certain risk of resistance in whiteflies to fluoxazolamide. Therefore, it is necessary to actively seek effective resistance control methods.

[0007] UDP-glucosyltransferase (UGT) is a key phase II metabolic enzyme in insects that catalyzes the biotransformation of various lipophilic compounds through glycosylation reactions.

[0008] UGT enzymes require a glycosyl donor to complete their catalytic reactions, and insect UGT enzymes use UDP-glucose (UDPG) as the glycosyl donor. In the insect UDPG synthesis pathway, UGPase catalyzes a key reaction at the end of the pathway, ensuring that insects can synthesize UDPG efficiently.

[0009] In the prior art, although there are reports on dsRNA for controlling bromonitrile whiteflies, there are no reports on dsRNA that is effective against both bromonitrile whiteflies and fluoxazolamide-resistant whiteflies. Summary of the Invention

[0010] To address the above problems, this invention provides an application of dsRNA in the control of insecticide-resistant whiteflies.

[0011] This invention discovers and confirms that in the insect UDPG synthesis pathway, BtUGPase The gene is closely related to UGT-mediated resistance in whiteflies to bromocyanamide and fluoxazolamide. When whiteflies feed on... BtUGPase dsRNA (ds BtUGPase After that, resistance to bromocyanamide and fluoxazolamide was significantly reduced.

[0012] The technical solution of the present invention is as follows:

[0013] The application of a dsRNA in the control of insecticide-resistant whiteflies includes any of the following applications:

[0014] (1) Control of bromocyanamide-resistant whiteflies;

[0015] (2) Control of fluoxazolamide-resistant whiteflies;

[0016] (3) Control of bromocyanamide and fluoxazolamide-resistant whiteflies;

[0017] (4) Preparation of reagents for controlling whiteflies;

[0018] Wherein, the dsRNA is BtUGPase dsRNA of genes BtUGPase The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0019] Preferred, BtUGPase The nucleotide sequence of the gene's dsRNA is shown in SEQ ID NO.2.

[0020] Preferably, the application method is through feeding whiteflies. BtUGPase The dsRNA of the gene reduces the resistance of whiteflies to bromocyanamide and fluoxazolamide.

[0021] Preferably, the feeding ds BtUGPase The method is: using a mixture containing BtUGPase Whiteflies were fed sucrose water containing the dsRNA of the gene.

[0022] Preferably, the concentration of sucrose in the sucrose water is 0.17~0.23 mg / L.

[0023] Preferably, the concentration of sucrose in the sucrose water is 0.2 mg / L.

[0024] Preferably, the BtUGPase The concentration of the gene's dsRNA in sucrose water was 250 ng / µL.

[0025] The beneficial effects of this invention are:

[0026] This invention has discovered and confirmed that BtUGPase The gene is closely related to UGT-mediated resistance in whiteflies to bromocyanamide and fluoxazolamide. When whiteflies feed on... BtUGPase dsRNA (ds BtUGPase After treatment, resistance to bromocyanamide and fluoxazolamide was significantly reduced. This invention provides molecular targets for the control of whitefly resistance and lays a solid theoretical and applied foundation for the development of new methods for controlling whiteflies resistant to bromocyanamide and fluoxazolamide. Attached Figure Description

[0027] Figure 1 The UGT enzyme activity of bromocyanamide-resistant and susceptible populations.

[0028] Figure 2 The activity of UGT enzyme in fluoxazolamide-resistant and susceptible populations.

[0029] Figure 3 For bromocyanamide-resistant populations (SG20) BtUGPase Efficiency of gene interference.

[0030] Figure 4 For fluoxazolamide-resistant population (FXM-R-22) BtUGPase Efficiency of gene interference.

[0031] Figure 5 The dose-response curves of brofentanil to brofentanil after interference with the brofentanil-resistant population (SG20).

[0032] Figure 6The dose-response curve of fluoxazolamide to fluoxazolamide after interference with the fluoxazolamide-resistant population (FXM-R-22). Detailed Implementation

[0033] The following description uses specific experimental examples, but the scope of protection of this invention is not limited thereto.

[0034] Unless otherwise specified in the examples, the procedures were performed under standard conditions; reagents or instruments used without a specified manufacturer were all commercially available products.

[0035] The tested populations are shown in Table 1.

[0036] Table 1

[0037]

[0038] Experimental methods

[0039] 1. Bioassay

[0040] The median lethal concentration (LC50) of cyanamide against whitefly resistant populations (SG20) and susceptible populations (QS) was determined using the agar moist leaf immersion method. 50 : Prepare a solution of 5×10 by dissolving bromocyanamide (94%, stock solution) in DMSO (dimethyl sulfoxide). 4 A stock solution of 200 mg / L was diluted with 0.5‰ Triton X-100 aqueous solution to create five concentration gradients: 200 mg / L, 100 mg / L, 33.3 mg / L, 11.1 mg / L, and 3.7 mg / L. The control was a 0.5‰ Triton X-100 aqueous solution. Cotton leaf discs (2.5 cm in diameter) were immersed in each solution for 10 seconds, air-dried for 15 minutes, and then placed face down on a 1% (0.01 g / mL) agar bed inside 50 mL centrifuge tubes. Each tube was used to feed whiteflies, with 15 adult whiteflies inoculated (the tube opening was sealed with an insect-proof net and a cap containing the leaf). Mortality was recorded after 48 hours, and the LC50 was calculated using PoloPlus software. 50And 95% confidence interval. Similarly, the toxicity of fluoxazolamide to the resistant (FXM-R-F22) and susceptible (FXM-S) whitefly populations was evaluated using the agar moist leaf immersion method: 9% fluoxazolamide formulation was prepared into a series of concentrations of 900 mg / L, 450 mg / L, 225 mg / L, 75 mg / L and 15 mg / L using 0.5‰ Triton X-100 aqueous solution, with 0.5‰ Triton X-100 aqueous solution as the control; 2.5 cm diameter cotton leaf discs were immersed in each concentration of the drug solution for 10 s, air-dried for 15 min, and then placed face down on a 1% (0.01 g / mL) agar bed in a 50 mL centrifuge tube cap. 15 adult whiteflies were inoculated into each treatment (the tube opening was double-sealed with an insect-proof net and a cap containing the leaf). Mortality data were recorded after 48 h, and LC50 was calculated using PoloPlus software. 50 The value and its 95% confidence interval.

[0041] 2. Synergist Experiment

[0042] To evaluate the synergistic effect of UGT enzyme inhibitors on insecticides, sulfinpyrazone (Sul) and 5-nitrouracil (5-Nul) were selected as inhibitors and prepared into working solutions with a concentration of 400 mg / L using acetone. The toxicity effects of the two inhibitors on the bromocyanamide-resistant population (SG20) and the fluoxazolamide-resistant population (FXM-R-F22) were determined using the agar moist leaf immersion method.

[0043] 3. UGT enzyme activity assay

[0044] UDP-glycosyltransferase (UGT) has the function of catalyzing the conversion of the mode substrate α-naphthol to α-naphthyl glucoside.

[0045] The UDP-glycosyltransferase (UGT) activities of bromocyanamide-resistant and susceptible whitefly populations (SG20 and QS), and fluoxazolamide-resistant and susceptible whitefly populations (FXM-R-F22 and FXM-S) were determined using enzyme kinetics. The insects of different populations were lysed with 0.05 M PBS (pH 7.8) and centrifuged at 12000 g for 15 min at 4 °C to obtain the supernatant enzyme solution. MgCl2, UDP-glucose, and fresh enzyme solution were added sequentially to 1.5 mL centrifuge tubes, and water was added to a final volume of 960 µL, resulting in a final concentration of 10 mM for MgCl2 and 1 mM for UDP-glucose. The mixture was aliquoted into 240 µL / well microplates, and finally, 10 µL of α-naphthol solution (final concentration 250 µM) was added and mixed thoroughly to obtain the enzyme reaction system. Infinite 200 microplates were used for the reaction. The Pro microplate reader continuously monitored the enzyme reaction system for 15 minutes under conditions of 287 nm excitation wavelength, 335 nm emission wavelength, and 5 nm slit width, detecting the fluorescence value of the enzyme reaction system. Simultaneously, a reaction system without α-naphthol was used as a negative control to measure the background fluorescence value. A standard curve of α-naphthyl glucoside and fluorescence value was plotted. The content of α-naphthyl glucoside in the enzyme reaction system was calculated based on the standard curve, and then the enzyme activity of UGT was calculated based on the α-naphthyl glucoside content. Three biological replicates were performed for each sample.

[0046] The specific calculation method for UGT enzyme activity is as follows: Based on the standard curve of α-naphthyl glucoside and fluorescence value, the fluorescence value of the enzyme reaction system is substituted into the standard curve to obtain the content of α-naphthyl glucoside; the content of α-naphthyl glucoside is divided by the monitoring time, and then divided by the protein content of the sample to obtain the enzyme activity value in "pmol / min / mg".

[0047] 4. Synthesize ds BtUGPase

[0048] Total RNA was extracted from whiteflies in the SG20 and FXM-R-F22 populations using Trizol reagent. First-strand cDNA was synthesized using the PrimeScript™ II 1st Strand cDNA Synthesis Kit. The cDNA was then amplified using ApexHF HS DNA Polymerase. BtUGPase The gene fragment was synthesized. Simultaneously, dsRNA (dsGFP) synthesized from green fluorescent protein served as a control group for subsequent RNAi experiments.

[0049] The PCR amplification system (50 μL) consisted of: ApexHF HS DNA Polymerase 25 μL, dsBtUGPase-F: 1 μL, dsBtUGPase-R: 1 μL, cDNA template 2 μL, and ddH2O 21 μL.

[0050] The PCR amplification program was as follows: 94℃ pre-denaturation for 1 min; 94℃ denaturation for 15 s, 55℃ annealing for 15 s, 72℃ extension for 10 s, for a total of 35 cycles; 72℃ for 1 min.

[0051] Using the above-synthesized BtUGPase Using gene fragments as templates, the TranscriptAid T7 HighYield Transcription Kit was used for synthesis. BtUGPase Double-stranded RNA (dsRNA);

[0052] The dsRNA synthesis system consisted of: 5 μL of 5×TranscriptAid Reaction Buffer, 2 μL each of ribonucleotides (A / G / C / U, 100 mM), 1 μg of DNA template, and 20 μL of enzyme-free water, all mixed thoroughly.

[0053] BtUGPase The nucleotide sequence of the gene is shown in SEQ ID NO.1;

[0054] The BtUGPase primer sequences are as follows: dsBtUGPase-F as shown in SEQ ID NO.3; dsBtUGPase-R as shown in SEQ ID NO.4.

[0055] The above dsRNA synthesis system was incubated at 37℃ for 4 h. After incubation, 2 μL of DNase I was added, and the mixture was incubated in a 37℃ water bath for 15 min. 2 μL of 0.5M EDTA was added, and the mixture was incubated at 65℃ for 10 min. Enzyme-free water was added to bring the volume to 500 μL, and then 200 μL of chloroform was added and gently mixed. The mixture was allowed to stand for 10 min. The mixture was centrifuged at 12000g and 4℃ for 15 min, and the supernatant was collected. The process was repeated with the addition of 200 μL of chloroform, centrifugation, and collection of the supernatant. Sodium acetate (3M, pH 10.5) was added to 1 / 10 volume of the supernatant. 5.2) Add 2.5 times the volume of ethanol to the supernatant and incubate at -80℃ for 2.5 h; centrifuge at 12000g, 4℃ for 30 min, discard the supernatant, add 1 mL of 75% ethanol, and resuspend the precipitate; centrifuge at 10000g, 4℃ for 10 min, discard the supernatant, and after the remaining ethanol in the tube has dried, dissolve it in 200 μL of enzyme-free water to obtain the target product dsRNA, i.e., ds BtUGPase The nucleotide sequence is shown in SEQ ID NO.2.

[0056] Simultaneously, dsRNA (ds) synthesizes green fluorescent protein (GFP). GFP () was used as a control group for subsequent RNA interference (RNAi) experiments.

[0057] 5. RNAi

[0058] ds BtUGPase and ds GFP Dissolve each dsRNA separately in 20% (0.2 mg / L) sucrose water to achieve a final concentration of 250 ng / µL, thus obtaining the feeding solution. The feeding solution was added to a feeding device, which was made of 50 mL centrifuge tubes. The tubes were sealed with insect-proof netting around the sides and bottom, and the top was sealed with a centrifuge tube cap. The feeding solution was separated from approximately 500 adult whiteflies through a polytetrafluoroethylene (PTFE) membrane. Using this method, the SG20 and FXM-R-F22 populations were fed separately. The whitefly mouthparts could pierce the PTFE membrane, allowing the aspirator to extract the feeding solution from the inside. After 72 hours of feeding, the surviving whiteflies were collected for subsequent qPCR and bioassay experiments.

[0059] 6. qPCR experiment

[0060] Detection of RNAi-surviving whiteflies in vivo using qPCR method BtUGPase Relative gene expression levels; total RNA was extracted from surviving whiteflies after RNAi using Trizol reagent, and then mRNA was reverse transcribed using the PrimeScript™ RT reagent Kit with gDNA eraser according to the kit instructions to synthesize first-strand cDNA; succinate dehydrogenase complex A (SDHA) and heat shock protein 40 (HSP40) were used as two internal control genes for mRNA expression analysis. Each treatment was performed in triplicate, using 2- ΔΔCt The relative expression of the BtUGPase gene in whiteflies after RNAi was analyzed using a relative quantitative method.

[0061] The qPCR reaction system (20 μL) consisted of the following: 10 μL TB Green Advantage Premix, 0.5 μL Primer-F, 0.5 μL Primer-R, 2 μL cDNA, and 7 μL ddH2O.

[0062] The qPCR amplification program is as follows: 95℃ pre-denaturation for 30s; 95℃ denaturation for 5s, 60℃ annealing and extension for 34s, 40 cycles.

[0063] 7. Bioassays after RNAi

[0064] Following the bioassay method described in step 1 above, bioassays were performed on surviving whitefly individuals in the RNAi-treated cyantraniliprole-resistant population (SG20) and the interference-treated fluoxazolamide-resistant population (FXM-R-F22). Mortality rates were recorded after 48 h, and LC was calculated using PoloPlus software. 50 The value and its 95% confidence interval.

[0065] Experimental results

[0066] 1. Bioassay and synergist results

[0067] Experimental results showed that the LC50 of the cyanamide-resistant population (SG20) 50 The toxicity of bromocyanamide against whiteflies was 33.8 times that of the susceptible population (QS). Meanwhile, the UGT enzyme inhibitors sulfinpyrazone (Sul) and 5-nitrouracil (5-Nul) both significantly enhanced the toxicity of bromocyanamide against whiteflies. In the SG20 resistant population, Sul and 5-Nul showed significant synergistic effects of 2.93 times and 2.31 times, respectively (Table 2). The LC50 of the fluoxazolamide-resistant population (FXM-R-F22) was significantly higher. 50 The synergistic effect of the whitefly on sulpera (FXM-S) was 103.3 times that of the susceptible population. In the FXM-R-F22 resistant population, the synergistic ratios of the potentiators sulfinpyrazone (Sul) and 5-nitrouracil (5-Nul) reached 1.8 and 1.5 times, respectively (Table 2). This finding suggests that the UGT enzyme in whiteflies may be involved in the formation of their resistance to bromocyanamide and fluoxazolamide.

[0068] Table 2. Toxicity assays of bromocyanamide and fluoxazolamide in their respective resistant and susceptible populations, and analysis of the synergistic effects of UGT inhibitors (Sul and 5-Nul).

[0069]

[0070] a. The number of whiteflies used in the bioassay;

[0071] Slope±SE represents the standard error of the regression slope;

[0072] b. 95% confidence interval;

[0073] c Chi-square value ( χ 2 ) and degrees of freedom df (Obtained via PoloPlus software.) The linear relationship between dose-mortality response was assessed using a chi-square test. p >0.05);

[0074] d Synergistic effect ratio = Lethal median concentration (LC50) of insecticides used alone (brofentanil or fluoxazolamide) 50 / Lethal median concentration (LC50) when insecticides and synergists are used in combination 50 );

[0075] e Resistance ratio (RR) = LC of the resistant population 50 / LC of sensitive populations 50 .

[0076] 2. Results of UGT enzyme activity assay

[0077] The UGT enzyme activity assay results showed that compared with the cyantraniliprole-sensitive population QS, the cyantraniliprole-resistant population (SG20) had a significantly increased UGT activity, with a 3.7-fold increase in UGT enzyme activity. Figure 1 Similarly, the UGT activity of the fluoxazolamide-resistant population (FXM-R-22) was significantly increased compared to that of the susceptible population (FXM-S), with a 2.1-fold increase in UGT activity. Figure 2 The above results further demonstrate that the enhancement of UGT enzyme activity may be a key factor leading to the formation of resistance in whiteflies, and this enhancement was significantly observed in different resistant populations.

[0078] 3. RNAi successfully knocked down the levels of phosphatidylcholine in whiteflies. BtUGPase Gene

[0079] The results showed that when the bromoxynil-resistant population (SG20) of whiteflies fed on ds BtUGPase Three days later, BtUGPase Expression decreased to 60% of the control group ( Figure 3 Similarly, when the fluoxazolamide-resistant population (FXM-R-22) feeds on ds BtUGPase Three days later, compared with the control group, BtUGPase The expression decreased by 70% ( Figure 4 The above results indicate that feeding dsRNA can achieve [results] in both resistant populations. BtUGPase Effective knockdown of genes.

[0080] 4. After successful RNAi knockdown, the resistance level of whiteflies decreased.

[0081] Investigating key genes for glycosyl donors using RNAi BtUGPase The effect of chlorantraniliprole on whitefly resistance, experimental results of the bromoxynil-resistant population (SG20) showed that, compared with ds GFP Compared with the control group, those who consumed foods containing ds BtUGPase The mortality rate of adult whiteflies in sucrose solution increased significantly at all five test concentrations after 72 hours. BtUGPase LC of the processing group 50 The value decreased to 24.1% of the control group, meaning the resistance decreased by 4.1 times (Table 3).Figure 5 Experimental results of the fluoxazolamide-resistant population (FXM-R-22) showed that, compared with ds GFP Compared with the control group, those who consumed foods containing ds BtUGPase The mortality rate of adult whiteflies in sucrose solution increased significantly at all five concentrations after 72 hours. The LC50 of the treatment groups was [not specified]. 50 The value decreased to 28.8% of the control group, meaning the resistance decreased by 3.5 times (Table 4). Figure 6 ).

[0082] Table 3. Determination of toxicity to cyananthramide in cyananthramide-resistant populations (SG20) after feeding with dsRNA.

[0083]

[0084] a. The number of whiteflies used in the bioassay;

[0085] Slope±SE represents the standard error of the regression slope;

[0086] b. 95% confidence interval;

[0087] c Chi-square value ( χ 2 ) and degrees of freedom df (Obtained via Polo Plus software.) The linear relationship between dose-mortality response was assessed using a chi-square test. p >0.05).

[0088] Table 4. Fluoxazolamide toxicity assay of fluoxazolamide-resistant population (FXM-R-22) after dsRNA feeding.

[0089]

[0090] a. The number of whiteflies used in the bioassay;

[0091] Slope±SE represents the standard error of the regression slope;

[0092] b. 95% confidence interval;

[0093] c Chi-square value ( χ 2 ) and degrees of freedom df (Obtained via Polo Plus software.) The linear relationship between dose-mortality response was assessed using a chi-square test. p >0.05).

[0094] The above experimental results show that BtUGPase The gene is closely related to UGT-mediated resistance in whiteflies to bromocyanamide and fluoxazolamide. When whiteflies feed on...BtUGPase dsRNA (ds BtUGPase After that, resistance to bromocyanamide and fluoxazolamide was significantly reduced.

Claims

1. The application of a dsRNA in the control of insecticide-resistant whiteflies, comprising any one of the following applications: (1) Control of fluoxazolamide-resistant whiteflies; (2) Control of whiteflies resistant to bromocyanamide and fluoxazolamide; The dsRNA is BtUGPase dsRNA of genes BtUGPase The nucleotide sequence of the gene is shown in SEQ ID NO.

1. BtUGPase The nucleotide sequence of the gene's dsRNA is shown in SEQ ID NO.

2.

2. The application as described in claim 1, characterized in that, The application method involves feeding whiteflies. BtUGPase The dsRNA of the gene reduces the resistance of whiteflies to bromocyanamide and fluoxazolamide.

3. The application as described in claim 2, characterized in that, The feeding method is as follows: using a feed containing... BtUGPase Whiteflies were fed sucrose water containing the dsRNA of the gene.

4. The application as described in claim 3, characterized in that, The concentration of sucrose in the sucrose solution is 0.17~0.23 mg / L.

5. The application as described in claim 3, characterized in that, The concentration of sucrose in the sucrose solution is 0.2 mg / L.

6. The application as described in claim 3, characterized in that, The BtUGPase The concentration of the gene's dsRNA in sucrose water was 250 ng / µL.

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