Application of dsRNA in prevention and treatment of pesticide-resistant bemisia tabaci

Abstract

The invention provides application of dsRNA in prevention and treatment of pesticide-resistant bemisia tabaci, belongs to the technical field of pesticides, particularly relates to application of dsRNA of a BtUGPase gene in prevention and treatment of cyantraniliprole and fluxazole amide-resistant bemisia tabaci, and discovers and verifies that the BtUGPase gene is closely related to resistance of UGT-mediated bemisia tabaci to cyantraniliprole and fluxazole amide. After the bemisia tabaci eats the dsRNA of the BtUGPase, the resistance to cyantraniliprole and fluxazole amide is obviously reduced.

Description

Technical Field

[0001] The present invention belongs to the technical field of pesticides, and specifically relates to the application of dsRNA in controlling insecticide-resistant Bemisia tabaci. Background Art

[0002] Bemisia tabaci Bemisia tabaci (Gennadius)(Hemipetra:Alteyrodidae) belongs to hemimetabolous 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] At present, the control measures for Bemisia tabaci still mainly rely on chemical control. Chemical control refers to spraying various insecticides on pests or plant leaves, which has the characteristics of quick effect and significant insecticidal effect.

[0004] Due to the long-term use of insecticides, it is inevitable that Bemisia tabaci has developed varying degrees of resistance to a variety of traditional insecticides (such as neonicotinoids and pyrethroids). The resistance of Bemisia tabaci develops rapidly and has become a major challenge in the current field of pest control. To address this problem, insecticides with novel action mechanisms and no cross-resistance are needed as alternative agents for resistance management. In this context, cyantraniliprole in diamide insecticides and flufiprole in novel isoxazoline insecticides have attracted much attention due to their unique action mechanisms and good control effects.

[0005] As the first diamide insecticide with good insecticidal activity against piercing-sucking mouthpart pests, the target of cyantraniliprole is the ryanodine receptor in insect muscle tissue, which can induce continuous contraction of the muscles of Bemisia tabaci, resulting in impaired motor function until death. Due to its unique action mechanism, it has no cross-resistance with other traditional insecticides and can be used to effectively control Bemisia tabaci in the field. However, due to repeated use, the field populations of Bemisia tabaci in some regions of China have developed obvious resistance to cyantraniliprole. Therefore, it is urgent to find new methods to delay the emergence of resistance.

[0006] Flufiprole represents a novel class of isoxazoline neurotoxins that interfere with the insect nervous system through both contact and stomach poisoning actions. This compound can specifically act on the γ-aminobutyric acid (GABA) receptor, interfere with the nerve conduction of Bemisia tabaci, and cause its overexcitation, spasm until death. In recent years, due to the increasing resistance of Bemisia tabaci to traditional insecticides, flufiprole has received extensive attention as an alternative agent. Through more than 20 generations of insecticide screening, a Bemisia tabaci population with more than 100-fold resistance to flufiprole was obtained, indicating that Bemisia tabaci has a certain resistance risk to flufiprole. Therefore, it is necessary to actively search for effective resistance management methods.

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

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

[0009] In the prior art, although there have been reports on dsRNA for controlling cyantraniliprole-resistant Bemisia tabaci, there have been no reports on dsRNA that is effective for controlling both cyantraniliprole-resistant Bemisia tabaci and fluazifop-resistant Bemisia tabaci. Summary of the Invention

[0010] In response to the above problems, the present invention provides a use of dsRNA in controlling insecticide-resistant whiteflies.

[0011] The present invention discovered and confirmed that in the UDPG synthesis pathway of insects, BtUGPase The gene is closely related to the UGT-mediated resistance of whitefly to cyantraniliprole and fluazifop-dapoxetine. BtUGPase dsRNA (ds BtUGPase ), the resistance to cyantraniliprole and fluazifop-butyl was significantly reduced.

[0012] The technical solutions of the present invention are as follows: A use of dsRNA in controlling insecticide-resistant whiteflies, comprising any one of the following uses: (1) Control of cyantraniliprole-resistant whiteflies; (2) Control of fluoxetine-resistant whiteflies; (3) Control of cyantraniliprole and fluazifop-resistant whiteflies; (4) Preparation of reagents for controlling whitefly; Wherein, the dsRNA is BtUGPase dsRNA of genes, BtUGPase The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0013] Preferably, BtUGPase The nucleotide sequence of the dsRNA of the gene is shown in SEQ ID NO.2.

[0014] Preferably, the application method is by feeding the whitefly BtUGPase The dsRNA of the gene reduces the resistance of Bemisia tabaci to cyantraniliprole and fluazifop-butyl.

[0015] Preferably, the method of feeding ds BtUGPase is as follows: feeding Bemisia tabaci with sucrose solution containing BtUGPase dsRNA of the gene.

[0016] Preferably, the concentration of sucrose in the sucrose solution is 0.17 - 0.23 mg / L.

[0017] Preferably, the concentration of sucrose in the sucrose solution is 0.2 mg / L.

[0018] Preferably, the BtUGPase concentration of dsRNA of the gene in the sucrose solution is 250 ng / µL.

[0019] Advantages of the present invention: The present invention discovers and confirms that BtUGPase the gene is closely related to the resistance of Bemisia tabaci to cyantraniliprole and fluxametamide mediated by UGT. When Bemisia tabaci feeds on BtUGPase dsRNA (ds BtUGPase ), its resistance to cyantraniliprole and fluxametamide is significantly reduced. The present invention provides support for the molecular targets of Bemisia tabaci resistance management and lays a good theoretical and application foundation for the development of new control methods for cyantraniliprole - and fluxametamide - resistant Bemisia tabaci. Description of the drawings

[0020] Figure 1 UGT enzyme activities of the cyantraniliprole - resistant population and the susceptible population.

[0021] Figure 2 UGT enzyme activities of the fluxametamide - resistant population and the susceptible population.

[0022] Figure 3 Interference efficiency of the gene in the cyantraniliprole - resistant population (SG20) BtUGPase Interference efficiency of the gene.

[0023] Figure 4 Interference efficiency of the gene in the fluxametamide - resistant population (FXM - R - 22) BtUGPase Interference efficiency of the gene.

[0024] Figure 5 Dose - response curve of the cyantraniliprole - resistant population (SG20) after interference with cyantraniliprole.

[0025] Figure 6 Dose - response curve of the fluxametamide - resistant population (FXM - R - 22) after interference with fluxametamide. Detailed implementation manners

[0026] The following is illustrated with specific experimental examples, but the protection scope of the present invention is not limited thereto.

[0027] For the content without specific conditions in the examples, conventional conditions are followed; for the reagents or instruments without specified manufacturers, they are all common commercially available products.

[0028] The test populations are shown in Table 1.

[0029] Table 1

[0030] Experimental method 1. Bioassay The agar moisturizing leaf dipping method was used to determine the median lethal concentration LC of cyantraniliprole against the resistant population (SG20) and the susceptible population (QS) of Bemisia tabaci: Cyantraniliprole (94%, stock solution) was dissolved in DMSO (dimethyl sulfoxide) to prepare a 5×10 50 mg / L mother liquor, and then diluted with 0.5‰ Triton X-100 aqueous solution to five concentration gradients of 200 mg / L, 100 mg / L, 33.3 mg / L, 11.1 mg / L, and 3.7 mg / L. The control was 0.5‰ Triton X-100 aqueous solution; Cotton leaf discs with a diameter of 2.5 cm were immersed in each medicinal solution for 10 s, air-dried for 15 min, and then placed with the back side down on a 1% (0.01 g / mL) agar bed in the lid of a 50 mL centrifuge tube for feeding Bemisia tabaci. 15 adult Bemisia tabaci were introduced into each tube (the tube mouth was sealed with an insect-proof net and the tube lid containing the leaf), and the mortality was counted after 48 h, and the LC 4 and 95% confidence interval were calculated by PoloPlus software. Similarly, the agar moisturizing leaf dipping method was used to evaluate the toxicity of fluxametamide against the resistant population (FXM-R-F22) and the susceptible population (FXM-S) of Bemisia tabaci: The 9% fluxametamide 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 with 0.5‰ Triton X-100 aqueous solution, and 0.5‰ Triton X-100 aqueous solution was used as the control; Cotton leaf discs with a diameter of 2.5 cm were immersed in each concentration of medicinal solution for 10 s, air-dried for 15 min, and then placed with the back side down on a 1% (0.01 g / mL) agar bed in the lid of a 50 mL centrifuge tube. 15 adults were introduced into each treatment (the tube mouth was double-sealed with an insect-proof net and the tube lid containing the leaf), and the mortality data were recorded after 48 h, and the LC 50 value and its 95% confidence interval were calculated using PoloPlus software. 50

[0031] 2. Synergist experiment 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 a working solution concentration of 400 mg / L by dissolving in acetone. Through the agar moisturizing leaf-dipping method, the toxicity effects of the two inhibitors on the cyantraniliprole-resistant population (SG20) and the fluxametamide-resistant population (FXM-R-F22) were measured respectively.

[0032] 3. Determination of UGT enzyme activity UDP-glycosyltransferase (UGT) has the function of catalyzing the model substrate α-naphthol to generate α-naphthyl glucoside.

[0033] The enzyme kinetics method was used to determine the UDP-glycosyltransferase (UGT) activities of the cyantraniliprole-resistant Bemisia tabaci population (SG20) and the susceptible Bemisia tabaci population (QS), as well as the fluxametamide-resistant Bemisia tabaci population (FXM-R-F22) and the susceptible Bemisia tabaci population (FXM-S): The bodies of different populations of insects were lysed with 0.05 M PBS (pH 7.8), and the supernatant enzyme solution was obtained by centrifugation at 12000 g for 15 min at 4°C; MgCl2, UDP-glucose, and fresh enzyme solution were added to a 1.5 mL centrifuge tube in turn, and then water was added to make up to 960 µL, so that the final concentration of MgCl2 was 10 mM and the final concentration of UDP-glucose was 1 mM to obtain a mixed system; the mixed system was dispensed into an enzyme-labeled plate at 240 µL / well, and finally 10 µL of α-naphthol solution with a final concentration of 250 µM was added and mixed evenly to obtain an enzyme reaction system; using an Infinite 200 Pro enzyme-labeled instrument, the fluorescence value of the enzyme reaction system was continuously monitored for 15 min under the conditions of an excitation wavelength of 287 nm, an emission wavelength of 335 nm, and a slit width of 5 nm, and the background fluorescence value was measured with a reaction system without α-naphthol as a negative control. A standard curve of α-naphthyl glucoside-fluorescence value was drawn, the content of α-naphthyl glucoside in the enzyme reaction system was calculated according to the standard curve, and then the enzyme activity of UGT was calculated according to the content of α-naphthyl glucoside. Each sample was set with 3 biological replicates.

[0034] Among them, the specific calculation method of the enzyme activity of UGT is as follows: According to the standard curve of α-naphthyl glucoside-fluorescence value, substituting the fluorescence value of the enzyme reaction system into the standard curve, the content of α-naphthyl glucoside can be obtained; dividing the content of α-naphthyl glucoside by the monitoring time and then by the protein content of the measured sample, the enzyme activity value in the unit of "pmol / min / mg" can be obtained.

[0035] 4. Synthesis of ds BtUGPase Total RNA of Bemisia tabaci populations SG20 and FXM-R-F22 was extracted using Trizol reagent, and the first-strand cDNA was synthesized using PrimeScript™ II 1st Strand cDNA Synthesis Kit; using the cDNA as a template, the gene fragment was amplified using ApexHF HS DNA Polymerase to obtain BtUGPase The dsRNA of green fluorescent protein (dsGFP) was synthesized as a control group for subsequent RNAi experiments.

[0036] Among them, the PCR amplification system (50 μL) was: ApexHF HS DNA Polymerase 25 μL, dsBtUGPase-F: 1 μL, dsBtUGPase-R: 1 μL, cDNA template 2 μL, ddH2O 21 μL.

[0037] The PCR amplification program was: pre-denaturation at 94 °C for 1 min; denaturation at 94 °C for 15 s, annealing at 55 °C for 15 s, extension at 72 °C for 10 s, for a total of 35 cycles; 72 °C, 1 min.

[0038] Using the fragment of the BtUGPase gene synthesized above as a template, double-stranded RNA (dsRNA) of BtUGPase was synthesized using TranscriptAid T7 HighYield Transcription Kit; Among them, the dsRNA synthesis system was: 5×TranscriptAid Reaction Buffer 5 μL, ribonucleotides (A / G / C / U, 100 mM) 2 μL each, DNA template 1 μg, and nuclease-free water was added to make up 20 μL, and mixed well.

[0039] BtUGPase The nucleotide sequence of the gene is shown in SEQ ID NO.1; Among them, the BtUGPase primer sequences: dsBtUGPase-F is shown in SEQ ID NO.3; dsBtUGPase-R is shown in SEQ ID NO.4.

[0040] Incubate the above dsRNA synthesis system at 37 °C for 4 h. After the incubation, add 2 μL of DNase I and incubate in a 37 °C water bath for 15 min; add 2 μL of 0.5 M EDTA and incubate at 65 °C for 10 min; make up to 500 μL with enzyme-free water, then add 200 μL of chloroform and mix gently, and let stand for 10 min; centrifuge at 12000 g at 4 °C for 15 min and take the supernatant; repeat adding 200 μL of chloroform, centrifuging, and taking the supernatant; add 1 / 10 volume of sodium acetate (3 M, pH 5.2) of the supernatant and 2.5 times the volume of ethanol of the supernatant, and place at -80 °C for 2.5 h; centrifuge at 12000 g at 4 °C for 30 min, discard the supernatant, add 1 mL of 75% ethanol to resuspend the precipitate; centrifuge at 10000 g at 4 °C for 10 min, discard the supernatant, and after the remaining ethanol in the tube is dried, add 200 μL of enzyme-free water to dissolve to obtain the target product dsRNA, i.e., ds BtUGPase , and its nucleotide sequence is shown in SEQ ID NO.2.

[0041] Meanwhile, synthesize dsRNA (ds GFP ) of green fluorescent protein (GFP) as a control group for subsequent RNA interference (RNAi) experiments.

[0042] 5. RNAi Dissolve ds BtUGPase and ds GFP separately in 20% (0.2 mg / L) sucrose water to make the final concentration of each dsRNA 250 ng / μL to obtain the feeding solution; add the feeding solution to the feeding device respectively. The feeding device is made of a 50 mL centrifuge tube, the periphery and bottom of the tube are enclosed by an insect-proof net, and the top is sealed by a centrifuge tube cap; separate the feeding solution from about 500 adult Bemisia tabaci through a polytetrafluoroethylene membrane. Feed the SG20 population and the FXM-R-F22 population respectively according to this method. The mouthparts of Bemisia tabaci can pierce the polytetrafluoroethylene membrane to absorb the feeding solution inside the membrane; after feeding for 72 hours, collect the surviving Bemisia tabaci for subsequent qPCR experiments and bioassay experiments.

[0043] 6. qPCR experiment Detect the relative expression level of the BtUGPase gene in the surviving Bemisia tabaci after RNAi by qPCR method; extract the total RNA of the surviving Bemisia tabaci after RNAi with Trizol reagent respectively, and then reverse transcribe mRNA with PrimeScript™ RT reagent Kit with gDNA eraser. The reverse transcription is carried out according to the kit instructions to synthesize the first-strand cDNA; use succinate dehydrogenase complex A (SDHA) and heat shock protein 40 (HSP40) as two internal reference genes for mRNA expression analysis. Each treatment is repeated three times biologically, and the 2-ΔΔCt Analysis of the relative expression of the BtUGPase gene in Bemisia tabaci after RNAi by relative quantification method.

[0044] Among them, the qPCR reaction system (20 μL) is as follows: TB Green Advantage Premix 10 μL, Primer-F 0.5 μL, Primer-R 0.5 μL, cDNA 2 μL, ddH2O 7 μL.

[0045] The qPCR amplification program is as follows: pre-denaturation at 95 °C for 30 s; denaturation at 95 °C for 5 s, annealing and extension at 60 °C for 34 s, 40 cycles.

[0046] 7. Bioassay after RNAi Refer to the bioassay method described in step 1 of the previous text to conduct a bioassay on the surviving Bemisia tabaci individuals of the cyantraniliprole-resistant population (SG20) and the fluxametamide-resistant population (FXM-R-F22) after RNAi treatment. Record the mortality data after 48 h, and use PoloPlus software to calculate the LC 50 value and its 95% confidence interval.

[0047] Experimental results 1. Bioassay and synergist results The experimental results show that the LC 50 of the cyantraniliprole-resistant population (SG20) is 33.8 times that of its susceptible population (QS). At the same time, the UGT enzyme inhibitors sulfapyrone (Sul) and 5-nitrouracil (5-Nul) both significantly enhanced the virulence effect of cyantraniliprole on Bemisia tabaci. 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 LC 50 of the fluxametamide-resistant population (FXM-R-F22) is 103.3 times that of its susceptible population (FXM-S). In the FXM-R-F22 resistant population, the synergistic ratios of the synergists sulfapyrone (Sul) and 5-nitrouracil (5-Nul) reached 1.8 and 1.5 times (Table 2). This finding suggests that the UGT enzyme of Bemisia tabaci may be involved in the formation of its resistance to cyantraniliprole and fluxametamide.

[0048] Table 2 Toxicity determination of cyantraniliprole and fluxametamide against their corresponding resistant and susceptible populations and analysis of the synergistic effects of UGT inhibitors (Sul and 5-Nul)

[0049] a The number of Bemisia tabaci used in the bioassay; Slope ± SE represents the standard error of the regression slope; b 95% confidence interval; c Chi-square value ( χ 2 ) and degrees of freedom ( df ) were obtained by software PoloPlus. The linear relationship of the dose-mortality response was evaluated by chi-square test ( p >0.05); d Synergistic ratio = median lethal concentration (LC 50 ) of the insecticide (cyantraniliprole or flonicamid) used alone / median lethal concentration (LC 50 ) when the insecticide was used in combination with the synergist; e Resistance ratio (RR) = LC 50 of the resistant population / LC 50 of the susceptible population.

[0050] 2. Results of UGT enzyme activity assay The results of UGT enzyme activity assay showed that compared with the UGT enzyme activity value of the cyantraniliprole-susceptible population QS population, the UGT activity of the cyantraniliprole-resistant population (SG20) increased significantly, and the UGT enzyme activity increased by 3.7 times ( Figure 1 ) Similarly, compared with the UGT enzyme activity value of the sensitive population FXM-S population, the UGT activity of the flonicamid-resistant population (FXM-R-22) increased significantly, and the UGT enzyme activity increased by 2.1 times ( Figure 2 ). The above results indicate again that the increase in UGT enzyme activity may be a key factor leading to the formation of Bemisia tabaci resistance, and this increase is significantly manifested in different resistant populations.

[0051] 3. RNAi successfully knocked down the BtUGPase gene in Bemisia tabaci The results showed that when Bemisia tabaci of the cyantraniliprole-resistant population (SG20) fed on ds BtUGPase for three days, BtUGPase expression decreased to 60% of the control group ( Figure 3 ). Similarly, when the flonicamid-resistant population (FXM-R-22) fed on ds BtUGPase for three days, compared with the control group, BtUGPase expression decreased by 70% ( Figure 4 ). The above results indicate that effective knockdown of the BtUGPase gene can be achieved in both resistant populations after feeding dsRNA.

[0052] 4. After successful RNAi knockdown, the resistance level of Bemisia tabaci decreased The key gene of glycosyl donor was explored by RNAi ( BtUGPase) on the resistance of Bemisia tabaci. The experimental results of cyantraniliprole-resistant population (SG20) showed that GFP Compared with the control group, the BtUGPase The mortality of whitefly adults exposed to sucrose solution for 72 hours increased significantly at all five test concentrations. BtUGPase LC of the treatment group 50 The value was reduced to 24.1% of the control group, that is, the resistance decreased by 4.1 times (Table 3, Figure 5 The experimental results of the fluoxetine-resistant population (FXM-R-22) showed that the ds GFP Compared with the control group, the BtUGPase The mortality of whitefly adults exposed to sucrose solution for 72 hours increased significantly at all five concentrations. 50 The value was reduced to 28.8% of the control group, that is, the resistance decreased by 3.5 times (Table 4, Figure 6 ).

[0053] Table 3 Toxicity test of cyantraniliprole in SG20 resistant population after feeding with dsRNA

[0054] a The number of whiteflies used in the bioassay; Slope±SE represents the standard error of the regression slope; b 95% confidence interval; c Chi-square value ( χ 2 ) and degrees of freedom ( df ) was obtained using Polo Plus software. The linear relationship of dose-mortality response was assessed by chi-square test ( p >0.05).

[0055] Table 4 Toxicity test of oxazolidinone-resistant population (FXM-R-22) after feeding dsRNA to oxazolidinone

[0056] a Number of whiteflies used in the bioassay; Slope±SE represents the standard error of the regression slope; b 95% confidence interval; c Chi-square value ( χ 2 ) and degrees of freedom ( df ) was obtained using Polo Plus software. The linear relationship of dose-mortality response was assessed by chi-square test ( p >0.05).

[0057] The above experimental results show that, BtUGPase the gene is closely related to the UGT-mediated resistance of Bemisia tabaci to cyantraniliprole and flonicamid. When Bemisia tabaci feeds on BtUGPase dsRNA (ds BtUGPase ), its resistance to cyantraniliprole and flonicamid is significantly reduced.

Claims

1. Use of a dsRNA in controlling insecticide-resistant Bemisia tabaci, including any one of the following applications: (1) Controlling chlorantraniliprole-resistant Bemisia tabaci; (2) Controlling fluxametamide-resistant Bemisia tabaci; (3) Controlling chlorantraniliprole- and fluxametamide-resistant Bemisia tabaci; (4) Preparing a reagent for controlling Bemisia tabaci; Among them, The dsRNA is BtUGPase dsRNA of the BtUGPase gene, and the nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, BtUGPase The nucleotide sequence of the dsRNA of the gene is shown in SEQ ID NO.

2.

3. The application according to claim 1, characterized in that, The application method is to feed Bemisia tabaci BtUGPase with dsRNA of the gene to reduce the resistance of Bemisia tabaci to cyantraniliprole and flonicamid.

4. The application according to claim 3, characterized in that, The feeding method is as follows: feeding Bemisia tabaci with sucrose water containing BtUGPase dsRNA of the gene.

5. The application according to claim 4, characterized in that, The concentration of sucrose in the sucrose solution is 0.17 - 0.23 mg / L.

6. The application according to claim 4, wherein, The concentration of sucrose in the sucrose solution is 0.2 mg / L.

7. The application according to claim 4, characterized in that, The BtUGPase dsRNA of the gene has a concentration of 250 ng / µL in the sucrose solution.

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