Agent for delaying development of drug resistance of brown planthopper and application thereof

Abstract

The invention discloses an agent for delaying development of drug resistance of brown planthopper and application thereof.The agent comprises 96.6% of pymetrozine original medicine and 12% of Zhongshengmycin original medicine, and the 96.6% of pymetrozine original medicine and the 12% of Zhongshengmycin original medicine are prepared through an organic solvent N, N-dimethyl formamide. The preparation method comprises the following steps: dissolving N, N-dimethylformamide analytically pure (DMF), diluting with distilled water to obtain a pymetrozine solution with the LC50 value of 3.80 mg / L and a zhongshengmycin solution with the LC50 value of 152.61 mg / L, and mixing the pymetrozine solution with the LC50 value of 3.80 mg / L and the zhongshengmycin solution with the LC50 value of 152.61 mg / L according to a volume ratio to prepare the medicament. The invention aims to provide an agent for preventing and treating brown planthopper and application of the agent for delaying development of drug resistance of brown planthopper, aiming at the defect that the chemical prevention and treatment effect is obviously reduced due to drug resistance of brown planthopper to conventional chemical insecticides.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to an agent for delaying the development of pesticide resistance of brown planthopper and application thereof. Background Art

[0002] Brown planthopper (Nilaparvata lugens ) belongs to the family Delphacidae in the order Hemiptera and is an important pest that harms rice in my country. Brown planthoppers not only cause mechanical damage by sucking rice phloem sap and laying eggs in the center of rice leaf sheaths, but also spread a variety of viral diseases, causing large-scale grain failure and production reduction in my country. How to safely and effectively prevent and control brown planthoppers is a bottleneck problem that needs to be solved in the current brown planthopper prevention and control work, and it is also the key to ensuring national food security.

[0003] Chemical pesticides have always been the main means of controlling rice pests. However, the irrational use of chemical pesticides in large quantities has caused brown planthoppers to develop a high degree of resistance, thereby reducing the effectiveness of pesticide control. Data from the Zhejiang Provincial Department of Agriculture and Rural Affairs on rice pest and disease resistance monitoring since 2013 show that the resistance of brown planthoppers in Jinhua rice areas to imidacloprid has increased sharply from 171.8 times to 3948.5 times, an increase of 23 times, and the resistance to thiamethoxam has increased from 101.8 times in 2013 to 3180.8 times in 2020, an increase of 31.2 times. In order to achieve better prevention and control effects, rice farmers often increase the concentration of pesticides used, resulting in a vicious cycle, leading to increased environmental pollution, excessive rice residues, and increased pressure on pests and diseases to develop resistance. The results of the 2017 test of neonicotinoid pesticide residues in rice field soil samples in Zhejiang Province showed that nine common neonicotinoid pesticides were commonly detected, among which imidacloprid had the highest detection rate of 96%; thiamethoxam had the highest residual concentration of 1.39-22.68μg / kg. In order to ensure food security and ecological security, it is urgent to fundamentally delay the development of chemical pesticide resistance.

[0004] Symbiosis between insects and microorganisms is a ubiquitous phenomenon, and the two have formed an inseparable relationship during the evolution process. Insects provide a stable microhabitat and nutrition for symbiotic bacteria, and insects rely on symbiotic bacteria to synthesize certain important nutrients that are lacking in food. At the same time, symbiotic bacteria also play a certain role in the metabolic detoxification of insects, such as synthesizing important nutrients for insects, participating in nitrogen cycle, and degrading exogenous substances in insects. The lack of symbiotic bacteria will directly cause insect growth and reproduction to be hindered, and the mortality rate to increase. Studies have shown that there are a variety of symbiotic bacteria in the intestines and fat bodies of brown planthoppers. These symbiotic bacteria can provide essential amino acids, steroids, proteins and other nutrients for host synthesis, make up for the imbalance of the nutritional structure of brown planthoppers, and ensure the normal growth and reproduction of brown planthoppers. Since brown planthoppers and symbiotic bacteria are so closely related in growth and reproduction, antibacterial substances can be used as adjuvants and chemical insecticides to achieve an insecticidal effect of 1+1 greater than 2. Moreover, since the target of antibacterial substances is not brown planthoppers, it may also delay the development of brown planthoppers' resistance.

[0005] Pymetrozine is a non-biocidal insecticide that targets TRPV channels in insect stretch receptors. It activates TRPV channels to cause behavioral disorders in insects, thereby affecting feeding and other behaviors, and ultimately killing insects. Pymetrozine was registered in my country for pest control in 2007. Current pymetrozine resistance monitoring data show that the resistance of brown planthopper field populations to it is increasing year by year. Most brown planthopper field populations have reached a high level of resistance, and there is a trend of further development.

[0006] The invention prepares a new formula by mixing the chemical insecticide pymetrozine and the conventional fungicide Zhongshengmycin in a certain ratio, and studies the insecticidal effect and drug resistance of the formula on brown planthoppers. The invention provides a new and more effective method for delaying the drug resistance of rice brown planthoppers. Summary of the invention

[0007] The purpose of the present invention is to provide a drug for controlling brown planthoppers and an application for delaying the development of brown planthoppers' resistance to conventional chemical insecticides, in view of the problem that brown planthoppers develop resistance to conventional chemical insecticides, resulting in a significant decrease in chemical control effects.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme: an agent for delaying the development of insecticide resistance of brown planthopper, comprising 96.6% pymetrozine original drug and 12% zhongshengmycin original drug,

[0009] The combined preparations described in the present invention are all combined preparations of pymetrozine and zhongshengmycin.

[0010] The 96.6% pymetrozine original drug was dissolved in an organic solvent, N,N-dimethylformamide analytical grade (DMF), and then diluted with distilled water to form LC 50 The value is 3.80mg / L of pymetrozine solution,

[0011] The 12% Zhongshengmycin original drug was dissolved in an organic solvent, N,N-dimethylformamide analytical grade (DMF), and then diluted with distilled water to form LC 50 The value of the Zhongshengmycin solution is 152.61 mg / L.

[0012] LC 50 The pymetrozine solution with a value of 3.80 mg / L and the LC 50 The zhongshengmycin solution with a value of 152.61 mg / L is mixed according to the volume ratio to prepare the medicine.

[0013] Preferably, the LC 50 The pymetrozine solution with a value of 3.80 mg / L and the LC 50 The volume ratio of the Zhongshengmycin solution with a value of 152.61 mg / L is 90:10 to 10:90.

[0014] Preferably, the LC 50 The pymetrozine solution with a value of 3.80 mg / L and the LC 50 The volume ratio of the Zhongshengmycin solution with a value of 152.61 mg / L is 80:20.

[0015] Preferably, the LC 50 The pymetrozine solution with a value of 3.80 mg / L and the LC 50 The volume ratio of the Zhongshengmycin solution with a value of 152.61 mg / L is 50:50.

[0016] The invention discloses an agent for delaying the development of insecticide resistance of brown planthopper in controlling brown planthopper in rice.

[0017] Therefore, the present invention has the following beneficial effects: the combined use of pymetrozine and zhongshengmycin not only improves the control effect of brown planthoppers, but also delays the occurrence of drug resistance of brown planthoppers. DETAILED DESCRIPTION

[0018] The present invention is further described below in conjunction with specific embodiments.

[0019] Embodiment 1:

[0020] The 96.6% pymetrozine technical drug of the present invention is provided by Jiangsu Weiyunate Fine Chemical Co., Ltd., and the 12% zhongshengmycin technical drug is provided by Fujian Kaili Biological Co., Ltd.; the rice varieties used are all TN1, and the brown planthopper is a susceptible variety that has been continuously raised in the laboratory for 20 generations.

[0021] 1. Biological activity assay

[0022] 96.6% pymetrozine and 12% zhongshengmycin were dissolved in an organic solvent, N, N-dimethylformamide analytical grade (DMF), and then diluted with distilled water in an equal proportion to form 5-7 series of mass concentration gradients. 20 nymphs of the third instar of brown planthoppers with the same growth were taken and placed in a plastic cup containing rice seedlings treated with pymetrozine or zhongshengmycin. Each treatment was repeated three times, and the water treatment was used as the control group. After inoculation, all treatment groups were placed in an artificial climate chamber with a temperature of 26±1℃, a photoperiod of L:D=16h:8h, and a relative humidity of 70-80%. The growth and development of brown planthoppers were observed every day, and the number of deaths of the test insects was recorded. The results of the pymetrozine treatment group were checked after 120h, and the results of the zhongshengmycin treatment group were checked after 72h.

[0023] 2. Construction of resistant strains

[0024] Starting with the indoor sensitive strain, six generations of resistance selection were carried out to obtain the pymetrozine-resistant strain (PyR) and the pymetrozine original drug and zhongshengmycin combination preparation resistant strain (MixR) brown planthopper. Another part of the test insects were not exposed to any pesticides and were raised in the same environment as the initial resistance screening strain (SS). According to the results of the previous generation of biological activity assays, the strain slightly below the LC 50 The concentration of pymetrozine and the compatible preparation solution was prepared, the pesticide was sprayed on fresh rice seedlings and then dried, and about 300 3rd-instar brown planthopper nymphs were inoculated. After 5 days, the surviving test insects were inoculated into fresh rice seedlings that had not been exposed to pesticides for further feeding and reproduction (the feeding conditions were 27±1℃, relative humidity 70-80%, and photoperiod of L:D=16:8h) to screen the next generation.

[0025] 3. Effects of the combination of zhongshengmycin and pymetrozine on population parameters of brown planthopper

[0026] 200 female and male adults of brown planthoppers were randomly selected and raised in plastic cups of untreated rice seedlings. After 2-3 days, all adults were taken out and placed in new plastic cups of rice seedlings for feeding. The rice seedlings after egg laying were observed every day. When brown planthopper nymphs hatched, the newly hatched nymphs with the same growth were selected and placed in test tubes of rice seedlings treated with different agents. Single head was raised in each test tube, and each treatment group was repeated 100 times. Each head and each tube was numbered and recorded, and the clean water treatment was the negative control group. The growth, development and survival of brown planthopper nymphs were observed. After the nymphs emerged, the female and male insects in each treatment group were paired one by one. If the male insect died during the feeding process, a male insect raised in the same treatment group was required. The rice seedlings were replaced every 24 hours, and the replaced rice seedlings continued to be cultured in new test tubes, recorded and numbered. The rice seedlings with eggs were observed every 24 hours to check whether the nymphs had hatched. Finally, the number of unhatched eggs in the rice seedlings was counted under a microscope. During the experiment, all test insects were raised in an artificial climate chamber (26±1°C, L:D=16h:8h, relative humidity 70-80%).

[0027] Statistical Analysis

[0028] Using Professor Qi Xin's bisexual life table theory and Twosex-MSChart software, the data collected above were statistically analyzed to calculate the developmental period values ​​of each age of brown planthoppers and various population parameters of brown planthoppers. Based on the above data, the population-specific age survival rate (lx), age-specific fecundity (mx), female age-age characteristic fecundity (fxj), age-age survival rate (Sxj) curves were drawn. DPS (7.5) data processing software was used for one-way analysis of variance (ANOVA) and Turkey method to analyze differences (P < 0.05). Sigma Plot 12.0 was used for plotting.

[0029] The test results obtained according to Example 1 are as follows:

[0030] (I) Results of indoor toxicity test of zhongshengmycin and pymetrozine against brown planthopper

[0031] The indoor toxicity of zhongshengmycin and pymetrozine to the third instar nymphs of brown planthopper was determined by the rice seedling dipping method. From the test results in Table 1, it can be seen that zhongshengmycin and pymetrozine have good insecticidal activity against the third instar nymphs of brown planthopper. Among them, the LC 50 The value was 152.61 mg / L, and the LC value was 152.61 mg / L after 120 h of pymetrozine treatment. 50 The value is 3.80mg / L.

[0032] Table 1 Toxicity of zhongshengmycin and pymetrozine to the third instar nymphs of brown planthopper

[0033]

[0034] (II) Combined effects of zhongshengmycin and pymetrozine on brown planthopper

[0035] According to the LC of zhongshengmycin and pymetrozine 50 The interaction and compatibility ratio of Zhongshengmycin and pymetrozine were screened and determined by the interactive determination method. The results are shown in Table 2. It was observed that LC 50 The value is 3.80mg / L pymetrozine solution and LC 50 The value was 152.61 mg / L. When the volume ratio of zhongshengmycin solution was 80:20 and 50:50, the toxicity ratio was 1.28 and 1.29, respectively, both of which were greater than 1.25. This showed that the combined action of zhongshengmycin and pymetrozine showed a synergistic effect on the third-instar nymphs of brown planthoppers; the toxicity ratios of combinations 2, 4, 5, 7, 8, 9, and 10 were all greater than 1 and less than 1.25, showing an additive effect; none of the combinations showed an antagonistic effect. The combined action of zhongshengmycin and pymetrozine had a synergistic and additive effect on the third-instar nymphs of brown planthoppers, but no antagonistic effect. Compared with the group treated with zhongshengmycin alone (combination 11), the actual mortality rates of all combined treatment groups were higher than those of combination 11; compared with the group treated with pymetrozine alone (combination 1), the actual mortality rates of most combined treatment groups were higher than those of combination 1. Compared with the use of zhongshengmycin or pymetrozine alone as a treatment method, the combination of zhongshengmycin and pymetrozine has reasonably and effectively reduced the dosage of chemical insecticides to a certain extent. In terms of control effect, the effect of the combination of zhongshengmycin and pymetrozine is significantly better than that of single-dose treatment, increasing the mortality rate of brown planthoppers.

[0036] Table 2 Toxicity of different volume ratios of zhongshengmycin and pymetrozine to the third instar nymphs of brown planthopper

[0037]

[0038] Note: The number of dead insects represented by a is the average of three repeated treatments calculated at DPS 7.0. Different letters indicate significant differences among treatment groups (P<0.05, Tukey's LSD test).

[0039] bExpected mortality rate = LC of drug A 50 Actual mortality rate × proportion of drug A + LC of drug B 50 Actual mortality rate × proportion of drug B

[0040]

[0041] Table 3 Toxicity of the mixed formulation of zhongshengmycin and pymetrozine to the third instar nymphs of brown planthopper

[0042]

[0043]

[0044] The combination of zhongshengmycin and pymetrozine with a toxicity ratio greater than 1.25 was selected for indoor toxicity determination. As shown in Table 3, when the mass ratio of pymetrozine to the active ingredient of zhongshengmycin was 1:10 (the volume ratio in Table 2 was 80:20), the LC50 value for the third instar nymph of brown planthopper was 15.10 mg / L (95% confidence limit was 8.67-26.31 mg / L), and the co-toxicity coefficient was 221.63; when the mass ratio of pymetrozine to the active ingredient of zhongshengmycin was 1:40 (the volume ratio in Table 2 was 50:50), the LC50 value for the third instar nymph of brown planthopper was 11.60 mg / L (95% confidence limit was 6.63-20.31 mg / L), and the co-toxicity coefficient was 672.87. The co-toxicity coefficients of the two different active ingredient mass ratios were all greater than 120, showing significant synergistic effects. The above experimental results show that the combination of zhongshengmycin and pymetrozine has an obvious synergistic effect in controlling the third-instar nymphs of brown planthopper.

[0045] (III) Breeding for resistance to brown planthopper

[0046] The rice seedling dipping method was used to carry out resistance breeding for six consecutive generations of indoor sensitive strains of brown planthoppers (SS), and the pymetrozine-resistant strains of brown planthoppers (PyR) and the compatibility agent-resistant strains of brown planthoppers (MixR) were obtained. The data analysis in Table 4 showed that the resistance level of brown planthoppers in the first four generations of MixR strains increased relatively slowly and was at a low level of resistance. Then the resistance level steadily increased to 127.4 times in the sixth generation, and the development trend of the entire resistance breeding process was relatively fast. The pymetrozine-resistant strains also showed a similar resistance development trend (Table 5), and the PyR strain reached a maximum resistance level of 754.6 times in the sixth generation. Comparison between the two found that the combination of pymetrozine and zhongshengmycin can alleviate the development of brown planthoppers' resistance to pymetrozine.

[0047] Test example: (Comparative test on the control effect and drug resistance development of the mixed preparation of Zhongshengmycin and pymetrozine of the present invention and pymetrozine alone)

[0048] Table 4 Resistance breeding of indoor populations of brown planthopper to pymetrozine and zhongshengmycin combination preparations

[0049]

[0050] Table 5 Resistance breeding of indoor populations of brown planthopper to pymetrozine

[0051]

[0052] Table 6 Effects of the combination of zhongshengmycin and pymetrozine on population parameters of brown planthopper

[0053]

[0054] Note: (1) A: 96.6% pymetrozine; B: 12% zhongshengmycin; C: 96.6% pymetrozine and 12% zhongshengmycin in a mass ratio of 1:40; W: clean water

[0055] After the analysis of the life table system of both sexes, Table 6 reflects the changes in the parameter values ​​and growth potential of the brown planthopper population after treatment with sinoprotinin, pymetrozine and the combination of the two. The average generation period (T) refers to the average time required for the offspring in the population to reproduce from the birth of the mother; the intrinsic growth rate (rm) refers to the maximum instantaneous growth rate of a biological population with a stable age group to maintain the optimal living standard under the optimal environmental conditions. Generally speaking, r-strategy species have a higher intrinsic growth rate but a shorter lifespan; the net reproduction rate (Ro) refers to the multiplication multiple of the insect population after one generation under certain conditions; the weekly growth rate (λ) refers to the total growth rate within a certain period of time.

[0056] The average generation periods of the negative control W (clear water), treatment groups A (96.6% pymetrozine), B (12% sinoprotinin), and C (pymetrozine: sinoprotinin = 1:40) were 22.74, 21.04, 21.36, and 20.05, respectively. The average generation period of group C was the shortest, indicating that the brown planthopper population treated with sinoprotinin and pymetrozine required the shortest time to complete a generation cycle; the intrinsic growth rates were 0.231, 0.188, 0.158, and 0.095, respectively, indicating that the brown planthopper population treated with sinoprotinin and pymetrozine The growth trend of the brown planthopper population treated with zhongshengmycin and pymetrozine was the slowest; the net reproduction rates were 191.41, 52.77, 29.02 and 6.68, respectively, indicating that the brown planthopper population treated with zhongshengmycin and pymetrozine had the smallest proliferation multiple after one generation cycle; the weekly growth rate was 1.260 / d, 1.207 / d, 1.171 / d and 1.100 / d, respectively, indicating that the brown planthopper population treated with zhongshengmycin and pymetrozine had the least number of offspring per individual under the condition of no resource limitation. In summary, the brown planthopper population treated with zhongshengmycin and pymetrozine had an effective inhibitory effect on its development, survival and offspring reproduction.

[0057] As can be seen from Tables 4-6, the combined use of pymetrozine and sinoprotinin not only improves the control effect of brown planthoppers, but also delays the occurrence of insecticide resistance in brown planthoppers.

Claims

1. A medicament for delaying the development of insecticide resistance of brown planthopper, characterized in that: Including 96.6% pymetrozine technical and 12% zhongshengmycin technical. The 96.6% pymetrozine original drug was dissolved in an organic solvent, N,N-dimethylformamide analytical grade (DMF), and then diluted with distilled water to form LC 50 The value is 3.80mg / L of pymetrozine solution, The 12% Zhongshengmycin original drug was dissolved in an organic solvent, N,N-dimethylformamide analytical grade (DMF), and then diluted with distilled water to form LC 50 The value of the Zhongshengmycin solution is 152.61 mg / L. LC 50 The pymetrozine solution with a value of 3.80 mg / L and the LC 50 The zhongshengmycin solution with a value of 152.61 mg / L is mixed according to the volume ratio to prepare the medicine.

2. The method for delaying the development of insecticide resistance of brown planthopper according to claim 1, characterized in that: The LC 50 The pymetrozine solution with a value of 3.80 mg / L and the LC 50 The volume ratio of the Zhongshengmycin solution with a value of 152.61 mg / L is 90:10 to 10:

90.

3. The agent for delaying the development of insecticide resistance of brown planthopper according to claim 2, characterized in that: The LC 50 The pymetrozine solution with a value of 3.80 mg / L and the LC 50 The volume ratio of the Zhongshengmycin solution with a value of 152.61 mg / L is 80:

20.

4. The agent for delaying the development of insecticide resistance of brown planthopper according to claim 2, characterized in that: The LC 50 The pymetrozine solution with a value of 3.80 mg / L and the LC 50 The volume ratio of the Zhongshengmycin solution with a value of 152.61 mg / L is 50:

50.

5. Use of the agent according to any one of claims 1 to 4 in controlling brown planthopper in rice.

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