Spinetoram compound pesticide composition as well as preparation method and application thereof

Abstract

The invention discloses a spinetoram compound pesticide composition as well as a preparation method and application thereof. The composition comprises an effective component A and an effective component B, the effective component A is spinetoram, and the effective component B is allicin, carvacrol or hydroxychloroquine. The composition is compounded by the spinetoram, the allicin, the carvacrol and the hydroxychloroquine, and has an obvious synergistic effect on prevention and treatment of gray mold, tea leaf spot and peach gummosis. Therefore, a preparation product containing the composition can improve the control effect on gray mold, tea leaf spot disease and peach gummosis of vegetables such as tomatoes, reduce the drug use frequency and delay the generation of drug resistance, and has a wide application prospect in prevention and treatment of plant pathogenic fungal diseases.

Description

Technical Field

[0001] The invention belongs to the technical field of pesticides, and specifically relates to a spinetoram compound pesticide composition and a preparation method and application thereof. Background Art

[0002] At present, the application of chemical fungicides is the most effective means of preventing and controlling crop diseases. However, with the excessive use of chemical fungicides and the long-term use of single agents, the increase in disease resistance and pesticide residues have become issues of widespread concern throughout the world. This has led researchers to continuously study new varieties and dosage forms of pesticides, as well as compounding and mixing of pesticides.

[0003] Spinetoram is a highly effective, low-toxic and environmentally friendly insecticide that can be used to control Lepidoptera pests such as Plutella xylostella, Noctuidae, Cydia pomonella, Grapholitamolesta, Tortricidae, as well as leafminers (Pegomyia hyosciami Panzer) and thrips (Thrips vulgatissimus) (Zhang Kai, Xu Yuanyuan, Gao Shang, Li Jiarong. Research progress of biological pesticide Spinetoram. Modern Pesticides. 2024, 23: 39-44). At present, Spinetoram is mostly used as an insecticide, but there is no report on the use of Spinetoram as a fungicide.

[0004] There are many studies and reports on the biocontrol activity of garlic (Allium sativum L.) extracts. Among them, allicin has a broad spectrum of microbial inhibitory activity in plants. Garlic crude extract has obvious antibacterial effects on cucumber wilt (Fusarum oxysporum sp.cucumerinum Owen) (Cheng Zhihui, Song Li, Meng Huanwen. Antibacterial and disease prevention effects of garlic bulb crude extract on cucumber wilt. Journal of Northwest Agriculture and Forestry University (Natural Science Edition), 2008, 36 (5): 113-118), cucumber black spot (Cladosporium cucumerinum) (Wang Yunfan, Wang Gang, Yang Shengyu, Cheng Xi, Yang Zhiwei. Preliminary study on the prevention and control of cucumber black spot by garlic extract. Journal of Northwest Agriculture and Forestry University (Natural Science Edition), 2005, 33 (10): 7-10), strawberry gray mold (Botrytis cinerea Pers.) (Geng Jianfeng, Kuroda Keli, Tanaka Kazuhisa. Effects of onion oil and garlic extract on gray mold. Chinese Vegetables, 2008, (5): 20-22), and some soil-borne pathogens. At present, there are no reports on the use of allicin to control tomato gray mold (B. cinerea), cocoa hairy spore (L. theobromae), D. segeticola, and tea pseudodisk hairy spore (Ps. camelliae-sinensis).

[0005] Carvacrol is the main component of essential oil extracts of oregano (Origanum vulgare L.) and marjoram (Origanum marjoram L.) from the Labiatae family. Carvacrol inhibits the transcription of biofilm synthesis-related genes ica A, cid A and sarA and the synthesis of intercellular polysaccharide adhesins, thereby inhibiting the growth of Staphylococcus aureus (Zhao.Y., Wang.Q., Wu,X., Jiang,M., Jin,H., Tao,K., Hou,T. Unraveling the polypharmacology of a natural antifungal product,eugenol,against Rhizoctonia solani.Pest Manag.Sci.2021,77:3469-3483), oregano essential oil and carvacrol can reduce the incidence and severity of tomato wilt caused by Fusarium oxysporum, and the minimum inhibitory concentrations against Fusarium oxysporum are 125μg / mL and 400μg / mL, respectively (Abbaszadeh, S., Sharifzadeh, A., Shokri, H., Khosravi, AR, Abbaszadeh, A. Antifungal efficacy of thymol, carvacrol, eugenol and menthol as alternative agents to control the growth of food-relevant fungi. J. Mycol. Med.2014,24:e51-6).Carvacrol showed excellent antibacterial activity against L. theobromae. Optical microscopy showed that the thickness of L. theobromae hyphae changed significantly, the hyphae swelled, and the hyphae showed abnormal growth. Scanning electron microscopy showed that the surface of some L. theobromae hyphae were wrinkled and collapsed (Yin. JY, Wu, S., Yang, YL, Wang, DL, Ma, Y., Zhao, YT, Sheth., S., Huang, HL, Song, BA, Chen, Z. In addition to damaging the plasma membrane, can bind to the minor groove of DNA of phytopathogenic fungi to potentially control tea leaf spot caused by Lasiodiplodia theobromae. Phytopathology 2024, 114: 700-716).

[0006] Hydroxychloroquine (HCQ) is a weakly alkaline 4-aminoquinoline derivative. HCQ and its prodrugs, quinine and chloroquine, are antimalarial drugs and have been used clinically. HCQ combined with artemisinin (AH therapy) can significantly improve the renal function of IgAN rats (Bai, L., Li, J., Li, H., Song, J., Zhou, Y., Lu, R., Liu, B., Pang, Y., Zhang, P., Chen, J., Liu, X., Wu, J., Liang, C., Zhou, J. Renoprotective effects of artemisinin and hydroxychloroquine combination therapy on IgAnephropathy via suppressing NF-κB signaling and NLRP3inflammasome activation by exosomes in rats. Biochem. Pharmacol. 2019, 169: 113619). HCQ can reduce the proteinuria level in IgAN patients who have poor responses to conventional immunosuppressive therapy (Tang, C., Lv, JC, Shi, SF, Chen, YQ, Liu, LJ, Zhang, H. Effect of hydroxychloroquine in patients with IgAnephropathy with insufficient responses to immunosuppressive therapy: a retrospective case-control study. BMC Nephrol. 2020, 21: 469). Hydroxychloroquine has anti-inflammatory, anti-infective, and anti-allergic effects and can be used to treat a variety of skin diseases.

[0007] Long-term continuous use of a single pesticide can easily lead to the development of resistance. Reasonable mixing of pesticides has many advantages, such as improving control efficiency, delaying the development of pest resistance and resistance, reducing drug use costs, and expanding the insecticide spectrum. At present, there are no reports on the combination of ethyl spinetoram and allicin, carvacrol, and hydroxychloroquine to control B. cinerea, L. theobromae, D. segeticola, and Ps. camelliae-sinensis. There are also relatively few types of active ingredients for controlling B. cinerea, L. theobromae, D. segeticola, and Ps. camelliae-sinensis. Research on new active ingredients and their combination and synergistic effects needs to be developed urgently. Summary of the invention

[0008] In view of this, one of the objects of the present invention is to provide a spinetoram compound pesticide composition, which comprises an active ingredient A and an active ingredient B, wherein the active ingredient A is spinetoram and the active ingredient B is allicin, carvacrol or hydroxychloroquine.

[0009] Preferably, the mass ratio of active ingredient A to active ingredient B is 1-2:1-2.

[0010] Preferably, the mass ratio of active ingredient A to active ingredient B is 1:1, 1:2 or 2:1.

[0011] The second object of the present invention is to provide a method for preparing the above-mentioned composition, comprising the following steps:

[0012] 1) Weigh 25% spinetoram water dispersible granules and add them to sterilized water to prepare a mother solution I with a spinetoram concentration of 50,000 μg / mL; weigh allicin, carvacrol or hydroxychloroquine and dissolve them in DMSO (dimethyl sulfoxide) to prepare a mother solution II with a concentration of 50,000 μg / mL;

[0013] 2) Mix the mother solution I and the mother solution II in proportion for compounding.

[0014] The third object of the present invention is to provide the use of the above composition in inhibiting plant pathogens.

[0015] Preferably, the plant pathogenic bacteria include any one or more of Botrytis cinerea, Lasiodiplodia theobromae, Didymella segeticola and Pseudopestalotiopsis camelliae-sinensis.

[0016] When the active ingredients are spinetoram and allicin, the best control effect for B. cinerea is the composite composition in which the mass ratio of spinetoram to allicin is 1:2; the best control effect for L. theobromae is the composite composition in which the mass ratio of spinetoram to allicin is 1:2; the best control effect for D. segeticola is the composite composition in which the mass ratio of spinetoram to allicin is 1:1; the best control effect for Ps. camelliae-sinensis is the composite composition in which the mass ratio of spinetoram to allicin is 1:1.

[0017] When the active ingredients are spinetoram and carvacrol, the best compound composition for controlling B. cinerea is one in which the mass ratio of spinetoram to carvacrol is 1:2; the best compound composition for controlling L. theobromae is one in which the mass ratio of spinetoram to carvacrol is 1:2; the best compound composition for controlling D. segeticola is one in which the mass ratio of spinetoram to carvacrol is 2:1; the best compound composition for controlling Ps. camelliae-sinensis is one in which the mass ratio of spinetoram to carvacrol is 1:2.

[0018] The active ingredients are spinetoram and hydroxychloroquine. The best effect for preventing and controlling B. cinerea is the composite composition in which the mass ratio of spinetoram to hydroxychloroquine is 2:1; the best effect for preventing and controlling L. theobromae is the composite composition in which the mass ratio of spinetoram to hydroxychloroquine is 1:1; and the best effect for preventing and controlling D. segeticola is the composite composition in which the mass ratio of spinetoram to hydroxychloroquine is 1:2.

[0019] In view of the shortcomings of the existing pesticide bactericides that cannot take into account both the delay of drug resistance and low toxicity and low residue, the spinetoram compound pesticide composition provided by the present invention is compounded with spinetoram, allicin, carvacrol and hydroxychloroquine as effective ingredients, which can effectively inhibit a variety of plant pathogens and has a significant synergistic effect on the prevention and treatment of gray mold, tea leaf spot disease and peach gummosis. The present invention can not only reduce and control the occurrence and harm of diseases, enhance plant stress resistance, reduce the number of drug applications, and reduce the actual application cost, but also delay the generation of pathogen resistance, expand the pesticide bactericidal spectrum, and reduce the pollution of pesticides to the environment, which is in line with my country's policy of reducing pesticides and has broad application prospects in the prevention and treatment of plant pathogenic fungal diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Schematic diagram of the antibacterial activity of spinetoram and allicin in different proportions on tomato fruit inoculated with B. cinerea mycelium;

[0021] Figure 2 Schematic diagram of the antibacterial activity of the combination of spinetoram and carvacrol in different proportions of the present invention on the mycelium of Ps. camelliae-sinensis in vivo;

[0022] Figure 3 Schematic diagram of the antibacterial activity of spinetoram and hydroxychloroquine in different proportions against the mycelium of L. theobromae in vivo;

[0023] Figure 4The antibacterial activity of the combination of spinetoram and allicin (1:1) of the present invention on tomato fruit 48 hours after inoculation with B. cinerea mycelium, scale bar = 1 cm;

[0024] Figure 5 The antibacterial activity of the combination of spinetoram and allicin (1:2) of the present invention on tomato fruit 48 hours after inoculation with B. cinerea mycelium, scale bar = 1 cm;

[0025] Figure 6 The antibacterial activity of the combination of spinetoram and allicin (2:1) of the present invention on tomato fruit 48 hours after inoculation with B. cinerea mycelium, scale bar = 1 cm;

[0026] Figure 7 The antibacterial activity of the combination of spinetoram and carvacrol (1:1) of the present invention on tea leaves 48 hours after inoculation with Ps. camelliae-sinensis hyphae, scale bar = 1 cm;

[0027] Figure 8 The antibacterial activity of the combination of spinetoram and carvacrol (1:2) of the present invention on tea leaves 48 hours after inoculation with Ps. camelliae-sinensis hyphae, scale bar = 1 cm;

[0028] Fig. 9 The antibacterial activity of the combination of spinetoram and carvacrol (2:1) of the present invention on tea leaves 48 hours after inoculation with Ps. camelliae-sinensis hyphae, scale bar = 1 cm;

[0029] Fig.10 The antibacterial activity of the combination of spinetoram and hydroxychloroquine (1:1) of the present invention on tea leaves 48 hours after inoculation with L. theobromae hyphae, scale bar = 1 cm;

[0030] Fig.11 The antibacterial activity of the combination of spinetoram and hydroxychloroquine (1:2) of the present invention on tea leaves 48 hours after inoculation with L. theobromae hyphae, scale bar = 1 cm;

[0031] Fig.12 The antibacterial activity of the combination of spinetoram and hydroxychloroquine (2:1) of the present invention on tea leaves 48 hours after inoculation with L. theobromae hyphae, scale bar = 1 cm. DETAILED DESCRIPTION

[0032] The present invention will be described in detail below with reference to the examples. These examples are only for illustrative purposes and are not limited to the scope of application of the present invention. The present invention is not limited to the following embodiments or examples. Any modifications and variations that do not violate the spirit of the present invention should be included in the scope of the present invention. The experimental materials used in the following examples are commercially available unless otherwise specified; the percentages mentioned in the following examples are all mass percentages unless otherwise specified; 25% ethyl spinosad water-dispersible granules in the following examples are provided by Corteva Agricultural Science and Technology Co., Ltd. (Pesticide Registration Certificate No.: PD20181527), 95% allicin was purchased from Shanghai McLean Biochemical Co., Ltd., 99% carvacrol and 98% hydroxychloroquine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; the strain used in the following examples is B.cinere a (strain number: GZFQ-1, preservation number: CGMCC3.20932), L. theobromae (strain number: GZHS-2017-010, preservation number: CGMCC3.20151), D. segeticola (strain number: GZSQ-4, preservation number: CGMCC3.20152), Ps. camelliae-sinensis (strain number: GZHS-2017-01, preservation number: CGMCC3.20199).

[0033] Example 1: Combined toxicity study of spinetoram and allicin

[0034] Preparation method: Weigh 0.6g of 25% ethyl spinetoram water dispersible granules, add 3mL of sterile water and mix well to prepare 50,000μg / mL stock solution. Weigh 0.25g of allicin original drug, dissolve in 5mL of DMSO to prepare 50,000μg / mL stock solution.

[0035] The mother solution of spinetoram and the mother solution of allicin were mixed in proportion to obtain the compound preparations with the mixed solutions of spinetoram and allicin in the ratio of 1:1, 1:2 and 2:1. The combined toxicity of the compound preparations of spinetoram and allicin in different ratios to B. cinerea, L. theobromae, D. segeticola and Ps. Camelliae-sinensis was determined by the growth rate method.

[0036] The specific test steps are:

[0037] Firstly, spinetoram and allicin were prepared in mass ratios of 1:0, 1:1, 1:2, 2:1, and 0:1 (where 1:0 and 0:1 are single agents of spinetoram and allicin, i.e., single doses), and the mycelium growth rate method was used to determine the in vitro antibacterial activity of each agent against B. cinerea, L. theobromae, D. segeticola, and Ps. Camelliae-sinensis using clean water treatment as a blank control. Take 250mL conical flasks, prepare 90mL of PDA medium in each bottle, put them into a high-pressure steam sterilizer and sterilize them at 121℃ for 20min, take out the sterilized PDA medium, add the prepared drugs into PDA respectively, so that the final concentrations of the drugs are: 0.0μg / mL, 10.0μg / mL, 20.0μg / mL, 40.0μg / mL, 60.0μg / mL, 80.0μg / mL, and the concentration gradient is for B. cinerea; 0.0μg / mL, 5.0μg / mL, 25.0μg / mL, 50.0μg / mL, 70.0μg / mL, 85.0μg / mL, 90.0μg / mL, 100.0μg / mL, 120.0μg / mL, 140.0μg / mL, 150.0μg / mL, 160.0μg / mL, 170.0μg / mL, 180.0μg / mL, 190.0μg / mL, 200.0μg / mL, 250.0μg / mL, 20 ... 5.0μg / mL, 100.0μg / mL, this concentration gradient is for L.theobromae; 0.0μg / mL, 10.0μg / mL, 20.0μg / mL, 50.0μg / mL, 100.0μg / mL, 200.0μg / mL, this concentration gradient is for D.segeticola; 0.0μg / mL, 5.0μg / mL, 25.0μg / mL, 50.0μg / mL, 75.0μg / mL, 100.0μg / mL, this concentration gradient is for Ps.Camelliae-sinensis. Then pour the mixed PDA medium containing the drug into the culture dish, and complete 5 biological replicates for each concentration. After the culture medium is cooled and solidified, use a sterilized 4mm diameter puncher to punch out bacterial cakes from the activated fresh colonies of B. cinerea, L. theobromae, D. segeticola, and Ps. camelliae-sinensis, and then inoculate the punched bacterial cakes onto the PDA culture medium. After inoculation, seal the culture dish with a sealing film and place it upside down in a 25°C constant temperature incubator for culture. When the colonies in the 0.0μg / mL agent culture medium grow to 3 / 4 of the culture dish area, measure the colony diameter using the cross method. Finally, use the following formula (1) to calculate the inhibition rate.

[0038]

[0039] DPS v 9.01 software (http: / / www.dpsw.cn / dps_eng / ) was used to calculate the regression equation and r 2 and EC 50The regression equation represents the relationship between mycelial growth rate and the dosage of the agent; EC 50 represents the median effective concentration.

[0040] The Wadley method was used to evaluate the biological activity of the mixture of A and B, and the SR (synergistic ratio) was calculated according to the following formulas (2) and (3). Where: a and b are the ratios of A and B in the mixture; EC (A)50 ,EC (B)50 The EC of A and B are 50 Measured value; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value. SR represents the interaction between A and B after mixing: SR < 0.5 represents antagonistic effect, 0.5 ≤ SR ≤ 1.5 represents additive effect, and SR > 1.5 represents synergistic or synergistic effect.

[0041]

[0042] After ethyl spinetoram and allicin were mixed in different ratios, the effects of single agent and mixed agent on the mycelial growth of B. cinerea were determined. The results are shown in Table 1. Different mixed ratios of ethyl spinetoram and allicin have obvious differences in the antibacterial effect on B. cinerea. When the mixed ratios of the two are 1:1, 1:2 and 2:1, EC 50 The values ​​were 45.53μg / mL, 30.29μg / mL, and 24.71μg / mL, respectively. When the compound ratio was 1:1, the antibacterial effect on B. cinerea was additive. When the compound ratio was 1:2 and 2:1, it had a strong antibacterial effect on B. cinerea and showed a synergistic effect. When the compound ratio was 1:2, EC 50 It is 30.29μg / mL, and the synergistic coefficient is the largest (2.28).

[0043] Table 1 Combined biological activity of spinetoram and allicin on B. cinerea

[0044]

[0045] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0046] Referring to the same method, spinetoram and allicin were mixed in different proportions, and the effects of single agent and mixed agent on the mycelial growth of L. theobromae were determined. The results can be seen from Table 2 that the antibacterial effect of spinetoram and allicin in different mixed ratios on L. theobromae was significantly different. When the mixed ratio of the two was 1:1, 1:2, and 2:1, EC 50 The values ​​were 55.57μg / mL, 26.21μg / mL, and 36.84μg / mL, respectively. It can be seen that the antibacterial effects of the combination of ethyl spinetoram and allicin are significantly different. When the combination ratio is 1:2, EC 50 It was 26.21 μg / mL, and the synergistic coefficient was the largest (2.05), showing a synergistic effect.

[0047] Table 2 Combined biological activity of ethyl spinetoram and allicin against L. theobromae

[0048]

[0049] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0050] Referring to the same method, ethyl spinetoram and allicin were mixed in different ratios to determine the effects of single and mixed agents on the mycelial growth of D. segeticola. The results are shown in Table 3. There is no significant difference in the antibacterial effect of ethyl spinetoram and allicin in different mixed ratios on D. segeticola. When the mixed ratios of the two were 1:1, 1:2 and 2:1, EC 50 The values ​​were 61.31μg / mL, 219.36μg / mL, and 76.16μg / mL, respectively. It can be seen that the combination of ethyl spinetoram and carvacrol did not show obvious antibacterial effect. When the combination ratio was 1:1, EC 50 The synergistic coefficient was 61.31 μg / mL and the maximum (1.30), showing an additive effect. When the compound ratio was 1:2, EC 50 The concentration of spinetoram and allicin was 219.36 μg / mL, and the synergistic coefficient was the smallest (0.35), showing an antagonistic effect. Based on the above test results, the combination of spinetoram and allicin had no obvious synergistic effect on D. segeticola at the treated concentration.

[0051] Table 3 Combined biological activity of ethyl spinetoram and allicin on D. segeticola

[0052]

[0053] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0054] Referring to the same method, spinetoram and allicin were mixed in different proportions to determine the effects of single and mixed agents on the mycelial growth of Ps. camelliae-sinensis. The results are shown in Table 4. There is no significant difference in the antibacterial effect of spinetoram and allicin in different mixed ratios on Ps. camelliae-sinensis. When the mixed ratio of the two is 1:1, 1:2, and 2:1, EC 50 The values ​​were 267.33 μg / mL, 2012.55 μg / mL, and 494.11 μg / mL, respectively. It can be seen that the combination of ethyl spinetoram and allicin had no obvious antibacterial effect on Ps. camelliae-sinensis, and they all showed antagonism when the combination ratio was 1:1, 1:2, and 2:1.

[0055] Table 4 Combined bioavailability of ethyl spinetoram and allicin combined with Ps. camelliae-sinensis

[0056]

[0057] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0058] Example 2: Combined toxicity study of spinosad and carvacrol

[0059] Preparation method: Weigh 0.6g of 25% spinosad water dispersible granules, add 3mL of sterile water and mix well to prepare a 50,000μg / mL stock solution. Weigh 0.25g of carvacrol original drug, dissolve in 5mL of DMSO to prepare a 50,000μg / mL stock solution.

[0060] The mother solution of spinetoram and the mother solution of carvacrol were mixed in proportion to obtain mixed solutions with a ratio of spinetoram to carvacrol of 1:1, 1:2, and 2:1. The growth rate method was used to determine the combined toxicity of the compound agents of spinetoram and carvacrol in different proportions to B. cinerea, L. theobromae, D. segeticola, and Ps. camelliae-sinensis. The specific experimental steps were as follows:

[0061] Firstly, spinetoram and carvacrol were prepared in mass ratios of 1:0, 1:1, 1:2, 2:1, and 0:1 (where 1:0 and 0:1 are single agents of spinetoram and carvacrol, i.e., single doses), and the mycelium growth rate method was used with clean water treatment as a blank control to determine the in vitro antibacterial activity of the compound agent against B. cinerea, L. theobromae, D. segeticola, and Ps. camelliae-sinensis. Take 250mL conical flasks, prepare 90mL of PDA medium in each bottle, put them into a high-pressure steam sterilizer and sterilize them at 121℃ for 20min, take out the sterilized PDA medium, add the prepared agents into PDA respectively, so that the final concentrations of the agents are 0.0μg / mL, 5.0μg / mL, 25.0μg / mL, 50.0μg / mL, 75.0μg / mL, 100.0μg / mL, respectively. The concentration gradient is for B. cinerea; 0.0μg / mL, 5.0μg / mL, 25.0μg / mL, 50.0μg / mL, 75.0μg / mL, 100.0μg / mL, this concentration gradient is for L.theobromae; 0.0μg / mL, 20.0μg / mL, 40.0μg / mL, 60.0μg / mL, 80.0μg / mL, 100.0μg / mL, this concentration gradient is for D.segeticola; 0.0μg / mL, 2.5μg / mL, 5.0μg / mL, 25.0μg / mL, 50.0μg / mL, 75.0μg / mL, this concentration gradient is for Ps.camelliae-sinensis. Then pour the mixed PDA medium containing the drug into the culture dish, and make 5 biological replicates for each concentration. After the culture medium is cooled and solidified, a sterilized 4 mm diameter puncher is used to punch the activated fresh B. cinerea, L. theobromae, D. segeticola, and Ps. camelliae-sinensis colonies to obtain bacterial cakes, which are then inoculated onto PDA culture medium. After inoculation, the culture dish is sealed with a sealing film and inverted into a 25° C. constant temperature incubator for culture. When the bacterial colonies in the 0 μg / mL agent culture medium grow to 3 / 4 of the culture dish area, the colony diameter is measured according to the cross method. Finally, the inhibition rate, synergistic ratio, etc. are calculated with reference to the method of Example 1.

[0062] Table 5 Combined biological activity of ethyl spinetoram and carvacrol against B. cinerea

[0063]

[0064] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th)EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0065] After spinetoram and carvacrol were mixed in different ratios, the effects of single agent and mixed agent on the mycelial growth of B. cinerea were determined. As shown in Table 5, the antibacterial effects of spinetoram and carvacrol in different mixed ratios on B. cinerea were significantly different. When the mixed ratios were 1:1, 1:2, and 2:1, the EC 50 The values ​​were 39.96μg / mL, 41.78μg / mL, and 38.55μg / mL, respectively. It can be seen that the combination of ethyl spinetoram and carvacrol showed obvious antibacterial effect, and the combination ratios of the two were 1:1, 1:2, and 2:1, which showed a synergistic effect. When the combination ratio was 1:2, EC 50 It is 41.78μg / mL and has the largest synergistic coefficient (2.76), showing a synergistic effect.

[0066] Table 6 Combined biological activity of ethyl spinetoram and carvacrol against L. theobromae

[0067]

[0068] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0069] After ethyl spinetoram and carvacrol were mixed in different ratios, the effects of single agent and mixed agent on the mycelial growth of L. theobromae were determined. As shown in Table 6, the antibacterial effect of ethyl spinetoram and carvacrol in different mixed ratios on L. theobromae was 1:1, 1:2, 2:1. 50 The values ​​were 33.06μg / mL, 30.19μg / mL, and 81.80μg / mL, respectively. It can be seen that the combination of ethyl spinetoram and carvacrol did not show obvious antibacterial effect. When the combination ratio was 1:2, the EC 50 The concentration of spinetoram and carvacrol was 30.19 μg / mL, and the synergistic coefficient was the largest (1.49), showing an additive effect. Based on the above test results, the combination of spinetoram and carvacrol did not show obvious synergistic effect at the treatment concentration.

[0070] Table 7 Combined biological activity of ethyl spinetoram and carvacrol on D. segeticola

[0071]

[0072]

[0073] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0074] After spinetoram and carvacrol were mixed in different ratios, the effects of single agent and mixed agent on the mycelial growth of D. segeticola were determined. As shown in Table 7, the antibacterial effects of spinetoram and carvacrol in different mixed ratios on D. segeticola were significantly different. When the mixed ratios were 1:1, 1:2, and 2:1, EC 50 The values ​​were 57.74μg / mL, 51.65μg / mL, and 45.35μg / mL, respectively. It can be seen that the combination of ethyl spinetoram and carvacrol showed obvious antibacterial effect. The SR values ​​of the three ratios were all greater than 1.5. When the combination ratio was 2:1, EC 50 It is 45.35μg / mL and has the largest synergistic coefficient (2.12), showing a synergistic effect.

[0075] Table 8 Combined biological activity of ethyl spinetoram and carvacrol against Ps. camelliae-sinensis

[0076]

[0077] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0078] After spinetoram and carvacrol were mixed in different ratios, the combined toxicity test on the mycelial growth of Ps. camelliae-sinensis was carried out. As shown in Table 8, the antibacterial effects of spinetoram and carvacrol in the tested ratios on Ps. camelliae-sinensis were significantly different. When the ratio of the two was 1:1, 1:2, and 2:1, the EC 50 The values ​​were 38.10μg / mL, 28.25μg / mL, and 66.58μg / mL, respectively. When the compounding ratio of the two was 1:1 and 1:2, the synergistic coefficients of the compounding results were all greater than 1.5. Therefore, the compounding of the two had a strong antibacterial effect on Ps. camelliae-sinensis and showed a synergistic effect. When the compounding ratio of ethyl spinetoram and carvacrol was 1:2, EC50 The value was 28.25 μg / mL, and the SR value was 3.77. The synergistic effect was the most obvious compared with other treatment groups, so it was finally determined as the optimal combination of ethyl spinetoram and carvacrol for subsequent experiments.

[0079] Example 3: Combined toxicity study of spinetoram and hydroxychloroquine

[0080] Preparation method: Weigh 0.6g of 25% spinosad water dispersible granules, add 3mL of sterile water and mix well to prepare a 50,000μg / mL stock solution. Weigh 0.25g of hydroxychloroquine original drug, dissolve in 5mL of DMSO to prepare a 50,000μg / mL stock solution.

[0081] The mother solution of spinetoram and the mother solution of hydroxychloroquine were mixed in proportion to obtain mixed solutions with a ratio of 1:1, 1:2, and 2:1 of spinetoram to hydroxychloroquine. The combined toxicity of the compound agents of spinetoram and hydroxychloroquine in different proportions to B. cinerea, L. theobromae, and D. segeticola was determined by the growth rate method. The specific experimental steps are:

[0082] First, ethyl spinetoram and hydroxychloroquine were mixed in a mass ratio of 1:0, 1:1, 1:2, 2:1, and 0:1, and the mycelium growth rate method was used with clean water as a blank control to determine the in vitro antibacterial activity of the compound agent against B. cinerea, L. theobromae, and D. segeticola. Take a 250mL conical flask, prepare 90mL of PDA culture medium in each bottle, put it in a high-pressure steam sterilizer and sterilize it at 121°C for 20 minutes, take out the sterilized PDA culture medium, add the prepared agents to the PDA, so that the final concentrations of the agents are 0.0μg / mL, 2.5μg / mL, 5.0μg / mL, 25.0μg / mL, 50.0μg / mL, and 75.0μg / mL, respectively. This concentration gradient is for B. cinerea a; 0.0μg / mL, 20.0μg / mL, 40.0μg / mL, 60.0μg / mL, 80.0μg / mL, 100.0μg / mL, this concentration gradient is for L.theobromae; 0.0μg / mL, 20.0μg / mL, 40.0μg / mL, 60.0μg / mL, 80.0μg / mL, 100.0μg / mL, this concentration gradient is for D.segeticola. Then pour the mixed PDA medium containing the drug into the culture dish, and do 5 biological replicates for each concentration. After the culture medium is cooled and solidified, use a sterilized 4 mm diameter puncher to punch the activated fresh B. cinerea, L. theobromae, and D. segeticola colonies to obtain bacterial cakes, and then inoculate the punched bacterial cakes onto the PDA culture medium, respectively. After inoculation, seal the culture dish with a sealing film and invert it in a 25° C. constant temperature incubator for culture. When the colonies in the 0.0 μg / mL agent culture medium grow to 3 / 4 of the culture dish area, measure the colony diameter according to the cross method. Finally, calculate the inhibition rate, synergistic ratio, etc. by referring to the method of implementation 1.

[0083] Table 9 Combined biological activity of ethyl spinetoram and hydroxychloroquine against B. cinerea

[0084]

[0085] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0086] After spinetoram and hydroxychloroquine were mixed in different ratios, the effects of single agent and mixed agent on the mycelial growth of B. cinerea were determined. As shown in Table 9, the antibacterial effect of spinetoram and hydroxychloroquine in different mixed ratios on B. cinereae was 1:1, 1:2, and 2:1. 50 The values ​​were 59.55 μg / mL, 823.93 μg / mL, and 40.27 μg / mL, respectively. It can be seen that the combination of ethyl spinetoram and hydroxychloroquine did not show obvious antibacterial effect. When the combination ratio was 2:1, EC 50 The synergistic coefficient was 40.27 μg / mL, and the maximum was 1.28, showing an additive effect. When the compound ratio was 1:2, EC 50 The concentration of hydroxychloroquine in the mixture of spinetoram and ethyl spinetoram was 823.93 μg / mL, and the synergistic coefficient was 0.09, showing an antagonistic effect. Based on the above test results, there was no obvious synergistic effect when spinetoram was combined with hydroxychloroquine at the treatment concentration.

[0087] Table 10 Combined biological activity of ethyl spinetoram and hydroxychloroquine against L. theobromae

[0088]

[0089] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC 50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0090] After spinetoram and hydroxychloroquine were mixed in different ratios, the effects of single agent and mixed agent on the mycelial growth of L. theobromae were determined. As shown in Table 10, the antibacterial effects of spinetoram and hydroxychloroquine in different mixed ratios on L. theobromae were significantly different. When the mixed ratios were 1:1, 1:2, and 2:1, the EC 50 The values ​​were 34.45μg / mL, 102.89μg / mL, and 46.54μg / mL, respectively. When the compound ratio was 1:1, the EC 50 It is 34.45μg / mL, and the synergistic coefficient is the largest (1.66), showing a synergistic effect. When the ratio is 1:2 and 2:1, the synergistic coefficients are 0.64 and 1.10, showing an additive effect.

[0091] Table 11 Combined biological activity of ethyl spinetoram and hydroxychloroquine combined with D. segeticola

[0092]

[0093] Note: “-” represents the antibacterial activity of a bactericidal active ingredient; EC50(th) EC for compound preparation 50 Theoretical value; EC 50(ob) EC for compound preparation 50 Measured value

[0094] After spinetoram and hydroxychloroquine were mixed in different ratios, the effects of single and mixed agents on the mycelial growth of D. segeticola were determined. As shown in Table 11, the antibacterial effects of spinetoram and hydroxychloroquine in different ratios on D. segeticola were as follows: 50 The values ​​were 173.76μg / mL, 181.01μg / mL, and 146.74μg / mL, respectively. It can be seen that the combination of spinetoram and hydroxychloroquine did not show obvious antibacterial effect, and the SR values ​​of the three ratios were all between 0.5 and 1.5. Based on the above test results, the combination of spinetoram and hydroxychloroquine did not show obvious synergistic effect at the treated concentration.

[0095] Experimental Example 4 In vivo antibacterial activity experiment of the compound combination against pathogenic bacteria

[0096] 1. Antibacterial activity of the combination of ethyl spinetoram and allicin against B. cinerea in vivo

[0097] The tomato fruit variety was the Huamang variety, and 5 replicates were set for each treatment. The front and back of the leaves were wiped clean with moist sterilized cotton and the surface moisture was naturally dried. 100, 250, 500, 1 000, and 1 500 μg / mL of ethyl spinetoram and allicin compound solutions were prepared. The tomato fruit was sprayed, and after the liquid on the surface of the fruit was naturally dried, a small triangular hole was punched in the middle of the fruit with a sterilized needle. Then, a sterilized 4mm diameter puncher was used to punch a bacterial cake on a fresh B. cinerea colony that had been cultured for 3 days, and then the beaten bacterial cake was inoculated with the mycelium facing down on the front puncture of the fruit. Among them, sterile PDA medium was used for inoculation as the negative control group, and sterile water was used for spraying as the positive control group. After 48 hours, the pathogenicity was observed and photographed.

[0098] Depend on Figure 1 (The horizontal axis represents the concentration of spinetoram combined with allicin, and the vertical axis represents the lesion inhibition rate of tomato fruit inoculated with B. cinerea after spinetoram combined with allicin) shows that after spraying the combination of spinetoram and allicin, the inhibition rate of lesions of the pathogen B. cinerea shows an upward trend as the concentration of the drug increases. Figure 4As shown in the figure, after spraying the 1:1 combination of ethyl spinetoram and allicin, the inhibition rate of the pathogen B. cinerea lesions showed an upward trend with the increase of the added concentration (A1: CK (after spraying water, connect blank PDA for 48 hours); B1: CK (no spraying, bacteria caused disease for 48 hours); C1: after spraying 100μg / mL compound solution, the bacteria caused disease for 48 hours; D1: after spraying 250μg / mL compound solution, the bacteria caused disease for 48 hours; E1: after spraying 500μg / mL compound solution, the bacteria caused disease for 48 hours; F1: after spraying 1 000μg / mL compound solution, the bacteria caused disease for 48 hours; G1: after spraying 1 500μg / mL compound solution, the bacteria caused disease for 48 hours). Figure 5 As shown in the figure, after spraying the 1:2 combination of ethyl spinetoram and allicin, the inhibition rate of the pathogen B. cinerea lesions showed an upward trend with the increase of the added concentration (A2: CK (after spraying water, connect blank PDA for 48 hours); B2: CK (no spraying, bacteria caused disease for 48 hours); C2: after spraying 100μg / mL compound solution, the bacteria caused disease for 48 hours; D2: after spraying 250μg / mL compound solution, the bacteria caused disease for 48 hours; E2: after spraying 500μg / mL compound solution, the bacteria caused disease for 48 hours; F2: after spraying 1 000μg / mL compound solution, the bacteria caused disease for 48 hours; G2: after spraying 1 500μg / mL compound solution, the bacteria caused disease for 48 hours). Figure 6 As shown in the figure, after spraying the combination of ethyl spinetoram and allicin in a ratio of 2:1, the inhibition rate of the pathogen B. cinerea lesions showed an upward trend with the increase of the added concentration (A3: CK (after spraying water, connected to blank PDA for 48 hours); B3: CK (no spraying, the bacteria caused disease for 48 hours); C3: after spraying 100μg / mL of the compound solution, the bacteria caused disease for 48 hours; D3: after spraying 250μg / mL of the compound solution, the bacteria caused disease for 48 hours; E3: after spraying 500μg / mL of the compound solution, the bacteria caused disease for 48 hours; F3: after spraying 1 000μg / mL of the compound solution, the bacteria caused disease for 48 hours; G3: after spraying 1 500μg / mL of the compound solution, the bacteria caused disease for 48 hours).

[0099] 2. Antibacterial activity of the combination of ethyl spinetoram and carvacrol against Ps. camelliae-sinensis in vivo

[0100] The tea tree variety used was Fuding Dabaicha (Camellia sinensis (L.) O. Kuntze), with a tree age of 3 years and a tea leaf age of 30 days. Five replicates were set for each treatment group. The second or third leaf of the tea tree was selected, and the front and back of the leaf were wiped clean with moist sterilized cotton and the surface moisture was naturally dried. 100, 250, 500, and 1 000 μg / mL of ethyl spinetoram and carvacrol compound solutions were prepared. The leaves were sprayed with the treatment, and after the liquid on the surface of the leaves was naturally dried, a triangular hole was punched in the middle of the fruit with a sterilized needle. Then, a sterilized 4 mm diameter puncher was used to punch a bacterial cake on a fresh colony of Ps. camelliae-sinensis that had been cultured for 5 days, and then the punched bacterial cake was inoculated on the front puncture of the fruit with the hyphae facing down. Among them, the sterile PDA medium was used for inoculation as the negative control group, and the sterile water was used for spraying as the positive control group. After 48 hours, observe and take photos of the pathogenic conditions.

[0101] Depend on Figure 2 (The horizontal axis represents the concentration of spinetoram combined with carvacrol, and the vertical axis represents the inhibition rate of lesions of tea leaves inoculated with Ps. camelliae-sinensis after spinetoram combined with carvacrol) shows that after spraying the combination of spinetoram and carvacrol, the inhibition rate of lesions of the pathogen Ps. camelliae-sinensis shows an upward trend as the concentration of the drug increases. Figure 7 As shown in the figure, after spraying the 1:1 combination of ethyl spinetoram and carvacrol, the inhibition rate of the pathogen Ps. camelliae-sinensis lesions showed an upward trend with the increase of the added concentration (A1: CK (after spraying, connect blank PDA for 48 hours); B1: CK (no spraying, bacteria caused disease for 48 hours); C1: after spraying 100μg / mL compound solution, the bacteria caused disease for 48 hours; D1: after spraying 250μg / mL compound solution, the bacteria caused disease for 48 hours; E1: after spraying 500μg / mL compound solution, the bacteria caused disease for 48 hours; F1: after spraying 1 000μg / mL compound solution, the bacteria caused disease for 48 hours). Figure 8 As shown in the figure, after spraying the 1:2 combination of spinetoram and carvacrol, the inhibition rate of the pathogen Ps. camelliae-sinensis lesions showed an upward trend with the increase of the drug concentration (A2: CK (after spraying, blank PDA was connected for 48 hours); B2: CK (no spraying, the bacteria caused disease for 48 hours); C2: after spraying 100μg / mL of the compound solution, the bacteria caused disease for 48 hours; D2: after spraying 250μg / mL of the compound solution, the bacteria caused disease for 48 hours; E2: after spraying 500μg / mL of the compound solution, the bacteria caused disease for 48 hours;

[0102] F2: After spraying 1 000 μg / mL compound solution, the fungus caused disease 48 hours later. Fig. 9 As shown in the figure, after spraying the combination of ethyl spinetoram and carvacrol in a ratio of 2:1, the inhibition rate of the pathogen Ps. camelliae-sinensis lesions showed an upward trend with the increase of the added concentration (A3: CK (after spraying, connected to blank PDA for 48 hours); B3: CK (no spraying, the bacteria caused disease for 48 hours); C3: after spraying with 100μg / mL of the compound solution, the bacteria caused disease for 48 hours; D3: after spraying with 250μg / mL of the compound solution, the bacteria caused disease for 48 hours; E3: after spraying with 500μg / mL of the compound solution, the bacteria caused disease for 48 hours; F3: after spraying with 1 000μg / mL of the compound solution, the bacteria caused disease for 48 hours).

[0103] 3. Antibacterial activity of the combination of spinetoram and hydroxychloroquine against L. theobromae in vivo

[0104] The tea tree variety used was Fuding Dabai, the tea tree age was 3 years, and the tea tree age was 30 days. Five replicates were set for each treatment group. The second or third leaf of the tea tree was selected, and the front and back of the leaf were wiped clean with moist sterilized cotton and the surface moisture was naturally dried. 100, 250, 500, 1 000, and 2 000 μg / mL of ethyl spinetoram and hydroxychloroquine compound solutions were prepared. The leaves were sprayed for treatment. After the liquid on the surface of the leaves was naturally dried, a small triangular hole was punched in the middle of the fruit with a sterilized needle. Then, a sterilized 4 mm diameter puncher was used to punch a bacterial cake on a fresh L. theobromae colony that had been cultured for 2 days, and then the beaten bacterial cake was inoculated with the hyphae facing down on the front puncture of the fruit. Among them, sterile PDA medium was used for inoculation as a negative control group, and sterile water was used for spraying as a positive control group. After 48 hours, the pathogenicity was observed and photographed.

[0105] Depend on Figure 3 (The horizontal axis represents the concentration of spinetoram combined with hydroxychloroquine, and the vertical axis represents the inhibition rate of the combined spinetoram and hydroxychloroquine on tea leaves inoculated with L. theobromae) As shown, after spraying the combined spinetoram and hydroxychloroquine, the inhibition rate of the pathogen L. theobromae lesions showed an upward trend as the concentration of the drug increased. Fig.10As shown in the figure, after spraying the 1:1 combination of ethyl spinetoram and carvacrol, the inhibition rate of the pathogen L. theobromae lesions showed an upward trend with the increase of the added concentration, but it was not very obvious (A1: CK (after spraying water, connect blank PDA for 48 hours); B1: CK (no spraying, bacteria caused disease for 48 hours); C1: after spraying 100μg / mL compound solution, the bacteria caused disease for 48 hours; D1: after spraying 250μg / mL compound solution, the bacteria caused disease for 48 hours; E1: after spraying 500μg / mL compound solution, the bacteria caused disease for 48 hours; F1: after spraying 1000μg / mL compound solution, the bacteria caused disease for 48 hours; G1: after spraying 2 000μg / mL compound solution, the bacteria caused disease for 48 hours). Fig.11 As shown in the figure, after spraying the 1:2 combination of ethyl spinetoram and carvacrol, as the concentration of the drug increased, the inhibition rate of the pathogen L. theobromae lesions showed an upward trend, but it was not very obvious (A2: CK (after spraying water, connected to blank PDA for 48 hours); B2: CK (no spraying, the bacteria caused disease for 48 hours); C2: after spraying 100μg / mL of the compound solution, the bacteria caused disease for 48 hours; D2: after spraying 250μg / mL of the compound solution, the bacteria caused disease for 48 hours; E2: after spraying 500μg / mL of the compound solution, the bacteria caused disease for 48 hours; F2: after spraying 1 000μg / mL of the compound solution, the bacteria caused disease for 48 hours; G2: after spraying 2 000μg / mL of the compound solution, the bacteria caused disease for 48 hours). Fig.12 As shown in the figure, after spraying the combination of ethyl spinetoram and carvacrol in a ratio of 2:1, the inhibition rate of the pathogen L. theobromae lesions showed an upward trend with the increase of the added concentration, but it was not very obvious (A3: CK (after spraying water, connected to blank PDA for 48 hours); B3: CK (no spraying, the bacteria caused disease for 48 hours); C3: after spraying 100μg / mL of the compound solution, the bacteria caused disease for 48 hours; D3: after spraying 250μg / mL of the compound solution, the bacteria caused disease for 48 hours; E3: after spraying 500μg / mL of the compound solution, the bacteria caused disease for 48 hours; F3: after spraying 1 000μg / mL of the compound solution, the bacteria caused disease for 48 hours; G3: after spraying 2 000μg / mL of the compound solution, the bacteria caused disease for 48 hours).

[0106] The conventional techniques and schemes not described in detail in the above embodiments are well known in the art, so they will not be described in detail here. The above embodiments and / or experimental examples describe the preferred embodiments of the present invention in detail, but the present invention is not limited to the specific details in the above embodiments. Within the scope of the technical concept of the present invention, the technical scheme of the present invention can be subjected to a variety of simple modifications, and these simple modifications all belong to the protection scope of the present invention.

Claims

1. A spinetoram compound pesticide composition, characterized in that: The composition comprises an active ingredient A and an active ingredient B, wherein the active ingredient A is spinetoram, and the active ingredient B is allicin, carvacrol or hydroxychloroquine.

2. The composition according to claim 1, characterized in that The mass ratio of active ingredient A to active ingredient B is 1-2:1-2.

3. The composition according to claim 2, characterized in that The mass ratio of active ingredient A to active ingredient B is 1:1, 1:2 or 2:

1.

4. The method for preparing the composition according to any one of claims 1 to 3, characterized in that: The steps include: 1) Weigh 25% spinetoram water dispersible granules and add them to sterilized water to prepare a mother solution I with a spinetoram concentration of 50,000 μg / mL; weigh allicin, carvacrol or hydroxychloroquine and dissolve them in DMSO to prepare a mother solution II with a concentration of 50,000 μg / mL; 2) Mix the mother solution I and the mother solution II in proportion for compounding.

5. Use of the composition according to any one of claims 1 to 3 in inhibiting plant pathogens.

6. The use according to claim 5, characterized in that The plant pathogenic bacteria include any one or more of Botrytis cinerea, Lasiodiplodia theobromae, Didymella segeticola and Pseudopestalotiopsis camelliae-sinensis.

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