CN114304150A - Tobacco aphid repellent and poisoning inhibitor and preparation method thereof - Google Patents
Tobacco aphid repellent and poisoning inhibitor and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a myzus persicae repellent and poison killing inhibitor and a preparation method thereof. When the active ingredients of dibutyl phthalate, diallyl disulfide and 2, 6-ditert-butyl-p-cresol are compounded for use, the contact killing and repellent activity to myzus persicae is obviously improved, and the activity of carboxylesterase can be obviously inhibited. The inhibitor can be directly used for controlling the occurrence and the harm of myzus persicae and the loss of tobacco leaves caused by aphid-borne virus diseases. Meanwhile, the use of chemical pesticides can be effectively reduced, the environmental pollution is reduced, and the safety of tobacco leaves and other plants is improved.
Description
Technical Field
The invention particularly relates to a method for avoiding and killing tobacco aphids by using main components of garlic root exudates and a mixture thereof.
Background
Myzus persicaeMyzus persicae(Sulzer), belonging to Hemiptera, Aphidae, also known as green peach aphid, is an important agricultural pest in China, mainly harms tobacco and various cruciferous vegetables and other plants (G. Weber, 1985), and occurs in various tobacco production areas in China with high harmfulness. Myzus persicae secretes honeydew to induce sooty mould, block photosynthesis, greatly affect the quality and yield of tobacco, reduce the yield of flue-cured tobacco, reduce the quality of flue-cured tobacco, and spread various tobacco virus diseases to indirectly damage tobacco plants, so that great loss is brought to the tobacco economic industry, and the economic loss rate of 1 myzus persicae is =0.0695% (Yuanfeng et al, 1994; iris loyalty dragon et al, 2011). In recent years, along with the large-area use of chemical pesticides, the tobacco aphids are induced to immunize against the chemical pesticides to generate drug resistance, for example, the tobacco aphids in most tobacco producing areas of Guizhou generate moderate-level resistance to acetamiprid, and a small area generates extremely-high-level resistance to deltamethrin (Boehu jade and the like, 2017; Xunzhu and the like, 2018), and meanwhile, the problems of reduction of natural enemies of the tobacco aphids, ecological imbalance and the like (Zhuxiuzhen and the like, 2011) are caused, and along with the improvement of environmental awareness of people, the use of biosynthetic pesticides is worth people's paradox. Root exudates are defined as the general term for organic substances released by living plants through different root parts into the root environment under certain environmental conditions (Xiaorong et al, 2021), garlic (garlic)Allium sativum L.) belongs to the family of Bulbus Allii Liliaceae, has certain antibacterial and bactericidal effects, and essential oil contained in Bulbus Allii has obvious poisoning effect on insects, such as fumigating essential oil of Bulbus Allii on Theobroma rusticana, and LC of Bulbus Allii essential oil on Theobroma rusticana adults within 24h500.7827uL/L (Yuanli maple et al, 2017). There have been many studies and reports at present: the garlic has a certain effect on controlling the tobacco anthracnose, and when the concentration of the extracts of the stem leaves and the root systems of the garlic is 0.5g/mL, the inhibition rate of the extracts on the tobacco anthracnose is 100 percent (Wangjun, etc., 2018); garlic has allelopathic effect on many vegetables, such as garlic andafter the rotation of the cucumber, the generation of cucumber blight is effectively reduced, and when the concentration of the purple garlic clove leaching liquor is 0.25mg/mL, the inhibition rate of the phytophthora melonis on the zoospore germination of the phytophthora melonis 98% (Wu Jia Qing et al, 2019); the garlic root secretion has obvious promotion effect on the length and fresh weight of the root of the tobacco, but has certain inhibition effect on the height of the seedling (Guoling and the like, 2019; Wuxiangting and the like, 2019). The use of the effective components in the garlic extract for preventing and controlling myzus persicae has become a hotspot for the research of botanical pesticides for preventing and controlling myzus persicae in China. However, the action of the effective components in the garlic root exudates, especially the mixture of the effective components on the aphid is not reported at present.
The level of carboxylesterase (CarE) and acetylcholinesterase (AchE) activity of myzus persicae is associated with its resistance and the level of resistance (William et al, 2000). The higher the detoxification enzyme activity of cnaphalocrocis medinalis, the lower its sensitivity to pesticides (royal clouds et al, 2017). The carboxylesterase activity is improved, the detoxification capability of the myzus persicae can be enhanced, and the carboxylesterase can be combined with a medicament to reduce the inhibition of the medicament on acetylcholinesterase (Wangkong et al, 2000; Songchunman and Liangfei, 2011).
Therefore, on the basis of analyzing the components of the garlic root secretion, 3 reagents of dibutyl phthalate, 2, 6-di-tert-butyl-p-cresol and diallyl disulfide are selected for testing. The influence of the 3 reagents and the mixing thereof on the repellent and poisoning activities of myzus persicae is measured by a leaf disc method; and the influence of the 3 reagents and the mixing thereof on the aphid detoxification enzyme is determined by adopting an ELISA detection kit, thereby providing scientific reference for developing a physiological and biochemical mechanism containing the mixture reagent for controlling the aphid and the control effect of the mixture reagent on the aphid.
Disclosure of Invention
The invention aims to explore main components (dibutyl phthalate, diallyl disulfide and 2, 6-ditertbutyl-p-cresol) of garlic root secretion and a mixture thereof for myzus persicaeMyzus persicae(Sulzer) repellent, poisoning effects and the influence of detoxification enzymes. [ METHOD ] dibutyl phthalate, diallyl disulfide, 2, 6-di-tert-butyl-p-cresol and mixtures thereof were determined by the leaf-dish methodThe influence of the aphid on the activity of repelling and killing the myzus persicae is matched, and the influence of the aphid on the antitoxic enzyme of the myzus persicae is measured by adopting an ELISA detection kit. (results) dibutyl phthalate, diallyl disulfide and 3 reagents of 2, 6-di-tert-butyl-p-cresol were used alone, and when the volume ratio was 1:10, the repellent and touch-killing effects on myzus persicae were more significant (P<0.05). Wherein, the repellent activity of diallyl disulfide and dibutyl phthalate to myzus persicae is higher and reaches 97.00 percent and 93.00 percent respectively; the contact killing activity of dibutyl phthalate and 2, 6-ditert-butyl-p-cresol to myzus persicae is high, and the corrected death rate reaches 93% and 92.00% respectively. When the three reagents are mixed, the reagent which is used independently compared with dibutyl phthalate, diallyl disulfide and 2, 6-di-tert-butyl-p-cresol has more obvious repellent activity to myzus persicaeP<0.05) and contact activity (P<0.05). Wherein, the three reagents are mixed simultaneously to have better contact killing and repellent activity to myzus persicae, and the repellent rate is between 81.00% and 93.00%; the mortality rate is between 95.00% and 98.00%. The research shows that the single reagent used in the mixture ratio of the three reagents has more obvious repellent and contact killing effects on myzus persicae. The detoxification enzyme activity of the three reagents and the mixture thereof on the myzus persicae is as follows: acetylcholinesterase>Carboxylic esterases>Glutathione S-transferase. The method provides scientific reference for developing a physiological and biochemical mechanism containing the mixture reagent for controlling myzus persicae and the control effect of the mixture reagent on myzus persicae.
In order to achieve the purpose, the invention adopts the following technical scheme:
a tobacco aphid repellent and poison killing inhibitor comprises the active components of dibutyl phthalate, diallyl disulfide and 2, 6-ditert-butyl-p-cresol. Wherein the volume ratio of dibutyl phthalate, 2, 6-di-tert-butyl-p-cresol and diallyl disulfide is 1: 0.2-1: 1, preferably 1: 1: 1.
further, preparing active ingredients of dibutyl phthalate, diallyl disulfide and 2, 6-di-tert-butyl-p-cresol into gradient solutions of 1:10, 1:20, 1:30, 1:40 and 1:50 according to the volume ratio of the ethanol solutions respectively; 3 when the active substances are mixed, selecting the active substances with the volume ratio of 1:10 dibutyl phthalate, diallyl disulfide and a volume ratio of 1: 10-50 parts of 2, 6-di-tert-butyl-p-cresol in a molar ratio of 1: 1:1, mixing.
The preparation method of the myzus persicae repellent and poison killing inhibitor comprises the following steps: dissolving active ingredients of dibutyl phthalate, diallyl disulfide and 2, 6-di-tert-butyl-p-cresol into an ethanol solution of a solvent, adding a proper amount of Tween 80, and stirring and mixing uniformly.
Further, the concentration of the solvent ethanol solution was 8 wt%.
Further, Tween 80 was used in an amount of 0.1 ml.
The invention has the advantages that:
acetylcholinesterase, carboxylesterase, glutathione S-transferase are important detoxifying metabolic enzymes in the body of insects. The method discovers that 4-year-old wingless myzus persicae is treated after three reagents, namely dibutyl phthalate, diallyl disulfide and 2, 6-di-tert-butyl-p-cresol, are mixed simultaneously, and that carboxylesterase activity in the myzus persicae is remarkably inhibited, so that the toxic substance detoxifying capability of the myzus persicae is reduced, and the myzus persicae is killed, but the three reagents have no inhibiting effect but promoting effect on the carboxylesterase activity when being used independently. Carboxylesterases can rapidly bind to toxic substances entering the insect body, blocking or degrading the toxic substances before reaching the target site of action. After the three reagents are mixed, 4-year-old wingless myzus persicae is treated, the activity of carboxylesterase is reduced, and the toxin degradation capability is reduced, so that the toxin can quickly reach a target action site to play a role. Glutathione S-transferase is one of the important conjugative enzyme systems in insect metabolism for pesticide or toxic substances, and can ensure that exogenous electrophilic groups and reduced glutathione in vivo generate conjugation metabolism to protect other nucleophilic centers in vivo. After the three reagents are mixed, the activity of the myzus persicae glutathione S-transferase is inhibited to a certain extent, and the degradation capability of the glutathione S-transferase to endogenous or exogenous toxins can be reduced. In conclusion, the activity inhibition of the three reagents on the myzus persicae carboxylesterase and the glutathione S-transferase after being mixed is related to the poisoning effect of the three reagents, the detoxifying metabolic capability of the insects is weakened, and the insects die finally. Carboxylesterase and glutathione S-transferase are important detoxification metabolic enzymes in insect bodies, and can be one of action targets of three reagents mixed for myzus persicae.
Drawings
FIG. 1 shows the result of acetylcholinesterase detection;
FIG. 2 shows the results of detection of carboxylesterase;
FIG. 3 shows the results of detection of glutathione S-transferase.
Detailed Description
In order to make the aforementioned and other features and advantages of the invention more comprehensible, embodiments accompanied with figures are described in detail below. The method of the present invention is a method which is conventional in the art unless otherwise specified.
Materials and methods
1.1 test materials
Tobacco: the flue-cured tobacco K326 is cultivated indoors, and no pesticide is used in the process for standby.
Myzus persicae: wingless myzus persicae collected from field tobacco plants of a test station of Taining branch company of tobacco company of Sanming city are bred indoors for 2-3 generations and then tested.
Reagent: dibutyl phthalate (99.5% purity, manufactured by Ningxing Chemicals Co., Ltd., Tianjin), 2, 6-di-tert-butyl-p-cresol (AR, manufactured by Michelin Biochemical technology Co., Ltd., Shanghai), diallyl disulfide (85% purity, manufactured by Michelin Biochemical technology Co., Ltd., Shanghai), ethanol (AR, manufactured by chemical reagents Co., Ltd., national drug group) and Tween 80 (manufactured by Sjongsu science Co., Ltd.).
Other materials: a micro-dropper (model PDE0003, manufactured by Shanghai gem Yu science instruments Co., Ltd.), a culture dish (diameter 9 cm), filter paper, absorbent cotton, distilled water, an electronic balance (AUY 120 manufactured by Shimaduza, Japan), a centrifuge tube (1.5 mL), a writing brush and a leaf dish; a high-speed refrigerated centrifuge (CR 22, manufactured by IKE, Germany), an electric heating constant temperature water tank (DK-80, manufactured by Heishai science and technology Co., Ltd.), an acetylcholinesterase ELISA test kit (manufactured by Jiangsu Bo-Shen Biol.), a glutathione S-transferase ELISA test kit (manufactured by Jiangsu Bo-Shen Biol.), a carboxylesterase ELISA test kit (manufactured by Jiangsu Bo-Shen Biol.).
Test method
1.2.1 determination of the biological Activity of a Single reagent on myzus persicae
1.2.1.1 determination of repellent Activity of Individual reagents on myzus persicae
The method is characterized in that a blade smearing method (Jiangsun et al, 2009) is adopted, dibutyl phthalate, 2, 6-di-tert-butyl-p-cresol and diallyl disulfide reagent are respectively dissolved by 8% ethanol and 0.1mL of Tween 80 solution, and then the solution is diluted into gradient solution with the volume ratio of 1:10, 1:20, 1:30, 1:40 and 1:50 for treatment. The tobacco leaves which are not polluted, fresh and clean and similar in size (length multiplied by width =8cm multiplied by 5 cm) are taken, the main vein in the middle is taken as a boundary, and the similar areas are drawn on the left and the right of the leaves. 1mL of treatment solution with different concentrations was evenly applied to the front and back of the leaf dish on one side with a brush, and the leaf dish on the other side was applied with 1mL of an equivalent 8% ethanol plus Tween solution (control). After the leaves were air-dried, they were placed in a petri dish (diameter 9 cm) with tobacco leaves while keeping the petioles moist with wet cotton. Each petri dish was placed 1 half leaf of each of the treated and control, i.e. 2 half leaf dishes (8 cm. times.5 cm). And then 4-year-old Aphis finosus which is starved for 4 hours before the test and is consistent in size is sequentially inoculated into a culture dish (in the middle of 2 half tobacco leaves). Each dish was inoculated with 10 myzus persicae and each treatment was repeated 5 times. The culture dish is sealed by a preservative film in time, and then 50 air holes with the same size are punctured by needles, so that the myzus persicae can normally breathe and can be prevented from escaping. Then, the seeds were kept in a greenhouse at 25. + -. 2 ℃. After 12h, 24h and 48h of treatment, the inhabitation number of tobacco aphids on the tobacco leaf control area and the treatment area in each culture dish is observed and recorded (Shangxiang Kun et al, 2008), and the repellent rate is calculated according to the formula 1.
Determination of contact killing activity of single reagent on myzus persicae
Three reagent solutions with a volume ratio of 1:10 are respectively prepared by adopting a drop method (Jianjing et al, 2009) and adopting a method in 1.2.1.1 for treatment. Carefully selecting 4-year-old healthy wingless myzus persicae with consistent size, dripping 1 μ L of test solution on the anterior chest back plate of the myzus persicae with a micro-dripping instrument (PDE 0003 model, manufactured by Shanghai gem Yu scientific instruments Co., Ltd.), transferring the test insects into a culture dish (diameter of 9 cm) with fresh tobacco leaves in a greenhouse of 25 + -2 deg.C, and feeding. Each treatment was 10 myzus persicae, replicated 5 times. After 12h, 24h and 48h of treatment, the death condition of the myzus persicae is observed and recorded (remarks: the myzus cannot crawl, overturn or slightly shake the body of the myzus persicae by touching the myzus persicae with a writing brush, all the myzus persicae are dead). The mortality and corrected mortality of myzus persicae was then calculated according to equations 2, 3.
Biological activity determination of three reagent mixed pair myzus persicae
On the basis of 1.2.1, the screened dibutyl phthalate and diallyl disulfide have good effects when the volume ratio is 1:10, and the 2, 6-di-tert-butyl-p-cresol has a good effect when the volume ratio is 1: 50. Therefore, whether the better effect can be achieved by mixing two components at the concentration is further explored.
Determination of repellent activity of three reagents mixed to myzus persicae
8% ethanol and two drops of Tween are adopted to sequentially prepare the dibutyl phthalate and the diallyl disulfide into solutions with the same volume according to the volume ratio of 1:10 and the volume ratio of 2, 6-di-tert-butyl-p-cresol according to the volume ratio of 1:50, the prepared solutions absorb 5mL of reagents with the same volume, and the two solutions are mixed in pairs (volume ratio of 1: 1) and the three solutions (volume ratio of 1: 1: 1). Equal amount of 8% ethanol plus tween was added dropwise to the control. The other methods are the same as those in 1.2.1.1.
Three reagents mixed and matched type contact killing activity determination of myzus persicae
The method comprises the steps of preparing solutions with the same concentration and volume by using 8% ethanol and two drops of Tween according to the volume ratio of 1:10, preparing solutions with the same concentration and volume by using two drops of Tween sequentially, sucking 5mL of reagents with the same volume, and mixing two by two (the volume ratio is 1: 1) and three (the volume ratio is 1: 1: 1). Control drops of 8% ethanol plus tween were added in equal amounts. The other methods are the same as those in 1.2.1.2.
Influence of three reagents and mixing on aphid fumigase
On the basis of 1.2.2 tests, solutions with a volume ratio of 1:10 and a better test effect are screened out, wherein the solutions are mixed in pairs (the volume ratio is 1: 1) and three solutions (the volume ratio is 1: 1: 1). The other methods are the same as those in 1.2.1.2.
Preparation of enzyme source
Adopting a belt worm immersion method (Yuan Jia Yu et al, 2020), selecting 30 aphids with the same size and activity and 4 ages in each treatment or contrast, immersing for 5s in 7 treatments (shown in table 6), taking out, sucking the medicinal liquid with filter paper, and putting into a centrifuge tube for grinding. Each process set 3 replicates. (1) Preparation of acetylcholinesterase (AChE) enzyme source: 1.5ML of 0.2mol/ML pre-cooled phosphate buffer (pH 7.0) was added to each centrifuge tube and homogenized before the temperature: 4 ℃, 10000 r.min-1Centrifuging for 20 min, and collecting supernatant as enzyme source. (2) Preparation of glutathione S-transferase (GSTs) enzyme source: respectively adding 0.05 mol.l which is pre-cooled-1Tris-HCl1.5 mL at pH 7.5 was then homogenized at 4 ℃ at 10000 r.min-1Centrifuging for 15min, and collecting the supernatant as enzyme source. (3) Preparation of Carboxylesterase (CES) enzyme source: respectively adding precooled 0.04 mol.L-1Tris-HCl1.5 mL at pH 7.5, homogenate at 4 ℃ 10000 r.min-1Centrifuging for 15min, and collecting the supernatant as enzyme source.
Acetylcholic ester AChE activity assay
According to the operating instruction of acetylcholinesterase of the kit of Jiangsu Boshen biological company, a 96-hole enzyme label plate is taken, standard substance holes and sample holes are sequentially arranged, 40 mu L of standard substances with different concentrations are sequentially added into each standard substance hole, 10 mu L of prepared acetylcholinesterase is taken and added into the sample holes, then 40 mu L of sample diluent is added, and particularly, no solution is added into blank holes. And adding 100 mu L of detection antibody marked by horseradish peroxidase (HRP) into each of the sample product hole and the standard product hole, sealing the reaction hole by using a sealing plate film after the operation is finished, and reacting for 60min in a 37 ℃ constant temperature box. After the test fully reacts, the sealing plate film is torn off, the liquid is patted off, part of residual liquid is patted dry on the absorbent paper, then the cleaning solution is added into each hole, after standing for about 1min, the cleaning solution is quickly thrown off, the liquid is patted dry on the absorbent paper, and the steps are repeated for 5 times in sequence. After the plate washing was completed, 50. mu.L each of the substrates A and B was added to each well in turn, and the OD of each well was measured at a wavelength of 450nm within 15min and the acetylcholinesterase AChE enzyme activity value was recorded.
Glutathione S-transferase GSTs enzyme activity assay
Refer to the kit glutathione S-transferase operating manual of Jiangsu Bo-Shen biological company. The method and the operation steps are the same as those in 1.2.3.2, and finally, the enzyme activity of glutathione S-transferase GSTs is calculated.
Carboxylesterase CarE enzyme activity assay
Refer to the kit carboxylesterase instructions of Jiangsu Bo-Shen-Bio Inc. The method and the operation steps are the same as those in 1.2.3.2, and finally, the activity of the carboxylesterase CarE enzyme is calculated.
Data processing
Statistical, followed by one-way analysis of variance (α = 0.05) LSD comparisons were performed using Excel2010 and SPSS22 software, with all data taken as mean ± standard error.
Results and analysis
2.1 biological Activity of Individual Agents on Aphis Nigris
The biological activity of the individual agents on myzus persicae is shown in tables 1-3.
Repellent activity of a single reagent on myzus persicae
Repellent activity of the individual agents against myzus persicae, as shown in table 1.
TABLE 1 repellent Activity of three reagents on myzus persicae
Note: the data in the table are mean ± sem; different lower case letters after the same column of data indicate significance of difference (one-way ANOVA, LSD test,P<0.05), the same as below.
As can be seen from table 1: taboo of three reagents to myzus persicaeThe activity of the compound is better. The highest repellent activity of the same reagent among different dilution times is diallyl disulfide at the concentration of 1:10 within 12h, and the difference is very obvious (F4,20=2.418, P<0.01) and the repellent rate of the pesticide on myzus persicae reaches 97.98 percent; within 24h and 48h, the repellent activity is still diallyl disulfide, the repellent rate is 98.60 percent and 100 percent respectively, and the difference reaches a significant level (F4,20=66.75, P<0.05；F4,20=777.39, P<0.01). Secondly, dibutyl phthalate is better for the aphid repellent activity, and 2, 6-di-tert-butyl-p-cresol is the lowest. In addition, under the treatment of different concentrations at the same time, the dibutyl phthalate and the diallyl disulfide both have stronger repellent activity to myzus persicae along with the reduction of the dilution times. The repellent effect is most pronounced at a concentration of 1: 10. Overall, the repellent activity of the three agents is in order from strong to weak: diallyl disulfide>Dibutyl phthalate>2, 6-di-tert-butyl-p-cresol.
Contact killing activity of single reagent on myzus persicae
The contact killing activity of the individual agents on myzus persicae is shown in table 2.
TABLE 2 contact killing Activity of three reagents on myzus persicae
As can be seen from table 2: all three reagents have stronger contact killing activity on myzus persicae. The same reagent has different dilution times, the higher contact activity is 2, 6-di-tert-butyl-p-cresol with the concentration of 1:10 in 12h and 24h, the corrected mortality rate respectively reaches 89.08 percent and 90.90 percent, and the difference is obvious (F 4,20=2095.54, P<0.01；F 4,20=3727.90, P<0.05); within 48 hours, the corrected death rate of the diallyl disulfide with better contact activity to myzus persicae reaches 100.00 percent, and the difference is very obvious (F 4,20=39.23, P<0.01). Therefore, under different concentration treatments at the same time, the mortality rate of the patients is gradually increased along with the reduction of the dilution times; are different from each otherThe longer the treatment time is, the higher the mortality rate is. Overall, the contact killing activity of the three reagents on myzus persicae is in the order of strong to weak: dibutyl phthalate>2, 6-di-tert-butyl-p-cresol>Diallyl disulfide.
Regression analysis of contact killing and repellent activity of three reagents with different concentrations on myzus persicae
Regression analysis of the contact killing and repellent activity of three reagents with different concentrations on myzus persicae is shown in table 3.
As can be seen from table 3: there is a significant correlation between the concentration of three different agents and their contact and repellent activities against myzus persicae ()P<0.05). Wherein, except that the repellent activity of the 2, 6-ditertbutyl-p-cresol to the myzus persicae is in negative correlation with the concentration of the reagent, the other repellent activity and the contact killing activity are respectively in positive correlation with the concentration of the reagent.
TABLE 3 regression analysis results of the contact killing and repellent activity of three reagents with different concentrations
Note: y1 and Y4 represent the repellent and contact activity equations of dibutyl phthalate respectively; y is2、Y5Respectively representing the repellent and contact activity equations of 2, 6-di-tert-butyl-p-cresol; y is3、Y6Respectively representing the repellent and contact activity equations for diallyl disulfide.
Biological activity determination of three reagent mixed pair myzus persicae
2.2.1 repellent Activity of three reagents mixed to myzus persicae
The repellent activity of three reagents mixed to myzus persicae is shown in table 4.
TABLE 4 repellent Activity of three reagents mixed on myzus persicae
As can be seen from table 4: the repellent activity of different reagents for mixing the two reagents to myzus persicae is stronger. The repellent activity to myzus persicae is within 12h, 24h and 48hThe repellent activities of the three reagents with the highest property on myzus persicae respectively reach 94.84%, 88.92% and 81.96% when the three reagents are used simultaneously, and the difference is very obvious (F 3,16=852.277, P<0.01；F 3，16=536.215, P<0.01；F 3，16=155.625, P<0.01). Among the two reagents mixed in pairs, the highest repellent rate to myzus persicae is dibutyl phthalate + diallyl disulfide, and the lowest repellent rate to myzus persicae is dibutyl phthalate + 2, 6-di-tert-butyl-p-cresol. The repellent activity of the three reagents on myzus persicae gradually decreases with time. In summary, the repellent activities of the different reagents in the compounding order from strong to weak are: dibutyl phthalate + diallyl disulfide + 2, 6-di-tert-butyl-p-cresol>Dibutyl phthalate + diallyl disulfide>Diallyl disulfide + 2, 6-di-tert-butyl-p-cresol>Dibutyl phthalate + 2, 6-di-tert-butyl-p-cresol.
Three reagents are mixed and matched for contact killing activity of myzus persicae
The three reagents were mixed for the contact killing activity of myzus persicae, as shown in table 5.
TABLE 5 contact killing Activity of three reagents in combination on myzus persicae
As can be seen from table 5: different reagents are mixed to have stronger contact killing activity on myzus persicae. Within 12h, 24h and 48h, the three reagents with the highest contact killing activity on myzus persicae are simultaneously mixed for use, the corrected mortality rates on the myzus persicae are 95.14%, 97.16% and 98.60%, and the difference is very obvious (the three reagents are used for killing the myzus persicae simultaneously)F 3，16=18.594, P<0.01；F 3，16=701.459, P<0.01；F 3，16=187.035, P<0.01). In the reagents mixed in pairs, the highest activity for killing the myzus persicae by contact is dibutyl phthalate + 2, 6-di-tert-butyl-p-cresol, and the lowest activity is dibutyl phthalate + diallyl disulfide. The contact killing activity of different reagents mixed to myzus persicae is gradually enhanced along with the increase of time. Contact killing activity for mixing different reagentsThe property is in the order from strong to weak: dibutyl phthalate + diallyl disulfide + 2, 6-di-tert-butyl-p-cresol>Dibutyl phthalate + 2, 6-di-tert-butyl-p-cresol>Diallyl disulfide + 2, 6-di-tert-butyl-p-cresol>Dibutyl phthalate + diallyl disulfide.
Influence of three reagents and mixing on activity of antitoxic enzyme of myzus persicae
The effects of the three reagents and compounding on the activity of the detoxification enzyme of myzus persicae are shown in table 6.
As can be seen from Table 6, the different reagents and their mixture pair, the aphid detoxification enzyme acetylcholinesterase: (F 6,13=4.837，P<0.01), a carboxylesterase (F 6,13=3.216，P<0.05) and glutathione S-transferase: (F 6,13=2.013，P<0.05) had a significant effect. Compared with a control, the activity of the acetylcholinesterase of the myzus persicae after different treatments is obviously improved. Wherein, the highest dibutyl phthalate has the enzyme activity of 1.066U/mg; secondly, dibutyl phthalate and dipropenyl disulfide are added, and the enzyme activity is 0.91U/mg; the lowest content is dibutyl phthalate + 2, 6 ditert-butyl-p-cresol + dipropenyl disulfide, and the enzyme activity is only 0.748U/mg. The 3 reagents and the combination thereof have induction effect on the myzus persicae acetylcholinesterase. Compared with a control, in the differently treated myzus persicae carboxylesterase, the enzyme activity of the combination of dibutyl phthalate, 2, 6 di-tert-butyl-p-cresol and dipropenyl disulfide is only 0.609U/mg, and the enzyme activity of the other six reagent combinations on myzus persicae carboxylesterase is higher than that of the control. Wherein, the highest carboxylesterase activity is dipropenyl disulfide, and the enzyme activity is 0.944U/mg; secondly, dibutyl phthalate and dipropylene disulfide are added, and the enzyme activity is 0.912U/mg; the lowest content is dibutyl phthalate + 2, 6 ditert-butyl-p-cresol, and the enzyme activity is only 0.677U/mg. The 3 reagents and the combination thereof have an induction effect on myzus persicae carboxylesterase. Compared with a control, the activity of the glutathione S-transferase of the myzus persicae after different treatments has four test combinations higher than that of the control, and has little difference compared with the control, the highest is dibutyl phthalate + dipropenyl disulfide, the enzyme activity is 0.410U/mg, and the second is dibutyl phthalate, the enzyme activity isThe force was 0.408U/mg; the minimum is dibutyl phthalate + 2, 6 di-tert-butyl-p-cresol + dipropenyl disulfide, and the enzyme activity is 0.345U/mg. The 3 reagents and the combination thereof have inhibition effect on myzus persicae glutathione S-transferase. In conclusion, the detoxification enzyme activities of different reagents and their mixed pairs of myzus persicae are shown as follows: the enzyme exhibits an inducing action on acetylcholinesterase and carboxylesterase of myzus persicae, and an inhibiting action on glutathione transferase.
TABLE 6 enzyme activities of three reagents and their blending against different detoxification enzymes of myzus persicae
3. Summary and discussion
The test results of this test show that: 3 reagents of dibutyl phthalate, diallyl disulfide and 2, 6-di-tert-butyl-p-cresol in the garlic root secretion have obvious repellent and contact killing activity on myzus persicae, and in addition to the repellent effect of the 2, 6-di-tert-butyl-p-cresol on the myzus persicae, the higher the concentration of the other two reagents is, the better the contact killing effect on the myzus persicae is. In the test concentration, the repellent and contact killing effects of different reagents on myzus persicae when two reagents and three reagents are mixed are better than the repellent and contact killing effects of one reagent when the reagents are used independently, and particularly the repellent and contact killing activities of the reagents on the myzus persicae are optimal when the three reagents are used simultaneously.
The repellent activity of three main reagents and the mixed reagents on myzus persicae is in the order from strong to weak: dibutyl phthalate + diallyl disulfide + 2, 6-di-tert-butyl-p-cresol > diallyl disulfide > dibutyl phthalate >2, 6-di-tert-butyl-p-cresol; the contact activity is from strong to weak: dibutyl phthalate + diallyl disulfide + 2, 6-di-tert-butyl-p-cresol > dibutyl phthalate + diallyl disulfide > diallyl disulfide + 2, 6-di-tert-butyl-p-cresol > dibutyl phthalate >2, 6-di-tert-butyl-p-cresol > diallyl disulfide. However, the mixing and use of the main components of the garlic root exudates are not reported at present, which provides a certain reference basis for developing plant-derived pesticides by using the main components of the garlic root exudates.
During the process of mutual evolution of insects and plants, certain self-regulation and reaction mechanisms are formed to resist adverse factors. Detoxification enzymes in the insect body play an important role in the physiological and biochemical balance metabolic reactions of the insect body, can participate in enzymatic reactions such as hydrolysis, oxidation and reduction, and can be discharged from the body to maintain the physiological balance in the body (Liu Chang, etc., 2019). When the three reagents are mixed, the activity of the antitoxic enzyme of the myzus persicae is as follows: acetylcholinesterase > carboxylesterase > glutathione S-transferase. The three reagents are mixed to obviously inhibit the activity of carboxylesterase, such as acetylcholinesterase, carboxylesterase and glutathione S-transferase, and are important metabolic enzymes for interpretation in the insect body. The method discovers that 4-year-old wingless myzus persicae is treated after three reagents, namely dibutyl phthalate, diallyl disulfide and 2, 6-di-tert-butyl-p-cresol, are mixed simultaneously, and that carboxylesterase activity in the myzus persicae is remarkably inhibited, so that the toxic substance detoxifying capability of the myzus persicae is reduced, and the myzus persicae is killed, but the three reagents have no inhibiting effect but promoting effect on the carboxylesterase activity when being used independently. Carboxylesterases can rapidly bind to toxic substances entering the insect body, blocking or degrading the toxic substances before reaching the target site of action. After the three reagents are mixed, 4-year-old wingless myzus persicae is treated, the activity of carboxylesterase is reduced, and the toxin degradation capability is reduced, so that the toxin can quickly reach a target action site to play a role. Glutathione S-transferase is one of the important conjugative enzyme systems in insect metabolism for pesticide or toxic substances, and can ensure that exogenous electrophilic groups and reduced glutathione in vivo generate conjugation metabolism to protect other nucleophilic centers in vivo. After the three reagents are mixed, the activity of the myzus persicae glutathione S-transferase is inhibited to a certain extent, and the degradation capability of the glutathione S-transferase to endogenous or exogenous toxins can be reduced. In conclusion, the activity inhibition of the three reagents on the myzus persicae carboxylesterase and the glutathione S-transferase after being mixed is related to the poisoning effect of the three reagents, the detoxifying metabolic capability of the insects is weakened, and the insects die finally. Carboxylesterase and glutathione S-transferase are important detoxification metabolic enzymes in insect bodies, and can be one of action targets of three reagents mixed for myzus persicae. The corresponding enzyme activity in the insect body can be activated or inhibited by foreign substances, so that the sensitivity of pests to the pesticide is changed, and therefore, the study on the aphid detoxication enzyme activity has important significance on the drug resistance of the aphid detoxication enzyme, and a certain reference value can be provided for the study on the pesticide.
The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.
Claims (6)
1. A tobacco aphid repellent and poison killing inhibitor is characterized in that the active ingredients of the inhibitor consist of dibutyl phthalate, diallyl disulfide and 2, 6-ditertbutyl-p-cresol.
2. The myzus persicae-avoiding and poisoning-inhibiting agent according to claim 1, wherein the volume ratio of dibutyl phthalate, 2, 6-di-tert-butyl-p-cresol, and diallyl disulfide is 1: 0.2-1: 1.
3. the myzus persicae-avoiding and poisoning-inhibiting agent according to claim 1, wherein the volume ratio of dibutyl phthalate, 2, 6-di-tert-butyl-p-cresol, and diallyl disulfide is 1: 1: 1.
4. the method for preparing a myzus persicae-avoiding and poisoning-inhibiting agent according to claim 1, wherein the active ingredients of dibutyl phthalate, diallyl disulfide and 2, 6-di-tert-butyl-p-cresol are dissolved in the ethanol solution of the solvent, and a proper amount of tween 80 is added, stirred and mixed uniformly.
5. The method according to claim 3, wherein the volume ratio of the ethanol solution as a solvent to the active ingredient is 10: 1.
6. the method according to claim 3, wherein the concentration of the ethanol solution as a solvent is 8 wt%.
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