CN110376341B - Method for determining photolysis of tomato gray mold control drug - Google Patents
Method for determining photolysis of tomato gray mold control drug Download PDFInfo
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Abstract
The invention discloses a method for determining the photolysis of a tomato gray mold control drug, which comprises the steps of mixing a bactericide drug and an agar culture medium to prepare a culture plate, carrying out illumination treatment to cause the photolysis of the drug, wherein the photolysis of the drug causes the activity of a light treatment product to change, and the photolysis of the drug is displayed by testing the reaction of tomato gray mold bacteria on the light treatment product. The implementation method comprises the following steps: 1) preparing an agar culture medium; 2) preparing a drug flat plate; 3) detecting the activity of the medicine before light treatment; 4) carrying out light treatment; 5) detecting the activity of the medicine after the light treatment; 6) and obtaining a photolysis result. The invention has the advantages that: 1) the whole technology is simple, and high-precision instruments and equipment or complex serology technology operation is not needed; 2) can reflect the relationship between the photolysis of the medicine and the drug effect; 3) information can be obtained that non-photolytic effects result in a change in drug activity.
Description
Technical Field
The invention relates to a detection technology of the photolysis of a bactericide, in particular to a method for determining the photolysis of a tomato gray mold control drug.
Background
The tomato gray mold is an important disease of a large number of vegetables and tomatoes, the disease prevalence can bring great loss to tomato production, and the prevention and the control of the disease mainly depend on pesticide prevention and control at present. The control agents for controlling pesticides are usually naturally degraded once applied to the field, and photodecomposition by light irradiation is most prominent. Therefore, the photolysis is closely related to the prevention and treatment effect of the drug and the drug residue. Obviously, understanding the photolysis law of the drug is of great significance to fully play the prevention and treatment effects of the drug and reduce the drug residue.
The detection technology of photolysis of drugs currently mainly utilizes physicochemical and serological means. The physicochemical means is to utilize the chemical property and physical characteristic of the medicine to display and identify the medicine in modern equipment and instruments, and the means is quick and sensitive, but needs modern large-scale precise instruments. Serological means, which convert a drug into an antigen and thereby produce an antibody, and which utilize an antigen-antibody specific reaction to display and recognize the drug, is also sensitive but requires the production of specific antibodies. The prior technical means have a common characteristic that the detection process only tracks the original molecular structure of the drug and does not relate to the biological activity of the drug on a control object, and consequently, the related information that the activity of a drug degradation product on the control object is enhanced or weakened cannot be obtained, and the relationship between the drug degradation and the drug effect cannot be reflected.
Whether the drug is photolyzed or not can directly affect the action activity of the drug on a control object, and the action activity can be displayed by affecting the growth and development of the control object, however, a detection technology which utilizes a biological activity means of the drug as the photolysis of the drug has not been provided so far.
Disclosure of Invention
The invention aims to provide a method for measuring the photolysis of a tomato gray mold control drug, which utilizes the photolysis of the drug to cause the change of the drug effect of a photo-treated product.
The technical scheme for solving the technical problems is as follows:
a method for determining the photolysis effect of a tomato gray mold control drug is characterized in that a bactericide drug is mixed in an agar culture medium plate and is subjected to illumination treatment, the drug in the agar plate is subjected to photodecomposition under the illumination effect, the action activity of an optical treatment product on botrytis cinerea is changed, the photolysis effect of the drug is displayed by testing the reaction of the botrytis cinerea on the optical treatment product, and the method for determining the photolysis effect of the drug comprises the following steps:
1. preparing an agar culture medium: preparing agar-containing culture medium by conventional method, and sterilizing by conventional method.
2. Preparing a drug flat plate: under the aseptic condition, fully mixing the medicines with the culture medium prepared in the step 1 in a proper proportion, and pouring the mixture into a flat plate by using a common culture dish to obtain a medicine flat plate; media plates treated in the same manner but without drug served as control plates.
3. Detection of drug activity before light treatment: quantitatively transplanting botrytis cinerea prepared by conventional culture into the medicine flat plate and the control flat plate prepared in the step 2; culturing at 23 deg.C, measuring the growth amount of Botrytis cinerea on the plate, and calculating to obtain the pre-light treatment inhibition rate I of the medicine on Botrytis cinerea0。
4. And (3) light treatment: and (3) sealing the medicine flat plate obtained in the step (2) by using a common sealing film, then placing the sealed medicine flat plate under a set illumination condition for illumination treatment, and packaging the same medicine flat plate by using double-layer tin foil paper as a dark contrast CK for treatment.
5. And (3) detecting the activity of the medicine after the light treatment: 4, after the light treatment plate is subjected to light treatment for a set time t, switching back to the aseptic condition for dismounting and sealing, and then performing the drug activity detection after the light treatment according to the operation of the drug activity detection before the light treatment in the step 3; the inhibition rate It1 of the drug on the botrytis cinerea after light treatment and the inhibition rate It2 of the drug on the CK treatment of the dark control are calculated.
6. Obtaining a photolysis result: i obtained according to step 30And calculating to obtain a photolysis result of the medicament with the It1 and It2 obtained in the step 5.
The invention has the advantages that:
1. the whole technology is simple, and large-scale high-precision instruments and equipment or complex serology technical operation are not needed.
2. The detection process depends on the action activity of the medicine and the photolysis product on the prevention and treatment object, and can obtain the information that the activity of the photolysis product is enhanced and weakened, and can reflect the relationship between the medicine photolysis and the medicine action effect.
3. Information can be obtained that non-photolytic effects result in a change in drug activity.
Detailed Description
The present invention will be further described with reference to the following examples.
The invention relates to a method for measuring the photolysis effect of a tomato gray mold control drug. The bactericide drug is mixed in the agar culture medium for light treatment, the light treatment causes the drug in the agar culture medium to carry out photolysis, and the photolysis causes the change of the action activity of the drug on the botrytis cinerea, so the result of the photolysis can be obtained by detecting the biological action activity of the light treatment product on the botrytis cinerea.
The culture medium used by the invention is an agar culture medium which can normally grow by culturing botrytis cinerea conventionally, and is sterilized at high temperature conventionally for later use. Mixing the prepared culture medium and the drug to be tested in a proper proportion, wherein the proper proportion refers to the concentration dose of the drug which is at the concentration dose sensitive to the response of botrytis cinerea after the drug is mixed with the culture medium. The mixture of drug and medium was poured onto a plate to prepare a drug-containing plate, and the plate poured after mixing sterile water with the medium was used as a control plate.
After preparing the drug-containing plate, taking part of the drug-containing plate to perform drug activity detection before light treatment. Quantitatively implanting Botrytis cinerea prepared by conventional culture into a drug-containing plate and a control plate thereof, and culturing at a suitable growth temperature of 23 deg.C for a suitable time. When the amount of hypha growth is measured, the time required for the hypha of the colony to grow on the plate is appropriate, and is usually 3 days; when the germination rate of spores is measured, it is appropriate to take a time required for the spores to sufficiently germinate, usually 12 hours. And after the culture time is up, taking out the culture plate, measuring the growth amount of botrytis cinerea on the plate, and calculating the inhibition rate of the medicament on botrytis cinerea.
The drug inhibition rate (control plate growth amount-drug plate growth amount)/control plate growth amount.
The calculated drug inhibition rate is the drug activity I before light treatment0。
At the same time of testing the activity of the drug before starting the light treatment, the same drug-containing flat plate is sealed by a common sealing film and then is put under the set light condition for the light treatment. The same drug plate was sealed with double-layer tinfoil paper in the dark and placed under the same light conditions for the treatment of the dark control CK. The light treatment time t is set according to the work requirement.
And (4) after the illumination treatment for t time, transferring the flat plate back to the aseptic condition for unsealing, and detecting the activity of the medicament after the illumination treatment according to the medicament activity detection operation before the illumination treatment. The drug inhibition rate It1 of the light treatment after the detection result obtains the light t time; and drug inhibition rate It2 for the corresponding dark control CK treatment. In comparison, after the treatment for the time t, the inhibitory activity of the bactericide drug is changed, which is called activity attenuation in the invention, and the attenuation amount can be calculated as follows:
amount of drug activity attenuation It3 after light treatment: it3 ═ I0-It1；
Dark CK drug activity attenuation amount after light treatment It 4: it4 ═ I0-It2；
Dark CK drug activity attenuation amount It4 after light treatment, which is practically a non-photolytic attenuation amount of drug activity; since the amount of drug activity attenuation It3 after the photo-treatment is actually the sum of the photolytic effect and the non-photolytic effect, the amount of drug activity attenuation It5 due to the photolytic effect is:
It5＝It3-It4；
hair brushIt is clear that the non-photolysis results in the drug activity decrement It4 and the drug activity I before the light treatment0The ratio, referred to as the rate of decay of activity due to non-photolytic events, P1, is:
P1＝It4/I0；
the invention leads the drug activity attenuation amount It5 caused by photolysis and the drug activity I before light treatment0The ratio, called the photolysis-induced decay rate of activity P2, is:
P2＝It5/I0。
example 1
The method for determining the photolysis of the tomato gray mold control drug is applied, under the illumination condition in a common laboratory, the gray mold bacterial strain Bc-5 is used as a material to determine the photolysis of the bactericide iprodione, and the operation is implemented according to the following steps:
1. preparing an agar culture medium: potato Dextrose Agar (PDA) suitable for growth of botrytis cinerea is used as a test culture medium, and the test culture medium comprises the following components in parts by weight: 200g of potato, 20g of glucose, 20g of agar and 900mL of water, and sterilizing at high temperature conventionally for later use.
2. Preparing a drug flat plate: it is known that the hyphal growth of the tomato Botrytis cinerea strain Bc-5 inhibits the sensitivity of iprodione at a medium concentration EC50It was 1. mu.g/mL. Under the aseptic condition, mixing the iprodione liquid medicine with the PDA culture medium prepared in the step 1 fully to prepare a medicine-containing culture medium with the iprodione proportion of 1 mug/mL, pouring the medicine plate by using a common culture dish to obtain a medicine plate, and mixing sterile water with the culture medium prepared in the step 1 fully and pouring the medicine plate to obtain a control plate.
3. Detection of drug activity before light treatment: quantitatively transplanting a hypha cake of a tomato botrytis cinerea strain Bc-5 prepared by conventional culture on the drug flat plate and the control flat plate prepared in the step 2, culturing for 3 days at the temperature of 23 ℃, and taking out and measuring the colony diameter of the botrytis cinerea on the flat plate. Calculating to obtain the pre-light treatment inhibition rate I of the medicament to botrytis cinerea0。
4. And (3) light treatment: and (3) sealing the medicine flat plate obtained in the step (2) by using a common sealing film, then placing the sealed medicine flat plate on a bright desktop close to a window in a common laboratory, performing natural illumination treatment under the illumination condition of natural light intensity of 1000Lux at noon, and encapsulating the same medicine flat plate by using double-layer tinfoil paper and performing dark contrast CK treatment under the same illumination condition.
5. And (3) detecting the activity of the medicine after the light treatment: 4, the light treatment plate is respectively subjected to natural light treatment for 7 days, 14 days and 21 days, then is switched to be disassembled and sealed under an aseptic condition, and then the inhibition activity detection of the drugs on botrytis cinerea after the light treatment is carried out according to the operation of the drug activity detection before the light treatment in the step 3; the inhibition rate It1 of the light treatment drug and the inhibition rate It2 of the dark CK treatment drug are calculated.
6. Obtaining a photolysis result: i obtained by step 30And It1 and It2 obtained in step 5, and the amount of photolysis-induced drug activity decay It5, the rate of non-photolysis-induced activity decay P1, and the rate of photolysis-induced activity decay P2 were calculated after obtaining the amounts of photolysis for 7 days, 14 days, and 21 days after the light treatment, as shown in Table 1.
TABLE 1 Change in inhibitory Activity of iprodione before and after light treatment of example 1
Example 2
The method for determining the photolysis of the tomato gray mold control drug is used for determining the photolysis of the bactericide iprodione under the illumination condition in a greenhouse by using the gray mold bacterial strain Bc-5 as a material. The results of the photolysis of iprodione were measured as shown in Table 2, except that the light treatment conditions of step 4 were not the light conditions of a natural light intensity of 1000Lux at noon around the ordinary indoor window, but the light conditions of a natural light intensity of 11000Lux at noon in the greenhouse, and the operations of step 1 to step 6 of example 1 were carried out.
TABLE 2 Change in inhibitory Activity of iprodione before and after light treatment of example 2
Claims (1)
1. A method for determining the photolysis effect of a tomato gray mold control drug is characterized in that a bactericide drug is mixed in an agar medium plate and is subjected to illumination treatment, the drug in the agar plate is subjected to photodecomposition under the illumination effect, the action activity of an optical treatment product on the tomato gray mold pathogen is changed, the photolysis effect of the drug is displayed by testing the reaction of the tomato gray mold pathogen on the optical treatment product, and the method for determining the photolysis effect of the drug comprises the following steps:
1) preparing an agar culture medium: preparing a culture medium containing agar according to a conventional method, and conventionally sterilizing for later use;
2) preparing a drug flat plate: under the aseptic condition, fully mixing the medicine with the culture medium prepared in the step 1) in a proper proportion, pouring the mixture into a plate by using a common culture dish to obtain a medicine plate, and treating the culture medium plate which does not contain the medicine by the same operation as a control plate;
3) detection of drug activity before light treatment: quantitatively transplanting botrytis cinerea prepared by conventional culture into the medicine flat plate and the control flat plate prepared in the step 2); culturing at 23 deg.C, measuring the growth amount of Botrytis cinerea on the plate, and calculating to obtain the pre-light treatment inhibition rate I of the medicine on Botrytis cinerea0；
4) And (3) light treatment: sealing the medicine flat plate obtained in the step 2) by using a common sealing film, then placing the sealed medicine flat plate under a set illumination condition for illumination treatment, and packaging the same medicine flat plate by using double-layer tin foil paper for dark contrast CK treatment;
5) and (3) detecting the activity of the medicine after the light treatment: after the light treatment plate in the step 4) is subjected to light treatment for a set time t, the plate is switched back to an aseptic condition for removing the seal, and then the drug activity detection after the light treatment is carried out according to the operation of the drug activity detection before the light treatment in the step 3); calculating the inhibition rate It1 of the drug on the botrytis cinerea after light treatment and the inhibition rate It2 of the drug on the CK treatment of the dark control;
6) obtaining a photolysis result: i obtained according to step 3)0And It1 and It2 obtained in the step 5), calculating to obtain the photolysis result of the drug.
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| CN103798291B (en) * | 2014-02-18 | 2016-01-20 | 中国水稻研究所 | A kind of Recompounded pesticide containing triazolone and Cupravit |
| CN104904723A (en) * | 2014-03-11 | 2015-09-16 | 江苏龙灯化学有限公司 | Sterilization composition |
| CN204694630U (en) * | 2015-06-18 | 2015-10-07 | 淮阴工学院 | A kind of device of Portable ultraviolet light degrading pesticide residues |
| CN106508944A (en) * | 2016-08-31 | 2017-03-22 | 柳州市惠农化工有限公司 | Pesticidal composition containing 2-methylsulfonyl-5-(2,4-dichlorophenyl)-1,3,4-oxadiazole and prochloraz |
| CN107219205B (en) * | 2017-06-05 | 2019-08-06 | 河海大学 | A kind of pesticide degradation bacteria activity test method based on Fluorescent Staining Observation |
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