CN101571532A - Novel method for measuring biological activity of biological pesticides - Google Patents
Novel method for measuring biological activity of biological pesticides Download PDFInfo
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- CN101571532A CN101571532A CNA2008100943700A CN200810094370A CN101571532A CN 101571532 A CN101571532 A CN 101571532A CN A2008100943700 A CNA2008100943700 A CN A2008100943700A CN 200810094370 A CN200810094370 A CN 200810094370A CN 101571532 A CN101571532 A CN 101571532A
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Abstract
The invention relates to a novel method for measuring the biological activity of biological pesticides, which belongs to a novel method for measuring the biological activity. The novel method is characterized in that an agaragar layer is taken as a short-time cultivation nutrient source and a moisturizing material of a plant excised part in the measurement of biological activity; and meanwhile, green peach aphids are adopted as biological detecting materials to detect the biological activity of a detecting sample, and the insecticidal activity measurement is carried out according to the average reproduction quantity and the average death rate of the green peach aphides and the relevance of biological pesticides to be detected. The method solves the problems that plant leaves are dried easily and dehydrated generally in the insect bioassay by utilizing excised plant leaves to further influence the bioassay result and improves the accuracy of the experimental result. The method has simple and convenient operation, short experimental period and low cost. The invention can be applied to the biological activity measurement of biological pesticides and other pesticides of current varieties and the screening researches of new pesticides and is especially suitable for the bioassay of biological pesticides to insects with sucking mouth parts, such as aphides, and the like.
Description
Technical field
The present invention relates to a kind of source of nutrition and material which can retain moisture of utilizing agar layer to cultivate as the stripped part short time of plant in the measuring biological activity of biological pesticides, utilize aphid and plant to exsomatize and partly carry out biological activity determination, especially be fit to biological pesticide bioactive mensuration of sucking mouth parts insect such as aphids.Belong to the pesticide bioassay field.
Background technology
The common pesticides activity determination method has two big classes in the R﹠D process of novel pesticide: chemical assay and bioassay method.Chemical assay is some special reactions according to chemicals and specimen generation, as: chromogenic reaction etc., by the activity of relatively coming the evaluation test sample of change in color and light absorption value size; And the method for biologicall test is to utilize biological living specimen activity under similar natural environmental condition.Bioassay method is the particularly the most general method of biological pesticide determination of activity of agricultural chemicals.
The condition determination that different biological activity determination methods requires has nothing in common with each other, and wherein environmental factor such as temperature, intensity of illumination and humidity etc. are the principal elements that influences measurement result.Intensity of illumination can be controlled by the adjusting artificial light source, and temperature also keeps stablizing easily, but utilizes plant to exsomatize when partly carrying out biological activity determination, the cultivation of stripped part short time of plant, the fresh-keeping and very difficult control of experiment humidity.In giving birth to the survey experiment, the general method that adopts filter paper or absorbent cotton to preserve moisture keeps humidity, but all there are some problems in these methods: at first the filter paper container that can not guarantee and test usefulness fits like a glove, less test insect is easy to be sandwiched in the slit of container and filter paper, causes unusual death; Space between the degreasing cotton-wool is easy to hinder the activity of insect: the nutrition of the stripped part short time cultivation of plant can not guarantee in addition, thus the result that influence is measured.Therefore being badly in need of a kind of biological activity determination new method solves these problems.
Summary of the invention
The purpose of this invention is to provide biological activity determination new methods such as a kind of biological pesticide, source of nutrition and material which can retain moisture that this method utilizes black peach aphid to cultivate as the stripped part short time of plant in the biological activity determination as examination worm, agar layer, solve the problem of bringing owing to method for moisturizing such as filter paper or absorbent cotton, improved the accuracy of biological activity test.This characteristic feature of an invention is to use growth, the breeding cycle is short and black peach aphid (Myzuspersicae) that easily raise as test material and the agar layer of using 1.6% (w/v) as the material of preserving moisture.
The present invention relates to a kind of is the biological activity determination method of tested object with black peach aphid (Myzus persicae).Black peach aphid belongs to Homoptera, Aphidiadae.Black peach aphid is a polyphagous pest-insect, and it is very wide to distribute in China, and its host plant has 285 kinds of 74 sections.Wherein the overwintering host plant mainly contains rose family fruit trees such as pears, peach, Lee, plum, cherry etc.; The host crop of living abroad mainly contains various crop such as Chinese cabbage, wild cabbage, radish, leaf mustard, rape, turnip, pimento, capsicum, spinach.Black peach aphid is the primary pest of pimento cultivation, is again the main communication media of virus.This invents discovery, and black peach aphid growth and breeding cycle weak point, breeding are easily raised soon, can be used as a kind of very desirable living test worm, is used for the determination of activity of the biological screening and the existing pesticide species of novel pesticide.
Method of the present invention may further comprise the steps:
1. the sterilization double dish that needs of preparing experiment.
2. the agar preparation of layer of preserving moisture: take by weighing an amount of (decide according to the needs of experiment) agar and place aqua sterilisa, make agar content at end reach 1.6% (w/v), heating and melting is poured in the double dish of sterilization, the about 8～10ml agar solution of each double dish.
3. the preparation of plant leaf blade: get a certain amount of sample solution to be tested, add a small amount of honey, because there is one deck wax coat on the plant leaf blade surface, (final concentration is about 1% to add a little honey, w/v) in measuring solution and contrast solution, make solution better adhere to the plant leaf blade surface.The solution for preparing is coated on the plant leaf blade of test equably, and dries up with hair-dryer.
4. the preparation of experimental insect: the used black peach aphid of an amount of (deciding) experiment of random choose according to the needs of experiment, put into an independent closed container, make hungry 12-24 hour of test aphid.
5. at random 1～2 blade of handling is placed on the agar layer in the double dish, 10 tests of picked at random black peach aphid places on the blade in addition, with sealing the membrane closure double dish, prevent that black peach aphid from climbing out of double dish, double dish is put into illumination box to be cultivated, condition of culture: daytime, 26 ℃ of illumination were 14 hours, and night, 18 ℃ of dark were 10 hours.After the black peach aphid activity basicly stable (about 3～4 hours), remove and seal film.
6. from experiment same day, observe the 3rd day and the 5th day respectively, the death toll of record black peach aphid, breeding number, and do statistical study, experimental result is with average mortality and on average breed number and represent.The computing method of average mortality are: (contrast or total dead borer population/contrast of handling or total examination worm radix of handling) * 100%; According to the mortality ratio of each repetition, analyze the correlativity of testing sample and black peach aphid mortality ratio with statistical analysis technique again; The computing method of average breeding number are: contrast or total breeding number/contrast of handling or the repunit of handling.According to the breeding number of each repetition, analyze the correlativity of testing sample and black peach aphid breeding number with statistical analysis technique again.
Embodiment
Further specified the present invention in order to illustrate better below by non-limiting example.
Example 1:
The effect that filter paper is preserved moisture and agar is preserved moisture gives birth in surveying at biological sample compares
Used biological sample (processing) is by a kind of protein of gene engineering method abduction delivering in bacterium in the example 1, is treated to the testing sample protein solution in this example, contrasts other protein of handling sample for not containing, 4 repetitions.Preserve moisture with filter paper one group, the control group mortality ratio is 22.5%, reaches 62.5% through the mortality ratio of bacterium liquid processed group, handles or the contrast difference repeats widely different; And preserve moisture with agar, zymocyte liquid processed group black peach aphid mortality ratio reaches 52.5%, and the mortality ratio of control group is 0% (table 1), it is little to handle or contrast different repetition differences, the result shows that the present invention uses agar and preserves moisture and can effectively reduce in the experiment owing to the unstable error of bringing of humidity, has guaranteed the accuracy of experimental result.
Two kinds of different method for moisturizing of table 1 are to the influence of black peach aphid mortality ratio
Annotate: contrast to not inducing the thalline crude extract; Be treated to the thalline crude extract of inducing 4h.
Example 2:
Measure the activity of biological pesticide to black peach aphid
2 pairs of a kind of bacterium amalgamation and expression protein and crude extracts thereof that can be used as biological pesticide of example have carried out the biologicall test of insecticidal activity.The biological activity test experimental design comprises 2 contrasts and 2 processing, 5 repetitions.Contrast 1 is a phosphate buffer; Contrast 2 is inducible strain extract not; Handling 1 is purifying protein enzyme inhibitor solution; Handle 2 for inducing 4h bacterial strain extract.Each processing or contrast amount to 50 black peach aphids.From experiment same day, the 3rd day and the 5th day observed and recorded contrast and handle mortality ratio, the breeding number that respectively repeats black peach aphid respectively, experimental result is with average mortality and on average breed number and represent.Experimental technique sees foregoing experimental technique of the present invention and step for details.Data to example 2 are carried out statistical study.Relevant experimental results sees Table 2.
Table 2 bacterium amalgamation and expression albumen is to the influence of black peach aphid breeding and mortality ratio
Annotate: contrast 1 is PBS (pH:7.4) phosphate buffer; Handling 1 is the inhibitor protein solution of purifying; Contrast 2 is not for inducing the thalline crude extract; Handling 2 is the thalline crude extract of inducing 4h.
Test findings shows: the black peach aphid mortality ratio of processed group all is significantly higher than the black peach aphid mortality ratio of control group; The black peach aphid of processed group on average breeds the breeding number (table 2) that number average significantly is lower than control group.The biological activity determination result shows that this bacterium amalgamation and expression protein has very strong inhibiting effect to growth and the breeding of black peach aphid, and this albumen also can significantly improve the mortality ratio of black peach aphid.Therefore, this bacterium amalgamation and expression albumen has a good application prospect on insect is administered.
The present invention gives birth to the survey method as a kind of new biological pesticide, has simple to operationly, experimental period is short, Cost is low, measures the accuracy advantages of higher. Compare with existing pesticide bioassay method, the present invention has as follows Characteristics:
1. the present invention uses the agar layer moisturizing, has solved the office that adopts the methods such as filter paper or absorbent cotton moisturizing Unstable and the problems such as test error brought of humidity in sex-limited and living survey, the effect of moisturizing is bright Show methods such as being better than the filter paper moisturizing, improved significantly the accuracy of biological activity determination;
2. compare with other biological examination material, the present invention is take black peach aphid as biological examination material, and the black peach aphid breeding amount is big, Breeding cycle is short and be easy to a large amount of group breedings, is easy to obtain, and therefore gives birth to the survey method with other It is low to compare the method cost;
Involved in the present invention to method only need culture dish, agar, a small amount of plant leaf blade, operation Easy;
Living survey method of the present invention can be after processing testing result in the short time, therefore make to give birth to and survey effect Rate obviously improves;
5. the present invention can embody the biology work that specimen shows under field conditions (factors) more preferably The property, especially be fit to biological pesticide to bioactive mensuration of sucking mouth parts insect such as aphids.
Claims (8)
1. measuring biological activity of biological pesticides method, it is characterized in that, this method utilizes agar layer to cultivate source of nutrition and material which can retain moisture as the stripped part short time of plant in the biological activity determination, utilize aphid and plant to exsomatize and partly carry out the biological activity determination of agricultural chemicals, especially be fit to biological pesticide bioactive mensuration of sucking mouth parts insect such as aphids.
2. method according to claim 1 is characterized in that, effectively utilizes the source of nutrition of agar layer as material which can retain moisture in the biological activity determination and the stripped part short time cultivation of plant.
3. method according to claim 1 is characterized in that, described specimen is the biological pesticide or the compound of existing desinsection kind of biological pesticide or unknown activity.
4. a black peach aphid (Myzus persicae) is used for the application of biological activity determinations such as biological pesticide.
5. certain density agar layer is used for the application of pesticide biological activity determination.
6. the plant part that exsomatizes is used for measuring biological activity of biological pesticides as blade etc.
7. one kind is utilized aphid and the application of the stripped part of plant in measuring biological activity of biological pesticides.
8. according to described method of claim 1-7 and application, this inventive features is: certain density agar layer is used for pesticide biological activity determination; Utilize black peach aphid and plant to exsomatize and partly carry out the biological activity determination of specimen; Described specimen is the compound sample of existing desinsection kinds such as biological pesticide or unknown pesticide bioactivity.
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| CNA2008100943700A CN101571532A (en) | 2008-04-29 | 2008-04-29 | Novel method for measuring biological activity of biological pesticides |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621278A (en) * | 2012-03-14 | 2012-08-01 | 山东省农业科学院植物保护研究所 | Method for quickly detecting sensibility of two-spotted spider mite to avermectin |
| CN103675255A (en) * | 2013-12-16 | 2014-03-26 | 中国农业大学 | Indoor biological assay method of aphides |
| CN104119550A (en) * | 2014-06-27 | 2014-10-29 | 中国农业大学 | High molecular polysaccharide film for biological assay of piercing-sucking mouthpart insect |
| CN104483447A (en) * | 2014-11-26 | 2015-04-01 | 河北省农林科学院植物保护研究所 | Method for screening nematicide by utilizing pluronic F127 and sweet potato stems |
| CN106754415A (en) * | 2016-12-28 | 2017-05-31 | 中国农业科学院植物保护研究所 | A kind of method for expanding numerous downy mildew of garpe opportunistic pathogen in vitro |
| CN113702626A (en) * | 2020-05-20 | 2021-11-26 | 中国农业大学 | Simple, convenient and efficient biological assay reagent and method |
-
2008
- 2008-04-29 CN CNA2008100943700A patent/CN101571532A/en active Pending
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102621278A (en) * | 2012-03-14 | 2012-08-01 | 山东省农业科学院植物保护研究所 | Method for quickly detecting sensibility of two-spotted spider mite to avermectin |
| CN102621278B (en) * | 2012-03-14 | 2014-12-17 | 山东省农业科学院植物保护研究所 | Method for quickly detecting sensibility of two-spotted spider mite to avermectin |
| CN103675255A (en) * | 2013-12-16 | 2014-03-26 | 中国农业大学 | Indoor biological assay method of aphides |
| CN104119550A (en) * | 2014-06-27 | 2014-10-29 | 中国农业大学 | High molecular polysaccharide film for biological assay of piercing-sucking mouthpart insect |
| CN104483447A (en) * | 2014-11-26 | 2015-04-01 | 河北省农林科学院植物保护研究所 | Method for screening nematicide by utilizing pluronic F127 and sweet potato stems |
| CN104483447B (en) * | 2014-11-26 | 2016-08-24 | 河北省农林科学院植物保护研究所 | A kind of method utilizing pluronic F127 and Sweet Potato screening nematicide |
| CN106754415A (en) * | 2016-12-28 | 2017-05-31 | 中国农业科学院植物保护研究所 | A kind of method for expanding numerous downy mildew of garpe opportunistic pathogen in vitro |
| CN113702626A (en) * | 2020-05-20 | 2021-11-26 | 中国农业大学 | Simple, convenient and efficient biological assay reagent and method |
| CN113702626B (en) * | 2020-05-20 | 2023-10-13 | 中国农业大学 | Simple and efficient biological assay reagent and method |
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Application publication date: 20091104 |