CN104483447B - A kind of method utilizing pluronic F127 and Sweet Potato screening nematicide - Google Patents
A kind of method utilizing pluronic F127 and Sweet Potato screening nematicide Download PDFInfo
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- CN104483447B CN104483447B CN201410697113.1A CN201410697113A CN104483447B CN 104483447 B CN104483447 B CN 104483447B CN 201410697113 A CN201410697113 A CN 201410697113A CN 104483447 B CN104483447 B CN 104483447B
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- nematicide
- sweet potato
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Abstract
The invention discloses a kind of method utilizing pluronic F127 and Sweet Potato screening nematicide, belong to pesticide screening test method field.The present invention with mass percent be 20～40% pluronic F127 solution as substrate, medicament to be measured and nematicide liquid are joined in substrate, at room temperature place 48～72h, it is inserted into Sweet Potato, cultivates 3～15d, take out Sweet Potato, nematodal accounting to intrusion Sweet Potato, calculates preventive effect.Compared with infusion process, the inventive method can the overall merit medicament to be measured lethal effect to nematicide, and the inhibitory action to the mobility of nematicide with infiltration capability, screening accuracy is high, highly reliable；Secondly, Sweet Potato and Ditylenchus destructor used by the inventive method are prone at indoor expanding propagation, and material is easy to get, suitable a large amount of Chemicals；Additionally, the inventive method is with plant nematode for test object, compared with conventional Caenorthaditis elegans ws123, its testing result is more accurately, reliably.
Description
Technical field
The invention belongs to pesticide screening test method field, be specifically related to one and utilize pluronic F127 and Rhizoma Dioscoreae esculentae
The method of stem screening nematicide.
Background technology
Plant nematode is one of main pathogen thing of agricultural production, produces the warp caused every year to world agriculture
Ji loss about 100,000,000,000 dollars.The prevention and controls of Plant nematode includes disease-resistant variety, crop rotation, water logging, exposure
Solarization, Biological control etc. (.Plant Nematology, the CABI Publishing.2013. such as Roland N.P.),
But chemical prevention is still that prevention and controls quick, cost-effective, is deeply favored by peasant.
Traditional nematicide screening is how right with Caenorthaditis elegans ws123 (Caenorhabditis elegans) for test
As, evaluate reagent agent to the virulence of this nematicide (the .Journal of Pesticide Science such as Ohba K,
1984,9:91-96).The nematicide of commercialization at present all carries out primary dcreening operation acquisition with this filtering mode,
Further with plant nematode for test object on the basis of primary dcreening operation, respectively with infusion process, pot-culture method
And field test etc. carries out multiple sieve and checking.
Nematicide has multiple (the .Pesticide Biochemistry and such as Kearn J to the model of action of nematicide
Physiology.2014), but owing to the resistance of nematicide is relatively strong, how insensitive to medicament, generally
Nematicide is poor to the lethal effect of nematicide, and nematicide is mainly by suppression egg hatching, suppression nematicide
Migrate, control nematicide intrusion, suppress the compound actions such as elegans development to reach to control purpose (Zou of nematicide
Refined new etc. Jouranl of Agricultural University of Hebei, 2011,34 (5): 78-81).Infusion process is screening nematicide
Main method, the Ye Shi China test bioactive standard method of nematicide (NY/T 1833.1-2009).
But infusion process is only capable of the test medicament lethal effect to nematicide, and cannot evaluate medicament and press down line eggs
Making and use and to nematicide mobility, the effect of infiltration capability, therefore infusion process can cause nematicide
The leakage sieve of active material.
(English name: Pluronic F127, chemical formula is pluronic F127: (C3H6O.C2H4O) x,
Have another name called blocked polyethers, polyethers, expoxy propane and ethylene oxide copolymer, polyoxyethylene polyoxypropylene,
Polyethylene Glycol grafting polypropylene glycol, poloxamer, polyoxyethylene polyoxypropylene copolymer, poloxamer188
Deng) be all widely used in medical science, pharmacy and cosmetic field.The pluronic F127 of 20%～25%
It is in a liquid state at low ambient temperatures, becomes semisolid at normal temperatures.Owing to this substrate is transparent, nematicide can be at this base
In matter freely movable, be suitable for Nematode behaviour research (the .Journal of Nematology such as Ko M.P.,
1988,20:478-485).Ohba K etc. thinks that pluronic F127 is suitably to substitute agar to observe nematicide
The substrate (the .Journal of Pesticide Science, 1984,9:91-96 such as Ohba K) of behavior；Congli etc.
Utilize the plants such as this substrate research Fructus Lycopersici esculenti, arabidopsis to the attraction of root-knot nematode activity (Congli W etc.,
Nematology,2009,Vol.11(3),453-464；The .Journal of Nematology such as Ko M.P., 1988,
20:478-485)。
Through retrieval, do not find the research report utilizing pluronic F127 to carry out nematicide screening.
Summary of the invention
The problem Lou sieved, mesh of the present invention is easily caused present in existing screening nematicide method
Be provide a kind of utilize pluronic F127 and Sweet Potato screening nematicide method.
For achieving the above object, technical scheme is as follows:
A kind of method utilizing pluronic F127 and Sweet Potato screening nematicide, comprises the steps:
(1), according to mass percent be 20～40% ratio pluronic F127 is put into distilled water
In, it is placed under 0～6 DEG C of low temperature and overnight dissolves, obtain pluronic F127 mother solution；
(2), with distilled water, Ditylenchus destructor (Ditylenchus destructor thorne) is joined
Make the liquid of 10～1000/mL, obtain nematicide liquid；
(3), according to the polarity acetone of medicament to be measured or water by medicament dissolution to be measured, then dilute with water
For the liquid medicine to be measured that mass volume ratio is 1～20g/L；
(4), with the Tween 80 that mass percent is 0.1% liquid medicine to be measured to step (3) gained
Carry out gradient dilution, then join step (1) gained together with the nematicide liquid of step (2) gained
In pluronic F127 mother solution, make final concentration of 18～30% (mass percent) of pluronic F127,
The content making nematicide liquid is 5～100/mL；Slowly vibrate mixing, and pour in 9cm culture dish, room
Temperature is lower places 48～72h；
(5), in the Sweet Potato section that culture dish central authorities intubating length is 0.5～2.0cm, under room temperature cultivate 3～
15d, then takes out Sweet Potato section, uses tray method to separate nematicide therein, counting；With without medicine
Agent is processed as comparison；Calculating the prevention effect of medicament to be measured, the prevention effect at medicament to be measured is more than 20%
Time, i.e. illustrate that medicament to be measured is active to nematicide under this test concentrations；
Prevention effect (%)=(control treatment invades nematode population-chemicals treatment and invades nematode population)
/ control treatment invades nematode population × 100%.
Low temperature described in said method step (1) is preferably 0～4 DEG C.
Medicament to be measured described in said method step (3) refers to chemical agent or biological metabolic product etc..
The mass percent of the pluronic F127 described in said method step (4) is preferably 23%.
Room temperature described in said method step (4) or (5) is 20～30 DEG C.
Standing time described in said method step (4) is preferably 48h.
Sweet Potato segment length described in said method step (5) is preferably 1.0cm.
Incubation time described in said method step (5) is preferably 5d.
Compared with prior art, the present invention has the advantage that and beneficial effect: used by (1) the inventive method
Substrate pluronic F127 have be in a liquid state at low temperatures, characteristic in solid-state under room temperature, can simulate
Soil environment carries out the screening of nematicide, can in the pluronic F127 be placed with host effectively by nematicide
Complete to migrate, invade, the process such as growth；Compared with traditional infusion process, the inventive method can be comprehensive
Evaluate the medicament to be measured lethal effect to nematicide, and the suppression to the mobility of nematicide with infiltration capability
Effect, screening accuracy is high, highly reliable.(2) Sweet Potato used by the inventive method and Rhizoma Solani tuber osi
Rot stem nematodes is prone at indoor expanding propagation, and material is easy to get, suitable a large amount of Chemicals.(3) side of the present invention
Method is with plant nematode Ditylenchus destructor for test object, owing to plant nematode is with saprophytic
Nematicide has larger difference on feeding habits and life cycle, therefore, and with traditional Caenorthaditis elegans ws123
(Caenorhabditis elegans) compares for test object, and its result is more accurately, reliably.
Detailed description of the invention
Embodiment 1 utilizes pluronic F127 and Sweet Potato screening nematicide contrast test
(1), EXPERIMENTAL DESIGN: select conventional nematicide 80% Aldicarb (Shandong Huayang pesticide chemical collection
Group company limited), 95% avilamycin (Hebei Veyong Biochemical Farm Chemical Co., Ltd.), 90% carbosulfan
(Jiangsu Jialong Chemical Co., Ltd.) is for supplying examination chemical agent, using the process without medicament as comparison.
(2), test method:
(1), 35g pluronic F127 is put in 100ml distilled water, be placed under 4 DEG C of low temperature overnight
Dissolve, obtain pluronic F127 mother solution.
(2), with distilled water, Ditylenchus destructor (Ditylenchus destructor) is configured to 100
The liquid of head/mL, obtains nematicide liquid.
(3), 80% Aldicarb, 95% avilamycin, 90% 3 kinds of medicaments of the former medicine of carbosulfan are divided
Not Yong acetone solution, be then diluted with water to the mother solution that w/v is 10g/L.
(4), with the Tween 80 that mass percent is 0.1% respectively by the Aldicarb of step (3) gained,
Avilamycin, carbosulfan mother solution carry out gradient dilution, then with the nematicide liquid one of step (2) gained
Rise in the pluronic F127 mother solution being added separately to step (1) gained, make the end of pluronic F127
Concentration is for for 23% (mass percent), and making nematode levels is 50/mL, makes Aldicarb, Avermectin
Element, the concentration of carbosulfan are respectively 320,160,80,40,20 and 10ug/ml；With without
The process of medicament is as comparison；Slowly vibrate mixing, and pour 20ml, room temperature into each 9cm culture dish
Lower placement 48h；
(5), in the Sweet Potato section that culture dish central authorities intubating length is 1.0cm, test kind is Ji potato 98,
72h is placed in 25 DEG C of dark culturing casees；Then Sweet Potato section is taken out, use tray method to separate therein
Nematicide, counting, calculate different agents process according to chemicals treatment and the infection thread borer population amount of control treatment right
The prevention effect that nematicide invades；The computing formula of prevention effect is:
Prevention effect (%)=(control treatment invades nematode population-chemicals treatment and invades nematode population)/
Control treatment nematicide intrusion volume × 100%
Infusion process: by Aldicarb, avilamycin, 3 kinds of medicament acetone solutions of carbosulfan, then
It is diluted with water to the mother solution that percentage by weight is 10g/L；It is the Tween 80 of 0.1% by mass percent again
Successively each medicament is diluted to 320,160,80,40,20 and 10ug/ml, with the place without medicament
Reason, as control treatment, joins in 9cm culture dish, and adds the mixing of nematicide liquid so that nematode population
It is 50/mL.Place 5d in 25 DEG C of dark constant incubators, check the nematode population of different disposal, and
According to the mortality rate of chemicals treatment with control treatment, calculate prevention effect.
Result (being shown in Table 1) uses Ji potato 98 (susceptible variety) to be nematicide host, at all drug concentrations
The preventive effect that in process, the inventive method obtains is above infusion process；Additionally, two kinds of methods all show Avermectin
Element is the highest to the virulence of nematicide, and under same concentrations treatment conditions, its preventive effect is significantly higher than Aldicarb and fourth sulfur
Carbofuran.The above results illustrates that the inventive method gained the selection result is sensitiveer, more accurately than infusion process institute.
The different method of testing result of the test to different agents concentration preventive effect of table 1
Embodiment 2 present invention utilizes pluronic F127 and Sweet Potato screening nematicide contrast test
(1), for reagent product: the conventional nematicide Aldicarb of selection, avilamycin, carbosulfan are
Reagent agent, the relatively impact on evaluating drug effect of the different detection times.
(2), concrete operation step with embodiment 1, respectively medicament contact with nematicide 5d, 6d, 7d,
Pharmacy control efficacy is checked after 8d.Insert the Sweet Potato section of Ji potato 98 (susceptible variety) the most in the methods of the invention
Check after 3d, 4d, 5d, 6d, check after infusion process processes 5d, 6d, 7d, 8d, above-mentioned for examination
Medicament Aldicarb, avilamycin, the test concentrations of carbosulfan are 200ug/mL.With without medicine
The process of agent is as comparison.
Result (being shown in Table 2) is in all testing times, and the preventive effect that the inventive method obtains is above infusion process
The preventive effect obtained；Additionally, two kinds of methods all show that avilamycin is the highest to the virulence of nematicide, identical dense
Under degree treatment conditions, its preventive effect is significantly higher than Aldicarb and carbosulfan processes.It addition, at all medicaments
In process, along with the prolongation of the time of process, each medicament is the most in rising trend to the preventive effect of nematicide.Above-mentioned knot
Fruit explanation the inventive method is more accurate, reliable to the Screening and Identification result of each pharmacy control efficacy.
The different testing time impact on different agents concentration preventive effect of table 2
Embodiment 3 utilizes the Sweet Potato screening nematicide of pluronic F127 and different cultivars to having a competition
Test
(1) EXPERIMENTAL DESIGN: select conventional nematode killing agent Aldicarb, avilamycin, carbosulfan for supplying
Reagent agent, using the process without medicament as control treatment.Relatively select different sweet potato variety to drug effect
The impact evaluated.
(2) test method: concrete operation step, with embodiment 1, selects Ji potato 98, Ji potato 4 respectively
Number, the Sweet Potato of Xushen21 well, slowly potato 25 as host, within the 5th day, check result；Above-mentioned reagent agent
Aldicarb, avilamycin, the test concentrations of carbosulfan are 150ug/mL, with without medicament
Process as comparison.
Result (being shown in Table 3) selects different sweet potato varieties identical for the evaluation result trend of drug effect, adopts
The nematicide preventive effect obtained by the inventive method is higher than the preventive effect using traditional infusion process to obtain, but selects anti-
Property stronger kind such as Ji potato 4 and the preventive effect that obtains of Xu's potato 25 less than select susceptible variety Ji potato 98,
The preventive effect that Xushen21 well obtains.Illustrate that the inventive method should use more sensitive sweet of potato rot nematode
Potato kind is as test material.
The contrast test that different agents concentration preventive effect is affected by the different sweet potato variety of table 3
Claims (6)
1. the method utilizing pluronic F127 and Sweet Potato screening nematicide, it is characterised in that
Comprise the steps:
(1), according to mass percent be 20～40% ratio pluronic F127 is put into distilled water
In, it is placed under low temperature and overnight dissolves, obtain pluronic F127 mother solution；Wherein said low temperature is 0～4 DEG C；
(2), with distilled water, Ditylenchus destructor (Ditylenchus destructor thorne) is joined
Make the liquid of 10～1000/mL, obtain nematicide liquid；
(3), according to the polarity acetone of medicament to be measured or water by medicament dissolution to be measured, then dilute with water
For the liquid medicine to be measured that mass volume ratio is 1～20g/L；
(4), with the Tween 80 that mass percent is 0.1% liquid medicine to be measured to step (3) gained
Carry out gradient dilution, then join step (1) gained together with the nematicide liquid of step (2) gained
In pluronic F127 mother solution, the mass percent making the final concentration of pluronic F127 is 18～30%,
The content making nematicide liquid is 5～100/mL；Slowly vibrate mixing, and pour in 9cm culture dish,
48～72h are placed at 20～30 DEG C；
(5), in the Sweet Potato section that culture dish central authorities intubating length is 0.5～2.0cm, at 20～30 DEG C
Cultivate 3～15d, then Sweet Potato section is taken out, use tray method to separate nematicide therein, counting；With
It is comparison without chemicals treatment；Calculate the prevention effect of medicament to be measured, in the prevention effect of medicament to be measured
During more than 20%, i.e. illustrate that medicament to be measured is active to nematicide under this test concentrations；
Prevention effect (%)=(control treatment invades nematode population-chemicals treatment and invades nematode population)
/ control treatment invades nematode population × 100%.
The most in accordance with the method for claim 1, it is characterised in that to be measured described in its step (3)
Medicament refers to chemical agent or biological metabolic product.
The most in accordance with the method for claim 1, it is characterised in that the Pu Lang described in its step (4)
The mass percent of Buddhist nun gram F127 is 23%.
The most in accordance with the method for claim 1, it is characterised in that the placement described in its step (4)
Time is 48h.
The most in accordance with the method for claim 1, it is characterised in that the Rhizoma Dioscoreae esculentae described in its step (5)
Stem segment length is 1.0cm.
The most in accordance with the method for claim 1, it is characterised in that the cultivation described in its step (5)
Time is 5d.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000511204A (en) * | 1997-03-12 | 2000-08-29 | ソシエテ・チユニジエンヌ・ダングレ・シミツク・エス・テー・ウー・セー | Bio-nematicide with effective ovicidal action against plant parasitic nematodes |
| CN101571532A (en) * | 2008-04-29 | 2009-11-04 | 中国农业科学院植物保护研究所 | Novel method for measuring biological activity of biological pesticides |
| CN102972355A (en) * | 2012-12-05 | 2013-03-20 | 华南农业大学 | Method for breeding, culturing and storing plant nematodes by using sweet potato calluses |
| CN103645191A (en) * | 2013-12-05 | 2014-03-19 | 中国科学院东北地理与农业生态研究所 | Method for realizing rapid detection of phenomenon of identifying injurious insect host plant roots by entomopathogenic nematodes |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20090082453A1 (en) * | 2007-09-25 | 2009-03-26 | New Biology, Inc. | Exogenous Methyl Dihydrojasmonate for Prevention and Control of Biotic Attack in Plants |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2000511204A (en) * | 1997-03-12 | 2000-08-29 | ソシエテ・チユニジエンヌ・ダングレ・シミツク・エス・テー・ウー・セー | Bio-nematicide with effective ovicidal action against plant parasitic nematodes |
| CN101571532A (en) * | 2008-04-29 | 2009-11-04 | 中国农业科学院植物保护研究所 | Novel method for measuring biological activity of biological pesticides |
| CN102972355A (en) * | 2012-12-05 | 2013-03-20 | 华南农业大学 | Method for breeding, culturing and storing plant nematodes by using sweet potato calluses |
| CN103645191A (en) * | 2013-12-05 | 2014-03-19 | 中国科学院东北地理与农业生态研究所 | Method for realizing rapid detection of phenomenon of identifying injurious insect host plant roots by entomopathogenic nematodes |
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