CN105137054B - A kind of method of wheat aphid indoor bioassay - Google Patents
A kind of method of wheat aphid indoor bioassay Download PDFInfo
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- CN105137054B CN105137054B CN201510435143.XA CN201510435143A CN105137054B CN 105137054 B CN105137054 B CN 105137054B CN 201510435143 A CN201510435143 A CN 201510435143A CN 105137054 B CN105137054 B CN 105137054B
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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- G01N33/5014—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing toxicity
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- G—PHYSICS
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Abstract
The present invention relates to plant protection art, a kind of method specifically providing wheat aphid indoor bioassay, use plastic dropper as biometric tool; and use Cotton Gossypii to seal; easy and simple to handle, operate the damage produced for examination aphid and error during saving space, and minimizing bioassay.In plastic dropper, additionally place wheat seedling, it is possible to the humidity of comparatively ideal holding wheat seedling, the accuracy of bioassay results is effectively ensured.The wheat aphid medicine embrane method indoor bioassay new method that the present invention provides, it is possible to understand wheat aphid field population and instruct field medication to the resistance level of different mechanism of action medicaments and distributionly thereof and then more scientific.
Description
Technical field
The present invention relates to plant protection art, specifically, a kind of method relating to wheat aphid indoor bioassay.
Background technology
Wheat aphid is one of important pests of global harm wheat crops, and in China, north and south Mai Qu all has generation, kind
Mainly include grain aphid, green bugs, rhopalosiphum padi etc..Wheat aphid can not only directly sting suction plant nutrient, and drain
A large amount of honeydews also have a strong impact on breathing and the photosynthesis of wheat seedling, are the most also the important media propagating plant virus, to agricultural
Produce and cause great economic loss.At present, wheat aphid relies primarily on chemical method preventing and treating, but owing to wheat aphid biocycle is short, numerous
The amount of growing is big and fast, and often, dosage is big and the unreasonable use of single dose, causes wheat aphid to create some conventional medicaments in preventing and treating
Drug resistance in various degree.
Pest resistance to insecticide monitoring is the important foundation that pest resistance is effectively administered.Bioassay is monitored as pest resistance to insecticide
Important technical, it can not only reflect insect creates resistance to which insecticide, and can accurately provide resistance journey
Degree, has important practice significance for adjusting Strategy of resistance management in time.
Drop method, taeniasis leaf dipping method, glass tubing medicine embrane method, blade medicine embrane method is mainly had about wheat aphid biometric techniques
Deng.Wherein drop method is since it is desired that accurate dropper, requires that operator have higher technology simultaneously, and therefore application is few.
Taeniasis leaf dipping method, the reason such as easily fall due to aphid and individuality is less causes for examination insect damage is big and matched group mortality rate is higher,
The raw accuracy surveying result of impact.Glass tubing medicine embrane method, although easy and simple to handle, but toxicity of tagging can only be measured, it is impossible to measure stomach
Poison resistance.Blade medicine embrane method is then because it is easy and simple to handle easy to implement, and is all suitable for contact stomach function regulating toxic agents, thus
Being used widely in the bioassay of aphid, little yet with wheat aphid individuality, host is mainly grass, and blade is easy
The reasons such as loss of moist cause the blade medicine embrane method used at present not ideal, and the culture dish of use as usual is as the leaf of container
Sheet medicine embrane method, its airtight space, aphid easily escapes, and the problem such as the big and humidity difficulty control of taking up room have impact on biometric
Determine the accuracy of result.
The Chinese patent application of Publication No. CN103918611A, discloses a kind of easy wheat aphid bioassay method,
Comprise the steps: that A water planting obtains the wheat seedling that root leaf is complete；B takes a branch of wheat seedling, and its root system is put into centrifuge tube, with moistening
Absorbent cotton parcel basal part of stem after fill in the sealed-off heart mouth of pipe, keep blade outside centrifuge tube, obtain the wheat seedling of band centrifuge tube；C is paving
There is the culture dish internal spraying moisturizing of filter paper, put into the wheat seedling of band centrifuge tube；D inoculates wheat on the blade of the wheat seedling of band centrifuge tube
Aphid, empirically design carries out processing and recording measurement result.But, this technical scheme takes band root and adds the side protecting absorbent cotton
Method, to play moisture-keeping function, but this technical scheme is to roll up wheat seedling to be positioned in culture dish, and wheat seedling cannot stretch, and is out
The environment put, in view of wheat aphid is the examination worm relatively enlivened, fugacity is strong, and bioassay results is also deposited by open environment
In certain impact.
Summary of the invention
In order to solve problems of the prior art, it is an object of the invention to provide a kind of wheat aphid indoor bioassay
Method.
In order to realize the object of the invention, technical scheme is as follows:
A kind of method of wheat aphid indoor bioassay, described method utilizes plastic dropper (plastics measuring pipette) to survey as raw
Vessel.
Further, described method comprises the steps:
1) plastic dropper being pricked out 3～6 apertures, bore dia is less than 2mm；
As preferably, bottom plastic dropper, prick hole；
2) wheat seedling is immersed insecticide 10 to be measured～20 seconds, takes out to put and dry at shady and cool ventilation, put in plastic dropper；
3) access aptery adult wheat aphid with the density of 7～8/mL, and will mould by breathability material (such as Cotton Gossypii, gauze etc.)
Gob channel closure；
4) plastic dropper is placed in thermostatic chamber, checks mortality rate after 48h, calculate toxicity test result.
Further, the capacity of described plastic dropper is more than or equal to 3mL.
As preferably, the capacity of described plastic dropper is 3mL.
In the detailed description of the invention of the present invention, the plastic dropper of use is purchased from the healthy China in the Jiangsu limited public affairs of medical supplies
The 3mL plastic dropper of department, trade name: suction pipe (sampling container).
Further, in plastic dropper, the quantity of wheat seedling is 2～3/mL.
Further, described wheat seedling selects to grow fine and be in the wheat seedling in young tender stage, preferably cultivates the wheat seedling of 10 days,
Cut from base portion.
Further, described wheat aphid is the wheat aphid that field Wheat Aphid Population obtained in indoor culture many generations.
Further, checking that the standard of mortality rate is: touch aphid, being designated as that only one leg is dynamic or the most motionless is dead
Die.
Further, calculating toxicity test result is: obtain LC by POLO computed in software50(Lethal
Concentration of 50%).
The beneficial effects of the present invention is:
The new method of the wheat aphid indoor bioassay that the present invention provides, for using plastic dropper as biometric tool
Wheat seedling medicine embrane method, and use Cotton Gossypii to seal, easy and simple to handle, operate during saving space, and minimizing bioassay for examination aphid
The damage of worm generation and error.In plastic dropper, additionally place wheat seedling, it is possible to the humidity of comparatively ideal holding wheat seedling, effectively protect
The accuracy of card bioassay results.The wheat aphid medicine embrane method indoor bioassay new method that the present invention provides, it is possible to understand wheat aphid
Field population instructs field medication to the resistance level of different mechanism of action medicaments and distributionly thereof and then more scientific.
The technical scheme that the present invention provides, had both maintained wheat seedling normal condition, in turn ensure that wheat seedling freshness, existed again simultaneously
The environment closed maintains breathability, and eliminates the impact on experimental result of the open external environment uncertain factor, meet
Biotic experiment keeps the principle of unitary variant.
Accompanying drawing explanation
The plastic dropper that Fig. 1 is used by bioassay of the present invention.
The plastic dropper of Cotton Gossypii sealing when Fig. 2 is bioassay of the present invention.
Fig. 3 is the bioassay schematic diagram of 12 porocyte culture plate blade medicine embrane methods.
The escape situation of wheat aphid when Fig. 4 is to use 12 porocyte culture plate blade medicine embrane method experiment.
The withered situation of experiment wheat seedling when Fig. 5 is to use 12 porocyte culture plate blade medicine embrane method experiment.
Detailed description of the invention
Following example are used for illustrating the present invention, but are not limited to the scope of the present invention.
The foundation to the sensitive Toxicity baseline of rhopalosiphum padi of the embodiment 1 flonicamid cyanogen
1, object of study
Laboratory rearing does not the most contact the rhopalosiphum padi population of any medicament.
2, method
Reagent agent is the former medicine of sulfoxaflor that concentration is more than 95%, uses plastic dropper blade medicine embrane method, carries out room
Interior toxicity test.
The preparation of 2.1 former medicine mother solutions and dilution
Former medicine being dissolved in acetone, deposits in ampoule after constant volume, sealed membrane seals, 4 DEG C of Refrigerator stores.Toxicity test
Before, with the distilled water diluting containing 0.01% (v/v) TritonX-100 to each concentration, standby.
The selection of 2.2 bioassay blades and use
Selecting the fresh wheat seedling that indoor water culture cultivates about 10 days, wheat seedling is cut from base portion with clean shears, and length is about
For about 10cm, each suction pipe prepares 8 wheat seedlings.Wheat seedling is immersed each medicament diluted concentration 15s, takes out at rearmounted shady and cool ventilation
After drying, being placed in the plastic dropper that 4 apertures pricked by bottom tip tweezers, wherein matched group wheat seedling is individually with 0.01%
The distilled water of TritonX-100 processes.
2.3 toxicity tests are for the selection of examination wheat aphid
Select the rhopalosiphum padi population not contacting any medicament of indoor feeding, the healthy consistent aptery one-tenth of picking
Aphid is tested, and places 20 aphids in each suction pipe, and Cotton Gossypii is sealed, under the normal rearing conditions in indoor (temperature 20～25 DEG C,
Relative humidity 50%～75%, illumination 16L:8D) raise.
2.4 result inspections and death standard judge
Checking result after 48h, touch rhopalosiphum padi with brush pen, the most only one leg moves or the most motionless being designated as
Extremely, to compare mortality rate less than 10% for effectively measuring, and it is corrected with comparison mortality rate.Each medicament 6～7 concentration,
Each concentration repeats for 3 times, and data process and use POLO to analyze software, calculate LC50.The flonicamid cyanogen virulence to rhopalosiphum padi
Carry out three secondary pollutants and repeat (result such as table 1).
The table 1 flonicamid cyanogen sensitive Toxicity baseline (48h) to rhopalosiphum padi
N represents the borer population used by toxicity test；* represent that rhopalosiphum padi sensitive measure by flonicamid cyanogen three is secondary
The meansigma methods that thing repeats.
From table 1 result and Fig. 1, Fig. 2 it can be seen that utilize the bioassay method of the present invention, the plastic dropper that used,
Wheat seedling (blade) medicine embrane method of Cotton Gossypii sealing, it is possible to effectively keep the humidity of wheat seedling for a long time, and take into account bioassay simultaneously
The key factor of breathability, plastic dropper has again joint space-efficient advantage, and the method has easy and simple to handle equally, repeatability
Good, the advantages such as result is accurate.
Embodiment 2
1, object of study
The wheat aphid field population in laboratory rearing many generations.
2, method
Reagent agent sulfoxaflor, uses 12 porocyte culture plate blade medicine embrane methods, carries out Toxicity Determination.
The preparation of 2.1 former medicine mother solutions and dilution
Former medicine being dissolved in acetone, deposits in ampoule after constant volume, sealed membrane seals, 4 DEG C of Refrigerator stores.Toxicity test
Before, with the distilled water diluting containing 0.01% (v/v) TritonX-100 to each concentration, standby.
The 2.2 raw selection surveying blade and uses
Selecting the fresh wheat seedling that indoor water culture cultivates about 10 days, wheat seedling is cut into about length 10cm with clean shears.
Wheat seedling is immersed each medicament diluted concentration 15s, takes out after drying at rearmounted shady and cool ventilation, be cut into about 2cm with clean shears, put
In 12 porocyte culture plates, be covered with bottom every hole 1ml 1% agar and the filter paper (such as Fig. 3) of a diameter of 2cm.
2.3 toxicity tests are for the selection of examination aphid
Selecting the wheat aphid field population of indoor feeding, picking is tested by healthy consistent aptery one-tenth aphid, and every hole places 20
Head aphid, rice paper bar seals, under indoor normal condition (temperature 20～25 DEG C, relative humidity 50%～75%, illumination 16L:
8D) raise.
2.4 result inspections and death standard judge
Checking result after 48h, touch aphid, foot is motionless to be designated as extremely, to compare mortality rate less than 10% for effectively measuring, and
It is corrected with comparison mortality rate.
Such as Fig. 4,5 and table 2 shown in, owing to the diameter of wheat seedling is far smaller than 12 porocyte culture plates, rhopalosiphum padi holds
The easy dehydration of wheat seedling that fugacity is on agar and in vitro, the mortality rate causing matched group rhopalosiphum padi is about 35%,
These explanation 12 porocyte culture plate blade medicine embrane methods are not suitable for wheat aphid.
The table 2 flonicamid cyanogen sensitivity (48h) to rhopalosiphum padi
Embodiment 2 shows, is currently used for the bioassay utensil of blade medicine embrane method, such as 12 porocyte culture plates etc., no
Being applicable to wheat aphid blade medicine embrane method bioassay, there is comparison mortality rate high, aphid escapes in the medium problem of agar, main cause
Also it is because utilizing other bioassay utensil and method cannot keep the humidity of wheat seedling and the ventilative of bioassay space simultaneously
Property, therefore use traditional medicine embrane method utilizing the vessel such as Tissue Culture Plate not to be suitable for wheat aphid, due to comparison mortality rate and place
Reason mortality rate is the highest, it is impossible to accurately judge experimental result.
Although, the present invention is described in detail the most with a general description of the specific embodiments, but
On the basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Cause
This, these modifications or improvements without departing from theon the basis of the spirit of the present invention, belong to the scope of protection of present invention.
Claims (8)
1. the method for a wheat aphid indoor bioassay, it is characterised in that described method utilizes plastic dropper as bioassay
Vessel；
Described method comprises the steps:
1) plastic dropper being pricked out 3～6 apertures, bore dia is less than 2mm；
2) wheat seedling is immersed insecticide 10 to be measured～20 seconds, takes out to put and dry at shady and cool ventilation, put in plastic dropper；
3) access aptery adult wheat aphid with the density of 7～8/mL, and by breathability material, plastic dropper is sealed；
4) plastic dropper is placed in thermostatic chamber, checks mortality rate after 48h, calculate toxicity test result.
Method the most according to claim 1, it is characterised in that the capacity of described plastic dropper is more than or equal to 3mL.
Method the most according to claim 2, it is characterised in that the capacity of described plastic dropper is 3mL.
Method the most according to claim 1, it is characterised in that in plastic dropper, the quantity of wheat seedling is 2～3/mL.
5. according to the method described in claim 1 or 4, it is characterised in that described wheat seedling selects grow fine and be in young tender rank
The wheat seedling of section, cuts from base portion.
Method the most according to claim 1, it is characterised in that described wheat aphid is that field Wheat Aphid Population is in indoor culture many generations
The wheat aphid obtained.
Method the most according to claim 1, it is characterised in that the standard of inspection mortality rate is: touch aphid, only one
What lower limb was dynamic or the most motionless is designated as death.
Method the most according to claim 1, it is characterised in that calculating toxicity test result is: obtain by POLO computed in software
Obtain LC50。
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| CN105137054B true CN105137054B (en) | 2016-09-21 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108902052B (en) * | 2018-06-29 | 2021-04-30 | 中国农业大学 | Indoor biological assay method for cotton aphids |
| CN114451362A (en) * | 2022-03-01 | 2022-05-10 | 中国农业大学 | Insect bioassay device, method and application |
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| CN101315360B (en) * | 2008-07-24 | 2013-04-17 | 中国农业科学院蔬菜花卉研究所 | Bioassay method for pest |
| CN103675255A (en) * | 2013-12-16 | 2014-03-26 | 中国农业大学 | Indoor biological assay method of aphides |
| CN103918611B (en) * | 2014-03-26 | 2016-08-24 | 中国农业科学院植物保护研究所 | A kind of easy wheat aphid bioassay method |
| CN104062413A (en) * | 2014-07-21 | 2014-09-24 | 中国农业科学院蔬菜花卉研究所 | Biological assay method for tetranychids and application of method |
| CN104770340B (en) * | 2015-03-27 | 2017-07-11 | 华南农业大学 | A kind of method that utilization easy device determines citrus fruit fly stomach toxicity virulence |
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