CN107410218A - A kind of rearing method of viral vector insect Bemisia tabaci - Google Patents
A kind of rearing method of viral vector insect Bemisia tabaci Download PDFInfo
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- CN107410218A CN107410218A CN201710356868.9A CN201710356868A CN107410218A CN 107410218 A CN107410218 A CN 107410218A CN 201710356868 A CN201710356868 A CN 201710356868A CN 107410218 A CN107410218 A CN 107410218A
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- 230000002045 lasting effect Effects 0.000 abstract description 3
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Classifications
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
- A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
- A01K67/033—Rearing or breeding invertebrates; New breeds of invertebrates
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G24/00—Growth substrates; Culture media; Apparatus or methods therefor
-
- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01G—HORTICULTURE; CULTIVATION OF VEGETABLES, FLOWERS, RICE, FRUIT, VINES, HOPS OR SEAWEED; FORESTRY; WATERING
- A01G31/00—Soilless cultivation, e.g. hydroponics
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8203—Virus mediated transformation
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- General Health & Medical Sciences (AREA)
- Cell Biology (AREA)
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Abstract
The invention discloses a kind of rearing method of viral vector insect Bemisia tabaci, comprise the following steps：To grow the vegetables of true leaf as host plant；Infectious clone bacterium solution is inoculated into host plant；Continue to cultivate, filter out susceptible plant and nontoxic plant；The disease plant that B Bemisia tabacis and Q Bemisia tabacis are inoculated into respectively the disease plant for being inoculated with B Bemisia tabacis is obtained on susceptible plant and non-toxic herb, is inoculated with Q Bemisia tabacis, it is inoculated with the nontoxic plant of B Bemisia tabacis, is inoculated with the nontoxic plant of Q Bemisia tabacis；Individually raising, is bred Bemisia tabaci；After raising for 6 generations, Bemisia tabaci insect-taking is carried out.The rearing method of the present invention can shorten the development time of insect, improve egg laying amount, ensure survival rate and stability, ensure lasting, the stable supply of Bemisia tabaci.
Description
Technical field
The present invention relates to technical field of insect feeding, more particularly to a kind of artificial feeding side of viral vector insect Bemisia tabaci
Method.
Background technology
Bemisia tabaci Bemisia tabaci, very serious loss is brought to vegetables and cereal crops etc., and China is main
There are two kinds of Bemisia tabacis of B and Q.Bemisia tabaci can propagate various plants virus, and harm caused by its transmitted virus significantly larger than itself takes
Harm caused by food.Want to control transmission via whitefly virus, first have to study the mechanism of transmission via whitefly virus, therefore just need
The Bemisia tabaci that the carrying that can continue to supply is viral and population is stable is established, and has the tobacco powder without poison raised under the same terms
Lice is as control.Meanwhile in order to eliminate the influence of Field Plants virus Combined Infection, it is necessary to the infectious clone of virus to planting
Thing is inoculated with, and ensures the unicity in malicious source.Furthermore, it is necessary to the host plant of Bemisia tabaci and virus is screened, can
Still maintained vigour under virus infection and the dual-pressure of Bemisia tabaci feeding.Due to being related to host plant, virus and Bemisia tabaci three
Individual key factor, cause the current technology slower development, as long as there is a link error, test period will be extended, hinder examination
Test process.
The content of the invention
The technical problem to be solved in the present invention is overcome the deficiencies in the prior art, there is provided one kind can obtain not of the same race simultaneously
The rearing method for taking viruliferous Bemisia tabaci and the Bemisia tabaci without poison of group, have the development time of insect short, produce
Ovum amount is high, and survival rate is high, ensures the advantages such as lasting, the stable supply of material to be tested.
In order to realize above-mentioned technique effect, the invention provides a kind of artificial feeding side of viral vector insect Bemisia tabaci
Method, comprise the following steps：
S1, to grow the vegetables of true leaf as host plant；Infectious clone bacterium solution is inoculated into host plant；
S2, continue to cultivate, and filter out susceptible plant and nontoxic plant；
S3, B Bemisia tabacis and Q Bemisia tabacis are inoculated on the susceptible plant and the non-toxic herb connect respectively
Plant the disease plant for there are B Bemisia tabacis, the nontoxic plant for being inoculated with the disease plant of Q Bemisia tabacis, being inoculated with B Bemisia tabacis, connect
Kind has the nontoxic plant of Q Bemisia tabacis；
S4, respectively by the disease plant for being inoculated with B Bemisia tabacis, be inoculated with Q Bemisia tabacis disease plant, inoculation
The nontoxic plant for having B Bemisia tabacis, the nontoxic plant for being inoculated with Q Bemisia tabacis are individually raised, and are bred Bemisia tabaci；
S5, raising 6 more than generation carry out Bemisia tabaci insect-taking.
Above-mentioned rearing method, it is preferred that host plant described in the S1 steps is to grow 3~4 true leaves
Vegetables.
Above-mentioned rearing method, it is preferred that the culture of the host plant comprises the following steps：Vegetable seeds is broadcast
Kind it is being matrix without in worm cage using peat, vermiculite, organic fertilizer and perlite, until vegetables grow 3~4 true leaves.
Above-mentioned rearing method, it is preferred that the no worm cage is framework cage made of aluminium alloy, on bottom and front
End is made of glass, top layer, both sides and is followed by being made of 100 mesh nylon wires, and positive lower end is made of 100 mesh nylon wires
Door.
Above-mentioned rearing method, it is preferred that the peat: vermiculite: organic fertilizer: the ratio of perlite is 10: 10:
10∶1。
Above-mentioned rearing method, it is preferred that in the S1 steps, infectious clone bacterium solution is inoculated into host and planted
Thing, concretely comprise the following steps：
S1-1, infectious clone activated on YEP solid mediums, the single bacterium colony on picking solid medium, juxtaposition
Culture obtains clone's bacterium solution of infectivity in YEP fluid nutrient mediums；
S1-2, precipitation will be taken after clone's bacterium solution centrifugation of the infectivity, suspension is obtained to the pellet resuspended
The Agrobacterium infectious clone bacterium solution of virus；The suspension includes 10mM MEs, 10mM MgCl2, 200mM acetosyringones
(acetosyringone)；
S1-3, the viral Agrobacterium infectious clone bacterium solution is expelled in host plant, continues culture until posting
Main plant is with 6~7 true leaves.MEs also known as 2- (N- morpholines)-ethylsulfonic acid.
Above-mentioned rearing method, it is preferred that described to make the specific steps that Bemisia tabaci is bred in the S4 steps
For：Illumination 14 hours, and temperature is 28 DEG C when controlling illumination；Dark 10 hours, and temperature is 25 DEG C when controlling dark, is raised
Humidity is maintained at 60% during in journey.
Above-mentioned rearing method, it is preferred that in the S5 steps, in the S5 steps, the Bemisia tabaci insect-taking bag
Include following steps：Bemisia tabaci is gathered using small insects pest sucking device, the insect of requirement is drawn, is then gently blocked with finger
Small insects pest sucking device tip, is transferred into centrifuge tube, carries out follow-up test.
Above-mentioned rearing method, it is preferred that the small insects pest sucking device includes the first pipette tips, the second pipette tips, breast
One end connection of sebific duct and gauze, the first pipette tips interface end and emulsion tube, gauze is provided with junction；Second rifle
The sophisticated of head is connected with the emulsion tube other end.Preferably, the emulsion tube is transparent emulsion tube.
Above-mentioned rearing method, it is preferred that the first pipette tips tip cuts off 1cm, and the second pipette tips tip is cut
0.5cm is gone to grow, a diameter of 1cm of the emulsion tube, length 20cm；The gauze is 100 mesh.
Compared with prior art, the advantage of the invention is that：
(1) the invention provides a kind of rearing method of viral vector insect Bemisia tabaci, difference can be obtained simultaneously
Population takes viruliferous Bemisia tabaci and the Bemisia tabaci without poison, has the following advantages that：1st, it can use the consistent cigarette that grows
Aleyrodid is studied, and compared with the environment of crop field, can shorten the development time of insect, improve egg laying amount, ensure survival rate, ensures to supply
Try lasting, the stable supply of material.2nd, individually raised with plant without toxic smoke aleyrodid and live box, isolation effect is good, ensures sample
Product purity.3rd, the consistent plant that grows is inoculated with infectious clone, periodic detection, ensures that malicious source supply is stable, band poison is equal
It is even.4th, healthy plant and persistently supplied with malicious plant, ensure Bemisia tabaci vigor.
(2) the invention provides a kind of rearing method of viral vector insect Bemisia tabaci, test plant to support clean
Without in worm cage, no worm cage is framework cage made of aluminium alloy, and specification is 60cm × 40cm × 80cm, and bottom and front upper portion are
It is made of glass, top layer, both sides and is followed by being made of 100 mesh nylon wires, positive lower end is door made of 100 mesh nylon wires,
Glass and the nylon wire construction of this no worm cage, had both met the light source and permeability needed in growing process, had prevented again
The escape of Bemisia tabaci, and aluminum alloy frame is sturdy and durable.
(3) the invention provides a kind of rearing method of viral vector insect Bemisia tabaci, with peat: vermiculite: organic
Fertilizer: it is matrix planting vegetable seed that the ratio of perlite is at 10: 10: 10: 1, both ensure that the permeability of soil, and in turn ensure that
The supply of fertilizer, plant health can be grown.
(4) the invention provides a kind of rearing method of viral vector insect Bemisia tabaci, during virus inoculation,
Using 50mL suspension (MEs containing 10mM, 10mM MgCl2, 200mM acetosyringones) to thalline carry out resuspension purpose be
Bacterial concentration is adjusted, its OD600 value is promoted the morbidity after virus inoculation in the range of 1.0~1.5, is made inoculation
Plant infection virus rate is higher.
(5) the invention provides a kind of rearing method of viral vector insect Bemisia tabaci, in the mistake of Bemisia tabaci insect-taking
Cheng Zhong, Bemisia tabaci is gathered using small insects pest sucking device, the emulsion tube for being 20cm with diameter 1cm, length is process, by 1ml
Blue electron gun head tip cuts off one small section of about 1cm length, the afterbody access blue electron gun head rear end of emulsion tube, and middle 100 mesh gauzes are isolated,
The head access blue electron gun head tip of emulsion tube, the insect of the blue electron gun head absorption requirement of latex tube head is directed at mouth, it is small-sized
Insect pest sucking device.The effect so designed is that Bemisia tabaci is sucked into a relatively small space by the effect of air-flow, can be incited somebody to action
A large amount of Bemisia tabaci short time are transferred in required test equipment, and prevent the injury during insect-taking to Bemisia tabaci, in
Between with 100 mesh gauzes isolate effect be to prevent in Bemisia tabaci suction nozzle.
Brief description of the drawings
To make the purpose, technical scheme and advantage of the embodiment of the present invention clearer, below in conjunction with the embodiment of the present invention
In accompanying drawing, clear, complete description is carried out to the technical scheme in the embodiment of the present invention.
Fig. 1 is the flow chart of artificial feeding in the embodiment of the present invention 1.
Fig. 2 is the structural representation of the middle-size and small-size insect insect-taking device of the embodiment of the present invention 1, wherein, 1：First pipette tips, 2：Second
Pipette tips, 3：Emulsion tube, 4：Gauze.
Embodiment
Below in conjunction with Figure of description and specific preferred embodiment, the invention will be further described, but not therefore and
Limit the scope of the invention.
Material and instrument employed in following examples and comparative example are commercially available.
Embodiment 1
A kind of rearing method of viral vector insect Bemisia tabaci of the invention, trained referring to Fig. 1, including host plant
The steps such as foster, virus inoculation, Bemisia tabaci inoculation, Bemisia tabaci are bred, insect-taking, it is specially：
(1) host plant is cultivated：
1.1st, host plant is that tomato, capsicum, cucumber etc. test Common Vegetables, and the present embodiment employs tomato, because kind
Eggplant is the host of various plants virus.
1.2nd, test plant is supported in clean no worm cage, and no worm cage is framework cage made of aluminium alloy, specification 60cm
× 40cm × 80cm, bottom and front upper portion are made of glass, top layer, both sides and are followed by being made of 100 mesh nylon wires, just
Face lower end be door made of 100 mesh nylon wires (be raise Bemisia tabaci it is conventional without worm cage).
1.3rd, peat, vermiculite, organic fertilizer and perlite are mixed, mixed according to mass ratio for 10: 10: 10: 1,
As the matrix of host plant culture.
1.4th, the matrix added in nutritive cube in step 1.3 is sowed, standby after plant grows 3~4 true leaves.
(2) virus inoculation：
2.1st, the preparation of infectious clone bacterium solution：By infectious clone first in YEP solid mediums (Kan containing 50mg/l
+ and 50mg/l Rif+) on activate, subsequent therefrom picking single bacterium colony, be placed in YEP fluid nutrient mediums (Kan+ containing 50mg/L with
50mg/l Rif+) in, at least 24h is cultivated under the conditions of 28 DEG C of 200rpm, so as to obtain clone's bacterium solution of infectivity.By bacterium solution
10min is centrifuged in room temperature in 4000rpm, then abandons supernatant, and with 50mL suspension ((2- (N- morpholines)-ethyls of MEs containing 10mM
Sulfonic acid), 10mM MgCl2, 200mM acetosyringones) to thalline carry out resuspension obtain infectious clone bacterium solution, it is quiet in room temperature
It is standby to put 2h.
2.2nd, the healthy plant of about 3 true leaves is chosen, with Dispoable medical syringe, takes the above-mentioned infectivity prepared
Bacterium solution is cloned, about 0.5mL bacterium solutions are injected to every plant, and not inject the plant of bacterium solution (nontoxic plant) as control.
The clean no worm cage that plant after injection is placed on to glasshouse is cultivated.When plant to be planted is with 6~7 true leaves, selection has
The plant of obvious disease symptom, is detected, band poisonous plant and non-toxic herb with PCR.Specifically detection method is：Will inspection
Test sample product are contrasted with positive control and negative control, purposeful band and band is single, the sample consistent with positive control
Corresponding plant is band poisonous plant, and without purpose band, or the corresponding plant of the sample consistent with negative control is nontoxic plant
Thing.Testing result is：Ratio >=80% with poisonous plant after inoculation infectious clone bacterium solution.
(3) Bemisia tabaci is inoculated with：
3.1st, B Bemisia tabacis and Q Bemisia tabacis are inoculated into band poisonous plant and non-toxic herb respectively, each Bemisia tabaci pair
The every kind of plant answered with 3 without worm cage, plant by the health for being each put into 4~6 detections plant to fall ill without worm cage or individually raising
Thing, 300 B Bemisia tabacis and Q Bemisia tabacis is taken to be put on the plant of morbidity and health respectively, each Bemisia tabaci is put into individually
Cage in, 3 cages put a phjytotron corresponding to each population, are put into the phjytotron isolated between 4 altogether.It is all
Bemisia tabaci it is interior in the controlled environment chamber breed, illumination condition is illumination in 14 hours, 10 hours dark, and the temperature of illumination in 14 hours is
28 DEG C, dark temperature is 25 DEG C within 10 hours, and humidity is held in 60% under illumination and dark condition.Obtain being inoculated with B tobacco powder
The disease plant of lice, the disease plant for being inoculated with Q Bemisia tabacis, the nontoxic plant for being inoculated with B Bemisia tabacis, it is inoculated with Q tobacco powder
The nontoxic plant of lice.
3.2nd, after raising for 6 generations, 10 Bemisia tabacis are randomly selected on every plant, Bemisia tabaci band poison rate and kind are detected with PCR
Group's purity.PCR testing result is shown：Band poison rate with seed culture of viruses group is 100%, while Bemisia tabaci purity is 100%.
(4) Bemisia tabaci insect-taking：Using small insects pest sucking device gather Bemisia tabaci, small insects insect-taking device be with diameter 1cm,
Length carries out transformation for the pipette tips of 20cm transparent emulsion tube and liquid-transfering gun and formed.Specifically remodeling method is：Take two 1ml rifles
Head (pipette tips are the matching used pipette tips of liquid-transfering gun), is named as the first pipette tips 1 and the second pipette tips 2.The tip of first pipette tips 1 is cut off
After 1cm, one end of emulsion tube 3 is inserted in the relative interface end in tip (i.e. that one end of pipette tips and liquid-transfering gun interface), and the
Isolated among one pipette tips 1 and emulsion tube 3 with 100 mesh gauzes 4.Second pipette tips 2 cut off sophisticated 0.5cm, and tip is inserted in into emulsion tube 3
The other end.During using small insects insect-taking device insect-taking, the interface end of the second pipette tips 2 is directed at mouth, and the first pipette tips 1 is sophisticated right
Quasi- insect, the insect of requirement is drawn, the tip of the first pipette tips 1 is then gently blocked with finger, is transferred into centrifuge tube.
Investigate the development time of the insect of Bemisia tabaci in embodiment 1 and crop field environment respectively, egg laying amount, survival rate is single
With malicious rate, Combined Infection rate, Bemisia tabaci stability etc..
Bemisia tabaci development time and survival rate：Under artificial feeding environment and under the environment of crop field, 30 plants are taken respectively with for the moment
Phase sowing, plant of the same size, leaf is chosen in the same position of each plant, it (is to survey to be put into a micro- worm cage
Determine Bemisia tabaci to grow conventional micro- worm cage), 10 pairs of Bemisia tabaci adults sprouted wings in 3 days are put into each micro- worm cage, treat it
Spawning removes all adults after 24 hours, counts under the microscope per the egg laying amount in cage, then observation ovum and nymph daily
Developmental state, the adult quantity that regular check is sprouted wings, until all adult eclosions, calculate survival rate of the Bemisia tabaci by ovum to adult
And development time.
Single head Bemisia tabaci egg laying amount：Under artificial feeding and crop field environment, 30 plants of same periods sowing and in the same size is taken respectively
Plant, choose leaf in the same position of each plant, be put into a micro- worm cage, be put into each micro- worm cage at the beginning of 1
The Bemisia tabaci female adult of emergence, the Bemisia tabaci in micro- worm cage is transferred on 1 plant of new plant leaf blade of the same size every 7 days,
Blade (when the plant under the environment of crop field is observed, is fetched reality by the egg laying amount for observing Bemisia tabaci on prophyll piece under the microscope simultaneously
Room is tested to be observed under the microscope), until Bemisia tabaci is dead, the total spawning quantity of every Bemisia tabaci of statistics.
The single band poison rate of Bemisia tabaci and Combined Infection rate：By Bemisia tabaci artificial feeding and that crop field is fetched, PCR method is used
Detect respectively and insect vector high in field incidence be Bemisia tabaci virus, as tomato yellow leaf curl virus (TYLCV), in
State's tomato yellow leaf curl virus (TYLCCNV), tomato chlorisis viral (ToCV) etc..Each Bemisia tabaci sample is entered with all viruses
Row detection, to determine Bemisia tabaci to single viral band poison rate and while carry one or more of viral Combined Infection rates.
Bemisia tabaci stability：Including Bemisia tabaci purity and Bemisia tabaci body length, sex ration, the measure of Bemisia tabaci purity
Method is with reference to step 3.2；Bemisia tabaci sex ration assay method is to carry out the Bemisia tabaci under the conditions of two kinds under the microscope respectively
Observation, female adult and the quantity of male worm are recorded, calculate sex ration；Bemisia tabaci body length measuring method is surveyed with the graduated scale on microscope
The body length of single head Bemisia tabaci is measured, is counted and is contrasted；Comprehensive three indexs, draw the stability of Bemisia tabaci.
Table 1：Rearing method and Bemisia tabaci quality versus's table in the environment of crop field
| Artificial feeding environment | Crop field environment | |
| Bemisia tabaci development time | 20~30 days | 25~35 days |
| Single head Bemisia tabaci egg laying amount | 100~200 | 50~150 |
| Bemisia tabaci survival rate | 90% | 10~70% |
| The single band poison rate of Bemisia tabaci | 100% | 0~80% |
| Bemisia tabaci Combined Infection rate | 0% | 50~100% |
| Bemisia tabaci stability | It is stable | It is unstable |
| The other factors such as agricultural chemicals influence | Nothing | Have |
As can be known from the results of Table 1：No matter the rearing method of the present invention is from development time, egg laying amount or Bemisia tabaci
Survival rate, with malicious rate, stability etc., be significantly better than that the natural feeding method of crop field environment.
It is described above, only it is the part of the embodiment of the present invention, any formal limitation not is made to the present invention.
Although the present invention is not limited to the present invention so that a part of preferred embodiment to be disclosed as above.It is any to be familiar with ability
The technical staff in domain, in the case where not departing from the Spirit Essence of the present invention and technical scheme, all using the side of the disclosure above
Method and technology contents make many possible changes and modifications to technical solution of the present invention, or are revised as the equivalent reality of equivalent variations
Apply example.Therefore, every content without departing from technical solution of the present invention, the technical spirit according to the present invention are done to above example
Any simple modification, equivalent substitution, equivalence changes and modification, still fall within the range of technical solution of the present invention protects.
Claims (10)
1. a kind of rearing method of viral vector insect Bemisia tabaci, it is characterised in that comprise the following steps：
S1, to grow the vegetables of true leaf as host plant；Infectious clone bacterium solution is inoculated into host plant；
S2, continue to cultivate, filter out susceptible plant and nontoxic plant；
S3, B Bemisia tabacis and Q Bemisia tabacis are inoculated on the susceptible plant and the non-toxic herb are inoculated with respectively
The disease plant of B Bemisia tabacis, the disease plant for being inoculated with Q Bemisia tabacis, the nontoxic plant for being inoculated with B Bemisia tabacis, it is inoculated with Q
The nontoxic plant of Bemisia tabaci；
S4, respectively by the disease plant for being inoculated with B Bemisia tabacis, be inoculated with the disease plant of Q Bemisia tabacis, be inoculated with B
The nontoxic plant of Bemisia tabaci, the nontoxic plant for being inoculated with Q Bemisia tabacis are individually raised, and are bred Bemisia tabaci；
S5, raising 6 more than generation carry out Bemisia tabaci insect-taking.
2. rearing method according to claim 1, it is characterised in that host plant described in the S1 steps is length
Go out the vegetables of 3~4 true leaves.
3. rearing method according to claim 2, it is characterised in that the culture of the host plant includes following step
Suddenly：It is being matrix without in worm cage using peat, vermiculite, organic fertilizer and perlite by vegetable seeds sowing, until vegetables grow 3
~4 true leaves.
4. rearing method according to claim 3, it is characterised in that the no worm cage is framework made of aluminium alloy
Cage, bottom and front upper portion are made of glass, top layer, both sides and are followed by being made of 100 mesh nylon wires, and positive lower end is
Door made of 100 mesh nylon wires.
5. rearing method according to claim 3, it is characterised in that the peat: vermiculite: organic fertilizer: pearl
The ratio of rock is 10: 10: 10: 1.
6. rearing method according to claim 1, it is characterised in that in the S1 steps, by infectious clone bacterium
Liquid is inoculated into host plant, concretely comprises the following steps：
S1-1, infectious clone activated on YEP solid mediums, the single bacterium colony on picking solid medium, be placed in YEP
Culture obtains clone's bacterium solution of infectivity in fluid nutrient medium；
S1-2, precipitation will be taken after clone's bacterium solution centrifugation of the infectivity, suspension is obtained into virus to the pellet resuspended
Agrobacterium infectious clone bacterium solution；The suspension includes 10mM MEs, 10mM MgCl2With 200mM acetosyringones；
S1-3, the viral Agrobacterium infectious clone bacterium solution is expelled in host plant, continues culture until host plants
Thing is with 6~7 true leaves.
7. rearing method according to claim 1, it is characterised in that described to enter Bemisia tabaci in the S4 steps
What row was bred concretely comprises the following steps：Illumination 14 hours, and temperature is 28 DEG C when controlling illumination；Dark 10 hours, and when controlling dark
Temperature is 25 DEG C, and humidity is maintained at 60% in feeding process.
8. rearing method according to claim 1, it is characterised in that in the S5 steps, the Bemisia tabaci insect-taking
Comprise the following steps：Bemisia tabaci is gathered using small insects pest sucking device, the insect of requirement is drawn, is then gently blocked up with finger
Firmly small insects pest sucking device tip, is transferred into centrifuge tube.
9. rearing method according to claim 8, it is characterised in that the small insects pest sucking device includes the first rifle
Head (1), the second pipette tips (2), emulsion tube (3) and gauze (4), the first pipette tips (1) interface end and one end of emulsion tube (3) connect
Connect, gauze (4) is provided with junction；Sophisticated in second pipette tips (2) is connected with the emulsion tube (3) other end.
10. rearing method according to claim 9, it is characterised in that a diameter of 1cm of the emulsion tube (3), length
For 20cm；The gauze (4) is 100 mesh.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109090046A (en) * | 2018-08-16 | 2018-12-28 | 云南农业大学 | A kind of the acquisition storage device and acquisition method of living body Bemisia tabaci |
| CN114946475A (en) * | 2022-07-06 | 2022-08-30 | 扬州大学 | Low-temperature environment portable plant and insect source raising device and using method thereof |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892201A (en) * | 2010-06-23 | 2010-11-24 | 江苏省农业科学院 | Method for inoculating tomato yellow leaf curl virus by using injector to inject |
| CN101946607A (en) * | 2010-08-12 | 2011-01-19 | 江苏省农业科学院 | Method for identifying resistance of tomato to tomato yellow leaf curl virus by toothpick inoculation method |
| CN102948336A (en) * | 2012-11-12 | 2013-03-06 | 北京市农林科学院 | Method for inoculating tomatoes with tomato yellow leaf curl viruses |
| CN104285906A (en) * | 2014-10-11 | 2015-01-21 | 中国农业科学院植物保护研究所 | Method for building bemisia tabaci populations which contain rickettisa bacteria and do not contain rickettisa bacteria |
| CN204443835U (en) * | 2015-02-05 | 2015-07-08 | 中国农业科学院植物保护研究所 | A kind of mouth sucking small insects pest sucking device |
| CN205093420U (en) * | 2015-11-17 | 2016-03-23 | 北京市农林科学院 | Adult ware of accomodating of fast acquisition tobacco powder louse |
-
2017
- 2017-05-19 CN CN201710356868.9A patent/CN107410218A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101892201A (en) * | 2010-06-23 | 2010-11-24 | 江苏省农业科学院 | Method for inoculating tomato yellow leaf curl virus by using injector to inject |
| CN101946607A (en) * | 2010-08-12 | 2011-01-19 | 江苏省农业科学院 | Method for identifying resistance of tomato to tomato yellow leaf curl virus by toothpick inoculation method |
| CN102948336A (en) * | 2012-11-12 | 2013-03-06 | 北京市农林科学院 | Method for inoculating tomatoes with tomato yellow leaf curl viruses |
| CN104285906A (en) * | 2014-10-11 | 2015-01-21 | 中国农业科学院植物保护研究所 | Method for building bemisia tabaci populations which contain rickettisa bacteria and do not contain rickettisa bacteria |
| CN204443835U (en) * | 2015-02-05 | 2015-07-08 | 中国农业科学院植物保护研究所 | A kind of mouth sucking small insects pest sucking device |
| CN205093420U (en) * | 2015-11-17 | 2016-03-23 | 北京市农林科学院 | Adult ware of accomodating of fast acquisition tobacco powder louse |
Non-Patent Citations (5)
| Title |
|---|
| 代惠洁 等: ""山东寿光地区Q型烟粉虱对番茄褪绿病毒的传播"", 《植物保护学报》 * |
| 宁雯熙: ""B型、Q型烟粉虱传播番茄黄化曲叶病毒的差异研究"", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
| 应丰泽 等: ""两个入侵烟粉虱隐种MEAM1与MED对番茄黄曲叶病毒水平传播能力的比较"", 《昆虫学报》 * |
| 张蕊蕊 等: ""媒介虫口密度、植株苗龄及温度对B型烟粉虱传播台湾番茄曲叶病毒的影响"", 《植物保护学报》 * |
| 朱启顺 等: "《基因沉默——原理与方法》", 31 July 2005, 云南科技出版社 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109090046A (en) * | 2018-08-16 | 2018-12-28 | 云南农业大学 | A kind of the acquisition storage device and acquisition method of living body Bemisia tabaci |
| CN109090046B (en) * | 2018-08-16 | 2023-06-20 | 云南农业大学 | Collecting and storing device and collecting method for living bemisia tabaci |
| CN114946475A (en) * | 2022-07-06 | 2022-08-30 | 扬州大学 | Low-temperature environment portable plant and insect source raising device and using method thereof |
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