CN110514782A - A quick and easy bioassay method for whitefly-like stomach poisonous insecticides - Google Patents

Abstract

The invention discloses a quick and simple bioassay method for whitefly stomach poisonous insecticides: preparing bioassay agents and devices; preparing feeding liquid; weighing insecticides and dissolving them in feeding liquid; preparing feeding pouches Get the leaves with whitefly adults on it, drive the whitefly adults into the feeding glass tube, and put them into the black tube sleeve; place the whole bioassay device in the incubator, and use the whitefly phototaxis to eat quickly; 6 ‑After 12 hours, count the results and calculate the efficacy of the pesticide. The feeding solution containing the medicament of the present invention is convenient to prepare, easy to carry and transport, and convenient to quickly conduct field tests; the detection time is shortened to 12 hours; there is no need to collect the leaves used in the "soaking method", and the result deviation caused by the difference of leaves is eliminated; the detection device More compact and airtight. Therefore, the invention can accurately, quickly and efficiently detect the insecticide resistance of whiteflies, provide direct guidance for field medication, and provide theoretical support for rational medication.

Description

A quick and easy bioassay method for whitefly-like stomach poisonous insecticides

technical field

The invention relates to a fast and simple biological assay method for a whitefly-like stomach poisonous insecticide, which belongs to the field of biology.

Background technique

The whitefly family Aleyrodidae belongs to the Hemiptera Hemiptera suborder Sternorrhyncha. It is a small herbivorous piercing-sucking insect. It is distributed in all major fauna in the world, mainly in the tropics and subtropics. It mainly stings with nymphs and adults. Many species are important agricultural and forestry pests by sucking the sap of host plants. At present, 160 species of whiteflies have been recorded in the world, belonging to 165 genera and 3 subfamilies, namely Aleyrodinae, Aleurodicinae and Udamoselinae. There are 2 subfamilies, 49 genera, and 248 species.

The host range of whitefly is very wide, which can harm crops, vegetables, flowers, fruit trees and green plants, etc., including not only monocots and dicotyledons, but also ferns. There are more than 420 kinds of host plants of Bemisia tabaci in 74 families, and later increased to more than 500 kinds, and new hosts are constantly being discovered. There are 46 kinds of whitefly economic pests listed in the world. The whitefly species that cause harm to agriculture in China include Bemisia tabaci , whitefly Trialeurodes vapoariorum in greenhouses, whitefly Aleurocanthus spiniferus , whitefly Dialeurodes citri , mulberry Whitefly Pealius mori , rice whitefly Vasdavidius indicus , etc.

Ways of whitefly damage: (1) Direct damage: Both adults and nymphs can suck the juice of the phloem of plants. Due to its rapid development and high fecundity, the population can often reach a high density in a short period of time, thereby absorbing a large amount of juice and causing the plant to weaken. (2) Indirect damage; adults and nymphs secrete honeydew and waxy substances to pollute plant organs and fruits, induce the occurrence of soot disease, hinder plant photosynthesis, cause leaves to shrink, wither and fall leaves early, and at the same time reduce the quality and quality of crops. decline. (3) Some whiteflies are important vectors of many viral diseases, and the plant viruses they transmit can cause plant spraying and fruit abortion, causing serious losses.

Whiteflies are mainly controlled by chemicals. The excessive use of insecticides has caused whiteflies to have varying degrees of resistance to various control drugs, especially neonicotinoids, which have been used the longest and have the widest range of applications for whitefly control. most resistant insecticides. For example, the resistance of whitefly to neonicotinoid insecticides was discovered for the first time in the horticultural production area of southern Spain. Shortly afterwards, nearly 1000 times the thiamethoxam-resistant population of whitefly whitefly was found in Israel and Spain, and Highly resistant populations had significant cross-resistance to other nicotinic agents. In recent years, there have been reports of Bemisia tabaci becoming resistant to neonicotinoids in Beijing, Jiangsu, Zhejiang, Xinjiang, Hubei and other provinces in China, and my country's north reaches Heilongjiang, south reaches Hainan, and west reaches Qinghai. Bemisia tabaci is distributed and harmed in most areas from , east to Shanghai, so drug resistance is one of the main reasons for the large outbreak of whitefly and it is difficult to control.

The traditional whitefly bioassay method has many influencing factors, and the equipment is inconvenient to carry and takes a long time.

Contents of the invention

In view of the above problems, the present inventors have invented a new bioassay method for whitefly gastric poisoning drugs through preliminary exploration, which provides a new method for the detection of drug resistance in the field. Accurate, rapid and efficient detection of whitefly resistance to insecticides provides direct guidance for field application, and can provide theoretical support for rational use of pesticides.

The technical scheme provided by the invention is: a quick and easy bioassay method of whitefly class stomach poison insecticide, the method comprises the following steps:

(1) Prepare bioassay reagents and devices, which include thin parafilm, feeding glass tubes, and black tube sleeves;

(2) To prepare a feeding solution, weigh 25-35 grams of sucrose and 3-8 grams of yeast extract and dissolve them in 80-120 ml of distilled water, mix well and filter, and store the filtrate in a freezer for later use;

(3) Weigh the insecticide, dissolve it in the feeding solution, and prepare different concentrations;

(4) Prepare a feeding pouch, spread a thin layer of parafilm on one end of the feeding glass tube, then take 80-120 microliters of the feeding solution containing the drug in step (2) and put it on the parafilm, and then Take a piece of parafilm and cover it lightly to form a feeding pouch;

(5) Take the leaf with whitefly adults on it, gently pat the back of the leaf with the other side of the glass tube, drive the whitefly adults into the feeding glass tube, then seal the mouth with a piece of parafilm, and put it into a black tube sleeve , with the side of the feeding pouch facing outwards;

(6) Place the entire bioassay device in the incubator, with the side of the feeding pouch facing the light source, and take advantage of the phototaxis of whiteflies to eat quickly;

(7) After 6-12 hours, count the results and calculate the efficacy of the insecticide.

In the method described above, preferably, in step (2), 30 grams of sucrose and 5 grams of yeast extract are weighed and dissolved in 100 milliliters of distilled water, mixed thoroughly and then filtered through a 0.2 micron bacterial filter, and the filtrate is frozen and stored for later use; In step (4), take 100 microliters of the feeding solution containing medicine in step (2) and put it on the sealing film; in step (5), drive whitefly adults into feeding glass tubes, each glass tube 15 -25 whitefly adults.

In the method described above, in step (3), different concentrations are prepared to be thiamethoxam 10000 mg/L, 500 mg/L, 250 mg/L, 125 mg/L, 62.5 mg/L, 31.25 mg/L, respectively. mg/L, 15.625 mg/L, 7.8125 mg/L, Abamectin 100 mg/L, 50 mg/L, 25 mg/L, 12.5 mg/L, 6.25 mg/L, 3.125 mg/L, 1.5625 mg /L and 0.78125 mg/L, the sensitivity of the agent to whitefly was calculated through the mortality rate of whitefly treated with different concentrations of the agent, so as to obtain the application effect of the agent.

Simultaneously, the present invention also provides a kind of device of the bioassay of quick and easy whitefly class stomach poison insecticide, and described device comprises thin parafilm, black tube cover and the feeding glass tube that is placed in black tube cover; One end of the feeding glass tube is provided with a feeding pouch, and the other end is sealed by a layer of thin parafilm; one end of the black tube sleeve is open, so that one side of the feeding pouch faces outwards towards the light source.

In the device described above, one end of the feeding glass tube is covered with a layer of thin parafilm, then the feeding solution is placed on the thin parafilm, and then a piece of thin parafilm is gently covered on it to form a Feed pouches.

The device, wherein the black sleeve is a black plastic sleeve.

The present invention has the following beneficial effects:

1. The feeding solution containing the medicament of the present invention is convenient to prepare, easy to carry and transport, and convenient to conduct field experiments quickly.

2. The detection time is shortened to 12 hours compared with 48 hours of the "immersion method", which saves a lot of time.

3. There is no need to collect the leaves used in the "immersion method", which eliminates the deviation of results caused by differences in leaves.

4. The detection device is more compact and airtight, which is convenient for field detection.

Description of drawings

Fig. 1 is a schematic diagram of the structure of the bioassay device of the present invention.

Detailed ways

The quick and easy bioassay method of whitefly adult stomach poison insecticide of the present invention, the operation steps are as follows:

1. First prepare the bioassay agent and device (including thin parafilm, feeding glass tube 2, black tube sleeve 1), check whether the agent is within the validity period, and whether the device is clean.

2. To prepare a feeding solution, weigh 30 grams of sucrose and 5 grams of yeast extract and dissolve them in 100 milliliters of distilled water, mix well, filter them with a 0.2-micron bacterial filter, and store them frozen for later use.

3. Take a certain amount of insecticide thiamethoxam (97.5%) 0.103 and dissolve it in 1mL of acetone, then use the feeding solution to prepare a 1000mg/L mother solution, then dilute it to 500, 250, 125, 62.5, 31.25, 15.625, 7.8125 mg/L, the same method to prepare different concentrations of abamectin 100, 50, 25, 12.5, 6.25, 3.125, 1.5625 and 0.78125 mg/L, dissolved in the feeding solution, prepared in different concentrations.

4. Prepare the feeding pouch 3, spread a thin layer of sealing film on one end of the feeding glass tube 2, then take 100 microliters of the feeding solution containing the medicine and put it on the sealing film, then take a piece of sealing film and gently Cover it with the ground to form a feeding pouch 3 .

5. Take whitefly adults, lightly pat the back of the blade with the other side of the glass tube, drive whitefly adults into feeding glass tube 2, and each glass tube has 15-25 (20 in this embodiment) whiteflies Adult, seal mouth with a piece of parafilm then, put into black plastic tube cover 1, feed pouch 3 one side outwards.

6. Place the entire bioassay device in a 25-degree incubator, with the side of the feeding pouch facing the light source, and take advantage of the phototaxis of whiteflies to eat quickly.

7. Count the results after 6-12 hours (12 hours in this embodiment), and calculate the efficacy of the insecticide.

In this example, Bemisia tabaci was used as a model insect, and two commonly used insecticides in the field: thiamethoxam and avermectin were selected as model agents, and the above-mentioned method of the present invention was used to perform a new bioassay.

Through the bioassay method of the present invention, the drug efficacy detection of thiamethoxam and abamectin is carried out to the target pest Bemisia tabaci, and the mortality rate of Bemisia tabaci is detected after 12 hours. After the statistics, the toxicity is calculated with polo software, and the results are as follows As shown in the table, the 95% confidence intervals of the lethal concentration (LC50) of whitefly to thiamethoxam and avermectin are very narrow, indicating that whitefly has high reliability in determining the toxicity of these two drugs, It also indirectly shows that the method of the present invention has a good linear relationship for the determination of the efficacy of stomach poisoning insecticides with different concentrations, and can be well used for the detection of the efficacy of stomach poisoning insecticides for such pests.

insecticide population N Slope (±SE) df LC₅₀ (mg L^-1) (95%FL) Thiamethoxam LQS 682 1.62 (± 0.12) 6 61.07 (47.09~78.18) Avermectin LQS 697 0.99 (± 0.08) 6 14.42 (10.39~20.69)

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Claims (8)

1. a kind of bioassay method of fast and convenient aleyrodid class stomach insecticide, this method comprises the following steps:

(1) prepare bioassay medicament and device, described device include thin sealed membrane, feed glass tube, black pipe sleeve；

(2) formulating and feeding liquid, weighs 25-35 grams of sucrose and 3-8 grams of yeast extract is dissolved in 80-120 milliliters of distilled water, It is filtered after mixing well, filtrate stored frozen is spare；

(3) insecticide is weighed, is dissolved in feeding liquid, is configured to different concentration；

(4) then preparation feeding pouch takes 80-120 microlitres of step in the sealed membrane that one end layer overlay of feeding glass tube is very thin Suddenly the feeding liquid of drug containing is placed on sealed membrane in (2), then one piece of sealed membrane is taken lightly to cover above, forms feeding pouch；

(5) blade for having adult whitefly is taken thereon, blade back is gently clapped with glass tube another side, adult whitefly is caught up with into feeding It in glass tube, then with one piece of ParafilmTM mouth, is put into black pipe sleeve, feeding pouch side is outwardly；

(6) it will entirely give birth to and survey device placement in the incubator, and feed pouch side towards light source, taken rapidly using aleyrodid phototaxis Food；

(7) statistical result after 6-12 hours, calculates the drug effect of insecticide.

2. according to the method described in claim 1, it is characterized by: weighing 30 grams of sucrose in step (2) and 5 grams of yeast leaching Object is dissolved in 100 milliliters of distilled water, is filtered after mixing well with 0.2 micron of bacterial filter, filtrate stored frozen is spare.

3. according to the method described in claim 1, it is characterized by: taking the feeding of drug containing in 100 microlitres of steps (2) in step (4) Liquid is fed to be placed on sealed membrane.

4. according to the method described in claim 1, it is characterized by: adult whitefly is caught up with into feeding glass tube in step (5) In, each glass tube 15-25 adult whiteflies.

5. according to the method described in claim 1, it is characterized by: in step (3), be configured to different concentration be configured to it is dense Degree respectively 10000 mg/L of Diacloden, 500 mg/L, 250 mg/L, 125 mg/L, 62.5 mg/L, 31.25 mg/L, 15.625 mg/L, 7.8125 mg/L, avermectin 100 mg/L, 50 mg/L, 25 mg/L, 12.5 mg/L, 6.25 mg/L, 3.125 mg/L, 1.5625 mg/L and 0.78125 mg/L.

6. the device that a kind of any one of claim 1 to 5 the method is used, it is characterised in that: described device includes thin sealing Film, black pipe sleeve and the feeding glass tube being placed in black pipe sleeve；It is equipped with feeding pouch in described feeding glass tube one end, separately One end thin ParafilmTM as described in one layer；Black pipe sleeve is open at one end, so that the feeding pouch side is outwardly, towards light Source.

7. device according to claim 6, it is characterised in that: be covered with described in one layer in one end of the feeding glass tube Then thin sealed membrane takes feeding liquid to be placed on thin sealed membrane, then one piece of thin sealed membrane is taken lightly to cover above, and it is small to form feeding Bag.

8. device according to claim 7, it is characterised in that: the black pipe sleeve is black plastic casing.