Drug-resistant predatory mite screening device and efficient screening method

Abstract

The invention discloses a screening device for drug-resistant predatory mites and a method for efficiently screening the drug-resistant predatory mites by adopting the screening device. The screening device comprises a lower container and an upper container, the lower container is of a big-end-down table structure, and the top of the lower container is open; the upper container comprises an inner cylinder and an outer cylinder which are sleeved inside and outside, the tops and the bottoms of the outer cylinder and the inner cylinder are open, the inner cylinder is also in a big-end-down table shape, and the bottom edge of the inner cylinder and the bottom edge of the outer cylinder are fixedly connected into a whole. An accommodating cavity is formed between the side wall of the inner cylinder body and the side wall of the outer cylinder body and is used for accommodating feed for predatory mites; the shape of the inner cylinder body is matched with that of the top of the lower container, and the inner cylinder body is sleeved on the top of the lower container. The screening device is simple in structure, ingenious in design, easy to manufacture, low in cost and convenient to use; compared with a traditional screening method, the established screening method for the drug-resistant predatory mites greatly accelerates the breeding process of the drug-resistant predatory mites.

Description

Technical Field

[0001] The invention relates to the technical field of biological natural enemy control of plant pests, and in particular to a device for screening drug-resistant predatory mites and an efficient screening method. Background Art

[0002] Predatory mites are Arachnida, Acarina, and are beneficial mites that specialize in preying on harmful mites, hence the name predatory mites. Predatory mites include the families Erythropodidae, Macroerythropodidae, Velvetidae, Longibaldiidae, and Phytoseioidea. The predatory mites that are currently being studied and applied more are Amblyseius cucumeri, Amblyseius barnesi, Neoseiulus cucumeri, Neoseiulus californicus, Neoseiulus barnesi, and Neoseiulus sternii. Predatory mites can prey on a variety of pests (mites) such as spider mites, greenhouse whiteflies, and thrips. They are safe and pollution-free, and have been widely used in foreign countries to control pests of economic crops such as fruit trees, tea, and vegetables. They are also natural enemy products that are promoted as green control of crop pests (mites) in my country.

[0003] There are many kinds of pests in economic crops such as fruit trees, tea and vegetables. Predatory mites cannot control all pests. Therefore, chemical control is still an indispensable pest control measure in production. The application of chemical pesticides inevitably leads to the contradiction between chemical control and biological control. In particular, the application of broad-spectrum insecticides such as organophosphorus and pyrethroids is extremely toxic to predatory mites. Large-scale use of pesticides will cause the death of predatory mites placed in the field. In order to coordinate the contradiction between chemical control and biological control, countries around the world have stepped up efforts to breed predatory mite resistant strains against broad-spectrum insecticides. Resistant predatory mites are a type of predatory mite with high resistance to chemical pesticides. This resistance allows predatory mites to survive in an environment where chemical pesticides are used, thereby maintaining their effectiveness as a biological control method. Compared with ordinary predatory mites, resistant predatory mites can solve the contradiction between chemical control and biological control to the greatest extent, so they have more obvious advantages and broader market prospects.

[0004] Indoor breeding of predatory mites usually uses flour mites (represented by the oval flour mite Aleuroglyphus ovatus) as their prey. The feed for oval flour mites is mainly sterilized wheat bran, supplemented with nutritional additives such as yeast protein. In the research on indoor breeding of predatory mites with pesticide resistance, the wheat bran-flour mite-predatory mite breeding system is usually used to raise predatory mites, and pesticide treatment is used to select predatory mites with pesticide resistance.

[0005] At present, the world mainly adopts the method of generation-by-generation resistance screening, and the resistance of predatory mites increases extremely slowly. At present, there are less than 10 species of resistant predatory mites reported at home and abroad. Therefore, it is necessary to establish an efficient screening technology for predatory mites. Summary of the invention

[0006] The object of the present invention is to provide a device and an efficient screening method for drug-resistant predatory mites in view of the above problems.

[0007] In order to achieve its purpose, the present invention adopts the following technical solution:

[0008] The first aspect of the present invention provides a device for screening pesticide-resistant predatory mites, comprising a lower container and an upper container, wherein the lower container is a platform structure that is small at the top and large at the bottom, and the top of the lower container is open; the upper container comprises an inner cylinder and an outer cylinder that are arranged inside and outside, and the top and bottom of the outer cylinder and the inner cylinder are both open, and the inner cylinder is also in the shape of a platform that is small at the top and large at the bottom, and the bottom edge of the inner cylinder is fixedly connected to the bottom edge of the outer cylinder to form a whole, and a accommodating cavity is formed between the side wall of the inner cylinder and the side wall of the outer cylinder to hold feed for predatory mites; the shape of the inner cylinder matches the shape of the top of the lower container, and the inner cylinder is sleeved on the top of the lower container, so that the upper container and the lower container are overlapped and sleeved together to be assembled into a device for screening pesticide-resistant predatory mites.

[0009] Preferably, the lower container and the inner cylinder are both frustums that are smaller at the top and larger at the bottom.

[0010] Preferably, the outer cylinder is in the shape of a cylinder or a platform with a larger upper part and a smaller lower part; the inner cylinder is arranged in the middle and lower part of the outer cylinder; and the upper container is formed in one piece.

[0011] Preferably, the outer cylinder is in the shape of a platform that is larger at the top and smaller at the bottom.

[0012] The second aspect of the present invention provides a method for efficiently screening insecticide-resistant predatory mites, which is carried out using any of the insecticide-resistant predatory mites screening devices described above, and comprises the following steps:

[0013] S1. Predatory mites prepared for spraying

[0014] Preparing a substrate containing a set population of predatory mites for spraying in the lower container in step S2;

[0015] S2. Chemical treatment

[0016] S2.1. Prepare the upper container

[0017] Adding a substrate containing predatory mite prey into the holding chamber of the upper container;

[0018] S2.2, spraying treatment

[0019] Place the prepared matrix containing predatory mites into the container below and use LC of the treatment agent to 60 ~ 90 Dosage (e.g. LC 60 , LC 70 , LC 80 , LC 90 The substrate in the lower container is sprayed with a dosage of 100 mg / mL (all of which are commonly used in screening experiments in this field), and the substrate is stirred while spraying until the substrate is evenly moistened. Then the upper container is placed on top of the lower container, and the surviving predatory mites will crawl up into the substrate in the upper container by themselves.

[0020] S3. Transfer culture

[0021] After the predatory mites are treated by spraying in step S2 for 22 hours (preferably 22 to 28 hours), the substrate containing the predatory mites and their prey in the upper container is moved to a predatory mite breeding box, in which a substrate containing predatory mite prey is placed, and the predatory mites are continued to be bred and multiplied until the population of the predatory mites in the breeding box reaches a set population;

[0022] S4. Repeated screening

[0023] The substrate containing predatory mites cultured in step S3 is placed in the lower container, and steps S2-S3 are repeated for multiple times until the resistance level of the predatory mites reaches a preset multiple, and then screening is stopped to obtain drug-resistant predatory mites.

[0024] In the above method and technical solution, the matrix is ​​wheat bran.

[0025] In the above method and technical scheme, the agent to be treated in step S2.2 is selected from chlorpyrifos, cypermethrin, lambda-cyhalothrin, avermectin, and amitraz, and the spraying amount is calculated based on 80-120 ml (preferably 90-110 ml) per 500 g feed.

[0026] In the above-mentioned method and technical solution, the population density of the predatory mites placed in the lower container for spraying treatment is 1,000 or more predatory mites per 10 grams of substrate.

[0027] In the above method and technical scheme, every 10 grams of the matrix containing predatory mite prey added to the upper container contains 5,000 to 10,000 predatory mite prey; for every 2,500 grams of the matrix containing predatory mites in the lower container, 500 grams of the matrix containing predatory mite prey is provided in the upper container.

[0028] In the above method and technical scheme, the predatory mite is selected from Neoseiulus cucumeri, Neoseiulus californicus, Neoseiulus barnesi, and Neoseiulus sternii; the predatory mite prey is selected from Aleuroglyphus ovatus.

[0029] The beneficial effects of the present invention are:

[0030] The traditional method of screening pesticide-resistant predatory mites is to directly spray the predatory mite breeding box with pesticides. The surviving predatory mites after spraying need to be manually picked out one by one, which is difficult to collect. In addition, the wheat bran in the breeding box is prone to mold after spraying, and the living environment of the predatory mites becomes worse, causing deaths caused by non-pesticides, affecting the accuracy of the experimental results. However, after the pesticide is sprayed with the device of the present invention, the surviving predatory mites automatically crawl into the upper container, without manual selection and collection, making full use of the motility of the predatory mites themselves, which improves the screening efficiency on the one hand, reduces the death of predatory mites caused by non-pesticide toxicity on the other hand, and improves the accuracy of the experimental results.

[0031] The screening device used in the present invention has a simple structure, ingenious design, easy manufacturing, low cost and convenient use. A set of efficient methods for screening drug-resistant predatory mites is established by using the device. Compared with the traditional screening method, the method of the present invention greatly accelerates the breeding process, and can quickly breed new strains of predatory mites with high levels of resistance to pesticides such as organophosphorus, pyrethroids and avermectins, providing drug-resistant predatory mite strains for field pest control, improving the control effect of biological natural enemy pests, reducing the use of chemical pesticides, and reducing environmental pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 It is a schematic diagram of an explosion of a device for screening insecticide-resistant predatory mites used in the method of the present invention.

[0033] Figure 2 It is a schematic diagram of the assembly of the device for screening insecticide-resistant predatory mites used in the method of the present invention.

[0034] Figure 3 It is a schematic diagram of the use of the device for screening insecticide-resistant predatory mites used in the method of the present invention.

[0035] In the figure, 1-lower container, 2-upper container, 21-outer cylinder, 22-inner cylinder, 23-accommodating chamber. DETAILED DESCRIPTION

[0036] The present invention will be further described below in conjunction with embodiments, but the present invention is not limited thereto.

[0037] The experimental methods in the following examples are conventional methods unless otherwise specified.

[0038] Example 1: A device for screening insecticide-resistant predatory mites

[0039] like Figure 1-2 The device for screening insecticide-resistant predatory mites of the present invention shown in the figure is mainly composed of a lower container 1 and an upper container 2. The lower container 1 is a platform structure with a small top and a large bottom, and the top of the lower container 1 is open; the upper container 2 includes an inner cylinder 22 and an outer cylinder 21 arranged inside and outside, and the tops and bottoms of the outer cylinder 21 and the inner cylinder 22 are open, and the inner cylinder 22 is also in the shape of a platform with a small top and a large bottom. The bottom edge of the inner cylinder 22 is fixedly connected to the bottom edge of the outer cylinder 21 to form a whole, and a receiving chamber 23 is formed between the side wall of the inner cylinder 22 and the side wall of the outer cylinder 21 for holding feed for predatory mites; the shape of the inner cylinder 22 matches the shape of the top of the lower container 1, and the inner cylinder 22 is sleeved on the top of the lower container 1, so that the upper container 2 and the lower container 1 are overlapped, sleeved together, and assembled as a whole to form an insecticide-resistant predatory mite screening device.

[0040] In some embodiments, the lower container 1 and the inner cylinder 22 are both truncated cones that are smaller at the top and larger at the bottom. Compared with polyhedral cones, truncated cones are easier to manufacture and assemble.

[0041] In some embodiments, the inner cylinder 22 is disposed in the lower middle portion of the outer cylinder 21, so as to facilitate the predatory mites to enter the feed holding cavity 23 of the upper container 2 from the lower container 1, thereby reducing the distance.

[0042] In some embodiments, the upper container 2 is formed in one piece and can be manufactured after the mold is opened, which is convenient to manufacture.

[0043] In some embodiments, the outer cylinder 21 is in the shape of a cylinder or a table with a larger top and a smaller bottom, which is convenient for processing and manufacturing.

[0044] In some embodiments, the outer cylinder 21 is in the shape of a platform with a larger upper portion and a smaller lower portion, which is beneficial for balancing the upper and lower forces of the assembled drug-resistant predatory mite screening device and preventing it from tipping over.

[0045] Example 2: Efficient screening method for drug-resistant predatory mites

[0046] 1 Experimental reagents and materials

[0047] Predatory mite: Neoseiulus barkeri;

[0048] Prey: Oval flour-eating mites;

[0049] Pesticide: Chlorpyrifos (chlorpyrifos, CAS No. 2921-88-2).

[0050] The feed in the embodiment of the present invention refers to wheat bran containing flour mites, and flour mites are prey for predatory mites.

[0051] 2 Experimental methods

[0052] 2.1 Method for screening insecticide-resistant predatory mites of the present invention

[0053] The method for screening insecticide-resistant predatory mites of the present invention is carried out using the insecticide-resistant predatory mite screening device of Example 1, and is performed according to the following steps:

[0054] S1. Predatory mites prepared for spraying

[0055] A predatory mite species that can be reared and bred on a large scale by wheat bran-agar mites is selected for the experiment. In this embodiment, Neoseiulus barkeri is selected for the experiment. Wheat bran is placed in a breeding box, and 5,000 oval agar mites and 300 adult Neoseiulus barkeri are inoculated with oval agar mites and Neoseiulus barkeri at a rate of 5000 per 10 g of wheat bran and 300 adult Neoseiulus barkeri. The oval agar mites and Neoseiulus barkeri are raised under the conditions of a light-dark ratio of 14h:10h, a temperature of 25±1°C, and a relative humidity of 80±5%. When the number of predatory mites in each 10 g of wheat bran reaches more than 1,000, the predatory mites are used for the spraying treatment in step S2.

[0056] S2. Chemical treatment

[0057] S2.1. Prepare the upper container

[0058] Add 500 grams of wheat bran feed into the upper container. Every 10 grams of wheat bran feed contains 5,000 to 10,000 oval flour mites.

[0059] S2.2, spraying treatment

[0060] 2500 g of wheat bran feed, which had been raised to contain more than 1000 predatory mites per 10 g of wheat bran, was placed in the lower container of the apparatus of Example 1, and the treated agent LC was used. 90 (or LC 60 , LC 70 , LC 80 ) dosage to treat predatory mites. The specific treatment method is to use a hand-held sprayer to spray the prepared liquid toward the feed, stirring the feed while spraying until the feed is evenly moistened (spray about 500ml of pesticide for 2500g of feed). Then immediately put the upper container on top of the lower container, and the surviving predatory mites will crawl up into the feed in the upper container by themselves.

[0061] LC of the drug to be processed 90 The dose is the concentration that kills 90% of the predatory mites. The LC value of the agent is determined by conventional methods before screening for resistant predatory mites. 90 In this example, the LC of the pesticide chlorpyrifos90 It is 260mg / L.

[0062] S3. Transfer culture

[0063] After the predatory mites are sprayed with pesticides for 24 hours in step S2 (usually, predatory mites that are still alive 24 hours after spraying will not be poisoned by pesticides), the feed containing predatory mites and flour mites in the upper container is moved to the predatory mite breeding box and the predatory mites are continued to be bred and bred using the wheat bran-elliptical flour mite system: the breeding box is provided with wheat bran feed containing elliptical flour mites (containing 1000 to 1500 flour mites / g wheat bran), and the predatory mites are raised under the conditions of a light-dark ratio of 14h:10h, a temperature of 25±1°C, and a relative humidity of 80±5% until the predatory mite population in the breeding box rises to a density of 1000 or more predatory mites per 10 grams of wheat bran.

[0064] S4. Repeated screening

[0065] Take the wheat bran feed cultured in step S3, in which the number of predatory mites in every 10 grams of wheat bran reaches 1000, and repeat steps S2-S3 for multiple times until the resistance level of predatory mites reaches an ideal multiple and then stop screening.

[0066] 2.2 Traditional methods for screening insecticide-resistant predatory mites

[0067] At present, the operating steps of the traditional method for screening insecticide-resistant predatory mites (i.e., the traditional generation-by-generation screening method) are as follows:

[0068] Determination of untreated predatory mite population (F 0 ) to establish a baseline of sensitivity to chlorpyrifos. Then, the adult mites were treated with a dose that killed about 60% to 70% of the population. The surviving individuals were artificially selected for further breeding. The offspring of the surviving individuals were treated successively in succession. The toxicity of chlorpyrifos to Neoseius pasteurii was measured every three generations to calculate the LC 50 and resistance multiples.

[0069] 2.3 Methods for determining the toxicity of predatory mites

[0070] The indoor toxicity determination method of predatory mites adopts the conventional determination method and is carried out according to the following steps:

[0071] 1. Dilute the agent to 5 to 7 concentration gradients within the range of 20% to 80% mortality of predatory mites, and use a Potter spray tower to evenly spray 1 mL of the test agent on a six-hole groove (groove diameter × depth = 15 mm × 2.2 mm) organic glass plate (length × width × height = 78 mm × 58 mm × 5 mm) at a pressure of 2 MPa to form a uniform drug film. At the same time, soak the cover glass in the corresponding agent for 30 seconds, then take it out and put it in a fume hood to dry naturally.

[0072] 2. Place 20 to 30 oval powder-eating mites and nymphs in each groove, and then place 5 2-3 day-old predatory mites, cover with a cover slip, and then use a wet brush to apply clean water around the cover slip to prevent the test mites from escaping. Place them in an artificial incubator for breeding, and check the number of dead female predatory mites under a stereo microscope after 24 hours (touch the mite body with a brush, and those that crawl less than one mite body are considered dead). Use clean water as a control, and the control group mortality rate below 10% is considered a valid determination, and calculate its mortality rate and corrected mortality rate.

[0073] 3. Each treatment was repeated 4 times with 100 heads. The corrected mortality rate was calculated according to the Abbott (1925) formula. The toxicity regression equation and different lethal concentrations LC were calculated using Excel 2016 software. 50 , LC 90 and its 95% confidence interval. Resistance multiple = LC of predatory mite population after treatment 50 / LC of predatory mite population before treatment 50 .

[0074] 3 Experimental results

[0075] The traditional method and the method of the present invention were used to screen the insecticide-resistant Neoseiulus barkeri, respectively, to improve the insecticide resistance of Neoseiulus barkeri, and the screening efficiency of the two screening methods was compared after 180 days of screening. The results are shown in Table 1. Through the screening method of the present invention, the resistance multiple of Neoseiulus barkeri to chlorpyrifos reached 620.03 times, while through the screening method of the present invention, the resistance multiple of Neoseiulus barkeri to chlorpyrifos was only 5.52 times. The results show that the screening efficiency of the resistance of Neoseiulus barkeri by the method of the present invention is much higher than that of the traditional generation-by-generation screening method.

[0076] Table 1 Comparison of different screening methods to improve resistance of Neoseiulus pasteurii

[0077]

Claims

1. A device for screening insecticide-resistant predatory mites, characterized in that: The device comprises a lower container and an upper container, wherein the lower container is a platform structure with a small top and a large bottom, and the top of the lower container is open; the upper container comprises an inner cylinder and an outer cylinder arranged inside and outside, the top and bottom of the outer cylinder and the inner cylinder are both open, the inner cylinder is also a platform shape with a small top and a large bottom, the bottom edge of the inner cylinder is fixedly connected to the bottom edge of the outer cylinder to form a whole, and a accommodating cavity is formed between the side wall of the inner cylinder and the side wall of the outer cylinder to hold feed for predatory mites; the shape of the inner cylinder matches the shape of the top of the lower container, and the inner cylinder is sleeved on the top of the lower container, so that the upper container and the lower container are overlapped and sleeved together to be assembled into a drug-resistant predatory mite screening device.

2. The device for screening insecticide-resistant predatory mites according to claim 1, characterized in that: The lower container and the inner cylinder are both frustums that are smaller at the top and larger at the bottom.

3. The device for screening insecticide-resistant predatory mites according to claim 1, characterized in that: The outer cylinder is in the shape of a cylinder or a platform with a larger top and a smaller bottom; the inner cylinder is arranged in the middle and lower part of the outer cylinder; and the upper container is formed in one piece.

4. The device for screening insecticide-resistant predatory mites according to claim 3, characterized in that: The outer cylinder is in the shape of a platform with a larger top and a smaller bottom.

5. A method for efficiently screening drug-resistant predatory mites, characterized in that: The method is carried out using the device for screening insecticide-resistant predatory mites according to any one of claims 1 to 4, comprising the following steps: S1. Predatory mites prepared for spraying Preparing a substrate containing a set population of predatory mites for spraying in the lower container in step S2; S2. Chemical treatment S2.

1. Prepare the upper container Adding a substrate containing predatory mite prey into the holding chamber of the upper container; S2.2, spraying treatment Place the prepared matrix containing predatory mites into the container below and use LC of the treatment agent to 60 ~ 90 Spray the substrate in the lower container with the appropriate dosage, stirring the substrate while spraying until the substrate is evenly moistened, then put the upper container on top of the lower container, and the surviving predatory mites will crawl up into the substrate in the upper container by themselves; S3. Transfer culture 22 hours after the spraying treatment of the predatory mites in step S2, the substrate containing the predatory mites and their prey in the upper container is moved to the predatory mite breeding box, in which the substrate containing the predatory mite prey is placed, and the predatory mites are continued to be bred and multiplied until the population of the predatory mites in the breeding box reaches the set population; S4. Repeated screening The substrate containing predatory mites cultured in step S3 is placed in the lower container, and steps S2-S3 are repeated for multiple times until the resistance level of the predatory mites reaches a preset multiple, and then screening is stopped to obtain drug-resistant predatory mites.

6. The method according to claim 5, characterized in that: The matrix is ​​wheat bran.

7. The method according to claim 5, characterized in that: The drug to be treated in step S2.2 is selected from chlorpyrifos, cypermethrin, lambda-cyhalothrin, avermectin, and amitraz, and the spraying amount is calculated based on 80-120 ml of the drug per 500 grams of feed.

8. The method according to claim 5, characterized in that: The population density of the predatory mites placed in the container below for spraying treatment is 1,000 or more predatory mites per 10 grams of substrate.

9. The method according to claim 8, characterized in that: The substrate containing predatory mites added to the upper container contains 5,000 to 10,000 predatory mites for every 10 grams of substrate; and 500 grams of substrate containing predatory mites in the upper container are provided for every 2,500 grams of substrate containing predatory mites in the lower container.

10. The method according to claim 5, characterized in that: The predatory mite is selected from Neoseiulus cucumeri, Neoseiulus californicus, Neoseiulus barnesi and Neoseiulus sternii; the predatory mite prey is selected from Aleuroglyphus ovatus.

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