A root mite diet, rearing method, and indoor bioassay method.

Abstract

This invention relates to a root mite feed, a rearing method, and an indoor bioassay method, belonging to the field of plant protection technology. The root mite feed includes crop seed powder, yeast extract, milk powder, vitamin C, choline chloride, inositol, a coagulant, a preservative, and water, wherein the crop seed powder is soybean flour and / or corn flour. The rearing method involves introducing root mites into the feed of this invention and rearing them at 23 to 27°C and 55% to 75% humidity. The indoor bioassay method includes: 1) preparing a bioassay tube; 2) obtaining a test solution; 3) cutting the feed into small pieces; 4) soaking the feed pieces in the test solution and then removing them; 5) placing the feed pieces treated with the test solution into the bioassay tube and then introducing root mites; 6) soaking the bioassay tube containing root mites in the test solution, removing it, and rearing it; 7) periodically detecting the number of dead mites and performing statistical analysis.

Description

Technical Field

[0001] This invention relates to the field of plant protection technology, and more specifically to an indoor bioassay method for evaluating whether a test substance has an impact on the safety of root mites. Background Technology

[0002] Rhizoglyphus robini Claparede, also known as the Robin's root mite, belongs to the genus Rhizoglyphus in the family Acaridae of the class Arachnida in the phylum Arthropoda. It is a globally important underground pest mite. It has a wide host range, infesting more than 30 kinds of plants, including onions, garlic, leeks, potatoes, eggplants, carrots, beans, wheat, gladiolus, hyacinths, and pinellia. In the field, it congregates in the underground parts of plants, feeding on plant tissues, inducing and stimulating the occurrence of pathogens, leading to yellowing leaves, stunted growth, wilting, rotting, and death of the plant. Reports indicate that in severely affected onions, the damage rate can reach 50%, and the mortality rate can exceed 30%. In severely affected gladiolus, the loss rate can reach 54% to 90%.

[0003] Early diagnosis of the Robinia pseudomallei is extremely difficult due to its small size and rapid reproduction rate. Symptoms typically only appear in crops during the later stages of infestation, by which time the mites have already swarmed and are feeding on the affected crops. The long-term, excessive, and inappropriate use of chemical pesticides by many farmers has led to strong pesticide resistance in the Robinia pseudomallei, creating a vicious cycle of re-outbreaks. This not only increases environmental pollution but also harms beneficial insects and reduces the quality and safety of agricultural products. Therefore, it is necessary to screen for effective pesticides against Robinia pseudomallei to provide better and more environmentally friendly control methods and strategies. Summary of the Invention

[0004] One aspect of the present invention provides a feed for root mite, comprising crop seed powder, yeast extract, milk powder, vitamin C, choline chloride, inositol, coagulant, preservative and water, wherein the crop seed powder is soybean powder and / or corn powder.

[0005] In one specific embodiment, the coagulant is agar powder.

[0006] In one specific embodiment, the preservative is sorbic acid.

[0007] In one specific embodiment, the ingredients are: 22 to 26 parts by weight of crop seed powder, 20 to 24 parts by weight of yeast extract, 3 to 8 parts by weight of milk powder, 1.5 to 5 parts by weight of vitamin C, 0.1 to 0.3 parts by weight of choline chloride, 0.05 to 0.2 parts by weight of inositol, 3 to 9 parts by weight of coagulant, 0.5 to 2 parts by weight of preservative, and 200 to 300 parts by weight of water.

[0008] In one specific embodiment, the feed is prepared by the following operation: the coagulant is mixed with water, boiled, and then the preservative, crop seed powder, yeast extract, milk powder, vitamin C, choline chloride and inositol are added, mixed evenly, and cooled to solidify to obtain the feed.

[0009] In one specific embodiment, the root mite is *Rhizoctonia rubescens*.

[0010] The second invention provides a method for raising root mites, which includes the following steps: feeding root mites into the feed described in any one of the first inventions, and raising them at 23 to 27°C and 55% to 75% humidity.

[0011] In one specific embodiment, the ratio of female to male root mites is (1 to 2):1.

[0012] In one specific embodiment, the root mite is *Rhizoctonia rubescens*.

[0013] The third invention provides a method for indoor bioassay of root mites, which includes the following steps:

[0014] 1) Cut off the part of the centrifuge tube below 1 / 3 of the tube opening, and weld the cut end with wire mesh to obtain the raw test tube;

[0015] 2) Obtain the solution of the reagent to be tested;

[0016] 3) Cut the feed according to any one of the present invention into small pieces to obtain feed blocks;

[0017] 4) Immerse the feed block in the test reagent solution, remove it, and obtain the feed block treated with the test reagent;

[0018] 5) The feed block treated with the test agent is placed into the test tube, then root mites are introduced, and the cap is closed to obtain the test tube containing root mites;

[0019] 6) Immerse the test tube containing root mites in the test solution, remove it, and place it in a humidified dish containing 2% agar for rearing;

[0020] 7) Regularly test the number of dead insects and perform statistical analysis.

[0021] In one specific embodiment, in step 3), the feed block is soaked in the test reagent solution for 8 to 15 minutes.

[0022] In one specific embodiment, in step 3), after the feed block is soaked in the test reagent solution and then removed, the solvent on the surface of the feed block is absorbed with filter paper to obtain the feed block treated with the test reagent.

[0023] In one specific embodiment, in step 6), the test tube containing root mites is immersed in the test agent solution for 8 to 15 seconds.

[0024] In one specific embodiment, in step 6), after the biotest tube containing root mites is immersed in the test reagent solution and then removed, the solvent at the bottom of the biotest tube is absorbed with filter paper and then placed in a humidified dish containing 2% agar.

[0025] In one specific embodiment, in step 6), the animals are raised in complete darkness with a photoperiod of 23 to 27°C and 55% to 75% humidity.

[0026] In one specific embodiment, the root mite is *Rhizoctonia rubescens*.

[0027] The beneficial effects of this invention are:

[0028] This invention provides an artificial feed for indoor rearing of root mites. This invention has found that inositol and choline chloride in the artificial feed can shorten the life cycle of root mite larvae and nymphs.

[0029] Furthermore, based on the artificial feed for root mites, this invention provides a method for indoor rearing of root mites and an indoor bioassay method for root mites. In this invention, through research on rearing containers, conditions, and feed, an artificial rearing method for root mites was established, meeting the requirements for insect use in conducting indoor bioactivity assays of root mites. Simultaneously, through the optimization of the containers, feed, and testing conditions used for indoor bioactivity assays of root mites, a method for conducting indoor bioactivity assays of root mites was established. Attached Figure Description

[0030] Figure 1 The bioassay tube of the present invention is shown for the indoor bioactivity assay of *Rhizoctonia solani*. Detailed Implementation

[0031] The present invention will be further described in detail below through preferred embodiments, but these embodiments do not constitute a limitation thereof.

[0032] Unless otherwise specified, all reagents used in the embodiments of this invention are commercially available.

[0033] Robinia root mite feed and rearing methods

[0034] Example 1

[0035] Feed preparation:

[0036] (1) Feed composition: 24g soybean meal, 22g yeast extract, 5g milk powder, 2.5g vitamin C, 0.2g choline chloride, 0.1g inositol, 6g agar powder, 1g sorbic acid, 250mL distilled water;

[0037] (2) Dissolve agar powder in distilled water, boil it in a microwave oven, add sorbic acid first, then add the other ingredients that are mixed evenly, stir well, pour it into a petri dish with a diameter of 9cm and let it cool and solidify naturally. Store it at 4℃ for later use.

[0038] Feeding methods:

[0039] Twenty male and twenty female adult mites from the Rosa robinia population collected from the field were introduced into the above-mentioned cooled and solidified artificial feed. The culture dish was covered, the population name and the date of introduction were marked, and the dish was placed in the insect rearing room for rearing. The temperature was controlled at 25°C and the relative humidity was controlled at 65%.

[0040] The growth and reproduction of Robinia pseudomallei were observed regularly. Each generation of Robinia pseudomallei lasted less than 20 days. After one year of rearing, the survival days of each stage of the offspring and the number of eggs laid by a single female were counted. The results are shown in Table 1.

[0041] Example 2

[0042] The feed preparation is the same as in Example 1.

[0043] The rearing temperature was 23°C and the relative humidity was 75%, and other conditions were the same as in Example 1.

[0044] The growth and reproduction of the Robinia pseudomallei were observed regularly. After one year of rearing, the survival days of each stage of the offspring and the number of eggs laid by a single female were recorded. The results are shown in Table 1.

[0045] Example 3

[0046] The feed preparation is the same as in Example 1.

[0047] The rearing temperature was 27°C and the relative humidity was 55%, and other conditions were the same as in Example 1.

[0048] The growth and reproduction of the Robinia pseudomallei were observed regularly. After one year of rearing, the survival days of each stage of the offspring and the number of eggs laid by a single female were recorded. The results are shown in Table 1.

[0049] Example 4

[0050] Replace soybean meal with corn meal in the feed formulation, otherwise the same as in Example 1.

[0051] The growth and reproduction of the Robinia pseudomallei were observed regularly. After one year of rearing, the survival days of each stage of the offspring and the number of eggs laid by a single female were recorded. The results are shown in Table 1.

[0052] Example 5

[0053] Replace soybean meal with corn meal in the feed formulation, otherwise the same as in Example 2.

[0054] The growth and reproduction of the Robinia pseudomallei were observed regularly. After one year of rearing, the survival days of each stage of the offspring and the number of eggs laid by a single female were recorded. The results are shown in Table 1.

[0055] Example 6

[0056] Replace soybean meal with corn meal in the feed formula, otherwise the same as in Example 3.

[0057] The growth and reproduction of the Robinia pseudomallei were observed regularly. After one year of rearing, the survival days of each stage of the offspring and the number of eggs laid by a single female were recorded. The results are shown in Table 1.

[0058] Comparative Example 1

[0059] The feed formulation is free of choline chloride and inositol, but otherwise the same as in Example 1.

[0060] The growth and reproduction of the Robinia pseudomallei were observed regularly. After one year of rearing, the survival days of each stage of the offspring and the number of eggs laid by a single female were recorded. The results are shown in Table 1.

[0061] Table 1

[0062]

[0063] Note: The data in the table are the average standard error. Different letters after the data in the same column indicate that the differences are significant at the 5% level as determined by Duncan's new multiple range test.

[0064] According to the data in Table 1, feeding Robinia pseudomallei with the feed formulations of Examples 1 to 6 can obtain a stable and consistent source of pests under a wide range of temperature and humidity conditions. It also has the advantages of readily available feed components, simple feeding methods, and low labor and raw material costs.

[0065] Indoor bioassay method for Rosa robinis

[0066] Example 7

[0067] Take a common 1.5mL plastic centrifuge tube from the laboratory, cut off the lower third of the tube from the opening, and weld a high-mesh wire mesh (enough to prevent Robinia pseudomallei mites from escaping) to the cut. Trim the wire mesh with scissors to obtain the bioassay tube. The prepared bioassay tube is shown below. Figure 1 As shown.

[0068] (1) 5% abamectin emulsion (Tianmen Spring Plant Protection Co., Ltd.) was diluted with sterile water to prepare 5 series of concentration gradients to obtain abamectin treatment solutions of different concentrations, wherein the concentrations were 100mg / L, 200mg / L, 400mg / L, 800mg / L and 1600mg / L respectively, and sterile water was used as the control group CK.

[0069] (2) Cut the feed prepared in Example 1 into squares of 0.5cm×0.5cm, and soak them in avermectin treatment solutions and sterilized water of various concentration gradients for 10 minutes. Then, dry the surface moisture of the feed on filter paper to obtain avermectin-treated feed blocks and control feed blocks.

[0070] (3) After marking the test tubes, use clean tweezers to put the feed blocks treated with abamectin and the control feed blocks into the test tubes respectively. Then, the control group is introduced first. That is, 30 adult robin mites of the offspring generation obtained by the feeding method in Example 1 are introduced into the test tubes using a No. 0 brush. Then, 30 adult robin mites of the offspring generation obtained by the feeding method in Example 1 are introduced into the test tubes one year after the control group is raised from low concentration to high concentration. Each test tube is a replicate, and the experiment is repeated 3 times.

[0071] (4) Immerse the bioassay tubes inoculated with Robinia pseudomallei in the corresponding concentration of the drug solution for 10 seconds, then blot the bottom of the bioassay tubes dry on filter paper and cover the top of the bioassay tubes. This step is also performed first as a control group, and then proceeded from low concentration to high concentration.

[0072] (5) Place the live test tubes in a humidified dish containing 2% agar, and then place them in an insect culture box (temperature 25±2℃, relative humidity 65%, photoperiod is complete darkness) for rearing;

[0073] (6) After 72 hours (which may be adjusted appropriately according to the characteristics of the test agent or the purpose of the test), check the number of dead mites. The results are shown in Table 2. Among them, the mites that only have one leg shaking at a low frequency or do not move at all when lightly touched are considered dead.

[0074] Table 2

[0075]

[0076] As can be seen from Table 2, there were no deaths in the CK group, indicating that the biopsy results were stable and accurate.

[0077] Comparative Example 2

[0078] The adult Robinia Root Mite offspring obtained one year after the rearing method in Example 1 were used as the test subject.

[0079] Eight concentration gradients of abamectin treatment solution were prepared, using the same method as in Example 7.

[0080] The toxicity assay was performed using the method described in Chen's 1990 article, "An improved method for determining the susceptibility of Rhizoglyphusrobini and R. setosus (Acarina: Acaridae) to pesticieds." Specifically, a filter paper was placed in a 30mm diameter petri dish. 200μL of avermectin at various concentrations was added to each dish. Twenty adult Rhizoglyphus robini mites were picked up with a size 0 brush and placed into each dish. The dish opening was then sealed with tape to prevent moisture evaporation. The dishes were placed in an insect incubator at 25±2℃, 65% relative humidity, and complete darkness. Results were checked after 72 hours. Mites that showed only a low-frequency twitching of one leg or remained completely still upon gentle touching were considered dead. The results are recorded in Table 3.

[0081] Table 3

[0082]

[0083] Data Comparison and Analysis between Example 7 and Comparative Example 2

[0084] Based on the data in Table 2, the b±SE and LC values ​​of *Rhizoctonia solani* from Example 7 were obtained using Polo Plus Version 1.0 software. 50 The values ​​and their 95% confidence intervals are shown in Table 4.

[0085] Based on the data in Table 3, the b±SE and LC values ​​of *Rhizoctonia solani* from Comparative Example 2 were obtained using Polo Plus Version 1.0 software. 50 The values ​​and their 95% confidence intervals are shown in Table 4.

[0086] LC 50 If the 95% confidence intervals overlap, the difference is considered not significant; if they do not overlap, the difference is considered significant.

[0087] Table 4

[0088] Example Slope (± standard error) LC50 (mg / L) (95% confidence interval) Example 7 1.000±0.147 306.483(222.813-404.046)a Comparative Example 2 0.887±0.110 11546.896(7235.577-22967.962)b

[0089] The results in Table 4 show that the test results for the same agent using the two different treatments, Example 7 and Comparative Example 2, are significantly different. Specifically, the LC50 of the bioassay using the method in Comparative Example 2 is significantly lower. 50 The value was significantly higher than the LC50 value when performing bioassays using the method described in Example 7. 50 Value, and due to the LC of Comparative Example 2 50 Values ​​exceeding 10000 mg / L would lead to the erroneous conclusion that abamectin has no insecticidal activity against *Rhizoctonia solani* based solely on the data from Comparative Example 2. Since *Rhizoctonia solani* feeds on the underground parts of plants, screening pesticides for its control must consider not only contact action but also stomach poisoning. The bioassay method of this invention takes both aspects into account; while Comparative Example 2 only considers contact action. Therefore, the bioassay method for *Rhizoctonia solani* of this invention is more scientific and reasonable, while the bioassay method of Comparative Example 2 is flawed and cannot reflect the true insecticidal activity of the pesticide.

Claims

1. A method for indoor bioassay of root mites, comprising the following steps: 1) cutting off the centrifuge tube below 1 / 3 of the tube opening, welding a wire mesh at the cut opening to obtain a bioassay tube; the wire mesh is a high-mesh wire mesh, and the high-mesh wire mesh is determined according to the fact that root mites cannot escape; 2) obtaining a solution of a test agent; 3) cutting the feed into small pieces to obtain feed pieces; the feed comprising crop seed powder, yeast extract, milk powder, vitamin C, choline chloride, inositol, coagulating agent, preservative and water, wherein, the crop seed powder is bean powder and / or corn powder; 4) soaking the feed block in the test agent solution for 8 to 15 min, taking it out to obtain a test agent-treated feed block; 5) loading the test agent-treated feed block into the bioassay tube, then introducing root mites, covering the lid on the top of the bioassay tube to obtain a bioassay tube containing root mites; 6) soaking the bioassay tube containing root mites in the test agent solution for 8 to 15 s, taking it out and placing it in a moist petri dish containing 2% agar for feeding; 7) regularly detecting the number of dead insects and performing statistical analysis.

2. The method of claim 1, wherein, In step 4), after soaking the feed block in the test agent solution and taking it out, the solvent on the surface of the feed block is absorbed dry with filter paper to obtain a test agent-treated feed block.

3. The method of claim 1, wherein, In step 6), after soaking the bioassay tube containing root mites in the test agent solution and taking it out, the solvent at the bottom of the bioassay tube containing root mites is absorbed dry with filter paper and then placed in a moist petri dish containing 2% agar.

4. The method of claim 1, wherein, In step 6), the feeding is carried out at a temperature and humidity of 23 to 27°C and 55% to 75%, and the light cycle is complete darkness.

5. The method of claim 1, wherein, The coagulant is agar powder; and / or the preservative is sorbic acid.

6. The method of claim 1, wherein, Crop seed powder 22 to 26 parts by mass, yeast extract 20 to 24 parts by mass, milk powder 3 to 8 parts by mass, vitamin C 1.5 to 5 parts by mass, choline chloride 0.1 to 0.3 parts by mass, inositol 0.05 to 0.2 parts by mass, coagulant 3 to 9 parts by mass, preservative 0.5 to 2 parts by mass, and water 200 to 300 parts by mass.

7. The method of claim 1, wherein, The feed is prepared by mixing the coagulant with water, boiling, then adding the preservative, crop seed powder, yeast extract, milk powder, vitamin C, choline chloride, and inositol, mixing uniformly, and cooling and coagulating to obtain the feed.

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