Method for improving resistance of natural enemy mites to beauveria bassiana

Abstract

The invention discloses a method for improving resistance of natural enemy mites to beauveria bassiana, and relates to the technical field of resistance research of biocontrol natural enemy. The method comprises the following steps: S1, feeding natural enemy mites, wherein the natural enemy mites are predatory mites; s2, carrying out starvation treatment on natural enemy mites; s3, after the natural enemy mites are subjected to starvation treatment, beta-1. 3-glucan is adopted for feeding; s4, detecting the resistance of the beauveria bassiana after feeding treatment in S3, determining the survival rate of the natural enemy mites, and verifying the improvement rate. The background mechanism is clear, up-regulation expression of a PGRP (Protein Growth Reduction Protein) of a Neoseiulus barkeri pattern recognition receptor is induced in a targeted manner based on beta-1. 3-glucan, so that immune response of a mite body is activated in advance, and the resistance to biocontrol fungi is obtained in a short time through an'immune memory 'principle; the operation time is short, and the fitness cost caused by long-term domestication is avoided; and the resistance of the predatory mites to biocontrol fungi can be accurately improved, so that the tolerance of the predatory mites in different batches is stable.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of resistance research of biocontrol natural enemies, and particularly relates to a method for improving the resistance of natural enemy mites to Beauveria bassiana. BACKGROUND

[0002] With the continuous development of ecological agriculture and biological control technology, insect pathogenic fungi as an important biological control agent have achieved remarkable results in many pest control fields. These fungi not only have toxic effects on pests, but also can reduce pest population, inhibit their reproduction and transmission, and reduce the use of chemical pesticides, thereby having less negative impact on the environment and ecosystem.

[0003] However, the application of insect pathogenic fungi also faces some challenges. The potential pathogenic and lethal risk of broad-spectrum insect pathogenic fungi to predatory mites is still a key bottleneck in the promotion of fungus-mite synergistic pest control.

[0004] Therefore, it is urgent to seek a method for improving the resistance of predatory mites to biocontrol fungi and reducing the side effects of biocontrol fungi on non-target predatory mites in fungus-mite synergistic control strategies, and a new solution to the above problems is needed. SUMMARY

[0005] The application aims to provide a method for improving the resistance of natural enemy mites to Beauveria bassiana, which aims to improve the tolerance of predatory mites to biocontrol fungi and reduce the lethal and pathogenic risk of biocontrol fungi to non-target predatory mites in fungus-mite synergistic control strategies, so as to solve the technical problems proposed in the background art.

[0006] To achieve the above-mentioned purpose, the application provides the following technical scheme: a method for improving the resistance of natural enemy mites to Beauveria bassiana, comprising at least the following steps:

[0007] S1: feeding natural enemy mites, i.e. predatory mites;

[0008] S2: starving the natural enemy mites;

[0009] S3: feeding the natural enemy mites with beta-1.3-glucan after starvation;

[0010] S4: detecting the resistance of the natural enemy mites to Beauveria bassiana after the feeding treatment of S3, determining the survival rate of the natural enemy mites, and confirming the improvement rate.

[0011] Further, S1 comprises at least the following steps:

[0012] The predatory mites take the prey mites as food, and put the food of the prey mites into a feeding container, and the prey mites are bred and propagated in a large scale under the environmental conditions of a temperature of 25±1℃, a relative humidity of 70%-80%, and a light cycle of 14L:10D;

[0013] The feeding container is a 5L transparent fresh-keeping box, a 10cm*6cm hole is formed in the center of the fresh-keeping box cover, and a 300-mesh screen is welded for ventilation to prevent the predatory mites from escaping;

[0014] The fresh-keeping box is placed on a tray with water to maintain the relative humidity in the feeding container, the food of the prey mites needs to be shaken every day, and the food of the prey mites is replaced every 7 days.

[0015] Further, the S2 at least includes the following steps:

[0016] A circular sponge with a smaller diameter is placed in a 15cm-diameter culture dish;

[0017] A circular black cloth with the same diameter as the circular sponge is placed on the circular sponge, and the circular sponge and the black cloth are completely soaked with tap water, and the water surface is flush with the top of the sponge;

[0018] A circular plastic film is placed on the black cloth, and the diameter of the circular plastic film is slightly smaller than that of the black cloth, but the difference between the diameters of the circular plastic film and the black cloth is not less than 10cm;

[0019] An 8cm-diameter culture dish is placed on the circular plastic film;

[0020] The mixture in the fresh-keeping box is poured out and sieved through a 60-mesh sieve to obtain a mixture of "predatory mites-prey mites-prey mite food", the mixture is swept into an 8cm-diameter culture dish with a soft brush, and 1,000 viable female predatory mite adults are selected and starved in the 8cm-diameter culture dish for 24h.

[0021] Further, the S3 at least includes the following steps:

[0022] S3.1: A 20% sucrose solution is used as a solvent to prepare a β-1,3-glucan solution with a concentration of 1000ng / μL;

[0023] S3.2: A trace amount of blue dye is added to the β-1,3-glucan solution obtained in S3.1 as a color developing indicator;

[0024] S3.3: The solution obtained in S3.2 is filtered through a 0.22μm filter membrane, and 50μL of the solution is used to feed the Phytoseiulus persimilis for 12h.

[0025] Further, the S4 at least includes the following steps:

[0026] The predatory mites are swept onto the plastic film using a soft brush;

[0027] Subsequently, 1.5 mL of a high-concentration 2*109 cell / mL Beauveria bassiana spore suspension is sprayed;

[0028] After the spore suspension is air-dried, a single female predatory mite with vitality is picked in an observation chamber with prey mites added;

[0029] The observation chamber is placed in a normal feeding environment, and the survival rate of the predatory mites is recorded every day, and the survival rate of the natural enemy mites is observed continuously for 9 days to confirm the promotion rate.

[0030] Further, the main body of the observation chamber is a central-punched rectangular transparent organic glass plate I, the central punch is a round hole, one side of the round hole is a 300-mesh stainless steel screen, the other side of the round hole is covered with a rectangular transparent organic glass plate II, and the rectangular transparent organic glass plate II is clamped and fixed with a dovetail clamp.

[0031] Compared with the prior art, the beneficial effects of the present application are:

[0032] 1. The background mechanism of the present application is clear, which is based on the up-regulation of PGRP protein of the model recognition receptor of Neoseiulus barkeri induced by targeting β-1.3-glucan, thereby pre-activating the immune response of the mite body, and obtaining resistance to biocontrol fungi in a short period through the principle of "immunological memory";

[0033] 2. The operation time of the present application is short, which avoids the fitness cost brought by long-term domestication;

[0034] 3. The present application can more accurately improve the resistance of predatory mites to biocontrol fungi, so that the tolerance of different batches of predatory mites is stable;

[0035] 4. The present application is based on the major bottleneck of the synergistic pest control strategy, and reduces the side effects of biocontrol fungi on non-target natural enemy predatory mites during the synergistic pest control of bacteria and mites. DETAILED DESCRIPTION

[0036] The technical solutions in the embodiments of the present application will be described below, obviously, the described embodiments are only a part of the embodiments of the present application, not all the embodiments.

[0037] The present application found in the previous study that fungal cell wall polysaccharide β-1.3-glucan can induce the up-regulation of PGRP protein of the model recognition receptor of Neoseiulus barkeri, and the expression of PGRP can improve the resistance of predatory mites to Beauveria bassiana, and the silencing of PGRP leads to greater susceptibility of predatory mites to Beauveria bassiana.

[0038] Therefore, the application is based on the principle of "immunological memory", and pre-treatment of predatory mites with β-1.3-glucan before the combination of biocontrol fungi and natural enemy mites can greatly improve the resistance of predatory mites to biocontrol fungi, which is of great significance to improving the synergistic control effect of fungi and mites.

[0039] Example one:

[0040] A method for improving the resistance of natural enemy mites to Beauveria bassiana, comprising at least the following steps:

[0041] S1: feeding natural enemy mites, i.e. predatory mites;

[0042] S2: starving the natural enemy mites;

[0043] S3: feeding the natural enemy mites with β-1.3-glucan after starvation;

[0044] S4: detecting the resistance of the natural enemy mites to Beauveria bassiana after the feeding treatment of S3, determining the survival rate of the natural enemy mites, and confirming the improvement rate.

[0045] S1 at least comprises the following steps:

[0046] The predatory mites take the prey mites as food, and the prey mites' food is put into the feeding container, and the scale-up breeding is carried out under the environmental conditions of temperature 25±1℃, relative humidity 70%-80%, and light cycle 14L:10D;

[0047] The feeding container is a 5L transparent fresh-keeping box, the central part of the fresh-keeping box cover is provided with a 10cm×6cm hole, and a 300-mesh screen is welded for ventilation to prevent the predatory mites from escaping;

[0048] The fresh-keeping box is placed on a tray with water to maintain the relative humidity in the feeding container, and the prey mites' food needs to be shaken every day, and the prey mites' food is replaced every 7 days.

[0049] S2 at least comprises the following steps:

[0050] A smaller diameter circular sponge is placed in a 15cm diameter culture dish;

[0051] A circular black cloth with the same diameter as the circular sponge is placed on the circular sponge, and the circular sponge and the black cloth are completely soaked with tap water, with the water surface level with the top of the sponge;

[0052] A circular plastic film is placed on the black cloth, and the diameter of the circular plastic film is slightly smaller than that of the black cloth, but the difference between the diameters of the circular plastic film and the black cloth is not less than 10cm;

[0053] An 8cm diameter culture dish is placed on the circular plastic film;

[0054] The mixture in the preservation box was poured out and sieved through a 60-mesh sieve to obtain a "predatory mite-prey mite-prey mite food" mixture, which was swept into a 8-cm-diameter culture dish with a soft brush, and 1,000 viable female adult predatory mites were picked into the 8-cm-diameter culture dish and starved for 24 h.

[0055] In different embodiments, 1000-2000 female adult predatory mites can be picked into the feeding chamber and starved for 24 h. The feeding device is composed of different types of transparent organic glass plates, 300-mesh stainless steel sieves, cowpea leaves and feeding chambers of dovetail clamps. The main body of the feeding chamber is a central perforated (diameter: 2.5 cm; height: 2 cm) rectangular transparent organic glass plate III (length x width x height: 4 cm x 4 cm x 2 cm), which is covered with a rectangular transparent organic glass plate IV (length x width x height: 3 cm x 3 cm x 0.3 cm) on the top, and is sequentially followed by a 300-mesh stainless steel sieve, a central perforated (diameter: 2.5 cm; height: 0.3 cm) rectangular transparent organic glass plate V (length x width x height: 4 cm x 4 cm x 0.3 cm), a cowpea leaf and a rectangular transparent organic glass plate VI (length x width x height: 4 cm x 4 cm x 0.3 cm) on the bottom, and finally clamped with a dovetail clamp.

[0056] S3 at least includes the following steps:

[0057] S3.1: A 20% sucrose solution was used as a solvent to prepare a β-1,3-glucan solution with a concentration of 1000 ng / μL;

[0058] S3.2: A trace amount of blue dye was added to the β-1,3-glucan solution obtained in S3.1 as a color developing indicator;

[0059] S3.3: The solution obtained in S3.2 was filtered through a 0.22 μm filter membrane, and 50 μL of the filtrate was used to feed the B. neesoni for 12 h.

[0060] S4 at least includes the following steps:

[0061] The predatory mites were swept onto the plastic film with a soft brush;

[0062] Subsequently, 1.5 mL of a high-concentration 2×109cell / mL Beauveria bassiana spore suspension was sprayed;

[0063] After the spore suspension was air-dried, a single viable female adult predatory mite was picked into an observation chamber with prey mites;

[0064] The observation chamber was placed in a normal feeding environment, and the survival rate of the predatory mites was recorded daily. The survival rate of the natural enemy mites was observed continuously for 9 days to confirm the increase rate.

[0065] The main body of the observation chamber is a central perforated rectangular transparent organic glass plate I. The central perforation is a circular hole. One side of the circular hole is a 300-mesh stainless steel screen. The other side of the circular hole is covered with a rectangular transparent organic glass plate II. The rectangular transparent organic glass plate II is clamped and fixed with a dovetail clamp.

[0066] Example Two:

[0067] This example is based on the above example and uses the predatory mite Neoseiulus barkeri as an example to illustrate the specific embodiment:

[0068] (1) Neoseiulus barkeri feeding

[0069] Neoseiulus barkeri was fed on oval flour mites that fed on wheat bran. The scale-up feeding and breeding was carried out under the environmental conditions of temperature 25±1℃, relative humidity 70-80%, and light cycle 14L:10D. The feeding container was a 5L transparent preservative box. The preservative box cover had a 10cm×6cm hole in the center, and a 300-mesh screen was welded for ventilation to prevent the predatory mites from escaping. The preservative box was placed on a tray with water to maintain the relative humidity in the incubator above 80%. The wheat bran needed to be shaken and mixed evenly every day, and the fresh wheat bran was replaced every 7 days.

[0070] (2) Starvation treatment of Neoseiulus barkeri

[0071] A circular sponge slightly smaller than the culture dish was placed in a 15cm diameter culture dish. A circular black cloth of the same size as the sponge was placed on the sponge. The sponge and black cloth were completely soaked with tap water, with the water level at the top of the sponge. A circular plastic film was placed on the black cloth, with a diameter slightly smaller than the black cloth but not less than 10cm. A 8cm diameter culture dish was placed on the plastic film. The wheat bran fed in the preservative box was sieved with a 60-mesh sieve to obtain a mixture of "predatory mites-flour mites-wheat bran powder". The mixture was swept into the 8cm diameter culture dish with a soft brush. 1,000 active female Neoseiulus barkeri adult mites were selected and starved for 24h in the feeding device

[0072] (3) β-1.3-glucan feeding of Neoseiulus barkeri: 20% sucrose solution was used as the solvent, and a concentration of 1000ng / μL of β-1,3-glucan solution was prepared, and a trace of blue dye was added as a color developing indicator. After filtration through a 0.22μm filter membrane, 50μL of the solution was used to feed Neoseiulus barkeri for 12h.

[0073] (4) Resistance detection of Neoseiulus barkeri to Beauveria bassiana: After feeding β-1,3-glucan for 12 hours, the Neoseiulus barkeri was swept onto the plastic film of the above-mentioned water culture device (sponge-black cloth-plastic film water culture device) using a soft brush. Then, 1.5 mL of high-concentration 2x109 cell / mL of Beauveria bassiana spore suspension was sprayed. After the spore suspension was air-dried, the active single-head female adult Neoseiulus barkeri was picked and placed in an observation chamber with oval Tyrophagus putrescentiae. The observation chamber was placed in a normal feeding environment, and the survival rate of Neoseiulus barkeri was recorded every day, and the observation was continuously conducted for 9 days. The main body of the observation chamber was a central-punched rectangular transparent organic glass plate I, one side of the round hole was a 300-mesh stainless steel screen, and the other side was covered with a rectangular transparent organic glass plate II, and then clamped with a dovetail clamp.

[0074] Resistance judgment: In the resistance detection of Neoseiulus barkeri to Beauveria bassiana, the survival rate of Neoseiulus barkeri pretreated with β-1,3-glucan was 55% on the 9th day, while the survival rate of the general female adult was 30%, and there was a significant difference between the two.

[0075] It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be regarded as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application.

Claims

1. A method of increasing the resistance of a predatory mite to Beauveria bassiana, characterized in that: At least comprising the following steps: S1: feeding the natural enemy mites, i.e. predatory mites; S2: starving the natural enemy mites; S3: feeding the natural enemy mites with β-1.3-glucan after starvation; S4: detecting the resistance of Beauveria bassiana after the feeding treatment of S3, determining the survival rate of the natural enemy mites, and confirming the promotion rate.

2. The method of claim 1, wherein the predatory mite species is selected from the group consisting of Amblyseius cucumeris, Amblyseius swirskii, and Phytoseiulus persimilis. The S1 at least comprises the following steps: The predatory mites take the prey mites as food, and put the prey mites and food into a feeding container, and the predatory mites are mass bred under the environmental conditions of temperature 25±1℃, relative humidity 70%-80%, and light cycle 14L:10D; The feeding container is a 5L transparent fresh-keeping box, a 10cm×6cm hole is opened in the center of the fresh-keeping box cover, and a 300-mesh screen is welded for ventilation to prevent the predatory mites from escaping; The fresh-keeping box is placed on a tray with water to maintain the relative humidity in the feeding container, and the food of the prey mites needs to be shaken every day, and the food of the prey mites is replaced every 7 days.

3. The method of claim 2, wherein the predatory mite species is selected from the group consisting of Amblyseius cucumeris, Amblyseius swirskii, and Phytoseiulus persimilis. The S2 at least comprises the following steps: A smaller diameter circular sponge is placed in a 15cm diameter culture dish; A circular black cloth with the same diameter as the circular sponge is placed on the circular sponge, and the circular sponge and the black cloth are completely soaked with tap water, and the water surface is flush with the top of the sponge; A circular plastic film is placed on the black cloth, the diameter of the circular plastic film is slightly smaller than that of the black cloth, but the difference between the diameters of the circular plastic film and the black cloth is not less than 10cm; An 8cm diameter culture dish is placed on the circular plastic film; The mixture in the fresh-keeping box is poured out, sieved through a 60-mesh sieve, and a mixture of "predatory mites-prey mites-prey mites food" is obtained, the mixture is swept into an 8cm diameter culture dish with a soft brush, and 1,000 viable female predatory mites are selected and starved for 24h in the 8cm diameter culture dish.

4. The method of claim 3, wherein the predatory mite species is selected from the group consisting of Amblyseius cucumeris, Amblyseius swirskii, and Phytoseiulus persimilis. The S3 at least comprises the following steps: S3.1: using 20% sucrose solution as solvent, preparing β-1,3-glucan solution with a concentration of 1000ng / μL; S3.2: adding a small amount of blue dye as a color developing indicator to the β-1,3-glucan solution obtained in S3.1; S3.3: filtering the solution obtained in S3.2 through a 0.22μm filter membrane, and feeding 50μL of the solution to the Phytoseiulus persimilis for 12h.

5. The method of claim 4, wherein the predatory mite species is selected from the group consisting of Amblyseius cucumeris, Amblyseius swirskii, and Phytoseiulus persimilis. The S4 at least comprises the following steps: The predatory mites are swept onto the plastic film with a soft brush; Then, 1.5mL of high-concentration 2×109cell / mL Beauveria bassiana spore suspension is sprayed; After the spore suspension is dried, a single viable female predatory mite is selected and placed in an observation chamber with prey mites; The observation chamber is placed in a normal feeding environment, the survival rate of the predatory mites is recorded every day, and the observation is continuously conducted for 9 days to determine the survival rate of the natural enemy mites and confirm the promotion rate.

6. The method of claim 5, wherein the predatory mite species is selected from the group consisting of Amblyseius cucumeris, Amblyseius swirskii, and Phytoseiulus persimilis. The main body of the observation chamber is a central perforated cuboid transparent organic glass plate I, the central perforation is a round hole, one side of the round hole is a 300-mesh stainless steel screen, the other side of the round hole is covered with a cuboid transparent organic glass plate II, and the cuboid transparent organic glass plate II is clamped and fixed with a dovetail clamp.