Method for testing tropism of thrips and natural enemies thereof to different volatile substances

Abstract

The invention relates to the technical field of agricultural pest control, in particular to a method for testing the tropism of thrips and natural enemies thereof to different volatile substances, which comprises the following steps: S1, sample collection and volatile substance extraction; s2, preparing and calibrating a test device; s3, test insect acquisition and pretreatment; s4, performing test operation and data recording; the method has the advantages that accurate volatile substance information can be obtained through strict and normative sample collection and substance extraction, the tropism of the thrips to the volatile substance information can be observed under the accurately controlled test condition, the olfaction reaction mechanism of the thrips can be deeply analyzed, a key behavioral basis is provided for a thrip prevention and treatment strategy, and the thrips can be prevented and treated. The method effectively improves the pertinence of prevention and control, facilitates screening of substances capable of attracting natural enemies, enhances the pest control effect of the natural enemies in a farmland ecosystem, promotes ecological balance, reduces dependence on chemical pesticides, reduces pollution risks caused by the chemical pesticides, and guarantees the quality safety of agricultural products and the stability of the ecological environment.

Description

Technical Field

[0001] The present invention relates to the technical field of agricultural pest control, and specifically provides a method for testing the preference of thrips and its natural enemies for different volatile substances. Background Art

[0002] Thrips are one of the common and severely harmful pests in agricultural production, causing damage to a variety of crops and affecting crop yield and quality. Although traditional chemical control methods can control the thrips population to a certain extent, they are prone to problems such as environmental pollution and pest resistance. Utilizing the olfactory behavioral characteristics of thrips and their natural enemies and conducting ecological regulation through volatile substances has become a research hotspot. However, there is currently a lack of a systematic, accurate, and repeatable test method to deeply explore the preference of thrips and its natural enemies for different volatile substances, which restricts the development and application in this field. Therefore, it is necessary to provide a scientific and effective test method to deeply explore the preference of thrips and its natural enemies for different volatile substances and provide strong support for the biological control of thrips. Summary of the Invention

[0003] The purpose of the present invention is to provide a method for testing the preference of thrips and its natural enemies for different volatile substances to fill the gaps in the existing technology and provide a scientific basis and effective means for the biological control of thrips.

[0004] To achieve the above purpose, the present invention provides the following technical solution: A method for testing the preference of thrips and its natural enemies for different volatile substances, comprising the following steps:

[0005] S1. Sample collection and extraction of volatile substances;

[0006] S2. Preparation and calibration of the test device;

[0007] S3. Acquisition and pretreatment of test insects;

[0008] S4. Test operation and data recording;

[0009] S5. Data analysis and result evaluation.

[0010] Preferably, the specific method in the step S1, sample collection and extraction of volatile substances, is as follows:

[0011] S1-1. Determine the target plants damaged by thrips, select samples of this plant with good growth status and no chemical contamination in the thrips occurrence area, use sterile scissors to collect the leaves, stems, and flowers of the plant, and quickly put them into a pre-cooled sterile sealed bag after collection, and make detailed marks, including information such as collection time, location, and plant parts. The temperature of the pre-cooled sterile sealed bag is maintained at 4-8°C, and the mass of the collected plant samples is not less than 50 grams to ensure that there is enough volatile substances for extraction;

[0012] S1-2. Transfer the collected plant samples into a clean glass container in a low-temperature environment. Using solid-phase microextraction technology, select a suitable extraction fiber coating, and perform the adsorption extraction of volatile substances under specific temperature and time conditions. After the extraction is completed, insert the fiber head into a gas chromatography-mass spectrometry (GC-MS) instrument and analyze it according to the standard operating procedures of the instrument. By comparing with the standard spectral library, identify and record the components and relative contents of volatile substances in the plant samples. The temperature for solid-phase microextraction is 35-40 °C, the adsorption time is 3-5 hours, and the extraction fiber coating is polydimethylsiloxane / divinylbenzene (PDMS / DVB) to ensure efficient extraction of plant volatile substances.

[0013] Preferably, the specific method in step S2, preparation and calibration of the test device, is as follows:

[0014] S2-1. Use polymethyl methacrylate material that is chemically resistant and odorless to make a four-arm olfactometer. Precision process each component according to the design specifications to ensure that the lengths of all arms are the same, the inner diameters are uniform, and the connection parts have good sealing. Set gas valves with adjustable flow rates at the entrances of each arm, and connect an insect collection device at the exits.

[0015] S2-2. Select high-precision electronic flow meters and pressure controllers, connect them to the four-arm olfactometer, and construct a gas delivery and control system. Calibrate the system with standard gases, adjust the gas flow rate and pressure, so that a stable and precisely controllable concentration gradient of volatile substances can be formed inside the four-arm olfactometer, and ensure that the concentration difference between all arms is within a predetermined range.

[0016] Preferably, the specific method in step S3, acquisition and pretreatment of test insects, is as follows:

[0017] S3-1. In the natural habitat or artificially reared population of thrips, select active thrips individuals with relatively consistent size, morphology, and physiological status. Use a suction tube to collect them into a breathable container containing a moist filter paper and an appropriate amount of fresh plant tissue, maintain suitable temperature and humidity conditions, and temporarily raise them for a period of time. The temperature inside the container for temporarily raising thrips is maintained at 22-25 °C, the relative humidity is 70%-80%, and the temporary raising time is 2-4 hours to make them adapt to the environment and maintain an active state.

[0018] S3-2. For the natural enemy insects of thrips, according to their ecological habits, select healthy individuals in the corresponding natural environment or breeding sites that are in the active predation or parasitism period, such as Orius spp. Use gentle capture tools to avoid damaging the natural enemy insects, transfer them to suitable breeding containers, provide their preferred food and living environment, and conduct short-term adaptive breeding. The environmental conditions in the containers for breeding natural enemy insects are customized according to their species. For example, the breeding temperature for Orius spp. is 25-28°C, the relative humidity is 60%-70%, the provided food is thrips nymphs or small aphids, and the breeding time is 1-3 days to ensure that they are in good physiological condition.

[0019] Preferably, the specific methods in step S4, test operation and data recording are as follows:

[0020] S4-1. Place the four-arm olfactometer in a laboratory with a constant temperature (25±1°C), relative humidity (60±5%), and no obvious air flow. Use anhydrous ethanol and deionized water to thoroughly clean and dry the inside of the instrument and the connecting pipes in turn, and then flush with pure air continuously for a period of time to make the instrument reach a state without odor and impurity residue.

[0021] S4-2. Use a micro syringe to accurately suck a certain amount of the extracted volatile substance solution, inject it into the designated volatile source part of the four-arm olfactometer, and control the release concentration of the volatile substance in each arm by adjusting the temperature and gas flow of the volatile source. At the same time, inject an equal amount of solvent into the control arm as a blank control. The injection volume accuracy of the volatile substance solution is controlled within 0.01 ml, the temperature adjustment range of the volatile source is 20-30°C, and the gas flow is controlled at 1-5 ml / min to ensure the stability and accuracy of the release of the volatile substance.

[0022] S4-3. In a low-light environment, carefully place a single thrips or natural enemy insect into the central area of the four-arm olfactometer from the release port, and use a high-definition camera device to record the movement trajectory and behavioral performance of the insect in the instrument in real time. Through image analysis software and a timer, accurately measure the first time the insect enters each arm, the total residence time in each arm, and the arm number where it finally chooses to stay, and automatically record the data into the computer database.

[0023] S4-4. After each group of tests is completed, immediately clean the four-arm olfactometer, replace all contact parts, reset the volatile substance concentration and control conditions, and introduce new thrips and natural enemy insect individuals for repeated tests. The number of repetitions for each group of tests is not less than 30 times to ensure the reliability and statistical validity of the data.

[0024] Preferably, the specific methods in step S5, data analysis and result evaluation are as follows:

[0025] S5-1. Use statistical analysis software to process the collected insect behavior data. Adopt methods such as analysis of variance and chi-square test to compare the significant differences in the behavioral choices of thrips and natural enemy insects at different concentrations of volatile substances and between different substances. Calculate the selection preference index and trend-related indicators.

[0026] S5-2. According to the data analysis results, draw charts to visually display the trend laws of thrips and natural enemy insects towards different volatile substances. Evaluate the repellent or attracting effects of each volatile substance on thrips and the attracting effect on natural enemy insects. Screen out volatile substances with potential application value, and conduct a preliminary analysis and prediction on their application prospects in thrips biological control.

[0027] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0028] 1. A method for testing the trend of thrips and its natural enemies towards different volatile substances provided by the present invention can obtain accurate volatile substance information through strict and standardized sample collection and substance extraction. Observe the trend of thrips towards them under precisely controlled experimental conditions, and can deeply analyze the olfactory response mechanism of thrips, providing a key behavioral basis for thrips control strategies and effectively improving the pertinence of prevention and control.

[0029] 2. This method reasonably obtains and processes natural enemy insects of thrips. Combining with a scientific experimental process, clearly presents the trend response of natural enemy insects to volatile substances, which helps to screen out substances that can attract natural enemies, enhance the pest control effect of natural enemies in the farmland ecosystem, promote ecological balance, and reduce the dependence on chemical pesticides.

[0030] 3. This patent provides a systematic experimental method, which helps to screen out volatile substances that have repellent or attracting effects on thrips and are environmentally friendly. These substances can be used to develop new green control products, reduce the pollution risk brought by chemical pesticides, and ensure the quality safety of agricultural products and the stability of the ecological environment. Specific Embodiments

[0031] Unless otherwise clearly specified in the context, nouns without quantity words and nouns modified by "the" include singular and plural referents.

[0032] As used in the specification and claims, the terms "comprising", "including", "having", "may", "containing" and their variants used herein refer to open transitional phrases, terms or words that require the presence of the specified components / steps and allow the presence of other components / steps. However, such descriptions should also be interpreted as describing the composition or method as "consisting of" and "substantially consisting of" the listed components / steps, which allows only the presence of the specified components / steps and any inevitable impurities that may result therefrom, and excludes other components / steps.

[0033] The numerical values in the specification and claims of this application should be understood to include the same numerical values when reduced to the same number of significant figures and numerical values that differ from the said values by less than the experimental error of the conventional measurement techniques used to determine the said values of the type described in this application.

[0034] All ranges disclosed herein include the indicated endpoints and can be combined independently (e.g., the range of "2 grams to 10 grams" includes the endpoints 2 grams and 10 grams, as well as all intermediate values).

[0035] The terms "about" and "approximate" can be used to include any numerical value that can vary without changing the basic function of the value. When used with a range, "about" and "approximate" also disclose the range defined by the absolute values of the two endpoints. For example, "about 2 to about 4" also discloses the range of "2 to 4". Generally, the terms "about" and "approximate" can refer to ±10% of the indicated number. However, for temperature, the term "about" means ±1 °C.

[0036] Unless otherwise clearly specified, the percentage of an element should be considered as the weight percentage of the said alloy.

[0037] This disclosure may relate to the temperature of certain method steps. It should be noted that these indicators generally refer to the temperature set by a heat source (such as a furnace), and not necessarily to the temperature that the heated material must reach.

[0038] The present invention provides a method for testing the tendency of thrips and their natural enemies towards different volatile substances, comprising the following steps:

[0039] S1. Sample collection and extraction of volatile substances;

[0040] S1-1. Determine the target plants damaged by thrips, select samples of this plant with good growth status and not chemically contaminated in the thrips occurrence area, use sterile scissors to collect the leaves, stems and flower parts of the plant, quickly put them into a pre-cooled sterile sealed bag after collection, and make detailed marks, including information such as collection time, location and plant parts, etc. Among them, the temperature of the pre-cooled sterile sealed bag is maintained at 4 - 8 °C, and the mass of the collected plant samples is not less than 50 grams to ensure that there is enough volatile substances available for extraction;

[0041] S1-2. Transfer the collected plant samples to a clean glass container under low-temperature conditions. Using solid-phase microextraction technology, select a suitable extraction fiber coating, and perform adsorption extraction of volatile substances under specific temperature and time conditions. After extraction, insert the fiber head into a gas chromatography-mass spectrometry (GC-MS) instrument and analyze it according to the standard operating procedures of the instrument. By comparing with the standard spectral library, identify and record the components and relative contents of volatile substances in the plant samples. The temperature for solid-phase microextraction is 35 - 40 °C, the adsorption time is 3 - 5 hours, and the extraction fiber coating is polydimethylsiloxane / divinylbenzene (PDMS / DVB) to ensure efficient extraction of plant volatile substances.

[0042] S2. Preparation and calibration of the test device;

[0043] S2-1. Fabricate a four-arm olfactometer using chemically resistant and odorless polymethyl methacrylate material. Precision machine each component according to the design specifications to ensure that the lengths of all arms are the same, the inner diameters are uniform, and the connection parts have good sealing. Install adjustable flow gas valves at the entrance of each arm and connect an insect collection device at the exit.

[0044] S2-2. Select high-precision electronic flow meters and pressure controllers, connect them to the four-arm olfactometer, and construct a gas delivery and control system. Calibrate the system using standard gases, adjust the gas flow and pressure to form a stable and precisely controllable concentration gradient of volatile substances inside the four-arm olfactometer, and ensure that the concentration differences between the arms are within the predetermined range.

[0045] S3. Acquisition and pretreatment of test insects;

[0046] S3-1. In the natural habitat or artificial breeding population of thrips, select active thrips individuals with relatively consistent size, morphology, and physiological status. Use a suction tube to collect them into a breathable container containing moist filter paper and an appropriate amount of fresh plant tissue, maintain suitable temperature and humidity conditions, and hold them for a period of time. The temperature inside the container for holding thrips is maintained at 22 - 25 °C, the relative humidity is 70% - 80%, and the holding time is 2 - 4 hours to allow them to adapt to the environment and remain active.

[0047] S3-2. For the natural enemy insects of thrips, according to their ecological habits, select healthy individuals in the corresponding natural environment or breeding sites that are in the active predation or parasitism period, such as Orius spp. Use gentle capture tools to avoid damaging the natural enemy insects, transfer them to suitable breeding containers, provide their preferred food and living environment, and conduct short-term adaptive breeding. The environmental conditions in the containers for breeding natural enemy insects are customized according to their species. For example, the breeding temperature for Orius spp. is 25-28°C, the relative humidity is 60%-70%, the provided food is thrips nymphs or small aphids, and the breeding time is 1-3 days to ensure that they are in good physiological condition.

[0048] S4. Experimental operation and data recording;

[0049] S4-1. Place the four-arm olfactometer in a laboratory with a constant temperature (25±1°C), relative humidity (60±5%), and no obvious air flow. Thoroughly clean and dry the inside of the instrument and the connecting pipes with anhydrous ethanol and deionized water in turn, and then flush with pure air continuously for a period of time to make the instrument reach a state without odor and impurity residue;

[0050] S4-2. Use a microsyringe to accurately aspirate a certain amount of the extracted volatile substance solution and inject it into the designated volatile source part of the four-arm olfactometer. By adjusting the temperature and gas flow rate of the volatile source, control the release concentration of the volatile substance in each arm. At the same time, inject an equal amount of solvent into the control arm as a blank control. The injection volume accuracy of the volatile substance solution is controlled within 0.01 ml, the temperature adjustment range of the volatile source is 20-30°C, and the gas flow rate is controlled at 1-5 ml / min to ensure the stability and accuracy of the release of the volatile substance;

[0051] S4-3. In a low-light environment, carefully place a single thrips or natural enemy insect into the central area of the four-arm olfactometer from the release port. Use a high-definition camera device to record the movement trajectory and behavioral performance of the insect in the instrument in real time. Through image analysis software and a timer, accurately measure the first time the insect enters each arm, the total residence time in each arm, and the arm number where it finally chooses to stay, and automatically record the data into the computer database;

[0052] S4-4. After each group of experiments is completed, immediately clean the four-arm olfactometer, replace all contact parts, reset the volatile substance concentration and control conditions, and introduce new thrips and natural enemy insect individuals for repeated experiments. The number of repetitions for each group of experiments is not less than 30 times to ensure the reliability and statistical validity of the data.

[0053] S5. Data analysis and result evaluation.

[0054] S5-1. Use statistical analysis software to process the collected insect behavior data. Adopt methods such as analysis of variance and chi-square test to compare the significant differences in the behavioral choices of thrips and natural enemy insects at different concentrations of volatile substances and between different substances. Calculate the selection preference index and related indicators of tendency.

[0055] S5-2. According to the data analysis results, draw charts to visually display the tendency laws of thrips and natural enemy insects towards different volatile substances. Evaluate the repellent or attracting effects of each volatile substance on thrips and the attracting effect on natural enemy insects. Screen out volatile substances with potential application value and conduct a preliminary analysis and prediction on their application prospects in the biological control of thrips.

[0056] Example 1:

[0057] I. Test background

[0058] Odontothrips loti is a pest that harms alfalfa (Medicago sativa, high-quality forage). The present invention aims to explore the tendency of thrips and its natural enemy Orius minutus towards different volatile substances, providing a basis for developing green pest control methods.

[0059] II. Test materials

[0060] Test insects: Odontothrips loti, Orius minutus (natural enemy of thrips).

[0061] Plant materials: Medicago sativa.

[0062] Volatile substances: 2-ethylhexyl salicylate, methyl linoleate, benzyl benzoate.

[0063] Instrument and equipment: Four-arm olfactometer, artificial climate chamber, insect rearing cage.

[0064] III. Test methods

[0065] Insect rearing: Raise Odontothrips loti and Orius minutus separately in insect rearing cages filled with fresh Medicago sativa. The rearing conditions are temperature (25±2)°C, relative humidity (60±5)%, and photoperiod 16L:8D.

[0066] Preparation of volatile substances: Prepare solutions of 2-ethylhexyl salicylate, methyl linoleate, and benzyl benzoate with concentrations of 0.1 mmol / L, 1 mmol / L, and 10 mmol / L respectively, using pure solvent as the control group (CK).

[0067] Tropism test: The test was carried out using a four-arm olfactometer. The olfactometer was placed in an artificial climate chamber, maintaining the temperature at (25±2)°C and the relative humidity at (60±5)%. Small bottles containing solutions of different volatile substances or control group solutions were placed at the end of each arm. 10 thrips or Orius sauteri were placed in the central release area of the olfactometer, and the residence time and selection times of the insects in each arm within 15 minutes were observed and recorded. Each treatment was repeated 5 times.

[0068] IV. Test Results

[0069] 2-Ethylhexyl salicylate: Compared with the control group, 2-ethylhexyl salicylate at concentrations of 0.1 mmol / L and 10 mmol / L showed significant repellent effects on thrips (P<0.05). The selection rate of thrips for 10 mmol / L 2-ethylhexyl salicylate was significantly higher than that for 0.1 mmol / L 2-ethylhexyl salicylate; Orius sauteri did not show an obvious preference for 0.1 mmol / L 2-ethylhexyl salicylate, and the selection rate for 10 mmol / L 2-ethylhexyl salicylate was significantly higher than that of the control group (P<0.05).

[0070] Methyl linoleate: Thrips showed an obvious preference for 10 mmol / L methyl linoleate and had no significant selection preference for 0.1 mmol / L methyl linoleate; Orius sauteri showed an obvious selection preference for 0.1 mmol / L methyl linoleate (P<0.05), and there was no significant selection preference between 10 mmol / L methyl linoleate and the control group.

[0071] Benzyl benzoate: Thrips had no significant selection preference for 1 mmol / L benzyl benzoate, and when facing the odor of 0.1 mmol / L benzyl benzoate, its selection rate decreased significantly; the selection rate of Orius sauteri for 0.1 mmol / L benzyl benzoate increased significantly (P<0.05), and there was no significant effect on the selection rate for 1 mmol / L benzyl benzoate.

[0072] V. Conclusion

[0073] 2-Ethylhexyl salicylate: It has an obvious repellent effect on thrips. It can be considered to set up a slow-release device containing high-concentration (such as 10 mmol / L) 2-ethylhexyl salicylate in the alfalfa planting area to keep thrips away from alfalfa plants and reduce damage. At the same time, utilize the tropism of Orius sauteri for 10 mmol / L 2-ethylhexyl salicylate. When releasing Orius sauteri for biological control, use this substance in combination to guide Orius sauteri to gather in the target area and improve the predation efficiency on thrips.

[0074] Methyl linoleate: Since thrips show a preference for 10 mmol / L methyl linoleate, it can be used as a bait component to design a trapping device. Traps are set up in the field, and lure cores containing 10 mmol / L methyl linoleate are placed inside to attract thrips for centralized killing, reducing the thrips population density. For Orius sauteri, 0.1 mmol / L methyl linoleate is attractive. In the area where Orius sauteri is released, slow-release materials containing methyl linoleate at this concentration are appropriately arranged, which helps attract Orius sauteri to settle and enhances the natural control ability against thrips.

[0075] Benzyl benzoate: 0.1 mmol / L benzyl benzoate has a repellent effect on thrips and an attractive effect on Orius sauteri. In practical applications, preparations containing 0.1 mmol / L benzyl benzoate can be arranged at intervals around or between rows in alfalfa fields. On the one hand, it repels thrips and makes it difficult for them to approach alfalfa plants; on the other hand, it attracts natural enemies such as Orius sauteri, creating an environment conducive to the survival and predation of natural enemies to achieve the purpose of controlling thrips.

[0076] Through this experimental method, the tropisms of thrips and Orius sauteri to volatile substances such as isooctyl salicylate, methyl linoleate, and benzyl benzoate were clarified. These results provide technical support for the ecological regulation of thrips by developing and utilizing volatile substances and have potential application value.

[0077] Example 2:

[0078] I. Preparation of experimental materials

[0079] Test insects: Odontothrips loti. After being reared on fresh alfalfa for multiple generations in an insect rearing room at a temperature of (25±2) °C, relative humidity of (60±5)%, and photoperiod of 16L:8D, it was used for the experiment. Orius sauteri was collected from the wild and used as a test natural enemy after being reared and domesticated with thrips for two generations under the same above conditions.

[0080] Volatile substances: Isooctyl salicylate, methyl linoleate, and benzyl benzoate were prepared, all of which were of analytical purity and were formulated into solutions with different concentration gradients of 0.1 mmol / L, 1 mmol / L, and 10 mmol / L using n-hexane.

[0081] Experimental equipment: Four-arm olfactometer (made of glass, each arm is 15 cm long, 2 cm in inner diameter, and the central intersection part is a circle with a diameter of 5 cm), odor source bottle (50 mL glass bottle), activated carbon filtered air device, insect rearing cage (40 cm×40 cm×40 cm).

[0082] II. Experimental steps

[0083] Olfactometer preparation: Clean the four-arm olfactometer with absolute ethanol. After drying, pass filtered pure air into each arm through an activated carbon air filtration device, with the flow rate controlled at 200 mL / min and stabilized for 30 min.

[0084] Odor source setting: Add 1 mL of volatile substance solutions with different concentrations into the odor source bottles respectively, connect one of the arms of the four-arm olfactometer to them, and connect the control group to the odor source bottle containing 1 mL of n-hexane.

[0085] Thrips tropism experiment: Select 50 healthy adult thrips that have emerged for 3 - 5 days from the insect rearing cage and place them in the release area at the center of the four-arm olfactometer. In a dark environment, let the thrips freely choose for 15 min, and then record the number of thrips entering each arm. Each experiment is repeated 10 times.

[0086] Orius sauteri tropism experiment: Select 30 healthy adult Orius sauteri that have emerged for 5 - 7 days and place them in the four-arm olfactometer for a choice experiment in the same method as the above thrips experiment. Record the number of Orius sauteri entering each arm after 15 min. Each experiment is repeated 10 times.

[0087] III. Experimental data processing

[0088] Use SPSS 22.0 statistical software to analyze the experimental data, and calculate the selection rates of thrips and Orius sauteri in different treatment groups (selection rate = number of insects entering a certain arm / total number of insects × 100%).

[0089] Use one-way analysis of variance (One-Way ANOVA) to compare the differences in selection rates between the treatment groups with different concentrations of volatile substances and the control group. When P < 0.05, the difference is considered significant.

[0090] IV. Experimental results

[0091] 2-Ethylhexyl salicylate: Compared with the control group, 2-ethylhexyl salicylate at concentrations of 0.1 mmol / L and 10 mmol / L showed significant repellent effects on thrips (P < 0.05), and the selection rate of thrips for 10 mmol / L 2-ethylhexyl salicylate was significantly higher than that for 0.1 mmol / L 2-ethylhexyl salicylate (P < 0.05). Orius sauteri did not show an obvious preference for 0.1 mmol / L 2-ethylhexyl salicylate, the selection rate of Orius sauteri for 10 mmol / L 2-ethylhexyl salicylate was significantly higher than that of the control group (P < 0.05), and there was no obvious preference between 0.1 mmol / L and 10 mmol / L 2-ethylhexyl salicylate.

[0092] Methyl linoleate: Thrips showed an obvious preference for 10 mmol / L methyl linoleate, and the selection rate was significantly higher than that at the concentration of 0.1 mmol / L (P<0.05), and there was no significant selection preference for 0.1 mmol / L methyl linoleate. Orius sauteri showed an obvious selection preference for 0.1 mmol / L methyl linoleate (P<0.05), and there was no significant selection preference between 10 mmol / L methyl linoleate and the control group.

[0093] Benzyl benzoate: Thrips had no significant selection preference for 1 mmol / L benzyl benzoate. When facing the odor of 0.1 mmol / L benzyl benzoate, the selection rate decreased significantly, and it significantly preferred to select 1 mmol / L benzyl benzoate between 0.1 mmol / L and 1 mmol / L benzyl benzoate (P<0.05). 1 mmol / L benzyl benzoate had no significant effect on the selection rate of Orius sauteri, the selection rate of 0.1 mmol / L benzyl benzoate increased significantly (P<0.05), and it significantly preferred to select 0.1 mmol / L benzyl benzoate (P<0.05).

[0094] V. Analysis of control potential

[0095] Isooctyl salicylate: Combining the results of Example 1 and this example, high-concentration isooctyl salicylate has a stable repellent effect on thrips. In actual farmland applications, repellents based on isooctyl salicylate, such as microcapsule preparations, can be developed. By spraying or spreading them in alfalfa fields, a repellent barrier can be formed. At the same time, an appropriate amount of high-concentration isooctyl salicylate can be placed near the natural enemy release area to enhance the attraction effect on natural enemies such as Orius sauteri and jointly control thrips.

[0096] Methyl linoleate: Utilizing the preference of thrips for 10 mmol / L methyl linoleate, highly efficient trapping products can be developed. For example, sticky trapping boards can be designed, with 10 mmol / L methyl linoleate added to the surface, and hung in alfalfa fields to improve the efficiency of trapping thrips. Regarding the tendency of Orius sauteri towards 0.1 mmol / L methyl linoleate, food supplement stations containing this concentration of methyl linoleate can be set up in natural enemy habitats or release areas to promote the reproduction and survival of Orius sauteri and enhance the long-term control ability of thrips.

[0097] Benzyl benzoate: According to the test results, 0.1 mmol / L benzyl benzoate has good repellent effects on thrips and attractive effects on Orius sauteri. Compound biological control products can be developed. For example, 0.1 mmol / L benzyl benzoate can be combined with the food source of Orius sauteri (such as thrips nymph mimics) to make slow-release granules, and these granules are sown in alfalfa fields to both repel thrips and attract Orius sauteri, achieving green and sustainable control of thrips.

[0098] Those skilled in the art should understand that the embodiments of the present invention described above and shown in the solutions are only examples and do not limit the present invention. The objectives of the present invention have been fully and effectively achieved. The functions and structural principles of the present invention have been demonstrated and explained in the embodiments. Without departing from the said principles, the embodiments of the present invention can have any deformation or modification. Although the embodiments of the present invention have been shown and described, those of ordinary skill in the art can understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the present invention. The scope of the present invention is defined by the appended claims and their equivalents. This specific embodiment is only an interpretation of the present application and does not limit the present application. Those skilled in the art can make modifications to this embodiment without creative contributions according to their needs after reading this specification, but as long as they are within the scope of the claims of the present application, they are protected by the patent law.

Claims

1. A test method for the tropism of thrips and its natural enemies to different volatile substances, characterized in that, It includes the following steps: S1. Sample collection and extraction of volatile substances; S2. Preparation and calibration of the test device; S3. Acquisition and pretreatment of test insects; S4. Test operation and data recording; S5. Data analysis and result evaluation.

2. The test method for the tendency of thrips and its natural enemies towards different volatile substances according to claim 1, characterized in that: The specific method in S1, the step of sample collection and extraction of volatile substances, is as follows: S1-1. Determine the target plants damaged by thrips. Select samples of this plant with good growth status and free from chemical contamination in the thrips occurrence area. Use sterile scissors to collect the leaves, stems and flowers of the plant. After collection, quickly put them into a pre-cooled sterile sealed bag and make detailed marks, including information such as collection time, location and plant parts. The temperature of the pre-cooled sterile sealed bag is maintained at 4-8°C, and the mass of the collected plant samples is not less than 50 grams to ensure that there are enough volatile substances for extraction; S1-2. Transfer the collected plant samples to a clean glass container in a low-temperature environment. Adopt solid-phase microextraction technology, select a suitable extraction fiber coating, and carry out adsorption extraction of volatile substances under specific temperature and time conditions. After extraction, insert the fiber head into a gas chromatography-mass spectrometry (GC-MS) instrument and analyze according to the standard operating procedures of the instrument. By comparing with the standard spectral library, identify and record the components and relative contents of volatile substances in the plant samples. The temperature of solid-phase microextraction is 35-40°C, the adsorption time is 3-5 hours, and the extraction fiber coating is polydimethylsiloxane / divinylbenzene (PDMS / DVB) to ensure efficient extraction of plant volatile substances.

3. A method for testing the tropism of thrips and its natural enemies to different volatile substances according to claim 1, characterized in that: The specific method in S2, the step of preparation and calibration of the test device, is as follows: S2-1. Use polymethyl methacrylate material resistant to chemical corrosion and odorless to make a four-arm olfactometer. Precision process each component according to the design specifications to ensure that the lengths of all arms are the same, the inner diameters are uniform, and the connection parts have good sealing performance. Set gas valves with adjustable flow rates at the entrances of each arm, and connect an insect collection device at the exits; S2-2. Select high-precision electronic flow meters and pressure controllers, connect them to the four-arm olfactometer, and construct a gas delivery and control system. Calibrate the system with standard gases, adjust the gas flow rate and pressure, so that a stable and precisely controllable concentration gradient of volatile substances can be formed in the four-arm olfactometer, and ensure that the concentration difference between each arm is within the predetermined range.

4. A test method for the tropism of thrips and its natural enemies to different volatile substances according to claim 1, characterized in that: The specific method in S3, the step of acquisition and pretreatment of test insects, is as follows: S3-1. In the natural habitat of thrips or an artificially reared population, select active thrips individuals with relatively consistent size, morphology and physiological status. Use a suction tube to collect them into a breathable container containing moist filter paper and appropriate fresh plant tissues, maintain suitable temperature and humidity conditions, and temporarily raise them for a period of time. The temperature in the container for temporarily raising thrips is kept at 22-25°C, the relative humidity is 70%-80%, and the temporary raising time is 2-4 hours to make them adapt to the environment and maintain an active state; S3-2. For natural enemy insects of thrips, according to their ecological habits, select healthy individuals in the corresponding natural environment or breeding sites that are in the active predation or parasitism period, such as Orius spp. Use gentle capture tools to avoid damaging the natural enemy insects, transfer them to suitable breeding containers, provide their preferred food and living environment, and conduct short-term adaptive breeding. The environmental conditions in the containers for breeding natural enemy insects are customized according to their species. For example, the breeding temperature for Orius spp. is 25-28°C, the relative humidity is 60%-70%, the provided food is thrips nymphs or small aphids, and the breeding time is 1-3 days to ensure that they are in good physiological condition.

5. A test method for the tropism of thrips and its natural enemies to different volatile substances according to claim 1, characterized in that: The specific methods in step S4, experimental operation and data recording, are as follows: S4-1. Place the four-arm olfactometer in a laboratory with a constant temperature (25±1°C), relative humidity (60±5%), and no obvious air flow. Thoroughly clean and dry the inside of the instrument and the connecting pipes successively with anhydrous ethanol and deionized water, and then flush with pure air continuously for a period of time to make the instrument reach a state without odor and impurity residue. S4-2. Use a micro syringe to accurately suck a certain amount of the extracted volatile substance solution and inject it into the designated volatile source part of the four-arm olfactometer. By adjusting the temperature and gas flow rate of the volatile source, control the release concentration of the volatile substance in each arm. At the same time, inject an equal amount of solvent into the control arm as a blank control. The injection volume accuracy of the volatile substance solution is controlled within 0.01 ml, the temperature adjustment range of the volatile source is 20-30°C, and the gas flow rate is controlled at 1-5 ml / min to ensure the stability and accuracy of the release of the volatile substance. S4-3. In a low-light environment, carefully place a single thrips or natural enemy insect into the central area of the four-arm olfactometer from the release port. Use a high-definition camera device to record the movement trajectory and behavioral performance of the insect in the instrument in real time. Through image analysis software and a timer, accurately measure the first time the insect enters each arm, the total residence time in each arm, and the arm number where it finally chooses to stay, and automatically record the data into the computer database. S4-4. After each group of tests is completed, immediately clean the four-arm olfactometer, replace all contact parts, reset the volatile substance concentration and control conditions, and introduce new thrips and natural enemy insect individuals for repeated tests. The number of repetitions for each group of tests is not less than 30 times to ensure the reliability and statistical validity of the data.

6. A method for testing the tendency of thrips and its natural enemies towards different volatile substances according to claim 1, characterized in that: The specific methods in step S5, data analysis and result evaluation, are as follows: S5-1. Use statistical analysis software to process the collected insect behavior data. Adopt methods such as analysis of variance and chi-square test to compare the significance of differences in behavioral choices between thrips and natural enemy insects at different volatile substance concentrations and different substances, and calculate the selection preference index and trend-related indicators. S5-2. According to the data analysis results, draw charts to visually display the tropism laws of thrips and natural enemy insects to different volatile substances, evaluate the repellent or attracting effects of each volatile substance on thrips and the attracting effect on natural enemy insects, screen out volatile substances with potential application value, and conduct a preliminary analysis and prediction on their application prospects in thrips biological control.