Application of (R)-Lavenderyl (R)-2-methylbutyrate in pheromone attractants and its attractant core

Abstract

The present invention belongs to the technical field of plant protection agents and relates to the application of (R)-lavandulyl (R)-2-methylbutyrate in pheromone attractants and an attractant core. The present invention extracts volatile substances from the surface of thrips by solid-phase microextraction technology. GC-MS analysis identifies that the volatile substances from the surface of thrips are composed of (R)-lavandulyl (R)-2-methylbutyrate. Furthermore, an indoor Y-type olfactometer test verifies the attractant effect of (R)-lavandulyl (R)-2-methylbutyrate on male and female adults of thrips. The components of the pheromone attractant for thrips are clarified, and the attractant can be used for dynamic detection of pest populations and prediction of occurrence patterns, providing technical support for the effective prevention and control of thrips.

Description

Technical Field

[0001] The invention belongs to the technical field of plant protection agents and relates to the application of (R)-lavenderyl (R)-2-methylbutyrate in pheromone attractants and an attractant core. Background Art

[0002] Thrips, a general term for insects of the order Thysanoptera, are numerous, widespread, and cause severe damage. They are currently one of the most important agricultural pests worldwide. Their high reproductive capacity, hidden nature, and strong pesticide resistance make control difficult, creating a major bottleneck restricting the high-quality development of the agricultural industry. Odontothrips loti Haliday is a dominant pest of alfalfa, widely distributed in major alfalfa-growing areas such as Xinjiang, Inner Mongolia, and Ningxia. Both larval and adult stages feed on the above-ground parts of plants, spreading plant viruses and causing leaf wilt and yellowing.

[0003] Currently, there are three main methods for controlling thrips: chemical, biological, and physical. However, due to their small size, ease of hiding, and high mobility, most sticky traps and insect traps are ineffective at attracting thrips, attracting non-target insects, and are relatively expensive. They also tend to develop resistance to most chemical pesticides, reducing their effectiveness and causing significant environmental pollution, food safety threats, and human health impacts. Consequently, chemical and physical control methods are not ideal. Given this current situation, the search for a pollution-free, sustainable biological control method for thrips is inevitable, addressing the health and environmental risks associated with existing technologies. Harnessing the insect's own pheromones for thrips control is one effective approach.

[0004] Currently, it is known that male adults of various thrips release aggregation pheromones. This can be a single component or a combination of multiple components released in a certain proportion, significantly attracting both sexes and serving as a key agent for information exchange within thrips populations. Prior art has effectively isolated and identified the following aggregation pheromone components that attract thrips: CN108902143B discloses an aggregation pheromone for Macrothrips spp., which is composed of geranyl acetate and farnesyl acetate. CN104381256B discloses an agent for attracting flower thrips, which is composed of (R)-lavender acetate and chonosyl (S)-2-methylbutyrate or its analogs: isobornyl valerate, isobornyl 2-methylbutyrate, isobornyl trimethylacetate, and lavender isovalerate, in a mass ratio of 1:8, exhibiting the strongest attracting ability. CN109730065B discloses an attractant composition for western flower thrips comprising (R)-lavender acetate and acenaphthenyl (S)-2-methylbutyrate. CN108041034B discloses an aggregation pheromone attractant for female tea thrips and a method for preparing the attractant core, wherein the aggregation pheromone attractant is 2,4,7,9-tetramethyl-5-decyne-4,7-diol.

[0005] The use of flower thrips aggregation pheromones for pest control has become a paradigm shift in the application of insect aggregation pheromones. Therefore, research on the aggregation pheromone of the flower thrips and the development of related application technologies could be widely applied in pest forecasting, trapping, and mating disruption. This could provide effective methods for monitoring flower thrips populations and conducting centralized trapping and control. Furthermore, it could effectively protect alfalfa varieties, mitigate the problems of increased resistance and incomplete control caused by chemical pesticides, and thus avoid serious environmental pollution, food safety threats, and ultimately human health. Summary of the Invention

[0006] The present invention aims to provide the use of (R)-lavanduloyl (R)-2-methylbutyrate in pheromone attractants and an attractant core. The present invention uses solid-phase microextraction technology to extract volatile substances from the surface of Botrytis cinerea. The volatile substances are separated and identified using a Shimadzu GC-MS QP2010 PLUS (gas chromatography-mass spectrometry, GC-MS). The attractant effect is further verified using an indoor Y-type olfactometer test. The components of the pheromone attractant for Botrytis cinerea are identified, and an aggregation pheromone with a significant attractant effect on both male and female Botrytis cinerea is developed, providing technical support for the effective prevention and control of Botrytis cinerea.

[0007] Based on the above objectives, the present invention addresses this need in the art by providing the use of (R)-lavenderoyl (R)-2-methylbutyrate in pheromone attractants and an attractant core.

[0008] In one aspect, the present invention relates to the use of (R)-lavandμLyl(R)-2-methylbutanoate in pheromone attractants.

[0009] Preferably, the use of (R)-lavenderoyl (R)-2-methylbutyrate in a pheromone attractant provided by the present invention includes using the pheromone attractant (R)-lavenderoyl (R)-2-methylbutyrate to attract male and female adults of Thrips spp.

[0010] The molecular structural formula of the (R)-lavenderoyl (R)-2-methylbutyrate is shown below:

[0011]

[0012] Preferably, the use of (R)-lavenderoyl (R)-2-methylbutyrate in a pheromone attractant provided by the present invention comprises the pheromone attractant (R)-lavenderoyl (R)-2-methylbutyrate having an attractant concentration of 0.5 μg / μL-2 μg / μL.

[0013] On the other hand, the present invention relates to an attractant core for attracting Botrytis cinerea, wherein the attractant core comprises: (R)-lavenderoyl (R)-2-methylbutyrate and paraffin oil.

[0014] On the other hand, the present invention relates to an attractant core for attracting Cypripedium spp., wherein (R)-lavanduloyl (R)-2-methylbutyrate is diluted to 0.5 μg / μL-2 μg / μL with paraffin oil.

[0015] Compared with the prior art, the present invention has the following beneficial effects or advantages:

[0016] The pheromone attractant for the thrips is a major pest that damages alfalfa. The present invention provides a pheromone attractant that can be used for the thrips, which can effectively attract both sexes of the thrips. The attractant is used together with paraffin oil to prepare an attractant core, which can effectively attract the thrips in alfalfa and has an excellent attracting effect, that is, high sensitivity. The attractant core can be used as one of the methods for quarantine departments to improve the recognition ability of the thrips, can be used for dynamic detection of pest populations and prediction and forecast of occurrence patterns, and for centralized trapping and killing of pests, thereby providing technical support for the effective prevention and control of the thrips, thereby achieving the purpose of maintaining and increasing production. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention.

[0018] Figure 1 These are the total ion chromatograms and mass spectra of volatiles from the body surface of male thrips. A: Total ion chromatogram of volatiles from the body surface of male thrips; B: Enlarged view of the peak at 27.37 min.

[0019] Figure 2 Comparison results of male body surface volatile mass spectra with the NIST mass spectrum library.

[0020] Figure 3 Behavioral responses of adults of the flower thrips Mysore to lavandulyl-2-methylbutyrate isomers.

[0021] Figure 4 The behavioral responses of adults of Cyprinus spp. to baits prepared from (R)-lavenderoyl (R)-2-methylbutyrate and paraffin oil. DETAILED DESCRIPTION

[0022] The technical solutions of the present invention are described below with reference to the following examples. However, the present invention is not limited to the following examples. The experimental methods and detection methods described in each example are conventional methods unless otherwise specified. The equipment and raw materials described are all commercially available unless otherwise specified.

[0023] The invention provides a pheromone attractant for thrips hornii. The (R)-lavenderoyl (R)-2-methylbutyrate (purity of 90%) and other enantiomers thereof are synthesized by the laboratory of Bian Qinghua, Department of Chemistry, College of Science, China Agricultural University, and are diluted to 0.5 μg / μL, 1 μg / μL and 2 μg / μL respectively with a solvent of liquid paraffin oil (analytical grade, S10187, SCRC).

[0024] Example 1

[0025] 1. Materials and Methods

[0026] This embodiment provides the separation, identification and application of volatiles from the surface of Cyprinus thrips.

[0027] 1.1 Origin of Thrips taurochoerus

[0028] Thrips spp. were collected from the Shangzhuang Experimental Station of China Agricultural University. Multiple generations of colonies were established in a laboratory incubator using fresh alfalfa leaves and lentils. The environment was maintained at a temperature of 26 ± 1°C, a humidity of 60%, and a photoperiod of 16:8 h.

[0029] 1.2 Collection of volatiles from the surface of Thrips taurochoerus using solid-phase microextraction (SPME)

[0030] Step 1: The solid phase microextraction adsorption needle needs to be inserted into the injection port of the gas chromatograph for 30 minutes before first use;

[0031] Step 2: Place 10 active male adults in a 5 ml sample bottle, then insert the adsorption needle into the sample bottle, rotate and extend the adsorption probe to collect the sample for 30 minutes;

[0032] Step 3: Repeat 3 times, 10 test insects each time, and all test insects are used for only one collection.

[0033] 1.3 Separation process:

[0034] The collected volatiles were separated and identified by Shimadzu GC-MS QP2010 PLUS. The chromatographic column used was Rtx-5MS (30.0 m × 0.25 mm × 0.25 μm);

[0035] The GC program for identifying volatiles from the surface of Cyprinus serratus was 32.5 minutes long. The starting temperature was 45°C, held for 1 minute, then increased at a rate of 5°C / min to 150°C, held for 1 minute; then increased at a rate of 10°C / min to 200°C, held for 1 minute; and finally increased at a rate of 20°C / min to 250°C, held for 1 minute. The inlet temperature was 250°C, and a splitless injection was used. The carrier gas was helium at a flow rate of 1 ml / min.

[0036] 1.4 Chemical composition identification and structural analysis of aggregation pheromones

[0037] According to the total ion current graph, the peak position of the target compound is determined, and then the mass spectrum of the target peak is compared with the NIST online database. The most matching result is selected according to the degree of consistency between the search matching degree and the mass spectrum peak type and characteristic ion peak information for preliminary qualitative analysis.

[0038] 1.5 The selection behavior of male and female adults of Thrips taurochoerus towards lavandulyl-2-methylbutyrate isomers was determined using a Y-type olfactometer.

[0039] Dilution of test samples:

[0040] (R)-Lavender (R)-2-methylbutyrate and other enantiomers were synthesized in the laboratory of Qinghua Bian, Department of Chemistry, College of Science, China Agricultural University, with a purity of 90%. They were diluted with liquid paraffin oil (analytical grade, S10187, SCRC) as a solvent. (R)-Lavender (R)-2-methylbutyrate and other enantiomers were diluted to 0.5 μg / μL. 50 μL of test sample was taken and placed in a sample loading bottle. At the same time, 50 μL of liquid paraffin oil was added to the corresponding setting of each isomer of (R)-lavender (R)-2-methylbutyrate as a control.

[0041] Y-type olfactometer measurement method:

[0042] The Y-shaped olfactometer was made of transparent glass. The main tube and two arms were 11 cm long, with an inner diameter of 1 cm and a 75° angle between the arms. The Y-shaped olfactometer was connected in sequence to the Y-shaped tube, a glass rotor flowmeter (LZB-3WB), a sample loading bottle (100 ml), distilled water (250 ml from a gas washing bottle), an activated carbon drying tower, and an air sampler. All interfaces were connected with Teflon tubing. The air sampler maintained a flow rate of 50 ml / min. For each dose, two sample loading bottles were used, each containing 50 μL of (R)-lavenderoyl (R)-2-methylbutyrate and its enantiomer, and 50 μL of a control sample (liquid paraffin oil) as the odor source. A 28W LED light board was placed at the Y end of the Y-shaped olfactometer as the sole induced light source. The test environment was maintained at 26 ± 1°C, and the experiments were conducted between 10:00 and 17:00 during the day.

[0043] For thrips, 3-5 day-old male and female adults were selected and introduced one by one into the center of the main Y-shaped tube, and the number entering each arm of the olfactometer was recorded. The timer began when the thrips were introduced into the main tube. Within 5 minutes, a thrip was considered to have chosen the odor source if it reached the halfway point of either arm and remained there for more than 10 seconds, or if it reached more than two-thirds of the way, and was considered to have chosen the odor source. Otherwise, it was considered to have not responded. To avoid position effects, the odor source was swapped after testing five thrips. To avoid the influence of residual thrip odor, a new Y-shaped tube was replaced after testing 10 thrips. The Y-shaped tube was cleaned with 75% ethanol and baked at 60°C for 30 minutes. The experiment was repeated 60 times, and all test insects were used only once.

[0044] 1.6 The selection behavior of male and female adults of Thrips taurochoerus towards baits prepared from (R)-lavenderoyl (R)-2-methylbutyrate and paraffin oil was measured using a Y-type olfactometer.

[0045] A preparation method for attracting thrips, comprising the following steps:

[0046] Step 1: Preparation of sustained-release carrier

[0047] Choose green natural rubber as the lure carrier, soak it in anhydrous ethanol for 24-48 hours, remove the rubber plug and place it in a 30-50℃ oven for 24-48 hours;

[0048] Step 2: Preparation of Aggregation Pheromone Attractants

[0049] Lures consisting of (R)-lavenderoyl (R)-2-methylbutyrate and paraffin oil were prepared. Three experimental groups were set up. (R)-lavenderoyl (R)-2-methylbutyrate was diluted to 0.5 μg / μL, 1 μg / μL, and 2 μg / μL with paraffin oil to prepare the lures. In each experimental group, 50 μL of paraffin oil was added as a control.

[0050] Step 3: Prepare the lure

[0051] Immerse the lure core carrier in the bottle containing the aggregation pheromone attractant prepared in step 2. Soak for 24 hours, then remove and air-dry in a cool, dark place. For the control group, soak only in paraffin oil. Wrap the prepared lure core in tinfoil and store in a -20°C refrigerator until ready to use.

[0052] The Y-type olfactometer measurement method is the same as the Y-type olfactometer measurement method in 1.5.

[0053] 1.7 Data Analysis

[0054] The chi-squared test was used to analyze behavioral selection. Other statistical analyses were completed using Office 2010 and SPSS 22.0, and Adobe Photoshop CC 2018 was used for drawing.

[0055] 2. Results and Analysis

[0056] 2.1 Identification of chemical components of aggregation pheromones

[0057] Figure 1 The results showed that a substance was found in the total ion current chromatogram of male adults of Cyprinus spp., with a peak time of 27.37 min. Figure 2 The results showed that the mass spectrum of this component, compared with the NIST mass spectral library, showed that the substance with the highest similarity was (R)-lavenderoyl (R)-2-methylbutyrate, with a similarity of 95%. Since (R)-lavenderoyl (R)-2-methylbutyrate has two chiral carbon atoms and therefore exists in four enantiomers, the volatile substance can be qualitatively identified as (R)-lavenderoyl (R)-2-methylbutyrate and its other enantiomers. To determine the specific structure of the volatile compound, the results of a Y-type olfactometer were used to analyze the selective behavior of the thrips occidentalis for (R)-lavenderoyl (R)-2-methylbutyrate and its other enantiomers.

[0058] 2.2 Behavioral responses of Thrips taurochoides to (R)-lavandinol (R)-2-methylbutyrate isomers

[0059] Since (R)-lavenderoyl (R)-2-methylbutyrate has two chiral carbon atoms, there are four enantiomers. We have verified the behavior of these four enantiomers and the racemate. Figure 3 The results showed that at a concentration of 0.5 μg / μL, only (R)-lavenderoyl (R)-2-methylbutyrate showed a significant attraction to male adults, and the volatile substance on the surface of the flower thrips was qualitatively identified as (R)-lavenderoyl (R)-2-methylbutyrate.

[0060] 2.3 Behavioral responses of Cyprinus spp. to lures prepared from (R)-lavenderoyl (R)-2-methylbutyrate and paraffin oil

[0061] Figure 4 Results of a behavioral choice test of Cyprinus spp. for lures prepared from (R)-lavandinyl (R)-2-methylbutyrate and paraffin oil. Figure 4 The results showed that the bait prepared with (R)-lavenderoyl (R)-2-methylbutyrate and paraffin oil showed strong attraction to male adults of Thrips taurus. (R)-lavenderoyl (R)-2-methylbutyrate diluted with paraffin oil at three concentration gradients of 0.5μg / μL, 1μg / μL, and 2μg / μL showed strong attraction to male and female adults of Thrips taurus. The attraction effect became stronger with increasing concentration, and male adults reacted more strongly than female adults.

[0062] When (R)-lavenderoyl (R)-2-methylbutyrate was diluted in paraffin oil to a concentration of 0.5 μg / μL, the prepared bait showed significant attraction to male adults, while there was no significant difference in female adults. When (R)-lavenderoyl (R)-2-methylbutyrate was diluted in paraffin oil to a concentration of 1 μg / μL, the prepared bait showed significant attraction to male adults, while there was no significant difference in female adults. When the concentration of (R)-lavenderoyl (R)-2-methylbutyrate was diluted with paraffin oil to 2 μg / μL, the prepared bait showed significant attraction to unmated female adults, female adults and male adults of the bullhorn flower thrips, indicating that the bait prepared with (R)-lavenderoyl (R)-2-methylbutyrate and paraffin oil had the best attracting effect when the concentration of (R)-lavenderoyl (R)-2-methylbutyrate was diluted with paraffin oil to 2 μg / μL, providing technical support for the preparation of pheromone attractants for attracting the bullhorn flower thrips.

[0063] As described above, the present invention can be better implemented. The above embodiments only describe the preferred implementation methods of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various changes and improvements made to the technical solutions of the present invention by ordinary technicians in this field should fall within the protection scope determined by the present invention.

Claims

1. Application of (R)-lavenderyl (R)-2-methylbutyrate in pheromone attractants, characterized in that: The application is that (R)-lavenderoyl (R)-2-methylbutyrate is used to attract male and female adults of Cyprinus thrips, and the attractant concentration of the (R)-lavenderoyl (R)-2-methylbutyrate is 0.5 μg / μL-2 μg / μL.

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