Application of terpinene and gamma-terpinene in inducing flower thrips

Abstract

The invention discloses an application of terpinene and gamma-terpinene in inducing flower thrips. According to the invention, volatile matters terpinene and gamma-terpinene which have a remarkable attraction effect on the flower thrips are separated and identified from the host plant mango flowers, and can be used for monitoring, preventing and treating the flower thrips. Compared with an existing color plate trapping and killing technology, the terpinene and the gamma-terpinene can trap and kill the flower thrips all day long, and the terpinene and the gamma-terpinene come from plants and are environmentally friendly, so that the terpinene and the gamma-terpinene can be used as an attractant of the flower thrips and have high application value.

Description

Technical Field

[0001] This invention belongs to the field of green control technology for flower thrips, specifically involving the application of terpinene and γ-terpinene in attracting flower thrips. Background Technology

[0002] Flower thrips Frankliniella instonsa Flower thrips is a small, polyphagous pest with a wide host range, damaging over 170 species of economic crops from 54 families. It is a significant agricultural pest in my country and globally. Thrips cause damage by ripping and sucking sap from flowers, young leaves, and young fruits, leading to wilting and fruit drop. They also spread various plant viruses such as TSWV and INSV. According to incomplete statistics, flower thrips damage can cause crop yield reductions of 15% to 30%, and in severe cases, complete crop failure.

[0003] In recent years, flower thrips have frequently broken out in tropical regions, especially damaging mangoes, and have become a significant factor restricting the healthy development of the mango industry. Therefore, it is necessary to develop green control agents and methods for flower thrips. Summary of the Invention

[0004] The purpose of this invention is to address the current outbreak of flower thrips in tropical regions. While physical and chemical induction techniques are important measures for controlling flower thrips, there is a lack of highly effective attractants. This invention aims to develop plant-derived attractants with significant attraction effects on flower thrips, and to provide the application of terpinene and γ-terpinene in attracting flower thrips.

[0005] This invention collects and identifies volatiles from mango flowers using solid-phase microextraction (SPME) coupled with gas chromatography-mass spectrometry (GC-MS), and screens and verifies volatile substances, terpinene and γ-terpinene, which have significant attraction effects on flower thrips using a Y-type olfactometer combined with field trapping experiments.

[0006] The first objective of this invention is to provide the use of terpinene in attracting flower thrips.

[0007] Preferably, the flower thrips is an adult flower thrips.

[0008] A second objective of this invention is to provide a method for attracting flower thrips, which utilizes terpinene to attract flower thrips.

[0009] Preferably, terpinene is used as the active ingredient of the attractant, and the concentration of terpinene in the attractant is 1 μg / μL~10 μg / μL. The attractant is adsorbed onto the sponge plate on the adhesive trap, and then the flower thrips are attracted.

[0010] Preferably, terpinene is used as the active ingredient of the attractant, the attractant is placed in a trap bottle, and then the flower thrips are attracted.

[0011] A third objective of this invention is to provide the application of γ-terpinene in attracting flower thrips.

[0012] Preferably, the flower thrips is an adult flower thrips.

[0013] A fourth objective of this invention is to provide a method for attracting flower thrips, which utilizes γ-terpinene to attract flower thrips.

[0014] Preferably, γ-terpinene is used as the active ingredient of the attractant, and the concentration of γ-terpinene in the attractant is 1 μg / μL~10 μg / μL. The attractant is adsorbed onto the sponge plate on the adhesive trap, and then the flower thrips are attracted.

[0015] Preferably, γ-terpinene is used as the active ingredient of the attractant. The attractant is placed in a trap bottle and then used to attract flower thrips.

[0016] This invention isolates and identifies volatile compounds, terpinene and γ-terpinene, from mango flowers, which have significant attractant effects on flower thrips. These compounds can be used for the monitoring and control of flower thrips, providing important technical support for the monitoring and physicochemical induction and control of flower thrips. Compared with current color-based trapping techniques, terpinene and γ-terpinene can attract and kill flower thrips around the clock. Furthermore, terpinene and γ-terpinene are derived from plants, making them environmentally friendly and suitable as attractants for flower thrips, thus possessing significant application value. Attached Figure Description

[0017] Figure 1 This is a total ion mass spectrum of mango flowers; where A is the blank control, B is the mango flower, C is the standard, and the numbers 1-6 represent the compounds myrcene, phellandrene, α-terpinene, γ-terpinene, terpinene, and ethyl benzoate, respectively.

[0018] Figure 2 This describes the selective behavior of thrips towards different concentrations of five volatile compounds and mixtures of volatile compounds; Note: ** indicates significant differences at the 0.01 level (GLM binomial distribution test).

[0019] Figure 3 This describes the selective behavior of flower thrips towards single and mixed agents of mango flower volatiles; Note: * and ** represent significant differences at the 0.05 and 0.01 levels, respectively (GLM binomial test).

[0020] Figure 4This study shows the attraction of different mango flower volatiles combined with transparent sticky insect traps to flower thrips in a cantaloupe field after 24 hours. Note: The concentrations of each volatile in the figure are 0.1 μg / μL, 1 μg / μL and 10 μg / μL from left to right. * indicates that the number of bean thrips trapped by this concentration of volatile is significantly different from the control (P<0.05) according to the Kruskal-Wallis test.

[0021] Figure 5 This study shows the attraction of different mango flower volatiles combined with transparent sticky insect traps to flower thrips in dragon fruit fields after 24 hours. Note: The concentrations of each volatile in the figure are 0.1 μg / μL, 1 μg / μL, and 10 μg / μL from left to right. * indicates that the number of bean thrips trapped by this concentration of volatile is significantly different from the control (P<0.05) according to the Kruskal-Wallis test. Detailed Implementation

[0022] The following embodiments are further illustrations of the present invention, but not limitations thereof.

[0023] Example 1 1. Collection and identification of volatile compounds from mango flowers Preheat the GC-MS to 200℃ at the injection port. Insert the SPME extraction tip into the injection port and age it for 30 min. Collect 2.0 g of fresh, pest-free mango blossoms and place them in a 20 mL headspace vial. Puncture the gasket on the top of the transparent headspace vial with the aged extraction tip, place it in a preheated 40℃ metal water bath, and after removing the adsorption coating, secure the handle to maintain stability (to prevent the adsorption layer from detaching due to shaking). Incubate at 40℃ for 1 h.

[0024] GC-MS program setup: The GC-MS model was Agilent GC-MS 7890A+5975C (column type: HP-5MS flexible quartz glass capillary column (30 m × 0.25 mm × 0.25 μm), and the gas chromatographic detector was a FID flame ionization detector). GC conditions: Carrier gas was high-purity nitrogen (>99.99%), flow rate 2 mL / min; auxiliary gas parameters were set to hydrogen 30 mL / min and air 400 mL / min; split injection mode (split ratio 10:1). The injection port temperature was constant at 250℃, and the column temperature was controlled using a gradient program: initial temperature of 40℃ for 4 min, increased to 160℃ at 5℃ / min, held for 5 min, then increased to 260℃ at a rate of 10℃ / min and held for 5 min. MS conditions: solvent delay time 3 min, transfer line temperature 280℃, quadrupole temperature 150℃, ion source temperature 230℃; ionization mode EI; ionization energy 70 eV; data acquisition via full scan, scan range 45-550 amu.

[0025] Each sample was manually injected at a rate of 1 μL. The content of each component was calculated using the peak area normalization method. The obtained total ion chromatogram was compared with the NIST14 (National Institute of Standards and Technology) database to obtain the name, peak time, molecular formula, CAS number, and relative content of each substance. Volatile substances with the same peak time and similarity ≥90% in multiple sets of replicate data, as well as volatile substances that appeared multiple times, were included in the preliminary list of volatile substances for identification. The peak time, molecular formula, similarity search, and mass spectrum were recorded. Volatile substance collection and identification were repeated 15 times. The volatile substance with the highest frequency and a matching degree greater than 90% in 5 replicates was identified as a mango flower volatile substance, and a standard was purchased for verification. The standard was diluted to 1 ppm with n-hexane. Using a 50 µL microsyringe, 10 µL of the diluted standard solution was taken and tested according to the same GC-MS procedure. The mass spectrum results of the standard were then compared and analyzed with the mass spectrum results of the volatile substance list to determine the types of volatile substances.

[0026] The volatiles of mango flowers were extracted and identified using SPME and GC-MS combined techniques. Figure 1(See Table 1) 27 highly matched components were obtained, including 24 terpenes, 1 aromatic hydrocarbon, and 2 esters. Myrcene, phellandrene, α-terpinene, terpinene, γ-terpinene, and ethyl benzoate were found to be present in high and stable amounts. Based on high matching and good reproducibility, six volatile compounds with high and stable content were selected: myrcene, phellandrene, α-terpinene, terpinene, γ-terpinene, and ethyl benzoate. By purchasing standards for these six volatile compounds and analyzing them on GC-MS, it was found that the retention times of these six volatile compounds were consistent with those of the standards. Therefore, the six volatile compounds were identified as myrcene, phellandrene, α-terpinene, terpinene, γ-terpinene, and ethyl benzoate.

[0027] Table 1. Identification results of the main active components of mango flower volatiles 2. Selective behavior of flower thrips towards mango flower volatiles A Y-shaped olfactometer was used to determine the selective behavior of the six volatile compounds screened above, identifying those that attracted thrips to crops. The Y-shaped olfactometer consisted of an air pump, a drying tower, a gas washing bottle, a scent source bottle, a flow meter, and a Y-shaped glass tube connected sequentially with Teflon tubing. The airflow velocity in both arms was controlled at 50 mL / min by the gas flow meter. Activated carbon was placed in the drying tower to purify the air, and two-thirds of the gas washing bottle was filled with distilled water for humidification. To avoid interference from uneven ambient light intensity on the selective behavior of thrips, the entire glass Y-shaped tube was covered with an opaque black cloth. The experiment was conducted at a room temperature of 26℃ and a relative humidity of 70%–75%, during the peak insect activity period (9:00–14:00).

[0028] The study included treatments for single-agent volatile compounds and blank controls, as well as treatments for single-agent volatile compounds and mixed volatile compounds. The mango flower volatile compound mixture was based on the results of previous identification. Figure 1The mixture was prepared by mixing the six volatile compounds (myrcene, phellandrene, α-terpinene, terpinene, γ-terpinene, and ethyl benzoate) as shown in Table 1 (i.e., the concentration of the volatile mixture was the total concentration of the six volatile compounds, with the molar ratio of myrcene, phellandrene, α-terpinene, terpinene, γ-terpinene, and ethyl benzoate being 1.45∶0.68∶2.55∶47.01∶0.33∶3.06). Thirty healthy, active 2-day-old adult flower thrips were collected and placed at the entrance of the main arm of a "Y"-shaped tube. Observation was conducted after 5 minutes. Thrips were considered to have responded to the substances in the sidewall if they entered 2 / 3 of the tube within 5 minutes and remained there for more than 30 seconds. Behavioral observation was terminated when the flower thrips did not make a choice after 5 minutes in the sidewall. To eliminate the influence of tube wall position effects, the positions of the two side arms were swapped for each test, and each treatment was repeated 5 times. Teflon tubing, gas washing bottles, and Y-shaped glass tubes were cleaned in ethanol and allowed to air dry after each treatment test to eliminate odor interference between different treatments. By comparing the attraction behavior of flower thrips to volatiles in relation to air and between pairs of volatiles, the volatiles with the strongest attraction to flower thrips were screened.

[0029] Because previous studies have reported the significant tropism of flower thrips towards myrcene using a Y-type olfactometer, no repeat measurements were performed in the volatile matter and blank control groups in this embodiment. The behavioral responses of flower thrips to terpinene, γ-terpinene, α-terpinene, phellandrene, and ethyl benzoate at concentrations of 10 μg / μL, 1 μg / μL, and 0.1 μg / μL were measured using a Y-type olfactometer. Figure 2 It was found that thrips exhibited significantly higher selectivity for terpinene, α-terpinene, phellandrene, and ethyl benzoate at various concentrations than for the paraffin control, indicating that these four substances had a significant attraction for thrips. Furthermore, it was also found that thrips showed significantly higher selectivity for γ-terpinene at concentrations of 10 μg / μL and 1 μg / μL than for the paraffin control, and the selectivity for the volatile mixture at concentrations of 10 μg / μL, 1 μg / μL, and 0.1 μg / μL was also significantly higher than that for the paraffin control. In summary, myrcene, terpinene, γ-terpinene, α-terpinene, phellandrene, and ethyl benzoate were selected for further experiments.

[0030] The selectivity of single-agent (1 μg / μL) and mixed-agent (1 μg / μL) mango flower volatiles that attracted thrips was further determined using a Y-type olfactometer. Figure 3The study found no significant difference in the selectivity of flower thrips for terpinene, ethyl benzoate, and mango flower volatiles mixtures. However, flower thrips showed significantly higher selectivity for γ-terpinene and phellandrene than for the mango flower volatiles mixture, while their selectivity for myrcene and α-terpinene was significantly lower. Based on the cost, convenience, and attraction effect of attractants on flower thrips, terpinene, ethyl benzoate, γ-terpinene, and phellandrene were selected as flower thrips attractants for subsequent field trials to verify their effectiveness.

[0031] 3. The attraction effect of mango flower volatiles on flower thrips in different host orchards Field trials were conducted at cantaloupe and dragon fruit farms. Based on volatiles identified in laboratory studies as having an attraction effect on thrips, these volatiles were diluted with paraffin oil to different concentrations (0.1 μg / μL, 1 μg / μL, and 10 μg / μL) for later use. Sticky traps, consisting of a transparent plastic plate coated with sticky adhesive and a fiber rod with crossbars, were placed 1.2 meters above the ground. A high-density sponge board was attached to the center of the transparent plastic plate, and then 100 μL of the volatile paraffin oil solution or a blank control (paraffin oil) was pipetted onto the sponge board. The sticky traps were randomly arranged in the center of each row, with 10 traps per row, at least one row between different concentrations, and at least 5 meters between each trap. Field trials validated the volatiles that attract thrips and their application concentrations. By combining different mango flower volatiles with transparent sticky insect boards, the attraction effects of four mango flower volatiles on adult flower thrips in cantaloupe and dragon fruit fields were determined. Figure 4 and Figure 5 The results showed that terpinene and γ-terpinene had good attraction effects on thrips in different orchards. Water phellandrene had an attraction effect on thrips in cantaloupe fields, but the number of thrips attracted in dragon fruit fields was not significantly different from the control. Based on the data from the two orchards, terpinene and γ-terpinene are suitable for the preparation of thrips attractants at concentrations of 1 μg / μL to 10 μg / μL.

Claims

1. Application of terpinene in attracting flower thrips.

2. The application according to claim 1, characterized in that, The flower thrips mentioned are adult flower thrips.

3. A method for attracting flower thrips, characterized in that, Flower thrips were attracted using terpineol oil.

4. The method according to claim 3, characterized in that, Terpinene was used as the active ingredient of the attractant, with a concentration of terpinene ranging from 1 μg / μL to 10 μg / μL. The attractant was adsorbed onto a sponge plate on an adhesive trap and then used to attract flower thrips.

5. The method according to claim 3, characterized in that, Terpinene was used as the active ingredient of the attractant. The attractant was placed in a trap bottle and then used to attract flower thrips.

6. Application of γ-terpinene in attracting flower thrips.

7. The application according to claim 6, characterized in that, The flower thrips mentioned are adult flower thrips.

8. A method for attracting flower thrips, characterized in that, Flower thrips were attracted using γ-terpinene.

9. The method according to claim 8, characterized in that, Using γ-terpinene as the active ingredient of the attractant, with a concentration of γ-terpinene in the attractant ranging from 1 μg / μL to 10 μg / μL, the attractant was adsorbed onto a sponge plate on an adhesive trap, and then used to attract flower thrips.

10. The method according to claim 8, characterized in that, Using γ-terpinene as the active ingredient of the attractant, the attractant was placed in a trap bottle and then used to attract flower thrips.

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