Volatile substance with attraction activity on tomato leaf miner in tomato

Abstract

The invention discloses a volatile substance with attraction activity on tomato leaf miners in tomatoes. The volatile substance comprises any one or a mixture of four of tomato volatile matters including cis-3-hexenol (leaf alcohol), beta-phellandrene, nonanal and 4-ethylbenzaldehyde. When the volatile substances are used as a mixture, cis-3-hexenol (leaf alcohol), beta-phellandrene, nonanal and 4-ethylbenzaldehyde are mixed according to the volume ratio of 3.5: 3.8: 1: 6.7, and the volatile substances have an attractive effect on adult tomato leaf miners. A Y-type olfactometer is used for determining behavior reactions of female and male adults of the tomato leaf miner to four EAD active compounds and mixtures thereof under different concentrations, and beta-phellandrene and the mixtures with the concentration reaching 10 mu g / mu l or above have a remarkable or extremely remarkable attraction effect on the female adults of the tomato leaf miner. The highest attraction rate of the leaf alcohol to female adult tomato leaf miners can reach 76.7% and 80%, the leaf alcohol with the concentration of 100 micrograms / microliter has a remarkable attraction effect on the female adult tomato leaf miners and the male adult tomato leaf miners, and the attraction rate of the leaf alcohol to the male adult tomato leaf miners can reach 70% and 73.3%.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological control, and specifically discloses volatile substances in tomatoes that have attractant activity to tomato leafminers. Background Art

[0002] The tomato leafminer, scientifically known as Tuta absoluta (Meyrick), also commonly known as the tomato wheat moth or tomato wheat miner, belongs to the Gelechiidae family of the order Lepidoptera and is a worldwide quarantine invasive pest. Immediately after hatching, the larvae of the tomato leafminer burrow into the tissues of tomato leaves, feeding on the mesophyll. They also bore into terminal buds, tender stems, flower buds, and fruit, posing a threat to tomatoes throughout their growing season and often causing significant economic losses. Due to the tomato leafminer's short life cycle, overlapping generations, and the cryptic feeding habits of its larvae, chemical pesticides are not only difficult to kill but also likely negatively impact its natural enemies and pollute the ecological environment. Reports of resistance to various insecticides have been reported in countries such as Chile, Brazil, and Argentina. Furthermore, its cryptic feeding habits make it difficult for parasitic and predatory natural enemies to effectively control it. Therefore, the development and utilization of effective, green, ecological, and sustainable control technologies are urgently needed.

[0003] Plant secondary metabolites are a variety of small organic compounds produced through secondary metabolism within plants. While they typically do not directly participate in primary biochemical reactions such as plant growth and development, their production and distribution are often species-, organ-, tissue-, and developmental-stage specific. Currently, over 100,000 plant secondary metabolites have been discovered, with structures of approximately 20% known. Plant secondary metabolites are characterized by their vast variety, diverse properties, and abundant sources. Based on their structural types, they are primarily classified into three categories: terpenoids, phenolics, and nitrogen-containing compounds. Plants orchestrate and deploy their complex secondary metabolites to survive in nature under adverse conditions. Secondary metabolites that affect insects can be divided into volatile and non-volatile types. Plant volatile secondary metabolites are crucial mediators of complex information exchange between plants and the external environment, influencing the preferences of herbivorous insects for key behaviors such as survival, feeding, mating, and oviposition. Previous studies have shown that 52 volatile compounds were identified in the headspace collection of three varieties, but it is unclear which specific compounds play a key role in attracting tomato leafminers and promoting their oviposition. In addition, ethyl heptanoate and 1-nonanol released by tomatoes can not only induce higher antennal potential (EAG) responses in female and male tomato leafminers. However, there is currently limited information on the effects of host plant volatiles on the behavioral responses of tomato leafminers.

[0004] The applicant used headspace collection experiments to obtain tomato secondary metabolite volatiles, and preliminarily screened the components in tomato secondary metabolite volatiles that had electrophysiological activity on the antennae of adult tomato leafminers through gas chromatography-electroantennary coupling (GC-EAD) and gas chromatography-mass spectrometry (GC-MS); using electroantenniotypic responses, Y-type olfactometer behavioral measurements, field oviposition preference responses, and field adult trapping experiments, the applicant finally identified the active volatiles in tomatoes that have an attractant effect on adult tomato leafminers. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to address the deficiencies of the above-mentioned prior art and provide a volatile substance in tomato volatiles that has attractant activity to tomato leafminers. The specific operation is as follows:

[0006] The present invention first discloses a volatile substance in tomatoes that has attractant activity to tomato leafminers, comprising:

[0007] Any one of cis-3-hexenol (leaf alcohol), β-phellandrene, nonanal, and 4-ethylbenzaldehyde, or a mixture of four of the above.

[0008] Furthermore, the mixture is:

[0009] A mixed liquid of cis-3-hexenol (leaf alcohol), β-phellandrene, nonanal and 4-ethylbenzaldehyde in a volume ratio of 3.5:3.8:1:6.7.

[0010] The present invention also discloses a biological preparation for preventing and controlling tomato leafminer, wherein:

[0011] The biological preparation includes the above-mentioned volatile substance.

[0012] The invention also discloses an application of the volatile substance in preventing and controlling tomato leafminer.

[0013] The invention also discloses application of the biological preparation in preventing and controlling tomato leafminer.

[0014] The present invention also discloses a method for preventing and controlling tomato leafminers, wherein:

[0015] The method comprises using the above-mentioned volatile substances for prevention and control.

[0016] The present invention also discloses a method for preventing and controlling tomato leafminers, wherein:

[0017] The method comprises using the above-mentioned biocontrol agent for prevention and control.

[0018] The present invention also discloses a method for collecting the volatile substances in tomatoes that have attractant activity to tomato leafminers, comprising:

[0019] (1) Headspace collection of tomato volatiles

[0020] After mechanically damaging the above-ground parts of fresh tomato plants, place them in a 250ml wide-mouth bottle. The bottle is sealed with a clean silicone stopper with an air inlet and outlet tubes, and 30mg of adsorbent Porapak Q is placed in a sampling tube. Silicone tubes are connected in the order of an atmospheric sampler, an activated carbon tube, a color-changing silicone tube, a glass rotor flowmeter, a wide-mouth bottle, and a glass rotor flowmeter. After checking that the device is airtight, the atmospheric sampler flow rate is set to 350ml / min. After evacuating the wide-mouth bottle for 15 minutes to completely expel the original gas in the wide-mouth bottle, the sampling tube is connected between the air outlet end of the wide-mouth bottle and the flowmeter. After checking that the device is airtight again, the headspace collection of volatiles released by the mechanically damaged tomato plants is formally carried out. After the collection is completed, the adsorbent with the tomato volatiles is eluted with dichloromethane at room temperature, and the eluate is stored at -20°C.

[0021] (2) GC-EAD analysis

[0022] The eluent was injected without splitting, with an injection volume of 2 μl each time, a solvent delay of 3 min, a detector temperature of 300 °C, an injection port temperature of 250 °C, a carrier gas of high-purity helium, and a flow rate of 1.5 mL / min. Active female adults of tomato leafminers aged 1-2 days were selected, and two glass capillaries with a length of 4-5 cm, neat ends and a pointed end were prepared. One glass tube was used as a measuring electrode, and its tip was slightly thicker than the end of the insect antennae. The other glass tube was used as a reference electrode. The two glass capillaries were aspirated with freshly prepared Ringer saline to a distance of 2-3 mm from the tail. At the same time, two clean silver wires with a length of 4.5 cm were prepared as electrode wires. The electrode wires were pulled from the glass capillaries. Insert the tail of the electrode holder, bend the end of the electrode wire 2mm and stabilize it to the tail of the holder, then insert the tip of the electrode wire from the end of the prepared glass capillary, and finally fix the two glass electrode holders to the measuring pole and reference pole of the PRG-3 electrode respectively; prepare adult insects with good activity, quickly cut the antennae from the base of the antennae with a scalpel under a dissecting microscope, and cut the antennae a little, remove the reference pole of the PRG-3 electrode, rely on the surface tension of physiological saline to suck the antennae base, then fix the reference pole, and at the same time connect the antennae to the measuring pole under a stereo microscope, record and analyze the data using GC-EAD software, and repeat the GC-EAD test 5 times;

[0023] (3) GC-MS analysis

[0024] The eluent was injected using splitless flow, with an injection volume of 2 μL per injection. The solvent delay was 3 min, the detector temperature was 300°C, the injection port temperature was 250°C, the carrier gas was high-purity helium, the flow rate was 1.5 mL / min, the quadrupole temperature was 150°C, the ion source temperature was 250°C, the ionization mode was EI, the mass spectrometer bombardment voltage was 70 eV, the scanning mass range was 45-500 m / z, and the scanning frequency was 5 / s. The qualitative analysis of the volatile components of damaged tomatoes was carried out by checking and supplementing the search results with the NISTO8 spectral library in the computer connected to the gas chromatography-mass spectrometry instrument and reference to standard spectra. The experiment was repeated three times.

[0025] (4) Electroantennographic responses of adult tomato leafminers to four standard volatiles and their mixtures

[0026] A clean filter paper strip containing 10 μl of a test solution is placed in a Pasteur tube, and both ends of the tube are sealed with sealing film. A clean filter paper strip containing 10 μl of paraffin oil is placed in a Pasteur tube as a control, and both ends of the tube are also sealed with sealing film. An adult tomato leafminer is taken, and the antennae of the adult tomato leafminer are cut off at the base with a scalpel under a stereomicroscope, and 1-2 flagella at the ends of the antennae are removed. The ends of the treated antennae are then connected to a reference electrode and a recording electrode, respectively, with conductive glue. The airflow system is controlled by an airflow distributor. During the test, each antennae is stimulated in the order of control paraffin oil, test solution from low to high concentration, and control paraffin oil. Only one antennae of each adult is tested, and six independent individuals of both sexes are tested.

[0027] (5) Olfactory behavioral responses of adult tomato leafminers to four standard volatiles and their mixtures

[0028] The Y-shaped tube of the Y-type olfactometer has a main arm length of 20 cm and an inner diameter of 2.5 cm. The two side arms are both 15 cm long and 2.5 cm in inner diameter. The angle between the side walls is 75°. A clean filter paper strip containing 10 μl of the test solution is placed on one side arm of the Y-shaped tube. A clean filter paper strip containing 10 μl of paraffin oil is placed on the other side arm as a control. The odor source is connected to a QC-1B atmospheric sampler. A single adult tomato leafminer is released into the base of the main arm of the Y-shaped tube. Each adult tomato leafminer is tested for 5 minutes. The adult tomato leafminer is placed into the test. The experimental group was selected if the adult tomato leafminer entered the control arm more than 10 cm and stayed for more than 30 seconds. The control group was selected if the adult tomato leafminer entered the control arm more than 10 cm and stayed for more than 30 seconds. If the adult did not make a choice after 5 minutes, it was considered as non-responsive. Each adult was tested only once, and each group of 10 females or males was repeated 3 times. After testing 5 insects, the positions of the two arms of the Y-shaped tube were swapped. After each group was tested, the Y-shaped tube was thoroughly cleaned with alcohol. After each test, the Y-shaped tube, washing bottle and connecting silicone tube were cleaned with acetone.

[0029] (6) Oviposition preference responses of adult tomato leafminers to four standard volatiles and their mixtures

[0030] 100 μL of 100 μg / μL leaf alcohol, β-phellandrene, and their mixture, and 100 μL of 10 μg / μL 4-ethylbenzaldehyde and nonanal, were placed in a rubber-tipped bait carrier. 100 μL of paraffin oil was placed in the bait as a control. The experiment was conducted in insect cages. Four healthy and intact potted tomato host plants, Solanum lycopersicum L., were placed in the four corners of the mesh cages, and the cages were spaced 10 m apart to avoid mutual interference. 100 μL of the test solvent was pipetted into a rubber stopper to prepare a flavor source. In the mesh cages of group 1, two tomato plants on one diagonal were selected and two flavor source rubber stoppers were fixed to the plants respectively as the experimental group. Two plants on the other diagonal were fixed with rubber stoppers containing paraffin oil as the control group. Three pairs of two-day-old male and female tomato leafminers were released into each cage, and the number of eggs on each tomato plant was counted after 48 hours.

[0031] (7) Preferential responses of tomato leafminer adults to four standard volatiles and their mixtures in the field

[0032] Using paraffin oil as the solvent, solutions of cis-3-hexen-1-ol, β-phellandrene, and their mixture were prepared at a concentration of 100 μg / μL⁻¹. Nonanal and 4-ethylbenzaldehyde were prepared at a concentration of 10 μg / μL⁻¹. 100 μL of each solution was added to a brown slow-release bottle (1 mL capacity, with a 1 mm diameter hole drilled in the center of the cap for odor release). An equal amount of paraffin oil was used as a control. The slow-release bottle was hung in the center of a white triangular trap (17.8 cm side length, 26.5 cm height) or embedded in a square hole (1 cm × 1 cm) in a blue sticky trap (25 cm × 20 cm). The traps were randomly placed at the base of tomato plants, spaced 20 meters apart. After 72 hours, the number of trapped tomato leafminers (Tuta absoluta) was counted, with three replicates for each group;

[0033] (8) Data Analysis

[0034] The experimental data were statistically analyzed using IBM SPSS Statistics 27.0.1 software. All results were expressed as mean ± SEM. Tukey's multiple range test in one-way analysis of variance was used to test the significance of EAG response values. The significant differences in the Y-type olfactometer bioassay data were compared by chi-square test. Tukey's multiple range test in one-way analysis of variance was used to analyze the egg-laying repellent index. Statistical significance was set at P < 0.05.

[0035] The present invention is beneficial in that:

[0036] The present invention discovers that tomato volatiles cis-3-hexenol, β-phellandrene, nonanal, 4-ethylbenzaldehyde, and their mixtures have attractant activity against adult tomato leafminers. Testing revealed that cis-3-hexenol (leaf alcohol), β-phellandrene, nonanal, and 4-ethylbenzaldehyde, starting at a dose of 1 μg / μL, elicited significant dose-dependent EAG responses in the antennae of male and female tomato leafminers. A Y-type olfactometer was used to measure the behavioral responses of male and female tomato leafminers to varying concentrations of the four EAG-active compounds and their mixtures. β-phellandrene and its mixtures at concentrations of 10 μg / μL and above exhibited significant or highly significant attractant effects on female tomato leafminers, with attraction rates reaching as high as 76.7% and 80% for female adults. Leaf alcohol at a concentration of 100 μg / μL exhibited significant attractant effects on both male and female adults, with attraction rates reaching 70% and 73.3% for both male and female adults. The four active compounds and their mixtures can be used as attractants for the prevention and control of tomato leafminers. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Schematic diagram of the Y-type olfactometer and air delivery system;

[0038] Figure 2 Simulated diagram for the egg-laying preference response test;

[0039] Figure 3 This is the GC-EAD reaction spectrum of the antennae of female adults of the tomato leafminer to tomato volatiles. Tuta abs-oluta is the tomato leafminer, Solanum lycopersicum L. is the tomato, cis-3-Hexen-1-ol is cis-3-hexen-1-ol, i.e., leaf alcohol, β-phellandrene is β-phellandrene, Nonanal is nonanal, and 4-Ethylbenzaldeh-yde is 4-ethylbenzaldehyde.

[0040] Figure 4The EAG responses of male and female adults of the tomato leafminer to four standard compounds and their mixtures are shown. Cis-3-Hexen-1-ol is cis-3-hexen-1-ol, i.e., leaf alcohol; β-phellandrene is β-phellandrene; Nonanal is nonanal; 4-Ethylbenzaldeh-yde is 4-ethylbenzaldehyde; and Blend is a mixture. The control group is Paraffinoil.

[0041] Figure 5 The behavioral responses of male and female adults of the tomato leafminer to four standard compounds and their mixtures are shown in red, while those of male adults are shown in blue. The treatment group includes cis-3-Hexen-1-ol, β-phellandrene, β-phellandrene, Nonanal, 4-Ethylbenzaldeh-yde, and Blend, while the control group includes paraffin oil.

[0042] Figure 6 This is a graph showing the oviposition preference responses of male and female adults of the tomato leafminer to four standard compounds and their mixtures. The treatment group includes: cis-3-Hexen-1-ol, cis-3-hexen-1-ol, β-phellandrene, β-phellandrene, Nonanal, 4-Ethylbenzaldeh-yde, and Blend, a mixture; the control group includes: Paraffin oil.

[0043] Figure 7 This figure shows the results of the trapping test of four standard compounds and their mixtures on tomato leafminer adults in the field. The treatment group: cis-3-Hexen-1-ol is cis-3-hexen-1-ol, i.e., leaf alcohol, β-phellandrene is β-phellandrene, Nonanal is nonanal, 4-Ethylbenzaldeh-yde is 4-ethylbenzaldehyde, and Blend is a mixture; the control group: Paraffin oil is paraffin oil. DETAILED DESCRIPTION

[0044] In order to make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. However, this should not be construed as limiting the present invention. The following description is only used to explain the present invention. Any modifications, substitutions or improvements that do not deviate from the spirit and principles of the present invention are within the scope of protection claimed by the present invention.

[0045] Example 1:

[0046] Volatile substances in tomatoes with attractant activity to tomato leafminer:

[0047] 1. Test compound:

[0048] Standard volatiles of cis-3-hexenol (>98%), nonanal (>96%), and 4-ethylbenzaldehyde (>97%) were purchased from Shanghai Aladdin Co., Ltd. (Shanghai, China), and β-phellandrene (>98%) was purchased from Toronto Research Chemicals (TRC), Canada.

[0049] Using paraffin oil (purchased from Sigma-Aldrich, USA) as a solvent, standard volatiles of cis-3-hexenol (leaf alcohol), β-phellandrene, nonanal, and 4-ethylbenzaldehyde were prepared at concentrations of 1 μg / μl, 10 μg / μl, and 100 μg / μl. A mixture of cis-3-hexen-1-ol: β-phellandrene: nonanal: 4-ethylbenzaldehyde was prepared at a ratio of 3.5:3.8:1:6.7 and prepared at concentrations of 1 μg / μl, 10 μg / μl, and 100 μg / μl using paraffin oil as a solvent.

[0050] 2. Test method:

[0051] (1) Headspace collection of tomato volatiles

[0052] On a clear, windless day, fresh, healthy, intact tomato plants were first washed with distilled water. The surface moisture was then blotted dry with clean filter paper strips. The above-ground portion of the plant was mechanically wound with clean scissors and gently placed in a clean 250 ml wide-mouth bottle. The bottle was filled to 2 / 5 of its capacity at a time and tightly capped with a clean silicone stopper with inlet and outlet tubes. 20 mg of the adsorbent Porapak Q (purchased from CNW Technologies) was placed in a sampling tube (12 cm long, 6 mm outer diameter, 4 mm inner diameter, purchased from Shanghai Anpu Laboratory Technology Co., Ltd.). Prior to the experiment, the adsorbent was activated by rinsing with 3 ml of acetone (≥99.5%, purchased from Tianjin Tianli Chemical Reagent Co., Ltd.) and 3 ml of methanol (≥99.5%, purchased from Tianjin Tianli Chemical Reagent Co., Ltd.), respectively. Silicone tubing was used to sequentially connect an air sampler (QC-1B model, purchased from the Beijing Institute of Labor Protection Science), an activated carbon tube (purchased from Shanghai Anpu Laboratory Technology Co., Ltd.), a color-changing silicone tube (purchased from Shanghai Anpu Laboratory Technology Co., Ltd.), a glass rotameter, a wide-mouth bottle, and the glass rotameter. After verifying the airtightness of the device, the air sampler was set to a flow rate of 350 ml / min. After evacuating the jar for 15 minutes to completely remove any remaining air, the sampling tube was connected between the jar's outlet and the flowmeter. After rechecking the airtightness of the device, headspace collection of damaged tomato volatiles was performed for 6 hours. After collection, the adsorbent containing the tomato volatiles was eluted with dichloromethane (≥99.5%, purchased from Tianjin Tianli Chemical Reagent Co., Ltd.) at room temperature, 300 μl per rinse, for five times. The eluate was placed in a 1.5 ml glass bottle and stored at -20°C for subsequent GC-EAD and GC-MS experiments. The experiment was repeated 6 times.

[0053] (2) GC-EAD analysis

[0054] A gas chromatography-electrophysiological antennal detection (GC-EAD) system was manufactured by Syntech, the Netherlands. The chromatographic column was an HP-5MS capillary column (length × inner diameter × film thickness = 30 m × 0.25 mm × 0.25 μm). The dichloromethane eluate of damaged tomato volatiles was injected splitlessly, with a 2 μL injection volume per injection. The column temperature program was as follows: an initial temperature of 50°C for 2 min, followed by a temperature increase of 4°C / min to 120°C for 2 min, and then a temperature increase of 20°C / min to 250°C for 4 min, for a total of 32 min. A solvent delay of 3 min was used. The detector temperature was 300°C, the inlet temperature was 250°C, and the carrier gas was high-purity helium at a flow rate of 1.5 mL / min. Select active female tomato leafminers (Cyprinus spp.) at 1-2 days old. Prepare two 4-5 cm long glass capillaries with neat, pointed ends. One glass capillary will serve as the measuring electrode, with its tip slightly thicker than the tip of the insect's antennae; the other will serve as the reference electrode, with its tip slightly thicker than the base of the antennae. Fill both capillaries with freshly prepared Ringer's saline until the tip is 2-3 mm from the tail. Also, prepare two 4.5 cm long clean silver wires as electrodes. Insert the electrode wires through the end of a glass electrode holder, bend the ends approximately 2 mm, and secure them to the end of the holder. Then, insert the electrode tips through the prepared glass capillaries. Finally, secure the two glass electrode holders to the measuring and reference electrodes of a PRG-3 electrode, respectively. Prepare active adults. Using a dissecting scalpel, quickly cut the antennae from the base and slightly slit the tips. The reference electrode of the PRG-3 electrode was removed, and the base of the antenna was held by suction by the surface tension of saline. The reference electrode was then carefully fixed in place, while the tip of the antenna was connected to the measuring electrode under a stereomicroscope. Data were recorded and analyzed using GC-EAD software (Version 2014 v1.2.5, Syntech). The GC-EAD experiment was repeated five times, all performed between 7:00 PM and 10:00 PM.

[0055] (3) GC-MS analysis

[0056] The gas chromatograph-mass spectrometer (GC-MS) was manufactured by Thermo Fisher Scientific, model Trace 1300. The chromatographic column was a VF-5MS capillary column (length × inner diameter × film thickness = 30 m × 0.25 mm × 0.25 μm). The dichloromethane eluate of damaged tomato volatiles was injected without splitting, with a 2 μL injection volume per injection. The column temperature program was the same as that for the GC-EAD. The solvent delay was 3 min. The detector temperature was 300°C, the inlet temperature was 250°C, and the carrier gas was high-purity helium at a flow rate of 1.5 mL / min. The quadrupole temperature was 150°C, the ion source temperature was 250°C, the ionization mode was EI, the mass spectrometer bombardment voltage was 70 eV, the scanning mass range was 45-500 m / z, and the scanning frequency was 5 / s. The qualitative analysis of the volatile components of damaged tomatoes was carried out by checking and supplementing the search results using the NISTO8 spectral library in the computer connected to the gas chromatography-mass spectrometry instrument and reference standard spectra. The experiment was repeated three times.

[0057] (4) Electroantennographic responses of adult tomato leafminers to four standard volatiles and their mixtures

[0058] The potentioantenometer was manufactured by Syntech in the Netherlands, and data were collected using the company's Spike software. A clean filter paper strip (5 mm × 50 mm) containing 10 μl of the test solution was placed in a Pasteur tube, sealed at both ends with Parafilm to reduce sample evaporation. A clean filter paper strip (5 mm × 50 mm) containing 10 μl of paraffin oil was placed in a Pasteur tube as a control, also sealed at both ends with Parafilm to reduce sample evaporation. An adult tomato leafminer was captured using a finger-shaped tube and frozen unconscious in a -20°C freezer. Under a stereomicroscope, the antennae of the adult tomato leafminer were dissected at the base with a scalpel, and one or two flagella at the distal ends were removed. The treated antennae were then connected to a reference electrode and a recording electrode using conductive glue (Spectra 360). The airflow system was controlled by an airflow distributor. The stimulation duration was 0.5 s, with a 30 s interval between stimulations to ensure full recovery of antennal activity. Each antenna was stimulated with control paraffin oil, the test solution from low to high concentration, and then control paraffin oil. Six replicates were performed on both male and female adults. EAG tests were conducted between 7:00 PM and 10:00 PM.

[0059] (5) Y-type olfactometer responses of tomato leafminer adults to four standard volatiles and their mixtures

[0060] The Y-shaped olfactometer test method was based on Yang et al. (2019). The main arm of the Y-shaped tube of the Y-shaped olfactometer is 20 cm long and has an inner diameter of 2.5 cm. The two side arms are both 15 cm long and have an inner diameter of 2.5 cm. The angle between the side walls is 75°. A clean filter paper strip (5 mm × 50 mm) containing 10 μl of the test solution was placed in one arm of a Y-shaped tube. A clean filter paper strip (5 mm × 50 mm) containing 10 μl of paraffin oil served as a control in the other arm. The odor source was connected to a QC-1B air sampler (Beijing Institute of Labor Protection Science). Airflow was filtered through activated carbon and humidified with distilled water before entering the odor source. The air flow rate of the air sampler was set at 350 mL / min. A single adult tomato leafminer was released into the base of the main arm of the Y-shaped tube. Each adult was tested for 5 minutes. Adults that entered the test arm more than 10 cm and remained for more than 30 seconds were considered the selective test group. Adults that entered the control arm more than 10 cm and remained for more than 30 seconds were considered the selective control group. Adults that did not make a choice after 5 minutes were considered non-responsive. Each adult was tested only once, and groups of 10 females or males were formed, with three replicates. After every five insects tested, the arms of the Y-tube were rotated. After each group, the Y-tube was thoroughly cleaned with alcohol. After each day's testing, the Y-tube, wash bottle, and connecting silicone tubing were cleaned with acetone. Olfactory behavior tests were conducted between 7:00 PM and 10:00 PM. Attraction index (%) = number of responses on the treated arm of the tomato leafminer / total number of tomato leafminers tested × %.

[0061] (6) Oviposition preference responses of adult tomato leafminers to four standard volatiles and their mixtures

[0062] The oviposition preference response test method was based on that of Wu et al. (2018). 100 μL of a mixture of leaf alcohol, β-phellandrene, and 100 μL of 4-ethylbenzaldehyde and nonanal at a concentration of 10 μg / μL were placed in a rubber-tipped bait carrier. 100 μL of paraffin oil was placed in the bait carrier as a control. The experiment was conducted in a 1 m × 1 m × 1 m cage. Four healthy and intact potted tomato plants (Solanum lycopersicum L.) were placed in the four corners of the cage (1 m × 1 m × 1 m). The cages were spaced 10 m apart to prevent cross-contamination. 100 μL of the test solvent was pipetted into a rubber stopper to prepare the flavor source. In Group 1 (G1), two tomato plants were selected from one diagonal of the cage and two flavor source rubber stoppers were attached to each plant to serve as the experimental group. Two plants on the other diagonal were fixed with rubber stoppers containing paraffin oil to serve as a control group. We also tested whether paraffin oil had any effect on egg laying in tomato leafminers. Therefore, in the mesh cages of group 2 (G2), two rubber stoppers containing 100 μL of paraffin oil were fixed to two tomato plants on one diagonal to serve as the experimental group. An empty rubber stopper was fixed to two plants on the other diagonal to serve as the control group. Three pairs of male and female adults (2 days old) were released into each cage, and the number of eggs on each tomato plant was counted after 48 hours. The experiment was repeated 6 times. The egg laying preference index = T / (C+T)×100%. C is the number of eggs on the control plants, and T is the number of eggs on the plants treated with the test compound.

[0063] (7) Preferential responses of tomato leafminer adults to four standard volatiles and their mixtures in the field

[0064] Using paraffin oil as the solvent, cis-3-hexen-1-ol, β-phellandrene, and their mixture were prepared at a concentration of 100 μg / μl. Nonanal and 4-ethylbenzaldehyde were prepared at a concentration of 10 μg / μl. 100 μl of each solution was added to a brown slow-release bottle (1 mL capacity, with a 1 mm diameter hole drilled in the center of the cap for odor release). An equal amount of paraffin oil was used as a control. The slow-release bottle was hung in the center of a white triangular trap (17.8 cm side length, 26.5 cm height) or embedded in a square hole (1 cm × 1 cm) on a blue sticky trap (25 cm × 20 cm). The traps were randomly placed at the base of tomato plants, 20 m apart. The number of tomato leafminers (Tuta absoluta) trapped was counted after 72 hours, with three replicates per group.

[0065] (8) Data Analysis

[0066] The experimental data were statistically analyzed using IBM SPSS Statistics 27.0.1 software. All results were expressed as mean ± SEM. Tukey's multiple range test in one-way analysis of variance was used to test the significance of EAG response values. The significant differences in the Y-type olfactometer bioassay data were compared by chi-square test. Tukey's multiple range test in one-way analysis of variance was used to analyze the egg-laying repellent index. Statistical significance was set at P < 0.05.

[0067] 3. Experimental results:

[0068] (1) Electrophysiological responses of adult tomato leafminers to tomato active volatiles

[0069] The electrophysiological responses of female tomato leafminers to tomato headspace volatiles were analyzed by GC-MS and GC-EAD. There are four compounds in tomato volatiles that can induce significant EAD responses, such as Figure 3 As shown, based on the order of GC analysis, the four compounds were identified as cis-3-hexenol, β-phellandrene, nonanal, and 4-ethylbenzaldehyde. Peak area normalization yielded a ratio of cis-3-hexen-1-ol: β-phellandrene: nonanal: 4-ethylbenzaldehyde of 3.5:3.8:1:6.7.

[0070] To further verify the electrophysiological activities of the four active compounds and their mixtures, the dose-EAG responses of the antennae of male and female tomato leafminers to each synthetic compound were measured. Figure 4 As shown, compared to the blank control, antennae of both male and female adults produced significant electroantennative responses to the active compound and its mixture at a concentration of 1 μg / μl. A clear dose-dependent response was observed in both male and female antennae to nonanal and the mixture, with significant differences in response values between different concentrations. At 100 μg / μl, female antennae produced the highest response to phellandrene (0.704±0.031 mV), while male antennae produced the highest response to the mixture (1.255±0.056 mV).

[0071] (2) Y-tube olfactometer to measure the attraction response of tomato leafminer adults to four active compounds and their mixtures

[0072] The behavioral responses of male and female adults of the tomato leafminer to four EAD active compounds at different concentrations were tested using a Y-tube olfactometer. Figure 5As shown in the data, nonanal at a concentration of 10 μg / μl and leaf alcohol at a concentration of 100 μg / μl had a significant attracting effect on male and female adults, but high concentrations (100 μg / μl) of nonanal and 4-ethylbenzaldehyde had a repellent effect on tomato leafminer adults; when the concentration reached 10 μg / μl and above, β-phellandrene had a significant or extremely significant attracting effect on male and female adults of tomato leafminer; the mixtures at the three concentrations all had a significant or extremely significant attracting effect on female adults of tomato leafminer.

[0073] (3) Effects of four active compounds and their mixtures on oviposition selection of tomato leafminer

[0074] The effects of four active compounds on the oviposition of tomato leafminers were studied in the presence of tomato plants. Figure 6 As shown in the results, the mixture at a concentration of 100 μg / μl, β-phellandrene and 10 μg / μl nonanal had extremely significant attracting effects on the egg-laying choice of female tomato leafminer adults, 4-ethylbenzaldehyde at a concentration of 10 μg / μl and leaf alcohol at a concentration of 100 μg / μl had significant attracting effects on the egg-laying choice behavior of female tomato leafminer adults, while solvent paraffin oil had no effect on egg-laying choice.

[0075] (4) Preferential responses of tomato leafminer adults to four standard volatiles and their mixtures in the field

[0076] The four active compounds and their mixtures screened were further verified for their attractant effects on adult tomato leafminers in the field. Figure 7 As shown, 4-ethylbenzaldehyde at a concentration of 10 μg / μl did not show significant attracting effect on tomato leafminer adults in the field; at a concentration of 10 μg / μl, nonanal had a significant attracting effect on tomato leafminer adults; β-phellandrene at a concentration of 100 μg / μl showed significant attracting effect on tomato leafminer adults in the field, and leaf alcohol and the mixture at the same concentration showed extremely significant attracting effect on tomato leafminer adults; while the solvent paraffin oil did not show any attracting effect on tomato leafminer in the field.

[0077] In summary, headspace collection of tomato volatiles, followed by GC-EAD, GC-MS, EAG, behavioral assays using a Y-type olfactometer, and field oviposition attractant testing, revealed cis-3-hexenol, β-phellandrene, nonanal, and 4-benzaldehyde, as well as mixtures in specific ratios. These four active compounds and their mixtures could potentially be used as attractants for the control of tomato leafminer.

Claims

1. Volatile substances in tomatoes that have attractant activity to tomato leafminers include: Tomato volatiles: cis-3-hexenol (leaf alcohol), β-phellandrene, nonanal, 4-ethylbenzaldehyde, or a mixture of any one of the four.

2. The volatile material according to claim 1, wherein: The mixture is: A mixed liquid of cis-3-hexenol (leaf alcohol), β-phellandrene, nonanal and 4-ethylbenzaldehyde in a volume ratio of 3.5:3.8:1:6.

7.

3. A biological agent for controlling tomato leafminer, wherein: The biological preparation comprises the volatile substance according to claim 1 or 2.

4. Use of the volatile substance according to claim 1 or 2 in controlling tomato leafminer.

5. Use of the biological preparation according to claim 3 in controlling tomato leafminer.

6. A method for controlling tomato leafminer, wherein: The method comprises using the volatile substance according to claim 1 or 2 for prevention and control.

7. A method for controlling tomato leafminer, wherein: The method comprises using the biocontrol agent according to claim 3 for control.

8. A method for collecting volatile substances in tomatoes that have attractant activity to tomato leafminers according to claim 1 or 2, comprising: Tomato volatiles were collected by headspace collection and tested for their attraction to tomato leafminer, from which single or combined volatiles were obtained.

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