Preparation method of 1-tridecanol as pheromone attractant for attracting female tomato leaf miner and lure

Abstract

The invention belongs to the field of plant protection, and discloses an application of 1-tridecanol as a pheromone attractant for attracting female tomato leaf miner. Researches show that 1-tridecanol with a specific concentration has a strong attraction effect on female tomato leaf miners. The 1, 1-tridecanol is used as pheromone to be applied to biological prevention and control of tomato leaf miners, and has the characteristics of no residue, safety to human, livestock and environment, difficulty in generating drug resistance, safety to natural enemies and the like.

Description

Technical Field

[0001] The present invention belongs to the field of plant protection, and discloses the application of 1-tridecanol as a pheromone attractant for attracting female Tuta absoluta. Background Art

[0002] The tomato leafminer, Tuta absoluta (Meyrick), belongs to the family Gelechiidae, order Lepidoptera. Also known as the South American tomato leafminer, tomato leafmining moth, and tomato gelechiid moth, it is a devastating invasive pest. The tomato leafminer mainly feeds on the phloem of tomato plants by mining the leaves, causing the plant leaves to turn white, affecting plant photosynthesis, and curbing the normal growth of the plants. When the population density is high, it can also directly bore into the tomato fruits, causing rotten fruits and fruit drop, seriously threatening tomato crop production. The tomato leafminer originated in Peru, South America. In 2006, it first left the South American continent and was introduced into Spain along with fresh tomatoes imported from Chile. Subsequently, it rapidly spread in many countries in Europe, North Africa, West Asia, etc. along the Mediterranean coast. In August 2017, Chinese researchers first detected the damage of the tomato leafminer in Yili Prefecture, Xinjiang. Subsequently, centered on Yunnan, the tomato leafminer rapidly spread across the country, seriously threatening China's tomato industry. In November 2023, the Ministry of Agriculture and Rural Affairs of China urgently included the tomato leafminer in the list of national first-class crop pests and diseases, and comprehensively strengthened the control.

[0003] Currently, globally, the control of the tomato leafminer mainly relies on chemical control, such as cartap, abamectin, pyrethroids (such as deltamethrin, permethrin), and organophosphates (such as methamidophos). Although pesticides can effectively reduce the damage level of pests, they can induce pests to develop resistance, and at the same time have negative impacts on non-target species and the ecological environment, thus exacerbating the degree of damage and the difficulty of control. In addition, due to the concealed "leaf-mining feeding" characteristics of its larvae, it is easy to escape the control of chemical pesticides, affecting the control efficacy, and then causing growers to increase the application frequency of pesticides, resulting in a rapid increase in resistance. Therefore, it is urgent to find effective means for the green control of new pests and diseases.

[0004] Insect behavior regulation technology is a green control technology developed by utilizing the behavioral responses of insects to stimuli such as vision, smell, touch, and hearing. Among them, olfactory behavior regulation technology is to specifically regulate the behavior of target pests by using various odor compounds derived from insects, plants, etc., and release the artificially synthesized semiochemicals slowly into the field by a releaser to attract pests to feed, lay eggs, and mate, directly trap and kill adults or interfere with pest mating, reducing the offspring population of target pests, so as to achieve the purpose of green pest control. Summary of the Invention

[0005] The inventors previously found in the field that the tomato leafminer preferred to lay eggs on TYLCV-infected plants, and this finding was verified in the laboratory. Further, volatile substances released by healthy plants and TYLCV-infected tomato plants were extracted by the dynamic headspace adsorption method, and the active components of the volatile substances were identified using a gas chromatography-mass spectrometer (GC-MS), and five differential volatiles were screened. By selecting 5 standard compound samples for electroantennogram (EAG) responses and simulated field trapping tests on the tomato leafminer, the experimental results showed that 1-tridecanol had a significant attracting effect on female adult tomato leafminers at a specific concentration.

[0006] The present invention provides the use of 1-tridecanol as a pheromone attractant for attracting female tomato leafminers.

[0007] The present invention also provides the use of 1-tridecanol in the preparation of a pheromone attractant for attracting female tomato leafminers.

[0008] The present invention further provides a method for preparing a lure core containing a pheromone attractant for attracting female tomato leafminers, which comprises the following steps:

[0009] 1) Volatile release device: Select a rubber stopper with a diameter of 5-15 mm, soak it in absolute ethanol for 24-64 h, take out the rubber stopper and place it in an oven at 40-60 °C overnight to obtain a blank lure core;

[0010] 2) Preparation or structure of the lure core: Mix 1-tridecanol with an organic solvent evenly, take the mixed liquid and place it in the groove of the blank lure core to obtain a pheromone attractant lure core for attracting female tomato leafminers.

[0011] Specifically, the concentration of 1-tridecanol is 180-220 ng / μL, preferably 198-202 ng / μL, specifically 200 ng / μL.

[0012] The organic solvent is selected from n-hexane or paraffin oil.

[0013] The present invention provides a method for attracting female tomato leafminers, which uses 1-tridecanol as a pheromone attractant to attract female tomato leafminers.

[0014] Specifically, it is attracted by the lure core obtained by the above preparation method, and the concentration of 1-tridecanol is 180-220 ng / μL, preferably 198-202 ng / μL, specifically 200 ng / μL.

[0015] The specific operation steps are as follows:

[0016] The lure core is used in combination with a trap. One end of a wire is passed through the lure core and fixed, and the other end of the wire passes through the middle gap at the top from inside the trap. Adjust the height of the lure core so that the lure core is 3 - 5 cm away from the bottom and fix it.

[0017] Hang the assembled triangular trap in a tomato greenhouse or in the field, and the hanging height is 80 - 120 cm from the ground.

[0018] During application, the lure core is replaced once every 4 - 6 weeks, and when the temperature is high and the damage is severe, it is replaced once every 2 - 3 weeks.

[0019] The glue board inside the trap is replaced every 4 - 7 days or in time when the glue board is full of pests.

[0020] The research of the present invention shows that at a specific concentration, 1 - tridecanol has a significant attracting effect on female tomato leafminer moths. As a pheromone, 1 - tridecanol is applied to the biological control of tomato leafminer moths and shows many advantages. First, 1 - tridecanol has no residue and will not pollute crops after use. Second, it has high safety for humans, livestock and the environment, and hardly causes negative impacts on the ecosystem during use. In addition, 1 - tridecanol is not easy to cause pest resistance, which helps to achieve a long - term and stable control effect. At the same time, it has little harmless impact on non - target organisms, including natural enemies of pests, can maintain ecological balance and promote biodiversity. Therefore, controlling pests based on plant - released pheromones can not only effectively control the number of pests, but also improve the control effect, reduce the use of chemical pesticides, has significant environmental - friendly advantages, and provides a green and sustainable prevention and control means for modern agriculture. Description of the Drawings

[0021] Figure 1 It is a schematic diagram of a free - choice device.

[0022] Figure 2 Showing the selection preference of newly - emerged male tomato leafminer moths for tomato plants infected with TYLCV.

[0023] Figure 3 Showing the selection preference of newly - emerged female tomato leafminer moths for tomato plants infected with TYLCV.

[0024] Figure 4 Showing the oviposition preference of gravid female moths for tomato plants infected with TYLCV.

[0025] Figure 5 Showing the EAG response of female tomato leafminer moths to 5 standard compounds at different concentrations.

[0026] Figure 6 Showing the EAG response of male tomato leafminer moths to 5 standard compounds at different concentrations.

[0027] Figure 7 Schematic diagram of the olfactory behavior device.

[0028] Figure 8 Showing the selectivity of the olfactory behavior device for male and female adult tomato leafminers to different concentrations of 1-tridecanol.

[0029] Figure 9 Showing the effect of 1-tridecanol on the oviposition of tomato leafminers. Detailed implementation manners

[0030] Experimental methods

[0031] I. Influence of TYLCV-infected tomato plants on the behavioral selection of tomato leafminers

[0032] To verify the host preference of tomato leafminers, TYLCV-infected tomato plants and healthy tomato plants were randomly placed at two diagonals of a free-choice device (free-choice experimental chamber: the main body is a cuboid with length, width and height of 100 cm, 40 cm and 45 cm respectively, and three fluorescent lamps provide continuous, stable and uniform light source at the top, and the test insects are always placed at the center point). Blue sticky cards were vertically hung 5 cm away from each plant. The device was placed in a dark room. At 9:00 am, 50 newly emerged male or female tomato leafminers were released at the center, and the number of moths captured on the sticky board was recorded after 48 hours. For the oviposition preference experiment of tomato leafminers, 6 pairs of male and female adults that had emerged for one day were taken and placed in a 50 mL centrifuge tube at the center point of the free-choice device. The centrifuge tube was opened at 19:00 pm so that the tomato leafminers could fly out freely, and the number of eggs on both sides of the plants was counted after 72 h.

[0033] II. Identification and analysis of tomato volatiles before and after TYLCV infection

[0034] 1. Dynamic headspace adsorption measurement method

[0035] The components of tomato volatiles were identified using the dynamic headspace adsorption method. The above-mentioned healthy and TYLCV-infected tomato plants were placed into the sampling cylinder. Before the experiment, the sampling cylinder and silica gel tube were cleaned with 75% ethanol and dried to remove residual odors. The soil pot part was completely wrapped with tin foil to reduce the interference of odors from parts other than the plants. The atmospheric sampler, carbon column, water column, and sampling cylinder were connected in sequence with a silica gel tube, and each connection was sealed with a sealing film. The air flow of the atmospheric sampler was set to 1 L / min. First, the air in the sampling cylinder was exhausted by running for about 20 min without connecting the adsorption column. Then, the adsorption column filled with 50 μg of Tenax adsorbent was connected to the device, and the odor was continuously collected for 6 h. After collection, 500 μL of n-hexane was slowly added to the adsorption column to elute the odor, and the eluate was collected in a sample vial. 50 μL was taken for subsequent GC-MS analysis, and the rest was sealed in the sample vial and stored at -20 °C for later use.

[0036] 2. GC-MS analysis of tomato volatiles

[0037] The differential volatiles of healthy plants, virus-infected plants, healthy plants and virus-infected plants after being fed by Tuta absoluta were analyzed and identified using GC-MS. The Rtx-5MS chromatographic column (Agilent) was used for GC-MS separation of volatile substances. The column length of the chromatographic column was 30 m, the diameter was 0.25 mm, and the film thickness was 0.25 μm. The GC-MS program was as follows: Helium was used as the carrier gas, the injection volume was 1 μL, the gas flow rate was 1 L / min, splitless injection was used, and the injection port temperature was 250 °C. The column oven was maintained at 50 °C for 2 min, then heated to 150 °C at a rate of 10 °C / min, and finally heated to 290 °C at a rate of 5 °C / min and maintained at 290 °C for 3 min. The mass range of mass spectrometry scanning was from 35 m / z to 500 m / z, and the scanning speed was 1666 u / s.

[0038] 3. EAG response of the antennae of Tuta absoluta

[0039] Based on the differential volatile single compounds screened from the above-mentioned healthy tomato plants and virus-infected tomato plants, they were diluted with n-hexane to 2, 20, 200, and 2000 ng / μL. The electroantennogram (EAG) instrument (Syntech, Netherlands) and EAG2000 software were used to further verify the electroantennogram responses of Tuta absoluta to odor substances at different concentrations. Healthy and active virgin adult Tuta absoluta were selected. Before the experiment, they were briefly stunned in a -20 °C refrigerator and then the antennae were quickly cut off from the base with a sharp scalpel, and a little was cut off from the end. The antennae were fixed on the electrodes by the surface tension of the conductive liquid to keep the antennae straight between the electrodes. The antennae were about 1 cm away from the air outlet of the glass tube. 10 μL of the sample solution was evenly applied to a filter paper strip (40 mm × 5 mm) and quickly placed into a Pasteur tube connected to the odor stimulation device. After the EAG baseline was stable, odor stimulation was carried out. The continuous air flow passing through the activated carbon and water column was set at 400 mL / min, the stimulation air flow was set at 300 mL / min, the stimulation time was 0.2 s, and the time interval between two stimulations was at least 30 s. The test order was from low to high concentration. Before and after testing each sample concentration, the antennae were tested with n-hexane as a control. Each treatment was tested 6 times. To ensure the experimental effect, the experiment was carried out from 17:00 to 22:00.

[0040] 4. Behavioral experiments of Tuta absoluta on tomato volatiles

[0041] Based on the EAG responses to 5 volatiles, we selected 1-tridecanol to test its attractiveness to male and female adult Tuta absoluta, using the improved olfactory behavior device of our team. The olfactory behavior device consisted of a central straight tube (40 cm long, 4 cm in diameter) and two cylindrical chambers (30 cm high, 18 cm in diameter) at both ends. A potted tomato plant with 6 - 7 leaves was placed on each side of each device. The volatile compound 1-tridecanol was diluted in hexane to concentrations of 2, 20, 200, and 2000 ng / μL. 20 μL of the 1-tridecanol solution was loaded into a green rubber lure and then fixed on the stem of the tomato plant. At the other end of the device, as a control, a rubber lure containing 20 μL of hexane was placed and fixed on the stem of another tomato plant ( Figure 7 ). Individual insects were introduced through the entrance of the main arm of the olfactometer and given 10 minutes to choose one of the two directions to enter. When the insect entered the cylindrical chamber containing the plant and stayed for more than 30 seconds, it was considered to have made a choice. Any other behavior was considered indeterminate. When using 5 insects each time, the device was replaced and rearranged to reduce the influence of the tube arm position and odor on behavior. A total of 20 insects were used in the olfactometer bioassay for each compound, and each insect was used only once.

[0042] Experimental results

[0043] 1. Effects of TYLCV-infected tomato plants on the behavioral choices of Tuta absoluta

[0044] The free-choice experimental chamber used ( Figure 1 ) has a main body structure that is a cuboid with a length, width, and height of 100 cm, 40 cm, and 45 cm respectively. The chamber is made of transparent acrylic. There are small doors on the left and right sides, each with a length of 15 cm and a height of 30 cm for placing plant samples. The top is provided with three fluorescent lamps to provide continuous, stable, and uniform light. At the center point, there is a round hole with a diameter of 20 cm, and a sleeve made of 120-mesh gauze is adhered to the outer circle for releasing test insects.

[0045] Randomly place TYLCV-infected tomato plants and healthy tomato plants ( Figure 2 ) on both sides of the free-choice device. Release 50 test insects at the center point of the free-choice device and check the selection situation after 48 h. The results show that only the newly emerged female adults of Tuta absoluta show a significant preference for TYLCV-infected tomato plants ( Figure 2 ), while male adults have no preference for either TYLCV-infected tomato plants or healthy plants ( Figure 3 ).

[0046] In the experiment on the oviposition preference of the Tuta absoluta population for TYLCV-infected tomato plants, the oviposition preference of Tuta absoluta for TYLCV-infected tomato plants for 72 h was detected. The results show that gravid female moths show a significant oviposition preference for TYLCV-infected tomato plants ( Figure 4 ).

[0047] 2. EAG and behavioral responses of Tuta absoluta to five standardized compounds

[0048] Five significantly different volatiles in healthy plants and TYLCV-infected tomato plants were identified by GC-MS, namely nicotine, nonadecane, 1-tridecanol, diethyl carbonate, and tetracosane. The EAG responses of male and female Tuta absoluta to different concentrations of 5 standard compound samples were further verified: 1-tridecanol showed a strong EAG response to female Tuta absoluta at high concentrations (200 ng / μL and 2000 ng / μL) ( Figure 5 ). At a concentration of 2 ng / μL, male insects showed almost no EAG response, and only at an extremely high concentration (2000 ng / μL) did they have a certain response, indicating that male antennae could hardly recognize this compound ( Figure 6 ).

[0049] Volatile slow-release device: Select a rubber stopper with a diameter of 10 mm, soak it in absolute ethanol for 48 h, take out the rubber stopper and place it in an oven at 50 °C overnight to obtain a blank lure core;

[0050] Preparation or structure of the lure core: 1-Tridecanol is added to an organic solvent such as n-hexane or paraffin oil and mixed evenly. The mixed liquid is taken and placed in the groove of the blank lure core, and thus the pheromone lure core for attracting female Tuta absoluta is prepared.

[0051] According to the EAG responses of male and female adult Tuta absoluta to 5 volatile compounds, we selected 1-tridecanol to test Tuta absoluta, and the test was carried out using the improved olfactory behavior device of our team ( Figure 7 ). For specific details, see Method 4. At the beginning of the experiment, a tomato plant with 6-7 leaves and a rubber stopper lure core hanging on it was placed in each cylindrical chamber on each side of the device. The volatile compound 1-tridecanol was diluted with n-hexane to 2, 20, 200, and 2000 ng / μL and added to the rubber stopper lure core fixed on the stem of a tomato plant as the treatment group, and another tomato plant added with n-hexane was used as the control. The test insects entered the device through the entrance of the main arm, and a 10-minute waiting period was set for them to make a choice. The results showed that female Tuta absoluta showed a significant attraction to 1-tridecanol at concentrations of 200 ng / μL and 2000 ng / μL. ( Figure 8 ).

[0052] Similarly, the oviposition preference experiment of gravid female Tuta absoluta was carried out using the above olfactory behavior device. Five mated female moths were introduced from the entrance of the main arm, and the number of eggs on the plants on both sides was calculated after 48 h. The results showed that female Tuta absoluta had a significant oviposition attraction effect on Tuta absoluta at a concentration of 200 ng / μL, while it showed avoidance at 2000 ng / μL ( Figure 9 ). Therefore, 1-tridecanol may be used as a long-distance oviposition attractant for Tuta absoluta.

Claims

Application of 1.1-tridecanol as a pheromone attractant for attracting female tomato leafminers. Application of 2.1-tridecanol in the preparation of pheromone attractants for attracting female tomato leafminers.

3. A method for preparing an attractant core containing a pheromone attractant for attracting female tomato leafminers, characterized in that: The steps include: 1) Volatile sustained-release device: Select a rubber stopper with a diameter of 5-15 mm, soak it in anhydrous ethanol for 24-64 hours, take out the rubber stopper and place it in a 40-60℃ oven overnight to obtain a blank lure; 2) Preparation or structure of the attractant core: 1-tridecanol is added to an organic solvent and mixed evenly, and the mixed liquid is placed into the groove of a blank attractant core to prepare a pheromone attractant core for attracting female tomato leafminers.

4. The preparation method according to claim 3, characterized in that: The concentration of 1-tridecanol is 180-220 ng / μL, preferably 198-202 ng / μL, specifically 200 ng / μL.

5. The preparation method according to claim 3, characterized in that: The organic solvent is selected from n-hexane or paraffin oil.

6. A method for attracting female tomato leafminers, characterized in that: The method uses 1-tridecanol as a pheromone attractant to attract female tomato leafminers.

7. The method according to claim 6, characterized in that The bait is obtained by the bait core prepared by the preparation method according to claim 3 or 4, and the concentration of 1-tridecanol is 180-220 ng / μL, preferably 198-202 ng / μL, specifically 200 ng / μL.

8. The method according to claim 6, characterized in that The specific steps are as follows: The lure is used in conjunction with the trap. One end of the wire is passed through the lure and fixed. The other end of the wire is passed through the middle notch at the top of the trap. The height of the lure is adjusted so that the lure is 3-5 cm away from the bottom and fixed. Hang the assembled triangular trap in the tomato greenhouse or field at a height of 80-120 cm from the ground.

9. The method according to claim 8, characterized in that The bait should be replaced every 4-6 weeks, or every 2-3 weeks when the temperature is high and the damage is serious; The plastic sheet in the trap should be replaced every 4-7 days or when it is full of pests.

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