Melon fruit fly attractant and preparation method and application thereof

Abstract

The present invention relates to the technical field of plant pest control, and specifically to a melon fruit fly attractant and a preparation method and application thereof, comprising a compound of 100µg / µL methyl isovalerate and 100µg / µL hexanal in a volume ratio of 1:1, which attracts female melon fruit flies. The melon fruit fly attractant of the present invention has main components derived from natural plant volatiles, is environmentally friendly, and is safe for non-target organisms, laying a foundation for field monitoring of melon fruit flies, and also providing a new idea for green control of melon fruit flies.

Description

Technical Field

[0001] The invention relates to the technical field of plant disease and insect pest control, and in particular to a melon fruit fly attractant and a preparation method and application thereof. Background Art

[0002] The volatiles of host plants play a vital role in the communication between herbivorous insects and host plants. The signals emitted by these volatile substances enable herbivorous insects to quickly and accurately identify and find host plants. Insect-inducing plant volatiles not only attract or repel herbivorous insects, but also have a certain impact on their growth, development and behavior. Using the volatiles of host plants to attract or repel herbivorous insects to study green and environmentally friendly control methods has gradually become a hot topic. The development of attractants based on plant volatiles to control pests has gradually become a new green control technology in recent years.

[0003] Melon fruit fly Bactrocera cucurbitae Coquillett belongs to the Trypetidae family of the order Diptera. It originated in India and is a major invasive pest. It has been included in the first list of national key alien species and is a Class II quarantine object in my country. The melon fruit fly has a wide range of hosts, strong adaptability, and a diverse diet. It can harm more than 120 kinds of vegetables and fruits from more than 20 families, among which Cucurbitaceae and Solanaceae plants are the most serious. The ovipositor of the female adult melon fruit fly pierces the fruit epidermis to lay eggs. In the early stage, the area around the pinholes on the epidermis rots, followed by large-scale yellowing, deformation and collapse, until the larvae feed on the entire host, and the fruits rot and fall, resulting in a decrease in fruit quality and yield. The melon fruit fly adults lay a large number of eggs, have a long egg-laying period, have a strong ability to fly and transfer, and have serious generation overlap, which makes it extremely difficult to prevent and control them.

[0004] At present, the control methods of melon fruit flies include chemical control, attractant killing, etc., and chemical control is its main control method. The frequent use of chemical pesticides brings many problems, such as the large-scale use of pesticides easily causes some populations to produce different degrees of resistance problems, and the quality of melons and fruits is reduced due to excessive pesticide residues. Melon fruit fly attractants include sex attractants and food attractants. Chinese patents CN1312992C and CN100431412C both provide sex attractants with fly attractant as the main component, which only kill some male insects, while female melon fruit flies can mate repeatedly, and those that mate only once can continuously lay eggs and cause harm for up to 20 days. The male attractant can only reduce the mating rate of female melon fruit flies. Chinese patent CN101411327B provides a melon fruit fly food attractant with melon fragrance as the main component, but the main components of the attractant are various and the preparation is relatively complicated.

[0005] Therefore, there is an urgent need for a new control method that is efficient, simple, environmentally friendly, friendly to the environment, ecology, and non-target organisms, and conducive to sustainable agricultural development. Summary of the invention

[0006] In order to achieve the above-mentioned object, the present invention provides a melon fruit fly attractant and a preparation method and application thereof. The attractant is made based on the volatiles of bitter melon, a host plant of the melon fruit fly, and lays a foundation for the development of a plant-based attractant for the melon fruit fly. The specific technical scheme is as follows:

[0007] A melon fruit fly attractant, the attractant is a compound containing 100µg / µL methyl isovalerate and 100µg / µL hexanal, wherein the volume ratio is set to 100µg / µL methyl isovalerate:100µg / µL hexanal=1:1.

[0008] A method for preparing the above-mentioned melon fruit fly attractant, comprising the following steps:

[0009] Step 1: Dissolve methyl isovalerate in n-hexane to prepare a 100 µg / µL methyl isovalerate solution;

[0010] Step 2: Dissolve hexanal in n-hexane to prepare a 100µg / µL hexanal solution;

[0011] Step 3: Mix 100µg / µL methyl isovalerate solution and 100µg / µL hexanal solution in a 1:1 volume ratio.

[0012] The above-mentioned melon fruit fly attractant is used as a melon fruit fly attractant. The attractant has an attracting effect on female melon fruit flies, but has no obvious attracting effect on male melon fruit flies.

[0013] A use of the above-mentioned melon fruit fly attractant is to prepare pesticides or attractant cores or attractant paper or attractants, or to be used in combination with hydrolyzed protein, biological pesticides or low-toxic chemical pesticides to enhance the trapping ability.

[0014] Beneficial effects of the present invention:

[0015] (1) The present invention studies the volatiles of bitter melon, the host plant of the melon fruit fly, from the aspects of electrophysiology and behavior, and proves that methyl isovalerate, methyl myristate, octadecene, squalene, styrene, and hexanal in the volatiles have an attracting or repelling effect on the melon fruit fly. Generally, as the concentration of volatile substances increases, the antennal response of insects increases, but the inventors have found that the antennal response of the melon fruit fly to different volatile substances has different thresholds. This provides theoretical and technical support for the screening and preparation of efficient plant-based attractants and repellents for the melon fruit fly.

[0016] (2) The present invention provides an attractant for attracting female melon fruit flies, and a preparation method and application thereof. The attractant of the present invention is used in combination with corresponding insecticidal means to kill female melon fruit flies, which can not only reduce the mating rate of melon fruit flies, but also reduce the ovulation of female melon fruit flies, thereby greatly reducing their reproductive capacity.

[0017] (3) The main components of the melon fruit fly attractant described in the present invention are all derived from natural plant volatiles, which are environmentally friendly and safe to non-target organisms. They lay the foundation for field monitoring of melon fruit flies and also provide new ideas for green prevention and control of melon fruit flies. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 This is a diagram of the antennal response of Bactrocera melonensis to volatiles of bitter melon of the present invention: the significant differences between males and females are represented by capital letters, and the significant differences between odor substances are represented by lowercase letters (P<0.05).

[0019] Figure 2 This is the EAG response diagram of Bactrocera melonensis to different doses of bitter melon volatiles of the present invention: the significant difference between different doses is represented by lowercase letters, the same letter represents no difference, and different letters represent significant difference (P<0.05).

[0020] Figure 3 Constructed for plastic cup traps.

[0021] Figure 4 Behavioral responses of female (a) and male (b) adults of Bactrocera melonensis to 100 µg / µL bitter melon volatiles.

[0022] Figure 5 Behavioral responses of female (a) and male (b) adults of Bactrocera melonensis to 10µg / µL bitter melon volatiles.

[0023] Figure 6 Behavioral responses of female (a) and male (b) adults of Bactrocera melonensis to 1µg / µL bitter melon volatiles.

[0024] Figure 7 Behavioral responses of female (a) and male (b) adults of Bactrocera melonensis to 0.1µg / µL bitter melon volatiles.

[0025] Figure 8 Behavioral responses of female (a) and male (b) adults of Bactrocera melonensis to 0.01µg / µL bitter melon volatiles.

[0026] Fig. 9 This is a graph showing the behavioral responses of male and female adults of Bactrocera melonensis to a mixture of 100µg / µL methyl isovalerate and 100µg / µL hexanal. DETAILED DESCRIPTION

[0027] The specific implementation modes of the present invention are described below so that those skilled in the art can understand the present invention. However, it should be clear that the present invention is not limited to the scope of the specific implementation modes. For those of ordinary skill in the art, as long as various changes are within the spirit and scope of the present invention as defined and determined by the attached claims, these changes are obvious, and all inventions and creations utilizing the concept of the present invention are protected.

[0028] Example 1 Extraction of volatiles from bitter melon using dynamic headspace technology

[0029] Dynamic headspace sampling (DHS) was used to collect volatiles from bitter melon. Before use, the sampling cylinder (radius 20 cm, height 60 cm), adsorption column, silicone tube and other instruments were cleaned with 75% anhydrous ethanol and heated at 180 °C for 6 h to eliminate odor. To collect volatiles, air flow was sucked in from the top of the tank and discharged from the bottom at a flow rate of 4 L / min. The adsorption column was loaded with 200 mg of PoraPak Q (80-100 mesh, Sigma Aldrich-Shang Hai) adsorbent. The experimental device sequence was atmospheric sampler, flow meter, carbon column, water column, sampling cylinder, adsorption column and atmospheric sampler. The connection of the silicone tube was sealed with a sealing film to prevent leakage of the collection device. The collection was continuous for 24 hours, and the sample and adsorption column were replaced every 8 hours. After the collection, the volatile substances in the three adsorption columns were eluted with 2 ml of chromatographic grade n-hexane, concentrated to 0.5 ml, and stored in a refrigerator at -20 °C for the next GC-MS and EAG experiments. Four sampling cylinders were used for collection each time, of which three were experimental groups and one was a blank control.

[0030] GC-MS was used to separate and identify the volatiles in bitter melon, and the results are shown in Table 1. Bitter melon contains 21 volatile components, including 6 alkenes, 6 esters, 6 alkanes, 2 aldehydes and 1 alcohol. Among them, hexadecene, methyl palmitate, octadecene and methyl stearate are the main components, with contents of 30.24%, 25.18%, 12.11% and 9.38% respectively.

[0031] Table 1. Identification of volatile components in bitter melon

[0032]

[0033] Note: a RI measured value, b RI database search value.

[0034] Example 2 Electroantennary (EAG) responses of male and female adults of Bactrocera melonensis to volatiles of bitter melon

[0035] Open the antenna potentiostat, insert the silver wire into the electrodes at both ends, insert one end into the reference electrode, and insert the other end into the recording electrode. Use the electrode puller PC-100 to prepare glass capillaries, select two glass capillaries, one of which is connected to the head of the melon fruit fly, and the other is cut at the front end to facilitate the connection of the contact angle. The conductive liquid is 0.1mol / L KCl prepared and used immediately, and the conductive liquid in the capillary does not exceed two-thirds of the volume. The device airflow is 400mL / min, the stimulation airflow is 200 mL / min, and the time interval between each stimulation is 0.2s. Use chromatographic pure n-hexane to dilute the odorant standard to 100µg / µL, 10µg / µL, 1µg / µL, 0.1µg / µL, and 0.01µg / µL in sequence, and use n-hexane as a blank control. Select male and female melon fruit flies within 3-7 days of emergence. Under a microscope, cut off the head of the melon fruit fly, and cut a small section at the end of one of the antennae to ensure good conductivity. Use a capillary to connect the head of the melon fruit fly to the reference electrode, and connect the beveled tip of the antenna to the recording electrode. No bubbles should be generated during the whole process. When the antennal potential baseline is stable, gently pinch the stimulating airflow and observe whether the EAG baseline fluctuates to detect whether the antennae are well connected. Drop 10µL of the odor compound standard solution on a 40mm×5cm filter paper strip, evaporate in the air for 20s, put it into a Pasteur tube and seal the tube mouth. After the EAG baseline is stable, insert the Pasteur tube into a metal tube 6cm away from the antennae, pass through the carbon column, water column, and blow to the antennae through the stimulating airflow. The stimulation order is: n-hexane, bitter melon volatiles, n-hexane, and the stimulation interval is at least 30s. At least 12 female or male antennae are tested for each standard sample.

[0036] EAG results Figure 1 As shown in the figure, among the 21 odorants selected, 10 odorants can induce EAG reactions in male and female adults of B. melonea, namely isopulegol, methyl stearate, hexanal, styrene, methyl myristate, decene, tetradecene, methyl isovalerate, octadecene and squalene. The antennal reactions of male and female adults induced by isopulegol, styrene, tetradecene and octadecene reached a significant level (isopulegol: F=12.366, P=0.001, df=1; methyl stearate: F=3.22, P=0.075, df=1; hexanal: F=2.234, P=0.134, df=1; styrene: F=13.034, P<0.001, df=1; methyl myristate : F=3.168, P=0.067, df=1; decene: F=4.985, P=0.059, df=1; tetradecene: F=7.221, P=0.008, df=1; methyl isovalerate: F=4.364, P=0.054, df=1; octadecene: F=7.109, P=0.009, df=1; squalene: F=2.694, P=0.117, df=1).

[0037] The antennal responses of B. melonii to 10 volatile substances of bitter melon at five concentration gradients (100µg / µL, 10 µg / µL, 1µg / µL, 0.1µg / µL and 0.01µg / µL) were tested. The experimental results are shown in Figure 2 The dose response of EAG-active volatiles is shown in Figure 2 As shown. In general, as the concentration of volatile substances increases from 0.01 to 100 µg / µL, the antennal response of insects increases. However, there are different thresholds for the antennal response of B. cucurbitae to different volatile substances. The antennal responses of male and female adults of B. cucurbitae to isopulegol, methyl stearate, squalene, tetradecene, styrene and hexanal reach the maximum value at 100 µg / µL, and the antennal responses to hexadecene, methyl myristate, octadecene and methyl isovalerate reach the maximum value at 100 µg / µL. =The maximum value was reached at 95.3 μg / μL (isopulegol: female F=45.804, P<0.001, male F=40.985, P<0.001, df=4; methyl stearate: female F=30.298, P<0.001, male F=30.664, P<0.001, df=4; hexanal: female F=32.358, P<0.001, male F=38.462, P<0.001, df=4; styrene: female F=21.332, P<0.001, male F=18.09, P<0.001, df=4; tetradecene: female F=20.136, P<0.001, male F=20.202, P<0.001, df=4 , df=4; squalene: female F=3.774, P=0.009, male F=3.514, P=0.013, df=4; methyl myristic acid ester: female F=14.171, P<0.001, male F=10.606, P<0.001, df=4; decene: female F=15.576, P<0.001, male F=18.454, P<0.001, df=4; methyl isovalerate: female F=7.902, P<0.001, male F=3.507, P=0.013, df=4; octadecene: female F=14.473, P<0.001, male F=11.073, P<0.001, df=4)).

[0038] Example 3 Behavioral responses of adults of Bactrocera melonensis to volatiles of bitter melon

[0039] The behavioral responses of volatile components of different concentrations to the melon fruit fly were tested through a double-trap experiment, and the behaviorally active components that can significantly attract the melon fruit fly were screened out.

[0040] Male and female cucurbit fruit flies (100 male and female cucurbit fruit flies) that were 3-7 days old and starved for 6 hours were introduced into insect cages with a size of 30 cm×30 cm×30 cm. The insect cages contained two 500 ml plastic cup traps (the plastic cup traps were constructed as shown in Figure 3 As shown in the figure, a small hole was made in the lid of the plastic cup, and a 5mL pipette tip was inserted to form an environment where the test insects could enter but not leave. Two 500ml plastic cup traps were set as volatile standard control trap treatment and n-hexane control treatment respectively: absorbent cotton containing 2ml sample and 2ml n-hexane were added to the plastic cups respectively, and the number of melon fruit flies entering the trap containing the odor source and the control trap was counted after 24 hours. Entering the standard trap was recorded as attraction choice, and entering the n-hexane trap was recorded as repellent choice. The experiment of each volatile standard at different concentrations was repeated 5 times.

[0041] Male and female adults of B. cucurbitae showed significant sex- and concentration-dependent responses to the 10 EAG-active substances. Methyl isovalerate had an attractive effect on male and female adults of B. cucurbitae at all tested concentrations at 100, 10, and 1 μg / μL, but had a repellent effect on male and female adults of B. cucurbitae at 0.1 and 0.01 μg / μL ( Figure 4-8 In addition, there are gender differences in the response of B. melonis to methyl myristic acid. Methyl myristic acid changes the effect of female B. melonis from repellent (10 μg / μL) to attractive (1-0.01 μg / μL), and changes the effect of male B. melonis from attractive (10 μg / μL) to repellent (1 μg / μL) ( Figure 4-7 In addition, hexanal had an attractive effect on female melon fruit flies at 100 and 0.1 μg / μL ( Figure 3 ), squalene at 10μg / μL has an attractive effect on both male and female adults of melon fruit flies ( Figure 4 ), octadecene changed from an attraction (0.1 μg / μL) to a repellent (0.01 μg / μL) effect on male B. melonensis, and styrene had a repellent effect on male B. melonensis at 0.1 and 0.01 μg / μL ( Figure 7-8 ).

[0042] Example 4 Experiment on the attracting effect of hexanal and methyl isovalerate combination on adults of Cucurbita melonensis

[0043] Step 1: Dissolve methyl isovalerate in n-hexane to prepare a 100 µg / µL methyl isovalerate solution;

[0044] Step 2: Dissolve hexanal in n-hexane to prepare a 100µg / µL hexanal solution;

[0045] Step 3: Mix 100µg / µL methyl isovalerate solution and 100µg / µL hexanal solution in a volume ratio of 1:1 to prepare a melon fruit fly attractant.

[0046] Male and female cucurbit fruit flies (100 male and female cucurbit fruit flies) that were 3-7 days old and starved for 6 hours were introduced into insect cages with a size of 30 cm×30 cm×30 cm. The insect cages contained two 500 ml plastic cup traps (the plastic cup traps were constructed as shown in Figure 3 As shown), a small hole was made on the lid of the plastic cup, and a 5mL gun tip was inserted to form an environment where the test insects could enter but not leave. Two 500ml plastic cup traps were set as the melon fruit fly attractant test treatment and the n-hexane control treatment respectively: absorbent cotton containing 2ml of melon fruit fly attractant and 2ml of n-hexane were added into the plastic cups respectively, and the number of melon fruit flies that entered the trap containing the odor source and the control trap were calculated after 24 hours. Entering the melon fruit fly attractant trap was recorded as attraction choice, and entering the n-hexane trap was recorded as repellent choice. Male and female adults were tested separately, and the experiment was repeated 5 times. The results show that the attractant of the present invention has an attraction effect on female melon fruit flies, but has no obvious attraction effect on male insects ( Fig. 9 )

[0047] The present invention adopts two techniques, antennal potential and behavior measurement, to compare the responses of male and female adults of Cucurbita cucurbita to volatiles of bitter melon, and finds that there are certain differences in the test results of electrophysiological responses and behavioral selection responses of Cucurbita cucurbita, although Cucurbita cucurbita has higher EAG response values ​​to methyl stearate and isopulegol, but the performance in the tendency behavior response is not significant. Herbivorous insects mainly perceive plant volatile substances through olfactory receptors. In this study, most of the 10 volatile substances have a certain luring or repelling effect on male and female adults of Cucurbita cucurbita, but the effects are different between males and females. This may be caused by the different roles played by male and female individuals of insects in locating hosts, finding feeding and laying eggs, and may also be related to the physiological structure of the olfactory receptors of Cucurbita cucurbita and the sensitivity to odors. The specific reasons need further study. The present invention studies 10 kinds of bitter melon volatiles from the aspects of electrophysiology and behavior, and proves that methyl isovalerate, methyl myristate, octadecene, squalene, styrene, and hexanal have an attracting or repelling effect on melon fruit flies, which can attract melon fruit flies and provide theoretical and technical support for the screening and preparation of efficient plant-based attractants and repellents for melon fruit flies. The present invention also discloses a plant-based attractant for attracting female melon fruit flies, a preparation method thereof, and an application thereof, laying a foundation for the green prevention and control of melon fruit flies.

[0048] The foregoing description of specific exemplary embodiments of the present invention is for the purpose of illustration and demonstration. The scope of protection disclosed by the present invention is not limited thereto, so that those skilled in the art can utilize various exemplary embodiments of the present invention and various selections and changes. The scope of the present invention is intended to be limited by the claims and their equivalents.

Claims

1. A melon fruit fly female insect attractant, characterized in that: The attractant is a compound containing methyl isovalerate and hexanal, and the attractant preparation method is as follows: Step 1: Dissolve methyl isovalerate in n-hexane to prepare a 100 µg / µL methyl isovalerate solution; Step 2: Dissolve hexanal in n-hexane to prepare a 100µg / µL hexanal solution; Step 3: Mix 100µg / µL methyl isovalerate solution and 100µg / µL hexanal solution in a 1:1 volume ratio.

2. A method for preparing the melon fruit fly female insect attractant according to claim 1, characterized in that: The preparation method is: Step 1: Dissolve methyl isovalerate in n-hexane to prepare a 100 µg / µL methyl isovalerate solution; Step 2: Dissolve hexanal in n-hexane to prepare a 100µg / µL hexanal solution; Step 3: Mix 100µg / µL methyl isovalerate solution and 100µg / µL hexanal solution in a 1:1 volume ratio.

3. The use of the melon fruit fly female insect attractant as claimed in claim 1, characterized in that: It can be used to prepare bait cores or bait paper, or used in combination with biological pesticides or low-toxic chemical pesticides to enhance the trapping ability.

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