Female bactrocera cucurbitae attractant based on volatile matters of bitter gourd fruits and leaves and application thereof

Abstract

The invention relates to the technical field of green prevention and control of agricultural pests, in particular to a female bactrocera cucurbitae attractant based on volatile matters of bitter gourd fruits and leaves and application of the female bactrocera cucurbitae attractant. The active components of the female bactrocera cucurbitae attractant come from volatile matters of fruits and leaves of bitter gourds, and comprise 4-terpilenol, verbenol, alpha-terpilenol, capraldehyde, beta-caryophyllene and perillaldehyde. The main components of the female bactrocera cucurbitae attractant are all derived from natural plant volatile matters, the attractant is environment-friendly, a foundation is laid for field monitoring of bactrocera cucurbitae, and a new thought is provided for green prevention and control of bactrocera cucurbitae.

Description

Technical Field

[0001] This invention relates to the field of plant disease and pest control technology, specifically to an attractant for female melon fly based on volatiles from bitter gourd fruit and leaves, and its application. Background Technology

[0002] melon fly ( Zeugodacus cucurbitae The melon fly (Solanum melonatum) is a globally invasive pest that severely damages cucurbitaceous crops, with its females laying eggs on the fruit, causing devastating economic losses. Developing efficient and environmentally friendly female attractants is crucial for accurately monitoring melon fly population dynamics, implementing mass trapping of females (directly reducing egg production), and disrupting their oviposition behavior; it is a core requirement of integrated pest management. However, developing attractants that are highly attractive to female melon flies has always faced significant challenges. Female behavior is typically more focused on finding suitable oviposition hosts than mating, and their olfactory response mechanisms are more complex, requiring extremely high sensitivity and specificity to pheromones. Currently, effective attractants for female melon fly are extremely scarce. Chinese patents CN1312992C and CN100431412C both provide sex attractants with clodinafop as the main component, which only kill some males. However, female melon flies can mate repeatedly, and even a single mating can result in continuous egg-laying and damage for up to 20 days. These male attractants only reduce the mating rate of female melon flies. While Chinese patent CN118592448B provides a female melon fly attractant and identifies 21 volatiles derived from bitter melon, it only vaguely mentions "bitter melon volatiles," resulting in an insufficient screening sample and potentially overlooking unique active ingredients in the leaves that are more attractive to melon flies.

[0003] Therefore, developing a long-lasting, more attractive, and environmentally friendly female attractant for the melon fly is of great significance for achieving green and efficient control of this pest. Summary of the Invention

[0004] The purpose of this invention is to provide a long-lasting, more attractive, and environmentally friendly female attractant for the melon fly. This attractant is based on the volatiles from the fruit and leaves of the bitter melon plant, the host plant of the melon fly, laying the foundation for the development of plant-derived attractants for the melon fly. The specific technical solution is as follows: This invention provides a female attractant for melon fly, wherein the active components of the female attractant for melon fly include: 4-terpineol, verbenatenol, α-terpineol, decanal, β-caryophyllene, and perillaldehyde; The method for preparing the female melon fly attractant is as follows: Step 1: Dissolve 4-terpineol in n-hexane to prepare a 10 µg / µL 4-terpineol solution; dissolve verbenol in n-hexane to prepare a 10 µg / µL verbenol solution; dissolve α-terpineol in n-hexane to prepare a 10 µg / µL α-terpineol solution; dissolve decanal in n-hexane to prepare a 10 µg / µL decanal solution; dissolve β-caryophyllene in n-hexane to prepare a 10 µg / µL β-caryophyllene solution; dissolve perillaldehyde in n-hexane to prepare a 10 µg / µL perillaldehyde solution. Step 2: Mix 10 µg / µL of 4-terpineol, verbenatenol, α-terpineol, decanal, β-caryophyllene and perillaldehyde in a volume ratio of 1:1:1:1:1:1 to obtain the female attractant for the melon fly.

[0005] The present invention also provides the use of the aforementioned attractant for female melon flies, for preparing lures or attractant paper, or for use in combination with biological pesticides or low-toxicity chemical pesticides to enhance the trapping and killing ability.

[0006] The beneficial effects of this invention are: (1) This invention studies the effects of volatiles from the fruit and leaves of bitter melon, the host plant of the melon fly, on the behavior of the melon fly from both electrophysiological and behavioral perspectives. It has been shown that 1, 10 and 100 µg / µL of 4-terpineol, verbenol, α-terpineol, decanal, β-caryophyllene and perillaldehyde have an attractive effect on female melon flies. This provides theoretical and technical support for the development of highly efficient plant-derived attractants for the melon fly.

[0007] (2) The present invention provides an attractant in which 10 µg / µL of 4-terpineol, verbenol, α-terpineol, decanal, β-caryophyllene and perillaldehyde are mixed evenly in a volume ratio of 1:1:1:1:1:1. The attractant of the present invention has no attraction effect on male insects, but specifically attracts female insects.

[0008] (3) The main components of the fruit fly attractant described in this invention are all derived from natural plant volatiles, which are environmentally friendly and safe for non-target organisms. This lays the foundation for field monitoring of female fruit flies and provides a new idea for the green control of fruit flies. Attached Figure Description

[0009] Figure 1 This is an antennal response diagram of the melon fly to volatiles from bitter melon fruit and leaves. Significant differences between odor substances are represented by lowercase letters. P <0.05).

[0010] Figure 2 This is an EAG response diagram of the melon fly to different doses of volatiles from bitter melon fruit and leaves. Significant differences between different doses are represented by lowercase letters. P<0.05), the volatiles of the bitter gourd fruit and leaves include: 4-terpineol, verbenaenool, 1-nonanol, myrtol, α-pinene, and 3-hexen-1-ol.

[0011] Figure 3 This is an EAG response diagram of the melon fly to different doses of volatiles from bitter gourd fruit and leaves. Significant differences between different doses are represented by lowercase letters (P < 0.05). The volatiles from bitter gourd fruit and leaves include: α-terpineol, 2-undecone, β-caryophyllene, 2-nonanone, and 1-decyl alcohol.

[0012] Figure 4 This is an EAG response diagram of the melon fly to different doses of volatiles from bitter gourd fruit and leaves. Significant differences between different doses are represented by lowercase letters (P < 0.05). The volatiles from bitter gourd fruit and leaves include decanal and perillaldehyde.

[0013] Figure 5 This is a structural diagram of an olfactory trap.

[0014] Figure 6 This is a diagram showing the behavioral response of female adult melon fly to volatiles from bitter melon fruit and leaves at a concentration of 1 µg / µL.

[0015] Figure 7 This is a diagram showing the behavioral response of female adult melon fly to volatiles from bitter melon fruit and leaves at a concentration of 10 µg / µL.

[0016] Figure 8 This is a diagram showing the behavioral response of female adult melon fly to volatiles from bitter melon fruit and leaves at a concentration of 100 µg / µL.

[0017] Figure 9 This study presents behavioral response diagrams of adult male and female melon fly to a compound attractant and compares the attraction activity of the compound attractant with that of a single volatile substance on adult female melon fly. Detailed Implementation

[0018] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0019] Example 1: Headspace solid-phase microextraction (HS-SPME) was used to collect volatiles from bitter melon fruits and leaves. Volatile compounds from bitter melon fruits and leaves were collected using headspace solid-phase microextraction (HS-SPME). Bitter melon leaves were cut into 5-7 cm lengths, and 10 g of each was placed in a 100 ml headspace vial and sealed with a PTFE membrane cap. Bitter melon fruits were vertically cut into strips 6-7 mm wide and 5 cm long, and 20 g of each strip was placed in a 100 ml headspace vial and sealed with a PTFE membrane cap. The extraction head was inserted into the top of the headspace vial, and extraction was performed at 40°C for 6 hours. The extractor was then removed and desorbed for analysis.

[0020] The volatile compounds of bitter melon were separated and identified by GC-MS, and the results are shown in Table 1. Forty-eight compounds were identified in the bitter melon fruit, and forty-four compounds were identified in the leaves, with 28 of them being identical volatile compounds. The fruit contained relatively high levels of 3-hexen-1-ol and myrtol, at 18.12% and 39.33%, respectively. The leaves contained relatively high levels of 3-hexen-1-ol, leaf acetate, and myrtol, at 18.12%, 23.3%, and 30.76%, respectively.

[0021] Table 1. Identification of volatile components in bitter melon fruit and leaves

[0022]

[0023]

[0024] Note: a RI measured value b RI database search value.

[0025] Example 2: Antennae potential (EAG) response of female adult melon fly to volatiles from bitter melon fruit and leaves The head of a melon fly was removed with a blade, and the tip of one antenna (1 mM) was cut off. A recording electrode was inserted, and the melon fly's head was gently inserted into a reference electrode. A glass electrode was filled with 0.1 mol / L KCl as a conductive solution. 10 µL of volatile sample was added to a filter paper strip (40 mM wide × 5 cm long) as an olfactory stimulus. The filter paper was placed in a 10 cm sample tube, one end of which was connected to an odor stimulation control device (CS-55, Syntech). Filtered air (400 mL / min) was continuously delivered to the antenna through a stainless steel delivery tube, with the outlet approximately 1 cm from the antenna. The odor sample was transmitted to the antenna through 0.5 s air pulses, with each stimulus spaced 30 s apart to ensure antennal recovery. The stimulation order was n-hexane, the volatile sample, and then n-hexane again. The relative EAG response value was calculated as (CT-CK) / CK, where CT is the EAG response value of the volatile sample and CK is the average EAG response value of the control group (n-hexane). Each compound was tested on at least 15 different female insects, and each insect was tested only once.

[0026] EAG results are as follows Figure 1 As shown, among all odorous substances, 13 substances elicited an EAG response in female melon fly insects. These were 4-terpineol, verbenaenool, α-terpineol, myrtol, α-pinene, 2-undecone, 1-nonanol, decanal, 1-decanol, 2-nonanone, 3-hexen-1-ol, β-caryophyllene, and perillaldehyde. Of these, 4-terpineol, myrtol, α-terpineol, 2-nonanone, 3-hexen-1-ol, β-caryophyllene, and α-pinene were common volatiles from both leaves and fruits; verbenaenool and perillaldehyde were volatiles from leaves; and 1-decanol, 2-undecone, 1-nonanol, and decanal were volatiles from fruits. Female insects showed a stronger EAG response to 4-terpineol, verbenaenool, and α-terpineol. P <0.05).

[0027] The antennal responses of the melon fly to 13 volatile compounds at three concentration gradients (100 µg / µL, 10 µg / µL, and 1 µg / µL) were tested. The experimental results are as follows: Figures 2 to 4 As the concentration increases, the stimulation of the antennae of the melon fly increases with the concentration of 4-terpineol, verbenol, 1-nonanol, myrtol, α-pinene, 3-hexen-1-ol, α-terpineol, 2-undecone, β-caryophyllene, 2-nonanone, and 1-decylol. Figures 2-3 Decanal and perillaldehyde had the greatest irritant effect on antennae at a concentration of 10 µg / µL. Figure 4 The three different concentrations of 4-terpineol, verbenatenol, α-terpineol, and myrtol showed significant differences in their stimulation of the female antennae (P < 0.05).

[0028] Example 3: Behavioral response of adult melon fruit flies to volatiles from bitter melon fruit and leaves The olfactory apparatus consists of three 30 × 30 × 30 cm cages connected by a 5 cm diameter circular hole. The middle cage (R) is used to release melon flies, while the two outer cages (T and C) contain volatiles and n-hexane, respectively (the olfactory apparatus is constructed as shown in the diagram). Figure 3 (As shown). Starting at 9:00 AM, 60 female melon fly larvae, which had emerged 15 days prior and been starved for 12 hours, were released into cage R. The number of melon fly larvae entering cage T or C was counted at 6:00 PM. All tests were repeated five times under identical conditions, with the positions of cages T and C interchanged during repetitions to avoid contamination and directional interference. All tests were conducted in darkness to avoid visual interference.

[0029] Behavioral results showed that 1, 10, and 100 µg / µL of 4-terpineol, verbenol, α-terpineol, decanal, β-caryophyllene, and perillaldehyde attracted female melon fly larvae, while 1-decyl alcohol and 2-nonanone repelled them. At concentrations of 10 and 100 µg / µL, myrtol and 2-undecone repelled females, 3-hexen-1-ol attracted them, while 1 µg / µL of myrtol and 2-undecone had no behavioral effect on females, and 3-hexen-1-ol repelled them. 1-Nonanol changed its effect on females from attraction (100 µg / µL) to repulsion (1 and 10 µg / µL). P <0.05) Figure 6-8 ).

[0030] Example 4: Experiment on the attraction effect of a mixture of six substances (4-terpineol, verbenaenool, α-terpineol, decanal, β-caryophyllene, and perillaldehyde) at a ratio of 10 µg / µL on adult melon fly. Step 1: Dissolve 4-terpineol in n-hexane to prepare a 10 µg / µL 4-terpineol solution; Step 2: Dissolve verbenacol in n-hexane to prepare a 10 µg / µL verbenacol solution; Step 3: Dissolve α-terpineol in n-hexane to prepare an α-terpineol solution of 10 µg / µL; Step 4: Dissolve decanal in n-hexane to prepare a 10 µg / µL decanal solution; Step 5: Dissolve β-caryophyllene in n-hexane to prepare a 10 µg / µL β-caryophyllene solution; Step 6: Dissolve perillaldehyde in n-hexane to prepare a 10 µg / µL perillaldehyde solution; Step 7: Mix 10 µg / µL of 4-terpineol, verbenatenol, α-terpineol, decanal, β-caryophyllene and perillaldehyde in a volume ratio of 1:1:1:1:1:1.

[0031] The olfactory apparatus consists of three 30 × 30 × 30 cm cages connected by 5 cm diameter holes. The middle cage (R) is used to release melon flies, while the two outer cages (T and C) contain a volatile mixture and n-hexane, respectively. 200 µL of n-hexane and 200 µL of the volatile mixture are added separately to 1.5 cm × 1.5 cm strips of filter paper, and then placed in 12 cm diameter petri dishes (olfactory apparatus construction as shown). Figure 5(As shown). Starting at 9:00 AM, 60 female and 60 male melon fly larvae, which had emerged 15 days prior and been starved for 12 hours, were released into cage R. The number of male and female melon fly larvae entering cage T or C was counted at 6:00 PM. All tests were repeated five times under identical conditions, with the positions of cages T and C interchanged during repetitions to avoid contamination and directional interference. All tests were conducted in darkness to avoid visual interference.

[0032] Behavioral response results such as Figure 9 As shown in Figure a, the formula had no significant attraction effect on male melon fly larvae, but had an attraction effect on female larvae. P <0.01).

[0033] Example 5: Comparative Experiment on the Attraction Effect of Compound Attractant and Single Component on Female Melon Fly First, following the method described in Example 1, 4-terpineol, verbenaenool, α-terpineol, decanal, β-caryophyllene, and perillaldehyde were dissolved in n-hexane to prepare monomer solutions with a concentration of 10 µg / µL. Then, following the method described in Example 4, the above six monomer solutions were mixed evenly in a volume ratio of 1:1:1:1:1:1 to prepare the compound attractant. Pure n-hexane was used as the control solution. The testing apparatus and operating procedure were the same as in Example 4. The experimental setup is as follows: Treatment group (T cage): 1.5cm × 1.5cm filter paper strips (200 µL per treatment) containing the above 6 single-component solutions or compound attractants were placed in a 12cm diameter petri dish.

[0034] Control group (Cage C): A 1.5cm × 1.5cm filter paper strip containing 200 µL of pure n-hexane was placed in a 12cm diameter petri dish.

[0035] For each test, 60 adult female melon flies, 15 days post-emergence and starved for 12 hours, were released into a central release cage (R cage). The experiment was conducted in darkness, starting at 9:00 AM and recording the number of flies entering each side cage at 6:00 PM. Each treatment (6 single components + 1 compound agent) was performed in 5 independent replicates, with the positions of cages T and C swapped between replicates to eliminate directional interference.

[0036] Behavioral response results such as Figure 9 As shown in b in the figure, statistical analysis revealed that the attraction rate of the compound attractant to adult female melon flies was significantly higher than that of any single volatile component (P<0.01). The different lowercase letters above the bars for different treatments in the figure indicate statistically significant differences in attraction rates between treatments. This result demonstrates that the six specific volatiles, when compounded according to the proportions of this invention, produce a significant synergistic effect, and their attractiveness to female flies is far beyond the simple sum of the individual components.

[0037] This invention employs both antennal potential (EP) and behavioral assay (EAA) techniques to investigate the responses of female melon fly adults to volatiles from bitter melon fruit and leaves. EAG identification revealed that 13 volatiles, including 4-terpineol, verbenol, α-terpineol, myrtol, α-pinene, 2-undecanone, 1-nonanol, decanal, 1-decanol, 2-nonanone, 3-hexen-1-ol, β-caryophyllene, and perillaldehyde, elicited EAG responses in female melon fly adults. Furthermore, the EAG responses of females to different doses of these volatiles varied. Except for perillaldehyde and decanal, the EAG responses of the melon fly to the other compounds increased with increasing concentration. In addition, all 13 volatiles exhibited behavioral effects on female melon fly adults at different doses. Specifically, 4-terpineol, verbenol, α-terpineol, decanal, β-caryophyllene, and perillaldehyde all showed attractiveness to female melon fly adults at various doses. The behavioral effects of host plant volatiles on insects are the result of the synergistic effects of multiple compounds. Based on the screened behaviorally active substances of the melon fly, six substances—4-terpineol, verbenol, α-terpineol, decanal, β-caryophyllene, and perillaldehyde—were selected at a concentration of 10 µg / µL and blended in a ratio of 1:1:1:1:1:1. Compared with individual volatiles, the mixture showed a stronger attraction to female melon flies.

[0038] In this invention, we identified the volatile components of bitter gourd fruit and leaves, the preferred hosts of the melon fly, screened out the behaviorally active substances of the melon fly through EAG and behavioral responses, and discovered a volatile compound female attractant, laying the foundation for the development of novel attractants for the melon fly.

[0039] The foregoing description of specific exemplary embodiments of the present invention is for illustrative and explanatory purposes. The scope of protection disclosed herein is not limited thereto, thereby enabling those skilled in the art to utilize various different exemplary embodiments and various alternatives and modifications of the invention. The scope of the invention is intended to be defined by the claims and their equivalents.

Claims

1. A female mediterranean fruit fly attractant, characterized by, The active components of the female Bactrocera cucurbitae attractant include: 4-terpineol, verbenol, alpha-terpineol, decanal, beta-caryophyllene and perilene; The preparation method of the female Bactrocera cucurbitae attractant is: Step 1: 4-terpineol is dissolved in n-hexane to prepare a 10 µg / µL 4-terpineol solution; verbenol is dissolved in n-hexane to prepare a 10 µg / µL verbenol solution; alpha-terpineol is dissolved in n-hexane to prepare a 10 µg / µL alpha-terpineol solution; decanal is dissolved in n-hexane to prepare a 10 µg / µL decanal solution; beta-caryophyllene is dissolved in n-hexane to prepare a 10 µg / µL beta-caryophyllene solution; perilene is dissolved in n-hexane to prepare a 10 µg / µL perilene solution; Step 2: 10 µg / µL of 4-terpineol, verbenol, alpha-terpineol, decanal, beta-caryophyllene and perilene are mixed uniformly at a volume ratio of 1:1:1:1:1:1 to prepare the female Bactrocera cucurbitae attractant.

2. The method for preparing the female Bactrocera cucurbitae attractant of claim 1, characterized in that, Step 1: 4-terpineol is dissolved in n-hexane to prepare a 10 µg / µL 4-terpineol solution; verbenol is dissolved in n-hexane to prepare a 10 µg / µL verbenol solution; alpha-terpineol is dissolved in n-hexane to prepare a 10 µg / µL alpha-terpineol solution; decanal is dissolved in n-hexane to prepare a 10 µg / µL decanal solution; beta-caryophyllene is dissolved in n-hexane to prepare a 10 µg / µL beta-caryophyllene solution; perilene is dissolved in n-hexane to prepare a 10 µg / µL perilene solution; Step 2: 10 µg / µL of 4-terpineol, verbenol, alpha-terpineol, decanal, beta-caryophyllene and perilene are mixed uniformly at a volume ratio of 1:1:1:1:1:1 to prepare the female Bactrocera cucurbitae attractant.

3. Use of the female Anastrepha obliqua insect attractant of claim 1, characterized in that, For preparing lure core or lure paper, or used in combination with biological pesticides or low-toxicity chemical pesticides to enhance the trapping and killing ability. For preparing lure core or lure paper, or used in combination with biological pesticides or low-toxicity chemical pesticides to enhance the trapping and killing ability.

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