Botanical attractant for Menochia sexmaculata and application of botanical attractant

Abstract

The invention discloses a botanical attractant for Menochia sexmaculata and application of the botanical attractant. The attractant is composed of an attraction composition, paraffin and a fiber cotton core, the attraction composition and the paraffin are mixed according to the volume ratio of 1: (5-10) and are filled in the fiber cotton core, and the volume fractions of (3E, 7E)-4, 8, 12-trimethyltridecane-1, 3, 7, 11-tetraene, beta-elemene, beta-damascenone, beta-myrcene, benzaldehyde and farnesone in the attraction composition are 40%, 10%, 10%, 15%, 5% and 20% respectively. According to the attractant disclosed by the invention, plant terpene volatile matters and green leaf volatile matters are induced by insect pests to attract, so that the reduction of chemical pesticides is promoted while the biological control effect is improved.

Description

Technical Field

[0001] The present invention relates to the field of green prevention and control of agricultural pests, and particularly to an odor attractant of the predatory natural enemy Menochilus sexmaculata and its application. Background Art

[0002] The Asian citrus psyllid (Diaphorina citri) is an important vector insect for transmitting Huanglongbing (HLB) in nature, and Huanglongbing is the most devastating disease in citrus production. Controlling the population of Asian citrus psyllids is one of the main measures to limit the spread of Huanglongbing. Currently, it mainly relies on chemical control. However, the extensive, large-scale and long-term use of chemical pesticides easily leads to serious drug resistance of Asian citrus psyllids and toxicity problems to natural enemy organisms. Therefore, reducing the use amount of chemical pesticides and using natural enemy insects for biological control play a very important role in the sustainable prevention and control of Huanglongbing.

[0003] Menochilus sexmaculata is an important predatory natural enemy that can prey on common pests in citrus orchards, including Asian citrus psyllids, citrus aphids, citrus red spiders, mites, scale insects, whiteflies, etc. Menochilus sexmaculata has a high predation ability on the nymphs and adults of Asian citrus psyllids. By feeding on Asian citrus psyllids, it can meet its generational development and has a significant control effect on the population of Asian citrus psyllids. However, during the biological control process, due to the insufficient dispersal ability of Menochilus sexmaculata, the predation rate is often not high. Therefore, it is necessary to develop a natural enemy attractant to improve its biological pest control effect.

[0004] When plants are damaged by phytophagous insects, they will release a large amount of herbivore-induced plant volatiles, which can attract natural enemy organisms (such as ladybugs) of phytophagous insects to prey, thus forming an indirect defense. Herbivore-induced plant volatiles mainly include terpene compounds and green leaf volatiles, and also include a small amount of nitrogen-containing and sulfur-containing compounds. For example, ladybugs use terpene compounds for long-distance orientation in the habitat and green leaf volatiles for short-distance localization of hosts or prey. These compounds can be used to attract natural enemies to the pest occurrence area and improve the natural enemy predation rate. Summary of the Invention

[0005] The purpose of the present invention is to provide a plant-derived attractant of Menochilus sexmaculata and its application. This attractant is to identify plant-derived active substances that can efficiently attract Menochilus sexmaculata from host plant volatile substances (including herbivore-induced volatiles and puncture site volatiles) through metabolomics and insect behavior, and evaluate its attracting effect through greenhouse experiments.

[0006] The present invention uses a gas chromatography-mass spectrometry (GC-MS) instrument to determine the volatile metabolomes of citrus psyllid nymphs feeding on, mechanically damaged, and untreated young citrus leaves. Based on the results of the volatile metabolome, 11 terpene volatile substances were screened out, standard curves were established using standard products, and then a fully automatic headspace solid-phase microextraction (HS-SPME) was used for sample extraction. The absolute content levels of terpene compounds upregulated by citrus psyllid nymph feeding were detected by GC-MS. These pest-induced terpene substances can be used as candidate compounds for attracting Menochilus sexmaculatus over long distances.

[0007] When citrus psyllids suck plant sap into their bodies, the compounds in the plant sap will be injected into the plant along with saliva secretion. These substances are often the source of volatiles at the feeding site and can attract citrus psyllids at close range. To identify candidate compounds for attracting at close range, a large amount of saliva secreted by citrus psyllid adults after feeding on host plants was collected, and then the compound components were identified using liquid chromatography-tandem mass spectrometry (LC-MS / MS). Insect behavior methods were used to identify active substances from pest-induced terpene substances and saliva volatiles, and a composite attractant was prepared to improve the predation rate of citrus psyllids and enhance its biological control effect.

[0008] The first object of the present invention is to provide a plant-derived composite attractant composition for Menochilus sexmaculatus, comprising (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, β-elemene, β-damascenone, β-myrcene, benzaldehyde, and farnesene ketone.

[0009] Preferably, the volume fractions of (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, β-elemene, β-damascenone, β-myrcene, benzaldehyde, and farnesene ketone in the composition are 40%, 10%, 10%, 15%, 5%, and 20%.

[0010] The second object of the present invention is to provide a composite attractant for Menochilus sexmaculatus, which is composed of the above-mentioned attractant composition, a solvent, and a carrier. The attractant composition and the solvent are mixed and filled in the carrier.

[0011] Preferably, the volume ratio of the attractant composition to the solvent is 1:5 to 10, specifically 1:5 and 1:10. More preferably, the volume ratio of the attractant composition to the solvent is 1:5.

[0012] Preferably, the solvent is paraffin wax.

[0013] Preferably, the carrier is a fiber cotton core.

[0014] Preferably, the fiber cotton core is cylindrical, with a diameter of 2.0 cm, a height of 3.0 cm, and a pore diameter of 500 microns.

[0015] The present invention discovers that (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, β-elemene, β-damascenone, β-myrcene, benzaldehyde, and farnesone can each attract Menochilus sexmaculatus alone. However, when these six attracting compounds are compounded in specific ratios, they can exert a synergistic effect and achieve a significant attracting effect on Menochilus sexmaculatus.

[0016] The present invention selects paraffin as the solvent, mainly considering the characteristic that paraffin can dissolve various organic volatile substances. The fiber cotton core selected in the present invention has good permeability and adsorption, can evenly adsorb the mixture of the composite attracting composition and paraffin, and can stably and evenly release it during implementation, greatly reducing the usage amount.

[0017] The third object of the present invention is to provide a method for preparing the above-mentioned composite attractant for Menochilus sexmaculatus, which is to add the above-mentioned attracting composition and solvent to the carrier and fully absorb and prepare it to obtain.

[0018] Specifically, it includes the following steps:

[0019] S1. Put solid paraffin into a container and heat it to 70 °C until it is completely dissolved to obtain paraffin liquid;

[0020] S2. Mix (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, β-elemene, β-damascenone, β-myrcene, benzaldehyde, and farnesone into the paraffin liquid to obtain a blend of the attracting compound mixture and paraffin liquid;

[0021] S3. Immerse the fiber cotton core into the blend of the attracting compound mixture and paraffin liquid until it is completely absorbed, and then take it out and let it cool and solidify to obtain.

[0022] The fourth object of the present invention is to provide the application of the above-mentioned attracting composition or attractant in attracting Menochilus sexmaculatus. Specifically, it is the application in attracting Menochilus sexmaculatus in the natural environment or artificially released.

[0023] The fifth object of the present invention is to provide the application of the above-mentioned attracting composition or attractant in controlling Diaphorina citri in orchards.

[0024] Preferably, the application method is: put the attractant into a non-woven fabric bag and hang it in the tree crown layer of fruit trees.

[0025] Preferably, the application method is: hang the attractant 7 - 10 days before the shoot stage of citrus plants (including spring shoots, summer shoots, autumn shoots, late autumn shoots, and winter shoots).

[0026] Compared with the prior art, the present invention has the following advantages:

[0027] (1) The present invention combines pest-induced plant terpenoid volatiles and green leaf volatiles, greatly enhancing the short-distance positioning of natural enemies and the long-distance searching effect of natural enemies. This attractant has good attracting effects in both laboratory and net room tests, with good repeatability and high stability.

[0028] (2) The attractant prepared by the present invention can be regularly hung for use according to the occurrence law of the Diaphorina citri population. After use, the application amount of chemical insecticides in the orchard can be reduced, effectively reducing the pesticide residues in the orchard, which conforms to the development direction of green agriculture.

[0029] (3) The present invention is prepared into a slow-release lure core, and the paraffin mixture is filled into the fiber cotton core. Its preparation method is simple, the production cost is low, and it is easy to use, suitable for large-scale popularization and application. Description of the Drawings

[0030] Figure 1 It is the technical process for the collection and determination of pest-induced plant volatiles. Among them, A is the technical process for determining the volatile metabolome of young citrus leaves with Diaphorina citri nymphs feeding, mechanically damaged, and untreated by using gas chromatography-mass spectrometry (GC-MS); B is the technical process for determining the green leaf volatiles in the saliva secreted by Diaphorina citri after feeding on plants by using liquid chromatography-tandem mass spectrometry (LC-MS / MS).

[0031] Figure 2 It is a schematic diagram of the layout of the net room attraction effect test.

[0032] Figure 3 It is the determination result of the net room attraction effect test. Detailed Embodiments

[0033] The present invention will be further described in detail below in combination with specific embodiments. The provided embodiments are only for clarifying the present invention, rather than limiting the scope of the present invention.

[0034] The following embodiments select solid paraffin and fiber cotton core as the slow-release lure core to explain the present invention. It should be noted that those skilled in the art can also process the lure core of the present invention into natural rubber, synthetic rubber, polyethylene, polyvinyl chloride, paper materials, resins or fiber matrices, etc. on the basis of the disclosed content of the present invention in combination with the conventional preparation process of the lure core of the attractant.

[0035] Example 1

[0036] Analysis of the saliva components of Diaphorina citri based on non-targeted metabolomics:

[0037] Green leaf volatiles released from the feeding sites of phytophagous insects can attract predators to locate phytophagous insects. To obtain the volatile compounds released from the feeding sites of Diaphorina citri, according to the method for collecting D. citri saliva in our previous study (Wu et al., 2020), adults were first collected from the D. citri population reared on Murraya exotica, and then transferred to artificial diet. After sufficient amounts were collected, non-targeted metabolomics analysis was performed.

[0038] Specifically, it included mixing the collected saliva extracts in equal amounts to form QC samples, performing non-targeted detection on the LC-QTOF-MS / MS platform, performing accurate qualitative determination based on the standard database MWDB (including secondary spectra and retention time RT), integrating the AI prediction library of the DB-all public database (including databases such as Metlin, HMDB, and KEGG), and MetDNA, and extracting information such as multi-ion pairs and retention time RT of the identified metabolites. Combining with the target database, a new library exclusive to the project was formed. Finally, in all samples, MRM accurate quantification of the metabolites in the new library was performed based on the Q-Trap instrument platform.

[0039] A large amount of D. citri saliva was collected using the sandwich method, and then the metabolites were identified. The results showed that there were 6 metabolites in the aldehyde, ketone, and ester categories (nootkatone, jasmonone, farnesene ketone, diethyl phosphate, diethyl oxalopropionate, 9-octadecene aldehyde), 7 indoles and their derivatives (indole, methoxyindoleacetic acid, indole-3-carboxaldehyde, indole-3-methanol, imidazoleacetic acid, indole-3-butyric acid, 1-acetylindole), 51 benzenes and their derivatives, 64 organic acids and their derivatives, and 18 organic acids and their derivatives.

[0040] Example 2

[0041] Analysis of the component content of D. citri saliva based on targeted metabolomics:

[0042] Appropriately weigh each standard product precisely, dissolve it with chloroform, and prepare a standard stock solution of 1 mg / mL; subsequently, dilute it with chloroform to standard series concentration solutions with concentrations of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 5.0 μg / mL, 10 μg / mL, and 20 μg / mL, and perform on-machine detection.

[0043] Accurately weigh about 0.2 g of the sample, add 5.0 mL of chloroform-methanol solution (1:1, volume ratio), vortex for 30 s, perform ultrasonic extraction in a water bath for 30 min, add 1 mL of water, let it stand for stratification, take the chloroform layer, repeat the extraction of the upper layer solution with chloroform twice, combine the chloroform, dry it under nitrogen, and then dissolve it with 1 mL of chloroform, filter it through a membrane, and perform on-machine determination.

[0044] Based on the results of saliva non-target metabolomics, potential active substances were screened (Table 1). By purchasing reference standards and establishing standard curves, the absolute content levels of compounds in the saliva secreted by Diaphorina citri after feeding on plants were then detected by liquid chromatography-tandem mass spectrometry (LC-MS / MS). After detection, the concentrations of the main compound components in the saliva secreted by Diaphorina citri after feeding were as follows: 2-hydroxycinnamic acid 77.20 ng / mL, benzaldehyde 80.40 ng / mL, nootkatone 110.80 ng / mL, indole-3-carboxaldehyde 151.60 ng / mL, indole-3-methanol 286.40 ng / mL, farnesene ketone 4456.00 ng / mL, methyl caffeate 32320.00 ng / mL (see Table 1).

[0045] Table 1. Targeted determination of saliva collected from Diaphorina citri after feeding on Murraya exotica

[0046]

[0047]

[0048] Example 3

[0049] Determination of the behavioral response of Menochilus sexmaculatus to candidate attracting compounds using a Y-tube olfactometer:

[0050] Using paraffin oil as a solvent, the reference standards were prepared into test solutions with concentrations of 0.01 g / mL (1%) and 0.1 g / mL (10%). 10 μL of the experimental reagent and paraffin oil were respectively placed in two odor source bottles. The behavioral response of Menochilus sexmaculatus to the candidate compounds to be determined was measured using a Y-tube olfactometer. The experimental method referred to CN 110583645 A. One adult of Menochilus sexmaculatus was used for each treatment, and the experiment was repeated 60 times. Only one adult of Menochilus sexmaculatus was released each time for the chemotaxis behavior selection. The adult was introduced from the straight wall of the Y-tube and timing started. If the insect could cross 1 / 3 of the two arms of the Y-tube and stay for 5 s within 5 min, it was recorded as having a selection; otherwise, it was recorded as having no response. After each experiment, the positions of the two test arms were swapped, and the experimental instrument was cleaned after each group of experiments.

[0051] The selection behavior of Menochilus sexmaculatus towards 18 candidate volatile compounds to be determined was measured using a Y-tube olfactometer as shown in Table 2. Four terpenoid volatiles ((3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, β-damascenone, β-elemene, β-myrcene), and two green leaf volatiles (benzaldehyde and farnesene ketone) could attract Menochilus sexmaculatus to varying degrees. Except for farnesene ketone, the attraction rates of other compounds at a concentration of 0.1 g / mL were greater than those at a concentration of 0.01 g / mL, which also indicated the existence of a dose effect to a certain extent.

[0052] Table 2. Selectivity of Menochilus sexmaculatus to candidate compounds

[0053]

[0054]

[0055]

[0056] Example 4

[0057] Determination of the behavioral response of Menochilus sexmaculatus to different formulated attractant mixtures using a Y-tube olfactometer:

[0058] Referring to the concentration ratios of the active components in the terpene volatiles induced by the feeding of Diaphorina citri nymphs and the green leaf volatiles secreted in the saliva after the feeding of Diaphorina citri adults, (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, β-damascenone, β-elemene, β-myrcene, benzaldehyde, and farnesene ketone were measured, and then mixed samples were prepared by setting different ratios (Table 3), dropped into paraffin oil and dissolved, and mixed evenly to prepare a composite attractant with a total mass-volume concentration of 0.1 g / mL. The results showed that the selection rate of Formula 5 was the highest (87.56%), followed by Formula 4 (85.16%). This indicates that after these compounds were compounded, there was a synergistic effect between the compounds.

[0059] Table 3. Selectivity analysis of different formulations for Menochilus sexmaculatus

[0060]

[0061] Note: This table shows volume fractions.

[0062] Example 5

[0063] Evaluation of the greenhouse effect of the compound attractant:

[0064] In this experiment, the formulation with a high attraction rate, that is, 40% of (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, 10% of β-elemene, 10% of β-damascenone, 15% of β-myrcene, 5% of benzaldehyde, and 20% of farnesene ketone, was selected as the measured composite attractant formulation, and paraffin was used as the solvent. The measured attractant compound mixture and paraffin were prepared according to volume ratios of 1:5, 1:10, 1:20, and 1:30.

[0065] The preparation steps of the sustained-release attractant are as follows:

[0066] S1. Put solid paraffin into a beaker, heat it to 70 °C and stir until the paraffin is completely dissolved to obtain a paraffin solution;

[0067] S2. Mix (3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene, β-damascenone, β-elemene, β-myrcene, benzaldehyde and farnesone into the paraffin liquid according to the formula ratio with high attraction rate, heat to 70 °C and stir evenly to obtain a blend of the attracting substance mixture formula and paraffin;

[0068] S3. Immerse the fiber cotton core column into the blend until it is completely absorbed (no bubbles emerge), and then take it out to cool and solidify.

[0069] The measurement location is arranged in the indoor test net of Zhongkai University of Agriculture and Engineering. The length × width × height of the net room is 80 cm × 80 cm × 80 cm; then divide the samples and the control diagonally ( Figure 2 ), and hang the attractant in the middle of the canopy of Murraya paniculata (at a height of 30 cm). The paraffin cotton core column without the attractant is used as the blank control, and 5 replicates are set for each ratio. Release 50 adults of Menochilus sexmaculatus each time, and record the number of Menochilus sexmaculatus in Murraya paniculata at 10:00 am the next day. The attraction rate = (the number of attracted insects / the total number of released insects) × 100%.

[0070] The results show that under the net room conditions, the selection rates of the parasitoid wasps for Formulas 1, 2, 3, and 4 are 64.0%, 61.6%, 47.2%, and 43.6% respectively, indicating that Formula 1 has the highest attracting effect on Menochilus sexmaculatus, followed by Formula 2, Formula 3, and Formula 4 ( Figure 3 ). This shows that the active attractant must have a dose effect.

[0071] In summary, according to the qualitative and quantitative results of terpenoid volatiles and green leaf volatiles in pest-induced compounds, the present invention determined the attraction rates of 18 candidate compounds for Menochilus sexmaculatus. The compound formula and behavioral tests were formulated with reference to the quantitative results of pest-induced compounds. Among them, the formula ((3E,7E)-4,8,12-trimethyltrideca-1,3,7,11-tetraene 40%, β-elemene 10%, β-damascenone 10%, β-myrcene 15%, benzaldehyde 5%, farnesone 20%) has the highest attracting effect on Menochilus sexmaculatus. Using the fiber cotton core as the carrier, with a volume ratio of the compound formula to paraffin of 1:5 for the slow-release attractant, it has a significant attracting effect in the net room.

[0072] The formula of the present invention is the first attractant for the natural enemy of Menochilus sexmaculatus. The attractant of the present invention is used in combination with the release of artificially reared Menochilus sexmaculatus, which can develop the natural enemy trapping technology, guide and continuously trap the released predatory natural enemies, can largely make up for the shortcoming of the artificial release technology in pest control, and can also trap a large number of Menochilus sexmaculatus in nature to the orchard for biological control.

[0073] Finally, it should also be noted that the above are only several specific embodiments of the present invention. Therefore, the present invention is not limited to the above embodiments and there can be many variations. For those of ordinary skill in the art, without departing from the concept of the present invention, several variations and improvements can still be made, and these all fall within the protection scope of the present invention.

Claims

1. A plant-derived composite attractant composition for six-spotted ladybirds, characterized in that: These include (3E,7E)-4,8,12-trimethyltridecane-1,3,7,11-tetraene, β-elemene, β-damascenone, β-myrcene, benzaldehyde and farnesenone.

2. The plant-derived composite attractant composition for six-spotted ladybirds according to claim 1, characterized in that: The volume fractions of (3E, 7E)-4,8,12-trimethyltridecane-1,3,7,11-tetraene, β-elemene, β-damascenone, β-myrcene, benzaldehyde and farnesenone in the composition are 40%, 10%, 10%, 15%, 5% and 20%.

3. A composite attractant for six-spotted ladybugs, characterized in that: The invention comprises the attractant composition according to claim 1 and a solvent, wherein the volume ratio of the attractant composition to the solvent is 1:5-10.

4. The composite attractant for six-spotted ladybirds according to claim 3, characterized in that: The solvent is paraffin.

5. The composite attractant for six-spotted ladybirds according to claim 3, characterized in that: Also included is a carrier.

6. The composite attractant for six-spotted ladybirds according to claim 5, characterized in that: The carrier is a fiber cotton core.

7. A method for preparing the composite attractant for Coccinella spheniscus as claimed in any one of claims 3 to 6, characterized in that: The attractant composition and solvent described in claim 1 are added to the carrier and fully absorbed to obtain the product.

8. Use of the attractant composition according to any one of claims 1 to 2 or the attractant according to any one of claims 3 to 6 in attracting Coccinella septempunctata.

9. Use of the attractant composition according to any one of claims 1 to 2 or the attractant according to any one of claims 3 to 6 in controlling citrus psyllids in orchards.

10. The use according to claim 9, characterized in that: The attractant is put into a non-woven bag and then hung on the canopy of the fruit tree.

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