Uses of volatile compounds from Mengla musk, insecticides, and methods for controlling agricultural pests.

Abstract

This invention relates to the uses, insecticides, and methods for controlling agricultural pests using volatiles from *Muscone mongholicus*, belonging to the field of agricultural pest control technology. The main technical solution is as follows: the volatiles from *Muscone mongholicus* are used as or in the preparation of insecticides for agricultural pests. These insecticides are effective against both omnivorous lepidopteran pests and specialized pests; the omnivorous lepidopteran pests include cotton bollworm, fall armyworm, and armyworm; the specialized pests include brown planthopper, gray planthopper, and white-backed planthopper. This invention primarily aims to propose a new use for the volatiles from *Muscone mongholicus* in the use or preparation of insecticides for agricultural pests, thereby finding new avenues for future green agricultural pest control.

Description

Technical Field

[0001] This invention relates to the field of agricultural pest control technology, and in particular to the uses of volatile substances from Mengla Mao Musk, an insecticide, and a method for controlling agricultural pests. Background Technology

[0002] Currently, the main method for controlling agricultural pests is through chemical pesticides, which have the advantages of low dosage, rapid effect, and significant short-term results. However, chemical pesticide control has the following disadvantages: pests are prone to rebound and become more severe, and pests develop resistance, rendering the pesticides ineffective. Furthermore, although areas where DDT pesticides are applied only account for a small portion of the land area, DDT has been found in plants and animals in Antarctica, far from where it was applied, indicating that these chemical pesticides have entered the biogeochemical cycle and polluted the environment. In addition, agricultural products such as fruits and vegetables often have large amounts of pesticide residues, which endanger human health.

[0003] Therefore, there is an urgent need for a naturally sourced insecticide that can be used to control agricultural pests, thereby protecting human health and the environment. Summary of the Invention

[0004] In view of this, the present invention provides a use of the volatiles of Mengla musk, an insecticide, and a method for controlling agricultural pests. The main purpose is to propose a new use of the volatiles of Mengla musk as an insecticide for agricultural pests or in the preparation of such an insecticide.

[0005] To achieve the above objectives, the present invention mainly provides the following technical solutions:

[0006] On one hand, embodiments of the present invention provide a use of the volatiles of *Muscone mongholicus*, wherein the volatiles of *Muscone mongholicus* are used as or in the preparation of an insecticide for agricultural pests.

[0007] Preferably, the agricultural pest repellent is used to control both omnivorous lepidopteran pests and specialized pests; wherein the omnivorous lepidopteran pests include cotton bollworm, fall armyworm, and armyworm; and the specialized pests include brown planthopper, gray planthopper, and white-backed planthopper.

[0008] Preferably, the volatile compounds of the Mengla musk include p-cymene, γ-terpinene, carvacrol methyl ether, carvacrol, α-thujone, α-pinene, myrcene, α-phellandrene, 3-carene, limonene, α-terpinene, α-terpineol, thymol, trans-caryophyllene, and humulene.

[0009] Preferably, the content of p-cymene is 290-12000 ng / g.FW; the content of γ-terpinene is 2500-66500 ng / g.FW; the content of carvacrol methyl ether is 100-12500 ng / g.FW; the content of carvacrol is 250-32000 ng / g.FW; the content of α-thujone is 100-2200 ng / g.FW; the content of α-pinene is 5-280 ng / g.FW; and the content of myrcene is 10-140 ng / g.FW. The content of phellandrene is 5-170 ng / g.FW; the content of 3-carene is 70-2700 ng / g.FW; the content of limonene is 100-3000 ng / g.FW; the content of α-terpineol is 5-260 ng / g.FW; the content of thymol is 10-620 ng / g.FW; the content of trans-caryophyllene is 3.0-240 ng / g.FW; the content of humulene is 20-1100 ng / g.FW; and the content of α-terpinene is 90-4750 ng / g.FW.

[0010] Preferably, the preparation method of the volatile compounds of Mengla musk includes the following steps:

[0011] Collection and adsorption steps: The above-ground parts or tissue parts of the *Muscari mongholica* plant are covered inside a plant cover; the plant cover is connected to an air inlet pipe and an air outlet pipe; an adsorption column is installed on the air outlet pipe; air enters the plant cover through the air inlet pipe and flows out through the air outlet pipe, and the adsorption column is used to collect the volatiles of *Muscari mongholica*.

[0012] Elution steps: Remove the adsorption column and elute with solvent to obtain the volatiles of Mengla musk.

[0013] Preferably, the tissue part of the *Muscari* plant is any one of the following: root, stem, fresh leaves, dried leaves, inflorescence, or fruit inflorescence.

[0014] Preferably, the plant or tissue parts of *Mussaenda mollissima* are extracted using essential oil extraction methods or water extraction methods to obtain *Mussaenda mollissima* volatiles.

[0015] On the other hand, embodiments of the present invention provide an insecticide, wherein the insecticide includes volatiles of muskrat; wherein the insecticide is an agricultural pest insecticide; preferably, the agricultural pest insecticide is used to control omnivorous lepidopteran pests and specialized pests; wherein the omnivorous lepidopteran pests include cotton bollworms and armyworms; and the specialized pests include brown planthoppers.

[0016] In another aspect, embodiments of the present invention provide a method for controlling agricultural pests, wherein the insecticide described above is sprayed on crops to control agricultural pests; preferably, the insecticide is sprayed on crops in the early stage of pest infestation.

[0017] In another aspect, embodiments of the present invention also provide a method for controlling agricultural pests, wherein crops are intercropped with the Mengla musk to control agricultural pests.

[0018] Compared with the prior art, the uses of the volatile substances of Mengla musk, the insecticide, and the method for controlling agricultural pests of the present invention have at least the following beneficial effects:

[0019] On the one hand, this invention proposes for the first time a new use for the volatiles of *Muscone mongholicus*, namely, that the volatiles of *Muscone mongholicus* can be used as or in the preparation of insecticides for agricultural pests. Here, since the volatiles of *Muscone mongholicus* are of natural origin, this provides a new approach for future green agricultural pest control.

[0020] On the other hand, embodiments of the present invention provide a method for controlling agricultural pests, mainly by spraying the volatile substances of *Muscone mongholicus* onto crops to control agricultural pests. Here, the method proposed in these embodiments not only controls agricultural pests, but also, because it is of a natural source, can protect human health and the environment.

[0021] On another front, this invention provides a method for controlling agricultural pests, primarily by intercropping crops with the aforementioned *Muscaria molica* (if the crops are crops or vegetables, intercropping them with *Muscaria molica*; if the crops are fruit trees, *Muscaria molica* can be planted under the trees, which is also a form of intercropping), in order to control agricultural pests. This method can control agricultural pests while also protecting human health and the environment.

[0022] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0023] Figure 1 This is a physical image of a collection and adsorption device for volatile substances from Mengla musk provided in an embodiment of the present invention.

[0024] Figure 2 This is a schematic diagram illustrating the effect of volatile compounds from *Muscaria molica* on the selection of female adult cotton bollworms and armyworms, according to an embodiment of the present invention. In particular, in... Figure 2 In the middle: CK: analytical grade n-hexane; EOs: concentration of 10 -3A solution of volatile components of Mengla musk. The error bar represents the standard error (n = 20-25), with different asterisks indicating significant differences. * p<0.05, ** p<0.01, *** p<0.001).

[0025] Figure 3 This diagram illustrates the antennal electrophysiological responses of female armyworm adults to the volatiles and main components of *Muscary mongholicus*. (a) Figure shows the response of armyworms to solutions of *Muscary mongholicus* volatiles at different concentrations. CK: n-hexane; Ab: *Muscary mongholicus* volatiles; (b) Figure shows the response of armyworms to four compound components in *Muscary mongholicus* volatiles. CK: n-hexane; Ab0.1: 0.1 μL / mL *Muscary mongholicus* volatile solution; Mix: a compound solution of four standards: p-cymene, carvacrol, carvacrol methyl ether (CME), and γ-terpinene. (c) Figure shows the antennal electrophysiological responses of armyworms to four standards: p-cymene, carvacrol, carvacrol methyl ether (CME), and γ-terpinene. Error bars represent standard errors (n = 25-30), and different asterisks indicate significant differences between the treatment and control groups. * p<0.05, ** p<0.01, *** p<0.001).

[0026] Figure 4 A schematic diagram of the setup for field experimental plots.

[0027] Figure 5 A comparison of insect populations in dryland rice clumps with and without the presence of *Muscarya mongholica*; among them, Figure 5 Figure (a) shows a comparison of brown planthopper numbers. Figure 5 Figure (b) shows a comparison of the number of gray planthoppers. Figure 5 Figure (c) shows a comparison of the number of rice leaf rollers. Figure 5 Figure (d) shows a comparison of the number of brown-footed horned-breasted leaf beetles. Figure 5 Figure (e) shows a comparison of the numbers of the black-shouldered green mirid bug. Figure 5 Figure (f) shows a comparison of spider numbers. The error bars represent standard errors, and different asterisks indicate significant differences. * p<0.05, ** p<0.01, *** p<0.001). Among them, Figure 5 CON in the text refers to planting only upland rice, while FG refers to the coexistence of upland rice and Mengla musk.

[0028] Figure 6 This is a schematic diagram illustrating the effect of volatile compounds from *Muscary mongholicus* on the feeding selectivity of brown planthoppers; the error bars represent standard errors (n=20), and different asterisks indicate significant differences. *p<0.05, ** p<0.01, *** p<0.001).

[0029] Figure 7 This is a schematic diagram illustrating the influence of volatile compounds from *Muscary mongholicus* on the oviposition site selection behavior of female adult brown planthoppers; the error bars represent the standard error (n=20), and different asterisks indicate significant differences. * p<0.05, ** p<0.01, *** p<0.001).

[0030] Figure 8 This describes the response of jasmonic acid-isoleucine in upland rice leaves to simulated insect pests induced by *Muscari mollissima*. Specifically, in... Figure 8 In the data, "Rice only" refers to the group where only upland rice was planted; "Rice / FG" refers to the group where *Muscaria molica* and upland rice were planted separately but placed in the same space with gas contact; and "Rice+FG" refers to the group where *Muscaria molica* and upland rice were planted in the same pot with contact between the roots and the above-ground parts. The error bar represents the standard error (n=5), and different asterisks indicate significant differences (p<0.05). Detailed Implementation

[0031] To further illustrate the technical means and effects adopted by the present invention to achieve the intended purpose, the specific embodiments, structures, features, and effects according to the present invention will be described in detail below with reference to the accompanying drawings and preferred embodiments. In the following description, different "an embodiment" or "an embodiment" do not necessarily refer to the same embodiment. Furthermore, specific features, structures, or characteristics in one or more embodiments can be combined in any suitable form.

[0032] Mengla hairy musk (Adenosma bracteosum Bonati), also known as flea grass, is an annual herb belonging to the genus Adenosma in the family Plantaginaceae. The entire plant is densely covered with glandular hairs, has a strong aroma, and is rich in volatile substances. It is mainly distributed in Southeast Asian countries and regions such as Vietnam, Laos, and Cambodia.

[0033] This invention, through long-term research, has for the first time discovered that the volatiles of *Muscone mongholicus* can be used as or in the preparation of insecticides for agricultural pests. These insecticides are used to control both omnivorous lepidopteran pests and specialized pests; the omnivorous lepidopteran pests include cotton bollworm, fall armyworm, and armyworm; the specialized pests include brown planthopper, gray planthopper, and white-backed planthopper.

[0034] The present invention will be described in detail below through specific experimental examples:

[0035] Example 1

[0036] This embodiment mainly prepares the volatiles of Mengla hairy musk and analyzes the volatiles. The specific methods are as follows:

[0037] 1. Plant materials:

[0038] In July 2018, uniformly growing *Mussaenda pubescens* plants were selected from the *Mussaenda pubescens* planting base in Xishuangbanna and transplanted to the glass greenhouse of the Kunming Institute of Botany, Chinese Academy of Sciences. After routine care until the fruit matured, the above-ground parts were harvested and placed in a low-temperature drying room (temperature 15℃, relative humidity 20%). After drying for 60 days, the fruit clusters were manually rubbed, passed through a 20-mesh sieve, and impurities and unripe seeds were removed using a blower to obtain plump, uniformly shaped seeds. These seeds were then placed in seed collection bags and stored in a cool, shady place at room temperature.

[0039] Seeds of *Muscari mongholicus* were sown in 32cm×21cm×3cm plastic pots. The cultivation substrate consisted of humus and vermiculite in a 5:1 ratio. The artificial light incubator was set at 30℃ for 12 hours of light and 20,000 LX of light intensity, followed by 28℃ for 12 hours of darkness. Once the seedlings reached the 4-true-leaf stage, they were transplanted to a glass greenhouse with a temperature of 20-30℃, a relative humidity of 40-60%, and natural lighting for further cultivation.

[0040] 2. Instruments, equipment, and reagents:

[0041] Main instruments and equipment: Gas chromatography-mass spectrometry (Agilent 7890A GC, 5975C MS, Agilent Technologies, USA), HP-5 column (50m×0.2mm×0.33μm, Agilent Technologies, USA), light incubator (model: GXZ–310D, Ningbo Southeast Instrument Co., Ltd.), and a dynamic headspace adsorption device designed and assembled in the laboratory.

[0042] Main reagents: Dichloromethane (chromatographic grade, Tianjin Fengchuan Chemical Reagent Technology Co., Ltd.), chloroform (chromatographic grade), anhydrous ethanol (analytical grade), n-hexane (analytical grade), purchased from Tianjin Damao Chemical Reagent Factory. Compound standards: C8-C20 n-alkanes (chromatographic grade), α-pinene (98%), thymol (98%), linalool (98%), p-cymene (97%), purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; γ-terpinene (97%), carvacrol (98%), carvacrol methyl ether (95%), purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0043] 3. Collection of volatile substances.

[0044] 3.1 Selection of different developmental stages of Mengla musk

[0045] *Mussa megistophylla*, an annual herbaceous plant, has tiny seeds, approximately 600 micrometers in length, and germinates from the cotyledons. The entire life cycle of *Mussa megistophylla*, from seed germination to maturity, takes approximately 180-240 days. Based on its growth and development characteristics, it can be divided into four key developmental stages: rosette stage, initial flowering stage, full bloom stage, and fruiting stage. The rosette stage begins with the 4-leaf stage, and enters a rapid vegetative growth phase at the 8-10 leaf stage. The flowering period can last for 2-3 months, with indeterminate spike-like inflorescences. When the fruit matures, the bracts remain, and the leaves wither but do not fall off. From the moment the cotyledons emerge, the leaves release an aromatic scent, which remains strong until the fruit matures and the leaves turn yellow. This example selected plants at four growth and developmental stages of *Mussa megistophylla* for research: the rosette stage (8-10 leaves), the initial flowering stage (2-4 flowers open), the full bloom stage (concentrated flowering period), and the fruiting stage (end of flowering, when the bracts turn from green to yellow).

[0046] 3.2 Processing of different tissue parts of Mengla musk

[0047] Fresh *Muscari* plants were taken, and the cultivation substrate was rinsed off with distilled water. The surface moisture of the roots was then absorbed with laboratory paper. Different tissue parts, including roots, stems, leaves, inflorescences, and fruit clusters, were separated using scissors. The fresh weight of each tissue part was weighed and recorded. The base of each tissue part was then wrapped with absorbent cotton soaked in distilled water and placed in a spherical flask for dynamic headspace adsorption. Depending on the developmental stage, the biological characteristics of *Muscari* leaves are as follows: during the vegetative growth stage, fresh green oval leaves predominate; during the flowering period, lanceolate leaves predominate. As reproductive growth progresses, the leaves gradually dry from the lower to the upper part of the plant, until all leaves are withered and yellowed during the fruiting stage, but do not fall off. Therefore, the peak flowering period can be divided into five tissue parts: roots, stems, fresh leaves, dried leaves, and inflorescences.

[0048] 3.3 Collection of volatile substances from Mengla musk

[0049] The volatiles of Mengla musk were collected using a dynamic headspace adsorption device, which was built by the inventor in the laboratory. See [link to relevant documentation]. Figure 1As shown, the system mainly consists of an air pump, an activated carbon steel column, a flow meter, a sealing plate, a support platform, a glass column (i.e., a plant cover), and an adsorption column 4 (the glass column is connected to an inlet pipe and an outlet pipe 3). The specific operation is as follows: Place the *Mussaenda mollissima* plant on the lifting platform and adjust the height so that the above-ground part 1 of the *Mussaenda mollissima* plant is completely covered within the transparent glass column 2 (60cm high, 15cm in diameter). Open the air pump valve and adjust the gas flow rate. After passing through the stainless steel column filled with activated carbon (50cm high, 10cm in diameter), air enters the glass column 2 at a flow rate of 0.6L / min. The gas flow rate of the adsorption column 4 (adsorption material: Polydimethylsiloxane, PDMS) connected to the glass column is 0.4L / min. The adsorption time is from 9:30 AM to 4:30 PM, with an experimental room temperature of 28℃, humidity of 60%, and supplemental lighting from natural and artificial light sources.

[0050] In addition, the collection of volatiles from different tissue parts of *Muscari mongholicus* is based on the same method as the collection of volatiles from the whole plant. The difference is that different tissue parts of *Muscari mongholicus* are placed in 250mL glass spherical bottles, with air and an adsorption column connected to each end.

[0051] After the volatiles were collected, the adsorption column was quickly removed and completely eluted with 400 μL of dichloromethane. The eluent was then transferred to a 1.5 mL brown bottle, sealed, and stored at low temperature. 100 μL of the eluent was transferred to a GC vial for GC-MS analysis.

[0052] 4. GC-MS Analysis Conditions

[0053] GC conditions: Gas chromatography column: Agilent HP-5 (50m × 0.2mm × 0.33μm); Temperature program: initial temperature 50℃, hold for 5 min, increase to 250℃ at a rate of 5℃ / min, hold for 5 min; then increase to 300℃ at a rate of 10℃ / min; total analysis and detection time: 55 min; Splitless injection; Carrier gas: high-purity helium (99.999%); Carrier gas flow rate: 2.1 mL / min; Solvent delay: 5 min.

[0054] MS conditions: Chromatography-mass spectrometry interface temperature 280℃; ion source: EI source; ion source temperature 230℃; electron impact energy: 70 eV; mass-to-nuclear ratio scan range: 50-550 m / z; quadrupole temperature 150℃; ionization mode: electron collision. MS database: NIST 17 mass spectrometry database (Adams 2007).

[0055] 5. Component analysis results of volatiles from Mengla musk:

[0056] Table 1 shows the volatile components and contents of *Musk deer* from Mengla at four developmental stages.

[0057] Table 1

[0058]

[0059] In Table 1, different letter labels indicate significant differences (P<0.05).

[0060] Table 2 shows the volatile components and contents of different tissue parts during the peak flowering period of *Musk Thunbergii var. mongolica*.

[0061] Table 2

[0062]

[0063] The above analysis revealed that the main components of volatiles from plants at different stages, or from different tissues of plants at the same stage, were p-cymene, γ-terpinene, carvacrol methyl ether, and carvacrol. Further components included: α-thujone, α-pinene, myrcene, α-phellandrene, 3-carene, limonene, α-terpinene, α-terpineol, thymol, trans-caryophyllene, and humulene.

[0064] Preferably, the contents of the above fifteen main components are as follows: p-cymene content is 290-12000 ng / g.FW; γ-terpinene content is 2500-66500 ng / g.FW; carvacrol methyl ether content is 100-12500 ng / g.FW; carvacrol content is 250-32000 ng / g.FW; α-thujone content is 100-2200 ng / g.FW; α-pinene content is 5-280 ng / g.FW; and myrcene content is 10-140 ng / g.FW. g.FW; α-phellandrene content is 5-170 ng / g.FW; 3-carene content is 70-2700 ng / g.FW; limonene content is 100-3000 ng / g.FW; α-terpineol content is 5-260 ng / g.FW; thymol content is 10-620 ng / g.FW; trans-caryophyllene content is 3.0-240 ng / g.FW; humulene content is 20-1100 ng / g.FW; α-terpinene content is 90-4750 ng / g.FW.

[0065] Here, the unit "ng / g.FW" is explained as follows: it refers to the content of volatiles per gram of fresh tissue weight.

[0066] Example 2

[0067] This embodiment mainly tests the resistance of volatile substances from Mengla musk to agricultural pests. Several experiments were conducted, as detailed below:

[0068] 1. Experimental materials:

[0069] The test insects, cotton bollworm and armyworm egg masses, were purchased from Jiyuan Baiyun Industrial Co., Ltd. in Henan Province. After hatching, they were reared with artificial feed in an artificial light incubator. The rearing conditions were: 15 hours of light, 9 hours of darkness, temperature (24±3℃, relative humidity 70±5%). Larvae were reared to different instars for experiments on feeding selectivity and growth adaptability; pupae were reared and stored separately by sex. Emerging male and female adults were placed in the same cage and mated for 12-24 hours before being used for oviposition selectivity experiments. Emerging adults were fed 10% (v / v) sucrose solution, and the egg masses were collected, hatched, and artificially reared for subsequent experiments.

[0070] The cultivated tobacco seeds K326 used in the laboratory were provided by the Yunnan Provincial Tobacco Agricultural Research Institute. The culture conditions were 16 hours of light, 8 hours of darkness, and a temperature of 25±1℃. Brown planthoppers (Nilaparvata lugens) were collected from a dryland rice planting base in Xishuangbanna and placed in mesh insect cages for rearing and population propagation using rice seedlings. Male and female adults were used for subsequent experiments.

[0071] 2. Instruments, equipment, and reagents:

[0072] (1) Main instruments and equipment

[0073] Artificial light incubator (GXZ–310D), purchased from Ningbo Southeast Instrument Co., Ltd.; “Y” type olfactometer and “H” type olfactometer, designed and installed in the laboratory; insect antennal potentiometer, purchased from Syntech, Netherlands; electronic balance and microscope.

[0074] (2) Main reagents

[0075] n-Hexane (analytical grade) was purchased from Tianjin Damao Chemical Reagent Factory; chemical standards: p-cymene (97%) was purchased from Shanghai Maclean Biochemical Technology Co., Ltd.; γ-terpinene (97%), carvacrol (98%) and carvacrol methyl ether (95%) were purchased from Shanghai Yuanye Biotechnology Co., Ltd.

[0076] 3. Experimental methods:

[0077] 3.1 Selectivity experiment of female adult cotton bollworms and armyworms

[0078] This experiment used a Y-shaped olfactory instrument to determine the oviposition site selection behavior of female adult mosquitoes. The experimental setup was modified and designed based on the work of Deng Wan (Deng Wan. Study on the repellent effect of plant essential oils on Aedes albopictus and Aedes aegypti [D]. Hangzhou: Zhejiang University, 2015.). A dark box was added to the glass arm of the Y-shaped olfactory instrument, and an attraction light source was set at the end of the support arm of the dark box. In the preliminary experiment, *Muscaria menglaensis* plants and tobacco plants were used as test objects. However, the physicochemical properties of live plants changed significantly during the experiment due to factors such as wilting, leaf damage, and litter. Therefore, this experiment used a solution of volatiles from *Muscaria menglaensis* plants at full bloom. GC-MS analysis showed that the amount of volatiles released by fresh *Muscaria menglaensis* plants at full bloom was approximately equal to that diluted with n-hexane at 1000 mg / L. -3 The solution of volatile compounds from Mengla musk was selected for this experiment, therefore a volume concentration of 10 was chosen. -3 A solution of volatile compounds from Mengla musk was used as the test sample.

[0079] The specific operating steps are as follows: (1) Introduce one female adult insect that has mated for 24 hours into the main arm of the "Y"-shaped tube for 3 minutes to adapt; (2) Connect the flavor source bottle a containing a solution of volatile volatiles of mollusc musk to one side arm, and connect the flavor source bottle b containing the same volume of n-hexane to the other side arm as a control; (3) Turn on the air generator, purify the air by activated carbon adsorption, humidify it with distilled water, and pass it through the flavor source bottle, the side arm and the main arm in sequence. The gas flow rate is 0.6 L / min. After the ventilation device is turned on, record the selection of the female adult insect within 5 minutes. Entering the 1 / 3 position of one side arm is considered a valid selection. Entering both sides of the side arm in sequence or not reaching the 1 / 3 position of the side arm within 5 minutes is considered an invalid selection. After testing 3 adult insects, replace the "Y"-shaped tube glass device with a clean one that has been cleaned and dried with anhydrous alcohol, and change the position of the side arm to eliminate the influence of geometric position and residual odor.

[0080] Experimental results: Figure 2 This is a schematic diagram illustrating the effect of volatile compounds from *Muscaria molica* on the selection of female adult cotton bollworms and armyworms. Among them, in... Figure 2 In the middle: CK: analytical grade n-hexane; EOs: concentration of 10 -3 A solution of volatile components of Mengla musk. The error bar represents the standard error (n = 20-25), with different asterisks indicating significant differences. * p<0.05, ** p<0.01, *** p<0.001).

[0081] Bioassays using a Y-shaped olfactory apparatus to assess the selectivity of mated female bollworms and armyworms revealed that, compared to the control group (hexane), the number of adults selecting *Muscaria mongholica* volatiles was significantly lower. The chi-square test results for the selection of *Muscaria mongholica* by female bollworms and armyworms were X0.05 and X0.05, respectively. 2=61.48,df=3,p-value<0.0001,X 2 =34.276, df=3, p-value<0.0001.

[0082] The above results indicate that the volatile solution of *Muscari mongholicus* has a certain repellent effect on the oviposition site selection of female adult cotton bollworms and armyworms.

[0083] 3.2 Electrophysiological response of armyworms to volatiles of *Muscaria mongholica*

[0084] Antennae are important sensory organs in insects, playing a crucial role in host orientation, mate selection, and oviposition site selection. As the primary distribution area of ​​olfactory receptors, antennae are essential for insects to sense and identify odors, and are also important organisms for detecting volatile compounds. Electroantennography (EAG) originated in the 1950s when German scientist Schneider discovered a potential difference between the base and tip of an insect's antenna when stimulated. Currently, EAG has become a fundamental method for studying insect odor recognition, primarily used to explore the response of insect antennae to odors. An insect electroantennography system mainly consists of a signal acquisition system, a microscopic operating system, an air supply system, and a signal recording system. This experiment uses isolated antennae; the specific experimental steps are as follows:

[0085] (1) Preparation of test solutions: Chemical standards for the volatiles of *Muscone moschata* and its main components (p-cymene, γ-terpinene, carvacrol, and carvacrol methyl ether) were prepared in hexane to obtain five concentration gradients: 0.001 μL / mL, 0.01 μL / mL, 0.1 μL / mL, 1 μL / mL, and 10 μL / mL. First, a stock solution with a concentration of 10 μL / mL was prepared, and then each stock solution was diluted 10 times to obtain samples of all concentrations. 10 μL of the test solution was added to a folded filter paper (1.5 cm long and 0.8 cm wide) using a micropipette and placed in a Pasteur pipette as a stimulant.

[0086] (2) Antennae potential measurement: Adult insects 3-5 days after emergence were taken, and their antennae were cut off at the base. A small incision was made at the base, and the two ends of the antennae were attached to the two sides of the electrodes. The connected electrodes were then connected to the EAG micromanipulation stage, and the entire EAG system was connected. After the signal of the entire system stabilized, the antennae potential response value was recorded. Before and after each group of experiments, hexane was used for solvent testing. The stimuli were inserted into the odor stimulation control device, and the stimulation time was 0.5s. The interval between two stimulations was 30s. Each group of experiments was set up with 20 biological replicates.

[0087] The experimental results are as follows:

[0088] (1) Electrophysiological response of the antennae of armyworms to the volatiles of *Muscarya mongholica*

[0089] Electrophysiological experiments on antennae revealed that, compared with the solvent control group, different concentrations of *Muscary mongholicus* volatiles all caused significant changes in the electrophysiological responses of the antennae of the armyworm. The results are as follows: Figure 3 As shown in Figure (a): Under the odor stimulation of pure musk volatiles, the potential amplitude of the detached antennae of the armyworm was 3.67522±0.38643mV; under the odor stimulation of musk volatile solutions of 100μL / mL, 10μL / mL, 1μL / mL and 0.1μL / mL, the potential response amplitudes of the detached antennae were 3.83814±0.31807mV, 3.33935±0.32068mV, 2.94149±0.22323mV and 2.75666±0.20709mV, respectively; under the odor stimulation of n-hexane, the potential response value of the detached antennae was 2.33784±0.12918mV.

[0090] The experimental data revealed that the electrophysiological response amplitude of the antennae of the armyworm was significantly higher than that of the control group (n-hexane) under stimulation with different concentrations of *Muscone moschata* volatile solutions. While the electrophysiological responses of the armyworm antennae varied under different concentrations of *Muscone moschata* volatile solutions, the electrophysiological response did not increase with increasing solution concentration. The peak electrophysiological response was not observed at the highest concentration (pure *Muscone moschata* volatile solution), but rather at a concentration of 100 μL / mL, where the electrophysiological response of the detached antennae was highest. Furthermore, the electrophysiological response decreased with decreasing volatile solution concentration. However, even at a concentration of 0.1 μL / mL (the lowest tested concentration), the stimulation of the detached antennae of the armyworm remained significantly different compared to the solvent control group. Therefore, in subsequent experiments, a 0.1 μL / mL concentration of *Muscone moschata* volatile solution was used as a positive control.

[0091] (2) Electrophysiological responses of armyworms to a compound solution containing four main components of volatile moss from Mengla hairy musk.

[0092] To further analyze which components in the volatiles of *Muscone mongholicus* are active against insects, the inventors selected chemical standards for four main components of the volatiles: p-cymene, carvacrol, carvacrol methyl ether, and γ-terpinene. Based on their proportions in the volatiles, four concentration gradient sample solutions (mixes) were prepared using n-hexane. Using n-hexane as a blank control and a 0.1 μL / mL solution of the volatiles of *Muscone mongholicus* as a positive control, the electrophysiological responses of armyworm antennae were measured under the stimulation of the mixed solutions of the main components of *Muscone mongholicus*. The results are as follows: Figure 3As shown in Figure (b). Compared with the solvent control group, all four concentrations of the treatment solution induced electrophysiological responses in the antennae of armyworms, and the response values ​​were significantly different. Under odor stimulation at four concentration gradients of 10 μL / mL, 1 μL / mL, 0.1 μL / mL, and 0.01 μL / mL, the potential amplitudes of the detached antennae of armyworms were 2.19877±0.08821 mV, 2.23689±0.09399 mV, 2.23637±0.08032 mV, and 2.32616±0.09096 mV, respectively. Compared with the response value under the odor stimulation of the control group (n-hexane) (1.88194±0.09323 mV), the potential response values ​​of the detached antennae were significantly increased, but compared with the response value of the positive control group (2.55397±0.09567 mV), they were significantly decreased. Specifically, regarding the relationship between treatment with different concentrations of the compound solution and the electrophysiological response of the antennae of armyworms, the potential response value is negatively correlated with the concentration of the Mix.

[0093] (3) Electrophysiological responses of the antennae of armyworms to chemical standards of four main components in the volatiles of *Muscaria mongholica*.

[0094] Analysis of antennal potential amplitude data of detached female adult armyworms under odor stimulation from four main components of volatile compounds from *Muscary mongholicus* is as follows: Figure 3 As shown in Figure (c). Under the odor stimulation of four standard solutions (0.1 μL / mL) of cymene, carvacrol, carvacrol methyl ether (CME), and γ-terpinene, the potential amplitudes of the detached antennae of armyworms were 2.84054 ± 0.1099 mV, 2.81501 ± 0.11636 mV, 2.81701 ± 0.121 mV, and 2.71894 ± 0.10382 mV, respectively. Compared with the control group's hexane odor stimulation (2.23946 ± 0.11067 mV), the potential response values ​​of the detached antennae of armyworms significantly increased under the stimulation of all four compound standards. The response amplitudes of the detached antennae of the armyworm to the standard solutions of these four compounds differed. The potential response amplitudes of the antennae to the odor stimulation of the first three standard solutions of compounds (cymene, carvacrol, and carvacrol methyl ether) were similar, while the response amplitude to the stimulation of γ-terpinene was reduced.

[0095] The experimental results above show that armyworms exhibit significant electrophysiological responses to the volatiles and main components of *Muscaria molica*. However, the volatile solution of *Muscaria molica* has a stronger stimulating effect on armyworms. This is because: firstly, the diverse compounds in the volatiles have a synergistic effect; secondly, certain components in the volatiles of *Muscaria molica*, present in lower concentrations, exert small amounts of highly effective insecticidal activity.

[0096] 3.3 Field Experiment of Intercropping Mengla Musk with Upland Rice

[0097] (1) Sample plot setup

[0098] This experiment was conducted from 2019 to 2021 in the traditional Hani dryland rice planting area of ​​Daka Lao Village, Mengla County, Xishuangbanna Dai Autonomous Prefecture. A 16m × 16m square plot was selected and then divided into nine 4m × 4m experimental plots, as shown below. Figure 4 As shown: in plots 1-5, *Muscari mongholica* was intercropped with upland rice, while in plots 6-9, only upland rice was sown. There was a 1-meter-wide isolation strip between each experimental plot, where only upland rice was sown.

[0099] (2) Field planting

[0100] In late April, at the beginning of the rainy season in Xishuangbanna, after leveling the land, plots 1-9 were physically separated using colored isolation strips. First, seedlings of *Mussaenda pubescens* were cultivated. A number of *Mussaenda pubescens* seeds were sown in moist soil and transplanted when they reached the 4-6 leaf stage. Second, upland rice was sown. A number of upland rice seeds were taken, impurities and unripe seeds were removed, and the seeds were sown using the traditional Hani planting method: a small hole was made in the soil with a long wooden stick with a conical iron end, about 3cm in diameter and 3-5cm deep. 3-5 upland rice seeds were placed in the hole, and the hole was sealed with soil. The row spacing was about 50cm. Third, intercropping was implemented. When the upland rice reached the 5-7 leaf stage, the *Mussaenda pubescens* seedlings were transplanted to plots 1-5, with a row of *Mussaenda pubescens* seedlings transplanted next to each row of upland rice, forming an intercropping pattern.

[0101] (3) Insect population survey statistics

[0102] During the 10-week period from mid-June to the end of September, the experimental fields were surveyed every two weeks to record the insect species and population numbers. The insect population counting method followed the approach described by Wang Wanwan (Wang Wanwan. Research on the Mechanism of Rice Resistance to Rice Planthopper Induced by Chemical Evotrons on Fluorophenoxyacetic Acid and Its Field Application Technology [D]. Hangzhou: Zhejiang University, 2019.). Parallel skip sampling was used in each experimental plot, with 10 clumps of upland rice sampled. The insect population was counted using a tray-tapping method, focusing on the number of rice pests and natural enemies. During the survey, a white rectangular flat-bottomed plastic tray (22cm long, 16cm wide, and 3cm high) soaked in oil was used. The base of the rice clumps was tapped from above the tray, and the species and number of insects in the tray were recorded and counted.

[0103] Experimental results:

[0104] See Figure 5As shown, in the experimental plots of intercropping *Muscari* and upland rice, the number of pests decreased, but the number of their natural enemies increased significantly. In the field experiment of intercropping *Muscari* and upland rice, a total of 23 insect species were investigated and counted, including: planthopper, rice leaf roller, black leaf beetle, brown planthopper, four-spotted leaf beetle, black-shouldered green mirid bug, ant, red leaf beetle, braconid wasp, tassel wasp, bridge-building insect, gray leaf beetle, spider, snail, small round leaf beetle, mantis, orange leaf beetle, plump round leaf beetle, aphid, two-spotted stink bug, leaf silkworm, cricket, and pygmy stink bug. Among them, the most numerous crop pests were rice planthopper, rice leaf roller, and leaf beetle. Statistical analysis of the number of major pests and beneficial insects in the experimental field revealed a significant change in the number of insects in a single rice clump in the intercropping plot of upland rice and *Muscari* compared to the experimental plot where only upland rice was sown. Among them, the numbers of drought-resistant rice pests such as brown planthopper (Nilaparvata lugens), gray planthopper (Laodelphax striatellus), rice leaf roller (Cnaphalocrocis medinalis), and brown-footed leaf beetle (Basilepta fulvipes) decreased significantly. Figure 5 (Figures a-d in the text); however, the numbers of natural enemies of pests, such as the black-shouldered green mirid bug (Cyrtorhinus lividipennis) and spiders, have increased significantly. Figure 5 (See Figures e-f in the original text). It can be seen that the volatiles of Mengla musk have the effect of repelling pests of dry rice or attracting natural enemies of pests.

[0105] 3.4 Selectivity Experiment of Brown Planthopper Feeding

[0106] Two healthy rice plants of similar growth were selected. A solution of volatile compounds from *Muscone mongholicus* was placed at the base of the stem in one pot, while the other pot contained the same volume of hexane solution as a control. The plants were covered with an "H"-shaped transparent plastic tube, with a circular hole left in the middle of the horizontal connecting tube. Twenty brown planthopper larvae were introduced through this hole, and the tube was then sealed with a sponge. The top of the vertical tube of the "H" was sealed with mesh to prevent the planthoppers from escaping. The number of planthoppers on both plants was observed and recorded at 0.5h, 1h, 2h, 4h, 8h, and 24h after inoculation. This experiment was conducted on sunny days from 8:00 AM to 8:00 PM. Valid selection was defined as the brown planthoppers moving from the horizontal tube of the "H" to the vertical tubes at both ends. Seven replicates were set up for each experiment, and the entire experiment was repeated three times.

[0107] Experimental results: such as Figure 6As shown, the volatiles of *Muscari mongholicus* significantly affect the feeding selection behavior of brown planthoppers. During the observation period from 1 hour to 24 hours post-inoculation, the number of brown planthoppers in the treatment group was significantly lower than that in the control group. Furthermore, the difference in selection gradually increased with observation time. Significant differences in selection were observed at 1 hour, 2 hours, and 4 hours post-inoculation, with the number of brown planthoppers in the treatment group decreasing by 36.94%, 31.85%, and 42.47% compared to the control group, respectively. The largest difference was observed at 24 hours, with the number of brown planthoppers in the treatment group decreasing by 53.08% compared to the control group.

[0108] 3.5 Selectivity Experiment of Oviposition by Female Adult Brown Planthoppers

[0109] The experimental setup and method for the selective oviposition experiment of adult brown planthoppers were the same as those for the selective feeding experiment of brown planthoppers. The test insects were replaced with oviparous adult brown planthoppers. The number of female planthoppers on the two potted plants was observed and recorded at 0.5h, 1h, 2h, 3h, 5h, 8h, and 12h after inoculation. Each experiment was repeated in 5 replicates, and the entire experiment was repeated 3 times.

[0110] Experimental results:

[0111] like Figure 7 As shown, the volatiles of *Muscari mongholicus* significantly repel the oviposition site selection behavior of female adult brown planthoppers. At 1h, 2h, 3h, 5h, 8h, and 12h after inoculation, the number of brown planthoppers in the selective treatment group was reduced by 46.58%, 44.01%, 44.76%, 53.44%, 48.55%, and 40.65% compared with the control group, respectively.

[0112] The volatiles of *Muscari mongholicus* significantly influenced the oviposition site selection behavior of female brown planthoppers. In the upland rice group treated with *Muscari mongholicus* volatiles, the number of egg-bearing female brown planthoppers was significantly lower than in the control group. This suggests that *Muscari mongholicus* volatiles may reduce the reproductive rate of brown planthoppers by influencing their oviposition site selection, thereby helping upland rice resist planthopper damage.

[0113] 3.6 Response of upland rice to simulated insect feeding under the co-existence of *Muscari mollissima* in Mengla

[0114] Take several healthy *Mussaenda pubescens* plants and several dryland rice plants, and plant them in long plastic flower pots (50×19×15cm in length, width, and height), dividing them into three groups for planting.

[0115] Group A: Plant 5 saplings of Mengla moss and 4 saplings of Mengla moss together in the same flowerpot.

[0116] Group B: Muscone and dryland rice were planted in different flower pots, with 5 dryland rice plants or 4 Muscone plants planted in each pot.

[0117] Group C: Only 5 dryland rice plants were planted in each pot. The three groups of plants were isolated and maintained in a greenhouse under the same conditions, with regular watering.

[0118] After 21 days of cultivation, on a sunny morning, the second complete leaf at the top of the upland rice plant in the three plant combinations was treated in a manner simulating insect feeding. The treatment method was mechanical damage plus application of insect oral secretions (two even lines were made on both sides of the leaf vein with a rolling pin to create mechanical damage, and then the pre-prepared bollworm oral secretions were evenly applied to the leaf). The leaves of the upland rice plants in each group that did not receive any treatment served as blank controls. 1.5 hours after treatment, the whole rice leaf was removed and quickly frozen in liquid nitrogen for subsequent determination of the endogenous hormone content of the upland rice leaf.

[0119] Experimental results:

[0120] An experiment conducted under greenhouse conditions, observing the response of upland rice to simulated insect pests while growing *Muscari mongholicus* in a co-op setting, revealed that under this condition, whether the rice was placed solely within a shared space or planted in the same pot, the levels of insect-resistant stress hormones in the rice leaves significantly increased. The results are as follows: Figure 8 As shown, under the conditions of coexistence with *Muscari* in Mengla, simulating insect feeding can significantly upregulate the endogenous insect-resistant hormone jasmonic acid-isoleucine (JA-Ile) in rice.

[0121] Regardless of the mode of co-occurrence of *Muscari* var. *menglabra*, upland rice leaves showed a significant response to simulated pests, manifested as a significant increase in the endogenous insect-resistant stress hormone JA-Ile. Compared with the control group, the JA-Ile content in the upland rice leaves increased by 39.04% in the *Muscari* var. *menglabra* and upland rice intercropping group, and by 55.37% in the upland rice leaves.

[0122] The above results indicate that regardless of whether there is root contact between *Muscaria menglaensis* and upland rice, it enhances the response of upland rice's own insect-resistant stress hormone JA-Ile to simulated insect pests. This suggests that *Muscaria menglaensis* can induce nearby plants to regulate endogenous hormones and produce an insect-resistant response simply by releasing volatile substances into the environment.

[0123] The experiment in Example 2 shows that:

[0124] (1) The terpenoid volatiles of Mengla moschata can resist insect pests; the diverse volatile compounds in Mengla moschata play a synergistic role, which can not only affect the feeding and oviposition behavior of pests, but also affect the electrophysiological response of insect antennae.

[0125] (2) The presence of Mengla moss can effectively help intercropped crops resist pests. Mengla moss plays an ecological role in helping crops resist pests by repelling crop pests, attracting natural enemies of pests, and inducing the crop's own insect resistance response.

[0126] In summary, this invention proposes a novel use for the volatiles of *Muscone mongholicus*, namely, that these volatiles can be used as or in the preparation of insecticides to control agricultural pests. Since *Muscone mongholicus* volatiles are of natural origin, this provides a new approach for future green agricultural pest control. Furthermore, this invention provides a method for controlling agricultural pests, primarily by intercropping crops with *Muscone mongholicus* to control agricultural pests. This method can control agricultural pests while also protecting human health and the environment.

[0127] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention shall still fall within the scope of the technical solution of the present invention.

Claims

1. The use of a volatile compound from *Adenosma bracteosum* Bonati, characterized in that... The volatiles of Mengla Mao Musk are used as or in the preparation of agricultural pest repellents; the agricultural pest repellents are used to control omnivorous lepidopteran pests and specialized pests; wherein the omnivorous lepidopteran pests are cotton bollworms and armyworms; and the specialized pests are brown planthoppers and gray planthoppers. The preparation method of the volatile compounds of Mengla musk includes the following steps: Collection and adsorption steps: The above-ground parts or tissue parts of the *Muscari mongholica* plant are covered in a dynamic headspace adsorption device; the dynamic headspace adsorption device is connected to an inlet pipe and an outlet pipe; an adsorption column is installed on the outlet pipe; air enters the dynamic headspace adsorption device through the inlet pipe and flows out through the outlet pipe, and the adsorption column collects the volatiles of *Muscari mongholica*. Elution steps: Remove the adsorption column and elute with solvent to obtain the volatiles of Mengla musk.

2. The use of the volatiles of *Adenosma bracteosum* Bonati according to claim 1, characterized in that, The volatile compounds of the Mengla musk include p-cymene, γ-terpinene, carvacrol methyl ether, carvacrol, α-thujone, α-pinene, myrcene, α-phellandrene, 3-carene, limonene, α-terpinene, α-terpineol, thymol, trans-caryophyllene, and humulene.

3. The use of the volatiles of *Adenosma bracteosum* Bonati according to claim 2, characterized in that, The content of p-cymene is 290-12000 ng / g.FW; the content of γ-terpinene is 2500-66500 ng / g.FW; the content of carvacrol methyl ether is 100-12500 ng / g.FW; the content of carvacrol is 250-32000 ng / g.FW; the content of α-thujone is 100-2200 ng / g.FW; the content of α-pinene is 5-280 ng / g.FW; myrcene The content of α-phellandrene is 10-140 ng / g.FW; the content of α-phellandrene is 5-170 ng / g.FW; the content of 3-carene is 70-2700 ng / g.FW; the content of limonene is 100-3000 ng / g.FW; the content of α-terpineol is 5-260 ng / g.FW; the content of thymol is 10-620 ng / g.FW; the content of trans-caryophyllene is 3.0-240 ng / g; the content of humulene is 20-1100 ng / g.FW; and the content of α-terpinene is 90-4750 ng / g.FW.

4. The use of the volatiles of *Adenosma bracteosum* Bonati according to claim 1, characterized in that, The tissue parts of the Mengla musk plant are any one of the following: root, stem, fresh leaves, dried leaves, inflorescence, and fruit inflorescence.

5. A method for controlling agricultural pests, characterized in that, The volatiles of Mengla musk as described in any one of claims 1-4 are sprayed onto crops for the control of agricultural pests.

6. The method for controlling agricultural pests according to claim 5, characterized in that, In the early stages of crop pest infestation, the volatile components of Mengla musk are sprayed onto the crops.

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