Product against tea tippy and its application

Abstract

The present application provides cis-jasmone and / or a pinocamphone-type mint (Mentha suaveolens Ehrhart ) In the preparation of anti-Pestalotiopsis theae product. The present application found that pinocamphone-type mint has the strongest antibacterial effect relative to the rest of the species of mint, significantly better than isomenthone-type mint, and extremely significantly better than other chemical types and thiophanate-methyl.

Description

Technical Field

[0001] This invention relates to the field of green pest control technology, and in particular to a product for treating tea leaf spot disease and its application. Background Technology

[0002] Currently, chemical control is still the primary method for controlling tea leaf spot disease in my country. This mainly involves synthetic fungicides such as 25% pyraclostrobin, 70% thiophanate-methyl, and triazoles. Pyraclostrobin is a highly effective mitochondrial respiration inhibitor. Although it is generally believed that short-term intake poses low health risks to the general population, data suggests that its true toxicity to mammals is underestimated. Toxicity tests on C57BL / 6 mice using an oil-based carrier at standard concentrations showed various adverse health outcomes. Thiophanate-methyl has been proven toxic to silkworms, bees, and aquatic animals, and also poses a potential threat to the ecological environment. Systemic triazole fungicides pose potential hazards to the soil environment and have been identified with various toxicological effects, including high plasma protein binding rates, inhibition of cytochrome and hepatotoxicity, neurotoxicity, and mutagenicity. They are also toxic to aquatic plants, fish, and mammals. Besides common environmental and human health problems, synthetic fungicides can easily lead to drug resistance in pathogens, necessitating increasingly higher dosages each year.

[0003] To address the aforementioned issues, plant volatile oils are increasingly attracting attention as alternatives to synthetic fungicides. The volatile oils of various *Mentha* species, including spearmint (*Mentha spicata* L.), field peppermint (*M. arvensis* L.), peppermint (*M. × piperita* L.), and European peppermint (*M. longifolia* (L.) Huds., have been shown to have good inhibitory activity against plant pathogenic fungi. Although there are no reports of their application in the treatment of tea leaf spot disease, the use of plant extracts for the green control of tea leaf spot disease is gradually gaining attention. Summary of the Invention

[0004] In view of this, the technical problem to be solved by the present invention is to provide a product and / or intercropping mode that resists tea leaf spot disease. The nepeta lactone type of peppermint of the present invention has a good effect on tea leaf spot disease.

[0005] This invention provides the use of cis-jasmone and / or nepeta lactone-type peppermint in the preparation of products resistant to *Pseudomonas aeruginosa*.

[0006] This invention also provides the use of cis-jasmone and / or nepeta lactone-type peppermint in the preparation of products for treating tea leaf spot disease.

[0007] The present invention also provides products resistant to *Pseudomonas aeruginosa*, comprising cis-jasmone and / or nepeta lactone-type peppermint.

[0008] In some specific embodiments, the nepetalactone-type peppermint includes the whole plant of nepetalactone-type peppermint, the tender branches of nepetalactone-type peppermint, or the volatile oil of nepetalactone-type peppermint.

[0009] In some specific embodiments, the volatile oil of the nepeta lactone type peppermint is a volatile oil containing caryophyllene oxide, calamusene, piperone, α-pinene, caryophyllene, menthol, and cis-jasmone.

[0010] In some specific embodiments, the method for preparing the volatile oil includes: Select plants that have been cultivated for more than 2 years, and use the five-point sampling method to collect clean, dew-free, and irrigation-free tender shoots within 15-20 cm from the top. Cut them into small sections of 2-3 cm in length, add water and reflux to extract, keep it at a gentle boil for 4-5 hours, stop heating for 0.5-1 hours, collect the upper volatile oil, seal and store it for later use.

[0011] The present invention provides a composition for green control of tea leaf spot disease, comprising caryophyllene oxide and calamusene.

[0012] In some specific embodiments, the mass ratio of caryophyllene oxide to calendulae is (1~100):(100~1).

[0013] The present invention provides a method for treating *Pseudomonas aeruginosa*, which includes administering the product described in any of the above-mentioned technical solutions, or administering the composition described in the above-mentioned technical solutions.

[0014] This invention provides a method for combating tea leaf spot disease, which includes intercropping tea plants with nepeta lactone-type peppermint.

[0015] Compared with the prior art, the present invention provides cis-jasmone and / or nepetalone type peppermint. (Mentha suaveolens Ehrhart ) Application in the preparation of products resistant to *Pseudomonas aeruginosa*. This invention found that nepeta lactone-type peppermint has the strongest antibacterial effect compared to other peppermint varieties, significantly better than isomenthone-type peppermint, and extremely significantly better than nine aromatic plants including marigold, rosemary, wormwood, Australian tea tree, lemongrass, and clove basil. It is also extremely significantly better than other chemical types and thiophanate-methyl. Attached Figure Description

[0016] Figure 1 Non-contact testing of the in vitro inhibitory activity of 10 plant volatiles against *Pseudomonas aeruginosa*. Figure 2 Chromatograms of volatile oils from five peppermint species; Figure 3 Differences in the in vitro inhibitory activity of different chemical types of peppermint oil against *Pseudomonas aeruginosa*; Figure 4 The difference lies in the antibacterial activity of peppermint essential oil at gradient concentrations; Figure 5 OPLS analysis of antibacterial active ingredients in peppermint oil; where A is the coefficient plot, B is the s-plot, and C is the VIP plot. Figure 6 EC 50 The differences in antibacterial activity of different peppermint components at different concentrations compared to nepeta lactone-type peppermint oil. Detailed Implementation

[0017] This invention provides a product for treating tea leaf spot disease and its application. Those skilled in the art can refer to the content of this document and appropriately modify the process parameters to achieve the desired result. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art and fall within the scope of protection of this invention. The methods and applications of this invention have been described through preferred embodiments. Those skilled in the art can clearly modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit, and scope of this invention to realize and apply the technology of this invention.

[0018] The main components of the volatile oil of the same plant can vary due to differences in origin, variety, and place of origin. The volatile oil of the plant can be classified into different chemical types according to the different main components. The antibacterial activity of the volatile oil of different chemical types of the same plant can also vary significantly.

[0019] Peppermint volatile oil is known to exist in several chemical forms, including L-menthone, carvone, and pulegone, and their inhibitory activities against tea leaf spot fungus are predicted to vary.

[0020] The inventors extracted volatile oils from five peppermint species produced in Zhangzhou using steam distillation. The tea leaf spot pathogen, isolated from diseased leaves, was used as the test strain. Gas chromatography-mass spectrometry (GC-MS) was used to identify the chemical types of the different peppermint volatile oils. The differences in in vitro antibacterial activity of different chemical types of peppermint oil against the pathogen were evaluated, and the core antibacterial components were identified based on OPLS analysis.

[0021] This invention provides the use of cis-jasmone and / or nepeta lactone-type peppermint in the preparation of products resistant to *Pseudomonas aeruginosa*.

[0022] The nepeta lactone type of peppermint of this invention includes, but is not limited to, apple mint. (Mentha suaveolens Ehrhart )。

[0023] The products containing *Pseudomonas aeruginosa* can take various forms, including fungicides and plant protectants. In practical applications, extracts of nepeta lactone-type peppermint can be used as active ingredients and prepared into emulsifiable concentrates, wettable powders, and suspensions using conventional formulation processes for easy application in tea gardens. For example, the above-ground parts (including stems and leaves) of nepeta lactone-type peppermint can be extracted through steam distillation and oil-water separation, and then mixed with suitable adjuvants (such as emulsifiers, dispersants, and carriers) to create a fungicide with good dispersibility and stability. Furthermore, cis-jasmone, a known compound, can also be directly combined with other agriculturally acceptable adjuvants to produce corresponding products containing *Pseudomonas aeruginosa*. The content of either nepeta lactone-type peppermint extract or cis-jasmone in the product can be adjusted according to actual needs to achieve the best anti-*Pseudomonas aeruginosa* effect.

[0024] This invention also provides the use of cis-jasmone and / or nepeta lactone-type peppermint in the preparation of products for treating tea leaf spot disease.

[0025] Tea leaf spot is an important tea tree disease caused by *Pestalotiopsis theae*, severely affecting tea yield and quality. This invention, based on the inhibitory effects of cis-jasmone and nepeta lactone-type peppermint on *Pestalotiopsis theae*, applies these inhibitors to the preparation of tea leaf spot-resistant products, providing a new approach for green disease control in tea gardens. These products can take various forms, including fungicides, plant protectants, and foliar fertilizers. In practical applications, nepeta lactone-type peppermint extract can be used as the active ingredient and prepared into emulsifiable concentrates, wettable powders, and suspensions using conventional formulation processes for easy application in tea gardens. For example, the above-ground parts (including stems and leaves) of nepeta lactone-type peppermint can be pulverized, extracted with ethanol, and concentrated to obtain the extract, which can then be mixed with suitable adjuvants (such as emulsifiers, dispersants, and carriers) to create a fungicide with good dispersibility and stability. Furthermore, cis-jasmone, as a known compound, can also be directly combined with other agriculturally acceptable excipients to produce corresponding anti-tea leaf spot products. The content of either nepeta lactone-type peppermint extract or cis-jasmone in the product can be adjusted according to actual needs to achieve the best anti-tea leaf spot effect.

[0026] The present invention also provides products resistant to *Pseudomonas aeruginosa*, comprising cis-jasmone and / or nepeta lactone-type peppermint.

[0027] In some specific embodiments, the nepetalactone-type peppermint includes the whole plant of nepetalactone-type peppermint, the tender branches of nepetalactone-type peppermint, or the volatile oil of nepetalactone-type peppermint.

[0028] When using the whole plant or tender branches of mint with nepeta lactone as raw material, the active ingredients can be obtained through various extraction processes.

[0029] When a product contains cis-jasmone, it can be either naturally extracted or synthetically produced. During formulation, it can be combined with extracts of nepeta lactone-type peppermint or alone with suitable adjuvants, depending on the formulation requirements. When using these different formulations, different application methods, such as spraying or broadcasting, can be selected based on the specific conditions of the tea garden and application habits to ensure that the active ingredients can effectively act on *Pseudomonas aeruginosa* and exert an anti-tea leaf spot effect.

[0030] In some specific embodiments, the volatile oil of the nepeta lactone type peppermint is a volatile oil containing caryophyllene oxide, calamusene, piperone, α-pinene, caryophyllene, menthol, and cis-jasmone.

[0031] In some specific embodiments, the volatile oil of the nepetalactone-type peppermint contains nepetalactone, d-limonene, β-pinene, α-pinene, 1-octen-3-ol, β-myrcene, caryophyllene, γ-terpinene, ethyl salicylate, 1-octen-1-ol acetate, calcein, isoterpinene, β-piperene, pentanol, caryophyllene oxide, humulene, eucalyptol, verbenatone, α-elemenol, cis-jasmone, 1,3,8-p-menthtriene, α-fennelene, eugenol, and β-calceinol.

[0032] In some specific embodiments, the volatile oil of the nepetalactone-type peppermint of the present invention comprises the following components and their mass percentages: nepetalactone 55.08%, d-limonene 16.14%, β-pinene 4.18%, α-pinene 3.54%, 1-octen-3-ol 3.08%, β-myrcene 2.47%, caryophyllene 5.74%, γ-terpinene 1.01%, ethyl salicylate 1.10%, 1-octen-1-ol acetate 1.27%, calomel 0.88%, isoterpinene 0.80%, β-piperene 0.75%, pentanol 0.61%, caryophyllene oxide 0.61%, humulene 0.50%, eucalyptol 0.48%, verbenaconone 0.38%, α-elemenol 0.29%, cis-jasmone 0.28%, 1,3,8 - 0.25% of menthol, 0.20% of α-fenestrate, 0.18% of eugenol, 0.18% of β-acorenool, with the remainder being undetected or trace components.

[0033] In some specific embodiments, the method for preparing the volatile oil includes: Select plants that have been cultivated for more than 2 years, and use the five-point sampling method to collect clean, dew-free, and irrigation-free tender shoots within 15-20 cm from the top. Cut them into small sections of 2-3 cm in length, add water and reflux to extract, keep it at a gentle boil for 4-5 hours, stop heating for 0.5-1 hours, collect the upper volatile oil, seal and store it for later use.

[0034] The present invention provides a composition for green control of tea leaf spot disease, comprising caryophyllene oxide and calamusene.

[0035] This invention creatively discovers that caryophyllene oxide and calamusene exhibit a synergistic effect in the green control of tea leaf spot disease. When used in combination, their inhibitory effect on the pathogen is significantly better than that of either component alone. This synergistic effect may stem from the complementary mechanisms of action of the two components. For example, caryophyllene oxide may primarily disrupt the cell membrane structure of the pathogen, while calamusene may inhibit the activity of key enzymes within the pathogen, thus working together to address multiple physiological processes of the pathogen and achieve better control. This composition can be prepared into various formulations such as emulsifiable concentrates, wettable powders, and suspensions. It can be applied to tea gardens through foliar spraying, soil treatment, or seed coating, effectively reducing the incidence of tea leaf spot disease and decreasing the use of chemical pesticides, thus meeting the requirements of green agricultural development.

[0036] In some specific embodiments, the mass ratio of caryophyllene oxide to calendulae is (1~100):(100~1).

[0037] The present invention provides a method for treating *Pseudomonas aeruginosa*, which includes administering the product described in any of the above-mentioned technical solutions, or administering the composition described in the above-mentioned technical solutions.

[0038] In practical applications, the appropriate application method and dosage can be selected based on the actual conditions of the tea garden and the severity of tea leaf spot disease. For example, when using foliar spraying, a composition containing effective concentrations of caryophyllene oxide and calamusene (such as emulsifiable concentrate or suspension) can be diluted with water in a certain proportion and sprayed evenly on both sides of the tea leaves, especially on new shoots and tender leaves, to ensure that the agent can fully contact the pathogen. It is generally recommended to start application at the early stage of tea leaf spot disease, spraying once every 7-10 days, for 2-3 consecutive applications, which can effectively control the spread of the disease. For soil treatment, the wettable powder composition can be mixed with an appropriate amount of fine soil or organic fertilizer, and then evenly spread on the soil surface around the roots of the tea trees, and then lightly tilled into the soil. This will inhibit the growth and infection of pathogens in the soil, thereby preventing tea leaf spot disease. Seed coating involves coating the surface of tea seedlings with a compound before sowing or transplanting, forming a protective film that provides continuous protection during seed germination and seedling growth, reducing early infection by pathogens. Furthermore, the dosage needs to be adjusted according to the selected formulation, the tea plant's growth stage, and the severity of the disease. Generally, the total effective ingredient content of caryophyllene oxide and calamusene in the applied agent can be controlled between 0.5% and 5% to ensure good control efficacy while avoiding phytotoxicity to the tea plants or environmental stress.

[0039] This invention provides a method for combating tea leaf spot disease, which includes intercropping tea plants with nepeta lactone-type peppermint.

[0040] Specifically, in tea garden planning, tea trees and nepeta lactone-type mint can be intercropped at a certain row ratio. Utilizing the traditional shrub-herb intercropping pattern, mint is planted around the root collar of the tea trees. 4-8 nepeta lactone-type mints are intercropped per tea tree, with a spacing of 5-20 cm between plants, ensuring root mulch coverage. This ensures good growth and maximizes their effectiveness. During the tea tree's growth period, nepeta lactone-type mint, through the secretion of specific allelopathic substances from its roots and the release of volatile components from its above-ground parts, can effectively inhibit the growth and reproduction of tea leaf spot pathogens, reducing the number of pathogens in the tea garden environment. Simultaneously, the planting of nepeta lactone-type mint can improve the microecological environment of the tea garden, increase biodiversity, attract beneficial insects, and further enhance the tea garden's natural disease control capabilities. Furthermore, the intercropped nepeta lactone-type mint can be harvested at appropriate times, and the harvested plants can be distilled to extract volatile oils for foliar spraying, achieving the goal of reducing fertilizer application and increasing efficiency in the tea garden. During intercropping, tea garden operations can be combined with routine management of nepeta lactone-type peppermint, such as timely watering and fertilization, to ensure its healthy growth and fully leverage its synergistic disease resistance with tea trees, thereby effectively reducing the incidence and severity of tea leaf spot disease.

[0041] Different chemical types of peppermint oil exhibit highly significant differences in their components, and their inhibitory activities against the same plant pathogenic fungi also show significant to highly significant differences. Among them, nepeta lactone-type peppermint oil demonstrates a particularly outstanding antibacterial effect, with an inhibition rate significantly superior to thiophanate-methyl. It is generally believed that the antibacterial activity of volatile oils originates from their most abundant components, while other trace components receive little attention. This study, based on OPLS analysis, is the first to identify cis-jasmone as the main inhibitory component against the target pathogen in nepeta lactone-type peppermint oil. This discovery provides a preliminary foundation for the application of cis-jasmone in the field of plant-derived antibacterial agents.

[0042] It should be understood that the expression “one or more of…” individually includes each of the objects described after the expression, as well as various different combinations of two or more of the described objects, unless otherwise understood from the context and usage. The expression “and / or” combined with three or more described objects should be understood to have the same meaning, unless otherwise understood from the context.

[0043] The terms “including,” “having,” or “containing,” including the use of their grammatical synonyms, should generally be understood as open-ended and non-restrictive, for example, not excluding other unstated elements or steps, unless otherwise specifically stated or understood from the context.

[0044] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural.

[0045] In this application, "at least one" means one or more, and "more than one" means two or more. "At least one of the following" or similar expressions refer to any combination of these items, including any combination of single or multiple items.

[0046] It should be understood that the order of the steps or the order in which certain actions are performed is not important as long as the invention remains operational. Furthermore, two or more steps or actions can be performed simultaneously.

[0047] The use of any and all instances or exemplary language such as “e.g.” or “including” in this document is merely intended to better illustrate the invention and is not intended to limit the scope of the invention unless the claims are made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0048] Furthermore, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as precisely as possible. However, any value inevitably contains standard deviations due to individual test methods. Therefore, unless explicitly stated otherwise, it should be understood that all ranges, quantities, values, and percentages used in this disclosure are modified with the word "approximately". Here, "approximately" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific value or range.

[0049] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0050] The embodiments and comparative examples of this invention describe some examples, in which the embodiments illustrate certain implementations of the invention. However, this does not mean that the effects of the invention can only be achieved in these examples.

[0051] To further illustrate the present invention, the following describes in detail, with reference to embodiments, a product for treating tea leaf spot disease and its application.

[0052] Example 1

[0053] 1. Materials and Methods

[0054] 1.1 Test Materials

[0055] 1.1.1 Plant Materials Water mint was introduced from Suining, Sichuan; apple mint, banana mint, Japanese mint, and grapefruit mint were introduced from Ningbo, Zhejiang. All were conserved at the Aromatic Plant Resource Nursery of the Subtropical Agriculture Research Institute of Fujian Academy of Agricultural Sciences, and were varieties cultivated in open fields for more than 2 years. They were identified as test varieties by Senior Agronomist Wu Weijian of the research group. Detailed information on the test plants is shown in Table 1.

[0056] Table 1 Information on tested plants

[0057] Table 1 Directory of tested plants

[0058] 1.1.2 The tested pathogen, *Pseudomonas aeruginosa* (GenBank accession number: PX362927), was isolated from tea leaves infected with leaf spot disease and preserved in the Plant Pathology Laboratory of the Subtropical Agriculture Research Institute, Fujian Academy of Agricultural Sciences. ITS full-sequence analysis and homology comparison of the sequencing results in the NCBI database confirmed it as the tested species. Following Koch's rule, the bacterial suspension was evenly sprayed onto the surface of tea leaves, with sterile water as a blank control, confirming the significant pathogenicity of the tested pathogen.

[0059] 1.1.3 Test culture medium: Potato glucose agar medium (PDA): 24 g potato glucose water, 20 g agar powder, and deionized water to a final volume of 1000 mL.

[0060] 1.1.4 Experimental Instruments and Chemical Reagents: Volatile oil analyzer (density < 1.0), spherical condenser, round-bottom flasks and other glassware, Zhangzhou Tengxin Chemical Glassware Instrument Co., Ltd.; XB. K. 25 hemocytometer, Shanghai Qiujing Biochemical Reagent Instrument Co., Ltd.; Heating mantle, Gongyi Yuhua Instrument Co., Ltd.; Basic-Q15-IT pure water system, Shanghai Hetai Instrument Co., Ltd.; GF54DA vertical automatic pressure steam sterilizer, Zhiwei (Xiamen) Instrument Co., Ltd.; MJ-250 constant temperature light incubator, Shanghai Yiheng Scientific Instrument Co., Ltd.; SW-CJ-1F clean bench, Suzhou Antai Air Technology Co., Ltd. (Suzhou Jingjing Group); Trace 1300 / TSQ9000 gas chromatograph-mass spectrometer, Thermo Fisher Scientific, USA; Potato glucose solution, technical agar powder, Guangdong Huankai Biotechnology Co., Ltd.; Thiophanate-methyl (≥98.0%), analytical standard, Sigma-Aldrich (Shanghai). Trading company; Dimethyl sulfoxide (≥99.5%), analytical grade, Xilong Scientific Co., Ltd.; Menthol ( dl- menthol) 99%, Shandong Keyuan Biochemical Co., Ltd.; α -pinene (α Caryophyllene oxide (98%), caryophyllene oxide (95%), Beijing Mairuida Technology Co., Ltd.

[0061] 1.2 Methods

[0062] 1.2.1 Extraction and Determination of Volatile Oil from Peppermint Oil

[32] The volatile oil content of different peppermint varieties was extracted and determined with slight modifications. For the test samples, plants cultivated for more than 2 years were uniformly selected. On sunny mornings for at least 3 consecutive days, at 11:00 AM, clean, dew-free, and unwatered tender shoots within 20 cm of the tip were collected using a five-point sampling method. These shoots were cut into small sections approximately 3 cm long and placed in 1000 mL hard-bottom round-bottom flasks, 200 g per flask. 200 mL of water was added, and the mixture was shaken and connected to the volatile oil analyzer and reflux condenser. Distilled water was added from the top of the condenser until it filled the graduated section of the volatile oil analyzer and overflowed into the flask. The flask was placed in a heating mantle and slowly heated until the liquid surface in the flask reached a gentle boil. This was maintained for 5 hours at a low boil. After stopping heating for 1 hour, the upper layer of volatile oil was collected and sealed in a brown reagent bottle at below 26 °C for later use.

[0063] 1.2.2 Determination of volatile oil chemical components, peppermint oil content, by gas chromatography-mass spectrometry (GC-MS). The gas chromatography method was slightly modified to use headspace sampling and GC-MS component analysis.

[0064] Gas chromatography conditions: Thermo TG-5SilMS GC polar column (30 m length, 0.25 mm inner diameter, 0.25 μm film thickness); injection port temperature: 250 ℃; detector temperature: 250 ℃; injection volume: 1.0 μL, split ratio 1:100; carrier gas: nitrogen. Temperature program: initial temperature 60 ℃, hold for 4 min, increase to 100 ℃ at a rate of 2 ℃ / min, then increase to 230 ℃ at a rate of 10 ℃ / min, hold for 1 min.

[0065] Mass spectrometry conditions: EI ionization mode; EI ion source temperature: 210 ℃; transfer line temperature: 250 ℃; electron multiplication voltage: 1847 V; ionization voltage: 70 eV; ion source temperature: 300 ℃; scan mass range (m / z): 30-550. Chemical components were determined through library retrieval and data analysis, combined with manual spectral interpretation from literature. The relative content of each component was calculated using peak area normalization based on the average peak area of ​​each chromatographic peak according to the total ion current, with two significant figures retained.

[0066] 1.2.3 Screening of antibacterial activity of volatile oils, EC50 and MIC determination of mycelial growth inhibition method The antibacterial activity of five peppermint volatile oils was determined, with slight modifications, and divided into two stages.

[0067] Phase 1: Under aseptic conditions: ① Add 10.0 μL of the test volatile oil to each Erlenmeyer flask, and bring the volume to 50 mL with sterile, thawed PDA medium to prepare a 0.20 ml / L drug-containing medium. Pour the medium into five 90 mm diameter sterile petri dishes to prepare drug-containing plates of the corresponding concentration. ② Add 300 μL of thiophanate-methyl stock solution to each Erlenmeyer flask, and bring the volume to 50 mL with sterile, thawed PDA medium to prepare a positive control (+1) containing 0.20 ml / L thiophanate-methyl. ③ Set PDA medium without any reagents as a blank control. ④ Set PDA medium with 300 μL of dimethyl sulfoxide (DMSO) added to 50 mL as a carrier control.

[0068] Phase 2: Based on the experimental results of Phase 1, a one-way ANOVA was performed on the experimental data to screen out the volatile oil chemical types with significantly superior antibacterial activity compared to the positive control. Using the method from Phase 1, five concentration gradients (0.12 ml / L, 0.16 ml / L, 0.20 ml / L, 0.24 ml / L, and 0.28 ml / L) were designed, and a virulence regression equation was established based on the antibacterial rate. y = a x + b; Using SPSS regression probability, predict the 50% effective concentration (EC50) of Fusarium oxysporum. 50 ) and 98% effective concentration (EC) 98 PDA medium without volatile oil was set as a blank control (CK). The effect concentration was verified by the concentration predicted by the software and the minimum complete inhibitory concentration (MIC) was determined.

[0069] Preparation of mycelium cake: Under aseptic conditions, take 200 μL of a 10... 6 ~10 7 The bacterial suspension of CFU / mL was evenly spread on PDA plates and incubated at 28℃ for 15 days. The bacterial cakes were then punched out with a 4 mm diameter punch and transferred to the center of each of the above-mentioned drug-containing and blank PDA culture dishes. The plates were placed in a constant temperature incubator at 28℃ and incubated upright in the dark for 24 h, and then inverted in the dark for 13 days.

[0070] Measurement and Data Analysis: When the hyphae of the control group approached the edge of the culture dish, the colony diameter (mm) was measured using the cross-sectional method. This was repeated 5 times, and the average value was taken. The inhibition rate was calculated as follows: Inhibition rate (%) = (Coronation diameter of blank control - Coronation diameter of treated colonies) / Coronation diameter of blank control × 100

[36] .

[0071] Thiophanate stock solution: Dissolve 200 mg of thiophanate-methyl in 10 ml of DMSO to prepare a thiophanate-methyl stock solution with an effective mass concentration of 20 ml / L.

[0072] 1.2.4 The method for identifying the main contributing components was also modified from the method in 1.2.3, namely the mycelial growth inhibition method, to determine the antibacterial activity of the main contributing volatile components.

[0073] Based on the GC-MS component analysis results and antibacterial activity screening results of five peppermint oils, orthogonal partial least squares discriminant analysis (OPLS) was performed on all components of the tested oils using Simca 14.1 to screen five predicted core antibacterial components. Under aseptic conditions, the solid components were first dissolved in DMSO to prepare stock solutions, and then dissolved in a solution containing EC... 50 The PDA of the best antibacterial volatile oil was used as a positive control (+2). PDA containing the same concentration of the test component was used as the treatment, and PDA containing the same concentration of DMSO was used as the carrier control. PDA culture medium without any reagents was used as a blank control (CK). The antibacterial activity of the test component was tested.

[0074] 1.3 Statistical Analysis

[0075] The mean inhibition rate and standard deviation were calculated using Microsoft Excel, OPLS analysis was performed using Simca 14.1, and one-way ANOVA, linear regression equations, and effect concentration regression probability statistics were performed using SPSS V13.0 software.

[0076] 2 Results and Analysis

[0077] 2.1 Comparison of the chemical types of volatile oils from different peppermint varieties

[0078] GC-MS analysis revealed significant differences in the chromatograms of the five peppermint volatile oils. Figure 1 Based on principal component analysis, the tested peppermint oils can be classified into five chemical types: nepetalactone, carvone, isomenthone, menthone, and linalool.

[0079] As shown in Table 2, the volatile components of the five peppermint oils differ greatly, with only a few being common to all of them. β- Myrcene β -myrcene、 d -Limonene d -limonene, eucalyptol, and caryophyllene.

[0080] Figure 2 Chromatograms of volatile oils from five peppermint species

[0081] Table 2 Chemical composition analysis of five peppermint volatile oils

[0082] Table 2 Chemical constituents of essential oils from 5 Menthas

[0083] Note: "—" indicates not detected.

[0084] Notes: “—” means not detected.

[0085] 2.2 Screening of antibacterial activity of peppermint volatile oils of different chemical types

[0086] From Table 3 and Figure 2 It was found that at a volume concentration of 0.20 ml / L, different chemical types of peppermint oil all exhibited in vitro inhibitory activity against *Pseudomonas aeruginosa*, with three peppermint oils showing significantly better antibacterial activity than thiophanate-methyl. p ≤ 0.05). The strongest inhibitory activity was observed in nepeta lactone-type peppermint (28.63 ± 2.65%), which was significantly better than isomenthone-type peppermint (25.49 ± 0.55%), and extremely significantly better than other chemical types and thiophanate-methyl. The weakest antibacterial activity was observed in linalool-type peppermint, which showed an in vitro inhibition rate of only 6.27% against *Pseudomonas aeruginosa* at a volume concentration of 0.20 ml / L, which was not significantly different from thiophanate-methyl.

[0087]

[0088] Figure 3 Different chemical types of peppermint oil showed differences in their in vitro inhibitory activity against *Pseudomonas aeruginosa*.

[0089] 2.3 In vitro EC50 assay of nepeta lactone-type peppermint volatile oil against *Pseudomonas aeruginosa* 50 and MIC

[0090] The results of the gradient concentration test are shown in Table 4 and Figure 4 , Figure 4 The difference in antibacterial activity was observed across gradient concentrations of peppermint essential oil. Based on the results in Table 4, SPSS software was used to predict the EC50 of carvone-type peppermint oil. 50 and EC 99 The concentration was determined and validated using the mycelial inhibition method, and the results are shown in Table 5. The R-squared value was close to 1.0, and the P-value was ≥0.05, proving that the equation fit was accurate and the data were reliable. Prediction of EC50 of target bacteria by nepeta lactone-type peppermint oil. 50 The concentration was 0.26 ml / L, predicting EC 98 The concentration was 0.42 ml / L. In vitro experiments confirmed that the MIC of nepeta lactone-type peppermint oil against *Pseudomonas aeruginosa* was 0.55 ml / L.

[0091]

[0092] 2.4 Mining of Main Contributing Components

[0093] according to Figure 3 As can be seen from the VIP plot, components with a VIP value > 1 are generally considered important variable components, and the prediction of caryophyllene oxide in peppermint oil is based on this. cis-β- Twenty-four components, including farnesene, cis-jasmone, linalyl butyrate, piperone, and piperone oxide, are important variables related to the antibacterial rate.

[0094] The coefficient plot shows the correlation between components and antibacterial rate; positive values ​​indicate a positive correlation, and negative values ​​indicate a negative correlation. Based on the key variable components in the VIP plot, it is predicted that 15 components, including caryophyllene oxide, piperonone, cis-jasmone, and caryophyllene, are positively correlated with the antibacterial rate.

[0095] The first quadrant of the S-plots shows the predicted components positively correlated with antibacterial activity; the further away from the origin, the greater the contribution and the stronger the correlation. Combining the two plots above, the main components in peppermint oil with inhibitory activity against *Pseudomonas aeruginosa* are predicted to be caryophyllene oxide, piperonone, etc. α- Pinene, caryophyllene, or cis-jasmone.

[0096] Figure 5 OPLS analysis chart of antibacterial active ingredients in peppermint oil

[0097] 2.5 The main contributing components are clearly defined.

[0098] Table 6 shows the differences in antibacterial activity between the five tested components and nepeta lactone-type peppermint oil at a volume concentration of 0.26 ml / L. Cis-jasmone showed the highest antibacterial rate, completely inhibiting the mycelial growth of the tested strains, significantly superior to other components and the positive control. At this concentration, α-pinene and caryophyllene showed no inhibitory activity against *Pseudomonas aeruginosa*.

[0099]

[0100] Figure 6 EC 50 Differences in antibacterial activity of different peppermint components at different concentrations compared to nepeta lactone-type peppermint oil

[0101] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Application of cis-jasmone and / or nepeta lactone-type peppermint in the preparation of products resistant to *Pseudomonas aeruginosa*.

2. Application of cis-jasmone and / or nepeta lactone-type peppermint in the preparation of products for treating tea leaf spot disease.

3. Products resistant to *Pseudomonas aeruginosa*, including cis-jasmone and / or nepetalactone-type peppermint.

4. The product according to claim 3, characterized in that, The nepetalactone-type peppermint includes the whole plant of nepetalactone-type peppermint, tender branches of nepetalactone-type peppermint, or volatile secondary metabolites of nepetalactone-type peppermint.

5. The product according to claim 3 or 4, characterized in that, The volatile oil of the nepeta lactone type peppermint contains caryophyllene oxide, calamusene, piperone, α-pinene, caryophyllene, menthol, and cis-jasmone.

6. The product according to claim 3 or 4, characterized in that, The method for preparing the volatile oil includes: Select plants that have been cultivated for more than 2 years, and use the five-point sampling method to collect clean, dew-free, and irrigation-free tender shoots within 15-20 cm from the top. Cut them into small sections of 2-3 cm in length, add water and reflux to extract, keep it at a gentle boil for 4-5 hours, stop heating for 0.5-1 hours, collect the upper volatile oil, seal and store it for later use.

7. A composition for green control of tea leaf spot disease, characterized in that, Including caryophyllene oxide and calamusene.

8. The composition according to claim 7, characterized in that, The mass ratio of caryophyllene oxide to calendulene is (1~100):(100~1).

9. A method for treating *Pseudomonas aeruginosa*, comprising administering the product of any one of claims 3 to 6, or the composition of claim 7 or 8.

10. A method for resisting tea leaf spot disease, comprising intercropping tea plants with nepeta lactone-type peppermint.

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