Application of spearmint oil and spearmint oil-containing compositions in the control of citrus Huanglongbing (HLB)

Abstract

This invention discloses the application of spearmint oil and spearmint oil-containing compositions in the control of citrus Huanglongbing (HLB), belonging to the field of agricultural disease control. This invention provides the application of spearmint oil in inhibiting the pathogen of citrus HLB. Addressing the problem of the lack of effective green control methods for citrus HLB, this invention screened a fungicide of the pathogen of citrus HLB from a natural plant-derived active substance. Spearmint oil not only has a significant direct inhibitory and killing effect on the pathogen of citrus HLB, but also induces systemic resistance in citrus plants and enhances tree vigor. The spearmint oil of this invention is derived from natural plants, has clearly defined main components, is biodegradable in the environment, has extremely low residue risk, and meets the requirements of green agriculture and safe agricultural production.

Description

Technical Field

[0001] This invention relates to the field of agricultural disease control, and in particular to the application of spearmint oil and a composition containing spearmint oil in the control of citrus Huanglongbing (HLB). Background Technology

[0002] Citrus Huanglongbing (HLB), caused by Bacillus phloem, is widely recognized as the most devastating disease affecting the global citrus industry, posing a significant threat to its sustainable development. Citrus trees infected with Bacillus phloem exhibit stunted growth, a sharp decline in yield, deformed fruit with severely degraded quality (e.g., bitter taste, uneven coloring, smaller and deformed fruit, such as "red-nosed fruit"), and may even die, causing irreversible and enormous economic losses to citrus yield and quality.

[0003] Currently, the control of Huanglongbing (HLB) still faces severe challenges. The pathogen of HLB cannot yet be cultured in vitro in a laboratory, a characteristic that fundamentally restricts the screening, evaluation, and development of highly effective, targeted chemical agents. The existing core control system mainly relies on the following strategies: timely removal of diseased trees, planting of disease-free seedlings, and strict control of vectors (citrus psyllids). These strategies can slow the spread of the disease to some extent, but given the complex field environment and continuous pathogen pressure, they are insufficient to achieve fundamental and effective control.

[0004] In the practice of controlling citrus Huanglongbing (HLB), existing major control measures all have significant limitations. Chemical control (such as the use of thiamethoxam and bifenthrin) can effectively suppress the population of the virus-transmitting vector, the citrus psyllid, but it cannot block the systemic transmission and spread of the pathogen within the plant. Agricultural control measures (including disease-free seedling propagation systems and timely removal of diseased trees) can delay the spread of the epidemic to some extent, but they lack the ability to reverse the spread of the disease in already infected plants. Biological control strategies (such as the use of endophytic antagonism or bacteriophage targeting technology) are easily constrained by environmental conditions, have limited scope of application, and their stability and applicability in field control still need to be improved.

[0005] Regions like Florida in the United States have attempted to introduce antibiotic interventions (such as streptomycin, oxytetracycline, and ampicillin sodium). A 2019 research paper published in *Phytopathology*, titled "The in Planta Effective Concentration of Oxytetracycline Against 'Candidatus Liberibacter asiaticus' for Suppression of Citrus Huanglongbing," indicated that oxytetracycline has a significant effect in inhibiting the pathogen of citrus Huanglongbing. A research paper published in *Journal of Plant Protection*, titled "Inhibitory Effects of Two Classes of Antibiotics on 'Newhall' Navel Orange Huanglongbing and Their Impact on Rhizosphere Bacterial Community Structure," pointed out that antibiotic use has triggered a series of secondary problems, including ecological and environmental risks such as soil microbial community imbalance and water pollution, potential threats to human health from drug residues and the spread of drug-resistant bacteria, and the potential for pathogens to develop drug resistance, raising questions about their long-term safety and sustainability.

[0006] Against this backdrop, various emerging technological strategies have gradually attracted attention. Control methods based on nanomaterials have shown promise. A research paper published in *Environment Science nano*, titled "Manganese-based nanozyme-enabled efficient mitigation of Huanglongbing-induced oxidative damage in Citrus," describes how the construction of manganese oxide nanozymes (MONPs) can scavenge and regulate reactive oxygen species metabolism in infected plants, alleviate oxidative stress damage, protect plant tissues, and alleviate Huanglongbing symptoms. A research paper published in the *Journal of Advanced Research*, titled "Fabrication of nanogels to improve the toxicity and persistence of cycloxaprid against Diaphorina citri, the vector of citrus huanglongbing," demonstrates that drug-loaded nanogels can significantly enhance the toxicity and persistence of insecticides against citrus psyllids. A research paper published in the *International Journal of Biological Macromolecules*, titled "A Two-in-One Molybdenum Disulfide-Chitosan Nanoparticles System for Activating Plant Defense Mechanisms and Reactive Oxygen Species to Treat Citrus Huanglongbing," develops a molybdenum disulfide-chitosan nanosystem capable of activating plant defense mechanisms and regulating reactive oxygen species to treat citrus Huanglongbing. On the other hand, plant-derived active ingredients also show significant application potential. Several Chinese invention patents (such as CN118415183A, CN118489678A, CN117770253A) have also confirmed the significant effects of natural compounds such as geraniol, eucalyptol, cinnamaldehyde, and chlorogenic acid in improving pesticide delivery efficiency or directly inhibiting pathogens.

[0007] Plant extracts are rich in various natural antibacterial active ingredients. Systematic screening and evaluation of their antibacterial mechanisms and field efficacy hold promise for overcoming the bottlenecks of existing chemical control methods and constructing a new, environmentally friendly, sustainable, and efficient integrated management technology system for Huanglongbing (HLB). This is of great significance for ensuring the healthy development of the global citrus industry. Against this backdrop, green control strategies based on natural substances have demonstrated relatively better advantages and potential. Scientific research has found that plant extracts contain abundant natural antibacterial active substances; systematic evaluation of their antibacterial activity holds promise for overcoming the bottlenecks faced by chemical control. This opens up a new path for developing a new, environmentally friendly, sustainable, and efficient HLB control technology system, which is of great significance for ensuring the health, safety, and long-term development of the global citrus industry. Summary of the Invention

[0008] The purpose of this invention is to provide an application of spearmint oil and a composition containing spearmint oil in the prevention and control of citrus Huanglongbing (HLB), so as to solve the above-mentioned problems in the background art.

[0009] To achieve the above objectives, the present invention provides the following technical solution:

[0010] One of the technical solutions of this invention is to provide the application of spearmint oil in inhibiting the pathogen of citrus Huanglongbing (HLB).

[0011] Preferably, the application is for the prevention and control of citrus Huanglongbing (HLB).

[0012] The second technical solution of the present invention provides a fungicide for the pathogen of citrus Huanglongbing, the raw material of which contains spearmint oil.

[0013] Preferably, the spearmint oil contains 45-70 wt% carvacrol, 12-30 wt% limonene, and 2-10 wt% eucalyptol.

[0014] Although the pathogen of citrus Huanglongbing (CLas) cannot be cultured in pure culture media using traditional media, current research can use in vitro host tissues to simulate the microenvironment in which the pathogen survives.

[0015] A phase-inversion method was used to prepare a water-in-oil emulsion of spearmint oil. The active ingredient, spearmint oil, along with emulsifiers and other additives, was used as the oil phase, and deionized water as the aqueous phase. The aqueous phase was poured into the oil phase, and the mixture was sheared and emulsified at 11,000 rpm for 5 minutes using a high-speed shear emulsifier to prepare a stable water-in-oil emulsion sample. Spearmint oil was diluted to a series of concentrations (0.1 mg / L, 0.5 mg / L, 1.0 mg / L, 1.5 mg / L, 2.0 mg / L) and added to the culture system. The antibacterial effect was evaluated by quantitatively detecting changes in the CLAs genome copy number using qPCR. The study found that spearmint oil, at concentrations ≥0.5 mg / L, exhibited significant in vitro inhibitory activity against *Citrus citrus Huanglongbing*, and significantly reduced the pathogen load (PCR positivity rate decreased to 0). Spearmint oil is a natural metabolite with no residual risk.

[0016] Carvone (45–70 wt%) in spearmint oil can directly disrupt the cell membrane of Huanglongbing fungus, playing a major antibacterial role. Limonene (12–30 wt%) assists carvone in penetrating the outer layer of the fungus, enhancing its antibacterial efficiency. Other components (such as caryophyllene) can prolong the effect and enhance the plant's own immunity. Carvone is the main active ingredient, but all the components in spearmint oil work synergistically, resulting in a more significant and longer-lasting effect. The effect of using carvone alone cannot reach that of spearmint oil.

[0017] The third technical solution of the present invention provides a method for controlling citrus Huanglongbing by applying the above-mentioned fungicide.

[0018] Preferably, the application method is impregnation, spraying, misting, drip irrigation, injection, rinsing, or smearing.

[0019] Preferably, the application method is trunk injection.

[0020] Preferably, the application is applied to diseased citrus trees infected with Huanglongbing (HLB) or to healthy trees in high-risk areas.

[0021] Preferably, the concentration of spearmint oil in the medicinal solution is ≥0.5 mg / L.

[0022] Spearmint oil can disrupt the cell membrane integrity of the pathogen causing Huanglongbing (HLB), leading to leakage of its contents, and has a significant therapeutic effect on HLB-infected trees at a concentration of ≥0.5 mg / mL.

[0023] Preferably, the concentration of spearmint oil in the medicinal solution is 0.5–2.0 mg / L, more preferably 1.0–1.5 mg / L (this concentration range ensures the antibacterial effect while taking into account both economy and plant safety).

[0024] Preferably, the medicinal solution further contains an emulsifier, which is one or more of AEO-3P, tea saponin, and soybean lecithin; the concentration of the emulsifier in the medicinal solution is 0.1-5 wt%.

[0025] The emulsifier added in this invention is to ensure that the hydrophobic spearmint oil forms a stable emulsion in water, which helps the liquid to be evenly distributed and absorbed on the leaves and inside the tree, and is safe for the environment and crops.

[0026] Preferably, the liquid medicine also contains a stabilizer, which is a low-temperature stabilizer, a preservative, an antioxidant, or a light stabilizer.

[0027] More preferably, the trunk injection method is as follows: drill a hole at a suitable location on the trunk of the diseased tree, install an infusion device, and inject the emulsified spearmint oil emulsion (concentration 1.0-1.5 mg / L) into the tree. The injection volume for each tree is 150-300 mL (the injection volume depends on the tree age, severity of disease, and tree size; 2-4 weeks after injection, the yellowing symptoms of the tree are significantly reduced, the newly sprouted branches and leaves are healthy, the pathogen content test shows a decrease of more than 50%, and the tree vigor continues to increase).

[0028] The technical principle of this invention is as follows:

[0029] This invention addresses the challenge of lacking effective green control methods for citrus Huanglongbing (HLB) by focusing on the screening of natural plant-derived active substances. Through pathogen proliferation inhibition experiments (qPCR detection) and in vivo treatment experiments on infected plants, the direct killing and inhibitory effects of spearmint oil on HLB pathogens were systematically verified, and its ability to stimulate plant defense responses was observed. Therefore, a trunk injection application technology based on spearmint oil was developed to achieve effective control of HLB.

[0030] Spearmint Oil (Main active ingredient: L-carvone, molecular formula: C) 10 H 14 Spearmint oil (Molecular weight: 150.22; CAS No.: 2244168), also known as green peppermint oil, is a volatile essential oil extracted from the whole herb of spearmint (Lamiaceae family) through steam distillation. The finished product is a pale yellow to green transparent liquid with a characteristic cool aroma. It is slightly soluble in water and readily soluble in ethanol and organic solvents. The main chemical component of spearmint oil is L-carvone (approximately 60%), which is the key component that gives it its unique aroma and biological activity. According to my country's "Standards for the Use of Food Additives" (GB 2760-2021), spearmint oil is a permitted edible natural flavoring and can be directly added to foods such as candies and chewing gum, as well as oral hygiene products such as toothpaste and mouthwash.

[0031] Oral LD50 of spearmint oil in rats 50 The LD50 is 5000 mg / kg, which falls into the low toxicity category (according to the WHO toxicity classification standards). 50 At doses above 2000 mg / kg, the product is practically non-toxic or low in toxicity, posing an extremely low risk of acute exposure. In chronic toxicity studies (emphysema model experiments), rats were administered spearmint oil via gavage at doses up to 100 mg / kg / day for four consecutive weeks, and mice up to 141 mg / kg / day, without reporting significant organ damage or death, and the product exhibited anti-inflammatory activity. In the EU feed additive assessment, the safe dose of spearmint oil for dogs is 5 mg / kg (feed addition), and for cats it is 1 mg / kg; no cumulative toxicity was found with long-term use.

[0032] According to the World Agrochemical Network, spearmint oil is mainly used for pest control by fumigation to control armyworms, diamondback moths, cotton bollworms, greenhouse whiteflies, and two-spotted spider mites. In the current technology, there are no research reports on the direct inhibitory effect of spearmint oil on Huanglongbing fungus or its synergistic mechanism in phloem delivery.

[0033] The purpose of this invention is to overcome the shortcomings of existing antibiotics in the prevention and control of citrus Huanglongbing, such as residues and environmental safety risks, and to provide a highly efficient, green, and safe plant-derived solution—spearmint oil and compositions rich in this substance—for the prevention and control of citrus Huanglongbing.

[0034] This invention, based on research into the antibacterial activity of various plant essential oils and considering the characteristics of Huanglongbing (HLB) of citrus, screened and identified spearmint oil as having significant inhibitory activity against HLB through systematic in vitro antibacterial tests and in vivo control efficacy evaluation. Spearmint oil not only directly inhibits the proliferation of the pathogen but also induces systemic resistance in citrus plants, enhancing tree vigor. The terpenes and other natural compounds in spearmint oil are recognized as "danger signals" by citrus plants. These signals activate internal defense signaling pathways, particularly those related to defense hormones such as salicylic acid (SA) and jasmonic acid (JA) / ethylene (ET).

[0035] Even distal tissues of the plant not directly exposed to spearmint oil (such as new leaves, branches, and even roots) enter a "vigilance state," developing systemically acquired resistance. In this state, the plant systematically enhances multiple defense responses: producing secondary metabolites with direct antibacterial activity; accumulating disease-related proteins such as chitinase and β-1,3-glucanase, which degrade pathogen cell walls; strengthening cell walls by depositing callosity and lignin to form a physical barrier against pathogen invasion; activating the antioxidant system by scavenging reactive oxygen species produced by disease stress and reducing oxidative damage; and comprehensively improving the tree's overall "physical condition" (antioxidant capacity, photosynthesis, and nutrition), making the tree stronger and more resistant to and tolerant of Huanglongbing (HLB). Spearmint oil is mainly extracted from the spearmint (Mentha picata L.) plant of the Lamiaceae family. It is a natural, low-toxicity, and easily degradable bioactive substance that is widely used in the food, daily chemical, and pharmaceutical industries. It has a wide range of sources, controllable costs, and significant feasibility for application in agricultural production.

[0036] Spearmint oil outperforms antibiotics such as oxytetracycline hydrochloride in controlling citrus Huanglongbing (HLB), primarily due to its unique multi-faceted mechanism of action, enhanced penetration, and induction of plant resistance. The main active components of spearmint oil, L-carvone and limonene, disrupt the integrity of the pathogen's cell membrane, leading to protoplasmic leakage, abnormal organelle (such as mitochondria) structure, and ultimately cell death. This physical disruption is particularly effective against the HLB pathogen (Bacillus asiaticus, CLAs). Unlike single antibiotics, spearmint oil synergistically inhibits bacterial growth through multiple pathways, including interfering with the pathogen's energy metabolism, respiration, and enzyme activity, reducing the risk of antibiotic resistance. Spearmint oil can activate the salicylic acid (SA) and jasmonic acid (JA) defense signaling pathways in citrus plants, promoting the synthesis of phytoalexins and pathogenesis-related proteins (such as chitinase), enhancing cell wall lignification, and forming a systemic immune barrier. This "plant vaccine" effect is a function that chemical antibiotics lack. While oxytetracycline hydrochloride works solely by inhibiting bacterial protein synthesis, long-term use can easily lead to drug resistance in pathogens. Spearmint oil, however, exhibits a multi-component synergistic effect that can delay the development of resistance. Terpenes in spearmint oil (such as limonene) are natural penetrants that can dissolve the waxy and cuticle layers of citrus leaf epidermis, significantly increasing the distribution depth of the pesticide in the phloem. Through a triple mechanism of "direct antibacterial action + induction of plant immunity + enhanced pesticide penetration," spearmint oil overcomes the technical bottleneck of oxytetracycline hydrochloride's inability to reach the phloem target and its singular action. Its natural component characteristics further avoid environmental residues and the risk of drug resistance, providing a more sustainable solution for the green control of Huanglongbing (HLB).

[0037] The beneficial technical effects of the present invention are as follows:

[0038] In vitro and in vivo tests have confirmed that spearmint oil has a significant direct inhibitory and bactericidal effect on the pathogen of citrus Huanglongbing (HLB). Foliar spraying and injection treatment can effectively reduce the pathogen content in diseased trees within several days to several weeks (the reduction can reach more than 50%), alleviate yellowing symptoms, and promote tree recovery. The effect is significantly better than that of single antibiotics (such as oxytetracycline hydrochloride) and is less likely to induce drug resistance. The spearmint oil of this invention is derived from natural plants, with clearly defined main components. It is biodegradable in the environment and has an extremely low residue risk. Toxicological evaluation shows that it has low toxicity to mammals (including humans), is safe for citrus trees, and meets the requirements of green agriculture and safe agricultural production. Spearmint is widely cultivated, the essential oil extraction process is mature, and the raw material cost is relatively low, which is conducive to large-scale agricultural production applications.

[0039] This invention provides a trunk injection method (mainly used for systemic treatment of severely affected trees) that can effectively control citrus Huanglongbing (HLB). This is the first time, both domestically and internationally, that the efficacy of spearmint oil and its formulations in controlling HLB has been systematically proposed and verified. It offers a novel and highly promising green solution for overcoming this disease, with broad application prospects. Attached Figure Description

[0040] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0041] Figure 1 A comparative study of leaf yellowing reversal before and after injection of spearmint oil emulsion in Example 1 into diseased trees in the Jianghua citrus production area of ​​Yongzhou, Hunan Province.

[0042] Figure 2 A comparative study of leaf yellowing reversal in diseased citrus trees from Xinning, Hunan Province, before and after injection of spearmint oil emulsion in Example 1 into the tree trunk. Detailed Implementation

[0043] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention. It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the present invention.

[0044] Furthermore, regarding the numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, are also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0045] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. It should be noted that any aspects of this invention not described in detail are conventional practices in the art and are not the focus of this invention.

[0046] The terms “comprising,” “including,” “having,” “containing,” etc., used in this invention are all open-ended terms, meaning that they include but are not limited to.

[0047] Spearmint oil's main component is carvone, a stereochemical natural product comprising both L-carvone and D-carvone. L-carvone, chemically known as 1-methyl-4-isopropen-6-cyclohexene-2-one, is the core component of spearmint oil and is widely used in food processing such as chewing gum. In the pharmaceutical field, it is used as a flavoring ingredient in poultices and mouthwashes, and as a flavoring raw material in products such as toothpaste. It is listed in the food flavoring list of the "Standards for the Use of Food Additives" (GB 2760-2021). The carvone molecule contains a chiral center, thus forming two racemic forms, L- and D-carvone, which exhibit different functions due to their different stereochemical structures. Pharmacological studies show that L-carvone possesses various activities, including antibacterial, antifungal, antiparasitic, antineuraminidase, antioxidant, anti-inflammatory, and anticancer activities. Its antibacterial mechanism is mainly related to its action on the cell membrane and alteration of ultrastructure. The research paper "Anti-virulence potential of carvone against Serratia marcescens" published in *Food & Medicine Homology* indicates that L-carvone can significantly inhibit the virulence factors of *Serratia marcescens* (such as biofilms, extracellular polysaccharides, and sclerotin), and can enhance oxidative damage by inhibiting antioxidant enzymes such as glutathione peroxidase, thereby improving antibiotic penetration. The article "Bioactivity and Control Efficacy of Five Terpenes and Synergistic Combinations against Peach Aphids" in the *Journal of Inner Mongolia Agricultural University (Natural Science Edition)* mentions that L-carvone achieved a repellency rate of 49.6% against peach aphids, with a median lethal concentration (LC50). 50The concentration was 0.56 mL / L. Liu Chenyu's dissertation, "Study on the Sublethal Effect of a Mixture of L-carvone and Cuminol on the Prickly Pear," showed that the mixture of L-carvone and Cuminol had a contact-killing effect on the Prickly Pear and the Peach Aphid. Research in "Food Industry Technology" (2016, 37(16)) confirmed that L-carvone has a strong antibacterial effect against Escherichia coli and Salmonella, with a minimum inhibitory concentration of 12.5 μg / mL. Chinese invention patent application CN109329410A discloses a fruit and vegetable bactericide and preservative with L-carvone as the main component, which can effectively inhibit postharvest diseases such as gray mold, anthracnose, and blue mold, and extend the storage period of fruits and vegetables. Chinese invention patent application CN115053900A discloses a fungicide for preventing and controlling avocado canker. Its active ingredient is composed of L-carvone and tebuconazole, tebuconazole or triadimefon. L-carvone plays a synergistic role in this process, which can reduce the amount of chemical pesticides used, reduce residues and reduce the environmental burden.

[0048] Dextrorotatory carvone, a stereoisomer of levorotatory carvone, exhibits different biological activities due to differences in its molecular spatial structure. Studies have shown that dextrorotatory carvone differs from its levorotatory counterpart in antibacterial spectrum and potency; for example, it shows more significant inhibitory effects against certain fungi, but research on its effects on Gram-negative bacteria is limited. Chinese invention patent application CN110226600B discloses a strong inhibitory effect of dextrorotatory carvone on Fusarium sulphureum, the fungus causing potato rot. The unique characteristics of the pathogen causing citrus Huanglongbing (such as dependence on the phloem environment for survival) mean that questions such as whether dextrorotatory carvone has an inhibitory effect, whether its mechanism of action differs from that of the levorotatory counterpart, and whether a synergistic effect occurs when the two are combined, all lack supporting research. Currently, there is no literature or patent record of dextrorotatory carvone used alone or in combination with other substances for the control of citrus Huanglongbing, and its application value in this field requires further exploration.

[0049] In summary, there are no research reports on the application of spearmint oil and L-carvone / D-carvone in the control of citrus Huanglongbing (HLB). Chinese invention patent application CN107372667A discloses a method using extracts from 15 traditional Chinese medicines, including Houttuynia cordata, Sichuan pepper, and neem bark, as the core, releasing active ingredients through enzymatic hydrolysis to inhibit pathogen proliferation. However, due to the complexity of its composition, it is impossible to directly identify which substance directly affects the HLB pathogen. Chinese invention patent application CN104920521A discloses a method using traditional Chinese medicines such as Coptis chinensis and Amomum villosum, along with copper sulfate and gypsum, utilizing the synergistic antibacterial effect of alkaloids and copper ions to reduce the incidence of disease in trees. However, this method is overly cumbersome, its mechanism of action is extremely unclear, and its field results are poor. Currently, there are no research reports on the direct inhibitory effect of spearmint oil and L-carvone / D-carvone on HLB pathogens or their synergistic mechanism in phloem delivery.

[0050] This invention discloses a fungicide for the pathogen of citrus Huanglongbing, the composition of which is shown in Table 1.

[0051] Table 1. Composition of antibacterial agents

[0052]

[0053] Furthermore, the bactericide is formulated as an emulsion, microemulsion, emulsifiable concentrate, soluble concentrate, soluble gel, oil, wettable powder, water-dispersible granules, suspension concentrate, microcapsule suspension, oil suspension, or dispersible oil suspension.

[0054] The present invention also discloses a method for controlling citrus Huanglongbing, which involves applying the aforementioned fungicide.

[0055] Furthermore, the application method is spraying or trunk injection.

[0056] Furthermore, the spraying method is as follows: spray the solution containing spearmint oil (concentration 0.5-2.0 mg / L) evenly on the canopy of citrus trees, focusing on the new shoots and the back of the leaves. Spray once every 15-20 days, for a total of 3-4 times (7-10 days after spraying, initial signs of tree vigor recovery can be observed, the new shoots turn green faster, and subsequent treatment can reduce the pathogen content).

[0057] Furthermore, the method of trunk injection is as follows: drill a hole at a suitable location on the trunk of the diseased tree, install an infusion device, and inject the emulsified spearmint oil emulsion (concentration 0.5-2.0 mg / L) into the tree. The injection volume for each tree is 150-300 mL (the injection volume depends on the age of the tree, the severity of the disease, and the size of the tree; 24 weeks after injection, the yellowing symptoms of the tree are significantly reduced, the newly sprouted branches and leaves are healthy, the pathogen content test shows a decrease of more than 50%, and the tree vigor continues to increase).

[0058] One month after trunk injection, the yellowing symptoms of diseased trees were reversed. Field trials showed that within a 3-month treatment period, repeated trunk injections (usually once a month for a total of 3 times) significantly turned the trees green, allowed them to bear fruit normally, and eliminated the red noses on the fruit.

[0059] The spearmint oil used in this invention was purchased from Hubei Maidehao Biotechnology Co., Ltd., and contained 60 wt% carvone, 35 wt% limonene, and 5 wt% eucalyptol.

[0060] Unless otherwise specified, "room temperature" in this invention refers to 10-30°C.

[0061] All raw materials used in the following embodiments and comparative examples of the present invention are commercially available products.

[0062] Example 1

[0063] A method for controlling Huanglongbing (HLB) of citrus was tested in a field trial in Jianghua County, Yongzhou City, Hunan Province, using spearmint oil to control HLB. The specific steps are as follows:

[0064] 1. Location: Jianghua citrus production area in Yongzhou, Hunan (incidence rate >10%), and Xinning citrus Huanglongbing high-incidence area in Hunan.

[0065] 2. Method:

[0066] Preparation of spearmint oil emulsion: A 10% spearmint oil aqueous emulsion was prepared using the phase inversion method. The active ingredient spearmint oil was added to the emulsifier (specifically AEO-3P) as the oil phase, and deionized water was used as the aqueous phase. The aqueous phase was poured into the oil phase, and the mixture was sheared and emulsified at 11000 r / min for 5 min using a high-speed shear emulsifier to obtain the spearmint oil emulsion (with a spearmint oil concentration of 10 mg / L and an emulsifier concentration of 1.0%).

[0067] Trunk injection: Dilute spearmint oil emulsion to a concentration of 0.5 mg / L, drill a hole at a suitable location on the trunk of the diseased tree, install an infusion device, and inject the 0.5 mg / L spearmint oil emulsion into the tree. The injection volume for each tree is 300 mL.

[0068] 3. The total sample size of the selected citrus trees was 30 trees with mild symptoms and 30 trees with severe symptoms (mild symptoms were defined as canopy yellowing rate <30% with new shoots emerging; severe symptoms were defined as canopy yellowing rate ≥60% with stagnant new shoots), and the trees were 5-8 years old (base diameter 8-12cm). The specific application plan is shown in Table 2.

[0069] Table 2 Application Plan

[0070]

[0071]

[0072] 4. Dynamic monitoring data of pathogen content

[0073] The copy number of CLas genome in the phloem of the above-mentioned mildly and severely affected trees before and after injection of spearmint oil emulsion was quantified using qPCR (target gene: 16S rRNA). The results are as follows:

[0074] Table 3 Dynamic monitoring data of pathogen content

[0075]

[0076] 5. Conclusion:

[0077] CLas inhibition rate was negatively correlated with initial bacterial load, with a more significant decrease observed in mildly infected trees (P<0.05). After 90 days, CLas content in mildly infected trees approached the healthy threshold (≤3.0 log copies / g).

[0078] Spearmint oil emulsion achieves highly effective control of Huanglongbing (HLB) through trunk injection. Its core advantages are:

[0079] It is delivered to the target point in the phloem to directly inhibit CLA proliferation; periodic injection (once a month × 3 times) matches the tree's metabolism and new shoot growth rhythm; the CLA inhibition rate of mildly affected trees can reach 50%, while the treatment course needs to be extended to 6 months for severely affected trees.

[0080] Figure 1 A comparative study of leaf yellowing reversal before and after injection of spearmint oil emulsion in Example 1 into diseased trees in the Jianghua citrus production area of ​​Yongzhou, Hunan Province.

[0081] Figure 2 A comparative study of leaf yellowing reversal in diseased citrus trees from Xinning, Hunan Province, before and after injection of spearmint oil emulsion in Example 1 into the tree trunk.

[0082] Figure 1 and Figure 2 The top figure shows the survey results before the injection of spearmint oil emulsion, and the bottom figure shows the survey results on the 90th day after the injection of spearmint oil emulsion of Example 1.

[0083] Example 2

[0084] A method for controlling Huanglongbing (HLB) of citrus was tested in a field trial in Xinning, Hunan Province, using spearmint oil to control HLB. The specific steps are as follows:

[0085] 1. Location: Citrus producing area of ​​Xinning, Hunan (morbidity rate >30%).

[0086] 2. Method:

[0087] Preparation of spearmint oil emulsion: A water-in-oil emulsion was prepared using the phase inversion method. The active ingredient spearmint oil was added to the emulsifier (specifically AEO-3P) as the oil phase, and deionized water was used as the aqueous phase. The aqueous phase was poured into the oil phase, and the mixture was sheared and emulsified at 11000 r / min for 5 min using a high-speed shear emulsifier to obtain a spearmint oil emulsion (with a spearmint oil concentration of 1.0 mg / L and an emulsifier concentration of 1.0%).

[0088] Trunk injection: Drill a hole at a suitable location on the trunk of the diseased tree, install an infusion device, and inject a 1.0 mg / L concentration of spearmint oil emulsion into the tree. The injection volume for each tree is 300 mL.

[0089] 3. The total sample size of the selected citrus trees was 30 trees with mild symptoms and 30 trees with severe symptoms (mild symptoms were defined as canopy yellowing rate <30% with new shoots emerging; severe symptoms were defined as canopy yellowing rate ≥60% with stagnant new shoots), and the trees were 5-8 years old (base diameter 8-12cm). The specific application plan is shown in Table 4.

[0090] Table 4 Application Plan

[0091]

[0092] 4. Dynamic monitoring data of pathogen content

[0093] The copy number of CLas genome in the phloem of the above-mentioned mildly and severely affected trees before and after injection of spearmint oil emulsion was quantified using qPCR (target gene: 16S rRNA). The results are as follows:

[0094] Table 5 Dynamic monitoring data of pathogen content

[0095]

[0096]

[0097] 5. Conclusion:

[0098] CLas inhibition rate was negatively correlated with initial bacterial load, with a more significant decrease observed in mild cases (P<0.05).

[0099] After 90 days, the CLas content of the mildly affected tree was close to the healthy threshold (≤3.0 log copies / g).

[0100] Spearmint oil emulsion achieves highly effective control of Huanglongbing (HLB) through trunk injection. Its core advantages are:

[0101] (1) Directly delivered to the target site in the phloem to directly inhibit CLAs proliferation;

[0102] (2) Periodic injection (once a month for 3 months) to match tree metabolism and new shoot growth rhythm;

[0103] (3) The inhibition rate of CLas in mild cases can reach 50%, while the treatment course for severe cases needs to be extended to 6 months.

[0104] It was observed that the CLas inhibition rate was negatively correlated with the initial bacterial load, with a more significant decrease observed in mildly infected trees (P<0.05). After 90 days, the CLas content in mildly infected trees approached the healthy threshold (≤3.0 log copies / g).

[0105] Effect verification

[0106] To evaluate the antibacterial effect of spearmint oil against CLAs, a pathogen that is difficult to culture in pure form, a system based on "semi-in vitro culture—drug treatment—qPCR verification" was established. The in vitro inhibitory activity of spearmint oil against CLAs was verified. Specific methods and results are as follows:

[0107] I. Experimental Design

[0108] 1. Pathogen amplification and experimental system construction

[0109] Using citrus shoot segments infected with CLas, new shoot growth was induced for 30 days in a modified medium containing 2,4-D, NAA, and 6-BA (MS basal salt and vitamin 4.43 g / L, sucrose 30 g / L, 2,4-D 0.5 mg / L, 6-BA 2.0 mg / L, NAA 0.5 mg / L, ascorbic acid 50 mg / L, activated charcoal 1.5 g / L, phytagel 2.5 g / L, pH 5.8). This induced an approximately 1600-fold increase in the CLas genome copy number in the phloem, overcoming the challenges of low pathogen levels and the inability to achieve pure culture.

[0110] 2. Drug treatment and control group setup

[0111] The spearmint oil emulsion from Example 1 was prepared at gradient concentrations of 0.1, 0.5, 1.0, 1.5, and 2.0 mg / L, with sterile water treatment serving as a blank control. Infected new shoots were immersed in each concentration of the emulsion for 10 minutes to ensure that the agent fully contacts the phloem pathogen.

[0112] 3. Multiple detection indicators

[0113] qPCR quantification: Absolute quantification of the CLas 16S rRNA gene copy number to calculate the pathogen load per unit of plant DNA.

[0114] Phenotypic records: Simultaneous monitoring of disease phenotypes such as yellowing of new shoots and growth retardation.

[0115] II. Criteria for Judging Results

[0116] Significant inhibition: defined as a decrease in pathogen load ≥90% in qPCR detection and a PCR positivity rate of 0 (i.e., complete clearance of pathogen).

[0117] III. Results and Conclusions

[0118] 1. qPCR quantification results

[0119] Treatment with 0.5 mg / L spearmint oil emulsion reduced the CLas genome copy number by more than 90% and resulted in a negative PCR test; treatment with 1.0 mg / L spearmint oil emulsion reduced the pathogen load to below the detection limit, demonstrating strong antibacterial activity.

[0120] 2. Phenotypic Consistency

[0121] The yellowing of new shoots in the treatment group was significantly reduced, and growth recovered, consistent with the downward trend in pathogen load.

[0122] This invention, through an independently established integrated experimental system of "pathogen proliferation-drug treatment-qPCR verification," scientifically demonstrates that spearmint oil can significantly inhibit CLas proliferation and destroy their cell structure at a concentration of ≥0.5 mg / L. This provides a reliable pathway for evaluating the efficacy of spearmint oil under conditions where CLas are difficult to culture, and also provides experimental evidence for the application of spearmint oil in the control of citrus Huanglongbing (HLB).

[0123] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of a spearmint oil in inhibiting Candidatus Liberibacter asiaticus, the pathogen of citrus Huanglongbing; the concentration of the spearmint oil in the pesticide solution is ≥ 0.5 mg / L. The proportion of carvone in the spearmint oil is 45-70 wt%, the proportion of limonene is 12-30 wt%, and the proportion of eucalyptol is 2-10 wt%.

2. Use according to claim 1, characterized in that, The application is in the prevention and control of citrus Huanglongbing.

3. A method for preventing and controlling citrus huanglongbing, characterized by, A fungicide is applied, the raw material of the fungicide contains spearmint oil; during the application, the concentration of the spearmint oil in the pesticide solution is ≥ 0.5 mg / L; the pathogen of the citrus Huanglongbing is Candidatus Liberibacter asiaticus; The proportion of carvone in the spearmint oil is 45-70 wt%, the proportion of limonene is 12-30 wt%, and the proportion of eucalyptol is 2-10 wt%.

4. The method of claim 3, wherein, The application is in the prevention and control of citrus Huanglongbing.

5. The method of claim 3, wherein, The concentration of the spearmint oil in the pesticide solution is 0.5-2.0 mg / L.

6. The method of claim 3, wherein, The pesticide solution also contains an emulsifier, the emulsifier is one or more of AEO-3P, tea saponin, and soybean lecithin; the concentration of the emulsifier in the pesticide solution is 0.1-5 wt%. The application is in the prevention and control of citrus Huanglongbing.

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