A combination of essential oils of Ledum palustre and Ophiopogon japonicus and its application in agricultural bacteriostasis

Abstract

The application provides a combination of essential oils of Ledum palustre and Ophiopogon japonicus and application thereof in agricultural bacteriostasis, and belongs to the field of extraction and application of plant essential oils. The essential oil combination is obtained by mixing essential oils of Ledum palustre and Ophiopogon japonicus; the mass ratio of the essential oils of Ledum palustre and Ophiopogon japonicus is 1:0.5-1.5; the essential oils of Ledum palustre and Ophiopogon japonicus are obtained by using a steam distillation method respectively; the steam distillation method is specifically as follows: Ledum palustre leaves and crushed Ophiopogon japonicus roots are placed in respective distillation equipment, water is added, and then the temperature is raised to boiling, and distillation is performed for 1-3 hours, so that the essential oils of Ledum palustre and Ophiopogon japonicus are obtained respectively. The essential oil combination is derived from plants, is a natural bactericide, has good bacteriostatic effect, is safe to crops, is environment-friendly, and has a wide application prospect.

Description

Technical Field

[0001] This invention relates to the field of plant essential oil extraction and application, and in particular to an essential oil combination of Rhododendron simsii and Ophiopogon japonicus and its application in agricultural antibacterial activity. Background Technology

[0002] In agriculture, pest control encompasses three distinct areas: diseases, insects, and weeds. For disease control, pyridine fungicides like Aminopyrifen are used to control rice blast and sheath blight, while quinoline fungicides like Ipflufenoquin are used to control rice false smut and other foliar diseases. For insect control, 25% cartap hydrochloride is used to control rice leaf rollers, while 58% imidacloprid and 30% thiamethoxam are used to control rice planthoppers and brown planthoppers. For weed control, 10% oxadiazon, 10% pyrimisulfuron, and 25% fenfluridine are used to control rice sedges and broadleaf weeds.

[0003] Of the three types of damage mentioned above, plant diseases account for approximately 12% of total crop yield reduction. For example, the prevalence of wheat stem rot can severely impact wheat production; control agents include 30% thiamethoxam suspension seed dressing + 6% tebuconazole suspension, 27% difenoconazole·fludioxonil·thiamethoxam suspension seed dressing, 25% cyazofamid suspension, and 30% tebuconazole·imidacloprid. Early blight of potatoes can severely affect potato yield and quality; control agents include mancozeb, chlorothalonil, captan, tebuconazole, difenoconazole, azoxystrobin, iprodione, and fludioxonil. Gray mold of cucumbers often causes flower and fruit rot; control agents mainly include sclerotinia sclerotinia, carbendazim, iprodione, and ethoxysulfuron.

[0004] It is evident that chemical agents remain the primary means of controlling plant diseases, and their unique advantages, such as rapid effectiveness, ease of use, variety, and low price, have allowed them to maintain a dominant position in the pesticide market for a long time.

[0005] However, the long-term, widespread, and unscientific use of chemical pesticides has led to many increasingly prominent problems, such as increased resistance in both harmful and target organisms, impacts on non-target organisms, environmental pollution, ecosystem damage, and the resurgence of pests and diseases. These issues have become major problems in current pesticide application and should attract widespread attention. Therefore, developing safe, environmentally friendly, and cost-effective novel biological antifungal agents has become a crucial task in the field of plant fungal disease control.

[0006] Natural pesticides, with their advantages of easy degradation, low residue, low pollution, high safety, and low susceptibility to resistance development in pests and diseases, have attracted much attention and become one of the key focuses and hot topics in current pesticide research. Plant essential oils are volatile secondary metabolites extracted from the flowers, buds, leaves, roots, and fruits of plants. Their chemical composition is complex, mainly including terpenes, alkaloids, flavonoids, lipids, phenols, and alkynes, among other compounds. Furthermore, plant essential oils extracted from different plants or different parts of the same plant can vary significantly.

[0007] Plant essential oils possess complex pharmacological activities due to their complex active ingredients and chemical structures, such as antibacterial, antifungal, anti-inflammatory, and antioxidant effects. Studies have shown that plant essential oils have excellent antibacterial effects against plant pathogens. For example, Ghabraie et al. found that 32 plant essential oils had good antibacterial effects against four pathogenic bacteria and Pseudomonas aeruginosa. Prieto et al.'s research showed that marigold essential oil and thyme essential oil can also effectively inhibit Fusarium head blight of potatoes.

[0008] Plant essential oils are not only green and safe, but also, due to their complex chemical structures, can act on pathogens at multiple sites, thus making it less likely for pathogens to develop drug resistance. However, at present, there is limited research both domestically and internationally on the use of plant essential oils to control various diseases, and reports on related formulations are also scarce. Moreover, the antibacterial effects of most essential oils are not ideal. Therefore, there is a need to develop a more effective combination of essential oils to provide a theoretical basis for better development and utilization of plant essential oils, and to provide new, green, and environmentally friendly natural pesticides for the control of agricultural diseases. Summary of the Invention

[0009] To address the aforementioned problems in existing technologies, this invention provides a combination of essential oils from *Rhodiola rosea* and *Ophiopogon japonicus* and their application in agricultural antibacterial treatment. The essential oil combination of this invention is derived from plants, is a natural bactericide, exhibits good antibacterial effects, is safe for crops, environmentally friendly, and has broad application prospects.

[0010] The technical solution of the present invention is as follows:

[0011] A blend of essential oils of Rhododendron simsii and Ophiopogon japonicus, wherein the essential oil blend is composed of Rhododendron simsii essential oil and Ophiopogon japonicus essential oil; wherein the mass ratio of Rhododendron simsii essential oil to Ophiopogon japonicus essential oil is 1:0.5-1.5.

[0012] Preferably, the essential oils of Rhododendron simsii and Ophiopogon japonicus are obtained by steam distillation.

[0013] More preferably, the steam distillation method is specifically operated as follows: put the leaves of Rhododendron simsii and the crushed root of Ophiopogon japonicus into their respective distillation equipment, add water and heat to boiling, and distill for 1-3 hours to obtain Rhododendron simsii essential oil and Ophiopogon japonicus essential oil respectively.

[0014] The present invention also provides the application of the aforementioned essential oil combination, namely, the application of the essential oil combination as an antibacterial agent in the prevention and control of agricultural diseases.

[0015] Furthermore, the antibacterial agent is composed of an essential oil blend and excipients; the excipients include one or more of organic solvents, surfactants, dispersants, defoamers, thickeners, and antifreeze agents.

[0016] Preferably, the antibacterial agent is formulated in the form of, but is not limited to, water-in-oil emulsions and suspensions.

[0017] More preferably, when the antibacterial agent is formulated as an emulsion, the emulsion comprises: 5-30% of *Eupatorium fortunei* essential oil, 5-30% of *Ophiopogon japonicus* essential oil; 10-60% of ethanol; 1-30% of emulsifier Tween-80, 1-20% of agricultural emulsion 500, and ultrapure water to make up to 100%.

[0018] More preferably, when the antibacterial agent is in the form of a suspension, the suspension comprises: 5-30% of *Eupatorium fortunei* essential oil, 5-30% of *Ophiopogon japonicus* essential oil; 1-5% of wetting agent T70, 1-10% of dispersant NP-10, 1-10% of dispersant D425, 0.1-5% of thickener xanthan gum, 0.1-5% of thickener magnesium aluminum silicate, 1-10% of antifreeze ethylene glycol, 0.1-5% of defoamer YS-0173, and ultrapure water to make up to 100%.

[0019] Furthermore, the agricultural diseases described in this invention include, but are not limited to, wheat stem rot, potato early blight, and cucumber gray mold.

[0020] The beneficial technical effects of this invention are as follows:

[0021] 1. This invention is the first to combine eucommia essential oil and ophiopogon essential oil and determine their combined antibacterial activity. This essential oil composition can give full play to the synergistic effect of the two essential oils, and the antibacterial activity is better than using one of them alone.

[0022] 2. This invention is the first to combine these two essential oils and apply them to the prevention and control of agricultural diseases, and to develop them into a pesticide formulation. This essential oil combination can be used to prevent and control a variety of agricultural diseases, and the control effect is better than using either one alone.

[0023] 3. The essential oil combination provided by this invention not only has the characteristics of being green and safe plant extracts, but also improves antibacterial activity while reducing the amount of essential oil used. Compared with using any one essential oil alone, it not only reduces the concentration and cost of use, but also effectively prevents and controls plant diseases, promoting green and high-quality agricultural development. Attached Figure Description

[0024] Figure 1 This is a process flow diagram for water-based emulsions;

[0025] Figure 2 This is a process flow diagram for the suspension preparation. Detailed Implementation

[0026] The present invention will now be described in detail with reference to the accompanying drawings and embodiments. Obviously, the described embodiments are merely some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without inventive effort are within the scope of protection of the present invention.

[0027] In the plant essential oil blend used in this invention, *Ledumpalustre L.*, an evergreen shrub belonging to the genus *Ledumpalus* in the family Ericaceae, is a traditional Chinese medicinal herb and an important spice plant. Plants of the genus *Ledumpalus* are distributed in the temperate or cold-temperate zones of the Northern Hemisphere. *Ledumpalustre* has wide applications in medicine; extracts from its tender branches and leaves have analgesic, expectorant, desensitizing, and diuretic effects.

[0028] Ophiopogon japonicus, a perennial evergreen herbaceous plant belonging to the Liliaceae family, is used in traditional Chinese medicine for its tuberous roots. These roots are believed to quench thirst, moisten the lungs, and relieve coughs. It is a traditional and valuable medicinal herb, and is listed in the Chinese Pharmacopoeia as a food and medicine homology product. Ophiopogon japonicus is mainly produced in Sichuan, Zhejiang, Fujian, and Jiangsu provinces. Originally wild, its cultivation has expanded due to high market demand. It has wide applications in medicine, nourishing yin and promoting body fluid production, moistening the lungs and clearing the heart. Clinically, it is frequently used to treat dry cough due to lung dryness, consumptive cough due to yin deficiency, sore throat, thirst due to fluid depletion, internal heat and thirst, irritability and insomnia, and constipation due to intestinal dryness.

[0029] Example 1: Preparation and antibacterial effect test of Rhododendron simsii essential oil and Ophiopogon japonicus essential oil

[0030] I. The preparation methods for Rhododendron simsii essential oil and Ophiopogon japonicus essential oil are as follows:

[0031] (1) Preparation of eucalyptus essential oil:

[0032] After processing 400g of Rhododendron simsii leaves, it was soaked in a supramolecular solvent for 6 hours, then dried and placed in a distillation apparatus (HOOLOO: DW hydrosol and essential oil machine). 12L of water was added, and the water was heated to boiling at 100℃. After distillation for 2 hours, Rhododendron simsii essential oil was obtained. The supramolecular solvent consisted of 10% decanoic acid, 15% ethanol, and 75% water.

[0033] (2) Preparation of Ophiopogon japonicus essential oil:

[0034] After crushing 300g of Ophiopogon japonicus into powder and passing it through a 200-mesh sieve, put it into a distillation apparatus and add 12L of water. Heat the water to boiling point (100℃) and distill for 2 hours to obtain Ophiopogon japonicus essential oil.

[0035] II. The steps for testing the antibacterial effect are as follows:

[0036] (1) Preparation of PDA culture medium:

[0037] Peel and wash 200g of potatoes, then shred them. Place the shredded potatoes in a beaker, add 1L of distilled water, and boil until the shredded potatoes are soft. Filter the mixture, add 20g of glucose and 20g of agar to the filtrate, boil until dissolved, and add water to bring the volume to 1000mL. Sterilize at 121℃ for 20 minutes and cool to 50℃ for later use.

[0038] (2) The antibacterial effect of essential oils was determined using the growth rate inhibition method:

[0039] Take ① 40 μg / mL of *Aristolochia debilis* essential oil, ② 40 μg / mL of *Ophiopogon japonicus* essential oil, and ③ 20 μg / mL of *Aristolochia debilis* essential oil and *Ophiopogon japonicus* essential oil at a 1:1 ratio and mix them well. Use PDA plates with an equal amount of sterile water as a control. Use a 6 mm diameter punch to make fungal cakes of wheat stem rot and potato early blight pathogens and place them in the center of the PDA medium. Each treatment has 3 replicates and is placed in a 28℃ constant temperature incubator for dark incubation.

[0040] The diameter of the pathogenic hyphae was measured after 3 and 7 days, and the hyphal growth inhibition rate was calculated. The formula for calculating the inhibition rate is as follows:

[0041] Inhibition rate = (control group hyphae diameter - treatment group hyphae diameter) / control group hyphae diameter × 100%.

[0042] (3) The experimental results are shown in Table 1 and Table 2:

[0043] Table 1. Antimicrobial effects of different treatments on wheat stem rot and potato early blight.

[0044]

[0045]

[0046] Table 2. Antimicrobial virulence of different treatments against pathogens causing wheat stem rot and potato rot.

[0047]

[0048] As shown in Tables 1 and 2, the essential oil combination of Aristolochia debilis and Ophiopogon japonicus has good control efficacy against wheat stem base rot and potato early blight, and the control efficacy is better than that of Aristolochia debilis and Ophiopogon japonicus essential oil alone. On day 3, the inhibition rates of the essential oil combination against wheat stem base rot and potato early blight were 70.90% and 74.59%, respectively; on day 7, the inhibition rates of the essential oil combination against wheat stem base rot and potato early blight were 93.27% and 88.13%, respectively.

[0049] Toxicity assays showed that the essential oil blend exhibited EC50-95% seroconversion rate against wheat stem rot and potato early blight. 50 The EC values ​​of 11.04 and 12.99 μg / mL, respectively, were both lower than those obtained by using Rhododendron simsii essential oil and Ophiopogon japonicus essential oil alone. 50 Based on the above bioassay results, this invention will further study the preparation of pesticide emulsions and suspensions using essential oil combinations.

[0050] Example 2:

[0051] This embodiment provides an agricultural antibacterial water-in-oil emulsion prepared from the essential oils of Rhododendron simsii and Ophiopogon japonicus, the specific preparation steps of which are as follows: Figure 1 As shown:

[0052] The oil phase is prepared by thoroughly mixing 10% *Rhododendron simsii* essential oil, 10% *Ophiopogon japonicus* essential oil, 20% ethanol, 5% Tween-80, and 7% agricultural emulsion 500. The mixture is then homogenized at high speed using a high-speed shear emulsifier at 12000 rpm. -1 Slowly add the aqueous phase dropwise to bring the volume to 100%. After the aqueous phase is completely added, shear for 15 minutes to obtain the water emulsion sample.

[0053] Example 3:

[0054] This embodiment provides an agricultural antibacterial suspension prepared from the essential oils of Rhododendron simsii and Ophiopogon japonicus, and the specific preparation steps are as follows: Figure 2 As shown:

[0055] (1) Add 10% of eucalyptus oil, 10% of ophiopogon japonicus oil, 5% of wetting agent T70, 2% of dispersant NP-10, 3% of dispersant D425, 0.15% of thickener xanthan gum, 0.5% of thickener magnesium aluminum silicate, 5% of antifreeze ethylene glycol, 1% of defoamer YS-0173, and ultrapure water to 100%, mix well, and set aside for use;

[0056] (2) After pre-dispersion by a high-speed shear emulsifier, it is then ground and pulverized by a sand mill to D. 90 ≤5μm is considered a suspension.

[0057] Test example:

[0058] (1) Select potted cucumber seedlings with uniform growth (at the two-leaf stage), and spray 10 mL of each dilution (800 times, 400 times, 200 times) of the drug solution with a sprayer. Use deionized water as a blank control and 40% carbendazim suspension as a positive control. Let them air dry naturally. After 24 hours, prepare the pre-cultured test colonies into mycelial cakes. Inoculate the mycelial cakes with the mycelium side down onto the cucumber leaves. Inoculate one mycelial cake per leaf, one plant per pot, and three pots per treatment, i.e., three replicates.

[0059] (2) After inoculation, the plants were cultured in a greenhouse and covered with plastic sheeting to keep them moist. When the lesions were clearly visible, the infection area of ​​gray mold on all leaves of each treatment plant was recorded after 7 days. The antibacterial effect was calculated based on the average infection area per leaf.

[0060] (3) The grading criteria for gray mold disease in cucumbers are as follows:

[0061] Level 0: No symptoms;

[0062] Grade 1: A few lesions appear on the inoculated leaves;

[0063] Grade 2: The lesions cover 1 / 3 of the leaf area;

[0064] Grade 3: The lesion area accounts for 1 / 3 to 1 / 2 of the leaf area;

[0065] Grade 4: The lesion area accounts for 1 / 2 to 2 / 3 of the leaf area;

[0066] Level 5: The lesion area covers more than 2 / 3 of the leaf area.

[0067] Disease index calculation:

[0068] Disease severity index (%) = [Σ(Disease level × Number of plants at that level) / (Total number of plants surveyed × Highest disease level)] × 100

[0069] Calculation of protective efficacy:

[0070] Prevention and control efficacy (%) = [(Control disease index - Treatment disease index) / Control disease index] × 100

[0071] (4) The results of the potted plant test are shown in Table 3.

[0072] Table 3. Control effects of different treatments on cucumber gray mold.

[0073]

[0074] The pot experiment described above shows that both the water-in-oil emulsion and suspension formulated with this essential oil combination have a certain control effect on cucumber gray mold. The water-in-oil emulsion, diluted 200 times, showed the best control effect, with an efficacy of 92.17%; the control effect at a dilution of 800 times was comparable to that of the carbendazim suspension, both exceeding 60%. Similarly, the suspension, diluted 200 times, showed the best control effect, with an efficacy of 91.87%; the control effect at a dilution of 800 times was comparable to that of the carbendazim suspension, both exceeding 60%. No adverse effects on cucumber leaves were observed during the experiment.

[0075] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, and for those of ordinary skill in the art, various changes, modifications, substitutions and variations can be made to these embodiments without departing from the principles and spirit of the present invention. Therefore, the present invention is not limited to the specific details without departing from the general concept defined by the claims and their equivalents.

Claims

1. A blend of essential oils from *Rhododendron simsii* and *Ophiopogon japonicus*, characterized in that, The essential oil blend is composed of a blend of Rhododendron simsii essential oil and Ophiopogon japonicus essential oil; the mass ratio of Rhododendron simsii essential oil to Ophiopogon japonicus essential oil is 1:0.5-1.5; the Rhododendron simsii essential oil and Ophiopogon japonicus essential oil are obtained by steam distillation respectively; In the preparation of eucalyptus essential oil, eucalyptus leaves are first soaked in a supramolecular solvent for 6 hours, and then prepared by steam distillation. The supramolecular solvent consists of 10% decanoic acid, 15% ethanol, and 75% water.

2. The essential oil blend according to claim 1, characterized in that, The specific operation of the steam distillation method is as follows: put the leaves of Rhododendron simsii and the crushed root of Ophiopogon japonicus into their respective distillation equipment, add water and heat to boiling, and distill for 1-3 hours to obtain Rhododendron simsii essential oil and Ophiopogon japonicus essential oil respectively.

3. The application of the essential oil combination according to claim 1, characterized in that, The essential oil combination is used as an antibacterial agent to prevent and control agricultural diseases, namely wheat stem base rot, potato early blight, and cucumber gray mold.

4. The application according to claim 3, characterized in that, The antibacterial agent is composed of an essential oil blend and excipients; the excipients include one or more of organic solvents, surfactants, dispersants, defoamers, thickeners, and antifreeze agents.

5. The application according to claim 3, characterized in that, The antibacterial agents are available in formulations including emulsions and suspensions.

6. The application according to claim 5, characterized in that, When the antibacterial agent is formulated as an emulsion, the emulsion comprises: 5-30% of *Eupatorium fortunei* essential oil, 5-30% of *Ophiopogon japonicus* essential oil; 10-60% of ethanol; 1-30% of emulsifier Tween-80, 1-20% of agricultural emulsion 500, and ultrapure water to make up to 100%.

7. The application according to claim 5, characterized in that, When the antibacterial agent is in the form of a suspension, the suspension comprises: 5-30% of *Eupatorium fortunei* essential oil, 5-30% of *Ophiopogon japonicus* essential oil; 1-5% of wetting agent T70, 1-10% of dispersant NP-10, 1-10% of dispersant D425, 0.1-5% of thickener xanthan gum, 0.1-5% of thickener magnesium aluminum silicate, 1-10% of antifreeze ethylene glycol, 0.1-5% of defoamer YS-0173, and ultrapure water to make up to 100%.

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