Anoectochilus formosanus open tissue culture efficient bacteriostatic agent based on natural plant extract
By preparing a highly efficient antibacterial agent for open tissue culture of Anoectochilus roxburghii based on natural plant extracts, the problems of environmental pollution and drug resistance caused by chemical pesticides have been solved, achieving a highly efficient and environmentally friendly antibacterial effect and the application of the finished product.
Patent Information
- Application Number
- CN202510862574.8
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-25
- Publication Date
- 2025-11-28
AI Technical Summary
Existing technologies using chemical pesticides to control leaf spot fungi, gray mold, and root rot fungi present problems such as environmental pollution and increased pathogen resistance. Therefore, there is a need to develop natural, efficient, and environmentally friendly antibacterial agents.
Using natural plant extracts such as Silphium styracifolium, Paris polyphylla, Vitex negundo, Fraxinus chinensis, Lysimachia christinae, and Ginkgo biloba nuts, a highly efficient antibacterial agent for open tissue culture of Anoectochilus roxburghii is prepared through reflux extraction, anaerobic fermentation, and vacuum distillation. This agent is then combined with waste sugar water and EM bacteria to form a complex plant-derived antibacterial agent.
It significantly improved the antibacterial effect against leaf spot fungus, gray mold, and root rot fungus, increased the seedling rate of Anoectochilus roxburghii tissue culture, reduced production costs, and avoided pesticide residues and resistance problems, meeting the requirements of green agriculture.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of plant tissue culture technology, and in particular relates to a highly efficient antibacterial agent for open tissue culture of Anoectochilus roxburghii based on natural plant extracts. Background Technology
[0002] Leaf spot fungi, gray molds, and root rot fungi are common and serious pathogens in agricultural production. Leaf spot fungi cause spots on plant leaves, severely affecting photosynthesis and reducing crop yield and quality; gray molds can infect the flowers, fruits, and leaves of various crops, causing rot and leading to significant yield reduction; root rot fungi damage plant roots, affecting water and nutrient absorption, and ultimately causing plant death. Currently, chemical pesticides are the main means of controlling these pathogens, but long-term use of chemical pesticides causes environmental pollution, pesticide residues, and increased pesticide resistance in pathogens. Therefore, developing natural, efficient, and environmentally friendly plant-derived antifungal agents is of significant practical importance.
[0003] Based on this, the present invention designs a highly efficient antibacterial agent for open tissue culture of Anoectochilus roxburghii based on natural plant extracts to solve the above problems. Summary of the Invention
[0004] To make the technical problems, technical solutions and advantages of the present invention clearer, a detailed description will be given below in conjunction with specific embodiments.
[0005] The specific technical solution of the present invention for a highly efficient antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts is as follows:
[0006] A highly effective antibacterial agent for open-culture *Anoectochilus roxburghii* based on natural plant extracts, characterized in that the natural plant extract is prepared from the following plants:
[0007]
[0008]
[0009] The antibacterial agent described herein has antibacterial effects against leaf spot fungi, gray mold, and root rot fungi.
[0010] Furthermore, the antibacterial agents extracted from natural plants are prepared from the following plants:
[0011]
[0012] The antibacterial agent described herein has antibacterial effects against leaf spot fungi, gray mold, and root rot fungi.
[0013] Furthermore, the natural plant extract includes the following steps:
[0014] Raw material pretreatment: Wash, dry and crush the following herbs: Silphium perfoliatum, Paris polyphylla, Vitex negundo, Fraxinus chinensis, Lysimachia christinae, and Ginkgo nuts, and pass them through a 40-60 mesh sieve.
[0015] Collect the filtrate: Add deionized water to the mixed powder, reflux at 70℃ for 2 hours, filter, collect the filtrate, and set aside the filter residue. Add 70% ethanol solution to the filter residue, reflux at 50℃~70℃ for 2~3 hours, filter, and obtain the mixed filtrate.
[0016] Anaerobic fermentation: Add 40g of waste sugar water to the mixed filtrate, then add 0.1% by weight of EM bacteria agent, and anaerobic ferment at 25℃ for 40 days in an anaerobic environment;
[0017] Extraction of concentrated liquid: Filter the fermentation broth with gauze, collect the filtrate, transfer the filtrate to a vacuum distillation apparatus, and concentrate it to 1 / 3 to 1 / 4 of the original volume at 40℃~50℃ to obtain concentrated liquid;
[0018] Refining: Add ethanol to the concentrate to achieve an ethanol concentration of 60%–80%, stir well, let stand for 36 hours, filter, and remove the precipitate. The filtrate is then subjected to vacuum distillation to remove the ethanol, yielding the refined plant antibacterial agent.
[0019] Furthermore, the reflux extraction temperature is 60°C, and the reflux extraction time is 2.5 hours.
[0020] Furthermore, the vacuum concentration temperature is 45°C, and the volume is concentrated to 1 / 3 of the original volume.
[0021] Furthermore, the ethanol concentration is 70%.
[0022] Furthermore, the powder is passed through a 50-mesh sieve.
[0023] Furthermore, the application of the antibacterial agent based on natural plant extracts in inhibiting leaf spot fungi, gray mold, and root rot fungi.
[0024] The present invention provides a highly effective antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts, which has the following advantages:
[0025] 1. Significant antibacterial effect: Natural plant extracts were prepared using *Silphium styracifolium*, *Paris polyphylla*, *Vitex negundo*, *Fraxinus chinensis*, *Lysimachia christinae*, and ginkgo nuts as raw materials. These natural plant extracts were then combined with waste sugar water and EM (Effective Microorganisms) to obtain an antibacterial agent. This antibacterial agent effectively improved the antibacterial efficiency of the plant extracts, exhibiting good inhibitory effects against leaf spot fungi, gray mold, and root rot fungi. It also significantly increased the seedling survival rate of *Anoectochilus roxburghii* tissue culture, reduced production costs, and can be widely applied in plant biotechnology fields such as rapid tissue culture propagation, seedling detoxification, and the establishment of regenerated plant lines.
[0026] 2. Natural and environmentally friendly: This antibacterial agent uses plants as raw materials and has natural ingredients. It will not cause pollution to the environment during use, nor will it cause pesticide residues in agricultural products to exceed the standard, which meets the requirements of green agriculture development.
[0027] 3. Less likely to develop drug resistance: Compared with chemically synthesized antibacterial agents, plant-derived antibacterial agents have complex and diverse components, making it difficult for pathogens to develop targeted drug resistance mechanisms, thus enabling sustainable and effective disease control. Detailed Implementation
[0028] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.
[0029] Example 1: Preparation of a Highly Effective Antibacterial Agent from Tissue Culture of Natural Plant Extracts
[0030] 1. Preparation of natural plant extracts:
[0031] S1 Take 20g of *Symplocos lucida*, 20g of *Paris polyphylla*, 15g of *Vitex negundo*, 15g of *Fraxinus chinensis*, 10g of *Lysimachia christinae*, and 10g of ginkgo nuts. Wash all the plant materials, then dry them in the sun. Finally, pulverize them using a Chinese medicine pulverizer and pass them through a 50-mesh sieve to obtain a mixed powder.
[0032] S2 Add ten times the weight of deionized water to the mixed powder, reflux at 70°C for 2 hours, filter, collect the filtrate, and keep the residue for later use.
[0033] S3 adds 8 times its weight of 70% ethanol solution to the filter residue, refluxes at 60°C for 2.5 hours, filters, and collects the filtrate.
[0034] 2. Preparation of antibacterial agents for high-efficiency open tissue culture of natural plants
[0035] S4 mixes the water ion extraction filtrate and the ethanol extraction filtrate to obtain a mixed filtrate.
[0036] S5 adds 40g of waste sugar water to the mixed filtrate, then adds 0.1% by weight of EM bacterial agent, and then puts it into a sealed container for anaerobic fermentation at 25℃ for 40 days.
[0037] After S6 fermentation is complete, the fermentation broth is filtered through gauze, the filtrate is collected, and the filtrate is transferred to a vacuum distillation apparatus and concentrated to 1 / 3 of the original volume at 45°C to obtain a concentrated solution.
[0038] S7 adds ethanol to the concentrate to achieve an ethanol concentration of 70%, stirs thoroughly, lets stand for 36 hours, filters, and removes the precipitate. The filtrate is then subjected to vacuum distillation to remove the ethanol, yielding a refined plant antibacterial agent.
[0039] Comparative Example 1
[0040] 1. Preparation of natural plant extracts:
[0041] S1 Take 40g of *Symplocos lucida*, 40g of *Paris polyphylla*, and 30g of *Vitex negundo*. Wash all the plant materials, dry them in the sun, and then pulverize them using a Chinese medicine pulverizer. Pass the powder through a 60-mesh sieve to obtain a mixed powder.
[0042] S2 Add ten times the weight of deionized water to the mixed powder, reflux at 70°C for 2 hours, filter, collect the filtrate, and keep the residue for later use.
[0043] S3 adds 8 times its weight of 70% ethanol solution to the filter residue, refluxes at 70°C for 3 hours, filters, and collects the filtrate.
[0044] 2. Preparation of antibacterial agents for high-efficiency open tissue culture of natural plants
[0045] S4 mixes the water ion extraction filtrate and the ethanol extraction filtrate to obtain a mixed filtrate.
[0046] S5 adds 40g of waste sugar water to the mixed filtrate, then adds 0.1% by weight of EM bacterial agent, and then puts it into a sealed container for anaerobic fermentation at 25℃ for 40 days.
[0047] After S6 fermentation is completed, the fermentation broth is filtered with gauze, the filtrate is collected, and the filtrate is transferred to a vacuum distillation apparatus and concentrated to 1 / 3 of the original volume at 50°C to obtain a concentrated solution.
[0048] S7 adds ethanol to the concentrate to achieve an ethanol concentration of 80%, stirs thoroughly, lets stand for 36 hours, filters, and removes the precipitate. The filtrate is then subjected to vacuum distillation to remove the ethanol, yielding a refined plant antibacterial agent.
[0049] Comparative Example 2
[0050] 1. Preparation of natural plant extracts:
[0051] S1 Take 20g of *Symplocos lucida*, 20g of *Paris polyphylla*, 15g of *Vitex negundo*, and 30g of *Fraxinus chinensis*. Wash all the plant materials, dry them in the sun, and then pulverize them using a Chinese medicine pulverizer. Pass the powder through a 40-mesh sieve to obtain a mixed powder.
[0052] S2 Add ten times the weight of deionized water to the mixed powder, reflux at 70°C for 2 hours, filter, collect the filtrate, and keep the residue for later use.
[0053] S3 adds 8 times its weight of 70% ethanol solution to the filter residue, refluxes at 50°C for 2 hours, filters, and collects the filtrate.
[0054] 2. Preparation of antibacterial agents for high-efficiency open tissue culture of natural plants
[0055] S4 mixes the water ion extraction filtrate and the ethanol extraction filtrate to obtain a mixed filtrate.
[0056] S5 adds 40g of waste sugar water to the mixed filtrate, then adds 0.1% by weight of EM bacterial agent, and then puts it into a sealed container for anaerobic fermentation at 25℃ for 40 days.
[0057] After S6 fermentation is complete, the fermentation broth is filtered through gauze, the filtrate is collected, and the filtrate is transferred to a vacuum distillation apparatus and concentrated to 1 / 3 of the original volume at 45°C to obtain a concentrated solution.
[0058] S7 adds ethanol to the concentrate to achieve an ethanol concentration of 70%, stirs thoroughly, lets stand for 36 hours, filters, and removes the precipitate. The filtrate is then subjected to vacuum distillation to remove the ethanol, yielding a refined plant antibacterial agent.
[0059] Comparative Example 3
[0060] 1. Preparation of natural plant extracts:
[0061] S1 Take 20g of *Symplocos lucida*, 20g of *Paris polyphylla*, 15g of *Vitex negundo*, and 20g of *Lysimachia christinae*. Wash all the plant materials, dry them in the sun, and then pulverize them using a Chinese medicine pulverizer. Pass the powder through a 50-mesh sieve to obtain a mixed powder.
[0062] S2 Add ten times the weight of deionized water to the mixed powder, reflux at 70°C for 2 hours, filter, collect the filtrate, and keep the residue for later use.
[0063] S3 adds 8 times its weight of 70% ethanol solution to the filter residue, refluxes at 60°C for 2.5 hours, filters, and collects the filtrate.
[0064] 2. Preparation of antibacterial agents for high-efficiency open tissue culture of natural plants
[0065] S4 mixes the water ion extraction filtrate and the ethanol extraction filtrate to obtain a mixed filtrate.
[0066] S5 adds 40g of waste sugar water to the mixed filtrate, then adds 0.1% by weight of EM bacterial agent, and then puts it into a sealed container for anaerobic fermentation at 25℃ for 40 days.
[0067] After S6 fermentation is complete, the fermentation broth is filtered through gauze, the filtrate is collected, and the filtrate is transferred to a vacuum distillation apparatus and concentrated to 1 / 4 of the original volume at 40°C to obtain a concentrated solution.
[0068] S7 adds ethanol to the concentrate to achieve an ethanol concentration of 70%, stirs thoroughly, lets stand for 36 hours, filters, and removes the precipitate. The filtrate is then subjected to vacuum distillation to remove the ethanol, yielding a refined plant antibacterial agent.
[0069] Comparative Example 4
[0070] 1. Preparation of natural plant extracts:
[0071] S1 Take 20g of *Symplocos lucida*, 20g of *Paris polyphylla*, 15g of *Vitex negundo*, and 20g of ginkgo nuts. Wash all the plant materials, dry them in the sun, and then grind them using a Chinese medicine pulverizer. Pass the powder through a 50-mesh sieve to obtain a mixed powder.
[0072] S2 Add ten times the weight of deionized water to the mixed powder, reflux at 70°C for 2 hours, filter, collect the filtrate, and keep the residue for later use.
[0073] S3 adds 8 times its weight of 70% ethanol solution to the filter residue, refluxes at 60°C for 2.5 hours, filters, and collects the filtrate.
[0074] 2. Preparation of antibacterial agents for high-efficiency open tissue culture of natural plants
[0075] S4 mixes the water ion extraction filtrate and the ethanol extraction filtrate to obtain a mixed filtrate.
[0076] S5 adds 40g of waste sugar water to the mixed filtrate, then adds 0.1% by weight of EM bacterial agent, and then puts it into a sealed container for anaerobic fermentation at 25℃ for 40 days.
[0077] After S6 fermentation is complete, the fermentation broth is filtered through gauze, the filtrate is collected, and the filtrate is transferred to a vacuum distillation apparatus and concentrated to 1 / 3 of the original volume at 45°C to obtain a concentrated solution.
[0078] S7 adds ethanol to the concentrate to achieve an ethanol concentration of 60%, stirs thoroughly, lets stand for 36 hours, filters, and removes the precipitate. The filtrate is then subjected to vacuum distillation to remove the ethanol, yielding a refined plant antibacterial agent.
[0079] Example 2: Antibacterial effect of natural plant tissue culture agents on bacterial cells
[0080] The antibacterial effects of the natural plant tissue culture antibacterial agents prepared in Example 1 and Comparative Examples 1-4 on bacteria (leaf spot fungi, gray mold, root rot fungi) in tissue culture contamination were studied.
[0081] The details are as follows:
[0082] Antibacterial activity test: Take an Oxford cup with dimensions of Φ8mm×7mm×11mm (outer diameter×inner diameter×height), autoclave at 125℃, dry and cool for later use. In a clean bench, separately coat leaf spot fungus, gray mold, and root rot fungus onto the surface of LB petri dishes (100mm diameter), each containing 200μl of a 10% concentration. 6 CFU / ml of test bacteria. Oxford cups were placed on the surface of each petri dish, and 250 μl of the antibacterial agent for plant tissue culture prepared in Example 1 was added to the Oxford cup. The dishes were then incubated at 35°C for 5 to 6 days. Each group was repeated in 3 replicates. LB petri dishes inoculated with bacterial cells but without antibacterial agent were used as controls.
[0083] The antibacterial effect was verified as follows:
[0084] The inhibition zone test results showed that the Oxford cup of the antibacterial agent for high-efficiency culture of natural plant open tissue prepared in Example 1 had obvious inhibition zones; the diameters of the inhibition zones against leaf spot fungus, gray mold, and root rot fungus were 18 mm, 20 mm, and 16 mm, respectively; the minimum inhibitory concentration (MIC) test results showed that the MICs against leaf spot fungus, gray mold, and root rot fungus were 0.5 mg / mL, 0.4 mg / mL, and 0.6 mg / mL, respectively.
[0085] The specific antibacterial effect data are shown in Table 1.
[0086] Table 1 shows the results of antibacterial treatment of different bacterial strains using the antibacterial agents for plant tissue culture prepared in Example 1 and Comparative Examples 1-4:
[0087]
[0088] Note: Indicates: Mycelial ring diameter 8-10mm, mildly sensitive; Indicates: Mycelial ring diameter 10-14mm, moderately sensitive; Indicates: The mycelial ring diameter is 14-20mm, indicating high sensitivity; Indicates: The diameter of the mycelial ring is >20mm, indicating extreme sensitivity.
[0089] Example 3: Antibacterial effect of natural plant tissue culture antibacterial agent on leaf spot fungus
[0090] In a clean bench, leaf spot fungus was applied to the surface of *Anoectochilus roxburghii* seedlings (tissue culture seedlings obtained through tissue culture), and then inoculated into culture media containing or without the antibacterial agent for natural plant tissue culture prepared in Example 1. The culture was then placed at 35°C, under light for 10 hours / day, and with a light intensity of 2500 lx for 4-5 days.
[0091] Experimental results: The tissue culture seedlings of Anoectochilus roxburghii containing antibacterial agents were significantly less contaminated by leaf spot fungi.
[0092] Example 4: Antibacterial effect of natural plant tissue culture antifungal agent against Botrytis cinerea
[0093] In a clean bench, apply gray mold to the surface of *Anoectochilus roxburghii* seedlings (tissue culture seedlings obtained through tissue culture), and inoculate them into culture media containing or without the antibacterial agent for natural plant tissue culture prepared in Example 1. Incubate at 35°C, under 10h / d light and 2500lx light intensity for 4-5 days.
[0094] Experimental results: The tissue culture seedlings of Anoectochilus roxburghii containing antibacterial agents were significantly less contaminated by Botrytis cinerea.
[0095] Example 5: Antibacterial effect of natural plant tissue culture antibacterial agent on root rot fungi. Root rot fungi were applied to the surface of *Anoectochilus roxburghii* seedlings (tissue culture seedlings obtained through tissue culture) in a clean bench. The seedlings were then inoculated into culture media containing or without the natural plant tissue culture antibacterial agent prepared in Example 1, and cultured at 35°C, under a light intensity of 2500 lx for 4-5 days with a light intensity of 10 h / d.
[0096] Experimental results: The tissue culture seedlings of Anoectochilus roxburghii containing antibacterial agents were significantly less contaminated by root rot fungi.
[0097] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit it. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that after reading this application specification, they can still modify or make equivalent substitutions to the specific implementation of the present invention, but these modifications or changes do not depart from the protection scope of the pending claims of the present invention.
Claims
1. A highly effective antibacterial agent for open-culture *Anoectochilus roxburghii* based on natural plant extracts, characterized in that, Natural plant extracts are prepared from the following plants: The antibacterial agent described herein has antibacterial effects against leaf spot fungi, gray mold, and root rot fungi.
2. The highly effective antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts according to claim 1, characterized in that, Natural plant-derived antibacterial agents are prepared from the following plants: The antibacterial agent described herein has antibacterial effects against leaf spot fungi, gray mold, and root rot fungi.
3. A highly effective antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts as described in claim 1 or 2, characterized in that, The natural plant extract includes the following steps: Raw material pretreatment: Wash, dry and crush the following herbs: Silphium perfoliatum, Paris polyphylla, Vitex negundo, Fraxinus chinensis, Lysimachia christinae, and Ginkgo nuts, and pass them through a 40-60 mesh sieve. Collect the filtrate: Add deionized water to the mixed powder, reflux at 70℃ for 2 hours, filter, collect the filtrate, and set aside the filter residue. Add 70% ethanol solution to the filter residue, reflux at 50℃~70℃ for 2~3 hours, filter, and obtain the mixed filtrate. Anaerobic fermentation: Add 40g of waste sugar water to the mixed filtrate, then add 0.1% by weight of EM bacteria agent, and anaerobic ferment at 25℃ for 40 days in an anaerobic environment; Extraction of concentrated liquid: Filter the fermentation broth with gauze, collect the filtrate, transfer the filtrate to a vacuum distillation apparatus, and concentrate it to 1 / 3 to 1 / 4 of the original volume at 40℃~50℃ to obtain concentrated liquid; Refining: Add ethanol to the concentrate to achieve an ethanol concentration of 60%–80%, stir well, let stand for 36 hours, filter, and remove the precipitate. The filtrate is then subjected to vacuum distillation to remove the ethanol, yielding the refined plant antibacterial agent.
4. The highly effective antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts according to claim 3, characterized in that, The reflux extraction temperature is 60℃, and the reflux extraction time is 2.5 hours.
5. The highly effective antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts according to claim 3, characterized in that, The vacuum concentration temperature is 45℃, and the volume is concentrated to 1 / 3 of the original volume.
6. The highly effective antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts according to claim 3, characterized in that, The ethanol concentration is 70%.
7. The highly effective antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts according to claim 3, characterized in that, The powder is passed through a 50-mesh sieve.
8. A highly effective antibacterial agent for open tissue culture of *Anoectochilus roxburghii* based on natural plant extracts according to claims 1-7, characterized in that, The application of the antibacterial agent based on natural plant extracts in inhibiting leaf spot fungi, gray mold, and root rot fungi.