A traditional Chinese medicine composition, a biocontrol preparation and application in the field of prevention and treatment of pearl leaf spot nematode disease

Abstract

The application belongs to the technical field of biocontrol preparation, and particularly relates to a traditional Chinese medicine composition, a biocontrol preparation and application in the field of pearl plant leaf spot nematode disease prevention and treatment. The leaf spot nematode disease is a main disease and pest of pearl plant planting, and there is no effective prevention and treatment method at present. The application provides a traditional Chinese medicine composition, which comprises wolfberry, leigongteng, peppermint, phellodendron, boerhaavia diffusa and chamomile. Water extracts of the above-mentioned medicinal ingredients can effectively reduce the number of nematodes in soil and improve the disease resistance of pearl plants when used for pearl plant irrigation. The research results of the application are helpful for restraining the occurrence of pearl plant leaf spot nematode disease through biological prevention and control means, and provide theoretical guidance for the development of pearl plant green industry.

Description

Technical Field

[0001] This invention belongs to the field of biocontrol agents, specifically relating to a traditional Chinese medicine composition, a biocontrol agent, and its application in the control of leaf spot nematode disease in *Philodendron chinense*. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Pearl Orchid ( Chloranthus spicatus (Thunb.) Makino Also known as Golden Chrysanthemum Orchid, Pearl Orchid, and Chicken Claw Orchid, it is a perennial evergreen herbaceous plant belonging to the genus *Chrysanthemum* in the family Chrysanthemumaceae. The inflorescence of *Chrysanthemum* is a conical shape, with small, pearl-like flowers that are ivory-yellow in color and have a delicate, rich fragrance. Due to its unique fragrance, *Chrysanthemum* flowers have been used to scent tea since the Qing Dynasty, organically combining the famous tea with the floral aroma to create *Chrysanthemum* flower tea, a superior product in color, aroma, and taste. It is considered one of the "Four Great Scented Flowers for Tea in China," along with jasmine, magnolia, and bitter orange. *Chrysanthemum* flower tea also has beautifying, calming, nourishing, pain-relieving, and weight-loss effects, making it particularly popular among female consumers. Furthermore, the stems, leaves, and roots of *Chrysanthemum* can be used medicinally, possessing properties that dispel wind and dampness, promote bone healing, break up accumulations, expel parasites, relieve pain, and stop bleeding.

[0004] However, with the increasing planting density of *Chloranthus spicatus*, pests and diseases occur frequently. Leaf spot nematode disease, caused by nematodes, is a major threat to *Chloranthus spicatus* cultivation. Nematodes invade the leaves of *Chloranthus spicatus* without damage through stomata. In the early stages of the disease, the leaf spots are pale yellow, later turning reddish-brown. The spots gradually enlarge, eventually becoming dark brown. Diseased leaves easily fall off prematurely. Infected plants produce small and few flowers, and severely infected plants lose most of their leaves, significantly impacting yield. Furthermore, nematodes spread extremely quickly within *Chloranthus spicatus* leaves and have the ability to actively infect. During the rainy months of March to May, nematodes can move rapidly with the help of water flow or spread through rain splash. The incidence of diseased leaves decreases from October to December, which is related to the nematodes gradually entering overwintering. Nematodes can overwinter in the axillary buds and dead diseased leaves of *Chloranthus spicatus*. If infected leaves that have not yet fallen off are not removed in time, the nematodes overwintering in the axillary buds or dead leaves will infect healthy plants the following year, causing a severe reduction in *Chloranthus spicatus* flower yield. The leaf spot nematode of *Chloranthus spicatus* is highly damaging and spreads rapidly. Once a plant is infected, the only way to remove it is by uprooting the entire plant, resulting in severe economic losses. To date, research on the pathogenesis of leaf spot nematode disease in *Chloranthus spicatus* is still lacking both domestically and internationally.

[0005] Chemical control has long been the primary means of controlling nematode diseases. Herbs such as *Euphorbia fischeriana*, *Phellodendron amurense*, *Euphorbia helioscopia*, and *Sophora flavescens* contain various types of bioactive substances (such as phenylpropenoids, lignins, coumarins, flavonoids, and terpenoids), which are highly effective in controlling crop pests and diseases, exhibiting strong killing activity against cabbage caterpillars, diamondback moths, aphids, and nematodes. Currently, research on the control of *Phyllostachys edulis* leaf spot nematode disease using traditional Chinese medicine is still in its infancy. Summary of the Invention

[0006] In view of the above-mentioned research status, this invention aims to improve the control effect of *Chloranthus spicatus* leaf spot nematode disease by applying biocontrol agents, and to expand the green and efficient control methods for *Chloranthus spicatus*. Specifically, it provides a traditional Chinese medicine composition and biocontrol agent with control effect on *Chloranthus spicatus* leaf spot nematode disease, and the specific scheme is as follows: In a first aspect, a traditional Chinese medicine composition is provided, comprising the following components in parts by weight: 25-35 parts of Euphorbia fischeriana, 18-22 parts of Tripterygium wilfordii, 8-12 parts of Mentha haplocalyx, 6-10 parts of Phellodendron chinense, 4-6 parts of Angelica sinensis, and 4-6 parts of Chrysanthemum indicum.

[0007] This invention first provides a traditional Chinese medicine composition for the prevention and treatment of diseases related to nematode infestation. The core ingredients of this composition are *Euphorbia fischeriana*, *Tripterygium wilfordii*, and *Boletus edulis*. The main active components of these three herbs are alkaloids, which are toxic to plant nematodes. *Mentha haplocalyx*, *Phellodendron chinense*, and *Chamomile* assist plant recovery and enhance the preventative effect. This invention has verified that irrigating infected *Chloranthus spicatus* with extracts of the above-mentioned herbs can effectively reduce the number of nematodes in the soil, while also increasing the chlorophyll and soluble sugar content in the plants, thus enhancing their disease resistance.

[0008] The sources and effects of each medicinal ingredient in the above composition are as follows: Wolfsbane, also known as Gelsemium elegans, is a plant of the Euphorbiaceae family. Euphorbia ebracteolata Hayata or Euphorbia lathyris Euphorbia fischeriana The dried root of Steud. is processed as raw wolfsbane, removing impurities, washing, moistening, slicing, and sun-drying. It is pungent and neutral in nature; toxic, and enters the liver and spleen meridians. It has the effects of dispersing nodules and killing parasites. In this invention, wolfsbane is used as the principal ingredient. Its alkaloids (such as gelsminoides) are highly toxic to plant nematodes, directly killing adult nematodes, larvae, and eggs in the soil, and are the source of the core active ingredient targeting nematodes.

[0009] Tripterygium wilfordii, a plant belonging to the Celastraceae family. Tripterygium wilfordiThe dried root of *Hook.f.*. Its properties are bitter, pungent, and cool; it is highly toxic. It enters the liver and kidney meridians. It has the effects of dispelling wind and dampness, promoting blood circulation and unblocking collaterals, reducing swelling and relieving pain, and killing parasites and detoxifying. It is mainly used for rheumatoid arthritis, rheumatic arthritis, glomerulonephritis, nephrotic syndrome, lupus erythematosus, leprosy, and scabies. (Refer to Shandong Provincial Standard for Traditional Chinese Medicine - 2012 Edition). In this invention, *Tripterygium wilfordii* is used as an assistant herb, containing triptolide and other components, which not only have a significant toxic effect on nematodes but also inhibit nematode reproduction, synergistically enhancing the nematode-killing efficacy with *Euphorbia fischeriana*.

[0010] Mint, a plant belonging to the Lamiaceae family. Mentha haplocalyx The dried aerial parts of *Briquette*. It is pungent and cool in nature, and enters the lung and liver meridians. It has the effects of dispersing wind-heat, clearing the head and eyes, relieving sore throat, promoting rash eruption, and soothing the liver and regulating qi. It is used for wind-heat colds, early stages of wind-heat syndrome, headache, red eyes, sore throat, mouth ulcers, urticaria, measles, and chest and rib distension. In this invention, the volatile components such as menthol in peppermint can, on the one hand, enhance the permeability of the medicinal solution, helping the nematicidal components to better penetrate the soil and contact nematodes; on the other hand, it can improve the soil microenvironment, reducing the conditions suitable for nematode survival.

[0011] Phellodendron bark, this product is the yellow bark tree (Phellodendron amurense), a plant of the Rutaceae family. Phellodendron chinense Dried bark of Schneid. It is bitter and cold in nature. It enters the kidney and bladder meridians. It has the effects of clearing heat and drying dampness, purging fire and eliminating steaming heat, detoxifying and healing sores. It is used for damp-heat diarrhea, jaundice with dark urine, leukorrhea and vulvar itching, painful urination due to heat, beriberi with weakness and paralysis, bone steaming fever, night sweats, seminal emission, sores and carbuncles, eczema and damp sores. Salt-processed Phellodendron bark nourishes yin and reduces fire. It is used for yin deficiency with excessive fire, night sweats and bone steaming fever. In this invention, the alkaloids and flavonoids contained in Phellodendron bark have the combined effects of killing nematodes and enhancing the immunity and stress resistance of *Symplocos edulis* plants, reducing the severity of disease after nematode infection, and promoting the recovery and growth of affected plants.

[0012] Boluohui, the poppy pod plant Boluohui. Macleaya cordata The dried whole herb of (Willd.) R.Br. It is bitter, pungent, and cold in nature, highly toxic, and enters the liver and lung meridians. It has the functions of dispersing blood stasis and relieving pain, clearing heat and detoxifying, and dispelling wind and killing parasites. It is used for traumatic injuries, rheumatic pain, carbuncles and boils, stubborn tinea, eczema, and insect and snake bites (refer to Hubei Provincial Standard for Quality of Traditional Chinese Medicine - 2018). In this invention, *Bo Luo Hui* is used as an adjuvant. Its alkaloids, such as sanguisorbin and celandine, have contact and stomach poison effects on various plant nematodes, supplementing the nematode-killing spectrum of the principal and assistant herbs and reducing nematode resistance.

[0013] Chamomile, also known as chamomile, is a plant belonging to the Asteraceae family. Matricaria chamomillaThe dried whole herb of *L.*. It has the effects of tonifying nerves, relieving pain and swelling, inducing sweating and bowel movements, and promoting urination and treating dysmenorrhea. It is used for abnormal putrefactive bodily fluids, persistent headaches, constipation, difficulty sweating, urinary retention, and amenorrhea (refer to the Xinjiang Traditional Chinese Medicine Uyghur Medicinal Herbs Processing Specifications (2010 Edition)). In this invention, chamomile contains volatile oils, flavonoids, and other components, which can directly damage the body wall of leaf spot nematodes and interfere with their metabolism and reproduction; it can also induce *Chloranthus spicatus* to synthesize disease-resistant substances and maintain photosynthetic nutrient absorption; at the same time, it optimizes the soil microecology, indirectly inhibiting nematode survival, thereby controlling *Chloranthus spicatus* leaf spot nematode disease.

[0014] Unless otherwise specified, the medicinal parts, properties, meridian tropism, and indications of the above-mentioned medicinal ingredients are all based on the 2025 edition of the Chinese Pharmacopoeia, Part I. The above formulation underwent thorough toxicity studies before being applied to diseased plants. Rat and mouse toxicity tests confirmed that the above-mentioned drug combination did not exhibit biological toxicity and would not cause adverse effects on production and users.

[0015] In one embodiment of the present invention, the weight proportions of each medicinal ingredient in the above-mentioned traditional Chinese medicine composition are as follows: Wolfsbane 28-32 parts, Tripterygium wilfordii 19-21 parts, Mentha haplocalyx 9-11 parts, Phellodendron amurense 7-9 parts, Bo Luo Hui 4-6 parts, and Chamomile 4-6 parts.

[0016] The most effective method is to use 30 parts of wolfsbane, 20 parts of tripterygium wilfordii, 10 parts of peppermint, 8 parts of phellodendron bark, 5 parts of clematis root, and 5 parts of chamomile.

[0017] In a second aspect, a biocontrol preparation is provided, the biocontrol preparation comprising a traditional Chinese medicine liquid prepared from the traditional Chinese medicine composition described in the first aspect, wherein the traditional Chinese medicine liquid is at a pharmaceutically acceptable active dose.

[0018] The aforementioned herbal liquid is an aqueous extract, alcoholic extract, water-soluble extract of the herbal composition of the first aspect, or water-soluble powder. In one embodiment verified by the present invention, the herbal liquid is an aqueous extract of the herbal composition, prepared as follows: *Euphorbia fischeriana*, *Tripterygium wilfordii*, *Phellodendron chinense*, and *Botrytis cinerea* are extracted with water, filtered, and the filtrate is retained. The filtrate is concentrated to obtain a concentrated solution. *Mentha haplocalyx* and *Chrysanthemum indicum* are added and soaked at 30-50°C for 30-50 minutes. Solid residue is filtered off to obtain the herbal liquid.

[0019] In some specific embodiments, the preparation method of the traditional Chinese medicine liquid is as follows: (1) First decoction: Put the wolfsbane, tripterygium wilfordii, phellodendron bark, and balsam pear back into the extraction tank, add 6 to 10 times the amount of distilled water (based on the total weight of the herbs), and soak for 20 to 40 minutes. First bring to a boil over high heat, then reduce to a simmer and decoct for 1 to 2 hours. Filter and collect the first filtrate.

[0020] (2) Second decoction: Add 3 to 6 times the amount of distilled water to the dregs, bring to a boil over high heat, then simmer over low heat for 0.5 to 1.5 hours, and filter again to collect the second filtrate.

[0021] (3) Combining and concentrating: Mix the two filtrates and concentrate them at 50~60℃ and vacuum degree 0.06-0.08MPa until the relative density of the filtrate reaches 1.05-1.10 (tested at 60℃). Stop the concentration to obtain the concentrated solution.

[0022] (4) Compound preparation: Add peppermint and chamomile to the concentrated liquid and soak at a constant temperature of 30-50℃ for 30-50 minutes. After soaking, filter to remove solid residue and obtain the Chinese medicine liquid, which can be spray-dried to obtain the corresponding powder.

[0023] In some embodiments of the present invention, the above-mentioned biocontrol agent further includes a carrier. The herbal liquid is mixed with the carrier and then applied to the soil around the roots of *Chloranthus spicatus* to retain the herbal liquid and prolong its release time. The carrier is an organic carrier, an inorganic carrier, or a synthetic / semi-synthetic carrier.

[0024] Among them, the aforementioned organic carriers mainly include decomposed organic fertilizers, plant-derived carriers, and by-products of agricultural and sideline product processing. Further examples include decomposed chicken manure, cow manure, sheep manure, compost, straw powder, sawdust, rice husk powder, coconut coir, decomposed sawdust, soybean meal powder, peanut cake powder, cottonseed cake powder, corn cob powder, wheat bran, etc.

[0025] The aforementioned inorganic carriers mainly include mineral carriers and inert carriers; further examples include talc, bentonite, diatomaceous earth, kaolin, zeolite powder, vermiculite, perlite, and quartz sand.

[0026] The aforementioned synthetic / semi-synthetic carriers include polymer carriers, porous material carriers, and further, sodium carboxymethyl cellulose (CMC), sodium alginate, chitosan, polyacrylamide, porous starch, activated carbon, porous ceramics, etc.

[0027] Thirdly, the application of the traditional Chinese medicine composition described in the first aspect or the biocontrol agent described in the second aspect in the field of controlling leaf spot nematode disease of *Plectranthus chinensis* is provided.

[0028] The application method includes any of the following: 1) Pearl Orchid plants used for prevention, improvement, or treatment of leaf spot nematode infection; 2) Used to prepare fertilizers, pesticides, etc. involved in the cultivation of pearl orchid.

[0029] In the above 2), when preparing pesticides, the pesticides are applied as follows: water once every 7 to 10 days, and apply continuously for 2 to 3 times. When watering, slowly pour the pesticide into the soil around the roots of the plant to ensure that the pesticide penetrates into the soil layer where the nematodes are active (5 to 15 cm deep).

[0030] Fourthly, the application of the traditional Chinese medicine composition described in the first aspect or the biocontrol agent described in the second aspect in increasing the yield of *Chlorophytum comosum* flowers is provided.

[0031] Compared with the prior art, the beneficial effects of the present invention are: Leaf spot nematode disease poses a serious threat to the cultivation of Chloranthus spicatus, and there are currently no effective control measures. This invention first provides a traditional Chinese medicine composition, the water extract of which contains abundant alkaloids, which can kill nematodes and inhibit their reproduction. In addition, the above composition also contains auxiliary ingredients to help the liquid penetrate and enhance the plant's disease resistance, effectively overcoming leaf spot nematode disease in Chloranthus spicatus.

[0032] In addition, the present invention has found that the above-mentioned Chinese herbal medicine composition can not only inhibit the leaf spot nematode disease of Chloranthus spicatus, but also effectively prolong the flowering period of Chloranthus spicatus, increase the number of flowers per plant, and improve the yield of Chloranthus spicatus flowers, which is expected to improve the economic benefits of Chloranthus spicatus cultivation. Attached Figure Description

[0033] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0034] Figure 1 The effect of biocontrol agents on the number of nematodes in the soil of *Chloranthus spicatus* cultivation; Figure 2 The effect of biocontrol agents on chlorophyll content in leaves of *Chlorophytum comosum*; Figure 3 The effect of biocontrol agents on malondialdehyde content in leaves of *Chloranthus spicatus*; Figure 4 The effect of biocontrol agents on the soluble sugar content in *Chloranthus spicatus* leaves. Detailed Implementation

[0035] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0036] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0037] In the context of this specification, the word "comprising" is considered to mean "especially including". It should not be interpreted as "consisting of only".

[0038] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0039] Example 1 In this embodiment, a traditional Chinese medicine composition for the prevention and control of leaf spot nematode disease in *Plectranthus verticillata* is provided, comprising the following ingredients in parts by weight: 30 parts of *Euphorbia fischeriana*, 20 parts of *Tripterygium wilfordii*, 10 parts of *Mentha haplocalyx*, 8 parts of *Phellodendron chinense*, 5 parts of *Botrytis cinerea*, and 5 parts of *Chrysanthemum indicum*.

[0040] In this embodiment, a biocontrol preparation based on the above-mentioned traditional Chinese medicine composition is also provided, and the preparation method is as follows: 1. Raw material pretreatment: (1) Screening of medicinal materials: Remove moldy and insect-infested parts of wolfsbane, thunder vine, and clematis; remove impurities from phellodendron bark and chamomile; and retain the intact stems and leaves of mint.

[0041] (2) Graded crushing: Wolfsbane, Tripterygium wilfordii and Phellodendron amurense are crushed to 30 mesh (particle diameter about 0.55 mm); peppermint and clematis are cut into 1-2 cm pieces, and chamomile is kept in its original state.

[0042] 2. Extraction and Concentration (1) First decoction: Put the wolfsbane, tripterygium wilfordii, phellodendron bark, and balsam pear back into the extraction tank, add 8 times the amount of distilled water (based on the total weight of the herbs), and soak for 30 minutes. First bring to a boil over high heat, then reduce to a simmer and decoct for 1.5 hours. Filter through a 200-mesh sieve and collect the first filtrate.

[0043] (2) Second decoction: Add 5 times the amount of distilled water to the dregs, bring to a boil over high heat, then simmer over low heat for 1 hour, and filter again to collect the second filtrate.

[0044] (3) Combining and concentrating: Mix the two filtrates and concentrate them at 55℃ and a vacuum of 0.06-0.08MPa until the relative density of the filtrate reaches 1.05-1.10 (tested at 60℃). Stop the concentration to obtain the concentrated solution.

[0045] (4) Compound preparation: Add peppermint pieces and chamomile to the concentrated liquid and soak at a constant temperature of 40°C for 40 minutes, stirring once every 10 minutes during the process. After soaking, filter with a 150-mesh filter to remove solid residue and obtain the Chinese medicine liquid, which can be spray-dried to obtain the corresponding powder.

[0046] Example 2 In this embodiment, another traditional Chinese medicine composition for the prevention and control of leaf spot nematode disease in *Plectranthus verticillata* is provided, comprising the following ingredients in parts by weight: 25 parts of *Euphorbia fischeriana*, 18 parts of *Tripterygium wilfordii*, 8 parts of *Mentha haplocalyx*, 6 parts of *Phellodendron chinense*, 4 parts of *Botrytis cinerea*, and 4 parts of *Chrysanthemum indicum*.

[0047] In this embodiment, a biocontrol preparation based on the above-mentioned traditional Chinese medicine composition is also provided, and the preparation method is as follows: 1. Raw material pretreatment: (1) Screening of medicinal materials: Remove moldy and insect-infested parts of wolfsbane, thunder vine, and clematis; remove impurities from phellodendron bark and chamomile; and retain the intact stems and leaves of mint.

[0048] (2) Graded grinding: Wolfsbane, Tripterygium wilfordii and Phellodendron amurense are ground to 30 mesh (particle diameter about 0.55 mm) to facilitate the dissolution of active ingredients; peppermint and clematis are cut back to 1-2 cm small pieces, and chamomile is kept in its original state (to avoid high temperature damage to active ingredients).

[0049] 2. Extraction and Concentration (1) First decoction: Put the wolfsbane, tripterygium wilfordii, phellodendron bark and balsamina back into the extraction tank, add 6 times the amount of distilled water (based on the total weight of the herbs), and soak for 20 minutes. First bring to a boil over high heat, then reduce to a simmer and decoct for 1 hour. Filter through a 200-mesh sieve and collect the first filtrate.

[0050] (2) Second decoction: Add 3 times the amount of distilled water to the dregs, bring to a boil over high heat, then simmer over low heat for 0.5 hours, and filter again to collect the second filtrate.

[0051] (3) Combining and concentrating: Mix the two filtrates and concentrate them at 50°C and a vacuum of 0.06-0.08 MPa until the relative density of the filtrate reaches 1.05-1.10 (tested at 60°C). Stop the concentration to obtain the concentrated solution.

[0052] (4) Compound preparation: Add peppermint pieces and chamomile to the concentrated liquid and soak at a constant temperature of 30°C for 50 minutes. After soaking, filter with a 150-mesh filter to remove solid residue and obtain the Chinese medicine liquid, which can be spray-dried to obtain the corresponding powder.

[0053] Example 3 In this embodiment, another traditional Chinese medicine composition for the prevention and control of leaf spot nematode disease in *Plectranthus verticillata* is provided, comprising the following ingredients in parts by weight: 35 parts of *Euphorbia fischeriana*, 22 parts of *Tripterygium wilfordii*, 12 parts of *Mentha haplocalyx*, 10 parts of *Phellodendron chinense*, 6 parts of *Botrytis cinerea*, and 6 parts of *Chrysanthemum indicum*.

[0054] (1) First decoction: Put the wolfsbane, tripterygium wilfordii, phellodendron bark, and balsam pear back into the extraction tank, add 10 times the amount of distilled water (based on the total weight of the herbs), and soak for 40 minutes. First bring to a boil over high heat, then reduce to a simmer and decoct for 2 hours. Filter and collect the first filtrate.

[0055] (2) Second decoction: Add 6 times the amount of distilled water to the dregs, bring to a boil over high heat, then simmer over low heat for 1.5 hours, and filter again to collect the second filtrate.

[0056] (3) Combining and concentrating: Mix the two filtrates and concentrate them at 60℃ and vacuum of 0.06-0.08MPa until the relative density of the filtrate reaches 1.05-1.10 (tested at 60℃). Stop the concentration to obtain the concentrated solution.

[0057] (4) Compound preparation: Add peppermint pieces and chamomile to the concentrated liquid and soak at a constant temperature of 50°C for 30 minutes. After soaking, filter to remove solid residue and obtain the Chinese medicine liquid, which can be spray-dried to obtain the corresponding powder.

[0058] Performance testing I. Experimental Materials and Research Methods The following experiment was conducted at a *Chloranthus spicatus* planting base in Shexian County, Anhui Province. Two-year-old *Chloranthus spicatus* plants were used, with several healthy plants and plants showing similar levels of disease selected. The herbal extract obtained in the above examples was used for root drenching of the *Chloranthus spicatus* plants, once a week at a volume of 500 mL, for three consecutive treatments. Samples were taken before treatment and on days 7, 14, 21, and 28 after treatment. Leaves from five *Chloranthus spicatus* plants from each treatment were collected for physiological and biochemical index determination, with three biological replicates.

[0059] The experimental groups are set up as follows: Control group (CK): Diseased plants were watered with the same amount of water; Negative control (JK): Healthy plants were watered with the same amount of water; Low concentration experimental group (T1): The drug was prepared by mixing 1 kg of raw drug with 50 kg of water for diseased plants. High concentration experimental group (T2): Prepared for diseased plants at a ratio of 1 kg of raw drug to 20 kg of water.

[0060] II. Research Methods The methods for measuring the indicators of the plants in the above experimental groups are as follows: 1. Determination of nematode content The Bellman funnel separation method was employed: a 10cm diameter funnel was fixed to a support, and a 10cm long rubber tube was connected to the bottom of the funnel. A stopcock was fitted to the middle of the tube to control the outflow of liquid. 5g of fresh soil sample was weighed, wrapped in double-layered gauze, and suspended inside a funnel containing sufficient water for 24 hours. The stopcock was then opened to release the suspension in the tube, and the number of nematodes in the suspension was observed and counted using a counting dish.

[0061] 2. Determination of chlorophyll content Select several leaves, remove the veins, and crush them thoroughly. Add 80% ethanol solution at a solid-liquid ratio of 0.2g:10ml. Seal the solution and place it in an 80℃ constant temperature water bath to extract chlorophyll. Stop extraction when the leaves have completely lost their green color, and you will get the chlorophyll extract.

[0062] The above chlorophyll extract was added to a cuvette with a 1 cm optical path. Using 80% ethanol as a blank control, the absorbance (A) values ​​of the extract were measured and recorded at two characteristic wavelengths, 663 nm and 645 nm.

[0063] The calculation method is as follows: Chlorophyll a content (Ca) = [12.7 × A] 663 - 2.69×A 645 ] × V / (W×1000) Chlorophyll b content (Cb) = [22.9 × A] 645 - 4.68×A 663 ] × V / (W×1000) Total chlorophyll content (Ct) = Ca + Cb = [8.02 × A 663 + 20.21×A 645 ] × V / (W×1000) In the above formula, V: the volume of the extract (ml), which is 25ml in this method; W: Fresh weight of leaf sample (g), which is 0.2g in this method; 12.7, 2.69, 22.9, 4.68, 8.02, 20.21: These are the derived coefficients of the molar absorptivity of chlorophyll a and b at specific wavelengths.

[0064] 3. Determination of malondialdehyde content Select representative leaves from the plant, remove the midrib, chop and mix thoroughly. Quickly weigh 0.5g and place in a pre-cooled mortar. Add 5ml of 0.1mol / L pH 7.8 phosphate buffer and grind in an ice bath to form a homogenate. Transfer the homogenate to a centrifuge tube and centrifuge at 8000r / min for 10min at 4℃. Collect the supernatant as the extraction solution. Take 2ml of the supernatant and mix with 2ml of 0.6% TBA solution (prepared with 10% trichloroacetic acid). Set up a blank control group (2ml buffer + 2ml TBA solution). Boil in a water bath for 15min, then cool in an ice bath and centrifuge at 4000r / min for 5min. Use a 1cm path length cuvette to measure the absorbance at 532nm and 600nm wavelengths, using the blank group as a reference.

[0065] The calculation method is as follows: MDA content = [ΔA × Vt × 1000] / (ε × Vs × W × L) where ΔA = A 532 - A 600 (Net absorbance after deducting non-specific absorption) ΔA: The difference in absorbance at wavelengths of 532nm and 600nm; Vt: Total volume of sample extract (ml), which is 5ml in this method; Vs: Volume of extract used for the reaction (ml), which is 2ml in this method; W: Fresh weight of leaf sample (g), which is 0.5g in this method; L: Optical path of the cuvette (cm), which is 1cm in this method; ε: Molar absorptivity of TMP (L / (mol)) cm), with a value of 155000.

[0066] 4. Determination of soluble sugar content (1) Sample preparation: Select representative leaves, remove the midrib, cut and mix them, quickly weigh 0.2g of sample, add 10ml of distilled water, extract in boiling water bath for 30min, shaking 2-3 times during the process. After cooling, centrifuge at 3000r / min for 10min, take the supernatant, and make up to 25ml with distilled water, which is the extract.

[0067] (2) Color reaction: Take 1 ml of extract into a test tube, add 4 ml of 0.2% anthrone-concentrated sulfuric acid reagent, shake quickly and then boil in a water bath for 10 min. After taking it out, cool it to room temperature in an ice bath.

[0068] (3) Absorbance measurement: Distilled water was used as a blank control, and absorbance (A) was measured at a wavelength of 620 nm using a 1 cm optical path cuvette.

[0069] (4) Standard curve plotting: Prepare 0-100 μg / ml glucose standard solution, perform colorimetric determination according to the above steps, plot the standard curve and obtain the regression equation (y = ax + b, where y is absorbance and x is glucose mass).

[0070] Soluble sugar content = (x × Vt × N) / (Vs × W × 1000) x: Glucose mass (μg) obtained from the regression equation of the standard curve based on the sample absorbance; Vt: Total volume of sample extract (ml), which is 25 ml in this method; N: Dilution factor of the extract (1 if undiluted); Vs: Volume of extract (ml) used for colorimetric reaction, which is 1 ml in this method; W: Fresh weight of leaf sample (g), which is 0.2g in this method; Dividing by 1000 converts μg to mg.

[0071] 5. Determination of the flowering period of *Philodendron simsii* Healthy two-year-old *Chlorophytum comosum* plants were selected and divided into a blank control group and a biocontrol agent treatment group (including a low-concentration experimental group T1 and a high-concentration experimental group T2). Five *Chlorophytum comosum* plants were taken from each treatment group, and the biocontrol agent obtained in Example 1 was used to drench the roots of the *Chlorophytum comosum* plants. The application was started 20 days before the expected budding of the *Chlorophytum comosum* plants, and the plants were drenched once a week at a rate of 3L per plant. The treatment was repeated for 3 consecutive times.

[0072] Negative control (JK): Healthy plants were watered with the same amount of water; Low concentration experimental group (T1): The drug was prepared by mixing 1 kg of raw drug with 50 kg of water for diseased plants. High concentration experimental group (T2): Prepared for diseased plants at a ratio of 1 kg of raw drug to 20 kg of water.

[0073] Indicator observation: Flowering period: The number of days from the date when the first flower petal of the first plant in each group fully opens to the date when the last flower completely withers is taken as the average of the group's values, which is the flowering period of each treatment group. Weight of a single flower: Starting from the beginning of the flowering period, each flower was picked and weighed 2-3 days after it was fully open until the end of the flowering period. The weight of each flower was accumulated to obtain the weight of a single flower, and the average value of the group was calculated.

[0074] III. Research Results 1. Changes in nematode populations Table 1. Changes in the number of nematodes in the soil Table 1 shows that there were basically no nematodes in the soil near healthy plants (JK), while the number of nematodes in the soil near infected plants (CK) did not change significantly from 0 to 28 days. The number of nematodes decreased to some extent from 21 to 28 days. This is mainly because after the Chloranthus spicatus was infected by nematodes, the roots and leaves were damaged to a certain extent and could no longer provide sufficient nutrition for the nematodes.

[0075] Both groups treated with the biocontrol agent showed a significant decrease in the number of nematodes in the soil after application. By day 28, the nematode population had dropped to single digits, essentially returning to normal levels. The high-dose group (T2) had the highest number of nematodes in the soil before treatment, but the nematode population rapidly decreased to normal levels after application, indicating that increasing the drug dosage helps improve the insecticidal effect.

[0076] 2. Changes in chlorophyll content in leaves after application of biocontrol agents Changes in chlorophyll content in *Spiritia jasminoides* leaves before and after application of biocontrol agents are as follows: Figure 2 As shown, chlorophyll a in the leaves of the negative control (JK) group showed a trend of increasing and then decreasing over time, which is consistent with the changes during the leaf growth and senescence periods. The change in chlorophyll b was relatively insignificant.

[0077] The chlorophyll content in the leaves of the leaf spot nematode (CK) group was significantly reduced overall, and the ratio of chlorophyll a to chlorophyll b did not show an upward trend, indicating that the light reaction efficiency of the leaves in the infected group was reduced, the leaves were damaged, and chlorophyll synthesis was affected.

[0078] The chlorophyll content in the leaves of *Chloranthus spicatus* treated with the above-mentioned biocontrol agents showed a significant rebound. In particular, compared with the JK group at the same time point, the chlorophyll a and b contents in the T1 group were generally at similar levels, with only a slight decrease from T1 to 7 days. After that, they gradually rebounded and approached those of the JK group. The chlorophyll a / b ratio fluctuated little and remained at around 3.0, indicating that the low concentration of biocontrol agents did not have a significant negative impact on chlorophyll synthesis and photosynthetic structure stability in the leaves.

[0079] The chlorophyll a and b contents in group T2 were slightly lower than those in the healthy group in the early stage (T2-0d to T2-14d), but significantly increased in the later stage (T2-21d to T2-28d). The chlorophyll a / b ratio continued to rise after T2-14d, indicating that the high concentration of biocontrol agent may have promoted the relative accumulation of chlorophyll a in the later stage.

[0080] The chlorophyll content in both experimental groups showed an upward trend, with the high-concentration group (T2) showing a more significant increase. This indicates that after a period of application, the above-mentioned biocontrol agents not only did not produce a sustained negative impact, but may have promoted the synthesis and accumulation of chlorophyll by inducing plant defense responses or improving physiological metabolism.

[0081] 3. Changes in malondialdehyde content in leaves The malondialdehyde (MDA) content in leaves directly reflects the peroxidation level of plant cell membranes. Elevated MDA levels usually indicate more severe membrane damage and decreased stress resistance. The measurement results are as follows: Figure 3As shown, the MDA content in the JK group remained at a low level of 0.02–0.03 μmol / g FW, with minimal fluctuations throughout the observation period, indicating low membrane lipid peroxidation in healthy *Plectranthus spicatus* leaves and a stable cell membrane system. The MDA content in the CK group showed a significant and continuous upward trend from CK-0d to CK-28d, increasing from approximately 0.03 μmol / g FW to nearly 0.05 μmol / g FW, indicating a significant increase in membrane lipid peroxidation over time and a continuous worsening of oxidative damage to the cell membrane.

[0082] Compared with the control group (CK) at the same time point, the MDA content in the T1 group was consistently significantly lower (e.g., 0.036 μmol / g FW at T1-28d, compared to 0.049 μmol / g FW at CK-28d), and the rate of increase was significantly slower. The MDA content in the T2 group was also significantly lower than that in the CK group at the same time point, and the MDA content in the later stages (T2-21d to T2-28d) was even slightly lower than that in the low-concentration group, showing a stronger inhibitory effect on oxidative damage. This indicates that high-concentration biocontrol agents are superior in enhancing the plant's antioxidant capacity and protecting cell membrane integrity.

[0083] 4. Changes in soluble sugar content in leaves after application of biocontrol agents The results of soluble sugar content in leaves are as follows: Figure 4 As shown, the soluble sugar content in the JK group remained at a low level of 0.15%~0.25%, with a slight increase only at JK-28d, indicating that the soluble sugar metabolism in healthy *Plectranthus spicatus* leaves was relatively stable, without significant stress-responsive accumulation. The soluble sugar content in the CK group showed a continuous upward trend, confirming the biological stress caused by nematode infestation.

[0084] The soluble sugar content in group T1 was significantly lower than that in the control group at the same time point, but slightly higher than that in group JK. This indicates that the low-concentration biocontrol agent effectively alleviated the stress on the plants, reduced the stress accumulation of soluble sugars, and maintained basic metabolic balance. The soluble sugar content in group T2 was also significantly lower than that in the control group, and the later levels were closer to those in the healthy group, indicating that the high-concentration biocontrol agent performed better in reducing plant stress response and maintaining metabolic homeostasis.

[0085] 5. Results of the blooming period determination of *Plectranthus spicatus* Table 2. Results of the determination of the effect of biocontrol agents on the flowering period of *Pistia striatum*. (a indicates that P < 0.05 between the low concentration treatment group and the negative control group, and b indicates that P < 0.05 between the low concentration treatment group and the high concentration treatment group).

[0086] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A traditional Chinese medicine composition, characterized in that, It includes the following ingredients in parts by weight: 25-35 parts of wolfsbane, 18-22 parts of tripterygium wilfordii, 8-12 parts of peppermint, 6-10 parts of phellodendron bark, 4-6 parts of clematis root, and 4-6 parts of chamomile.

2. The traditional Chinese medicine composition according to claim 1, characterized in that, The weight proportions of each herb in the herbal composition are as follows: Wolfsbane 28-32 parts, Tripterygium wilfordii 19-21 parts, Mentha haplocalyx 9-11 parts, Phellodendron amurense 7-9 parts, Bo Luo Hui 4-6 parts, and Chamomile 4-6 parts.

3. The traditional Chinese medicine composition as described in claim 2, characterized in that, The weight proportions of each herb in the herbal composition are as follows: 30 parts of wolfsbane, 20 parts of tripterygium wilfordii, 10 parts of peppermint, 8 parts of phellodendron bark, 5 parts of clematis root, and 5 parts of chamomile.

4. A biocontrol agent, characterized in that, The herbal liquid prepared from the herbal composition according to any one of claims 1-3 in the biocontrol agent is a pharmaceutically acceptable active dose.

5. The biocontrol agent as described in claim 4, characterized in that, The herbal liquid is an aqueous extract of a herbal composition. The preparation method is as follows: extract the herbs, including wolfsbane, tripterygium wilfordii, phellodendron chinense, and clematis chinensis, with water, filter and retain the filtrate. Concentrate the filtrate to obtain a concentrated liquid. Add peppermint and chamomile and soak at 30-50℃ for 30-5 minutes. Filter out the solid residue to obtain the herbal liquid.

6. The biocontrol agent as described in claim 5, characterized in that, The preparation method of the herbal liquid is as follows: (1) First decoction: Put the wolfsbane, thunder vine, phellodendron bark and bougainvillea back into the extraction tank, add 6 to 10 times the amount of distilled water based on the total weight of the herbs, soak for 20 to 40 minutes, decoct for 1 to 2 hours, filter, and collect the first filtrate; (2) Second decoction: Add 3 to 6 times the amount of distilled water to the dregs, decoct for 0.5 to 1.5 hours, filter again and collect the second filtrate; (3) Combining and concentrating: Mix the two filtrates and concentrate them at 50~60℃ and vacuum degree 0.06-0.08MPa until the relative density of the filtrate reaches 1.05-1.

10. Stop the concentration to obtain the concentrated solution. (4) Compound preparation: Add peppermint and chamomile to the concentrated liquid and soak at a constant temperature of 30-50℃ for 30-50 minutes; after soaking, filter to remove solid residue and obtain Chinese medicine liquid.

7. The biocontrol agent as described in claim 4, characterized in that, The biocontrol agent also includes a carrier. The traditional Chinese medicine liquid is mixed with the carrier and then applied to the soil around the roots of the pearl orchid. The carrier is an organic carrier, an inorganic carrier, or a synthetic / semi-synthetic carrier.

8. The biocontrol agent as described in claim 7, characterized in that, The organic carrier is selected from one or a combination of several of the following: well-rotted chicken manure, cow manure, sheep manure, compost, straw powder, sawdust, rice husk powder, coconut coir, well-rotted sawdust, soybean meal powder, peanut cake powder, cottonseed cake powder, corn cob powder, and wheat bran. The inorganic carrier is one or a combination of several of the following: talc, bentonite, diatomite, kaolin, zeolite powder, vermiculite, perlite, and quartz sand. The synthetic / semi-synthetic carrier is one or a combination of several of the following: sodium carboxymethyl cellulose, sodium alginate, chitosan, polyacrylamide, porous starch, activated carbon, and porous ceramics.

9. The application of the traditional Chinese medicine composition according to any one of claims 1-3 or the biocontrol agent according to any one of claims 5-8 in the field of controlling *Plectranthus spp.* leaf spot nematode disease, characterized in that, The application method includes any of the following: 1) Pearl Orchid plants used for prevention, improvement, or treatment of leaf spot nematode infection; 2) Used to prepare fertilizers and pesticides for the cultivation of *Chloranthus chinensis*; when using pesticides, the pesticides are applied as follows: water once every 7 to 10 days, for 2 to 3 consecutive times. When watering, slowly pour the pesticide into the soil around the roots of the plant to ensure that the pesticide penetrates to a depth of 5 to 15 cm.

10. The use of the traditional Chinese medicine composition according to any one of claims 1-3 or the biocontrol agent according to any one of claims 5-8 in increasing the yield of *Chlorophytum comosum*.

Images