Microbial composition for preventing and treating root-knot nematode disease of traditional Chinese medicinal materials and application thereof

Abstract

This invention relates to a microbial composition for controlling root-knot nematode disease in traditional Chinese medicinal materials and its application, belonging to the field of biotechnology. The composition comprises the following components: *Beauveria bassiana* (CGMCC No. 21047), lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, and trace elements. Through the synergistic effect of specific functional microbial communities, the composition significantly promotes the growth of the underground parts of traditional Chinese medicinal materials, increases root biomass and root vigor, and enhances the plant's efficiency in nutrient absorption and utilization, particularly nitrogen utilization. Simultaneously, this preparation effectively controls root-knot nematode disease, significantly reducing the number of root-knot nematode infestation sources in the soil and the severity of root disease, achieving a high natural nematode reduction rate. Furthermore, this preparation activates the plant's own defense system, induces the synthesis of disease-resistant substances, and significantly enhances the activity of various defense enzymes. It also significantly improves the soil microecological environment, enhances the diversity and richness of the soil bacterial community, and is suitable for various crop planting systems.

Description

Technical Field

[0001] This invention belongs to the field of microbial composition technology, specifically, it relates to a microbial composition for preventing and treating root-knot nematode disease in traditional Chinese medicinal materials and its application. Background Technology

[0002] Root-knot nematode disease ( Meloidogyne Nematode disease (SPP) is one of the most destructive soil-borne diseases in global agricultural production. This pathogenic nematode invades plant roots, forming giant cells that cause abnormal swelling of root tissue, resulting in "root knots." This severely hinders water and nutrient absorption, causing plant wilting, stunted growth, and yield loss. Traditional control strategies have significant shortcomings and are insufficient to meet the sustainable development needs of green agriculture: commonly used fumigants (such as methyl bromide) and non-fumigation chemical nematicides (such as abamectin and thiazophos) are effective in the short term, but they have problems such as highly toxic residues, damage to soil microbial communities, environmental pollution, and rapid evolution of nematode resistance. Some highly toxic agents have been banned by international conventions. Commercially available nematode-resistant crop varieties cover a limited range of species, and resistance genes are easily overcome by new pathogenic nematodes, making it impossible to cope with complex field variations. Long crop rotation and fallow cycles (usually 3-5 years) pose a significant conflict with intensive production; solar-powered soil disinfection is constrained by climate and has high energy consumption.

[0003] To address the aforementioned bottlenecks, integrated biological control technologies have become a research hotspot in recent years. Faced with the limitations of traditional control methods, developing environmentally friendly and sustainable green control strategies is imperative. Biological control, especially the use of beneficial microorganisms and their metabolites, and strategies that enhance disease resistance by activating the plant's own immune system (inducing systemic resistance, ISR), has become a research hotspot and a future development direction. An ideal biological control agent should not only effectively inhibit pathogenic microorganisms but also improve the overall health and resistance of plants. This approach integrates three mechanisms: biological parasitism, physical and chemical killing, and systemic resistance induction, covering the entire life cycle of nematodes. Functional fungi, under the synergistic effect of potassium humate and trace elements, exhibit significantly enhanced stress resistance and colonization capabilities, overcoming the poor environmental stability of single-agent formulations. This rapidly reconstructs the soil microbial community, forming a sustainable nematode-suppressing environment, achieving integrated control and remediation. All components are naturally biodegradable, with no risk of chemical residues, meeting organic agriculture standards and providing technical support for solving pesticide residue problems in agricultural products. Summary of the Invention

[0004] The purpose of this invention is to address the problems of poor efficacy and high environmental risks associated with the use of chemical fertilizers and pesticides in the cultivation of medicinal herbs, and to provide a microbial composition and its application for preventing and controlling root-knot nematode disease in medicinal herbs. The microbial composition provided by this invention can significantly promote the growth of medicinal herbs, increase root biomass and root vitality, enhance the plant's efficiency in nutrient absorption and utilization, promote the formation of stable soil aggregates, activate key mineral nutrients fixed in the soil, activate the plant's own defense system, induce the synthesis of disease-resistant substances and significantly enhance the activity of various defense enzymes, improve the soil microecological environment, and increase the diversity and richness of soil bacterial communities, thereby preventing and controlling root-knot nematode disease in medicinal herbs.

[0005] To achieve the above objectives, the first aspect of the present invention provides a microbial composition for preventing and controlling root-knot nematode disease in traditional Chinese medicinal materials, the microbial composition comprising: *Beauveria bassiana* strain with preservation number CGMCC No. 21047. Beauveria pseudobassiana The ingredients are: lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe; wherein the weight ratio of Beauveria bassiana, lecithin, xanthan gum, wood vinegar, soybean peptides, and potassium humate is 0.8-1.2:2-3:0.8-1.2:8-12:20-30:0.4-0.8.

[0006] Furthermore, the microbial composition comprises: *Beauveria bassiana* with accession number CGMCC No. 21047, lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe; wherein the weight ratio of *Beauveria bassiana*, lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe is 0.8-1.2:2-3:0.8-1.2:8-12:20-30:0.4-0.8:4-6:0.25-0.45:0.04-0.07:1.0-1.8:0.003-0.006:0.02-0.04.

[0007] Furthermore, the lecithin contains 0.5-1.5% by weight of water; and / or the xanthan gum contains 8-12% by weight of water; and / or the wood vinegar contains 80-90% by weight of water; the organic acid content is 5%-10% by weight, the phenolic compound content is 1%-3% by weight, and other contents are 0.1-1% by weight. And / or the soybean peptides contain 5-8% by weight of water; on a dry matter basis, the peptide content is 70-90% by weight, the free amino acid content is 5-15% by weight, the ash content is 5-10% by weight, and other contents are 1-2% by weight; and / or the potassium humate contains 5-10% by weight of water; on a dry matter basis, the fulvic acid content is 50-95% by weight; the K2O content is 8-15% by weight; and the ash content is 5-20% by weight.

[0008] The second aspect of this invention provides the above-mentioned microbial composition for the prevention and control of root-knot nematode disease in Chinese medicinal herbs, improving the nitrogen utilization rate of Chinese medicinal herbs, increasing proline content and the activities of polyphenol oxidase (PPO) and superoxide dismutase (SOD), and improving the dry weight and fresh weight of Chinese medicinal herb roots, as well as increasing the content of aggregates with a particle size >0.25 mm, mineral nutrients and microbial diversity index in the soil.

[0009] Furthermore, the medicinal herbs mentioned are Gentiana macrophylla, Angelica sinensis, and Aucklandia lappa, and the pathogen causing root-knot nematode disease in these medicinal herbs is the southern root-knot nematode (Gynostemma pentaphyllum). Meloidogyne incognita ).

[0010] The third aspect of this invention provides a method for promoting the prevention and control of root-knot nematode disease in Chinese medicinal herbs, improving the nitrogen utilization rate of Chinese medicinal herbs, and / or increasing proline content and the activities of polyphenol oxidase (PPO) and superoxide dismutase (SOD), and / or increasing the dry weight and fresh weight of Chinese medicinal herb roots, and / or increasing the content of aggregates with a particle size >0.25 mm, mineral nutrients, and microbial diversity index in the soil. The method includes applying the above-mentioned microbial composition to the rhizosphere soil of crops, wherein the amount of the microbial composition is 300-500 g / mu, and the application frequency is 2-3 times per year.

[0011] Through the above technical solution, the present invention can achieve at least the following beneficial effects:

[0012] (1) The microbial composition provided by this invention can promote the formation of stable soil aggregate structure, increase the organic carbon content of rhizosphere soil of Chinese medicinal herbs, effectively activate key mineral nutrients fixed in the soil, increase the content of exchangeable calcium, exchangeable magnesium, available iron, available copper and available zinc in the rhizosphere soil of Chinese medicinal herbs, and improve their plant availability. It also significantly improves the soil micro-ecological environment and enhances the diversity (Chao 1 index) and richness (Simpson index) of soil bacterial communities.

[0013] (2) The microbial composition provided by the present invention can significantly promote the growth of the underground parts of Chinese medicinal materials, increase root biomass (increase the dry weight and fresh weight of Chinese medicinal materials roots), induce the synthesis of plant resistance substances (proline) and improve root vitality, and enhance the plant's absorption and utilization efficiency of nutrients, especially nitrogen utilization, thereby activating the plant's own defense system, inducing the synthesis of disease-resistant substances and significantly enhancing the activity of various defense enzymes such as polyphenol oxidase (PPO) and superoxide dismutase (SOD).

[0014] (3) This microbial composition, through mechanisms such as microbial activation, plant resistance induction, soil ecological regulation, and nematode behavior interference, is suitable for various Chinese medicinal herb planting systems. It has comprehensive benefits including promoting growth, enhancing resistance, improving soil, and restoring the ecosystem. It can inhibit the hatching of nematode eggs and kill nematodes, thereby significantly reducing the incidence of southern root-knot nematodes ( Meloidogyne incognita The method can reduce the number of second-instar larvae and the disease index, improve the nematode reduction rate, and has a significant effect on the prevention and control of root-knot nematode disease in Chinese medicinal materials, with broad application prospects.

[0015] Biological Preservation

[0016] The *Beauveria bassiana* species used in this invention is classified and named as follows: Beauveria pseudobassiana The strain was deposited on November 9, 2020, at the China General Microbiological Culture Collection Center (CGMCC), located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, with accession number CGMCC No. 21047. The preservation information of the strain has been published in the prior invention patent application CN202210486895.9. Detailed Implementation

[0017] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

[0018] In this invention, Beauveria bassiana 2-8F2 and Beauveria bassiana CGMCC No. 21047 are the same strain, and they have the same meaning. Their names (numbers) can be used interchangeably.

[0019] The inventors of this invention isolated a strain of Beauveria bassiana during their research. Beauveria pseudobassiana The sample, named 2-8F2, was deposited at the China Center for Type Culture Collection (CGMCC) on November 9, 2020, with accession number CGMCC No. 21047.

[0020] The inventors of this invention also discovered in their research that a microbial composition prepared by mixing wood vinegar, soybean peptides, potassium humate, and trace elements (sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe) in a certain proportion, when applied to the rhizosphere soil of medicinal herbs, can significantly promote the growth of medicinal herbs, increase root biomass and root vitality, enhance the plant's efficiency in nutrient absorption and utilization, promote the formation of stable soil aggregate structure, activate key mineral nutrients fixed in the soil, activate the plant's own defense system, induce the synthesis of disease-resistant substances and significantly enhance the activity of various defense enzymes, improve the soil microecological environment, enhance the diversity and richness of soil bacterial communities, and prevent root-knot nematode disease in medicinal herbs.

[0021] Further research revealed that when Beauveria bassiana 2-8F2 is applied together with wood vinegar, soybean peptides, and potassium humate to the rhizosphere soil of medicinal herbs, it can significantly promote the growth of medicinal herbs, increase root biomass and root vitality, enhance the plant's efficiency in nutrient absorption and utilization, promote the formation of stable soil aggregate structure, activate key mineral nutrients fixed in the soil, activate the plant's own defense system, induce the synthesis of disease-resistant substances and significantly enhance the activity of various defense enzymes, improve the soil microecological environment, enhance the diversity and richness of soil bacterial communities, and prevent root-knot nematode disease in medicinal herbs.

[0022] Based on the above findings, the first aspect of this invention provides a microbial composition for preventing and controlling root-knot nematode disease in traditional Chinese medicinal materials, the microbial composition comprising: *Beauveria bassiana* strain with preservation number CGMCC No. 21047. Beauveria pseudobassiana The ingredients are: lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe; wherein the weight ratio of Beauveria bassiana, lecithin, xanthan gum, wood vinegar, soybean peptides, and potassium humate is 0.8-1.2:2-3:0.8-1.2:8-12:20-30:0.4-0.8.

[0023] Further research revealed that when Beauveria bassiana 2-8F2 is applied together with wood vinegar, soybean peptides, potassium humate, and trace elements (sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe) to the rhizosphere soil of medicinal herbs, it can significantly promote the growth of medicinal herbs, increase root biomass and root vitality, enhance the plant's efficiency in nutrient absorption and utilization, promote the formation of stable soil aggregate structure, activate key mineral nutrients fixed in the soil, activate the plant's own defense system, induce the synthesis of disease-resistant substances and significantly enhance the activity of various defense enzymes, improve the soil microecological environment, enhance the diversity and richness of soil bacterial communities, and prevent root-knot nematode disease in medicinal herbs.

[0024] Based on the above findings, the present invention further provides a microbial composition comprising: *Beauveria bassiana* (accession number CGMCC No. 21047), lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe; wherein the weight ratio of *Beauveria bassiana*, lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe is 0.8-1.2:2-3:0.8-1.2:8-12:20-30:0.4-0.8:4-6:0.25-0.45:0.04-0.07:1.0-1.8:0.003-0.006:0.02-0.04.

[0025] In this invention, there are no particular restrictions on the raw materials used in the above-mentioned microbial compositions (such as lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, and trace elements). They can be any relevant products in the art that can be used to prepare microbial compositions, either commercially available products or products prepared by oneself according to existing technology.

[0026] The inventors discovered in their research that the control effect of root-knot nematode disease was better when certain raw materials were used. Therefore, according to a preferred embodiment of the present invention, the lecithin contains 0.5-1.5% by weight of water, preferably 0.8-1.5% by weight, and more preferably 1.2-1.5% by weight.

[0027] According to a preferred embodiment of the present invention, the xanthan gum contains 8-12% by weight of water, preferably 9-12% by weight, and more preferably 10-12% by weight.

[0028] According to a preferred embodiment of the present invention, the wood vinegar contains 80-90% by weight of water; 5%-10% by weight of organic acids; 1%-3% by weight of phenolic compounds; and 0.1-1% by weight of other substances.

[0029] More preferably, the wood vinegar contains 85-90% water by weight; 8-10% organic acid by weight; 2-3% phenolic compounds by weight; and 0.5-1% other substances by weight.

[0030] According to a preferred embodiment of the present invention, the soybean peptides contain 5-8% water by weight; on a dry matter basis, the peptide content is 70-90% by weight, the free amino acid content is 5-15% by weight, the ash content is 5-10% by weight, and other contents are 1-2% by weight.

[0031] More preferably, the soybean peptides contain 6-8% water by weight; on a dry matter basis, the peptide content is 80-90% by weight, the free amino acid content is 12-15% by weight, the ash content is 6-10% by weight, and other contents are 1.5-2% by weight.

[0032] According to a preferred embodiment of the present invention, the potassium humate contains 8-10% water by weight; 60-95% fulvic acid by weight (dry matter); 10-15% K₂O by weight; and 5-15% ash by weight.

[0033] The present invention further provides a method for preparing the above-mentioned microbial composition. According to a preferred embodiment of the present invention, the method includes concentrating the fermentation broth of Beauveria bassiana and then adding lecithin and xanthan gum (component 1) to obtain a microbial agent containing Beauveria bassiana (which may be solid or liquid), uniformly mixing wood vinegar, soybean peptides, potassium humate and trace elements (component 2), and then uniformly mixing component 1 and component 2 according to the aforementioned amounts to obtain the composition.

[0034] In this invention, the specific storage method of the microbial composition is to store the prepared component 1 (which can be solid or liquid) and the composition prepared from component 2 separately. Since the raw materials used in this microbial composition are primarily solid or liquid, the preparation method involves simply mixing the components in proportion and dissolving them in water. Therefore, when using this microbial composition, the bacterial agent is mixed with a certain amount of water to prepare a liquid or slurry (suspension) for use.

[0035] The second aspect of this invention provides Beauveria bassiana and, or the microbial composition described in the first aspect, its application in promoting the growth of Chinese medicinal herbs, increasing root biomass and root vitality, enhancing the plant's efficiency in nutrient absorption and utilization, and / or promoting the formation of stable soil aggregate structure, activating key mineral nutrients fixed in the soil, and / or activating the plant's own defense system, inducing the synthesis of disease-resistant substances and significantly enhancing the activity of various defense enzymes, and / or improving the soil microecological environment, increasing the diversity and richness of soil bacteria, and / or preventing and controlling root-knot nematode disease in Chinese medicinal herbs.

[0036] According to a preferred embodiment of the present invention, the Chinese medicinal materials are Gentiana macrophylla, Angelica sinensis, and Aucklandia lappa.

[0037] In this invention, root biomass refers to the dry and fresh weight of the roots of medicinal herbs; nutrient absorption and utilization efficiency refers to nitrogen utilization rate; soil aggregate structure refers to the content of aggregates with a particle size >0.25 mm in the soil; key mineral nutrients refer to the content of exchangeable calcium, exchangeable magnesium, available iron, available copper, available zinc, and soil organic carbon; disease-resistant substances refer to the content of proline; defense enzymes refer to polyphenol oxidase (PPO) and superoxide dismutase (SOD); and soil bacterial diversity refers to the diversity (Chao1 index) and richness (Simpson index) of soil bacterial communities.

[0038] In this invention, the prevention and control of root-knot nematode disease in Chinese medicinal materials refers to preventing or reducing the occurrence of diseases in Chinese medicinal materials, or reducing the losses caused by diseases after they occur.

[0039] Preferably, the disease affecting the medicinal herbs is root-knot nematode disease of Gentiana macrophylla, Angelica sinensis, and Aucklandia lappa, and the pathogen causing the root-knot nematode disease is the southern root-knot nematode (Gynostemma pentaphyllum). Meloidogyne incognita )

[0040] The specific characteristics of the microbial composition used in the method provided by this invention are as described above and will not be repeated here.

[0041] In this invention, there are no particular restrictions on the specific amount of the microbial composition used, as long as it can play a role in preventing and controlling root-knot nematode disease.

[0042] According to some preferred embodiments of the present invention, when the microbial composition is applied to the plants, the dosage is 300-500 g / mu / time. For example, it can be 300 g / mu / time, 350 g / mu / time, 400 g / mu / time, 450 g / mu / time, 500 g / mu / time, or any intermediate value between any two of the above values.

[0043] Preferably, the amount of the microbial composition used is such that the amount of Beauveria bassiana applied to the rhizosphere of the plant is 1 × 10⁻⁶. 8 -3×10 9The composition is applied 2-3 times per year, with each plant being treated with CFU / plant / time.

[0044] The present invention will be described in detail below through embodiments. It should be understood that the following embodiments are only used to further explain and illustrate the present invention, and are not intended to limit the present invention.

[0045] The lecithin used in the following examples was purchased from Xi'an Miaoguo Biotechnology Co., Ltd., xanthan gum from Jiangsu Liangpu Biotechnology Co., Ltd., wood vinegar and potassium humate from Shandong Hengke Biotechnology Co., Ltd., soybean peptides from Shenzhen Antai Biotechnology Co., Ltd., sugar alcohol chelated calcium from Shandong Voith Agricultural Technology Co., Ltd., amino acid chelated zinc from Zhengzhou Huafeng Food Technology Co., Ltd., boric acid from Henan Hongzi Chemical Products Co., Ltd., magnesium sulfate from Shandong Henghua Chemical Co., Ltd., and EDTA-Cu and EDTA-Fe from Shenzhen Lefu Biotechnology Co., Ltd.

[0046] Example 1: Preparation of Beauveria bassiana inoculum

[0047] This embodiment illustrates the preparation process of the Beauveria bassiana inoculant of the present invention. It includes the following steps:

[0048] (1) Beauveria bassiana with preservation number CGMCC No.21047 was cultured in solid medium to obtain test tube culture;

[0049] (2) Prepare liquid seed culture medium and inoculate test tube seeds in it for liquid culture to obtain liquid seeds;

[0050] (3) Prepare a solid fermentation medium and inoculate it with liquid seeds for fermentation.

[0051] In step (1), the Beauveria bassiana is inoculated by slant inoculation, and the solid culture is cultured at a temperature of 25±1℃ for 48-72h; the solid culture medium includes: 200g / L peeled potato, 20g / L glucose, 15-20g / L agar, and natural pH.

[0052] In step (2), the test tubes are inoculated into liquid seed culture medium, and the liquid culture is treated at a temperature of 25±1℃ and a rotation speed of 150-250r / min for 48-72h; the liquid seed culture medium includes: corn flour 20g / L, glucose 10g / L, soybean meal powder 15g / L, yeast powder 5g / L, KH2PO4 0.5g / L, MgSO4 0.5g / L, and natural pH.

[0053] In this step, the liquid seed culture medium is preferably sterilized at 120-125℃ for 20-30 minutes, and after cooling, 0.5-1.5 cm of the culture medium is inoculated into 100 mL of liquid seed culture medium.2 The in vitro culture was cultured and then incubated at 25±1℃ on a shaker at a speed of 150-250r / min for 48-72h to obtain liquid seeds.

[0054] In step (3), the liquid seed is inoculated in a solid fermentation medium at a volume ratio of 0.05-0.1:1 and treated for 8-12 days at a temperature of 25-28℃ and a humidity of 90-95%. The solid fermentation medium includes: wheat bran that has passed through 80 mesh, rice husk, 2% sucrose, 0.5% peptone, and a water content of 50-55%, and fermented for 8-12 days.

[0055] In this step, the solid fermentation medium is preferably sterilized at 120-125℃ for 20-30 minutes, cooled, and then inoculated with liquid seed, and preferably treated at 25-28℃ for 8-12 days.

[0056] The above preparation steps also include: after fermentation, separating the spore powder using a 200-mesh vibrating sieve, and adding lecithin and xanthan gum sequentially according to the proportions in Table 1, and drying to obtain a viable count of approximately 2 × 10⁻⁶. 10 CFU / g Beauveria bassiana solid inoculum.

[0057] Table 1. Formulation of Beauveria bassiana solid inoculum

[0058]

[0059] Example 2

[0060] Table 2 Material Composition

[0061]

[0062] Table 2 shows the test results of the raw materials used in the preparation of the microbial composition. Among them, water content was determined by drying method, organic acid was determined by high performance liquid chromatography, phenolic compounds were determined by Folin-Ciocalteu colorimetric method, peptides were determined by biuret method, free amino acids were determined by automatic amino acid analyzer, fulvic acid was determined by alkaline extraction and acid precipitation method, K2O was determined by flame atomic absorption spectrometry, and ash content was determined by high temperature ignition method.

[0063] Table 3 Microbial Composition Formulations (by Mass)

[0064]

[0065] *4 (J1) means adding 4 parts by weight of Beauveria bassiana solid inoculant with the code J1, and so on for the rest;

[0066] According to a preferred embodiment of the present invention, the microbial composition is applied 2-3 times per year.

[0067] To achieve better disease prevention effects, according to some preferred embodiments of the present invention, the microbial composition is applied to Chinese medicinal materials once at the time of transplanting (from mid-to-late April to May), then once in September-October, and a third time 15 days after the second application, for a total of 3 applications throughout the year.

[0068] According to the formulation of the microbial composition in Table 3, apply according to the application rate in Table 4 (100 plants per experimental group); investigate the control effect of root-knot nematode disease at the harvest period, determine the number of second-instar root-knot nematode larvae in the soil, determine the dry weight and fresh weight of plant roots and nitrogen use efficiency, determine the content of exchangeable calcium, exchangeable magnesium, available iron, available copper, available zinc and soil organic carbon in the rhizosphere soil, as well as the content of soil aggregate structure (aggregates with a particle size >0.25 mm in the soil), and microbial diversity, determine the root vigor, proline content and the activities of polyphenol oxidase (PPO) and superoxide dismutase (SOD) of the medicinal materials.

[0069] Grading standards for root-knot nematode disease in Chinese medicinal herbs:

[0070] Level 0, no root knots on the root system;

[0071] Grade 1, mild infection, with only a few small root knots, and a root knot rate of less than 3%;

[0072] Grade 3, with numerous root knots, the root knot rate is 25% to 50%;

[0073] Grade 4, with secondary root knots on the root nodules, and 50%–75% of the root system having root knots;

[0074] Level 5: Root knots are interconnected to form root knot clusters, with more than 75% of the root system having root knots.

[0075] The following two formulas are used to calculate the prevention and control effect:

[0076] Disease index = [(Σ(number of disease-grade plants × representative grade)) / (total number of plants × highest representative grade value)] × 100

[0077] Prevention and control efficacy (%) = [(Disease index of control group - Disease index of treatment group) / Disease index of control group] × 100%

[0078] Crop root activity and enzyme activity assays: Crop root activity, root peroxidase (POD), and superoxide dismutase (SOD) activities were determined using a kit purchased from Suzhou Greens Biotechnology Co., Ltd. The usage instructions were followed. Crop proline content was also determined using a kit purchased from Suzhou Greens Biotechnology Co., Ltd. The usage instructions were followed. Determination of key soil mineral nutrients: Exchangeable calcium (mg / kg) and exchangeable magnesium (cmol (1 / 2Mg) content were determined. 2+ The content of available copper (mg / kg) was determined by atomic absorption spectrophotometry, the content of available zinc (mg / kg) was determined by a fertilizer rapid tester, the content of available iron (mg / kg) was determined by flame atomic absorption spectrometry, the content of available iron (mg / kg) was determined by the o-phenanthroline spectrophotometric method, and the content of soil organic carbon was determined by potassium dichromate oxidation-spectrophotometry.

[0079] The method for determining and calculating soil aggregate structure (the content of aggregates with a particle size > 0.25 mm in the soil) is as follows: The content of aggregates with a particle size > 0.25 mm in the soil = the total weight of soil aggregates with a particle size greater than 0.25 mm / the total weight of soil aggregates.

[0080] Methods for determining nitrogen uptake and utilization rate (NRE) of plants: The nitrogen uptake and utilization rate of the experimental treatment groups (G...) were measured separately. N ) and control group (G O The amount of nitrogen absorbed by crop roots was recorded, and the amount of the composition applied in each treatment (N) was also recorded. N The nitrogen uptake and utilization rate (NRE) of the plant was determined using the Kjeldahl method, and calculated using the following formula: NRE (%) = (G) / (G) N -G O ) / N N ×100.

[0081] Soil microbial diversity determination: Total DNA from soil microorganisms was extracted using the OMEGA Soil Total DNA Extraction Kit according to the instructions, requiring an A260 / A280 ratio of 1.8–2.0 for DNA concentration and purity. Qualified DNA was sent to Meiji Biotechnology Co., Ltd. for high-throughput sequencing. Primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) were used to amplify the V3–V4 regions of 16S rRNA from rhizosphere soil bacteria. Primer ITS1 (5′-TCCGTAGGTGAACCTGCGG-3′) was used. The raw sequencing data underwent question sequence identification, inspection, and removal of chimeric sequences to obtain valid sequences from the samples. The sequence with the highest abundance in each OTU was used as the representative sequence for that OTU. Based on 97% sequence similarity, OTUs were merged and divided to construct an OTU abundance matrix for each sample. Based on the OTU abundance matrix results, the number of OTUs in each sample was calculated; bacterial community diversity was evaluated using the Chao 1 richness index and the ACE index; and bacterial community richness was evaluated using the Shannon diversity index and the Simpson diversity index.

[0082] As can be seen from the data in Tables 4, 5, 6, 7, 8, 9, and 10, the application of the microbial composition provided by this invention significantly enhanced the control effect against root-knot nematode disease in medicinal herbs compared to the control. The effects on increasing the dry weight, fresh weight, nitrogen utilization rate, proline content, and the activities of polyphenol oxidase (PPO) and superoxide dismutase (SOD) in the roots of medicinal herbs were also significantly stronger than the control. Furthermore, the effects on increasing the content of aggregates with a particle size >0.25 mm, mineral nutrients, and microbial diversity index in the soil were also stronger than the control. This indicates that the microbial composition is suitable for various crop planting systems, possessing comprehensive benefits in promoting growth, enhancing resistance, improving soil, and restoring the ecosystem, making it a highly efficient and environmentally friendly agricultural input.

[0083] Table 4. Test conditions and results of the experiment on the control effect of microbial compositions on root-knot nematode disease of Chinese medicinal herbs.

[0084]

[0085] *The control group was a blank control group that did not receive any fungicides, while the pesticide group received conventional commercially available pesticides (the main ingredient of which is thiazophos).

[0086] Table 5. Effects of microbial compositions on trace element content in rhizosphere soil of medicinal herbs.

[0087]

[0088] *The control group was a blank control group that did not receive any fungicides, while the pesticide group received conventional commercially available pesticides (the main ingredient of which is thiazophos).

[0089] Table 6. Effects of microbial compositions on proline content, root activity, and nitrogen uptake and utilization rate of cultivated medicinal herbs.

[0090]

[0091] *The control group was a blank control group that did not receive any fungicides, while the pesticide group received conventional commercially available pesticides (the main ingredient of which is thiazophos).

[0092] Table 7. Effects of microbial compositions on the content of organic carbon and aggregates in the root soil of Chinese medicinal herbs.

[0093]

[0094] *The control group was a blank control group that did not receive any fungicides, while the pesticide group received conventional commercially available pesticides (the main ingredient of which is thiazophos).

[0095] Table 8. Test conditions and results of the microbial composition on the activity of root defense enzymes in cultivated medicinal herbs.

[0096]

[0097] *The control group was a blank control group that did not receive any fungicides, while the pesticide group received conventional commercially available pesticides (the main ingredient of which is thiazophos).

[0098] Table 9. Test conditions and results of the experiment on the diversity of rhizosphere soil bacteria in cultivated medicinal herbs using microbial compositions.

[0099]

[0100] *The control group was a blank control group that did not receive any fungicides, while the pesticide group received conventional commercially available pesticides (the main ingredient of which is thiazophos).

[0101] Table 10 Test conditions and results of the effect of microbial compositions on the dry and fresh weight of roots of cultivated medicinal herbs.

[0102]

[0103] *The control group was a blank control group that did not receive any fungicides, while the pesticide group received conventional commercially available pesticides (the main ingredient of which is thiazophos).

[0104] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail through the above preferred embodiments, those skilled in the art should understand that various changes can be made to it in form and detail without departing from the scope defined by the claims of the present invention.

Claims

1. A microbial composition for preventing and controlling root-knot nematode disease in traditional Chinese medicinal materials, characterized in that: The microbial composition includes *Beauveria bassiana* with accession number CGMCC No. 21047. Beauveria pseudobassiana The ingredients include lecithin, xanthan gum, wood vinegar, soybean peptides, potassium humate, sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu, and EDTA-Fe; wherein the weight ratio of Beauveria bassiana, lecithin, xanthan gum, wood vinegar, soybean peptides, and potassium humate is 0.8-1.2:2-3:0.8-1.2:8-12:20-30:0.4-0.8; the medicinal materials are Gentiana macrophylla, Angelica sinensis, and Aucklandia lappa; the pathogen of root-knot nematode disease is Southern root-knot nematode.

2. The microbial composition according to claim 1, characterized in that: The weight ratio of Beauveria bassiana, lecithin, xanthan gum, wood vinegar, soybean peptide, potassium humate, sugar alcohol chelated calcium, amino acid chelated zinc, boric acid, MgSO4, EDTA-Cu and EDTA-Fe is 0.8-1.2:2-3:0.8-1.2:8-12:20-30:0.4-0.8:4-6:0.25-0.45:0.04-0.07:1.0-1.8:0.003-0.006:0.02-0.

04.

3. A method for preventing and controlling root-knot nematode disease in traditional Chinese medicinal materials, characterized in that: The microbial composition according to any one of claims 1-2 is applied to the rhizosphere soil of Chinese medicinal materials, wherein the amount of the microbial composition is 300-500g / mu, and the application frequency is 2-3 times per year.

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