A microbial agent for preventing and treating angelica root knot nematode disease and its application

Abstract

The present invention relates to a microbial agent for preventing and treating angelica root knot nematode disease and application thereof. The microbial agent comprises the following components: Bacillus velezine with a preservation number of CCTCC No: M2022923 ( Bacillus velezensis ), Bacillus mycoides with a deposit number of CCTCC No: M2022924 ( Bacillus Paramycoid ), Toosendan extract, amino oligosaccharides, fulvic acid and trace element fertilizers. The microbial agent can significantly improve root vitality, root enzyme activity and plant enzyme activity and proline content in the process of Angelica sinensis planting, improve crop resistance to nematodes; significantly increase soil pH, trace elements, enzyme activity and microbial diversity index, significantly reduce soil phenolic acid content, improve soil environment; and significantly reduce southern root-knot nematodes ( Meloidogyne incognita ) The number of second-instar larvae and the disease index can be reduced, and the nematode reduction rate can be increased. It has a significant effect on the prevention and control of southern root-knot nematode disease of Angelica sinensis and has broad application prospects.

Description

Technical Field

[0001] The invention belongs to the technical field of microorganisms and organic fertilizers, and in particular relates to a microbial agent for preventing and treating angelica root knot nematode disease and application thereof. Background Art

[0002] Angelica Angelica sinensis ) is a plant of the angelica family, Umbelliferae. It is a traditional Chinese medicine for both medicine and food, with a long history of medicinal use and cultivation. It has the effects of invigorating qi and replenishing blood, moistening the intestines and relieving constipation, regulating menstruation and relieving pain. At present, more than 300 compounds have been isolated and identified from roots, stems and leaves, including volatile oils, polysaccharides, organic acids, etc., with anti-inflammatory, antibacterial, antioxidant, anti-Alzheimer's disease, anti-depression and other pharmacological effects. With the continuous development of science and technology, its medicinal value and nutritional value have been gradually discovered, which has strongly promoted the development of the angelica industry.

[0003] In recent years, with the increasing demand for angelica, its planting area has continued to expand. However, the root-knot nematode disease in angelica production is serious and frequent, which has a great impact on angelica production and caused serious economic losses, becoming a difficulty that restricts the leapfrog development of the angelica industry. According to the survey, the incidence of angelica root-knot nematode disease is more than 15%. If the continuous cropping period is long, the incidence rate can reach more than 50%, and in severe cases it can reach 80%, or even a total crop failure, which seriously affects the quality of medicinal materials and the economic income of farmers. Due to the difficulty in finding root-knot nematode disease, the severity of the disease increases year by year, the long treatment period, the variety of nematodes and their tenacious vitality, the rapid reproduction rate, the great impact of the soil environment, and the limitations of the drug, the prevention and control effect is poor. The occurrence and prevalence of root-knot nematode disease is closely related to soil quality. Factors such as microbial communities, pH, humidity and temperature in the soil will affect the survival and reproduction of root-knot nematodes. Due to the irrational use of pesticides, the imbalance of the application ratio of N, P, and K fertilizers has destroyed the microecological balance of the soil and promoted the occurrence and prevalence of root-knot nematode disease. In addition, the occurrence and prevalence of root-knot nematode disease is closely related to host resistance. Plant resistance responses include the perception of nematode invasion, activation of defense mechanisms, and resistance to nematode effectors. Although traditional agricultural and chemical control have achieved certain results, their pathogens and pathogenesis have not been clearly elucidated. Therefore, a green and efficient biological control technology is urgently needed to promote the healthy growth of Angelica sinensis. Summary of the invention

[0004] The purpose of the present invention is to provide a microbial agent and application for preventing and treating Angelica root-knot nematode disease in view of the problems that chemical fertilizers and pesticides in the prior art have poor effects and high environmental risks in the process of Angelica planting. The microbial agent provided by the present invention can significantly improve the root vitality, root enzyme activity and plant enzyme activity and proline content of crops, increase the pH, trace element content, enzyme activity and microbial diversity in the soil, reduce the phenolic acid content in the soil, and thus reduce the southern root-knot nematode ( Meloidogyne incognita ) The number of second-instar larvae and the disease index were reduced, and the nematode reduction rate was increased, which had a significant effect on the prevention and control of southern root-knot nematode disease in Angelica sinensis.

[0005] To achieve the above object, the present invention provides a microbial agent in a first aspect, comprising the following components:

[0006] Bacillus velezine with a deposit number of CCTCC No: M2022923 ( Bacillus velezensis ), Bacillus mycoides with a deposit number of CCTCC No: M2022924 ( Bacillus paramycoides ), Toosendan fruit extract, amino oligosaccharides, fulvic acid and medium and trace element fertilizers.

[0007] Furthermore, the amount of Bacillus Velez in the bacterial agent is such that the content of Bacillus Velez in the bacterial agent is 2×10 8 -3×10 8 cfu / g; the amount of Bacillus mycoides in the bacterial agent is such that the content of Bacillus mycoides in the bacterial agent is 2×10 8 -3×10 8 cfu / g.

[0008] Further, the weight ratio of the Toosendan fruit extract, amino oligosaccharide, fulvic acid and medium and trace element fertilizer is 10-15:5-10:70-82:3-5. The medium and trace element fertilizer includes H2SiO3 fertilizer, CaO fertilizer, MgO fertilizer, CuSO4 fertilizer, ZnSO4 fertilizer and FeSO4 fertilizer, wherein the weight ratio of H2SiO3 fertilizer, CaO fertilizer, MgO fertilizer, CuSO4 fertilizer, ZnSO4 fertilizer and FeSO4 fertilizer is 10-20:51-71:10-15:4-6:3-5:2-3.

[0009] Furthermore, the total dosage of the Bacillus Velezii and Bacillus mycoides agents is 4-6% by weight.

[0010] Further, the water content of the Toosendan fruit extract is 5-20% by weight; based on dry matter, the content of toosendanin is 1-5% by weight, the content of alkaloids is 80-90% by weight, the content of kaempferol is 1-5% by weight, the content of resin is 1-5% by weight, and the content of tannin is 1-5% by weight. And / or, the water content of the amino oligosaccharide is 5-10% by weight. And / or, the water content of the fulvic acid is 5-10% by weight.

[0011] The second aspect of the present invention provides the use of the above-mentioned microbial agent in improving crop root vitality and root enzyme activity, and / or, improving plant enzyme activity and proline content, and / or, increasing soil pH, trace element content, enzyme activity and microbial diversity, and / or, reducing soil phenolic acid content, and / or, preventing and controlling crop root-knot nematode disease.

[0012] Furthermore, the crop is Angelica sinensis, and the pathogen causing the Angelica sinensis root knot nematode disease is Meloidogyne incognita ( Meloidogyne incognita ).

[0013] The third aspect of the present invention provides a method for improving the root vitality and enzyme activity of crops, and / or improving the enzyme activity and proline content of plants, and / or improving the pH, trace element content, enzyme activity and microbial diversity in the soil, and / or reducing the phenolic acid content in the soil, and / or preventing and controlling root-knot nematode disease in crops. The method comprises applying the above-mentioned bacterial agent to the rhizosphere soil of the crop, and the amount of the bacterial agent applied to the rhizosphere soil of the crop is 1000-2000g / 366.67m 2 / times, application frequency 3 times during the entire reproductive period.

[0014] Through the above technical solution, the present invention can at least achieve the following beneficial effects:

[0015] (1) The microbial agent provided by the present invention can produce abundant derivatives and multiple functional enzymes during the process of being applied to the soil, which can not only significantly improve the root vitality, significantly improve the activities of root enzymes such as catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), but also significantly increase the activities of cytokinin oxidase (CKO / CKK) and indoleacetic acid oxidase (IAAO) and proline content in crop plants, thereby improving the ability of crop immune response, further enhancing the resistance of crops to nematodes, and improving the ability to resist nematode infection.

[0016] (2) The bacterial agent provided by the present invention can effectively supplement Ca in the soil 2+ Mg 2+ , Cu 2 + and Fe 2+ It can increase the content of trace elements such as catalase, polyphenol oxidase, glutaminase, laccase and nuclease, and significantly improve the diversity index of soil microorganisms such as ACE, Chao I, Simpson, Shannon, etc., thereby promoting the transformation of soil nutrients, improving crop nutrient utilization, promoting crop growth, and enhancing the ability to resist nematode disease infection.

[0017] (3) This technology uses composite bacterial community technology, and utilizes the rapid occupancy effect and secretion of antimicrobial peptides of Bacillus Velezii and Bacillus mycoides to inhibit the hatching of nematode eggs and kill nematodes, thereby significantly reducing the southern root-knot nematode ( Meloidogyne incognita ) The number of second-instar larvae and the disease index can be reduced, and the nematode reduction rate can be increased. It has a significant effect on the prevention and control of southern root-knot nematode disease of Angelica sinensis and has broad application prospects.

[0018] Biological Deposit

[0019] The Bacillus Velezii used in the present invention is classified and named Bacillus velezensis SH-1471 was deposited in the China Center for Type Culture Collection on June 20, 2022, at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with a deposit number of CCTCC No: M2022923. The deposit information of the strain has been disclosed in the prior invention patent application number: CN202211479280.X.

[0020] The pseudomycoides bacillus used in the present invention is classified and named Bacillus paramycoides SH-1464 was deposited in the China Center for Type Culture Collection on June 20, 2022, at 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, Wuhan University, with a deposit number of CCTCC No: M2022924. The deposit information of the strain has been disclosed in the prior invention patent application number: CN202211189532.5. DETAILED DESCRIPTION

[0021] The endpoints and any values ​​of the ranges disclosed in this article 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 each range, the endpoint values ​​of each range and the individual point values, and the individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed in this article.

[0022] In the present invention, Bacillus Velezii SH-1471 and Bacillus Velezii CCTCC No: M2022923 are the same strain, and the two have the same meaning, and their names (numbers) can be used interchangeably. Bacillus pseudomycoides SH-1464 and Bacillus pseudomycoides CCTCC No: M2022924 are the same strain, and the two have the same meaning, and their names (numbers) can be used interchangeably.

[0023] The inventors of the present invention isolated and obtained a strain of Bacillus Velezii ( Bacillus from Velez), named SH-1471, was deposited in China Center for Type Culture Collection on June 20, 2022, with the deposit number CCTCC No: M2022923. And a strain of Bacillus mycoides ( Bacillus paramycoides ), named SH-1464, and deposited in the China Center for Type Culture Collection on June 20, 2022, with the deposit number CCTCC No: M2022924. Studies have found that strains SH-1471 and SH-1464 have antibiotic synthesis genes such as srfA, fenB, ituA, ituD, and bymA, which can inhibit the hatching of nematode eggs and kill nematodes, and have a strong killing effect on a variety of plant nematodes.

[0024] The inventors of the present invention also found in their research that applying a bacterial agent made by mixing Toosendan fruit extract, amino oligosaccharides, humic acid and medium and trace element fertilizers in a certain proportion to the rhizosphere soil of crops can significantly improve root vitality and root enzyme activity, and can also significantly improve enzyme activity and proline content in crop plants, and significantly improve pH, medium and trace element content, enzyme activity and microbial diversity index in the soil, thereby promoting the transformation of soil nutrients, improving crop nutrient utilization, and promoting crop growth.

[0025] After further research, the inventors cleverly discovered that applying Bacillus Velezii SH-1471 and Bacillus pseudomycoides SH-1464 alone or together with the above-mentioned bacterial agent to the rhizosphere soil of crops can further significantly improve root vitality and root enzyme activity, enzyme activity and proline content in the plant, soil enzyme activity and microbial diversity index, while further significantly reducing southern root-knot nematodes ( Meloidogyne incognita ) The number of second-instar larvae and disease index increased, the nematode reduction rate was improved, and the control effect on southern root-knot nematode disease of Angelica sinensis was further enhanced.

[0026] Based on the above findings, the present invention provides a bacterial agent on one hand, the bacterial agent comprising:

[0027] Component 1: optional Bacillus Velezii SH-1471 and Bacillus mycoides SH-1464;

[0028] Component 2: Toosendan fruit extract, amino oligosaccharides, fulvic acid and medium and trace element fertilizers.

[0029] That is, the bacterial agent formula provided by the present invention is any one of the following:

[0030] (1) Bacillus Velezii and Bacillus mycoides, Toosendan fruit extract, amino oligosaccharides, fulvic acid and medium and trace element fertilizers;

[0031] (2) Toosendan fruit extract, amino oligosaccharides, fulvic acid and medium and trace element fertilizers.

[0032] The inventors found in their research that when the components of the microbial agent are in a specific weight ratio, the root-knot nematode control effect is better. Therefore, according to a preferred embodiment of the present invention, the total amount of the Bacillus Velezii and Bacillus pseudomycoides microbial agents is 2-6% by weight, preferably 3-6% by weight, and more preferably 4-6% by weight, based on the total weight of the microbial agent.

[0033] Preferably, the amount of the bacterial agent is such that the contents of Bacillus Velezii and Bacillus mycoides in the bacterial agent are 0.5×10 8 -1×10 8 cfu / g. Preferably 1×10 8 -2×10 8 cfu / g. More preferably 2×10 8 -3×10 8 cfu / g.

[0034] According to a preferred embodiment of the present invention, the weight ratio of Toosendan fruit extract, amino oligosaccharide, fulvic acid and trace element fertilizer is 1-5:3-5:88-95:1-2, preferably 5-10:5-8:79-88:2-3, and more preferably 10-15:8-10:78-82.5:3-5.

[0035] According to a preferred embodiment of the present invention, in the medium and trace element fertilizers, the weight ratio of H2SiO3 fertilizer, CaO fertilizer, MgO fertilizer, CuSO4 fertilizer, ZnSO4 fertilizer and FeSO4 fertilizer is 20-30:14-46:20-25:7-15:5-10:2-6, preferably 15-25:32-40:15-20:5-10:4-8:1-5, and more preferably 10-20:51-71:10-15:4-6:3-5:2-3.

[0036] According to a preferred embodiment of the present invention, the weight ratio of component 1 to component 2 is 2-3:97-98, preferably 3-5:95-97, and more preferably 4-6:94-96.

[0037] In the present invention, there is no particular limitation on the raw materials used as the above-mentioned microbial agent (such as Toosendan fruit extract, amino oligosaccharides, fulvic acid and medium and trace element fertilizers), which can be any related product in the art that can be used to prepare microbial agents, and can be either commercially available related products or related products prepared by themselves according to existing technologies.

[0038] The inventors found in their research that when specific raw materials are used, the root-knot nematode control effect is better. Therefore, according to a preferred embodiment of the present invention, the water content in the Toosendan fruit extract is 5-20% by weight; preferably 8-15%, and more preferably 5-10%.

[0039] Preferably, in the Toosendan fruit extract, based on dry matter, the content of toosendanin is 1-5% by weight, the content of alkaloids is 70-90% by weight, the content of kaempferol is 1-5% by weight, the content of resin is 1-5% by weight, and the content of tannin is 1-5% by weight.

[0040] More preferably, in the Toosendan fruit extract, based on dry matter, the content of toosendanin is 3-5% by weight, the content of alkaloids is 70-88% by weight, the content of kaempferol is 3-5% by weight, the content of resin is 3-5% by weight, and the content of tannin is 3-5% by weight.

[0041] According to a preferred embodiment of the present invention, the water content of the amino oligosaccharide is 5-10% by weight, preferably 4-8% by weight, and more preferably 3-5% by weight.

[0042] According to a preferred embodiment of the present invention, the water content in the fulvic acid is 5-10% by weight, preferably 4-8% by weight, and more preferably 3-5% by weight.

[0043] The present invention further provides a method for preparing the above-mentioned bacterial agent. According to a preferred embodiment of the present invention, the method comprises uniformly mixing the Toosendan fruit extract, amino oligosaccharides, fulvic acid and trace element fertilizer of the above-mentioned component 2 to obtain a basic bacterial agent, and adding optional raw materials such as Bacillus Velezii and Bacillus mycoides (or its bacterial agent) to the basic bacterial agent according to the above-mentioned dosage ratio and mixing them evenly.

[0044] In the present invention, there is no particular restriction on the specific storage and use form of the microbial agent. Since the raw materials used in the microbial agent are basically solid, the preparation method is only to mix the components in proportion. Therefore, the microbial agent can be directly stored or used in the form of a solid preparation. Considering the differences in different crops or planting methods and planting conditions, the microbial agent can also be mixed with a certain amount of water to prepare a liquid or slurry (suspension) preparation for storage or use.

[0045] The Bacillus Velezii and Bacillus pseudomycoides provided in the second aspect of the present invention, or the bacterial agent described in the first aspect, improve root vitality and root enzyme activity, and / or, improve enzyme activity and proline content in plants, and / or, improve pH, trace element content, enzyme activity and microbial diversity index in soil, and / or, reduce southern root-knot nematode ( Meloidogyne incognita) The number of second instar larvae and the disease index, and / or, application in the control of root-knot nematode disease in crops.

[0046] According to a preferred embodiment of the present invention, the crop is Angelica sinensis.

[0047] In the present invention, the crop root enzymes refer to catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD), the crop plant enzymes refer to cytokinin oxidase (CKO / CKK) and indoleacetic acid oxidase (IAAO), and the trace elements in the soil refer to Ca 2+ Mg 2+ , Cu 2 + and Fe 2+ , soil enzymes refer to catalase, polyphenol oxidase, glutaminase, laccase and nuclease; soil microbial diversity refers to Chao1, ACE, Shannon and Simpson indices of bacterial and fungal communities; soil phenolic acids refer to water-soluble phenolic acids, complex phenolic acids and total phenolic acids.

[0048] In the present invention, preventing and controlling crop root diseases refers to preventing or reducing the occurrence of crop diseases, or reducing the losses caused by the diseases after the diseases occur.

[0049] Preferably, the crop disease is selected from root-knot nematode disease, preferably angelica root-knot nematode disease.

[0050] According to some preferred embodiments of the present invention, the pathogen causing the root-knot nematode disease of Angelica sinensis is Meloidogyne incognita ( Meloidogyne incognita ).

[0051] The third aspect of the present invention provides a method for preparing and using a microbial agent for preventing and controlling root-knot nematode disease in crops. The method comprises applying the agent described in the first aspect to the rhizosphere soil of the crops.

[0052] That is, the above method may include the following ways:

[0053] (1) Applying Bacillus Velez and / or Bacillus mycoides directly to the rhizosphere soil of crops (usually, this can be done by directly applying a solid bacterial agent to the rhizosphere soil of crops, or culturing Bacillus Velez and / or Bacillus mycoides and then applying the culture to the soil);

[0054] (2) Applying a microbial agent that does not contain Bacillus Velezii and / or Bacillus mycoides to the rhizosphere soil of crops;

[0055] (3) Applying a microbial agent containing Bacillus Velezii and / or Bacillus mycoides to the rhizosphere soil of crops.

[0056] In the present invention, the crop is Angelica sinensis.

[0057] Preferably, the pathogen causing the Angelica root-knot nematode disease is the southern root-knot nematode ( Meloidogyne unknown ).

[0058] The specific characteristics of the Bacillus Velezii and / or Bacillus mycoides and the bacterial agent used in the method provided by the present invention are as described above and will not be repeated here.

[0059] In the present invention, there is no particular limitation on the specific dosage of Bacillus Velezii and / or Bacillus mycoides and the bacterial agent, as long as the root-knot nematode disease can be prevented and treated.

[0060] According to a preferred embodiment of the present invention, in the above method (1), the amount of Bacillus Velezii and / or Bacillus mycoides is such that the bacterial content in the bacterial agent is 2×10 8 -3×10 8 The dosage of Bacillus Velez and / or Bacillus mycoides is calculated according to the application amount of the bacterial agent and the amount of Bacillus Velez and / or Bacillus mycoides contained therein, wherein the liquid bacterial agent is converted according to 1 g / mL.

[0061] The inventors of the present invention have also found in their research that when planting different crops, the dosage of the aforementioned Bacillus Velezii and / or Bacillus mycoides or the bacterial agent is also adjusted accordingly, which can further improve the systemic resistance and disease prevention effects of the crops.

[0062] According to some preferred embodiments of the present invention, for the above method (1):

[0063] When applied to the rhizosphere soil of crops, the total amount of the Velez subspecies and / or the pseudomycoides subspecies is not less than 2×10 8 cfu / strain / time. Preferably 2×10 8 -2×10 9 cfu / strain / time. More preferably 2×10 8 -8×10 8 cfu / strain / time. More preferably, 3×10 8 -6×10 8 cfu / plant / time. .

[0064] The bacterial agent provided by the present invention has a synergistic effect when used together with Bacillus Velez subtilis and / or Bacillus pseudomycoides. Therefore, when adopting the above-mentioned method (1), in order to obtain a better effect, the amount of Bacillus Velez subtilis and / or Bacillus pseudomycoides used can be appropriately increased compared with the amount of Bacillus Velez subtilis and / or Bacillus pseudomycoides used with the bacterial agent in method (3).

[0065] Preferably, for the above method (3):

[0066] When applied to crops, the total amount of the Velez subtilis and / or the mycoides subtilis is not less than 2×10 10 cfu / strain / time, preferably 2.5×10 10 cfu / strain / time, more preferably 3.0×10 10 cfu / strain / time, more preferably 3.5×10 10 cfu / plant / time.

[0067] According to some preferred embodiments of the present invention, for the above methods (2) and (3), the microbial agent is applied to the crop planting soil in an amount of 1000-2000 g / 366.67 m 2 / time. For example, it can be 1000g / 366.67m 2 / time, 1200g / 366.67m 2 / time, 1400g / 366.67m 2 / time, 1600g / 366.67m 2 / time, 1800g / 366.67m 2 / time, 2000g / 366.67m 2 / times, or it can be any intermediate value between any two of the above values.

[0068] The present invention will be described in detail below by way of examples. It should be understood that the following examples are only used to further explain and illustrate the present invention, and are not intended to limit the present invention. Example

[0069] The Toosendan fruit extract used in this example was purchased from Xi'an An'ao Biotechnology Co., Ltd., amino oligosaccharides were purchased from Qingdao Bozhi Huili Biotechnology Co., Ltd., fulvic acid was purchased from Nanjing Kezheng Chemical Co., Ltd., and trace element fertilizers: H2SiO3 fertilizer, CaO fertilizer, MgO fertilizer, CuSO4 fertilizer, ZnSO4 fertilizer and FeSO4 fertilizer were purchased from Shandong Mingwei Chemical Co., Ltd. The microbial agent was a liquid culture obtained by expanding Bacillus Velezii SH-1471 (see CN 202211479280.X for the acquisition method) and Bacillus pseudomycoides SH-1464 (see CN202211189532.5 for the acquisition method), wherein the content of Bacillus Velezii was 2.5×10 8 cfu / g, the content of Bacillus mycoides was 2.5×10 8 cfu / ml.

[0070] Table 1 shows the test results of raw material components used in the preparation of basic bacterial agents, among which the contents of toosendanin, alkaloids, kaempferol, resin and tannin were determined by high performance liquid chromatography, the content of H2SiO3 was determined by spectrophotometry, the content of calcium and magnesium was determined by EDTA titration, the content of copper and zinc was determined by flame atomic absorption spectrometry, and the content of iron was determined by o-phenanthroline colorimetry.

[0071] Table 1 Basic organic fertilizer material composition

[0072]

[0073] The microbial agent was prepared according to the formula in Table 2. The specific preparation method included: weighing the raw materials according to the proportions in Table 2, and mixing them evenly to obtain the microbial agent (solid preparation).

[0074] Table 2 Microbial agent formula

[0075]

[0076] According to a preferred embodiment of the present invention, the application frequency of the Bacillus Velezii and Bacillus mycoides inoculants is 1-3 times per crop.

[0077] In order to obtain better disease prevention effect, according to some preferred embodiments of the present invention, when the Bacillus Velezii and Bacillus mycoides agents are applied to Angelica sinensis, fertilizer is applied every 10-15 days starting from the time of planting, for a total of 3 applications.

[0078] According to the formula of microbial agent in Table 2, 1000-2000g / 366.67m 2 / times (100 plants were planted in each test group), so that the total amount of Bacillus velezium and Bacillus mycoides of the microbial agent was not less than 3×10 8 cfu / plant / time; investigate the control effect of root-knot nematode disease at the harvest period, determine the number of second-instar larvae of root-knot nematodes in the soil, and determine the Ca content of the rhizosphere soil of the plants. 2+ Mg 2+ , Cu 2 + and Fe 2+ The content of trace elements in crops, root vitality, root enzyme activity and plant enzyme activity, enzyme activity of rhizosphere soil, microbial diversity, and phenolic acid content in soil were determined.

[0079] Angelica root-knot nematode disease survey grading standards:

[0080] Level 0, no root knots on the root system;

[0081] Grade 1, mild infection, only a few small nodules, and the nodule rate is less than 3%;

[0082] Grade 3: more root knots, with a root knot rate of 25% to 50%;

[0083] Level 4: secondary root knots on the nodules, 50% to 75% of the roots have root knots;

[0084] Level 5: The root knots are interconnected into root knot clusters, and more than 75% of the root systems have root knots.

[0085] The control effect is calculated using the following formula:

[0086] Disease index = [(Σ(number of diseased plants × representative level)) / (total number of plants × highest representative level)] × 100

[0087] Control effect (%) = [(control group disease index - treatment group disease index) / control group disease index] × 100%

[0088] Crop root activity and enzyme activity determination: Angelica root activity, root catalase (CAT), peroxidase (POD) and superoxide dismutase (SOD) activities were determined using kits purchased from Suzhou Grace Biotechnology Co., Ltd. The usage method refers to the instructions.

[0089] Determination of enzyme activity and proline content in crop plants: The activities of cytokinin oxidase (CKO / CKK) and indoleacetic acid oxidase (IAAO) and the proline content in Angelica sinensis plants were determined using kits purchased from Suzhou Gres Biotechnology Co., Ltd. The usage method refers to the instructions.

[0090] Determination of soil pH and trace elements: Soil pH was determined by potentiometric method, calcium and magnesium contents were determined by EDTA titration method, copper and zinc contents were determined by flame atomic absorption spectrometry, and iron content was determined by o-phenanthroline colorimetry.

[0091] Soil enzyme activity determination: Soil catalase, polyphenol oxidase, glutaminase, laccase and nuclease enzyme activities were determined using kits purchased from Suzhou Gres Biotechnology Co., Ltd. The usage method refers to the instructions.

[0092] Determination of soil microbial diversity: Soil microbial total DNA was extracted using the OMEGA soil total DNA extraction kit according to the operating instructions. The quality of DNA extraction was detected by 0.8% agarose gel electrophoresis, and DNA was quantified by UV spectrophotometer. Qualified DNA was sent to Meiji Biotechnology Co., Ltd. for high-throughput sequencing. The original sequencing data was identified, checked and chimera sequences were removed by query sequence to obtain valid sequences of the samples. The sequence with the highest abundance in each OTU was used as the representative sequence of the OTU. OTUs were merged and divided based on 97% sequence similarity to construct the OTU abundance matrix in each sample. According to the results of the OTU abundance matrix, the number of common OTUs in each sample was calculated; the Chao1 richness index and ACE index were used to evaluate the richness of the microbial community; the Shannon diversity index and Simpson diversity index were used to evaluate the uniformity of the microbial community.

[0093] Determination of soil phenolic acid content: The contents of water-soluble phenolic acid, complex phenolic acid and total phenolic acid were determined by high performance liquid chromatography (HPLC).

[0094] From the data in Tables 3, 4, 5, 6, 7, 8, 9 and 10, it can be seen that when the microbial agent provided by the present invention is applied, the control effect on Angelica root-knot nematode disease is significantly higher than that of the control; the improvement of Angelica root vitality, enzyme activity and plant enzyme activity and proline content is also stronger than that of the control; the improvement of pH, trace elements, enzyme activity and microbial diversity index in the soil is also stronger than that of the control; in addition, the application of the microbial agent provided by the present invention significantly reduces the phenolic acid content in the soil, thereby facilitating the control of Angelica root-knot nematode disease.

[0095] Table 3 Test conditions and results of the control effect of microbial agents on Angelica root knot nematode disease

[0096]

[0097] *The control group is a blank control group without any bacterial agent, and the fertilizer group is a control group with the chemical fertilizer of component 2 in the example.

[0098] Table 4 Test conditions and results of microbial agents on root vitality and enzyme activity of Angelica sinensis

[0099]

[0100] *The control group is a blank control group without any bacterial agent, and the fertilizer group is a control group with the chemical fertilizer of component 2 in the example.

[0101] Table 5 Test conditions and results of microbial agents on enzyme activity and proline in cultivated Angelica sinensis plants

[0102]

[0103] *The control group is a blank control group without any bacterial agent, and the fertilizer group is a control group with the chemical fertilizer of component 2 in the example.

[0104] Table 6 Test conditions and results of microbial agents on the physical and chemical properties of soil for planting Angelica sinensis

[0105]

[0106] *The control group is a blank control group without any bacterial agent, and the fertilizer group is a control group with the chemical fertilizer of component 2 in the example.

[0107] Table 7 Test conditions and results of microbial agents on soil enzyme activity in Angelica sinensis

[0108]

[0109] *The control group is a blank control group without any bacterial agent, and the fertilizer group is a control group with the chemical fertilizer of component 2 in the example.

[0110] Table 8 Test conditions and results of microbial agents on soil bacterial diversity in Angelica sinensis

[0111]

[0112] *The control group is a blank control group without any bacterial agent, and the fertilizer group is a control group with the chemical fertilizer of component 2 in the example.

[0113] Table 9 Test conditions and results of microbial agents on fungal diversity in soil planted with Angelica sinensis

[0114]

[0115] *The control group is a blank control group without any bacterial agent, and the fertilizer group is a control group with the chemical fertilizer of component 2 in the example.

[0116] Table 10 Test conditions and results of microbial agents on phenolic acid content in soil for planting Angelica sinensis

[0117]

[0118] *The control group is a blank control group without any bacterial agent, and the fertilizer group is a control group with the chemical fertilizer of component 2 in the example.

[0119] Finally, it should be noted that the above preferred embodiments are only used to illustrate the technical solutions of the present invention rather than 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 in form and details without departing from the scope defined by the claims of the present invention.

Claims

1. A microbial agent for preventing and treating Angelica sinensis root knot nematode disease, characterized in that: The bacterial agent comprises the following components: Bacillus Velezii with a preservation number of CCTCC No: M2022923 ( Bacillus velezensis ), Bacillus mycoides with a deposit number of CCTCCNo: M2022924 ( Bacillus paramycoides ), Toosendan fruit extract, amino oligosaccharide, fulvic acid and medium and trace element fertilizer; the amount of Bacillus Velez in the bacterial agent is such that the content of Bacillus Velez in the bacterial agent is 2×10 8 -3×10 8 cfu / g; the amount of Bacillus mycoides in the bacterial agent is such that the content of Bacillus mycoides in the bacterial agent is 2×10 8 -3×10 8 cfu / g.

2. The microbial agent for preventing and treating Angelica sinensis root knot nematode disease according to claim 1, characterized in that: The weight ratio of the Toosendan fruit extract, amino oligosaccharides, fulvic acid and medium and trace element fertilizers is 10-15:5-10:70-82:3-5; the medium and trace element fertilizers include H2SiO3 fertilizer, CaO fertilizer, MgO fertilizer, CuSO4 fertilizer, ZnSO4 fertilizer and FeSO4 fertilizer, wherein the weight ratio of H2SiO3 fertilizer, CaO fertilizer, MgO fertilizer, CuSO4 fertilizer, ZnSO4 fertilizer and FeSO4 fertilizer is 10-20:51-71:10-15:4-6:3-5:2-3.

3. The microbial agent for preventing and treating angelica root-knot nematode disease according to claim 1, characterized in that: The total dosage of the Bacillus Velezii and Bacillus mycoides is 4%-6% of the total weight of the bacterial agent.

4. A microbial agent for preventing and treating Angelica root-knot nematode disease according to any one of claims 1 to 3, characterized in that: The water content of the Toosendan fruit extract is 5-20% by weight; based on dry matter, the content of toosendanin is 1-5% by weight, the content of alkaloids is 80-90% by weight, the content of kaempferol is 1-5% by weight, the content of resin is 1-5% by weight, and the content of tannin is 1-5% by weight; and / or, the amino oligosaccharide has a water content of 5-10% by weight; And / or, the fulvic acid has a water content of 5-10% by weight.

5. Use of the microbial agent according to any one of claims 1 to 3 in improving crop root vitality and root enzyme activity, and / or, improving plant enzyme activity and proline content, and / or, improving soil pH, trace element content, enzyme activity and microbial diversity, and / or, reducing soil phenolic acid content, and / or, preventing and controlling crop root-knot nematode disease.

6. The use according to claim 5, characterized in that: The crop is Chinese angelica, and the pathogen causing the root-knot nematode disease of Chinese angelica is southern root-knot nematode ( Meloidogyne incognita ).

7. A method for improving crop root vitality and root enzyme activity, and / or, improving plant enzyme activity and proline content, and / or, improving soil pH, trace element content, enzyme activity and microbial diversity, and / or, reducing soil phenolic acid content, and / or, preventing and controlling crop root-knot nematode disease, characterized in that: The method comprises applying the bacterial agent described in any one of claims 1 to 3 to the rhizosphere soil of the crop, wherein the amount of the bacterial agent applied to the rhizosphere soil of the crop is 1000-2000 g / 366.67 m 2 / times, and the application frequency is 3 times during the whole growth period; among them, root enzymes refer to catalase, peroxidase and superoxide dismutase; plant enzymes refer to cytokinin oxidase and indoleacetic acid oxidase; enzymes in the soil refer to catalase, polyphenol oxidase, glutaminase, laccase and nuclease.

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