Preparation method of functional compound microbial agent for preventing and treating plant root-knot nematode

Abstract

The invention relates to a preparation method of a functional compound microbial agent for preventing and treating plant root-knot nematodes, and belongs to the technical field of agricultural microbial agents. The preparation method comprises the following steps: uniformly mixing and stirring compound microorganisms, glucose, calcium hydroxide, monopotassium phosphate and magnesium sulfate to obtain a compound microbial agent; adding a selenium-containing insecticidal synergist into the compound microbial agent, and continuously stirring and mixing to obtain a functional compound microbial agent; the functional compound microbial agent is mixed with soil and then applied into the soil; the selenium-containing insecticidal synergist disclosed by the invention is prepared from fluensulfone, papain, allyl phenyl selenium and sodium bicarbonate. According to the selenium-containing insecticidal synergist prepared by the invention, the capability of killing or inhibiting the growth of root-knot nematodes is remarkably improved.

Description

Technical Field

[0001] The invention relates to the technical field of agricultural microbial agents, in particular to a method for preparing a functional composite microbial agent for preventing and controlling plant root-knot nematodes. Background Art

[0002] Root-knot nematodes have a wide range of hosts and can harm more than 130 crops from 39 families. Root-knot nematodes are very small and difficult to see with the naked eye. They are mostly distributed in the soil of 0-20 cm, especially in the soil of 3-9 cm where the largest number of root-knot nematodes are found. Root-knot nematodes are dioecious, with female adults being pear-shaped and male adults being thread-like. They often overwinter or the following year as eggs or second-instar larvae left in the soil or manure with plant debris. When the environment is suitable, second-instar larvae invade through tender roots to reproduce and cause damage. Root-knot nematodes can be spread through worm-infected soil or seedlings and irrigation water. There are many ways to control plant root-knot nematodes, including agricultural control, physical control, and chemical control.

[0003] The patent with publication number CN110724640B discloses a tomato root-knot nematode biocontrol bacterium, its preparation and its application. The purpurogenous spores selected by the present invention have strong pathogenicity to tomato root-knot nematodes and can be used as a tomato root-knot nematode biocontrol bacterium; the tomato root-knot nematode biocontrol preparation is applied to the soil in the form of soil mixing, which can effectively control tomato root-knot nematodes; the purpurogenous spores can also be used to extract or prepare high-yield and high-quality ash bacteriostatin.

[0004] The patent with publication number CN115005217B belongs to the field of pesticide technology, and specifically relates to a nematicidal composition for preventing and treating pepper root-knot nematode disease. A nematicidal composition for preventing and treating pepper root-knot nematode disease, wherein the active ingredient is a binary compound of fludioxonil and tolfenpyrad, flufenacet or cyclohexapride.

[0005] Patent publication number CN114467578B provides a method for controlling plant nematodes, which comprises feeding plant root-knot nematodes with phytosterols, wherein the phytosterols do not contain cholesterol and sitosterol.

[0006] The nematode control methods in the above patents and prior art have disadvantages such as low nematode mortality, slow effect and unstable effect. Summary of the invention

[0007] In order to solve the above problems, the present invention provides a method for preparing a functional composite microbial agent for controlling plant root-knot nematodes, wherein the steps are as follows: S1: Weigh 18-36 parts of composite microorganisms, 38-52 parts of glucose, 1-5 parts of calcium hydroxide, 22-28 parts of potassium dihydrogen phosphate, and 1-5 parts of magnesium sulfate, mix and stir evenly to obtain a composite microbial agent; S2: adding 1-3 parts of selenium-containing insecticide synergist to the composite microbial agent, and continuing to stir and mix to obtain a functional composite microbial agent; S3: Mix the functional compound microbial agent with soil and apply it into the soil.

[0008] The selenium-containing insecticide synergist is prepared by reacting fluthiazolin, ethanol, papain, allylphenyl selenium and sodium bicarbonate.

[0009] Preferably, the composite microorganism composition comprises, by weight: 12-18 parts of Pseudomonas lilacinus, 5-8 parts of Bacillus laterus, and 1-5 parts of Bacillus subtilis. Pseudomonas lilacinus, Bacillus laterus and Bacillus subtilis are mixed and air flow-pulverized according to the above weight parts to obtain the composite microorganism. Preferably, the Pseudomonas lilacinus is obtained by solid fermentation process and low-temperature drying, and the effective viable count is ≥ 20 billion CFU / g.

[0010] Preferably, the live bacteria mixture containing rich extracellular hydrolases and toxic proteins produced by the Brevibacillus laterus through three-stage liquid deep fermentation and high-efficiency centrifugal spray drying has an effective live bacteria count of ≥50 billion CFU / g.

[0011] Preferably, the live bacteria mixture rich in multiple induced enzymes and antimicrobial lipopeptides produced by the Bacillus subtilis through three-stage liquid fermentation and drying has an effective live bacteria count of ≥100 billion CFU / g.

[0012] Preferably, the components of the selenium-containing insecticide synergist include, by weight: 12-16 parts of fluthiazolinone, 100-200 parts of ethanol, 3-6 parts of papain, 0.003-0.04 parts of allylphenyl selenium, and 2-5 parts of sodium bicarbonate. Fluthiazolinone, ethanol, papain, allylphenyl selenium and sodium bicarbonate are mixed according to the above parts by weight, reacted at 40-50° C. for 2-4 hours, and then ethanol is removed by reduced pressure distillation to obtain the selenium-containing insecticide synergist.

[0013] Mechanism and Effect 1. Reaction mechanism Thiol-olefin addition reaction: The thiol (-SH) in papain undergoes a thiol-olefin addition reaction with the carbon-carbon double bond (C=C) in fluthiamethoxam and allylphenylselenium. Sodium bicarbonate acts as an alkaline catalyst to promote this reaction. The specific reaction formula is as follows: For fluthiazolin: R-CH=CH2+R'-SH→R-CH(SR')-CH3; For allylphenylselenide: Ph-CH=CH2+R'-SH→Ph-CH(SR')-CH3.

[0014] Generation of selenium-containing fluthiazolinone: Through the above reaction, a fluthiazolinone derivative containing selenium is generated. This derivative combines the insecticidal properties of fluthiazolinone with the biological activity of selenium, thereby enhancing its insecticidal effect.

[0015] 2. Molecular structure and cytotoxicity Molecular structure: Fluthiamethoxam itself is a highly effective insecticide. Its molecular structure contains specific functional groups that enable it to interfere with the nervous system of insects, causing their death.

[0016] Selenium is one of the essential trace elements for organisms, and has multiple biological activities such as anti-oxidation and immunity enhancement. Introducing selenium into the fluthiamethoxam molecule can change its original electron distribution and spatial configuration, thus affecting its biological activity.

[0017] Cytotoxicity: Selenium-containing fluthiazolinone insecticide synergists may have toxic effects on nematodes through multiple mechanisms. First, it interferes with the nematode's nervous system and affects its normal physiological functions. Second, the introduction of selenium increases the antioxidant properties of the compound, further disrupting the redox balance in the nematode's body, leading to cell damage or death.

[0018] In addition, the enhancer also affects key life processes of nematodes such as energy metabolism and protein synthesis, thereby inhibiting their growth and reproduction.

[0019] 3. Mechanism of synergistic effect Effects of Bacillus lilacinus and Bacillus brevis: Pseudomonas lilacinus can parasitize nematode eggs and female worms, destroying their structure and causing death.

[0020] The extracellular hydrolases secreted by Bacillus can hydrolyze collagen and the nematode body wall, making the nematodes more vulnerable to attack by other microorganisms.

[0021] Effects of Bacillus subtilis: Bacillus subtilis can secrete a variety of substrate-induced enzymes and antimicrobial lipopeptides, which can improve the soil environment and inhibit the growth and reproduction of nematodes.

[0022] Addition of selenium-containing fluthiazolin insecticide synergist: Adding a selenium-containing fluthiasulfone insecticide synergist to the above can further improve the ability to kill or inhibit the growth of nematodes. Due to the synergistic effect of the synergist and the enzymes and antibacterial substances produced by the above microorganisms, the physiological functions and defense mechanisms of the root-knot nematodes are destroyed together.

[0023] At the same time, the introduction of selenium can also enhance the permeability and stability of the compound, making it easier for it to accumulate and exert its effect in the body of root-knot nematodes.

[0024] In summary, the selenium-containing fluthiamethoxam insecticide synergist significantly improves the ability to kill or inhibit the growth of nematodes through the combination of fluthiamethoxam and selenium, as well as the synergistic effect with microorganisms such as Bacillus lilacinus, Bacillus laterus and Bacillus subtilis. DETAILED DESCRIPTION

[0025] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the following is a detailed description in combination with embodiments and comparative examples: Mortality determination: Take 1g of the composite microbial agent and add it to 10mL of sterile water, shake at 28℃, 180rpm for 30min to obtain a composite microbial suspension; take root-knot nematodes and dilute them to 100 / mL with sterile water to obtain a suspension containing nematodes; add 1mL of the suspension containing root-knot nematodes to 1mL of the composite microbial suspension, place it in a 28℃ incubator in the dark and culture it for 72h, then observe the number of dead root-knot nematodes and the total number of root-knot nematodes with a dissecting microscope at room temperature, and calculate the mortality rate: mortality rate = (number of dead nematodes / total number of nematodes) × 100%. The determination results are shown in Table 1.

[0026] Example 1 A method for preparing a functional composite microbial agent for preventing and controlling plant root-knot nematodes, the operating steps of which are as follows: S1: Weigh 18g of composite microorganism, 38g of glucose, 1g of calcium hydroxide, 22g of potassium dihydrogen phosphate, and 1g of magnesium sulfate, mix and stir evenly to obtain a composite microbial agent; S2: Add 1 g of selenium-containing insecticide synergist to the composite microbial agent, continue stirring and mixing, and obtain a functional composite microbial agent; S3: Mix the functional compound microbial agent with soil and apply it into the soil.

[0027] The composite microorganism composition comprises: 12g of Pseudomonas lilacinus, 5g of Brevibacillus laterus and 1g of Bacillus subtilis. Pseudomonas lilacinus, Brevibacillus laterus and Bacillus subtilis are mixed according to the above weight proportions and air flow crushed to obtain the composite microorganism. The purpurogenous fungus is obtained by solid fermentation process and low-temperature drying, and the effective viable bacteria count is ≥20 billion CFU / g.

[0028] The Brevibacillus laterus is produced through three-stage liquid deep fermentation and high-efficiency centrifugal spray drying to produce a live bacteria mixture rich in extracellular hydrolases and toxic proteins, with an effective live bacteria count of ≥50 billion CFU / g.

[0029] The Bacillus subtilis is produced through three-stage liquid fermentation and drying to produce a live bacteria mixture rich in multiple induced enzymes and antimicrobial lipopeptides, with an effective live bacteria count of ≥100 billion CFU / g.

[0030] The selenium-containing insecticide synergist comprises: 12g of fluthiazolinone, 100g of ethanol, 3g of papain, 0.003g of allylphenyl selenium and 2g of sodium bicarbonate. Fluthiazolinone, ethanol, papain, allylphenyl selenium and sodium bicarbonate are mixed according to the above weight proportions, reacted at 40°C for 2h, and then ethanol is removed by reduced pressure distillation to obtain the selenium-containing insecticide synergist.

[0031] Example 2 A method for preparing a functional composite microbial agent for preventing and controlling plant root-knot nematodes, the operating steps of which are as follows: S1: Weigh 24g of composite microorganisms, 45g of glucose, 2g of calcium hydroxide, 24g of potassium dihydrogen phosphate, and 2g of magnesium sulfate, mix and stir evenly to obtain a composite microbial agent; S2: Add 2 g of selenium-containing insecticide synergist to the composite microbial agent, continue stirring and mixing, and obtain a functional composite microbial agent; S3: Mix the functional compound microbial agent with soil and apply it into the soil.

[0032] The composite microorganism components include, by weight: 14g of Pseudomonas lilacinus, 6g of Bacillus laterus and 2g of Bacillus subtilis. Pseudomonas lilacinus, Bacillus laterus and Bacillus subtilis are mixed and air flow crushed according to the above weight proportions to obtain the composite microorganism. The purpurogenous fungus is obtained by solid fermentation technology and low-temperature drying, and the effective viable bacteria count is ≥20 billion CFU / g.

[0033] The Brevibacillus laterus is produced through three-stage liquid deep fermentation and high-efficiency centrifugal spray drying to produce a live bacteria mixture rich in extracellular hydrolases and toxic proteins, with an effective live bacteria count of ≥50 billion CFU / g.

[0034] The Bacillus subtilis is produced through three-stage liquid fermentation and drying to produce a live bacteria mixture rich in multiple induced enzymes and antimicrobial lipopeptides, with an effective live bacteria count of ≥100 billion CFU / g.

[0035] The selenium-containing insecticide synergist comprises: 13g fluthiamethoxam 140g ethanol, 4g papain, 0.01g allylphenyl selenium and 3g sodium bicarbonate. Fluthiamethoxam, ethanol, papain, allylphenyl selenium and sodium bicarbonate are mixed according to the above weight proportions, reacted at 45°C for 3h, and then ethanol is removed by reduced pressure distillation to obtain the selenium-containing insecticide synergist.

[0036] Example 3 A method for preparing a functional composite microbial agent for preventing and controlling plant root-knot nematodes, the operating steps of which are as follows: S1: Weigh 33g of composite microorganism, 48g of glucose, 4g of calcium hydroxide, 26g of potassium dihydrogen phosphate, and 4g of magnesium sulfate, mix and stir evenly to obtain a composite microbial agent; S2: Add 2 g of selenium-containing insecticide synergist to the composite microbial agent, continue stirring and mixing, and obtain a functional composite microbial agent; S3: Mix the functional compound microbial agent with soil and apply it into the soil.

[0037] The composite microorganism composition comprises: 16g of Pseudomonas lilacinus, 7g of Brevibacillus laterus and 4g of Bacillus subtilis. Pseudomonas lilacinus, Brevibacillus laterus and Bacillus subtilis are mixed according to the above weight proportions and air flow crushed to obtain the composite microorganism. The purpurogenous fungus is obtained by solid fermentation technology and low-temperature drying, and the effective viable bacteria count is ≥20 billion CFU / g.

[0038] The Brevibacillus laterus is produced through three-stage liquid deep fermentation and high-efficiency centrifugal spray drying to produce a live bacteria mixture rich in extracellular hydrolases and toxic proteins, with an effective live bacteria count of ≥50 billion CFU / g.

[0039] The Bacillus subtilis is produced through three-stage liquid fermentation and drying to produce a live bacteria mixture rich in multiple induced enzymes and antimicrobial lipopeptides, with an effective live bacteria count of ≥100 billion CFU / g.

[0040] The selenium-containing insecticide synergist comprises: 15g fluthiazolinone, 180g ethanol, 5g papain, 0.03g allylphenyl selenium, and 4g sodium bicarbonate. Fluthiazolinone, ethanol, papain, allylphenyl selenium and sodium bicarbonate are mixed according to the above weight proportions, reacted at 45°C for 3h, and then ethanol is removed by reduced pressure distillation to obtain the selenium-containing insecticide synergist.

[0041] Example 4 A method for preparing a functional composite microbial agent for preventing and controlling plant root-knot nematodes, the operating steps of which are as follows: S1: Weigh 36g of composite microorganisms, 52g of glucose, 5g of calcium hydroxide, 28g of potassium dihydrogen phosphate, and 5g of magnesium sulfate, mix and stir evenly to obtain a composite microbial agent; S2: Add 3 g of selenium-containing insecticide synergist to the composite microbial agent, continue stirring and mixing, and obtain a functional composite microbial agent; S3: Mix the functional compound microbial agent with soil and apply it into the soil.

[0042] The composite microorganism composition comprises: 18g of Pseudomonas lilacinus, 8g of Brevibacillus laterus and 5g of Bacillus subtilis. Pseudomonas lilacinus, Brevibacillus laterus and Bacillus subtilis are mixed according to the above weight proportions and air flow crushed to obtain the composite microorganism. The purpurogenous fungus is obtained by solid fermentation process and low-temperature drying, and the effective viable bacteria count is ≥20 billion CFU / g.

[0043] The Brevibacillus laterus is produced through three-stage liquid deep fermentation and high-efficiency centrifugal spray drying to produce a live bacteria mixture rich in extracellular hydrolases and toxic proteins, with an effective live bacteria count of ≥50 billion CFU / g.

[0044] The Bacillus subtilis is produced through three-stage liquid fermentation and drying to produce a live bacteria mixture rich in multiple induced enzymes and antimicrobial lipopeptides, with an effective live bacteria count of ≥100 billion CFU / g.

[0045] The selenium-containing insecticide synergist comprises: 16g of fluthiazolinone, 200g of ethanol, 6g of papain, 0.04g of allylphenyl selenium, and 5g of sodium bicarbonate. Fluthiazolinone, ethanol, papain, allylphenyl selenium and sodium bicarbonate are mixed according to the above weight proportions, reacted at 50°C for 4h, and then ethanol is removed by reduced pressure distillation to obtain the selenium-containing insecticide synergist.

[0046] Comparative Example 1 No selenium-containing insecticide synergist was added, and the other procedures were the same as in Example 1.

[0047] Comparative Example 2 The other steps were the same as in Example 1 except that fluthiazolin was not added.

[0048] Comparative Example 3 Allylphenylselenium was not added, and the other steps were the same as in Example 1.

[0049] Table 1 Test results of embodiments and comparative examples

[0050] Through the data analysis of the above examples and comparative examples, the selenium-containing insecticide synergist prepared by the present invention significantly improves the ability to kill or inhibit the growth of root-knot nematodes.

[0051] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a functional composite microbial agent for controlling plant root-knot nematodes, characterized in that: The operation steps are as follows: by weight, S1: Weigh 18-36 parts of composite microorganisms, 38-52 parts of glucose, 1-5 parts of calcium hydroxide, 22-28 parts of potassium dihydrogen phosphate, and 1-5 parts of magnesium sulfate, mix and stir evenly to obtain a composite microbial agent; S2: adding 1-3 parts of selenium-containing insecticide synergist to the composite microbial agent, and continuing to stir and mix to obtain a functional composite microbial agent; S3: Mix the functional composite microbial agent with soil and apply it into the soil; The selenium-containing insecticide synergist is prepared by reacting fluthiazolin, ethanol, papain, allylphenyl selenium and sodium bicarbonate.

2. The method for preparing a functional composite microbial agent for controlling plant root-knot nematodes according to claim 1, characterized in that: The composite microorganism components include, by weight: 12-18 parts of Pseudomonas lilacinus, 5-8 parts of Bacillus laterus and 1-5 parts of Bacillus subtilis. Pseudomonas lilacinus, Bacillus laterus and Bacillus subtilis are mixed and air flow crushed according to the above weight parts to obtain the composite microorganism.

3. The method for preparing a functional composite microbial agent for controlling plant root-knot nematodes according to claim 2, characterized in that: The purpurogenous fungus is obtained by solid fermentation technology and low-temperature drying, and the effective viable bacteria count is ≥20 billion CFU / g.

4. The method for preparing a functional composite microbial agent for controlling plant root-knot nematodes according to claim 2, characterized in that: The Brevibacillus laterus is a live bacteria mixture rich in extracellular hydrolases and toxic proteins produced by three-stage liquid deep fermentation and high-efficiency centrifugal spray drying, with an effective live bacteria count of ≥50 billion CFU / g.

5. The method for preparing a functional composite microbial agent for controlling plant root-knot nematodes according to claim 2, characterized in that: The Bacillus subtilis is a live bacteria mixture rich in multiple induced enzymes and antimicrobial lipopeptides produced by three-stage liquid fermentation and drying, and the effective live bacteria count is ≥100 billion CFU / g.

6. The method for preparing a functional composite microbial agent for controlling plant root-knot nematodes according to claim 1, characterized in that: The components of the selenium-containing insecticide synergist include, by weight: 12-16 parts of fluthiazolinone, 100-200 parts of ethanol, 3-6 parts of papain, 0.003-0.04 parts of allylphenyl selenium, and 2-5 parts of sodium bicarbonate. Fluthiazolinone, ethanol, papain, allylphenyl selenium and sodium bicarbonate are mixed according to the above parts by weight, reacted at 40-50° C. for 2-4 hours, and then ethanol is removed by reduced pressure distillation to obtain the selenium-containing insecticide synergist.

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