Complex microbial inoculant sustained-release granules for preventing and treating stem nematode disease of sweet potatoes and preparation method of complex microbial inoculant sustained-release granules

Abstract

The invention relates to the field of agricultural microbial preparations, in particular to a complex microbial inoculant sustained-release granule for preventing and treating sweet potato stem nematode disease and a preparation method of the complex microbial inoculant sustained-release granule. Comprising the following components in parts by weight: 15-25 parts of paecilomyces lilacinus viable bacterium powder, 10-20 parts of bacillus thuringiensis viable bacterium powder, 10-20 parts of streptomyces viable bacterium powder, 5-15 parts of bacillus subtilis viable bacterium powder, 15-25 parts of charcoal, 5-15 parts of sodium alginate, 5-15 parts of humic acid, 5-10 parts of chitosan, 3-8 parts of calcium silicate, 1-5 parts of mannitol and 1-5 parts of sodium lignin sulfonate. Four microorganisms with complementary functions, namely paecilomyces lilacinus, bacillus thuringiensis, streptomycete and bacillus subtilis, are adopted, the sweet potato stem nematode disease is synergistically controlled through multiple action mechanisms, and the control effect and the stability are remarkably improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of agricultural microbial preparations, in particular to a compound microbial agent slow-release granule for preventing and treating sweet potato stem nematode disease and a preparation method thereof. BACKGROUND

[0002] Sweet potato stem nematode disease (also known as "furfuraceous heart disease" or "empty box disease") is a devastating disease in sweet potato production, which is caused by Ditylenchus destructor. The nematode mainly damages the stem base, pith and tuber of sweet potato, leading to brown of the stem base, furfuraceous rot of the pith, formation of furrows and dark brown halo on the surface of the tuber, formation of furfuraceous heart in the internal tissue, and even 30%-80% reduction in yield or even no yield in severe cases. Due to the strong adaptability of Ditylenchus destructor, it can survive at low temperature of-15°C, and can survive in soil for more than 3 years without host, and the transmission route is extensive, which makes the prevention and control work face great challenges.

[0003] At present, the main methods for preventing and treating sweet potato stem nematode disease include agricultural prevention and control, chemical prevention and control and biological prevention and control. Agricultural prevention and control mainly includes crop rotation, cultivation of disease-free seedlings, etc.; chemical prevention and control mainly uses chemical pesticides such as phoxim and methyl isothion; biological prevention and control mainly uses microorganisms or their metabolites to inhibit the growth and reproduction of nematodes.

[0004] In addition, it is mentioned in the article "Research Progress of Purpureocillium lilacinum and Its Biocontrol Effect and Mechanism on Diseases and Pests" published in Shaanxi Agricultural Science, 2018, 64(09): 81-88 that the single use of Purpureocillium lilacinum has certain prevention and control effect, but the prevention and control effect is unstable and the effective period is short.

[0005] The prior art has the following disadvantages: first, the single microbial strain has limited and unstable prevention and control effect; second, the existing preparation lacks effective slow-release mechanism, leading to rapid inactivation of microorganisms in soil; third, the carrier material is single, which cannot meet the needs of microbial growth and slow release at the same time; fourth, the preparation process is complex and the cost is high, which is not conducive to industrial application.

[0006] Therefore, it is urgent to develop a compound microbial agent with synergistic effect of multiple functional strains, an environmentally friendly slow-release carrier and an efficient preparation process to achieve long-term prevention and control of sweet potato stem nematode disease. SUMMARY

[0007] The purpose of the present application is to provide a compound microbial agent slow-release granule for preventing and treating sweet potato stem nematode disease and a preparation method thereof, which utilizes the synergistic effect of multiple functional strains, combines with an environmentally friendly slow-release carrier, and realizes long-term prevention and control of sweet potato stem nematode, reduces the frequency of pesticide application and reduces environmental pollution.

[0008] In order to achieve the above-mentioned purpose, the application provides a complex microbial agent slow-release granule for preventing and treating sweet potato stem nematode disease, which comprises the following components in parts by weight: 15-25 parts of Paecilomyces lilacinum live bacteria powder, 10-20 parts of Bacillus thuringiensis live bacteria powder, 10-20 parts of Streptomyces live bacteria powder, 5-15 parts of Bacillus subtilis live bacteria powder, 15-25 parts of biochar, 5-15 parts of sodium alginate, 5-15 parts of humic acid, 5-10 parts of chitosan, 3-8 parts of calcium silicate, 1-5 parts of mannitol and 1-5 parts of sodium lignosulfonate.

[0009] Preferably, the Paecilomyces lilacinum is Purpureocillium lilacinum CGMCC No. 3.17493; the Bacillus thuringiensis is Bacillus thuringiensis CGMCC No. 1.6942; the Streptomyces avermitilis is Streptomyces avermitilis CGMCC No. 4.2072; and the Bacillus subtilis is Bacillus subtilis CGMCC No. 1.308.

[0010] Preferably, the number of live bacteria in the Paecilomyces lilacinum live bacteria powder is not less than 5.0*10 9 CFU / g, the number of live bacteria in the Bacillus thuringiensis live bacteria powder is not less than 1.0*10 10 CFU / g, the number of live bacteria in the Streptomyces avermitilis live bacteria powder is not less than 5.0*10 9 CFU / g, and the number of live bacteria in the Bacillus subtilis live bacteria powder is not less than 1.0*10 10 CFU / g.

[0011] Preferably, the biochar is obtained by pyrolyzing rice husks or wood chips at 500-600°C for 2-3 hours; the humic acid is a weathered coal extract containing not less than 70% humic acid; the chitosan has a deacetylation degree of not less than 90%; and the calcium silicate, mannitol and sodium lignosulfonate are all industrial grades.

[0012] Preferably, the particle size of the granule is 2-4 mm, the water content is less than 10%, the water resistance is not less than 2 hours, and the particle hardness is 1.5-3.0 kg / cm².

[0013] The application also provides a preparation method of the above-mentioned complex microbial agent slow-release granule for preventing and treating sweet potato stem nematode disease, which comprises the following steps:

[0014] 1) Solid-state fermentation culture of functional bacteria: inoculate Paecilomyces lilacinum, Bacillus thuringiensis, Streptomyces and Bacillus subtilis into corresponding solid-state culture media respectively, and culture at a suitable temperature until the bacterial reproduction reaches a maximum value;

[0015] 2) Preparation of live bacterial powder: The solid-state fermentation products of each bacterial species obtained in step 1) were dried at 45-50°C to a moisture content of less than 10%, and crushed to a size of less than 100 mesh to obtain live bacterial powder of each bacterial species;

[0016] 3) Carrier material pretreatment: Grind biochar, humic acid, and calcium silicate to less than 100 mesh respectively;

[0017] 4) Compound inoculant mixing: According to the formula ratio, mix the live bacterial powder of each strain, biochar, humic acid, calcium silicate and mannitol evenly, and stir at 25-30°C for 20-30 minutes;

[0018] 5) Preparation of binder solution: Dissolve chitosan in 1% acetic acid solution to a concentration of 2%, add sodium alginate and sodium lignin sulfonate to prepare a binder solution;

[0019] 6) Granule preparation: The mixture obtained in step 4) is mixed with the binder solution in step 5) in a weight ratio of 1:0.5-0.8, and wet granules with a diameter of 2-4 mm are formed by extrusion granulation equipment;

[0020] 7) Granule drying and stabilization: The wet granules were dried at 40-45°C until the moisture content was less than 10%. After cooling to room temperature, they were sprayed with 2% calcium chloride solution to solidify the sodium alginate.

[0021] 8) Product packaging: The dried granules are screened to remove powder and fragments, and then packaged and sealed to produce the slow-release granules of the composite bacterial agent for treating sweet potato stem nematode disease.

[0022] Preferably, in the step 1), the solid culture medium of Paecilomyces lilacinus consists of wheat bran, brown rice flour and corn steep liquor in a weight ratio of 5:3:2, a water content of 55-60%, a pH value of 6.5-7.0, a culture temperature of 26±1°C, and a culture time of 8-10 days; the solid culture medium of Bacillus thuringiensis consists of soybean meal, corn flour and calcium carbonate in a weight ratio of 6:3:1, a water content of 50-55%, a pH value of 7.0-7.5, a culture temperature of 30±1°C, and a culture time of 8-10 days. The culture medium for Bacillus subtilis is composed of wheat flour, corn steep liquor and fish meal in a weight ratio of 6:3:1, a water content of 50-55%, a pH value of 6.8-7.3, a culture temperature of 32±1°C, and a culture time of 3-4 days.

[0023] Preferably, in the step 2), the drying of each bacterial solid-state fermentation product is performed by using a hot air circulation drying method, with a drying temperature of 45-50°C and a drying time of 6-12 hours; and the dried fermentation product is crushed by using a high-speed crusher to a particle size of no more than 150 μm.

[0024] Preferably, in the step 6), the granulation is performed by using a double-screw extrusion granulator, with a working temperature of 20-25°C, an extrusion speed of 10-15 rpm, an extrusion pressure of 0.5-1.0 MPa, and the extruded product is cut into granules with a length of 2-4 mm.

[0025] Preferably, in the step 7), the drying of the wet granules is performed by using a fluidized bed dryer, with an inlet air temperature of 40-45°C, an outlet air temperature of 30-35°C, and a drying time of 1.5-2.5 hours; and the calcium chloride solution spraying treatment is performed by using a spraying amount of 5-10% of the weight of the granules, and the granules are allowed to stand at room temperature for 2-4 hours after spraying, so that a calcium alginate cross-linked network structure is formed on the surface of the granules, thereby enhancing the strength of the granules and improving the slow-release performance.

[0026] Advantages of the present application:

[0027] 1. Synergistic effect of multiple functional strains: The present application uses four functional complementary microorganisms, i.e., Paecilomyces lilacinus, Bacillus thuringiensis, Streptomyces and Bacillus subtilis, to synergistically control sweet potato stem nematode disease through multiple action mechanisms, thereby significantly improving the control efficiency and stability.

[0028] 2. Environment-friendly slow-release carrier system: A composite slow-release carrier system is constructed by using environment-friendly materials such as biochar, humic acid, chitosan and sodium alginate, which not only protects the activity of the microorganisms, but also realizes the sustained release of the active ingredients and prolongs the period of control efficiency.

[0029] 3. Optimized preparation process: Through solid-state fermentation technology and sodium alginate-chitosan cross-linking system, the present application realizes the efficient production and slow-release embedding of the microorganisms, and the prepared granules have good mechanical strength and slow-release performance.

[0030] 4. Comprehensive regulation of soil health: The product of the present application not only effectively controls the sweet potato stem nematode disease, but also improves the soil structure, promotes the balance of soil microbial community, and improves the disease resistance of crops.

[0031] 5. Advantages of product form: The granule form makes the product convenient to store, transport and apply, and is suitable for field popularization and application. DETAILED DESCRIPTION

[0032] The present application will be further described in detail below in conjunction with specific examples, but the scope of protection of the present application is not limited to the following examples.

[0033] The sweet potato stem nematode disease complex microbial agent slow-release granules of the present application adopts four functionally complementary microbial strains: Purpureocillium lilacinum, Bacillus thuringiensis, Streptomyces avermitilis and Bacillus subtilis. The four strains synergistically control the sweet potato stem nematode disease through different mechanisms of action:

[0034] Purpureocillium lilacinum is an important nematode parasitic fungus that can infect nematode eggs, larvae and adults. The strain produces chitinase, protease and other extracellular enzymes that can degrade the cuticle structure of nematodes and produce a series of toxins to inhibit the movement and reproduction of nematodes. In the present application, the CGMCC No. 3.17493 strain preserved by the China Microbial Culture Collection Committee is selected, which has strong pathogenicity to the sweet potato stem nematode.

[0035] Bacillus thuringiensis is a widely used biocontrol bacillus that can produce Cry protein toxins. These toxins can specifically bind to the receptors of the nematode digestive tract epithelial cells, form pores, destroy cell structure, and cause nematode death. The CGMCC No. 1.6942 strain selected in the present application has good toxic effect on nematodes.

[0036] Streptomyces is an important group in the Actinobacteria phylum, which can produce various antibiotics and bioactive substances. The Streptomyces avermitilis CGMCC No. 4.2072 strain used in the present application can produce avermectin substances, which can specifically act on the nervous system of nematodes, inhibit nerve conduction, and cause nematodes to die of paralysis.

[0037] Bacillus subtilis is a common growth-promoting bacterium. The CGMCC No. 1.308 strain selected in the present application can produce various antibacterial substances and biosurfactants, which can inhibit the growth of pathogenic microorganisms on the one hand, and enhance the penetration ability of other strains to nematodes on the other hand, while promoting plant growth and enhancing plant disease resistance.

[0038] In order to protect the above microbial strains and achieve their slow-release effect, the present application uses a series of environmentally friendly carrier materials:

[0039] Biochar is a solid material obtained by pyrolysis of biomass under limited oxygen conditions, which has a porous structure and a large specific surface area, and can provide a good attachment and reproduction site for microorganisms. At the same time, biochar can also adsorb harmful substances in the soil, improve the soil environment and promote plant growth.

[0040] Humic acid is an important component of soil organic matter, which can promote microbial growth, enhance plant disease resistance and improve soil aggregate structure. In the present invention, humic acid not only acts as a carrier material, but also has a synergistic effect with microorganisms to enhance the control effect.

[0041] Sodium alginate is a natural polysaccharide that can form a gel network structure in the presence of calcium ions to encapsulate microorganisms. This structure can slowly degrade in soil, gradually releasing microorganisms and achieving long-term control.

[0042] Chitosan is a natural polysaccharide extracted from the exoskeleton of crustaceans, which has good biocompatibility and antibacterial activity. In the present invention, chitosan forms a composite microcapsule structure with sodium alginate, not only protecting microorganisms, but also having its own disease resistance effect, which can induce plants to produce disease resistance.

[0043] Auxiliary materials such as calcium silicate, mannitol and sodium lignosulfonate further optimize the performance of the preparation: calcium silicate adjusts soil pH and enhances plant cell wall strength; mannitol as a microbial protective agent improves the survival rate of strains during drying and storage; sodium lignosulfonate as a dispersant enhances the dispersibility of particles in water and improves the uniformity of active ingredient release.

[0044] The preparation method of the present invention includes the following steps: solid-state fermentation culture of functional strains, preparation of live bacteria powder, pretreatment of carrier materials, mixing of composite microbial agents, preparation of adhesive solution, preparation of granules, drying and stabilization of granules, and product packaging. This preparation process fully considers the growth characteristics of microorganisms and the physical and chemical properties of carrier materials, ensuring the high activity and stability of the product.

[0045] The technical solutions of the present invention will be described in detail through specific examples.

[0046] Example 1

[0047] A composite microbial agent slow-release granule for controlling sweet potato stem nematode disease, by weight, includes the following components: 15 parts of Paecilomyces lilacinus live bacteria powder, 10 parts of Bacillus thuringiensis live bacteria powder, 10 parts of Streptomyces live bacteria powder, 5 parts of Bacillus subtilis live bacteria powder, 15 parts of biochar, 5 parts of sodium alginate, 5 parts of humic acid, 5 parts of chitosan, 3 parts of calcium silicate, 1 part of mannitol and 1 part of sodium lignosulfonate.

[0048] The preparation method of the granule includes the following steps:

[0049] 1) Solid-state fermentation culture of functional strains:

[0050] Purpureocillium lilacinum: the Purpureocillium lilacinum CGMCC No. 3.17493 strain was inoculated into a solid-state culture medium composed of wheat bran, brown rice powder and corn syrup (weight ratio of 5:3:2), with a water content of 55%, and the pH value was adjusted to 6.5, and cultured at 26°C for 8 days to obtain a Purpureocillium lilacinum solid-state fermentation product;

[0051] Bacillus thuringiensis: the Bacillus thuringiensis CGMCC No. 1.6942 strain was inoculated into a solid-state culture medium composed of soybean meal, corn flour and calcium carbonate (weight ratio of 6:3:1), with a water content of 50%, and the pH value was adjusted to 7.0, and cultured at 30°C for 4 days to obtain a Bacillus thuringiensis solid-state fermentation product;

[0052] Streptomyces avermitilis: the Streptomyces avermitilis CGMCC No. 4.2072 strain was inoculated into a solid-state culture medium composed of wheat bran, rice bran and sucrose (weight ratio of 6:3:1), with a water content of 45%, and the pH value was adjusted to 7.2, and cultured at 28°C for 6 days to obtain a Streptomyces avermitilis solid-state fermentation product;

[0053] Bacillus subtilis: the Bacillus subtilis CGMCC No. 1.308 strain was inoculated into a solid-state culture medium composed of wheat flour, corn syrup and fish meal (weight ratio of 6:3:1), with a water content of 50%, and the pH value was adjusted to 6.8, and cultured at 32°C for 3 days to obtain a Bacillus subtilis solid-state fermentation product;

[0054] 2) Live bacteria powder preparation: the solid-state fermentation products of each strain were dried at 45°C to a water content of less than 10% for 6 hours, and then crushed to 100 mesh using a high-speed crusher to obtain live bacteria powder of each strain. The live bacteria count was determined to be: 5.2 x 10 9 CFU / g for Purpureocillium lilacinum, 1.3 x 10 10 CFU / g for Bacillus thuringiensis, 5.5 x 10 9 CFU / g for Streptomyces avermitilis, and 1.2 x 10 10 CFU / g for Bacillus subtilis;

[0055] 3) Carrier material pretreatment: rice husk was selected as the raw material and pyrolyzed at 500°C for 2 hours, and then crushed to 100 mesh to obtain biochar; humic acid extracted from weathered coal (containing 75% humic acid) was crushed to 100 mesh; chitosan with a degree of deacetylation of 90% was crushed to 100 mesh; and industrial-grade calcium silicate was crushed to 100 mesh;

[0056] 4) Compound microbial inoculant mixing: mix the live bacteria powder of each strain, biochar, humic acid, calcium silicate and mannitol according to the formula proportion, and mix uniformly at 25°C for 20 minutes;

[0057] 5) Adhesive solution preparation: 5 parts of chitosan are dissolved in 250 parts of 1% acetic acid solution to prepare a 2% chitosan solution, 5 parts of sodium alginate and 1 part of sodium lignosulfonate are added and stirred uniformly to prepare an adhesive solution;

[0058] 6) Granule preparation: mix the mixture obtained in step 4) with the adhesive solution of step 5) at a weight ratio of 1:0.5, and pass through a double screw extrusion granulator (working temperature 20°C, extrusion speed 10 rpm, extrusion pressure 0.5 MPa) to prepare wet granules with a diameter of 2 mm;

[0059] 7) Granule drying and stabilization treatment: dry the wet granules in a fluidized bed dryer at 40°C for 1.5 hours, reduce the water content to 8%, cool to room temperature, and then spray with a 2% calcium chloride solution at a spray amount of 5% of the weight of the granules, and stand at room temperature for 2 hours after spraying;

[0060] 8) Product packaging: screen the dried granules to remove powder and broken pieces, package and seal to obtain a compound microbial inoculant slow-release granule for sweet potato stem nematode disease.

[0061] Example 2

[0062] A compound microbial inoculant slow-release granule for preventing and treating sweet potato stem nematode disease, comprising the following components by weight: 20 parts of live bacteria powder of Purpureocillium lilacinum, 15 parts of live bacteria powder of Bacillus thuringiensis, 15 parts of live bacteria powder of Streptomyces, 10 parts of live bacteria powder of Bacillus subtilis, 20 parts of biochar, 10 parts of sodium alginate, 10 parts of humic acid, 7 parts of chitosan, 5 parts of calcium silicate, 3 parts of mannitol and 3 parts of sodium lignosulfonate.

[0063] The preparation method of the granule comprises the following steps:

[0064] 1) Solid state fermentation culture of functional strains:

[0065] Purpureocillium lilacinum: inoculate the strain of Purpureocillium lilacinum CGMCC No. 3.17493 into a solid culture medium composed of wheat bran, brown rice powder and corn syrup (weight ratio 5:3:2) with a water content of 57% and a pH value adjusted to 6.8, and culture at 26°C for 9 days to obtain a solid state fermentation product of Purpureocillium lilacinum;

[0066] Bacillus thuringiensis: Bacillus thuringiensis CGMCC No. 1.6942 strain was inoculated in a solid-state culture medium (weight ratio of 6:3:1) composed of soybean meal, corn meal and calcium carbonate, with a water content of 52%, and the pH value was adjusted to 7.2, and cultured at 30°C for 4.5 days to obtain Bacillus thuringiensis solid-state fermentation product;

[0067] Streptomyces: Streptomyces avermitilis CGMCC No. 4.2072 strain was inoculated in a solid-state culture medium (weight ratio of 6:3:1) composed of wheat bran, rice bran and sucrose, with a water content of 48%, and the pH value was adjusted to 7.5, and cultured at 28°C for 7 days to obtain Streptomyces solid-state fermentation product;

[0068] Bacillus subtilis: Bacillus subtilis CGMCC No. 1.308 strain was inoculated in a solid-state culture medium (weight ratio of 6:3:1) composed of wheat flour, corn syrup and fish meal, with a water content of 52%, and the pH value was adjusted to 7.0, and cultured at 32°C for 3.5 days to obtain Bacillus subtilis solid-state fermentation product;

[0069] 2) Preparation of live bacteria powder: The solid-state fermentation products of each strain were dried at 47°C to a water content of less than 8% for 8 hours, and then pulverized to 100 mesh using a high-speed pulverizer to obtain live bacteria powder of each strain. The live bacteria count was measured as follows: Paecilomyces lilacinus 6.5×10 9 CFU / g, Bacillus thuringiensis 1.5×10 10 CFU / g, Streptomyces 6.2×10 9 CFU / g, Bacillus subtilis 1.4×10 10 CFU / g;

[0070] 3) Pretreatment of carrier material: Wood chips were pyrolyzed at 550°C for 2.5 hours, and then pulverized to 100 mesh to obtain biochar; humic acid extracted from weathered coal (containing 80% humic acid) was pulverized to 100 mesh; chitosan with a degree of deacetylation of 92% was pulverized to 100 mesh; and industrial-grade calcium silicate was pulverized to 100 mesh;

[0071] 4) Mixing of composite microbial agent: The live bacteria powder, biochar, humic acid, calcium silicate and mannitol of each strain were mixed according to the formula ratio, and stirred for 25 minutes at 28°C;

[0072] 5) Preparation of adhesive solution: 7 parts of chitosan were dissolved in 350 parts of 1% acetic acid solution to prepare a 2% chitosan solution, 10 parts of sodium alginate and 3 parts of sodium lignosulfonate were added and stirred uniformly to prepare an adhesive solution;

[0073] 6) Granule preparation: the mixture obtained in step 4) and the binder solution of step 5) are mixed in a weight ratio of 1:0.6, and wet granules with a diameter of 3 mm are prepared by a double-screw extrusion granulator (working temperature 22 °C, extrusion speed 12 rpm, extrusion pressure 0.7 MPa);

[0074] 7) Granule drying and stabilization treatment: the wet granules are dried in a fluidized bed dryer at 42 °C for 2 hours, and the water content is reduced to 7%, and after cooling to room temperature, the granules are treated with a 2% calcium chloride solution, and the spraying amount is 7% of the weight of the granules, and after spraying, the granules are left at room temperature for 3 hours;

[0075] 8) Product packaging: the dried granules are screened to remove powder and broken pieces, and are packaged and sealed to obtain the compound microbial agent slow-release granules for the control of sweet potato stem nematode disease.

[0076] Example 3

[0077] A compound microbial agent slow-release granule for the control of sweet potato stem nematode disease, comprising the following components in parts by weight: 25 parts of Purpureocillium lilacinum live bacteria powder, 20 parts of Bacillus thuringiensis live bacteria powder, 20 parts of Streptomyces live bacteria powder, 15 parts of Bacillus subtilis live bacteria powder, 25 parts of biochar, 15 parts of sodium alginate, 15 parts of humic acid, 10 parts of chitosan, 8 parts of calcium silicate, 5 parts of mannitol, and 5 parts of sodium lignosulfonate.

[0078] The preparation method of the granule comprises the following steps:

[0079] 1) Solid-state fermentation culture of functional bacteria:

[0080] Purpureocillium lilacinum: the Purpureocillium lilacinum strain CGMCC No. 3.17493 is inoculated into a solid-state culture medium composed of wheat bran, brown rice powder and corn syrup (weight ratio of 5:3:2), the water content is 60%, the pH value is adjusted to 7.0, and the culture is carried out at 26 °C for 10 days to obtain the Purpureocillium lilacinum solid-state fermentation product;

[0081] Bacillus thuringiensis: the Bacillus thuringiensis strain CGMCC No. 1.6942 is inoculated into a solid-state culture medium composed of soybean meal, corn flour and calcium carbonate (weight ratio of 6:3:1), the water content is 55%, the pH value is adjusted to 7.5, and the culture is carried out at 30 °C for 5 days to obtain the Bacillus thuringiensis solid-state fermentation product;

[0082] Streptomyces: Streptomyces avermitilis CGMCC No. 4.2072 strain was inoculated in a solid-state culture medium consisting of wheat bran, rice bran and sucrose (weight ratio of 6:3:1), with a water content of 50%, and the pH value was adjusted to 7.8, and cultured at 28°C for 8 days to obtain Streptomyces solid-state fermentation product;

[0083] Bacillus subtilis: Bacillus subtilis CGMCC No. 1.308 strain was inoculated in a solid-state culture medium consisting of wheat flour, corn syrup and fish meal (weight ratio of 6:3:1), with a water content of 55%, and the pH value was adjusted to 7.3, and cultured at 32°C for 4 days to obtain Bacillus subtilis solid-state fermentation product;

[0084] 2) Preparation of live bacteria powder: The solid-state fermentation products of each strain were dried at 50°C to a water content of less than 6% for 12 hours, and then crushed to 100 mesh using a high-speed crusher to obtain live bacteria powder of each strain. The live bacteria count was determined to be 7.8 x 10 9 CFU / g for Paecilomyces lilacinus, 1.8 x 10 10 CFU / g for Bacillus thuringiensis, 7.2 x 10 9 CFU / g for Streptomyces, and 1.7 x 10 10 CFU / g for Bacillus subtilis;

[0085] 3) Pretreatment of carrier material: A mixture of rice husk and sawdust (weight ratio of 1:1) was selected as raw material, pyrolyzed at 600°C for 3 hours, and then crushed to 100 mesh to obtain biochar; humic acid extracted from weathered coal (containing 85% humic acid) was crushed to 100 mesh; chitosan with a degree of deacetylation of 95% was crushed to 100 mesh; and industrial-grade calcium silicate was crushed to 100 mesh;

[0086] 4) Mixing of compound microbial agent: The live bacteria powder of each strain, biochar, humic acid, calcium silicate and mannitol were mixed according to the formula proportion, and stirred for 30 minutes at 30°C;

[0087] 5) Preparation of adhesive solution: 10 parts of chitosan were dissolved in 500 parts of 1% acetic acid solution to prepare a 2% chitosan solution, 15 parts of sodium alginate and 5 parts of sodium lignosulfonate were added and stirred uniformly to prepare an adhesive solution;

[0088] 6) Preparation of granules: The mixture obtained in step 4) and the adhesive solution of step 5) were mixed according to a weight ratio of 1:0.8, and then wet granules with a diameter of 4mm were prepared by a double-screw extrusion granulator (working temperature 25°C, extrusion speed 15rpm, extrusion pressure 1.0MPa);

[0089] 7) Particle drying and stabilization treatment: the wet particles were dried in a fluidized bed dryer at 45°C for 2.5 hours until the water content was reduced to 5%, and then cooled to room temperature. The particles were then sprayed with a 2% calcium chloride solution at a spray amount of 10% of the weight of the particles, and then left to stand at room temperature for 4 hours;

[0090] 8) Product packaging: the dried particles were sieved to remove powder and broken pieces, and then packaged and sealed to obtain the compound microbial agent slow-release granules for the control of sweet potato stem nematode disease.

[0091] Example 4

[0092] A compound microbial agent slow-release granule for the control of sweet potato stem nematode disease, comprising the following components in parts by weight: Paecilomyces lilacinus live bacteria powder 18 parts, Bacillus thuringiensis live bacteria powder 12 parts, Streptomyces live bacteria powder 12 parts, Bacillus subtilis live bacteria powder 8 parts, biochar 18 parts, sodium alginate 8 parts, humic acid 8 parts, chitosan 6 parts, calcium silicate 4 parts, mannitol 2 parts, and sodium lignosulfonate 2 parts.

[0093] The preparation method of the granule is basically the same as in Example 2, except that in step 6), the mixture and the binder solution are mixed at a weight ratio of 1:0.7 to form wet particles with a diameter of 3.5 mm; and in step 7), the wet particles are dried at a temperature of 43°C for 2.2 hours.

[0094] Example 5

[0095] A compound microbial agent slow-release granule for the control of sweet potato stem nematode disease, comprising the following components in parts by weight: Paecilomyces lilacinus live bacteria powder 22 parts, Bacillus thuringiensis live bacteria powder 18 parts, Streptomyces live bacteria powder 18 parts, Bacillus subtilis live bacteria powder 12 parts, biochar 22 parts, sodium alginate 12 parts, humic acid 12 parts, chitosan 8 parts, calcium silicate 6 parts, mannitol 4 parts, and sodium lignosulfonate 4 parts.

[0096] The preparation method of the granule is basically the same as in Example 3, except that in step 3), the biochar is prepared by pyrolyzing sawdust as a single raw material at 580°C for 2.8 hours; and in step 6), the mixture and the binder solution are mixed at a weight ratio of 1:0.75 to form wet particles with a diameter of 3.8 mm.

[0097] Comparative Example 1

[0098] A compound microbial agent slow-release granule for the control of sweet potato stem nematode disease, comprising the following components in parts by weight: Paecilomyces lilacinus live bacteria powder 20 parts, biochar 20 parts, sodium alginate 10 parts, humic acid 10 parts, chitosan 7 parts, calcium silicate 5 parts, mannitol 3 parts, and sodium lignosulfonate 3 parts.

[0099] The comparative example lacks Bacillus thuringiensis viable powder, Streptomyces viable powder and Bacillus subtilis viable powder compared with Example 2, and only contains a single Paecilomyces lilacinus strain. The preparation method is the same as that of Example 2, and only the viable powder of the other three strains is not added in the mixed bacteria agent mixing step.

[0100] Comparative Example 2

[0101] A slow-release granule of a mixed bacteria agent for preventing and treating sweet potato stem nematode disease comprises, by weight, the following components: 20 parts of Paecilomyces lilacinus viable powder, 15 parts of Bacillus thuringiensis viable powder, 15 parts of Streptomyces viable powder, 10 parts of Bacillus subtilis viable powder, 10 parts of humic acid, 7 parts of chitosan, 5 parts of calcium silicate, 3 parts of mannitol and 3 parts of sodium lignosulfonate.

[0102] The comparative example lacks two carrier materials, biochar and sodium alginate, compared with Example 2. The preparation method is basically the same as that of Example 2, and the difference is that: in step 3), biochar is not prepared; in step 4), biochar is not added; in step 5), sodium alginate is not added; and in step 7), calcium chloride solution spraying treatment is not performed.

[0103] Comparative Example 3

[0104] A slow-release granule of a mixed bacteria agent for preventing and treating sweet potato stem nematode disease comprises, by weight, the following components: 20 parts of Paecilomyces lilacinus viable powder, 15 parts of Bacillus thuringiensis viable powder, 15 parts of Streptomyces viable powder, 10 parts of Bacillus subtilis viable powder, 20 parts of biochar, 10 parts of sodium alginate, 10 parts of humic acid, 5 parts of calcium silicate, 3 parts of mannitol and 3 parts of sodium lignosulfonate.

[0105] The comparative example lacks chitosan compared with Example 2. The preparation method is basically the same as that of Example 2, and the difference is that: in step 5), chitosan is not used, and sodium alginate and sodium lignosulfonate are directly dissolved in water to prepare a binder solution.

[0106] In order to evaluate the prevention effect and slow-release performance of the product of the present application, the following test experiments were carried out:

[0107] Experiment 1: Inhibition of nematode activity test in vitro

[0108] Experimental method: The slow-release granules of the mixed bacteria agent of each example and comparative example were ground and prepared into a suspension (1% concentration), and then added to a culture dish containing 100 adult sweet potato stem nematodes, and cultured at 25°C. The survival of the nematodes was observed after 24 hours, 48 hours and 72 hours, respectively, and the mortality rate was calculated. A water control was also set up, and each treatment was repeated 3 times.

[0109] Experiment 2: Granule slow-release performance test

[0110] Experimental method: The slow-release granules of the complex microbial agent of each example and comparative example were placed in a buffer solution with pH value of 6.5, and slightly oscillated at 25°C. Samples were taken after 1 day, 3 days, 7 days, 14 days and 21 days, respectively, and the viable cell count of each strain in the solution was determined to calculate the cumulative release rate. Each treatment was repeated 3 times.

[0111] Experiment three: potting prevention effect test

[0112] Experimental method: Select flowerpots with a diameter of 30 cm, fill them with sterilized soil, and inoculate 10,000 sweet potato stem nematodes per pot. According to the dosage of 20 g / pot, the slow-release granules of the complex microbial agent of each example and comparative example were applied, and the treated flowerpots were placed at 25°C and 70% relative humidity for 7 days. Then, one sweet potato seedling (variety: Xushu No. 18) was planted in each pot. At the same time, blank controls without any agent and controls with commercially available chemical nematocide (10% abamectin emulsion, diluted 2000 times) were set up. Each treatment was repeated 5 times. On the 30th and 60th day after planting, the growth status and root nematode infection of the sweet potato seedlings were investigated, and the prevention effect was calculated.

[0113] Experiment four: field prevention effect test

[0114] Experimental method: Select a field where the sweet potato stem nematode disease occurs seriously, and set up 6 treatments: Example 1, Example 2, Example 3, Comparative Example 1, Comparative Example 2 and chemical pesticide control (10% abamectin emulsion, diluted 2000 times). Each plot area is 30 m², with random block design and 3 repetitions. At the time of sweet potato transplanting, the agents of each treatment were applied into the planting ditch at a dosage of 25 kg / ha, and then sweet potato seedlings (variety: Xushu No. 18) were transplanted. On the 30th, 60th and 90th day after transplanting, the occurrence of sweet potato stem nematode disease was investigated, and the prevention effect and yield were calculated.

[0115] The test results are as follows:

[0116] Experiment one: indoor nematode inhibition activity test results

[0117] Table 1 Inhibition activity of each treatment on sweet potato stem nematode (unit: %)

[0118] Treatment 24 hours 48 hours 72 hours Example 1 42.5 68.3 85.6 Example 2 45.7 72.4 91.2 Example 3 48.2 75.6 93.8 Example 4 44.1 70.8 89.5 Example 5 47.3 74.2 92.6 Comparative Example 1 25.3 42.7 59.8 Comparative Example 2 38.2 58.5 72.4 Comparative Example 3 35.6 54.2 68.5 Water control 3.2 5.6 7.8

[0119] As can be seen from Table 1, each of the examples shows good inhibition activity on sweet potato stem nematode, and the inhibition rate after 72 hours reaches 85.6%-93.8%. In comparison, the inhibition activity of Comparative Example 1 containing only a single strain is obviously lower, indicating that the compound strain has a synergistic effect; the inhibition activity of Comparative Example 2 lacking biochar and sodium alginate and Comparative Example 3 lacking chitosan is also significantly lower than that of the examples, indicating that these carrier materials are crucial for the protection and play of microbial activity.

[0120] Experiment II: Test results of particle slow-release performance

[0121] Table 2: Cumulative release rate of each treatment (unit: %)

[0122] Treatment 1 day 3 days 7 days 14 days 21 days Example 1 15.3 28.7 45.6 68.2 82.5 Example 2 12.6 25.3 42.8 65.4 80.7 Example 3 10.2 22.5 38.7 62.3 78.4 Example 4 13.5 26.8 43.9 66.8 81.2 Example 5 11.4 23.6 40.5 63.7 79.5 Comparative Example 1 17.2 34.5 58.3 85.6 96.2 Comparative Example 2 32.8 58.6 87.4 98.5 100 Comparative Example 3 25.6 47.2 76.5 94.3 99.7

[0123] As can be seen from Table 2, each of the examples shows good slow-release performance, and the cumulative release rate after 21 days is 78.4%-82.5%, and the release curve is relatively flat, which is conducive to the sustained release and long-acting effect of microorganisms in the soil. In comparison, the release rate of Comparative Example 1 is slightly faster, while the release rate of Comparative Examples 2 and 3 is significantly accelerated, especially Comparative Example 2 is almost completely released after 14 days, lacking sustained slow-release effect. This indicates that the combined use of carrier materials such as biochar, sodium alginate and chitosan is crucial for the slow release of microorganisms.

[0124] Experiment III: Test results of potting prevention effect

[0125] Table 3: Prevention effect of each treatment under potting conditions (unit: %)

[0126] Treatment 30-day control effect 60-day control effect Seedling height (cm) Root development index Example 1 78.5 72.3 65.2 3.8 Example 2 85.2 80.6 68.7 4.2 Example 3 89.6 85.3 72.5 4.5 Example 4 83.7 78.5 67.3 4 Example 5 87.4 83.2 70.8 4.3 Comparative Example 1 60.3 45.8 52.6 2.7 Comparative Example 2 72.5 42.3 58.5 3.2 Comparative Example 3 68.7 40.2 56.3 3 Chemical pesticide 92.5 68.3 60.2 3.5 Blank control - - 42.3 1.8

[0127] Note: The root development index is evaluated by 1-5 levels, 1 indicating extremely poor root system, and 5 indicating extremely good root system.

[0128] As can be seen from Table 3, each of the examples shows good prevention effect under potting conditions, and the 30-day prevention effect is 78.5%-89.6%, and the 60-day prevention effect is 72.3%-85.3%, with long prevention effect duration. In comparison, the prevention effect of Comparative Examples 1, 2 and 3 is lower, especially the 60-day prevention effect is significantly decreased, indicating that these comparative examples lack sustained prevention effect. It is worth noting that although the chemical pesticide has slightly higher prevention effect than the examples at 30 days, the prevention effect is significantly decreased at 60 days, while the prevention effect of the examples is relatively stable. In addition, the growth status and root development of sweet potato seedlings treated by each of the examples are better than those of the comparative examples and the chemical pesticide treatment, indicating that the slow-release granules of the compound microbial agent of the present application not only have the effect of preventing nematodes, but also can promote plant growth.

[0129] Experiment IV: Test results of field prevention effect

[0130] Table 4. Control effect and yield of each treatment under field conditions

[0131] Treatment 30-day control effect (%) 60-day control effect (%) 90-day control effect (%) Yield (kg / ha) Yield increase rate (%) Example 1 76.2 73.5 68.7 28750 32.8 Example 2 83.6 80.2 75.8 30250 39.7 Example 3 87.5 84.6 80.2 31500 45.4 Comparative Example 1 58.3 42.5 30.2 24250 12 Comparative Example 2 70.2 38.6 25.3 23500 8.5 Chemical pesticide 90.3 65.2 42.5 26750 23.6 Blank control - - - 21650 -

[0132] As can be seen from Table 4, each embodiment under field conditions shows good control effect and yield-increasing effect. Compared with the comparative examples and chemical pesticides, the control effect of the embodiments decreases slowly over time, and still maintains a high level of control effect after 90 days, indicating that the compound microbial agent slow-release granules have long-term control characteristics. In terms of yield, the yield-increasing rate of each embodiment is 32.8%-45.4%, which is significantly higher than that of the comparative examples and chemical pesticide treatment, which further proves the comprehensive benefits of the product of the application.

[0133] The sweet potato stem nematode disease compound microbial agent slow-release granules of the application achieves high-efficiency control of sweet potato stem nematode disease through the following mechanisms:

[0134] 1. Multi-strain synergistic effect: The four functionally complementary microbial strains synergistically control sweet potato stem nematode disease through different mechanisms. Paecilomyces lilacinus can parasitize nematode eggs, larvae and adults, produce chitinase and protease and other extracellular enzymes to destroy the nematode cuticle structure; Bacillus thuringiensis produces Cry protein toxin to damage the nematode digestive tract; Streptomyces produces avermectin substances to act on the nematode nervous system, causing the nematode to paralyze and die; Bacillus subtilis produces various antibacterial substances and biosurfactants to enhance the penetration ability of other strains to nematodes, while promoting plant growth and enhancing plant disease resistance. Experimental results show that the control effect of Comparative Example 1 lacking any one of the strains is significantly reduced, confirming the importance of this synergistic effect.

[0135] 2. Environment-friendly carrier composite slow-release system: The application uses biochar, humic acid, sodium alginate, chitosan and other environmentally friendly materials to construct a composite slow-release carrier system. The porous structure of biochar provides a place for microorganisms to attach and reproduce; humic acid promotes microbial growth and enhances plant disease resistance; sodium alginate and chitosan form a microcapsule structure to protect microorganisms and achieve slow-release effect; calcium silicate adjusts soil pH and enhances plant cell wall strength; mannitol protects the activity of microorganisms during drying and storage; sodium lignosulfonate enhances the dispersibility of the granules in water. Experimental results show that the slow-release performance of Comparative Examples 2 and 3 lacking key carrier materials decreases significantly, and the control effect lasts for a short time, confirming the necessity of this composite carrier system.

[0136] 3. Improve the soil environment: the complex bacterial agent slow-release granules of the application not only can effectively prevent and cure sweet potato stem nematode disease, but also can improve soil structure, promote soil microbial community balance and improve crop disease resistance. The experimental results show that the growth status and root system development of sweet potato seedlings treated by the product of the application are significantly better than those of the control, and the yield-increasing effect is obvious, which proves the comprehensive soil health regulation effect of the product of the application.

[0137] The sweet potato stem nematode disease complex bacterial agent slow-release granules of the application have the following application advantages:

[0138] 1. High efficiency: through the synergistic effect of multiple functional strains, the sweet potato stem nematode disease can be effectively prevented and treated, and the control effect can reach more than 85%;

[0139] 2. Durability: through the environmentally friendly carrier complex slow-release system, the slow release of microorganisms is realized, and the control effect lasts for 90 days;

[0140] 3. Safety: the microorganism strains and carrier materials used in the product are environmentally friendly, non-toxic and non-residual, and meet the requirements of green agricultural development;

[0141] 4. Comprehensive benefits: not only can prevent and treat sweet potato stem nematode disease, but also can improve soil environment, promote plant growth and increase yield significantly;

[0142] 5. Convenient to use: the granule form makes the product convenient to store, transport and apply, and is suitable for large-area popularization and application.

[0143] Therefore, the product of the application has broad application prospect and important practical value in the field of prevention and treatment of sweet potato stem nematode disease.

[0144] The above description is only the preferred embodiment of the application and is not used to limit the application. For those skilled in the art, the application can have various modifications and changes. Any modification, equivalent replacement, improvement, etc. within the spirit and principles of the application shall be included in the protection scope of the application.

Claims

1. A composite bacterial agent slow-release granule for preventing and treating sweet potato stem nematode disease, characterized in that: Calculated by weight, it includes the following components: 15-25 parts of live bacteria powder of Paecilomyces lilacinus, 10-20 parts of live bacteria powder of Bacillus thuringiensis, 10-20 parts of live bacteria powder of Streptomyces, 5-15 parts of live bacteria powder of Bacillus subtilis, 15-25 parts of biochar, 5-15 parts of sodium alginate, 5-15 parts of humic acid, 5-10 parts of chitosan, 3-8 parts of calcium silicate, 1-5 parts of mannitol and 1-5 parts of sodium lignin sulfonate.

2. The slow-release granules of the composite bacterial agent for sweet potato stem nematode disease according to claim 1, characterized in that The Paecilomyces lilacinum is Purpureocillium lilacinum CGMCC No.3.17493; the Bacillus thuringiensis is Bacillusthuringiensis CGMCC No.1.6942; the Streptomyces is Streptomyces avermitilis CGMCC No.4.2072; and the Bacillus subtilis is Bacillus subtilis CGMCC No.1.

308.

3. The slow-release granules of the composite bacterial agent for treating sweet potato stem nematode disease according to claim 1, characterized in that: The number of live bacteria in the live fungus powder of Paecilomyces lilacinus is not less than 5.0×10 9 CFU / g, the number of live bacteria in the Bacillus thuringiensis live bacteria powder is not less than 1.0×10 10 CFU / g, the number of viable bacteria in the Streptomyces live bacteria powder is not less than 5.0×10 9 CFU / g, the number of viable bacteria in the Bacillus subtilis live bacteria powder is not less than 1.0×10 10 CFU / g.

4. The slow-release granules of the composite bacterial agent for sweet potato stem nematode disease according to claim 1, characterized in that The biochar is prepared by pyrolyzing rice husks or sawdust at 500-600°C for 2-3 hours; the humic acid is extracted from weathered coal and contains no less than 70% humic acid; the deacetylation degree of the chitosan is no less than 90%; and the calcium silicate, mannitol and sodium lignin sulfonate are all industrial grade.

5. The slow-release granules of the composite bacterial agent for treating sweet potato stem nematode disease according to claim 1, characterized in that: The granules have a particle size of 2-4 mm, a water content of less than 10%, a water resistance of not less than 2 hours, and a particle hardness of 1.5-3.0 kg / cm².

6. A method for preparing the slow-release granules of the composite bacterial agent for treating sweet potato stem nematode disease according to any one of claims 1 to 5, characterized in that: The following steps are involved: 1) Solid-state fermentation culture of functional bacteria: Paecilomyces lilacinus, Bacillus thuringiensis, Streptomyces, and Bacillus subtilis were inoculated into the corresponding solid culture medium and cultured under suitable temperature conditions until the bacterial growth reached its maximum value; 2) Preparation of live bacterial powder: The solid-state fermentation products of each bacterial species obtained in step 1) were dried at 45-50°C to a moisture content of less than 10%, and crushed to a size of less than 100 mesh to obtain live bacterial powder of each bacterial species; 3) Carrier material pretreatment: Grind biochar, humic acid, and calcium silicate to less than 100 mesh respectively; 4) Compound inoculant mixing: According to the formula ratio, mix the live bacterial powder of each strain, biochar, humic acid, calcium silicate and mannitol evenly, and stir at 25-30°C for 20-30 minutes; 5) Preparation of binder solution: Dissolve chitosan in 1% acetic acid solution to a concentration of 2%, add sodium alginate and sodium lignin sulfonate to prepare a binder solution; 6) Granule preparation: The mixture obtained in step 4) is mixed with the binder solution in step 5) in a weight ratio of 1:0.5-0.8, and wet granules with a diameter of 2-4 mm are formed by extrusion granulation equipment; 7) Granule drying and stabilization: The wet granules were dried at 40-45°C until the moisture content was less than 10%. After cooling to room temperature, they were sprayed with 2% calcium chloride solution to solidify the sodium alginate. 8) Product packaging: The dried granules are screened to remove powder and fragments, and then packaged and sealed to produce the slow-release granules of the composite bacterial agent for treating sweet potato stem nematode disease.

7. The preparation method according to claim 6, characterized in that In the step 1), the solid culture medium of Paecilomyces lilacinus consists of wheat bran, brown rice flour and corn steep liquor in a weight ratio of 5:3:2, a water content of 55-60%, a pH value of 6.5-7.0, a culture temperature of 26±1°C, and a culture time of 8-10 days; the solid culture medium of Bacillus thuringiensis consists of soybean meal, corn flour and calcium carbonate in a weight ratio of 6:3:1, a water content of 50-55%, a pH value of 7.0-7.5, a culture temperature of 30±1°C, and a culture time of 8-10 days. The solid culture medium for Streptomyces is composed of wheat bran, rice bran and sucrose in a weight ratio of 6:3:1, a water content of 45-50%, a pH value of 7.2-7.8, a culture temperature of 28±1°C, and a culture time of 6-8 days; the solid culture medium for Bacillus subtilis is composed of wheat flour, corn steep liquor and fish meal in a weight ratio of 6:3:1, a water content of 50-55%, a pH value of 6.8-7.3, a culture temperature of 32±1°C, and a culture time of 3-4 days.

8. The preparation method according to claim 6, characterized in that In the step 2), the solid-state fermentation products of each strain are dried by hot air circulation drying at a drying temperature of 45-50° C. for 6-12 hours; the dried fermentation products are crushed by a high-speed crusher to a particle size of no more than 150 μm.

9. The preparation method according to claim 6, characterized in that In step 6), the granules are prepared using a twin-screw extruder at a working temperature of 20-25° C., an extrusion speed of 10-15 rpm, and an extrusion pressure of 0.5-1.0 MPa. After extrusion, the granules are cut into granules of 2-4 mm in length.

10. The preparation method according to claim 6, characterized in that In step 7), the wet granules are dried in a fluidized bed dryer at an inlet air temperature of 40-45° C., an outlet air temperature of 30-35° C., and a drying time of 1.5-2.5 hours. The calcium chloride solution is sprayed in an amount of 5-10% by weight of the granules. After spraying, the granules are allowed to stand at room temperature for 2-4 hours to form a cross-linked calcium alginate network structure on the surface of the granules, thereby enhancing the granule strength and improving the sustained-release performance.