Auxiliary agent for drying microbial fermentation products and its application

Abstract

The present invention is related to the field of biological pesticides. Disclosed are an adjuvant for drying microbial fermentation broth and its application. The adjuvant is added to the microbial fermentation broth and directly spray-dried into a mother powder, thereby avoiding the loss of effective ingredients caused by measures such as centrifugation, high temperature, concentration, and filtration, which affects the activity of the mother powder, while fully exerting the synergistic effect of the synergistic substances. The use of the adjuvant and preparation method provided by the present invention can improve the control effect and storage performance of Bacillus thuringiensis mother powder for controlling plant parasites, has good economic and ecological benefits, has broad prospects for application and promotion, and is conducive to food safety and sustainable development of agriculture.

Description

Technical Field

[0001] The invention belongs to the field of biological pesticides, and particularly relates to an auxiliary agent for drying microbial fermentation products and its application. Background Art

[0002] Plant-parasitic nematodes are obligate parasites of plants, primarily feeding on underground parts such as roots and tubers. They form root knots and egg masses, causing plants to wither, wilt, stunt, and die. They also provide pathways for pathogens such as bacteria, fungi, and viruses to infect, facilitating plant damage. Plant-parasitic nematodes that pose a significant threat to both plant economics and the plant ecosystem include root-knot nematodes (Meloidogynes spp.), pine wood nematodes (Bursaphelenchus xylophilus spp.), stem nematodes (Ditylenchus spp.), and cyst nematodes (Heteroderas spp. / Globodera spp.). Of these, root-knot and cyst nematodes are the most devastating.

[0003] Current methods for controlling plant-parasitic nematodes have significant limitations. Traditional methods for controlling plant-parasitic nematodes, such as physical control, are safe and environmentally friendly, but they are not only time-consuming and labor-intensive, with high economic costs, but also generally ineffective. Chemical control is relatively cost-effective, but chemical pesticides are difficult to degrade, have certain toxicity, and can enter the bodies of humans and animals through various channels, having a serious impact on the natural environment and human health, causing a series of problems such as pesticide residues, pesticide resistance, soil degradation, and the ecological environment. Compared with physical or chemical control, biological control is more environmentally friendly and more easily accepted by people. By using microorganisms with nematode control functions, nematode control can be achieved, soil can be improved, the rich microbial population in the soil can be restored, the immunity of plant roots can be enhanced, and the number of nematodes can be maintained at a low level. Therefore, the development of functional microorganisms is a research hotspot for solving nematode diseases.

[0004] Bacillus thuringiensis (Bt), currently the world's most produced and widely used microbial insecticide, is widely used to control agricultural pests, forest and fruit tree pests, and stored pests. Different Bt strains produce toxins of varying types and properties, exhibiting varying insecticidal activity. Bt strains reportedly active against a variety of nematodes produce parasporal crystal proteins (Cry proteins) that differ from those of conventional Bt strains. For example, the B. thuringiensis strain HAN055 (CN108070535A) screened by Huazhong Agricultural University possesses the nematicidal crystal proteins Cry6Aa2 and Cry55A. In addition to Cry protein toxins, existing research indicates that B. thuringiensis also produces a range of nematicidal compounds, such as thuringin, hemolysin, lecithinase, and trans-aconitic acid, which have varying effects on larvae and eggs. Synergistic compounds such as autoinducer inhibitors, biphasic acid, and accessory proteins are also produced.

[0005] However, Bacillus thuringiensis for nematode control also has some problems in its production and application, which lead to its nematocidal activity being weakened or even lost, and its residual effect being short. (1) Centrifugal concentration leads to the loss of soluble small molecules and synergistic components in the supernatant; (2) The spray drying process leads to its activity being reduced. Spray drying is an important method for producing high-potency Bt mother powder. In industrial production, the fermentation broth is generally spray-dried to produce mother powder, which is then processed into the required dosage form. However, high-temperature drying can easily lead to the denaturation of Bt active ingredients such as parasporal crystals, inactivation of spores, and loss of other heat-sensitive active ingredients, which will inevitably lead to a decrease in the insecticidal activity of the preparation. (3) The ultrafiltration concentration process has low selectivity for Bt metabolites, wastes some active substances, and cannot directly obtain dry powder preparations. (4) The process for recovering soluble synergistic substances of Bacillus thuringiensis is relatively cumbersome, costly, and has not improved its efficacy. Its stability is poor and the rate of foreign bacteria is high. (5) Adding mica powder to the Bt fermentation liquid for direct spraying recovery can simplify the process steps, but the resulting Bt mother powder is easy to absorb moisture, has high viscosity, and has poor properties. (6) During field application, the residual effect is short, the effect is unstable, and the cost-effectiveness is not high. This is because the effective active substances such as nematode crystal protein are degraded by soil microorganisms, and the Bt spores cannot germinate and cannot play a role. Therefore, ensuring the stability of effective active substances such as crystal protein in the soil and improving the survival rate of Bt spores are the primary factors to ensure the field control effect of Bt preparations for nematode control.

[0006] Research on applying related technologies to enhance the nematode control efficacy of Bacillus thuringiensis preparations has yet to be reported. Furthermore, most published nematode control technologies only test their effectiveness against larvae. However, in the infection cycle of plant-parasitic nematodes, the control rate of egg masses and egg particles is also an important factor in improving nematode efficacy.

[0007] Therefore, there is an urgent need for a Bacillus thuringiensis preparation and a preparation method thereof with a simple production process, high nematicidal activity and recovery rate of synergistic components, stable quality and efficacy, so as to improve the effect of controlling plant parasitic nematodes, including the effect of controlling parasitic nematode egg masses and egg grains. Summary of the Invention

[0008] In order to solve at least one of the above technical problems, the present invention provides an auxiliary agent for drying microbial fermentation products and application thereof.

[0009] According to a first aspect of the present invention, an auxiliary agent for drying microbial fermentation products is provided, wherein the auxiliary agent comprises one or more of the following components: ammonium salt, white carbon black, kaolin, and trehalose.

[0010] In some embodiments, the auxiliary agent includes one or more of the following components: calculated on a mass basis of the microbial fermentation product, 0.1% to 1.0% ammonium salt, 1.5% to 4.5% white carbon black, 1.0% to 3.0% kaolin, and 1.0% to 2.0% trehalose.

[0011] More preferably, the auxiliary agent comprises one or more of the following components: calculated by mass fraction of the microbial fermentation product, 1% ammonium molybdate, 3% white carbon black, 2% kaolin, and 2% trehalose.

[0012] In some embodiments, the ammonium salt includes one or more of the following: ammonium sulfate, ammonium thiocyanate, ammonium nitrate, diammonium phosphate, ammonium chloride, ammonium molybdate, and ammonium bicarbonate.

[0013] In some embodiments, the ammonium salt is ammonium molybdate.

[0014] In some embodiments, the auxiliary agent includes one or more of the following components: 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9% or 1.0% ammonium salt based on the mass fraction of the microbial fermentation product.

[0015] In some embodiments, the auxiliary agent includes one or more of the following components: 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9%, 3.0%, 3.1%, 3.2%, 3.3%, 3.4%, 3.5%, 3.6%, 3.7%, 3.8%, 3.9%, 4.0%, 4.1%, 4.2%, 4.3%, 4.4% or 4.5% of white carbon black, based on the mass fraction of the microbial fermentation product.

[0016] In some embodiments, the auxiliary agent includes one or more of the following components: 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9%, 2.0%, 2.1%, 2.2%, 2.3%, 2.4%, 2.5%, 2.6%, 2.7%, 2.8%, 2.9% or 3.0% kaolin, based on the mass fraction of the microbial fermentation product.

[0017] In some embodiments, the auxiliary agent includes one or more of the following components: 1.0%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2.0% trehalose based on the mass fraction of the microbial fermentation product.

[0018] In some embodiments, the auxiliary agent includes one or more of the following components: calculated on a mass basis of the microbial fermentation product, 1% ammonium salt, 3% white carbon black, 2% kaolin, and 2% trehalose.

[0019] In some embodiments, the microorganism comprises bacteria, actinomycetes, yeast, or mold.

[0020] In some embodiments, the microorganism comprises bacteria.

[0021] In some embodiments, the microorganism comprises Bacillus.

[0022] In some embodiments, the microorganism comprises one or more of the following: Bacillus subtilis, Bacillus licheniformis, Bacillus cereus, Bacillus megatherium, Bacillus thuringiensis.

[0023] In some embodiments, the microorganism comprises Bacillus thuringiensis.

[0024] In some embodiments, the microorganism comprises Bacillus thuringiensis having the function of controlling plant parasitic nematodes.

[0025] According to a second aspect of the present invention, a microbial mother powder is provided, wherein the microbial mother powder contains the auxiliary agent according to the first aspect.

[0026] In some embodiments, the microbial mother powder is Bacillus mother powder.

[0027] In some embodiments, the microbial mother powder includes Bacillus subtilis mother powder, Bacillus licheniformis mother powder, Bacillus cereus mother powder, Bacillus megaterium mother powder, Bacillus thuringiensis mother powder, etc.

[0028] In some embodiments, the microbial mother powder is Bacillus thuringiensis mother powder.

[0029] In some embodiments, the Bacillus thuringiensis has the function of preventing and controlling plant parasites.

[0030] In some embodiments, the plant parasite comprises a nematode.

[0031] In some embodiments, the nematodes include one or more of the following: Meloidogyne, Bursa phelenchus xylophilus, Ditylenchus, Heterodera, pratylenchus, radopholus, Aphelenchoides, bursaphelenchus.

[0032] In some specific embodiments, the nematodes include one or more of the following: Meloidogyne, Bursaphelenchus xylophilus, Ditylenchus, and Heterodera.

[0033] In some embodiments, the number of spores in the mother powder is not less than 30×10 9 cfu / g, protein crystals not less than 1.5%, water content not higher than 5%, and miscellaneous bacteria rate not higher than 2%.

[0034] In some specific embodiments, the Bacillus thuringiensis is Bacillus thuringiensis HAN055, which is deposited in the China Center for Type Culture Collection (CCTCC) of Wuhan University with a deposit number of CCTCC NO: M 2015608.

[0035] In some specific embodiments, the Bacillus thuringiensis is Bacillus thuringiensis KN63, which is deposited with the China Agricultural Culture Collection Center (ACCC) with a deposit number of ACCC NO: 62944.

[0036] In some specific embodiments, the Bacillus thuringiensis is derived from a commercially available 4000 IU / mg Bacillus thuringiensis suspension seed coating agent produced by Jiamusi Xingyu Biotechnology Development Co., Ltd. in Heilongjiang Province, and the target of prevention and control is soybean cyst nematode.

[0037] According to a third aspect of the present invention, a method for preparing a microbial mother powder is provided, the method comprising the following steps: adding the auxiliary agent according to the first aspect to a microbial fermentation product and mixing the mixture.

[0038] In some embodiments, the solid content of the microbial fermentation product is 1% to 6% by mass.

[0039] In some embodiments, the solid content of the microbial fermentation product is 1%, 2%, 3%, 4%, 5% or 6%.

[0040] In some specific embodiments, the solid content of the microbial fermentation product is 5%.

[0041] In some embodiments, the auxiliary agent of the first aspect is added to the microbial fermentation product and mixed to obtain a mixture, and the solid content of the mixture is not less than 10%.

[0042] In some embodiments, the auxiliary agent described in the first aspect is added to the microbial fermentation product and mixed to obtain a mixture, and the solid content of the mixture is not less than 10%, not less than 11%, not less than 12%, not less than 13%, not less than 14%, not less than 15%, not less than 16%, not less than 17%, not less than 18%, not less than 19% or not less than 20%.

[0043] In some embodiments, the microorganism comprises bacteria, actinomycetes, yeast, or mold.

[0044] In some embodiments, the microorganism comprises bacteria.

[0045] In some embodiments, the microorganism comprises Bacillus.

[0046] In some embodiments, the microorganism comprises one or more of the following: Bacillus subtilis, Bacillus licheniformis, Bacillus cereus, Bacillus megatherium, Bacillus thuringiensis.

[0047] In some embodiments, the microorganism comprises Bacillus thuringiensis.

[0048] In some embodiments, the Bacillus thuringiensis is a Bacillus thuringiensis capable of preventing and controlling plant parasitic nematodes.

[0049] In some specific embodiments, the Bacillus thuringiensis is Bacillus thuringiensis HAN055, which is deposited in the China Center for Type Culture Collection (CCTCC) of Wuhan University with a deposit number of CCTCC NO: M 2015608.

[0050] In some specific embodiments, the Bacillus thuringiensis is Bacillus thuringiensis KN63, which is deposited with the China Agricultural Culture Collection Center (ACCC) with a deposit number of ACCC NO: 62944.

[0051] In some specific embodiments, the Bacillus thuringiensis is derived from a commercially available 4000 IU / mg Bacillus thuringiensis suspension seed coating agent produced by Jiamusi Xingyu Biotechnology Development Co., Ltd. in Heilongjiang Province, and the target of prevention and control is soybean cyst nematode.

[0052] In some embodiments, the method comprises adding the auxiliary agent of the first aspect to the microbial fermentation product, mixing the mixture, and then spray drying the mixture.

[0053] In some embodiments, the spray drying is set as follows: inlet air temperature is 150-300°C; outlet air temperature is 50-100°C; the bottom powder outlet is connected to a cooler to cool the powder to below 20-40°C.

[0054] In some embodiments, the spray drying is set as follows: the inlet air temperature is 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, 220°C, 230°C, 240°C, 250°C, 260°C, 270°C, 280°C, 290°C or 300°C.

[0055] In some embodiments, the spray drying is set as follows: the outlet air temperature is 50°C, 60°C, 70°C, 80, 90°C or 100°C.

[0056] In some embodiments, the base powder outlet is connected to a cooler to cool the powder outlet to below 20°C, 25°C, 30°C, 35°C or 40°C.

[0057] According to a fourth aspect of the present invention, a microbial mother powder is provided, which is prepared using the preparation method described in the third aspect.

[0058] According to a fifth aspect of the present invention, a microbial agent is provided, wherein the microbial agent comprises the microbial mother powder according to the second aspect or the fourth aspect.

[0059] In some embodiments, the microbial agent further comprises an additive.

[0060] In some embodiments, the additives include one or more of the following: a dispersant, an antifreeze agent, a thickener, an antifoaming agent, a pH adjuster, a film former, a wetting agent, an anti-caking agent, a disintegrant, and a filler or water.

[0061] In some embodiments, the additives include one or more of the following: based on the mass fraction of the bacterial agent, 3-20% of a dispersant, 1-5% of an antifreeze agent, 0.1-5% of a thickener, 0.1-0.8% of a defoaming agent, 0.1-5% of a pH regulator, 1-10% of a film-forming agent, 1-10% of a wetting agent, 3-5% of an anti-caking agent, 1-5% of a disintegrant, and a filler or water.

[0062] In some embodiments, the bacterial agent includes one or more of the following components: calculated by mass fraction of the bacterial agent, 1-20% of Bacillus thuringiensis mother powder, 5-20% of dispersant, 1-5% of antifreeze agent, 0.1-2% of thickener, 0.1-0.8% of defoaming agent, 0.1-5% of pH regulator, 1-10% of film-forming agent and water.

[0063] In some embodiments, the bacterial agent includes one or more of the following components: calculated by mass fraction of the bacterial agent, 1-50% of Bacillus thuringiensis mother powder, 3-10% of a dispersant, 1-5% of a thickener, 3-5% of a film-forming agent, 1-10% of a wetting agent, 3-5% of an anti-caking agent, 1-5% of a disintegrant, and a filler.

[0064] In some embodiments, the dispersant is selected from one or more of polycarboxylates coded as D425, LG-3, GY-D1252, GY-D1256, SNWGF-01, lignin sulfonates coded as 201107, 201108, alkylphenol polyoxyethylene ether methyl ether condensate sulfate, alkyl sulfonate calcium salt, naphthalenesulfonic acid formaldehyde condensate sodium salt, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ester, fatty amine polyoxyethylene ether, and glycerol fatty acid ester polyoxyethylene ether.

[0065] In some embodiments, the wetting agent is selected from one or more of sodium lauryl sulfate, calcium dodecylbenzenesulfonate, lakai powder BX, wetting penetrant F, alkylbenzenesulfonate polyoxyethylene triphenyl phenyl phosphate, soapberry powder, silkworm feces, and soapberry powder.

[0066] In some embodiments, the disintegrant is selected from one or more of bentonite, urea, ammonium sulfate, aluminum chloride, citric acid, succinic acid, and sodium bicarbonate.

[0067] In some embodiments, the thickener is selected from one or more of xanthan gum, carboxymethyl cellulose, carboxyethyl cellulose, methyl cellulose, magnesium aluminum silicate, and polyvinyl alcohol.

[0068] In some embodiments, the stabilizer is selected from one or more of sodium citrate and resorcinol.

[0069] In some embodiments, the antifreeze agent is selected from one or more of ethylene glycol, propylene glycol, and glycerol.

[0070] In some embodiments, the defoaming agent is selected from one or more of silicone oil, silicone compounds, C10-20 saturated fatty acid compounds, and C8-10 fatty alcohols.

[0071] In some embodiments, the filler is selected from one or more of kaolin, diatomaceous earth, bentonite, attapulgite, white carbon black, starch, and light calcium carbonate.

[0072] In some embodiments, the microbial agent is selected from one or more of the following dosage forms: powder, granules, large granules, fine granules, microgranules, microcapsule granules, wettable powder, oil-dispersible powder, water-dispersible granules, dispersible granules, emulsifiable granules, effervescent granules, dispersible tablets, effervescent tablets, sustained-release agents, sustained-release blocks, sustained-release tubes, sustained-release particles, soluble powders, soluble granules, soluble tablets, soluble solutions, aqueous solutions, soluble gels, oils, film-spreading oils, ultra-low volume liquids, ultra-low volume microcapsule suspensions, emulsifiable concentrates, latexes, dispersible liquids, pastes, concentrated gels, emulsions in water, oil emulsions, microemulsions, ointments, suspensions, microcapsule suspensions, oil suspensions, suspoemulsions, dispersible powders for seed treatment, soluble powders for seed treatment, seed treatment liquids, seed treatment emulsions, seed treatment suspensions, suspended seed coatings, and microcapsule suspensions for seed treatment.

[0073] In some embodiments, the microbial agent is in the following dosage forms: powder, wettable powder, water-dispersible granules, dispersible granules, granules, dispersible powder for seed treatment, suspension, and suspension for seed treatment.

[0074] In some embodiments, the microbial inoculant comprises a bacterial inoculant.

[0075] In some embodiments, the microbial inoculant comprises a Bacillus inoculant.

[0076] In some embodiments, the microbial inoculant comprises a Bacillus thuringiensis inoculant.

[0077] According to a sixth aspect of the present invention, there is provided a method for preparing the microbial agent according to the fifth aspect, the method comprising mixing the microbial mother powder according to the second aspect or the fourth aspect with an additive.

[0078] In some embodiments, the preparation method comprises the following steps:

[0079] 1) Mix the additives evenly;

[0080] 2) Add the microbial mother powder described in the second aspect or the fourth aspect and mix well.

[0081] In some embodiments, the mixing in step 1) comprises shear mixing.

[0082] In some embodiments, the preparation method comprises the following steps:

[0083] 1) Mix all the components evenly;

[0084] 2) Crush.

[0085] In some specific embodiments, the pulverization in step 2) is performed in a jet mill.

[0086] In some embodiments, the preparation method comprises the following steps:

[0087] 1) Mix all the components evenly;

[0088] 2) Crushing, kneading, granulating, drying and screening.

[0089] In some specific embodiments, the step 2) comprises pulverizing with an ultrafine airflow pulverizer, kneading, and then adding to a fluidized bed granulation dryer for granulation, drying, and screening.

[0090] In some specific embodiments, the step 2) includes pulverizing the product with an ultrafine airflow mill, feeding the product into a disc granulator, and then feeding the product into a fluidized bed granulation dryer for granulation, drying, and screening.

[0091] In some embodiments, the microbial mother powder includes bacterial mother powder.

[0092] In some embodiments, the microbial mother powder includes Bacillus mother powder.

[0093] In some embodiments, the microbial mother powder includes Bacillus subtilis mother powder, Bacillus licheniformis mother powder, Bacillus cereus mother powder, Bacillus megaterium mother powder, Bacillus thuringiensis mother powder, etc.

[0094] In some embodiments, the microbial mother powder includes Bacillus thuringiensis mother powder.

[0095] According to a seventh aspect of the present invention, a microbial agent is provided, wherein the microbial agent is prepared using the preparation method described in the sixth aspect.

[0096] According to the eighth aspect of the present invention, provided is the use of the adjuvant described in the first aspect, the Bacillus thuringiensis mother powder described in the second aspect, the preparation method described in the third aspect, the Bacillus thuringiensis mother powder described in the fourth aspect, the Bacillus thuringiensis agent described in the fifth aspect, and the preparation method described in the sixth aspect in controlling plant parasites.

[0097] In some embodiments, the plant parasite comprises a nematode.

[0098] In some embodiments, the nematodes include one or more of the following: Meloidogyne, Bursa phelenchus xylophilus, Ditylenchus, Heterodera, pratylenchus, radopholus, Aphelenchoides, bursaphelenchus.

[0099] In some specific embodiments, the nematodes include one or more of the following: Meloidogyne, Bursaphelenchus xylophilus, Ditylenchus, and Heterodera.

[0100] The use of the adjuvant provided by the present invention to prepare Bacillus thuringiensis mother powder reduces the loss of active substances related to the prevention and control of plant parasites, can fully recover metabolites related to the prevention and control of plant parasites, and improves the prevention and control effect of Bacillus thuringiensis on plant parasites and the storage performance.

[0101] The method for preparing the microbial mother powder provided by the present invention is used to prepare Bacillus thuringiensis mother powder, which can fully recover active substances related to the prevention and treatment of plant parasites. Compared with the existing technology, the production process is simpler, the cycle is shorter, the discharge of waste residues and wastewater is greatly reduced, and it is suitable for industrial production.

[0102] The Bacillus thuringiensis mother powder provided by the present invention has a high recovery rate of spores and crystals, a high content of active substances related to preventing and controlling nematodes, strong activity, a low rate of foreign bacteria, low moisture content, good stability of quality and efficacy, is easy to store and transport, and has a good effect in preventing and controlling plant parasites.

[0103] The method of using the Bacillus thuringiensis mother powder provided by the present invention to control plant parasites has a better control effect than the existing technology at the same spore concentration. It is an ideal biological control method with good economic and ecological benefits, broad prospects for application and promotion, and is conducive to food safety and sustainable development of agriculture. DETAILED DESCRIPTION

[0104] To make the objectives, technical solutions, and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the following examples. The specific embodiments described herein are intended only to illustrate the present invention and are not intended to limit the present invention in any way. In addition, in the following description, descriptions of known structures and technologies are omitted to avoid unnecessary confusion about the concepts of the present disclosure. Such structures and technologies are also described in many publications.

[0105] The present invention aims to provide an adjuvant for spray drying of Bacillus thuringiensis fermentation products. The adjuvant is used to prepare Bacillus thuringiensis mother powder. The production process is simple, no waste residue or wastewater is generated, nematicidal activity and synergistic components can be efficiently recovered, the quality and efficacy are stable, and the control effect of plant parasitic nematodes can be improved.

[0106] The preparation method of the present invention comprises the following steps:

[0107] 1) Selecting a Bacillus thuringiensis strain capable of controlling plant nematodes:

[0108] In some embodiments, the Bacillus thuringiensis strain is derived from Bacillus thuringiensis HAN055 disclosed in patent number CN108070535A, and its deposit number is CCTCC NO: M 2015608.

[0109] In some embodiments, the Bacillus thuringiensis strain is derived from Bacillus thuringiensis KN63 screened and isolated by Wuhan Kono Biotechnology Co., Ltd., which has high activity against root-knot nematodes and has a deposit number of ACCC NO: 62944.

[0110] In some embodiments, the Bacillus thuringiensis is derived from a commercially available 4000 IU / mg Bacillus thuringiensis suspension seed coating agent produced by Jiamusi Xingyu Biotechnology Development Co., Ltd. in Heilongjiang Province, and the target of prevention and control is soybean cyst nematode.

[0111] 2) The Bt fermentation medium formula and fermentation conditions used are derived from the method disclosed in the document with application number 202111593497.9 in some embodiments. Unless otherwise specified, it is a known disclosed method. The present invention provides a full recovery process for Bacillus thuringiensis mother powder for controlling nematodes to increase the recovery rate and improve the effect of Bacillus thuringiensis mother powder on nematodes. The specific steps are fermentation broth acidification, adjuvant mixing, spray drying, mixed screening, and quality inspection. A more specific operating process is as follows:

[0112] 24 tons of Bt fermentation liquid with 5% solid content and spore count of 7.5×10 9cfu / mL, with a crystal content of 0.38%. Citric acid was added to acidify the fermentation broth to a pH of 4.5. The following additives were added and mixed to a solids content of approximately 13%. Stirring was maintained during the process to prevent sedimentation of the added materials. The inlet air temperature was 200±10°C; the outlet air temperature was 80±5°C. A cooler was connected to the bottom powder outlet to cool the powder to below 35°C before bagging. Mixing and screening were performed for 30 minutes, followed by a 25-minute return to the bottom. The mixed material was sieved through a 30-mesh sieve and bagged to obtain the mother powder.

[0113] The auxiliary agent added to the Bt fermentation liquid comprises the following components: calculated based on the mass fraction of the Bt fermentation liquid, 0.1-1.0% of ammonium salt, 1.5-4.5% of white carbon black, 1.0-3.0% of kaolin and 1.0-2.0% of trehalose.

[0114] As a preferred embodiment, the ammonium salt includes one or more of the following: ammonium sulfate, ammonium thiocyanate, ammonium nitrate, diammonium hydrogen phosphate, ammonium chloride, ammonium molybdate and ammonium bicarbonate.

[0115] As a preferred embodiment, the additives added to the Bt fermentation liquid with a solid content of 5% include the following components: 1% ammonium molybdate, 3% white carbon black, 2% kaolin, and 2% trehalose, calculated based on the mass fraction of the Bt fermentation liquid.

[0116] 3) Quality evaluation of Bacillus thuringiensis mother powder for nematode control

[0117] The quality of the obtained mother powder was evaluated in the following aspects, such as spore count, protein crystal content determination, bacterial count, nematode in vitro test, recovery rate and stability tracking. The test results showed that the final spore count was 64.2×10 9 cfu / g, spore recovery rate was 110.57%, protein crystals were 3.21%, crystal recovery rate was 109.11%, and the mother powder was diluted to 1.0×10 8 cfu / mL concentration, the 24-hour corrected mortality rate of the second-instar larvae of the southern root-knot nematode reached 96.8%, which is significantly better than the existing public technology. The moisture content is less than 2.8%, and the contaminant rate is less than 0.5%. After 12 months of sealed storage at room temperature (22±2)℃, the spore count and protein crystal content of the mother powder decreased by less than 3%. When the dilution concentration was 1.0×10 8 cfu / mL, the mortality rate of the second-instar larvae was 90.2%, and the decline rate was 3.41%. The quality and efficiency of the mother powder were stable, and no waste water or waste residue was generated.

[0118] 4) In actual use, the Bacillus thuringiensis mother powder of the present invention can be prepared using known methods into any formulation suitable for agricultural use, preferably a wettable powder, water-dispersible granules (also known as dispersible granules or dry suspension concentrates), granules, dispersible powders for seed treatment, suspension concentrates, and suspension concentrates for seed treatment. The total content of the active ingredient in the formulation is preferably 1% to 50%, with the remainder being auxiliary ingredients permitted and acceptable in pesticides.

[0119] The specific implementation scheme of the mother powder of the present invention to prepare the pesticide formulation of the microbial agent is as follows:

[0120] The microbial agent is a suspension agent, and its components by mass fraction are: 1-20% Bacillus thuringiensis mother powder; 5-20% dispersant; 1-5% antifreeze; 0.1-2% thickener; 0.1-0.8% defoamer; 0.1-5% pH adjuster; the balance is made up with water. The specific production steps of the suspension agent are as follows: first, the other additives are mixed, and then the mixture is uniformly mixed by high-speed shearing. Then, the Bacillus thuringiensis mother powder for the microbial agent for controlling nematodes is added, and the mixture is uniformly stirred in a stirred tank. The suspension agent of the present invention is then divided into portions, filled with nitrogen, and sealed.

[0121] The microbial agent is a seed treatment suspension agent, and its components by mass fraction are: 1-20% Bacillus thuringiensis mother powder; 1-10% film-forming agent; 5-20% dispersant; 1-5% antifreeze agent; 0.1-2% thickener; 0.1-0.8% defoamer; 0.1-5% pH adjuster; the balance is made up with water. The specific production steps of the seed treatment suspension agent are as follows: first, the other additives are mixed, and then the mixture is uniformly mixed by high-speed shearing. Then, the Bacillus thuringiensis mother powder for controlling nematodes is added to the microbial agent, and the mixture is uniformly stirred in a stirred tank. The mixture is then divided into portions, filled with nitrogen, and sealed. The seed treatment suspension agent of the present invention is thus produced.

[0122] The microbial agent is a wettable powder, and its components by mass fraction are: 1-50% Bacillus thuringiensis mother powder; 3-10% dispersant; 1-5% wetting agent, with the remainder being made up by filler. The specific production steps of the wettable powder are as follows: according to the above formula, the Bacillus thuringiensis mother powder for controlling nematodes, the dispersant, the wetting agent, and the filler are mixed, uniformly stirred in a stirred tank, and then mixed uniformly in a jet mill to produce the wettable powder microbial agent of the present invention.

[0123] The microbial agent is a dispersible powder for seed treatment, comprising the following components by mass: 1-50% Bacillus thuringiensis mother powder; 3-10% dispersant; 1-5% wetting agent; 3-5% film-forming agent; and 3-5% anti-caking agent; the remainder being filler. The specific production steps for the dispersible powder for seed treatment are as follows: the Bacillus thuringiensis mother powder for nematode control, the dispersant, wetting agent, film-forming agent, anti-caking agent, and filler are mixed according to the above formula, uniformly stirred in a stirred tank, and then mixed uniformly in a jet mill to produce the dispersible powder for seed treatment of the present invention.

[0124] The bacterial agent is a water-dispersible granule, and its components by mass fraction are: 1-50% Bacillus thuringiensis mother powder; 3-10% dispersant; 1-10% wetting agent; 1-5% disintegrant; and the balance is made up of fillers. The specific production steps of the water-dispersible granules are as follows: according to the above formula, the Bacillus thuringiensis mother powder for the bacterial agent for controlling nematodes is uniformly mixed with the dispersant, wetting agent, disintegrant, and filler; pulverized with an ultrafine airflow mill, kneaded, and then added to a fluidized bed granulation dryer for granulation, drying, sieving, and sampling and analysis, thereby producing the water-dispersible granule bacterial agent of the present invention.

[0125] The microbial agent is a granular formulation, and its components by mass fraction are: 1-50% Bacillus thuringiensis mother powder; 1-10% wetting agent; 1-5% disintegrant; 1-5% thickener; and the remainder is made up of fillers. The specific production steps of the water-dispersible granules are as follows: the Bacillus thuringiensis mother powder for controlling nematodes is uniformly mixed with the wetting agent, disintegrant, thickener, and filler according to the above formula; the mixture is pulverized in an ultrafine airflow mill, placed in a disc granulator, and then placed in a fluidized bed granulation dryer for granulation, drying, and screening to produce the microbial agent granules of the present invention.

[0126] The additives used in the preparation process of the above-mentioned microbial agent include solvents, cosolvents, dispersants, emulsifiers, wetting agents, stabilizers, antifreeze agents, disintegrants, thickeners, defoaming agents, fillers, etc. and other known substances that are beneficial to the stability of the active ingredients in the microbial agent and the efficacy of the active ingredients. They are all various components commonly used or permitted for use in pesticide microbial agents and are not particularly limited. The specific components and amounts are determined through experiments according to the formulation requirements.

[0127] The dispersant is selected from one or more of polycarboxylates coded as D425, LG-3, GY-D1252, GY-D1256, and SNWGF-01, lignin sulfonates coded as 201107 and 201108, alkylphenol polyoxyethylene ether methyl ether condensate sulfate, alkyl sulfonate calcium salt, naphthalenesulfonic acid formaldehyde condensate sodium salt, alkylphenol polyoxyethylene ether, fatty acid polyoxyethylene ester, fatty amine polyoxyethylene ether, and glycerol fatty acid ester polyoxyethylene ether.

[0128] The wetting agent is selected from one or more of sodium lauryl sulfate, calcium dodecylbenzenesulfonate, lakai powder BX, wetting penetrant F, alkylbenzenesulfonate polyoxyethylene triphenyl phenyl phosphate, soapberry powder, silkworm feces, and soapberry powder.

[0129] The disintegrant is selected from one or more of bentonite, urea, ammonium sulfate, aluminum chloride, citric acid, succinic acid, and sodium bicarbonate.

[0130] The thickener is selected from one or more of xanthan gum, carboxymethyl cellulose, carboxyethyl cellulose, methyl cellulose, magnesium aluminum silicate, and polyvinyl alcohol.

[0131] The stabilizer is selected from one or more of sodium citrate and resorcinol.

[0132] The antifreeze agent is selected from one or more of ethylene glycol, propylene glycol and glycerol.

[0133] The defoaming agent is selected from one or more of silicone oil, silicone compounds, C10-20 saturated fatty acid compounds, and C8-10 fatty alcohols.

[0134] The filler is selected from one or more of kaolin, diatomaceous earth, bentonite, attapulgite, white carbon black, starch, and light calcium carbonate.

[0135] The plant parasitic nematodes include one or more of the following: root knot nematodes (Meloidogyne), pine wood nematodes (Bursa phelenchusxylophilus), stem nematodes (Ditylenchus), and cyst nematodes (Heterodera).

[0136] Compared with the prior art, the present invention has the following advantages:

[0137] (1) Adding the adjuvant provided by the present invention to the fermentation broth and directly spray-drying it into a Bacillus thuringiensis mother powder, thereby avoiding the loss of nematicidal active ingredients caused by centrifugation, high temperature, concentration, filtration, etc., which affects the activity of the mother powder, and at the same time giving full play to the synergistic effect of the synergistic substances;

[0138] (2) The spore recovery rate and crystal recovery rate of the Bacillus thuringiensis mother powder provided by the present invention are as high as over 100%. Most importantly, the effect of preventing and controlling plant parasites is better than other technologies at the same spore concentration;

[0139] (3) The Bacillus thuringiensis mother powder provided by the present invention has a low rate of foreign bacteria and low moisture content, and is superior to other technologies in terms of storage stability, providing a product with excellent quality and stable effect;

[0140] (4) The effect of Bacillus thuringiensis on the prevention and control of nematodes is significantly improved, and it has a significant synergistic effect, which improves the prevention and control effect of Bacillus thuringiensis products for the prevention and control of nematodes.

[0141] (5) The production process is simple and the amount of wastewater and waste residue discharged is small.

[0142] definition

[0143] Unless otherwise defined, all technical and scientific terms used in the present invention have the same meaning as commonly used in the field to which the present invention belongs. For the purpose of interpreting this specification, the following definitions will apply, and where appropriate, terms used in the singular will also include the plural form, and vice versa.

[0144] As used herein, the articles "a," "an," and "an" include plural referents unless the context clearly dictates otherwise.

[0145] As used herein, the expression "about" is understood by one of ordinary skill in the art and varies within certain limits depending on the context in which it is used. If the use of the term is not understood by one of ordinary skill in the art based on the context in which it is used, "about" will mean up to plus or minus 10% of the specified value.

[0146] In the present invention, the term "Bacillus thuringiensis" refers to the Bacillus thuringiensis species of the genus Bacillus, abbreviated as Bt. Bacillus thuringiensis can produce toxins that are harmful to a variety of insects or other invertebrates. It has toxic activity against a variety of insects in various phyla of invertebrates and various orders of arthropods. It is non-toxic to humans and livestock, harmless to natural enemies and plants, and is not susceptible to pest resistance. It is low-cost, easy to industrialize, and does not pollute the environment. It is widely used in the biological control of various agricultural and forestry pests worldwide. The "Bacillus thuringiensis" mentioned herein is specifically understood to be Bacillus thuringiensis that is effective in controlling plant parasites.

[0147] For the purposes of this invention, the term "plant-parasitic nematodes" refers to nematodes that parasitize plants, accounting for approximately 10% of all nematodes. There are approximately 200 genera and over 5,000 species of plant nematodes documented worldwide, the majority of which belong to the Tylenchina and Aphelenchina suborders within the class Pleuronectiformes, while a smaller number belong to the Longidoridae and Trichodoridae families within the class Apleuronectiformes. Plant-parasitic nematodes severely harm the agricultural economy, food safety, and ecological environment due to their impacts on their hosts—reduced yields, decreased quality, and even mortality. Among them, the most serious damage to plant economy and plant ecological environment includes but is not limited to root-knot nematodes (Meloidogyne), pine wood nematodes (Bursa phelenchus xylophilus), stem nematodes (Ditylenchus), cyst nematodes (Heterodera), short-bodied nematodes (pratylenchus), radopholus nematodes, Aphelenchoides, bursaphelenchus, etc.

[0148] In the present invention, the term "fermented product" refers to a mixture produced by cell fermentation that has not undergone or has undergone minimal recovery and / or purification. For example, a mixture formed when a microbial culture is allowed to synthesize proteins and secrete proteins into a cell culture medium. The fermented liquid may contain unfractionated or fractionated contents of the fermented material obtained at the end of the fermentation. The fermented liquid contains spent cell culture medium, various extracellular metabolites, and viable and / or inactive microbial cells. Fermented products include solid-state fermented products, liquid fermented products, or semi-solid fermented products. Among them, liquid fermented products are also called "fermented liquid."

[0149] In the present invention, the term "fermentation broth" refers to liquid fermentation product.

[0150] In the present invention, the term "solid content" is measured by the loss on drying method, 100%×the dry mass of the fermentation liquid / the mass of the fermentation liquid.

[0151] In this application, the term "mother powder" refers to the powder containing a high concentration of microorganisms or active substances obtained directly from the fermentation product after processing. In actual use, it is diluted to a working concentration as needed or prepared into various formulations, such as wettable powders, water-dispersible granules, dispersible granules, granules, dispersible powders for seed treatment, suspension concentrates, and suspension concentrates for seed treatment.

[0152] In the present invention, the term "crystal or spore recovery rate" refers to 100% × (dry powder weight × crystal content or spore content) / (fermentation liquid weight before spraying × crystal content or spore content)

[0153] In the present invention, the term "adjuvant" or "additive" refers to any single component or multiple components added to the product, in addition to the active ingredient, which does not have the product activity and active ingredient function itself, but can or helps to enhance or improve the physical and chemical properties of the product.

[0154] The "adjuvant" mentioned in the present invention is specifically understood to be any single component or multiple components added to the microbial fermentation broth, in addition to the active ingredients, which do not have the same function as the active ingredients themselves but can or help to enhance or improve the performance of the microorganisms, such as inorganic salts, mineral fillers, organic matter, etc.

[0155] In some embodiments of the present invention, "adjuvant" refers to any single component or multiple components added to the Bacillus thuringiensis fermentation broth, in addition to the active ingredient, which does not itself have the activity of preventing and controlling plant parasites and the function of the active ingredient, but can or helps to enhance or improve the effect of preventing and controlling plant parasites, wherein the plant parasites are preferably nematodes, and the effects of the adjuvant vary significantly depending on the type of Bacillus thuringiensis, the type of pest and the concentration of the adjuvant.

[0156] The "additives" mentioned in the present invention are specifically understood to be any single component or multiple components added in the process of preparing the Bacillus thuringiensis mother powder into a microbial agent, in addition to the active ingredient, which does not itself have the activity of preventing and controlling plant parasites and the function of the active ingredient, but can or can help the active ingredient to be stable in the microbial agent and exert its efficacy.

[0157] For the purposes of this invention, the term "spray drying" refers to a thermal process involving the rapid evaporation of the solvent in a liquid material through spraying into a hot air stream, transforming it into a dry powder. This process disperses the material into small droplets, creating a large evaporation area, making it particularly suitable for drying heat-sensitive materials. Spray drying is currently the primary method for producing high-efficiency Bacillus thuringiensis powders both domestically and internationally. Depending on the atomization method, spray drying equipment can be categorized into three types: airflow, pressure, and centrifugal. Airflow is the most widely used method.

[0158] In the present invention, the term "microbial agent" refers to a stable product made from bacterial biopesticide technical, parent drug, parent powder or raw powder and suitable adjuvants, or processed by biological fermentation, plant extraction and other methods.

[0159] In some embodiments of the present invention, the "microbial agent" refers to a stable product processed from Bacillus thuringiensis mother powder and appropriate additives.

[0160] In the present invention, the term "adjusted mortality rate" is calculated as follows:

[0161] Corrected mortality (%) = 100% × (treatment mortality - control mortality) / (1 - control mortality),

[0162] The mortality rate (%) = 100% × number of dead insects / total number of insects treated.

[0163] The following examples are provided to facilitate understanding of the present invention. However, it should be understood that these examples are intended to illustrate the present invention only and are not intended to limit the present invention in any way. The actual scope of the present invention is set forth in the claims. It should be understood that any modifications and variations may be made without departing from the spirit of the present invention.

[0164] The following specific examples further illustrate the present invention. The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Unless otherwise specified, the experimental methods used in the following examples are conventional methods. The materials and reagents used in the following examples are all commercially available unless otherwise specified. Unless otherwise specified, the ratios mentioned below are all weight fraction ratios.

[0165] Example 1 Preparation of Bacillus thuringiensis mother powder for controlling nematodes using different processes and adjuvants

[0166] 1. The selected strain is Bacillus thuringiensis HAN055 disclosed in patent No. 108070535A, which has the function of controlling nematodes and has a deposit number of CCTCC NO: M 2015608.

[0167] 2. The Bt fermentation medium formula and fermentation conditions used for controlling nematodes are derived from the method disclosed in the document with application number 202111593497.9.

[0168] 3. 0.12 tons of Bt-HAN055 fermentation liquid for controlling nematodes with a solid content of 5% was obtained, and the number of spores was 7.5×10 9 cfu / mL, crystal content 0.38%, after adding citric acid to acidify the fermentation broth to pH 4.5, it was treated according to the following treatment methods 1 to 6:

[0169] Treatment 1: Add an additive containing 2% kaolin, stirring constantly during the process to prevent sedimentation. Inlet air temperature: 200±10°C; outlet air temperature: 80±5°C. A cooler is connected to the bottom powder outlet to cool the powder to below 35°C before bagging. Mix and screen for 30 minutes, return to the bottom after 25 minutes, and sieve the mixed material through a 30-mesh sieve. Bag the resulting masterbatch.

[0170] Treatment No. 2: The additive formula was replaced with 0.5% ammonium molybdate, 1.5% white carbon black, 2% kaolin, and 1.5% trehalose. Other ingredients were the same as those in Treatment No. 1.

[0171] Treatment No. 3: Add an additive containing 2% mica powder and process according to the process described in the patent document with authorization number ZL200610019609.9 to obtain a mother powder. (As control 1)

[0172] Treatment No. 4: The supernatant was concentrated under reduced pressure to prepare a mother powder according to the process reported by Yang Ziwen, Wang Kaimei, Wu Jixing, et al., "Recovery of Soluble Synergistic Substances from Bacillus thuringiensis" [J]. Chinese Journal of Biological Control, 2001, 17(3): 129-132. Specific steps included centrifugation, waste liquid, vacuum concentration, spray drying, and the supernatant synergistic powder was compounded with the original powder to obtain the mother powder (as control 2).

[0173] Treatment No. 5: No additives were added, and other conditions were the same as Treatment No. 1.

[0174] Treatment No. 6: Perform traditional centrifugal spray drying according to the method disclosed in patent document with application number 202111593497.9 to obtain mother powder.

[0175] The spore count was determined by the gradient dilution coating method, and the crystal protein content was detected by SDS-PAGE. The specific results are shown in Table 1.

[0176] Table 1 Results of spray drying tests on Bt fermentation broth for nematode control using different processes and adjuvant formulations

[0177]

[0178] From the results in Table 1, it can be seen that the mother powder prepared by treatment No. 2 has a moisture content of 2.45%, a contaminant bacteria rate of 0.84%, a crystal recovery rate and a spore recovery rate of 101.20% and 102.41% respectively. The total spore count, yield, mother powder quality and properties are significantly better than those of traditional centrifugation processes and publicly available technologies.

[0179] Example 2 Preliminary screening of the activity of Bacillus thuringiensis mother powder against southern root-knot nematodes using different adjuvant formulations and processes

[0180] (1) Activity test of southern root-knot nematode egg masses: Southern root-knot nematode egg masses were picked from the cucumber roots infected with southern root-knot nematodes, disinfected with 1% H2O2 and 0.25% KI disinfectant, and then the biological activity was determined on a 24-well plate. The above-mentioned Bacillus thuringiensis mother powder was diluted to 1×10 8 cfu / mL, a blank control was set up with sterile deionized water, and a control was set up with Bt fermentation liquid for controlling nematodes. Each treatment was repeated 4 times, and 5 egg masses with the same growth, color and size were picked from each well and cultured in an incubator at 25℃ for 5 days. The hatching inhibition rate was calculated.

[0181] (2) Test for southern root-knot nematode eggs: Cucumber roots infected with southern root-knot nematodes were collected, washed, and chopped thoroughly. 1% sodium hypochlorite solvent was added to lyse the plant cells. After stirring, the roots were rinsed and sieved through 160-mesh and 400-mesh sieves, and the filtrate mixture was collected on the 400-mesh sieve and rinsed repeatedly with clean water to obtain a nematode egg suspension. The biological activity was determined on a 96-well plate: the above-mentioned Bacillus thuringiensis mother powder was diluted to 1×10 8 cfu / mL. A blank control was set up with sterile deionized water, and a control was set up with Bt fermentation broth for nematode control. Each treatment was replicated four times. Approximately 40 to 50 eggs were aspirated into each well and incubated in an incubator at 25°C for 5 days. The number of hatched eggs was counted and the hatching inhibition rate was calculated.

[0182] (3) Test on the second-instar larvae of the southern root-knot nematode: The egg mass obtained in step (1) was sterilized and then placed in an incubator at 25°C for 3-5 days. The second-instar larvae of the southern root-knot nematode hatched from the egg mass were stored for future use. The bioactivity assay was performed on a 96-well plate: the above-mentioned Bacillus thuringiensis mother powder was diluted to 1×10 8 cfu / mL. A blank control was set up with sterile deionized water, and a control was set up with Bt fermentation broth for nematode control. Each treatment was replicated four times. Approximately 40–50 second-instar larvae were aspirated into each well and incubated in an incubator at 25°C for 24 hours. The number of dead larvae was counted and the corrected mortality rate was calculated.

[0183] The above test results are shown in Table 2. The results show that the nematode control Bt mother powder prepared with Treatment No. 2 had the highest egg mass hatching inhibition rate, egg granule hatching inhibition rate, and corrected mortality rate of second-instar larvae. Treatment No. 2 also showed superior performance in indicators such as crystal and spore recovery rates as shown in Table 1, and its performance was significantly improved compared to the original fermentation broth. However, the performance of mother powders prepared using publicly available recovery technologies was inferior to that of the original fermentation broth. Because nematode control Bacillus thuringiensis produces a wide variety of metabolites, existing technologies for Bacillus thuringiensis adjuvants are ineffective against nematode control Bacillus thuringiensis. This may be due to differences in the strains and insecticidal active substances between nematode control Bacillus thuringiensis and conventional Bacillus thuringiensis.

[0184] Table 2 The effect of different adjuvant formulas and processes on the control of nematodes by Bacillus thuringiensis mother powder at 1×10 8 Preliminary screening of activity against southern root-knot nematodes at cfu / mL concentrations

[0185] deal with Egg mass hatching inhibition rate (%) Egg hatching inhibition rate (%) Corrected mortality of second-instar larvae (%) 1 48.78 55.42 71.32 2 64.22 69.54 82.84 3 45.36 51.77 72.56 4 40.28 50.28 75.21 5 38.54 44.68 65.02 6 24.72 32.54 51.48 fermentation broth 46.45 52.05 76.42

[0186] Example 3: Optimization of Bacillus thuringiensis adjuvant formula for controlling nematodes

[0187] Based on treatment No. 2, the dosage of ammonium molybdate, white carbon black, kaolin, and trehalose was changed. The experimental design is shown in Table 3. The preparation and detection methods of Bacillus thuringiensis fermentation liquid and mother powder for controlling nematodes are the same as those in Example 1. The mortality rate of second-instar larvae was determined at a spore concentration of 1×10 8 CFU / mL, the method was the same as in Example 2.

[0188] Table 3 Test results of different additive formulations

[0189]

[0190]

[0191] The test results are shown in Table 3. The crystal and spore recovery rates for Treatments 1 to 10 in Table 3 all exceeded 100%. After treatment with the specific ingredients and specific amounts of the adjuvants listed in Table 3, the bacterial cells achieved results superior to those achieved by the mother powders obtained by other treatment methods, including Treatments 1 and 3 to 6, in Example 1, and also superior to those achieved by the fermentation broth in Example 2. Furthermore, the crystal and spore recovery rates for Treatments 1 to 10 in Table 3, which added ammonium salts, were higher than those for Treatment 11, which did not.

[0192] The results in Table 3 show that within a certain range, the combination of additives can achieve the effect of improving the crystal recovery rate and inhibiting the rate of foreign bacteria. For example, the combination of 0.1% to 1.0% ammonium molybdate, 1.5% to 4.5% white carbon black, 1.0% to 3.0% kaolin, and 1.0% to 2.0% trehalose. When the additive formula is 1% ammonium molybdate, 3% white carbon black, 2% kaolin, and 2% trehalose, the spore recovery rate and crystal recovery rate are as high as 110.57% and 109.11%, respectively. The rate of foreign bacteria is 0.48%, the water content is 2.16%, and at a spore concentration of 1×10 8 CFU / mL, the corrected mortality rate of the second-instar larvae of the southern root-knot nematode can reach 96.8%, which is significantly better than the effect of the fermentation liquid in Example 2 of 76.42%.

[0193] Example 4 Comparison of the performance of additives in preparing mother powders of Bacillus thuringiensis fermentation broth with different solid contents for controlling nematodes

[0194] 1. The selected strain is Bacillus thuringiensis HAN055 disclosed in patent No. 108070535A, which has the function of controlling nematodes and has a deposit number of CCTCC NO: M 2015608.

[0195] 2. Bt-HAN055 fermentation liquids for controlling nematodes with a solid content of 1% to 6% were obtained by fermentation in different culture media. The contents of spores and crystals in each fermentation liquid are shown in Table 4.

[0196] 3. Add citric acid to acidify the fermentation broth to pH 4.5, add the best-performing additives of formula No. 10 in Example 3 (ammonium molybdate 1%, white carbon black 3%, kaolin 2%, trehalose 2%), and then prepare the mother powder according to the production process in Example 1. The mortality rate of the second-instar larvae was measured. The spore concentration was 1×10 8 CFU / mL, the method was the same as in Example 2.

[0197] Table 4 Comparison of the performance of mother powders of Bacillus thuringiensis fermentation broth with different solid contents for nematode control after adding optimized adjuvants

[0198]

[0199] As shown in Table 4, when the solid content of the Bacillus thuringiensis fermentation broth for controlling nematodes was between 1% and 6%, the addition of an adjuvant comprising 1% ammonium molybdate, 3% white carbon black, 2% kaolin, and 2% trehalose increased the mortality rate of second-instar nematodes by 20.21% to 26.67%. When the solid content was 5%, the improvement reached 26.67%.

[0200] Example 5 Comparison of the performance and activity of adjuvants in preparing mother powders of different Bacillus thuringiensis strains for controlling nematodes

[0201] 1. All Bt strains used have the function of preventing and controlling plant nematodes:

[0202] Bacillus thuringiensis HAN055 disclosed in patent document CN108070535A, with a deposit number of CCTCC NO: M 2015608, was selected as treatment 1.

[0203] Bacillus thuringiensis KN63, which was screened and isolated from Wuhan Kono Biotechnology Co., Ltd. and has high activity against root-knot nematodes and cyst nematodes, and its preservation number is ACCC NO: 62944, was selected as treatment 2.

[0204] The commercially available 4000 IU / mg Bacillus thuringiensis suspension seed coating agent produced by Heilongjiang Jiamusi Xingyu Biotechnology Development Co., Ltd. was selected to control soybean cyst nematodes, which was used as treatment 3.

[0205] 2. The above strains were prepared into fermentation liquid with a solid content of 5%, and citric acid was added to acidify the fermentation liquid to pH 4.5. The mother powder was prepared according to the production process of treatment No. 1 in Example 1, wherein the auxiliary agents were replaced with the auxiliary agents shown in Table 5. The corresponding Bacillus thuringiensis mother powders 1 to 3 were obtained, and the above Bacillus thuringiensis mother powders were diluted to 1×10 8 CFU / mL, and the mortality of second-instar larvae of root-knot nematodes was determined according to the method described in Example 2.

[0206] 3. Cyst nematode assay method: Separate cysts from soil with a severe soybean cyst nematode infestation using the floatation method. Soak the cysts in a ZnSO4 solution for 24 hours. After the soybean cyst nematode larvae hatch at room temperature, dilute the suspension to approximately 200 second-instar larvae per mL for microscopic examination. Perform a toxicity bioassay in a commonly used 24-well plate: aspirate approximately 200 second-instar soybean cyst nematode larvae into each well, add spores at a concentration of 1×10 8 CFU / mL, and for blank treatment, an equal amount of sterile deionized water was added to make the total volume of each well 2 mL, which was then made up to the total volume with sterile deionized water. The number of dead heads was counted after 24 hours.

[0207] Table 5 Comparison of the performance of the mother powder of different Bacillus thuringiensis strains for the control of nematodes and the control effect on two nematodes

[0208]

[0209] The test results can be seen from Table 5 that the different nematode control functions of Bacillus thuringiensis strains, after fermentation and acidification, and the addition of optimized adjuvants, the mother powder prepared was compared with the original fermentation liquid at a concentration of 1×10 8CFU / mL, the mortality rate of the second-instar larvae of root-knot nematodes increased by 25.91% to 31.97%, and the mortality rate of the second-instar larvae of cyst nematodes increased by 18.42% to 30.44%. It can be seen that the adjuvant formula disclosed in this application has a significant positive effect on the preparation of Bacillus thuringiensis mother powder with high activity in controlling nematodes.

[0210] Example 6: Quality determination of different Bacillus thuringiensis mother powders for controlling nematodes

[0211] The Bacillus thuringiensis mother powders 1 to 3 for controlling nematodes prepared in Example 5 were selected and centrifuged and concentrated by spray powdering according to the method disclosed in the patent document with application number 202111593497.9 as the control powder. They were placed at room temperature (22±2)°C for 12 months, and the moisture content, bacteria rate, spore stability, crystal stability, and nematicide stability before and after placement were tested. The method was referred to Example 2.

[0212] Table 6 Quality parameters of mother powder of different Bacillus thuringiensis strains for controlling nematodes

[0213]

[0214] The results shown in Table 6 show that after the optimized spraying process, the quality of the Bacillus thuringiensis mother powder for controlling nematodes after 12 months of storage, such as crystal content, spore count, moisture content, contaminant rate, and nematode mortality, is significantly better than that of the mother powder produced by the traditional centrifugal concentration spraying process.

[0215] Example 7 Determination of the Activity of Bacillus thuringiensis Mother Powder against Plant Parasitic Nematodes

[0216] The nematode control Bacillus thuringiensis mother powders 1 to 3 prepared in Example 5 were selected. The fermentation broths of the respective strains were used as controls, namely fermentation broths 1 to 3. The plant parasitic nematodes tested included southern root knot nematode, soybean cyst nematode, pine wood nematode, and stem nematode.

[0217] The method for determining root knot nematode activity is shown in Example 2, and the method for determining cyst nematode activity is shown in Example 5.

[0218] Pine wood nematode: Inoculate PDA medium with Pestalotia sp. and incubate at 26°C for 7-10 days. Once the colonies have completely filled the plate, inoculate the pine wood nematode under sterile conditions and incubate at 26°C for 10 days. Once the mycelium has completely disappeared, separate and collect the nematodes using the Baermann funnel method. Disinfect the collected nematodes three times with a disinfectant (0.002% cycloheximide + 0.1% streptomycin sulfate) and rinse three times with sterile water to prepare a pine wood nematode suspension for testing. A virulence bioassay is performed in a conventional 24-well plate: approximately 100 pine wood nematodes are aspirated into each well. Each treatment is replicated four times. After incubation at 25°C for 24 hours, the number of dead nematodes is counted and the corrected mortality rate is calculated.

[0219] Ditylenchus destructor: Potato stem rot nematode (Ditylenchus destructor) was provided by the laboratory. Virulence bioassays were performed in a standard 24-well plate. Approximately 100 Ditylenchus nematodes were aspirated into each well, with four replicates per treatment. The concentration settings are shown in Table 6. For the blank treatment, an equal volume of sterile deionized water was added, resulting in a total volume of 2 mL per well. The volume was then made up with sterile deionized water. After incubation at 25°C for 24 hours, the number of dead Ditylenchus nematodes was counted, and the adjusted mortality rate was calculated.

[0220] Table 7 Activity determination of different nematode-control Bacillus thuringiensis strain mother powders against plant parasitic nematodes

[0221]

[0222] From the results in Table 7, it can be seen that the high-performance mother powder of Bacillus thuringiensis for controlling nematodes has a significantly better mortality rate against southern root-knot nematode, soybean cyst nematode, pine wood nematode and stem nematode than the fermentation liquid of each strain at the same concentration.

[0223] Example 8 Preparation of a High-Performance Bacillus thuringiensis Suspension for Nematode Control and Evaluation of Its Effect on Root-Knot Nematodes

[0224] 1. Preparation of high-performance Bacillus thuringiensis suspension for nematode control

[0225] The mother powder 1 prepared in Example 5 was used to prepare a microbial inoculant. The microbial inoculant was a suspension agent, comprising the following components by mass: 10% Bacillus thuringiensis mother powder; 5% D425; 3.5% ethylene glycol; 0.2% xanthan gum; 0.1% silicone oil; 0-10% penetration enhancer; 0.5% citric acid; the remainder made up with water. The specific production steps of the suspension agent were as follows: first, the other additives were mixed and uniformly mixed by high-speed shearing, then the high-performance mother powder of Bacillus thuringiensis for nematode control was added, and the mixture was uniformly stirred in a stirred tank. The suspension agent of the present invention was then divided into portions, filled with nitrogen, and sealed. A mother powder obtained by conventional centrifugal concentration and spraying was used as a control. Approximately 5% of the control Bacillus thuringiensis mother powder was added, with the other additives at the same levels, to maintain the same microbial inoculant spore count.

[0226] 2. Field test methods for southern root-knot nematodes

[0227] 1. Specific steps of the test:

[0228] (1) The Bacillus thuringiensis suspension concentrate of the present invention was used at three treatment concentrations: 2 kg / mu, 3 kg / mu, and 4 kg / mu; a control Bacillus thuringiensis inoculum was also used at three treatment concentrations: 2 kg / mu, 3 kg / mu, and 4 kg / mu. Two controls, water and thiazophos (2 kg / mu), were also used, for a total of five treatments. Three plots were set for each treatment, for a total of 24 plots, each with an area of ​​0.1 mu, and the plots were randomly arranged.

[0229] (2) The experiment was conducted in a vegetable greenhouse in Shouguang, Shandong Province in September 2022. The tomato seedlings were transplanted into the greenhouse one month after seedling cultivation.

[0230] (3) Dilute the Bacillus thuringiensis agent with water and use it to irrigate the roots at the time of transplanting and one week after transplanting.

[0231] (4) Investigate the control effect after 45 days.

[0232] 2. Root-knot nematode damage rating standards and control effectiveness calculation:

[0233] (1) According to the agricultural industry standard (NY / T 1858.8-2010), nematode damage was rated and the disease index and control effect were calculated. The nematode damage rating is as follows:

[0234] Level 0: There are no knots on all roots;

[0235] Level 1: 0% < the number of roots with root knots accounts for <10% of the total root system;

[0236] Level 3: 10% or less: the number of roots with root knots accounts for <25% of the total root system;

[0237] Level 5: 25% ≤ The number of roots with root knots accounts for <50% of the total root system;

[0238] Level 7: 50% ≤ The number of roots with root knots accounts for <75% of the total root system;

[0239] Level 9: 75% ≤ The number of roots with root knots accounts for ≤ 100% of the total root system.

[0240] (2) Calculation method of prevention effect:

[0241] Disease index (%) = ∑ (number of diseased plants at each level × disease level) × 100% / (total number of plants surveyed × highest disease level).

[0242] Control effect (%) = (disease index of control area - disease index of treated area) × 100% / disease index of control area.

[0243] 3. The test results are shown in Table 8.

[0244] Table 8 Field efficacy test results of Bacillus thuringiensis suspension concentrate of the present invention for controlling southern root-knot nematodes

[0245]

[0246] Example 9: Preparation of Bacillus thuringiensis wettable powder for nematode control and evaluation of its effect on root-knot nematodes

[0247] The mother powder 1 prepared in Example 5 was selected to prepare the bacterial agent.

[0248] The microbial agent is prepared as a wettable powder, the components of which are as follows: 30% Bacillus thuringiensis mother powder; 55% D425; 1-5% sodium lauryl sulfate; and the balance kaolin. The wettable powder is produced by mixing the nematode-control Bacillus thuringiensis mother powder, a dispersant, a wetting agent, and a filler according to the aforementioned formula, stirring the mixture uniformly in a stirred tank, and then passing the mixture through a jet mill for further mixing. The wettable powder microbial agent is then prepared. A mother powder obtained by conventional centrifugal concentration and spraying is used as a control. Approximately 15% of the control Bacillus thuringiensis mother powder is added, with the remaining additives at the same levels, to maintain the same microbial spore count.

[0249] The field test was conducted in Liaozhong District, Liaoning Province in September 2022. The test method was referred to Example 8, and the test concentration setting and control effect were shown in Table 9.

[0250] Table 9 Field efficacy test results of Bacillus thuringiensis wettable powder of the present invention for controlling southern root-knot nematode

[0251]

[0252] The test results in Examples 8 and 9 show that the Bacillus thuringiensis suspension concentrate and wettable powder of the present invention both achieved over 65% control efficacy against southern root-knot nematodes. This is significantly superior to the field efficacy of conventional microbial agents at the same dosage. The tests also demonstrated significant control efficacy against southern root-knot nematodes, with a medium-dose efficacy exceeding 70% and a high-dose efficacy exceeding 80%. This efficacy is significantly superior to the commercial 30% thiazothiazolyl microcapsule suspension (Shandong Lushi Pesticide Co., Ltd.). See Tables 8 and 9.

[0253] Example 10: Comparison of performance of different ammonium salts on preparation of mother powder of Bacillus thuringiensis fermentation broth for controlling nematodes

[0254] 1. Different ammonium salt compounds were selected to test the performance of the mother powder, including ammonium sulfate ((NH4)2SO4), ammonium thiocyanate (NH4CNS), ammonium nitrate (NH4NO3), diammonium hydrogen phosphate (NH4H2PO4), ammonium chloride (NH4Cl), ammonium molybdate ((NH4)2MoO4), and ammonium bicarbonate ((NH4HCO3).

[0255] 2. The method in Example 4 was selected for preparation and testing. The solid content of the fermentation broth was 5%, 120 kg of fermentation broth, and the additive formula was 1% ammonium salt, 3% white carbon black, 2% kaolin, and 2% trehalose. The results are shown in Table 10.

[0256] Table 10 Test results of different ammonium salt compounds on the performance of prepared mother powder

[0257]

[0258] From the test results in Example 10, it can be seen that different ammonium salts prepared mother powder at 1×10 8 The nematicidal effect can be improved at all CFU / mL concentrations, with the improvement efficiency ranging from 3.48% to 27.72%, among which the effect of ammonium molybdate reached 27.72%; at the same time, ammonium molybdate treatment can increase the crystal and spore recovery rates to 112.58% and 107.83%.

[0259] The above shows that the present invention has a significantly improved control effect on Bacillus thuringiensis for controlling nematodes, and has high application value and huge application potential in controlling a variety of plant parasitic nematodes that cause serious damage.

[0260] The technical solution of the present invention is not limited to the above-mentioned specific embodiments. Any technical variations made according to the technical solution of the present invention fall within the protection scope of the present invention.

Claims

1. An auxiliary agent for spray drying of microbial fermentation products, characterized in that: The auxiliary agent is composed of the following components: based on the mass fraction of the microbial fermentation product, 0.1% to 1.0% of ammonium molybdate, 1.5% to 4.5% of white carbon black, 1.0% to 3.0% of kaolin, and 1.0% to 2.0% of trehalose; the microorganism is Bacillus thuringiensis ( Bacillus thuringiensis ).

2. The auxiliary agent according to claim 1, characterized in that The auxiliary agent is composed of the following components: calculated by mass fraction of microbial fermentation products, 1% of ammonium molybdate, 3% of white carbon black, 2% of kaolin and 2% of trehalose.

3. A microbial mother powder, characterized in that: The microbial mother powder contains the adjuvant according to claim 1 or 2, and the microbial mother powder is Bacillus thuringiensis mother powder.

4. The microbial mother powder according to claim 3, characterized in that The spore count of the Bacillus thuringiensis mother powder is not less than 30×10 9 cfu / g, protein crystals not less than 1.5%, water content not higher than 5%, and miscellaneous bacteria rate not higher than 2%.

5. A method for preparing microbial mother powder, comprising the following steps: The auxiliary agent according to claim 1 or 2 is added to the microbial fermentation product, mixed, and spray-dried. The microorganism is Bacillus thuringiensis ( Bacillus thuringiensis ).

6. A microbial mother powder, characterized in that: The microbial mother powder is prepared using the preparation method according to claim 5, and the microbial mother powder is Bacillus thuringiensis mother powder.

7. The microbial mother powder according to claim 6, characterized in that: The spore count of the Bacillus thuringiensis mother powder is not less than 30×10 9 cfu / g, protein crystals not less than 1.5%, water content not higher than 5%, and miscellaneous bacteria rate not higher than 2%.

8. A microbial agent, characterized in that: The microbial agent comprises the microbial mother powder according to any one of claims 3 to 4 or 6 to 7, and the microbial agent is a Bacillus thuringiensis agent.

9. The microbial agent according to claim 8, characterized in that The microbial agent further comprises additives.

10. The microbial agent according to claim 9, characterized in that The additives include one or more of the following: a dispersant, an antifreeze agent, a thickener, an antifoaming agent, a pH adjuster, a film former, a wetting agent, an anti-caking agent, a disintegrant, and a filler or water.

11. The microbial agent according to claim 9, characterized in that The additives include one or more of the following: based on the mass fraction of the bacterial agent, 3-20% of a dispersant, 1-5% of an antifreeze agent, 0.1-5% of a thickener, 0.1-0.8% of a defoaming agent, 0.1-5% of a pH regulator, 1-10% of a film-forming agent, 1-10% of a wetting agent, 3-5% of an anti-caking agent, 1-5% of a disintegrant, and a filler or water.

12. The microbial agent according to claim 8, characterized in that The microbial agent is in the following dosage forms: powder, wettable powder, water-dispersible granules, dispersible granules, granules, dispersible powder for seed treatment, suspension or suspension for seed treatment.

13. A method for preparing a microbial agent, comprising mixing the microbial mother powder according to any one of claims 3 to 4 or 6 to 7 with an additive.

14. A microbial agent, characterized in that The microbial agent is prepared using the preparation method described in claim 13.

15. Use of the adjuvant according to claim 1 or 2, the microbial mother powder according to any one of claims 3 to 4 or 6 to 7, the preparation method according to claim 5, the microbial agent according to any one of claims 8 to 12, and the preparation method according to claim 13 in controlling plant parasites.

16. The use according to claim 15, characterized in that The plant parasites include nematodes ( Nematode ).

17. The use according to claim 16, characterized in that The nematodes include one or more of the following: root-knot nematodes ( Meloidogyne ), pine wood nematode ( Bursaphelenchus xylophilus ), stem nematodes ( Ditylenchus ), cyst nematodes ( Heterodera ).

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