WO2015096608A1

WO2015096608A1 - Mosquito-killing bacillus thurigiensis suspended slow release formulation and preparation method thereof

Info

Publication number: WO2015096608A1
Application number: PCT/CN2014/093158
Authority: WO
Inventors: 张灵玲; 关雄; 黄志鹏; 张小娟; 吴松青; 沙莉; 潘晓鸿; 黄天培; 邵恩斯; 黄必旺; 陈慧成; 朱小丽
Original assignee: 福建农林大学
Priority date: 2013-12-27
Filing date: 2014-12-05
Publication date: 2015-07-02
Also published as: ZA201503344B; CN103719163A; CN103719163B

Abstract

The invention relates to a mosquito-killing Bacillus thuringiensis suspended slow release formulation and a preparation method thereof. According to the preparation method of the present invention, using sodium alginate as a carrier and calcium chloride as a crosslinking agent, a mosquito-killing Bacillus thuringiensis raw powder is added as an effective active ingredient, and the slow release formulation has a high-efficiency mosquito-killing activity; the formulation has a suspended slow release effect via hollow glass beads, thus resisting ultraviolet radiation and prolonging the longevity of residues. The formulation has a reasonable formula, high insecticidal toxicity, good control effect, strong ultraviolet radiation resistance and long longevity of residues in the wild. The experiment indicates that the product of the invention has a good effect on killing mosquito larvae in water, and as the treatment time is prolonged, the average relative death rate of test insects is gradually increased until the relative death rate of test insects within 192 hours reaches 100%; and the product of the invention has a high mechanical property and long-acting suspension property, and the suspension begins to precipitate at 144 hours, and the suspension rate is kept to be 58.3% for 192 hours.

Description

Mosquito-killing strain suspension-release agent and preparation method thereof

Technical field

The invention relates to a biological pesticide sustained release agent and a preparation method thereof, in particular to a mosquito suspension suspension sustained release agent and a preparation method thereof, and belongs to the technical field of biological pesticides.

Background technique

Bacillus thuringiensis (Bt) is a Gram-positive bacterium that forms one or more parasporal crystals with protein properties in the bacteria while forming spores. Also known as Insecticidal Crystal Proteins, ICPs) or δ-endotoxin, for Lepidoptera, Diptera, Coleoptera, Hymenoptera, Homoptera, Orthoptera, Eriocheir, Odonata, and Odonata, as well as flukes and flagellates, Nematodes and cockroaches have strong poisoning effects, and overcome the shortcomings of traditional chemical pesticides polluting the environment, harming humans and animals, easily producing resistance, etc., and have the advantages of strong selectivity, safety, and simple use of raw materials. The most widely used microbial pesticide in the world. Bt thuringiensis subsp.israelensis (Bti) has high activity against a variety of mosquitoes and has been successfully developed as a biocide for the control of mosquitoes and other health pests. In mosquitoes such as malaria, dengue fever and yellow fever. The control of vector diseases has played an important role. However, the Bt bacteria currently produced and used in the use of active spores and crystals are susceptible to the effects of ultraviolet rays in the environment, which affects their insecticidal effect and long-lasting period, and has become one of the main limiting factors for the difficulty in popularizing Bt insecticides.

At present, there are two main ways to improve the anti-ultraviolet radiation ability of Bt insecticides. One is to use a dosage form to study or add an auxiliary method, and to reduce the amount of the ultraviolet absorber and the protective agent by adding a preparation such as a package or a microbial agent. Ultraviolet radiation to the cells. Mainly colorants and fluorescent whitening agents, including Congo red, sodium fluorescein, escin, berberine, ascorbic acid, benzotriazole, skim milk, lignin, melanin, humic acid, p-amino More than 100 kinds of UV protectants such as benzoic acid and syrup. These additives have a good UV protection effect on the Bt preparation, which greatly prolongs the expiration date of the Bt preparation. At present, Bt dosage forms that have been marketed and commercialized are mainly classified into solid type and liquid type, solid powders, granules, powders, and pellets; liquids have suspensions, emulsions, capsules, and the like. These dosage forms give Bt bacteria a good protection. The other is a gene regulation method to construct a multifunctional engineering strain by transducing the anti-ultraviolet radiation functional gene, thereby improving the anti-ultraviolet radiation ability of the Bt strain. Although the transgenic multifunctional engineering bacteria also have a good anti-ultraviolet radiation effect, they have the disadvantage of poor stability. The Bt strain obtained by the induction means has problems such as a slow fermentation rate and a decrease in insecticidal activity. The dosage form not only makes the insecticide stable in transportation and preservation, but also has adverse environmental factors such as purple. External radiation has a good protective effect, and adding some additives such as ultraviolet protection to the dosage form can also have a good ultraviolet protection effect and greatly prolong the expiration date of the Bt preparation.

The microencapsulated suspension sustained release agent is the best way to solve the wild use of Bt bacteria, which can protect it from ultraviolet radiation, and slow release and prolong the residual period of use of Bt bacteria. At present, there are three most commonly used methods for microencapsulation of sodium alginate: 1. The method of instillation of acute pores, that is, the sodium alginate solution containing microorganisms is dropped into the divalent cross-linking solution through needles with different pore diameters, and the size of the microcapsules depends on the size. The size of the needle and the viscosity of the sodium alginate solution. 2. Emulsification coagulation method is an oil-in-water emulsification technique. The size and height of the microcapsules obtained by this method are highly dependent on the stirring speed and the rate of addition of the cross-linking solution. 3. The complex agglutination method is based on the principle that when a mixture of oppositely charged colloidal solutions is used, agglomeration occurs due to charge neutralization. However, the latter two methods are complicated in process and high in cost. The first method is simple in process and convenient in operation. In this paper, the acute hole instillation method and the addition of some UV protective agents, attractants and insecticides not only play a protective role, but also enhance the virulence of the preparation and prolong the residual effect period.

Summary of the invention

The object of the present invention is to provide a suspension-sustaining release agent of Bacillus thuringiensis and a preparation method thereof.

In order to achieve the above object, the technical solution of the present invention is as follows:

The mosquito suspension suspension release agent of the present invention is characterized in that it consists of the original powder of Bacillus thuringiensis, sodium alginate, calcium chloride, hollow glass microbeads, melanin, sodium benzoate, fish meal, natural pyrethroid and water; The weight percentage of various raw materials in the suspension slow release agent is as follows:

The preparation method of the mosquito-killing suspension-release agent of the Bacillus thuringiensis of the invention is characterized in that the operation steps are as follows:

(1) taking sodium alginate, original powder of Bacillus thuringiensis, hollow glass microbeads, melanin, sodium benzoate solution, fish meal, natural pyrethroid and water, and preparing a mixed solution, each of which accounts for the weight percentage of the suspension sustained release agent 1% to 5%, 1% to 5%, 1% to 2%, 0.3% to 0.5%, 0.1% to 0.5%, 0.5% to 2.5%, 0.1% to 0.5%, and 84% to 95%;

(2) preparing a calcium chloride solution having a concentration by weight of 1% to 5%;

(3) adding the mixed solution of the step (1) dropwise to the calcium chloride solution of the step (2) with a sterile syringe, the droplets forming granular beads and solidifying;

(4) The droplet of the step (3) is hardened in the calcium chloride solution for not less than 4 hours, the calcium chloride solution is discarded, the hardened granular microbeads are washed with distilled water, air-dried, and then stored at room temperature.

The sodium alginate is chemically pure, calcium chloride and sodium benzoate are analytically pure; the Bacillus thuringiensis is a Bt islet subspecies (disclosed, the strain is deposited as: CGMCC 1.1754); the original powder of the Bacillus thuringiensis, the purity is 5400 ITU/mg; hollow glass microspheres are available in OLH-D with a particle size range of 10-125 microns; all of the above materials are commercially available.

Advantages and technical principles of the present invention: The present invention uses sodium alginate as a carrier and calcium chloride as a crosslinking agent. The original powder of Bacillus thuringiensis is added as an effective active ingredient and has high mosquito killing activity. The hollow glass microbeads are used to achieve a sustained release effect of the suspension, which resists ultraviolet radiation and prolongs the residual period. The addition of melanin to the UV protection additive also enhances the UV radiation resistance of the bacteria. In order to avoid the microbial deterioration and mildew of the suspension slow release agent in the water, the virulence of the bacteria is decreased, and the sodium benzoate is added to protect the strain. The addition of fishmeal has an attracting effect, induces larvae to forage, improves the insecticidal effect, and avoids the phenomenon that the dosage form sinks in the water for a long time and prolongs the residual effect period. The use of natural pyrethroids is internationally recognized as the safest, pollution-free natural insecticide, a contact insecticide that enhances the insecticidal effect of the dosage form. The formula of the mosquito-killing Bacillus thuringiensis is closely related to its insecticidal effect in the wild, which determines the actual mosquito killing effect and economic benefit of the product. The preparation of the invention has the advantages of reasonable formula, high insecticidal virulence, good control effect, strong anti-ultraviolet radiation ability and long residual period in the field, and provides convenience for the development, promotion and popularization of the biological preparation of Bacillus thuringiensis.

detailed description

In order to fully disclose the preparation method of a suspension-sustaining release agent of Bacillus thuringiensis of the present invention, the following description will be given in conjunction with the examples.

Example 1: A suspension-release agent of Bacillus thuringiensis, from sodium alginate, calcium chloride, hollow glass beads, black The pigment, sodium benzoate, fish meal and natural pyrethroid composition, the weight percentage of various raw materials in the suspension slow release agent is as follows:

Example 2: A method for preparing a suspension-released strain of Bacillus thuringiensis, comprising the following steps:

(1) taking sodium alginate, original powder of Bacillus thuringiensis, hollow glass microbeads, melanin, sodium benzoate solution, fish meal, natural pyrethroid and water, and preparing a mixed solution, each of which accounts for the weight percentage of the suspension sustained release agent 3%, 3%, 1.5%, 0.3%, 0.3%, 1.5%, 0.4% and 85%;

(2) preparing a calcium chloride solution having a concentration of 5% by weight;

(3) adding the mixed solution of the step (1) dropwise to the calcium chloride solution of the step (2) with a 10 mL sterile syringe, the droplets forming a pellet and solidifying;

(4) The droplet of the step (3) was hardened in the calcium chloride solution for 4 hours, the calcium chloride solution was discarded, and the hardened granular microbeads were washed twice with distilled water, air-dried, and then stored at room temperature.

1 g of the suspension-released Bacillus thuringiensis suspension of the present invention was weighed into 8 L of water, and 500 larvae of C. sinensis were added, and the experiment was repeated 3 times and compared. Observe the 24h mortality rate. When the mortality rate exceeds 50%, replace the fresh water and transfer the new test insects; replace the fresh water and fresh test insects every 24 hours. The insecticidal effect of the suspension-released strain of Bacillus thuringiensis suspension of the present invention is shown in Table 1, and the suspension ratio thereof is shown in Table 2.

Table 1 The average corrected mortality rate of C. sinensis larvae at different ages after treatment with Bacillus thuringiensis suspension sustained release agent

Note: The above data is the average of 3 replicates.

Table 2 Average Suspension Rate in Different Time after Treatment with Suspension

Note: The above data is the average of 3 replicates.

As shown in Table 1, after treatment with Suspension S. mobilis suspension sustained release agent, the average relative mortality of the test worms gradually increased with the prolongation of treatment time, and the relative mortality of the test worms reached 100% at 192 h. As shown in Table 2, the suspension-released strain of Bacillus thuringiensis has good mechanical properties and long-term suspension. It did not begin to sink until 144h, and the suspension rate remained at 58.3% at 192h.

The above are only the preferred embodiments of the present invention, and all changes and modifications made to the scope of the present invention should fall within the scope of the present invention.

Claims

A method for preparing a suspension-sustaining release agent of Bacillus thuringiensis, characterized in that the operation steps are as follows:

(1) taking sodium alginate, original powder of Bacillus thuringiensis, hollow glass microbeads, melanin, sodium benzoate solution, fish meal, natural pyrethroid and water, and preparing a mixed solution, each of which accounts for the weight percentage of the suspension sustained release agent 1% to 5%, 1% to 5%, 1% to 2%, 0.3% to 0.5%, 0.1% to 0.5%, 0.5% to 2.5%, 0.1% to 0.5%, and 84% to 95%;

(2) preparing a calcium chloride solution having a concentration by weight of 1% to 5%;

(3) adding the mixed solution of the step (1) dropwise to the calcium chloride solution of the step (2) with a sterile syringe, the droplets forming granular beads and solidifying;

(4) The droplet of the step (3) is hardened in the calcium chloride solution for not less than 4 hours, the calcium chloride solution is discarded, the hardened granular microbeads are washed with distilled water, air-dried, and then stored at room temperature.
The method for preparing a suspension-released strain of Bacillus thuringiensis according to claim 1, wherein the operation steps are as follows:

(1) taking sodium alginate, original powder of Bacillus thuringiensis, hollow glass microbeads, melanin, sodium benzoate solution, fish meal, natural pyrethroid and water, and preparing a mixed solution, each of which accounts for the weight percentage of the suspension sustained release agent 3%, 3%, 1.5%, 0.3%, 0.3%, 1.5%, 0.4% and 85%;

(2) preparing a calcium chloride solution having a concentration of 5% by weight;

(3) adding the mixed solution of the step (1) dropwise to the calcium chloride solution of the step (2) with a 10 mL sterile syringe, the droplets forming a pellet and solidifying;

(4) The droplet of the step (3) was hardened in the calcium chloride solution for 4 hours, the calcium chloride solution was discarded, and the hardened granular microbeads were washed twice with distilled water, air-dried, and then stored at room temperature.
A suspension-release agent of Bacillus thuringiensis prepared by the method of claim 1 or 2.