阅读说明：本技术 一种水田中使用的球形芽孢杆菌杀虫剂及其制备方法 (Bacillus sphaericus insecticide used in paddy field and preparation method thereof ) 是由 陈宏伟 钱贞舟 吕海光 杨泰然 冯晶 于 2021-08-10 设计创作，主要内容包括：本发明属于生物农药领域,提供了一种水田中使用的球形芽孢杆菌杀虫剂及其制备方法,该杀虫剂包括球形芽孢杆菌以及营养载体,营养载体为含有营养盐及有机糖类的琼脂凝胶载体；其中,球形芽孢杆菌均匀分布于营养载体的内部。该球形芽孢杆菌杀虫剂通过在细菌周围构筑一个适应细菌生存的营养载体,能够有效的解决细菌源农药环境制约大,受环境影响杀虫效果不佳的问题。(The invention belongs to the field of biological pesticides, and provides a bacillus sphaericus pesticide used in paddy fields and a preparation method thereof, wherein the pesticide comprises bacillus sphaericus and a nutrient carrier, and the nutrient carrier is an agar gel carrier containing nutrient salt and organic saccharides; wherein, the bacillus sphaericus is uniformly distributed in the nutrient carrier. The bacillus sphaericus insecticide can effectively solve the problems that the environment of a bacterial source pesticide is greatly restricted and the insecticidal effect is poor due to the influence of the environment by constructing a nutrient carrier which is suitable for the survival of bacteria around the bacteria.)

1. A Bacillus sphaericus insecticide for use in paddy fields, comprising: the bacillus sphaericus and the nutrient carrier are agar gel carriers containing nutrient salts and organic saccharides;

wherein the bacillus sphaericus is uniformly distributed in the nutrient carrier;

when the bacillus sphaericus kills insects, the nutrient carrier provides energy for the bacillus sphaericus and improves the insecticidal effect of the bacillus sphaericus.

2. The Bacillus sphaericus insecticide for use in paddy fields of claim 1, wherein the Bacillus sphaericus is further distributed on the surface of a nutrient carrier.

3. The bacillus sphaericus insecticide for use in paddy fields according to claim 1, wherein the mass ratio of bacillus sphaericus to a nutrient carrier is 1-3: 1000.

4. The Bacillus sphaericus insecticide for use in a paddy field of claim 1, wherein the nutrient salt is a phosphate or a sulfate, and the organic saccharide is a monosaccharide or a disaccharide.

5. The Bacillus sphaericus insecticide for use in paddy fields of claim 4, wherein the nutrient salt is one or more of dipotassium hydrogen phosphate, copper sulfate, zinc sulfate or manganese sulfate.

6. A preparation method of a Bacillus sphaericus insecticide for use in paddy fields based on any one of claims 1 to 5, comprising the steps of:

activating and shake culturing strains of bacillus sphaericus, centrifuging at a high speed to form bacterial pulp, and storing the prepared bacterial pulp at 2-4 ℃ for later use;

adding nutrient salt and organic sugar into sterile water, and stirring until the nutrient salt and the organic sugar are completely dissolved to prepare a nutrient carrier aqueous solution;

stirring and dispersing agar powder in cold water uniformly until no lumps exist, heating and dissolving, and then keeping the dissolved agar warm for later use;

adding the prepared bacterial slurry into the nutrient carrier aqueous solution, stirring, adding dissolved agar, cooling to obtain a solid nutrient carrier, and cutting and packaging the nutrient carrier to obtain the bacterial pesticide.

7. The method for preparing a Bacillus sphaericus insecticide for use in paddy fields according to claim 6, wherein the dissolving temperature of the agar powder when heated for dissolution is 85 to 90 ℃, and the temperature when the dissolved agar is added is 47 to 50 ℃.

8. The preparation method of the bacillus sphaericus insecticide for the paddy fields according to claim 6, wherein the added nutrient salt and the organic sugar account for 10-20% by mass of the nutrient carrier, and the mass ratio of the nutrient salt to the organic sugar is 1: 25-35.

9. The method for preparing a Bacillus sphaericus insecticide for use in paddy fields according to claim 6, wherein the method for preparing the insecticide of bacterial origin further comprises the steps of: after the solid nutrient carrier is prepared, the bacterial pulp is added again to distribute the insecticidal bacteria on the surface of the nutrient carrier.

10. The method for preparing a Bacillus sphaericus insecticide for use in paddy fields according to claim 6, wherein the agar incubation is a water bath incubation.

Technical Field

The invention relates to the field of biological pesticides, and particularly relates to a bacterial source pesticide and a preparation method thereof.

Background

Modern agricultural production is increasingly pursuing requirements of organic green, no pollution and the like, but the use of chemical pesticides always has the problem of chemical residue, pesticides are not used, and the yield of agricultural production cannot be ensured, so that the search for a safe and efficient pesticide without chemical residue is particularly important, and the biopesticide is a preparation which can prevent plant diseases and insect pests and weeds and regulate plant growth and is prepared by utilizing active ingredients generated by living organisms or organisms and chemically synthesized substances with natural compound structures. Because the biological living body is utilized, all the substances are naturally existing substances in nature, the problem of chemical residue is not generated; the bacteria source material is one of biological pesticides, which can destroy the metabolic balance of pests by bacteria producing specific toxins, or achieve the insecticidal effect by causing epidemic diseases by the propagation of trophosome in the pest body. Mosquito and mosquito larvae in paddy fields are the main insect pest problems of crops in paddy fields, bacillus sphaericus has obvious killing capacity to culex larvae, but the bacillus sphaericus has certain environmental requirements on the environment, and in an unsuitable environment, the bacillus sphaericus can show reduced biological activity, reduce the insecticidal effect, is not beneficial to the utilization and development of pesticides, and particularly in the complicated and variable environment of the paddy fields, the insecticidal effect of the bacillus sphaericus is more dependent on the surrounding environment.

Disclosure of Invention

Aiming at the defects in the prior art, the invention aims to provide a bacillus sphaericus insecticide used in paddy fields and a preparation method thereof, which solve the problem of environmental restriction of bacillus sphaericus, improve the activity of a bacteria-derived pesticide and further improve the insecticidal effect of the bacillus sphaericus insecticide.

To achieve the above objects and other advantages in accordance with the present invention, a first object of the present invention is to provide a bacillus sphaericus insecticide for use in paddy fields, comprising: the bacillus sphaericus and a nutrient carrier, wherein the nutrient carrier is an agar gel carrier containing nutrient salt and organic saccharides;

wherein, the bacillus sphaericus is uniformly distributed in the nutrient carrier.

Preferably, the bacillus sphaericus is also distributed on the surface of the nutrient carrier.

Preferably, the mass ratio of the bacillus sphaericus to the nutrient carrier is 1-3: 1000.

Preferably, the nutrient salt is phosphate or sulfate, and the organic saccharide is monosaccharide or disaccharide.

Preferably, the nutrient salt is one or more of dipotassium hydrogen phosphate, copper sulfate, zinc sulfate or manganese sulfate;

the second objective of the invention is to provide a preparation method of the bacillus sphaericus insecticide used in the paddy field, which comprises the following steps:

activating and shake culturing strains of bacillus sphaericus, centrifuging at a high speed to form bacterial pulp, and storing the prepared bacterial pulp at 2-4 ℃ for later use;

adding nutrient salt and organic sugar into sterile water, and stirring until the nutrient salt and the organic sugar are completely dissolved to prepare a nutrient carrier aqueous solution;

stirring and dispersing agar powder in cold water uniformly until no lumps exist, heating and dissolving, and then keeping the dissolved agar warm for later use;

adding the prepared bacterial slurry into the nutrient carrier aqueous solution, stirring, adding dissolved agar, cooling to obtain a solid nutrient carrier, and cutting and packaging the nutrient carrier to obtain the bacterial pesticide.

Preferably, the dissolving temperature of the agar powder during heating and dissolving is 85-90 ℃, and the temperature of the agar powder during adding and dissolving is 47-50 ℃.

Preferably, the added nutrient salt and the organic sugar account for 10-20% of the nutrient carrier by mass, and the mass ratio of the nutrient salt to the organic sugar is 1: 25-35.

Preferably, the preparation method of the bacterial insecticide further comprises the following steps: after the solid nutrient carrier is prepared, the bacterial pulp is added again to distribute the insecticidal bacteria on the surface of the nutrient carrier.

Preferably, the agar incubation is a water bath incubation.

Compared with the prior art, the invention has the beneficial effects that:

compared with the prior art that bacteria are directly used as insecticides, the bacillus sphaericus insecticide used in paddy fields provided by the invention provides a growing environment for insecticidal bacteria, combines the insecticidal bacteria with a nutrient carrier, and provides an additional nutrient environment for the bacteria, so that on one hand, the activity of a bacterial source insecticide in the transportation process can be improved, and the insecticidal effect is ensured; on the other hand, in the using process, environmental factors in the using process can be less considered, the environmental adaptability of bacteria is worried about, an initial propagation environment of the bacteria is provided, and the insecticidal effect of the bacterial insecticide is further ensured. Finally, the bacillus sphaericus is coated with a nutrient carrier, so that the bacillus sphaericus can be fixed in a range to a certain extent, and the insecticide cannot be enriched in the same region due to the flowing of water in a paddy field.

Detailed Description

In order to understand the present invention, the following examples are given to further illustrate the present invention. The following description is only a preferred embodiment of the present invention, and is only for the purpose of describing the present invention, and should not be construed as limiting the scope of the present invention. It should be understood that any modification, equivalent replacement, and improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The bacterial source pesticide provided by the invention is prepared by the following method:

s1, activating and shake culturing strains of bacillus sphaericus, centrifuging at high speed to form bacterial pulp, and storing the prepared bacterial pulp at 2-4 ℃ for later use;

s2 adding nutritive salt and organic saccharide into sterile water, stirring to dissolve completely, and making into nutrient carrier water solution. Wherein the mass of the added nutrient salt and organic saccharide is 20-40 g, the mass ratio of the nutrient salt to the organic saccharide is 1: 25-35, and the mass of the prepared nutrient carrier aqueous solution is 500 g; in the step, the added nutrient salt is phosphate or sulfate, specifically one or more of dipotassium hydrogen phosphate, copper sulfate, zinc sulfate or manganese sulfate; the organic saccharide is monosaccharide or disaccharide, specifically one or more of glucose, fructose, arabinose, rhamnose, galactose, sucrose and maltose;

s3, stirring and dispersing 26g of agar powder in 480mL of cold water until no lumps exist, heating to dissolve the agar powder at 85-90 ℃, and then keeping the dissolved agar in a water bath with 47-50 ℃ of water for later use;

s4, adding 1-3 g of prepared bacterial pulp into the nutrient carrier aqueous solution, stirring, adding dissolved agar into the nutrient carrier aqueous solution, cooling, changing the nutrient carrier aqueous solution into a fixed nutrient carrier, and cutting and subpackaging the nutrient carrier to obtain the bacterial source pesticide.

In some preferred embodiments, the method further comprises step S5, after the solid nutrient carrier is prepared, adding bacterial slurry to the solid nutrient carrier again to distribute the pesticidal bacteria on the surface of the nutrient carrier.

According to the bacterial insecticide prepared by the method, biological pesticide insecticidal bacteria are used as insecticidal effective substances, so that the method is environment-friendly, is not easy to generate chemical residues, cannot cause secondary problems, cannot generate sweating antibodies to pests, and is easy to protect biological diversity. In particular, the invention takes the bacillus sphaericus as an effective insecticidal substance, has high-efficiency insecticidal effect on mosquitoes in paddy fields, has no toxicity on non-target organisms and human and livestock, is easy to degrade in nature and does not pollute the environment.

In addition, the insecticide places the insecticidal bacteria in the nutrient carrier, so that the problem of large environmental restriction of the bacteria-derived biopesticide is solved, the nutrient carrier can provide a living environment for the bacteria-derived biopesticide when the biopesticide is transported and used, a buffering time for the bacteria after the environment is changed is provided, the bacteria survival is facilitated, and the insecticidal effect of the bacteria-derived biopesticide is ensured.

In addition, in the microcapsule pesticide, the nutrient substances are nutrient salts and organic saccharides, the nutrient salts are phosphate or sulfate, and particularly dipotassium hydrogen phosphate, copper sulfate, zinc sulfate or manganese sulfate, which not only provides inorganic nonmetallic elements such as phosphorus and sulfur required by microorganism growth, but also provides inorganic metallic elements such as potassium and copper required by microorganism growth. Compared with polysaccharide, the organic saccharide can be directly utilized by bacteria or consumes less energy to convert the disaccharide into monosaccharide for utilization by the bacteria, and is more favorable for the survival of the bacteria.

The bacteria source pesticide prepared by the invention also distributes the insecticidal bacteria on the surface of the nutrient carrier, compared with the prior art that the bacteria source pesticide has slow effect, the insecticidal bacteria can utilize the attached nutrient substances to grow rapidly, the pesticide can take effect rapidly when being applied, the effect taking period of the pesticide is shortened, the bacterial source pesticide is applied when the plant diseases and insect pests are found, the plant diseases and insect pests can be controlled in a short time, and the loss is reduced.

Moreover, the weight of the bacillus sphaericus can be increased by coating the agar outside the bacillus sphaericus, and the problems that the pesticide is concentrated in one area due to water flow in a paddy field, so that the pesticide concentration in part of the area is too high, and the pesticide concentration in part of the area is too low, so that the pesticide effect is not ideal can be avoided.

The advantages of the bacterially derived pesticide are illustrated by the following examples, which are described in more detail below, and by the test of pesticidal effect of the examples and comparative examples.

Example 1

S11, activating and shake culturing strains of bacillus sphaericus, centrifuging at high speed to form bacterial pulp, and storing the prepared bacterial pulp at 2-4 ℃ for later use;

s12 nutrient salt and organic sugar are added into 480mL sterile water, and the mixture is stirred until the nutrient salt and the organic sugar are completely dissolved to prepare the nutrient carrier water solution. Wherein the mass of the added nutrient salt and the organic saccharide is 20g, the nutrient salt is dipotassium hydrogen phosphate, the adding amount is 0.8g, the organic saccharide is glucose, and the adding amount is 19.2 g.

S13, stirring and dispersing 26g of agar powder in 480mL of cold water until no lumps exist, heating and dissolving at 85 ℃, and then keeping the dissolved agar powder in a water bath with water of 47-50 ℃ for later use;

s14, adding the prepared bacterial slurry into a nutrient carrier aqueous solution, wherein the mass of the added bacterial slurry is 1g, stirring, adding dissolved agar into the mixed solution, cooling, and then changing the nutrient carrier aqueous solution into a fixed nutrient carrier, and cutting and subpackaging the nutrient carrier to obtain the bacterial source pesticide.

Example 2

S21, activating and shake culturing strains of bacillus sphaericus, centrifuging at high speed to form bacterial pulp, and storing the prepared bacterial pulp at 2-4 ℃ for later use.

S22 nutrient salt and organic sugar are added into 470mL sterile water, and stirred to be completely dissolved, thus obtaining the nutrient carrier water solution. Wherein the mass of the added nutrient salt and the organic saccharide is 30g, the nutrient salt is copper sulfate, the addition amount is 1g, the organic saccharide is fructose, and the addition amount is 29 g.

S23, stirring and dispersing 26g of agar powder in 480mL of cold water until no lumps exist, heating to dissolve the agar powder at 88 ℃, and then keeping the dissolved agar in a water bath with water of 47-50 ℃ for later use;

s24, adding the prepared bacterial slurry into a nutrient carrier aqueous solution, wherein the mass of the added bacterial slurry is 2g, stirring, adding dissolved agar into the mixed solution, cooling, and then changing the nutrient carrier aqueous solution into a fixed nutrient carrier, and cutting and subpackaging the nutrient carrier to obtain the bacterial source pesticide.

Example 3

S31, activating and shake culturing strains of bacillus sphaericus, centrifuging at high speed to form bacterial pulp, and storing the prepared bacterial pulp at 2-4 ℃ for later use.

S32 nutrient salt and organic sugar are added into 460mL sterile water, and the mixture is stirred until the nutrient salt and the organic sugar are completely dissolved, so that the nutrient carrier water solution is prepared. The mass of the added nutrient salt and the added organic sugar is 40g, wherein the nutrient salt is zinc sulfate, the adding amount is 1.1g, the added organic sugar is arabinose, and the adding amount is 29.9 g.

S33 stirring and dispersing 26g of agar powder in 480mL of cold water until no lumps exist, heating and dissolving at 90 ℃, and then keeping the dissolved agar in a water bath with water of 47-50 ℃ for later use.

S34, adding the prepared bacterial slurry into the nutrient carrier aqueous solution, wherein the mass of the added bacterial slurry is 2g, stirring, adding the dissolved agar, cooling, changing the nutrient carrier aqueous solution into a fixed nutrient carrier, and cutting and subpackaging the nutrient carrier.

S35, uniformly dispersing 1g of the prepared bacterial slurry into the cut and subpackaged nutrient carrier to disperse the bacteria on the surface of the nutrient carrier, and finally preparing the bacterial insecticide.

Example 4

S41, activating and shake culturing strains of bacillus sphaericus, centrifuging at high speed to form bacterial pulp, and storing the prepared bacterial pulp at 2-4 ℃ for later use;

s42 nutrient salt and organic sugar are added into 480mL sterile water, and the mixture is stirred until the nutrient salt and the organic sugar are completely dissolved to prepare the nutrient carrier water solution. Wherein the mass of the added nutrient salt and the organic saccharide is 20g, the nutrient salt is dipotassium hydrogen phosphate and manganese sulfate, the adding amount is 0.4g, the organic saccharide is galactose, sucrose and maltose, and the adding amount is 5g, 5g and 9.2g respectively.

S43, stirring and dispersing 26g of agar powder in 480mL of cold water until no lumps exist, heating and dissolving at 85 ℃, and then keeping the dissolved agar powder in a water bath with water of 47-50 ℃ for later use;

s44, adding the prepared bacterial slurry into a nutrient carrier aqueous solution, wherein the mass of the added bacterial slurry is 3g, stirring, adding dissolved agar into the mixed solution, cooling, and then changing the nutrient carrier aqueous solution into a fixed nutrient carrier, and cutting and subpackaging the nutrient carrier to obtain the bacterial source pesticide.

And (3) testing the insect killing and expelling effect:

the insecticidal and anthelmintic effect was characterized by applying a certain amount of insecticide to the plants with the pests, and the test tested the number of dead insects at 3 and 7 days by applying the same mass of insecticide.

The death rate (%) < dead insect number/total insect number x 100%

Comparative example 1 is a blank control group without any pesticide application, comparative example 2 is the result of application of a bio-pesticide of bacterial origin cultured by a conventional method, which employs a bacterium of bacillus sphaericus, and the applied mass of the bacterial sludge after final culture is 2 g. The test results are shown in table 1.

TABLE 1

Through the comparative example and the comparative example 1, it is obvious that the pesticide provided by the invention has good insecticidal effect, the insecticidal efficiency of the pesticide reaches 31 percent in 3 days, and the insecticidal efficiency is about 5 times of the natural death of the pesticide without application; the insecticidal efficiency is 51.7% in 7 days, which is about 4 times of the natural death of the pesticide, and shows that the bacterial source insecticide has good insecticidal effect and can effectively prevent and treat field diseases and insect pests. Compared with the comparative example and the comparative example 2, the insecticidal effect of the example is obviously better than that of the comparative example 2, and the insecticidal effect of the insecticide is almost 2 times that of the traditional bacterial insecticide, so that the insecticide provided by the invention provides a suitable living environment for the bacterial insecticide, the activity of bacteria in the bacterial insecticide can be effectively improved, and the insecticidal effect of the bacterial insecticide is further improved. By comparing example 1 and example 3, the insecticidal effect of example 3 is better than that of example 1, and it is shown that the insecticidal effect of the bacterial insecticide can be further improved by coating bacteria to form a nutrient carrier and then attaching the bacteria to the surface of the nutrient carrier.

In conclusion, a living environment suitable for bacteria is arranged around the bacteria source pesticide, a living space is provided for the insecticidal bacteria, the insecticidal effect of the insecticidal bacteria can be effectively improved, and the problems that the insecticidal bacteria in a common environment are poor in environmental tolerance and the insecticidal effect is greatly influenced by the environment can be solved.

The foregoing is merely a preferred embodiment of the invention and is not intended to limit the invention in any manner; as will be apparent to those skilled in the art from this disclosure, the present invention may be practiced without these specific details; however, those skilled in the art should appreciate that they can readily use the disclosed conception and specific embodiments as a basis for designing or modifying other structures for carrying out the same purposes of the present invention without departing from the scope of the invention as defined by the appended claims; meanwhile, any changes, modifications, and evolutions of the equivalent changes of the above embodiments according to the actual techniques of the present invention are still within the protection scope of the technical solution of the present invention.