Application of hydrogel in improving insecticidal activity of Bacillus thuringiensis

Abstract

The present invention relates to the use of a hydrogel in improving the insecticidal activity of Bacillus thuringiensis. The hydrogel is folic acid-Zn 2+ Hydrogel, in which folic acid ions and Zn 2+ The mass ratio is 1:2.

Description

Technical Field

[0001] The invention relates to the field of biocides, and in particular to application of hydrogel in improving the insecticidal activity of Bacillus thuringiensis. Background Art

[0002] South American tomato leafminer ( Absolutely safe ), also known as the tomato leafminer, belongs to the family Gelechiidae of the order Lepidoptera, and is native to South America. Its larvae mainly feed on the mesophyll of tomatoes, and can also bore into fruits, tops, as well as tender shoots, young stems and side branches. Severe damage often causes a 80% to 100% reduction in tomato production.

[0003] Bacillus thuringiensis ( Bacillus thuringiensis Bacillus thuringiensis (Bt) is a Gram-positive bacterium widely distributed in nature. Its outstanding feature is that it can produce parasporal crystal proteins during spore formation. Parasporal crystal proteins are composed of one or more proteins and have highly specific insecticidal activity. This protein is usually called insecticidal crystal protein. Insecticidal crystal protein exists in the form of protoxin. When it enters the digestive tract of sensitive insects, it dissolves in an alkaline environment and is activated by proteases into toxic polypeptides. The activated toxic polypeptides bind to receptors on the marginal membrane of the insect's midgut and form pores on the cell membrane, destroying the osmotic balance of the cell, causing cell lysis, and ultimately leading to the death of the larvae. Therefore, as a new technical means of controlling agricultural and forestry pests, a series of microbial pesticide preparations represented by Bacillus thuringiensis have received widespread attention worldwide.

[0004] At present, the Bt insecticides available on the market are mainly made by enriching the fermentation products in the Bt fermentation liquid and then making them into wettable powders. However, the unstable efficacy and short duration of the Bt preparations have affected the promotion and use of this type of biological pesticides. Summary of the invention

[0005] One of the present invention provides the use of a hydrogel in improving the insecticidal activity of Bacillus thuringiensis, wherein the hydrogel is folic acid-Zn 2+ Hydrogel.

[0006] In one embodiment, in the hydrogel, folate ions and Zn 2+ The mass ratio is 1:2.

[0007] In a specific embodiment, the Bacillus thuringiensis is Bacillus thuringiensis G033A strain.

[0008] In a specific embodiment, the content of Bacillus thuringiensis in the hydrogel is 1.25 to 20 g / L.

[0009] The second aspect of the present invention provides a method for preparing a Bacillus thuringiensis hydrogel, which comprises the following steps:

[0010] 1) mixing folic acid and potassium hydroxide in water to prepare a folic acid-potassium hydroxide neutralization solution;

[0011] 2) suspending Bacillus thuringiensis in water to prepare a Bacillus thuringiensis water suspension;

[0012] 3) dissolving zinc nitrate in water to obtain an aqueous solution of zinc nitrate;

[0013] 4) Mixing the folic acid-potassium hydroxide neutralization solution, the Bacillus thuringiensis aqueous suspension and the zinc nitrate aqueous solution in sequence, mixing them evenly and then standing them to obtain the Bacillus thuringiensis hydrogel.

[0014] In one embodiment, in step 4), the folate ions in the mixed product and Zn 2+ The mass ratio is 1:2.

[0015] In a specific embodiment, the content of Bacillus thuringiensis in the hydrogel is 1.25 to 20 g / L.

[0016] In a specific embodiment, the concentration of folate ions in the folic acid-potassium hydroxide neutralization solution is 0.1 M.

[0017] In a specific embodiment, the concentration of zinc nitrate in the aqueous solution of zinc nitrate is 0.1 M.

[0018] Beneficial effects of the present invention: The present invention finds that folic acid-Zn 2+ Hydrogels can enhance the insecticidal activity of Bacillus thuringiensis. DETAILED DESCRIPTION

[0019] The above contents of the present invention are further described in detail below in the form of preferred implementation cases, but they do not constitute a limitation to the present invention.

[0020] Unless otherwise specified, the strains and reagents in the examples of the present invention can be purchased through commercial channels.

[0021] Bt G033A fermentation raw powder was provided by Wuhan Kono Company. Example 1

[0022] 1) Add 2.207 g of folic acid (0.01 Mol) and 50 mL of ultrapure water into a beaker, then add 50 mL of 0.5 M KOH solution and stir evenly to obtain a folic acid-KOH neutralized solution, in which the concentration of folate ions in the folic acid-KOH neutralized solution is 0.1 M.

[0023] 2) Weigh the Bt G033A fermentation raw powder and disperse it in 50 ml of ultrapure water to prepare a Bt G033A aqueous suspension.

[0024] 3) Dissolve 18.9 g zinc nitrate in 1 L ultrapure water to prepare a 0.1 M Zn(NO3)2 aqueous solution.

[0025] 4) The folic acid-KOH neutralization solution, the aqueous suspension of Bt G033A and the aqueous solution of Zn(NO3)2 were pipetted into the test tube in the order of folic acid-KOH neutralization solution and Zn(NO3)2 solution according to the concentration of folic acid ions and Zn in the final gel. 2+ The mass ratio of Bt G033A was 1:2, and the tube was vortexed for 1 min using an XH-C vortex mixer, and the tube was allowed to stand for about 30 min. The tube was inverted, and if there was no liquid flow, it proved that the gel was formed, and finally the G033A hydrogel 1# loaded with Bt G033A was obtained, wherein the loading amount of G033A in the G033A hydrogel 1# was 20 g / L. Example 2

[0026] The amount of Bt G033A fermentation raw powder in step 2) was changed, and the rest was the same as in Example 1, and finally BtG033A-loaded G033A hydrogel 2# was prepared, wherein the loading amount of G033A in G033A hydrogel 2# was 10 g / L. Example 3

[0027] The amount of Bt G033A fermentation raw powder in step 2) was changed, and the rest was the same as in Example 1, and finally BtG033A-loaded G033A hydrogel 3# was prepared, wherein the loading amount of G033A in G033A hydrogel 3# was 5 g / L. Example 4

[0028] The amount of Bt G033A fermentation raw powder in step 2) was changed, and the rest was the same as in Example 1, and finally BtG033A-loaded G033A hydrogel 4# was prepared, wherein the loading amount of G033A in G033A hydrogel 4# was 2.5 g / L. Example 5

[0029] The amount of Bt G033A fermentation raw powder in step 2) was changed, and the rest was the same as in Example 1, and finally BtG033A-loaded G033A hydrogel 5# was prepared, wherein the loading amount of G033A in G033A hydrogel 5# was 1.25 g / L. Comparative Example 1

[0030] Step 2 is omitted), and the rest is the same as in Example 1, and finally hydrogel D1# is obtained. Comparative Example 2

[0031] The Bt G033A fermentation raw powder was dispersed in sterile water to prepare test solutions with gradient concentrations, wherein the concentrations of the Bt G033A fermentation raw powder in each test solution were 20 g / L, 10 g / L, 5 g / L, 2.5 g / L, and 1.25 g / L.

[0032] Insecticidal activity test

[0033] Tomato leaves of the same size were immersed in G033A hydrogels 1# to 5#, hydrogel D1# and each test solution for 30 s on both sides. The petioles were wrapped with water-containing absorbent cotton. After the leaves were naturally air-dried, they were placed in a 9 cm diameter plastic culture dish containing qualitative filter paper. 17 active 2nd-instar tomato leafminer larvae were inoculated in each culture dish. Each treatment was repeated 3 times, and clean water was used as a negative control. They were placed in an incubator with a temperature of 25±1 degrees Celsius and a humidity of 60±5%. The effects of temperature and humidity on the larvae were observed every day. The number of live and dead insects was recorded at 72 h, and the mortality rate and the corrected mortality rate were calculated based on the negative control. The results showed that there was no significant difference between the mortality rate of hydrogel D1# and the mortality rate of the negative control, indicating that the hydrogel itself did not have insecticidal activity. Polo plus was further used to calculate the LC values ​​of G033A hydrogel and G033A original powder against 2nd-instar larvae of tomato leafminer at 72 h. 50 The results are as follows: LC of G033A hydrogel 50 is 5.840 g / L, with a 95% confidence limit of 4.612 to 7.495 g / L; the LC of G033A raw powder 50 The insecticidal activity of G033A hydrogel was 1.90 times that of G033A original powder.

Claims

1. Application of a hydrogel in improving the insecticidal activity of Bacillus thuringiensis, wherein the hydrogel is folic acid-Zn 2+ hydrogel; in the hydrogel, folic acid ions and Zn 2+ The mass ratio is 1:2; among them, The method for preparing the Bacillus thuringiensis hydrogel comprises the following steps: 1) mixing folic acid and potassium hydroxide in water to prepare a folic acid-potassium hydroxide neutralization solution; 2) suspending Bacillus thuringiensis in water to prepare a Bacillus thuringiensis water suspension; 3) dissolving zinc nitrate in water to obtain an aqueous solution of zinc nitrate; 4) Mixing the folic acid-potassium hydroxide neutralization solution, the Bacillus thuringiensis aqueous suspension and the zinc nitrate aqueous solution in sequence, mixing evenly and then standing to obtain the Bacillus thuringiensis hydrogel, wherein the folic acid ions in the mixed product and the Zn 2+ The mass ratio is 1:

2.

2. The use according to claim 1, characterized in that: The Bacillus thuringiensis is the Bacillus thuringiensis G033A strain.

3. The use according to claim 1, characterized in that: The content of the Bacillus thuringiensis in the hydrogel is 1.25 to 20 g / L.

4. A method for preparing a Bacillus thuringiensis hydrogel, comprising the following steps: 1) mixing folic acid and potassium hydroxide in water to prepare a folic acid-potassium hydroxide neutralization solution; 2) suspending Bacillus thuringiensis in water to prepare a Bacillus thuringiensis water suspension; 3) dissolving zinc nitrate in water to obtain an aqueous solution of zinc nitrate; 4) Mixing the folic acid-potassium hydroxide neutralization solution, the Bacillus thuringiensis aqueous suspension and the zinc nitrate aqueous solution in order, mixing them evenly and then standing them to obtain the Bacillus thuringiensis hydrogel, wherein: The folic acid ions and Zn in the mixed product 2+ The mass ratio is 1:

2.

5. The method according to claim 4, characterized in that The Bacillus thuringiensis is the Bacillus thuringiensis G033A strain.

6. The method according to claim 4, characterized in that The content of the Bacillus thuringiensis in the hydrogel is 1.25 to 20 g / L.

7. The method according to claim 4, characterized in that The concentration of folate ions in the folic acid-potassium hydroxide neutralization solution is 0.1 M.

8. The method according to claim 4, characterized in that The concentration of zinc nitrate in the aqueous solution of zinc nitrate is 0.1 M.