Method for preparing biopesticide preparation for preventing and controlling <i>Solenopsis invicta</i>

Abstract

A method for preparing a biopesticide preparation for preventing and controlling Solenopsis invicta comprising: a: preparing an agent containing pathogenic microorganisms for preventing and controlling Solenopsis invicta, the agent comprises Beauveria bassiana and sodium chloride, with a Beauveria bassiana spores to sodium chloride ratio of (1-8)×20 billion spores: 4.0-7.5 g; b: placing the agent into a solid container, and adding a corresponding amount of solution in a liquid container; inserting the solid container into the liquid container, wherein a retaining ring is stuck at the opening of the liquid container, a rubber gasket is pressed against the retaining ring, and a lid covers the rubber gasket and the retaining ring and is fixed to the opening of the liquid container.

Claims

1. A method for preparing a biopesticide preparation for preventing and controlling Solenopsis invicta, wherein the method comprises the following steps: providing a mixer comprising a liquid container, a solid container, a rubber gasket and a lid; the solid container is a container with an upper end having an opening and a lower end sealed with an aluminum foil composite film, and a retaining ring is disposed at an edge of the opening of the solid container; the solid container is inserted into an opening of the liquid container, so that the retaining ring is stuck at the opening of the liquid container, the rubber gasket is pressed against the retaining ring, and the lid covers the rubber gasket and the retaining ring and is fixable to the opening of the liquid container; a diversion hole is disposed at a top of the lid; a: preparing an agent containing pathogenic microorganisms for preventing and controlling Solenopsis invicta, wherein the agent containing the pathogenic microorganisms for preventing and controlling Solenopsis invicta is a water dispersible granule formulation, comprising by mass of 100%, totally 30% of a Beauveria bassiana powder formulation and sodium chloride wherein a mass ratio of the Beauveria bassiana powder formulation to sodium chloride is 1-8:4.0-7.5 and the Beauveria bassiana powder formulation contains 20 billion Beauveria bassiana spores per gram, 8% of wetting agent butylnaphtalenesulfonic acid sodium salt, 18% of dispersant sodium lignosulfonate, 5% of disintegrant calcium chloride, 2% of stabilizer disodium hydrogen phosphate, 5% of binder sodium polyacrylate, and 32% of carrier diatomite; and b: placing the agent containing the pathogenic microorganisms for preventing and controlling Solenopsis invicta into the solid container, and adding an amount of water in the liquid container; inserting the solid container into the liquid container, wherein the retaining ring is stuck at the opening of the liquid container, the rubber gasket is pressed against the retaining ring, and the lid covers the rubber gasket and the retaining ring and is fixed to the opening of the liquid container.

2. The method for preparing a biopesticide preparation for preventing and controlling Solenopsis invicta according to claim 1, wherein a rubber washer is provided at the opening of the liquid container, and the retaining ring is pressed against the rubber washer.

3. The method for preparing a biopesticide preparation for preventing and controlling Solenopsis invicta according to claim 1 wherein the Beauveria bassiana spores to sodium chloride ratio is 5×20 billion spores: 4.38-7.30 g.

4. The method for preparing a biopesticide preparation for preventing and controlling Solenopsis invicta according to claim 3, wherein the Beauveria bassiana spores to sodium chloride ratio is 5×20 billion spores: 7.30 g.

5. The method for preparing a biopesticide preparation for preventing and controlling Solenopsis invicta according to claim 1 wherein in the step of placing the agent containing the pathogenic microorganisms for preventing and controlling Solenopsis invicta into the solid container and adding an amount of water in the liquid container, the agent containing pathogenic microorganisms for preventing and controlling Solenopsis invicta and the sodium chloride are provided in an amount that, after the agent containing the pathogenic microorganisms for preventing and controlling Solenopsis invicta and the sodium chloride are dissolved in the water, the water contains 1×10.sup.8/mL of Beauveria bassiana spores and 75-125 mmol/L of sodium chloride.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows the cumulative mortality of Solenopsis invicta after applying agents in different concentrations in Embodiment 1 for 96 hours, wherein, “Beauveria bassiana”: 5 g/L aqueous solution of Beauveria bassiana; 1: Beauveria bassiana+25 mmol/L NaCl; 2: Beauveria bassiana+75 mmol/L NaCl; 3: Beauveria bassiana+100 mmol/L NaCl; 4: Beauveria bassiana+125 mmol/L NaCl; 5: Beauveria bassiana+150 mmol/L NaCl; 6: Beauveria bassiana+200 mmol/L NaCl; 7: Beauveria bassiana+250 mmol/L NaCl; 8: Beauveria bassiana+300 mmol/L NaCl; CK: sterile water; NaCl: 125 mmol/L aqueous solution of NaCl.

(2) FIG. 2 shows the cumulative mortality of Solenopsis invicta after applying different aqueous solutions of Beauveria bassiana/NaCl in Embodiment 2 for 96 hours, wherein, “Beauveria bassiana”: Beauveria bassiana group; 1: Beauveria bassiana+25 mmol/L NaCl+adjuvant; 2: Beauveria bassiana+75 mmol/L NaCl+adjuvant; 3: Beauveria bassiana+100 mmol/L NaCl+adjuvant; 4: Beauveria bassiana+125 mmol/L NaCl+adjuvant; 5: Beauveria bassiana+150 mmol/L NaCl+adjuvant; 6: Beauveria bassiana 200 mmol/L NaCl+adjuvant; 7: Beauveria bassiana+250 mmol/L NaCl+adjuvant; 8: Beauveria bassiana+300 mmol/L NaCl+adjuvant; CK: sterile water+adjuvant; NaCl: 125 mmol/L aqueous solution of NaCl+adjuvant.

(3) FIG. 3. shows a split structure of the mixer of the preparation for preventing and controlling Solenopsis invicta.

(4) FIG. 4. is a sectional view of the overall structure of the mixer of the preparation for preventing and controlling Solenopsis invicta.

(5) Reference signs: 1. diversion hole; 2. lid; 3. rubber gasket; 4. solid container; 5. aluminum foil composite film; 6. rubber washer; 7. opening of the liquid container; 8. liquid container; 9. retaining ring.

DETAILED DESCRIPTION OF THE EMBODIMENTS

(6) Beauveria bassiana is a widely-used entomopathogenic fungus, which is sold by all major collection centers such as the Guangdong Microbial Culture Collection Center, or can be purchased from a company that produces Beauveria bassiana, such as Jiangxi Tianren Ecology Co., Ltd.

(7) The following embodiments are intended to further describe the present invention, but not to limit the present invention.

Embodiment 1: Laboratory Virulence Test of the Combination of Beauveria bassiana and Sodium Chloride Against Solenopsis invicta Worker Ants

(8) In the laboratory virulence test, 5 g of Beauveria bassiana powder formulation was dissolved in 1 L of water and mixed well to give a Beauveria bassiana solution. The used Beauveria bassiana powder formulation contained 20 billion/g of Beauveria bassiana spores. 9 Beauveria bassiana solutions, 100 mL each, were taken from the IL Beauveria bassiana solution, and respectively added with NaCl so that NaCl concentrations in the 9 Beauveria bassiana solutions were: 0 mmol/L, 25 mmol/L, 75 mmol/L, 100 mmol/L, 125 mmol/L, 150 mmol/L, 200 mmol/L, 250 mmol/L, and 300 mmol/L, and thereby Beauveria bassiana/sodium chloride aqueous dispersions were obtained, wherein the Beauveria bassiana spores were present in a concentration of 1×10.sup.8/mL.

(9) The Beauveria bassiana solution, the Beauveria bassiana/sodium chloride aqueous solution, a sodium chloride solution (125 mmol/L), and sterile water were respectively tested for virulence against healthy Solenopsis invicta worker ants, with 3 replicates for each treatment, 1 petri dish in each replicate, and 100 Solenopsis invicta worker ants in each petri dish. The Solenopsis invicta worker ants were firstly dipped in the solution for about 5 seconds and then taken out; after air dried indoors, they were placed in a breeding box, fed with fresh honey water every day, and kept at 26±1° C., a humidity of 70-90%, and a photoperiod of 14 L/10 hD; 96 hours after the treatment, survival of the Solenopsis invicta worker ants were observed to calculate the mortality. Data statistical analysis was performed using SPSS 13.0 software, significance testing between samples was performed using the Duncan's method, wherein different lowercase letters indicate a significant difference (P<0.05) in cumulative mortality of the treated Solenopsis invicta.

(10) Results were as shown in FIG. 1. As can be seen from FIG. 1, for the Solenopsis invicta treated with merely NaCl, no significant difference in the 96 h cumulative mortality was observed as compared with the blank control (Solenopsis invicta treated with sterile water, 15.91±0.72%), suggesting that the use of merely NaCl had no killing effect on Solenopsis invicta. For the Solenopsis invicta treated with merely Beauveria bassiana solution, significant differences in the 96 h cumulative mortality were observed as compared with the use of merely NaCl and the blank control, suggesting that the use of merely Beauveria bassiana exhibited a certain pesticidal effect, while the mortality was low. When sodium chloride was mixed in a concentration of 75 mmol/L, 100 mmol/L, or 125 mmol/L with Beauveria bassiana (i.e., the solutions contained 1×10.sup.8/mL of spores and 75-125 mmol/L of NaCl), significant differences in the 96 h cumulative mortality were observed as compared with the use of merely Beauveria bassiana and the use of merely NaCl, suggesting that the combination of sodium chloride with Beauveria bassiana in a certain ratio would significantly increase the pesticidal effect against Solenopsis invicta, and thereby demonstrating that the mixture exhibited a significant synergism in pesticidal effect as compared with the use of merely NaCl or Beauveria bassiana.

(11) The inventors had also conducted the above test with calcium chloride, potassium chloride, magnesium chloride, ammonium sulfate, and ferric chloride instead of sodium chloride. Results showed that, none of calcium chloride, potassium chloride, magnesium chloride, ammonium sulfate, and ferric chloride provided a synergism that, addition of these compounds did not largely affect the pesticidal effect of Beauveria bassiana on Solenopsis invicta, and no significant difference was observed.

Embodiment 2: Laboratory Virulence Test of the Beauveria bassiana/Sodium Chloride Aqueous Dispersion Against Solenopsis invicta Worker Ants

(12) As shown in FIG. 3 and FIG. 4, in the instant embodiment, the mixer of the preparation for preventing and controlling Solenopsis invicta comprised a liquid container 8, a solid container 4, a rubber gasket 3 and a lid 2; the solid container 4 is a container with an upper end having an opening and a lower end sealed with an aluminum foil composite film 5, and a retaining ring 9 is disposed at an edge of the opening of the solid container; a rubber washer 6 is provided at an opening 7 of the liquid container; the solid container is inserted into the opening 7 of the liquid container through the rubber washer 6, so that the retaining ring 9 is pressed against the rubber washer 6, the rubber gasket 3 is pressed against the retaining ring 9, and the lid 2 covers the rubber gasket 3, the retaining ring 9 and the rubber washer 3 and is fixed to the opening 7 of the liquid container; a diversion hole 1 is disposed at a top of the lid.

(13) A totally 30 g of Beauveria bassiana powder formulation and sodium chloride (wherein a mass ratio of the Beauveria bassiana powder formulation to sodium chloride included 5:1.46, 5:4.38.sub.; 5:5.84, 5:7.30, 5:8.77, 5:11.69, 5:14.61 g, and 5:17.53, and the Beauveria bassiana powder formulation contained 20 billion Beauveria bassiana spores per gram), 8 g of wetting agent butylnaphtalenesulfonic acid sodium salt, 18 g of dispersant sodium lignosulfonate, 5 g of disintegrant calcium chloride, 2 g of stabilizer disodium hydrogen phosphate, 5 g of binder sodium polyacrylate, and 32 g of carrier diatomite, were weighed out and mixed according to a method for preparing a water dispersible granule formulation (1. The raw materials were mixed evenly according to the formula. 2. The materials were ground using a sand mill after mixed evenly, and subjected to spray granulation after determined to be acceptable. 3. The products were screened using a vibrating screen after the spray granulation, products with unacceptable appearance were returned to the sand mill to be ground again, and products with acceptable appearance were further subjected to quality inspection wherein products with unacceptable quality were also returned to the sand mill to be ground again. 4. Products with acceptable quality were packaged. 5. Packaged water dispersible granule formulation were put into storage), to eventually produce water dispersible granule formulations containing 30% by mass of Beauveria bassiana/sodium chloride (an agent containing pathogenic microorganisms for preventing and controlling Solenopsis invicta).

(14) The above water dispersible granule formulations containing 30% by mass of Beauveria bassiana/sodium chloride were respectively placed in different solid containers, and liquid containers were filled with 1 L of sterile water. A rubber washer was provided at an opening of each liquid container. Then the solid containers were inserted into the liquid containers, wherein the retaining ring was stuck at the rubber washer, the rubber gasket was pressed against the retaining ring, and the lid covered the rubber gasket, the retaining ring and the rubber washer and was fixed to the opening of the liquid container. With such configuration, the formulation was convenient for storage and transportation; when applied, it was not necessary to unscrew the lid, but instead, a needle of a syringe was inserted directly into the diversion hole at the top of the lid to pierce the aluminum foil composite film at the lower end of the solid container so that the water dispersible granule formulation for preventing and controlling Solenopsis invicta fell into the solution in the liquid container, and then the mixture was shaken well to give an aqueous dispersion of the water dispersible granule formulation. In each aqueous dispersion of water dispersible granule formulation, the concentration of Beauveria bassiana spores was 1×10.sup.8/mL, while the concentration of NaCl was respectively 25 mmol/L: (FIG. 2, 1), 75 mmol/L (FIG. 2, 2), 100 mmol/L (FIG. 2, 3), 125 mmol/L (FIG. 2, 4), 150 mmol/L (FIG. 2, 5), 200 mmol/L (FIG. 2, 6), 250 mmol/L (FIGS. 2, 7), and 300 mmol/L (FIG. 2, 8).

(15) An aqueous solution containing 1×10.sup.8 mL.sup.−1 of Beauveria bassiana spores (a Beauveria bassiana solution) was taken as control, wherein the aqueous solution further comprised adjuvants of the concentrations identical to those in the above test group (the wetting agent butylnaphtalenesulfonic acid sodium salt, the dispersant sodium lignosulfonate, the disintegrant calcium chloride, the stabilizer disodium hydrogen phosphate, the binder sodium polyacrylate, and the carrier diatomite), and was named as a Beauveria bassiana group (FIG. 2, Beauveria bassiana).

(16) An aqueous solution containing 125 mmol/L of sodium chloride was taken as control, wherein the aqueous solution further comprised adjuvants of the concentrations identical to those in the above test group (the wetting agent butylnaphtalenesulfonic acid sodium salt, the dispersant sodium lignosulfonate, the disintegrant calcium chloride, the stabilizer disodium hydrogen phosphate, the binder sodium polyacrylate, and the carrier diatomite), and was named as a NaCl group (FIG. 2, NaCl).

(17) Sterile water was also taken as control (CK), wherein the water further comprised adjuvants of the concentrations identical to those in the above test group (the wetting agent butylnaphtalenesulfonic acid sodium salt, the dispersant sodium lignosulfonate, the disintegrant calcium chloride, the stabilizer disodium hydrogen phosphate, the binder sodium polyacrylate, and the carrier diatomite), and was named as a CK group (FIG. 2, CK).

(18) The Beauveria bassiana solution (the Beauveria bassiana group), the aqueous dispersions of Beauveria bassiana/sodium, chloride water dispersible granule formulations, the sodium chloride solution (the NaCl group), and the sterile water (CK) were respectively tested for virulence against healthy Solenopsis invicta worker ants, with 3 replicates for each treatment, 1 petri dish in each replicate, and 100 Solenopsis invicta worker ants in each petri dish. The Solenopsis invicta worker ants were firstly dipped in the solution for about 5 seconds and then taken out; after air dried indoors, they were placed in a breeding box, fed with fresh honey water every day, and kept at 26±1° C., a humidity of 70-90%, and a photoperiod of 14 L/10 hD; 96 hours after the treatment, survival of the Solenopsis invicta worker ants were observed to calculate the mortality. Data statistical analysis was performed using SPSS 13.0 software, significance testing between samples was performed using the Duncan's method, wherein different lowercase letters indicate a significant difference (P<0.05) in cumulative mortality of the treated Solenopsis invicta.

(19) Results were as shown in FIG. 2. As can be seen from FIG. 2, for the Solenopsis invicta treated with merely NaCl, no significant difference in the 96 h cumulative mortality was observed as compared with the blank control (Solenopsis invicta treated with sterile water), suggesting that the use of merely NaCl had no killing effect on Solenopsis invicta. For the Solenopsis invicta treated with merely Beauveria bassiana solution, significant differences in the 96 h cumulative mortality were observed as compared with the use of merely NaCl and the blank control, suggesting that the use of merely Beauveria bassiana exhibited a certain pesticidal effect, while the mortality was low. When sodium chloride was mixed in a concentration of 75 mmol/L, 100 mmol/L, or 125 mmol/L with Beauveria bassiana, significant differences in the 96 h cumulative mortality were observed as compared with the use of merely Beauveria bassiana and the use of merely NaCl, suggesting that the combination of sodium chloride with Beauveria bassiana in a certain ratio would significantly increase the pesticidal effect against Solenopsis invicta, and thereby demonstrating that the mixture exhibited a significant synergism in pesticidal effect as compared with the use of merely NaCl or Beauveria bassiana.

(20) In addition as can be seen from FIG. 2, the Beauveria bassiana/sodium chloride aqueous dispersions, which were formed of Beauveria bassiana, sodium chloride and adjuvants, exhibited a pesticidal effect similar to those of the Beauveria bassiana/sodium water solution without the adjuvants.

Embodiment 3

(21) In the laboratory virulence test, 1 g of Beauveria bassiana powder formulation and 7.5 g of sodium chloride were dissolved in 1 L of water and mixed well to give a Beauveria bassiana solution, wherein the Beauveria bassiana powder formulation contained 20 billion/g of Beauveria bassiana spores.

(22) The laboratory virulence test was conducted according to the method of embodiment 1, and results showed that the Beauveria bassiana solution of the instant embodiment exhibited a high killing effect on Solenopsis invicta.

Embodiment 4

(23) In the laboratory virulence test, 8 g of Beauveria bassiana powder formulation and 4.0 g of sodium chloride were dissolved in 1 L of water and mixed well to give a Beauveria bassiana solution, wherein the Beauveria bassiana powder formulation contained 20 billion/g of Beauveria bassiana spores.

(24) The laboratory virulence test was conducted according to the method of embodiment 1, and results showed that the Beauveria bassiana solution of the instant embodiment exhibited a high killing effect on Solenopsis invicta.

Embodiment 5

(25) As shown in FIG. 3 and FIG. 4, in the instant embodiment, the mixer of the preparation for preventing and controlling Solenopsis invicta comprised a liquid container 8, a solid container 4, a rubber gasket 3 and a lid 2; the solid container 4 is a container with an upper end having an opening and a lower end sealed with an aluminum foil composite film 5, and a retaining ring 9 is disposed at an edge of the opening of the solid container; a rubber washer 6 is provided at an opening 7 of the liquid container; the solid container is inserted into the opening 7 of the liquid container, so that the retaining ring 9 is pressed against the rubber washer 6, the rubber gasket 3 is pressed against the retaining ring 9, and the lid 2 covers the rubber gasket 3, the retaining ring 9 and the rubber washer 3 and is fixed to the opening 7 of the liquid container; a diversion hole 1 is disposed at a top of the lid.

(26) A totally 30 g of Beauveria bassiana powder formulation and sodium chloride (wherein a mass ratio of the Beauveria bassiana powder formulation to sodium chloride was 5:7.30, and the Beauveria bassiana powder formulation contained 20 billion Beauveria bassiana spores per gram), 8 g of wetting agent butylnaphtalenesulfonic acid sodium salt, 18 g of dispersant sodium lignosulfonate, 5 g of disintegrant calcium chloride, 2 g of stabilizer disodium hydrogen phosphate, 5 g of binder sodium polyacrylate, and 32 g of carrier diatomite, were weighed out and mixed according to a method for preparing a water dispersible granule formulation (1. The raw materials were mixed evenly according to the formula. 2. The materials were ground using a sand mill after mixed evenly, and subjected to spray granulation after determined to be acceptable. 3. The products were screened using a vibrating screen after the spray granulation, products with unacceptable appearance were returned to the sand mill to be ground again, and products with acceptable appearance were further subjected to quality inspection wherein products with unacceptable quality were also returned to the sand mill to be ground again. 4. Products with acceptable quality were packaged. 5. Packaged water dispersible granule formulation were put into storage), to eventually produce water dispersible granule formulations containing 30% by mass of Beauveria bassiana/sodium chloride.

(27) 41 g of the above water dispersible granule formulation containing 30% by mass of Beauveria bassiana/sodium chloride was placed in a solid container, and a liquid container was filled with 1 L of sterile water. A rubber washer was provided at an opening of the liquid container. Then the solid container was inserted into the liquid container, wherein the retaining ring was stuck at the rubber washer, the rubber gasket was pressed against the retaining ring, and the lid covered the rubber gasket, the retaining ring and the rubber washer and was fixed to the opening of the liquid container, With such configuration, the formulation was convenient for storage and transportation; when applied, it was not necessary to unscrew the lid, but instead, a needle of a syringe was inserted directly into the diversion hole at the top of the lid to pierce the aluminum foil composite film at the lower end of the solid container so that the water dispersible granule formulation for preventing and controlling Solenopsis invicta fell into the solution in the liquid container, and then the mixture was shaken well to give an aqueous dispersion of the water dispersible granule formulation. Distilled water was taken as control (CK).

(28) The above aqueous dispersion of the water dispersible granule formulation containing 30% by mass of Beauveria bassiana/sodium chloride was injected according to a method described in Embodiment 6 of the Chinese Patent 201510103303.0 with a title as AN INJECTION-IN-NEST METHOD FOR BIOLOGICAL PREVENTION AND CONTROL OF SOLENOPSIS INVICTA. Results were as shown in Table 1.

(29) TABLE-US-00001 TABLE 1 Results of injection-in-nest prevention and control of Solenopsis invicta 4 days 10 days 20 days 30 days Dosage Decreasing after after after after Treatment (ml/nest) rate (%) treatment treatment treatment treatment aqueous dispersion of 500 Decreasing rate of 60 66.67 80 93.33 water dispersible nests after granule formulation treatment (%) containing 30% by 500 Decreasing rate of 84.38 90.37 96.03 98.35 mass of Beauveria worker ants after bassiana/sodium treatment (%) chloride (injection method) Blank (CK) 500 Decreasing rate of 0 0 0 0 nests/worker ants (%)