CN110184197B - Beauveria bassiana oil suspending agent - Google Patents
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Abstract
The invention provides a beauveria bassiana oil suspending agent, and relates to the field of microbial pesticides. The beauveria bassiana oil suspending agent is obtained by adding beauveria bassiana spores into a mixture of a surfactant and solvent oil and uniformly mixing, wherein the concentration of the spores is 90-110 hundred million/mL, and the beauveria bassiana oil suspending agent also contains 5-10g/L trehalose, 2-8g/L glycerol and 0.2-1g/L xanthan gum. The beauveria bassiana oil suspending agent provided by the invention has the advantages that the heat resistance of beauveria bassiana spores is obviously improved, and the beauveria bassiana oil suspending agent can be used for preparation, preservation and field application of beauveria bassiana preparations.
Description
Technical Field
The invention relates to the field of microbial pesticides, and particularly relates to a beauveria bassiana oil suspending agent.
Background
Beauveria bassiana (Beauveria bassiana) belonging to Ascomycota, Hypocreales, Cordyceps and Beauveria genus. Beauveria bassiana is one of the insecticidal fungi widely used for biological control of pests at home and abroad, and is considered as one of the insect disease pathogenic fungi with the most development potential.
Beauveria bassiana is a broad-spectrum insecticidal fungus, and researchers at home and abroad use the Beauveria bassiana to control agricultural and forestry pests such as tea leafhoppers, corn borers, pine caterpillars, sugarcane borers, Chinese toons, grain elephants, citrus red spiders, aphids and the like. In particular to the biological control of tea leafhoppers, corn borers and pine caterpillars, which are used as conventional means in China for years. The beauveria bassiana can effectively control the number of insect population, does not harm other natural enemy insects and beneficial organisms, completely accords with the purpose of comprehensive treatment of pests, and has wide application prospect because the beauveria bassiana is easy to produce in large scale and has competitive prevention cost.
Conidia (conidiospore) are a common type of asexual spores in fungi, are extracellular, and are called exospores. Conidia are grown on differentiated conidiophores (conidiophores) or small peduncles with certain shapes, and some conidia of fungi are grown on the top of hyphae.
During the preparation, preservation and use processes of the beauveria bassiana spore preparation, the beauveria bassiana spore preparation can be thermally damaged, which can accelerate the degradation of effective components in the pesticide, slow down the drug effect, reduce the drug effect or cause the instability of the control effect. Heat comes from the environment during drying, storage during preparation, sun exposure or host activity during use (field application). Therefore, the improvement of the heat resistance of the beauveria bassiana spores is of great importance for improving the insecticidal rate and the biological control effect of the beauveria bassiana spores.
Disclosure of Invention
The invention aims to provide a beauveria bassiana oil suspending agent, which obviously improves the heat resistance of beauveria bassiana spores and can be used for preparation, preservation and field application of beauveria bassiana preparations.
The purpose of the invention is realized by adopting the following technical scheme.
The beauveria bassiana oil suspending agent is obtained by adding beauveria bassiana spores into a mixture of a surfactant and solvent oil and uniformly mixing, wherein the concentration of the spores is 90-110 hundred million/mL, and the beauveria bassiana oil suspending agent also contains 5-10g/L trehalose, 2-8g/L glycerol and 0.2-1g/L xanthan gum.
In the invention, the beauveria bassiana oil suspending agent also contains 0.5-2g/L of sodium diphenylethene biphenyl disulfonate and 0.5-2g/L of 2-hydroxy-4-methoxybenzophenone.
In the invention, the Beauveria bassiana is a Beauveria bassiana (Beauveria bassiana) ZD-1 strain, and the preservation number is CGMCC NO: 16500.
in the invention, the surfactant is one or more than two of polyglycerol ricinoleate, fatty alcohol-polyoxyethylene ether, alkylphenol ethoxylates and tween.
In a preferred technical scheme, the surfactant is polyglycerol polyricinoleate.
In the invention, the solvent oil is one or a mixture of more than two of soybean oil, rapeseed oil, cottonseed oil, palm oil, sesame oil, corn oil and polymerized linseed oil.
In a preferred technical scheme, the solvent oil is polymerized linseed oil.
In the present invention, the volume ratio of the surfactant to the solvent oil is 6: 94.
the beauveria bassiana oil suspending agent provided by the invention has the advantages that the trehalose, the glycerol and the xanthan gum are added, so that the heat resistance of beauveria bassiana spores is obviously improved. After the stilbene biphenyl disulfonic acid sodium and the 2-hydroxy-4-methoxy benzophenone are added into the beauveria bassiana oil suspending agent, the ultraviolet resistance of beauveria bassiana spores is obviously improved, and the beauveria bassiana oil suspending agent is favorable for preventing the beauveria bassiana from being damaged by ultraviolet rays when used in fields. The oil suspending agent disclosed by the invention reduces adverse effects of high temperature and ultraviolet rays on beauveria bassiana in preparation, preservation and field use processes of spores, and is beneficial to improving the survival rate of the beauveria bassiana spores, prolonging the shelf life and improving the field use effect of products. The beauveria bassiana oil suspending agent can be used for preventing and treating tea leafhoppers.
Detailed Description
The solvent used in the medium of the present invention is water. Gas-liquid ratio is the ventilation per minute (m)3) Volume of fermentation broth (m)3) The ratio of. The ventilation volume is the ventilation volume per minute (m)3) Volume of solid fermentation medium (m)3) The ratio of.
Example 1 obtaining of spore powder of Beauveria bassiana ZD-1
This example describes the obtention of a spore powder of Beauveria bassiana ZD-1.
1. Screening and identification of beauveria bassiana strain
(1) Screening of Beauveria bassiana strains
This example describes the screening of beauveria bassiana ZD-1 strain, using tea leafhoppers as test subjects.
Test strains: XY-201805 strain, YN-201803 strain, ZD-1 strain, KM-4 strain, ZA-7-3 strain, B3 strain, B822 strain, PE-55 strain, PE-23 strain.
A test insect source: collecting lesser leafhoppers on a tea base of agricultural science research institute in Yunnan province from late 4 th to late 4 th in 2018. Putting the picked lesser leafhopper into a wide-mouth bottle, and feeding the lesser leafhopper indoors by using fresh tea tree branches with tender leaves and buds.
Inoculating each test strain on Sasa medium SDAY (Sasa medium)SDAY formulation: 4% glucose, 1% peptone, 1% yeast powder, 2% agar), and culturing at 25 + -1 deg.C for 8 days to obtain spores of each strain. The spores were washed with sterile water containing 0.05% Tween80 and formulated into 1.0X 107Spore suspension of one/mL for use.
And selecting the lesser leafhoppers with the same individual size, and inspecting the pathogenicity of the spore suspension of each strain to the lesser leafhoppers. The specific method comprises the following steps: soaking the lesser leafhopper in the spore suspension for 10s, taking out, placing on filter paper to absorb excessive water, and transferring into an artificial feeding box. 50 test insects were treated with each spore suspension and repeated 3 times. Sterile water soaked with 0.05% Tween80 was used as a control and fed normally under the same conditions. In the feeding process, dead insects are transferred into a sterile culture dish for culture in time. Counting the death number on the 6 th day, the 9 th day and the 12 th day after the treatment of the lesser leafhopper, and effectively killing hypha or spore powder growing on the dead body. Specific results are shown in table 1.
TABLE 1 cumulative mortality of Empoasca vitis after treatment with different Beauveria bassiana strains
As can be seen from the data in Table 1, various spores of Beauveria bassiana have different degrees of pathogenicity on the lesser leafhoppers. But ZD-1 strain has the best insecticidal effect, and the accumulated mortality rate can reach 95.33% on day 12.
(2) Identification
The ZD-1 strain grows slowly on a potato glucose agar medium (PDA), is cultured for 14 days at 28 ℃ in the dark, has a colony diameter of 58-61 mm, is white at the initial stage, gradually becomes light yellow, is fluffy to flocculent, and has concentric ring lines and radial lines; the back of the colony is beige to light yellow brown, and has no water-soluble pigment.
ZD-1 strain vegetative hypha is thin-walled, transparent and smooth, is commonly branched, and has a width of 0.7-2.0 μm, conidiophores are directly planted on the vegetative hypha, conidiophores cluster is planted on the conidiophores, and is nearly spherical to elliptical, and single spore, thin-walled, transparent and smooth, and has a diameter of 23.5-43.2 multiplied by 2.2-4.3 μm.
The rRNA gene sequence of the ZD-1 strain is shown in SEQ ID NO:1, and the ZD-1 strain is Beauveria bassiana through sequence comparison, is named as Beauveria bassiana (Beauveria bassiana) ZD-1 strain, and is abbreviated as Beauveria bassiana ZD-1 strain.
Beauveria bassiana (Beauveria bassiana) ZD-1 strain which is preserved.
The classification is named as: beauveria bassiana (balsamo) Vuillemin
Beauveria bassiana。
According to the biological materials (strains): ZD-1.
The preservation date is 11 months and 19 days in 2018.
The preservation unit is called China general microbiological culture Collection center (CGMCC) for short.
The address of the depository: xilu No.1, Beijing, Chaoyang, Beijing, and institute for microbiology, China academy of sciences.
The preservation number is: CGMCC NO. 16500.
2. Culture of Beauveria bassiana ZD-1 strain
(1) Beauveria bassiana ZD-1 strain spore activation
The strain ZD-1 slant seeds of beauveria bassiana are inoculated on a Sasa medium SDAY (4% glucose, 1% peptone, 1% yeast powder and 2% agar), and cultured for 8 days at 25 +/-1 ℃ to obtain the activated spores of the beauveria bassiana ZD-1 strain.
(2) Preparation of Beauveria bassiana ZD-1 strain first-order seed liquid
The above activated beauveria bassiana ZD-1 strain spores were inoculated into a 250mL Erlenmeyer flask containing 50mL of a liquid SDAY medium so that the spore concentration was 3X 106one/mL. Culturing at 25 + -1 deg.C and 150rpm under shaking for 36 hr to obtain spore with spore content of 30 × 108One seed/mL of the primary seed solution.
Wherein the liquid Sasa culture medium SDAY contains 40g/L glucose, 10g/L peptone and 10g/L yeast powder.
(3) Preparation of Beauveria bassiana ZD-1 strain secondary seed liquid
In a 100L fermenter, a medium was prepared containing the following components: starch10g/L, 15g/L glucose, 5g/L peptone, 10g/L yeast powder, 0.2g/L potassium dihydrogen phosphate and 0.25g/L magnesium sulfate. The solvent is water. Sterilizing at 121 deg.C for 30min, cooling to room temperature, inoculating the primary seed solution obtained in title 2 of this example at an inoculum size of 2.5%, culturing at 25 + -1 deg.C and 200rpm at a gas-liquid ratio of 1:1 for 36h to obtain secondary seed solution with spore content of 1.3 × 109one/mL.
(4) Beauveria bassiana ZD-1 strain three-stage liquid fermentation culture
And respectively inoculating the second-stage seed liquid of the beauveria bassiana ZD-1 strain into a 3000L fermentation tank according to the inoculation amount of 1% (V/V) for culturing to obtain a third-stage seed liquid. The culture medium and culture conditions used in the fermenter were as follows.
The culture medium contains the following components: 20g/L of rice flour, 10g/L of glucose, 5g/L of sucrose, 5g/L of soybean cake powder, 5g/L of silkworm chrysalis powder, 7.5g/L of yeast powder, 0.25g/L of monopotassium phosphate, 0.2g/L of magnesium sulfate, 0.3g/L of calcium chloride and pH 7.0. The solvent is water.
The culture conditions are as follows: the stirring speed of the fermentation tank is 150rpm, and the aeration and the temperature during the culture process are controlled as follows: 0 h-8 h: the gas-liquid ratio is 0.5:1, and the temperature is 28 ℃; 8 h-24 h: the gas-liquid ratio is 0.8:1, and the temperature is 25 ℃; 24 h-48 h: the gas-liquid ratio is 1:1 and the temperature is 25 ℃. Culturing for 44h until the spore content of the third-level seed liquid reaches 885 × 108one/mL.
(5) And (5) solid fermentation culture.
Adding water into the solid culture medium, mixing, sterilizing at high temperature under high pressure, flatly spreading in a clean room tray, wherein the thickness of the solid culture medium is 7cm, and cooling to room temperature. Uniformly inoculating the third-level seed liquid of the beauveria bassiana ZD-1 strain into a shallow tray for further fermentation culture.
Culture medium: weighing 30 parts of rice, 10 parts of corn flour, 1 part of glucose, 27 parts of rice husk, 18 parts of wheat bran, 8.5 parts of silkworm chrysalis meal, 5.5 parts of bean cake meal and 150 parts of water according to the mass parts, and uniformly mixing.
The culture conditions are as follows: the inoculum size was 15%, and the post-inoculation culture conditions were controlled as follows: and (3) 0-2 days: the ventilation volume is 0.3:1, the temperature is 27.5-28.5 ℃, and the humidity is 65%; 3-7 days: the ventilation volume is 0.5:1, the temperature is 24.5-25.5 ℃, and the humidity is 60%; 8-9 days: the ventilation volume is 0.5:1, the temperature is 28-29 ℃, and the humidity is 45%.
When the fermentation time is 216h, the spore content reaches 1150 × 108The germination rate of the spores is not less than 90 percent per gram. Drying the fermentation culture, and extracting spores by using a cyclone separation spore collector to obtain beauveria bassiana spore powder.
Example 2 thermal protectant of Beauveria bassiana spores
1. Determination of Heat resistance
The beauveria bassiana spore powder prepared in the example 1 is prepared into 5 multiplied by 10 by normal saline8Spore suspension per mL served as control.
The controls were treated separately as follows and the effect of each substance on the heat resistance of beauveria bassiana spores was examined.
Treatment 1: at 5X 108Trehalose was added to the spore suspension at a concentration of 8 g/L.
And (3) treatment 2: at 5X 108Glycerol was added to the spore suspension at a final concentration of 5 g/L.
And (3) treatment: at 5X 108Xanthan gum was added to the spore suspension at a final concentration of 0.5 g/L.
And (4) treatment: at 5X 108Trehalose and glycerol were added to the spore suspension so that the final concentration of trehalose was 8g/L and the final concentration of glycerol was 5 g/L.
And (4) treatment 5: at 5X 108Xanthan gum and glycerol were added to the spore suspension so that the final concentration of xanthan gum was 0.5g/L and the final concentration of glycerol was 5 g/L.
And (6) treatment: at 5X 108Xanthan gum and trehalose were added to the spore suspension per mL so that the final concentration of xanthan gum was 0.5g/L and the final concentration of trehalose was 8 g/L.
And (7) treatment: at 5X 108Trehalose, glycerol and xanthan gum are added into each/mL spore suspension to ensure that the final concentration of the trehalose is 8g/L, the final concentration of the glycerol is 5g/L and the final concentration of the xanthan gum is 0.5 g/L.
Samples were taken from control, treatment 1, treatment 2 and treatment 3, respectively, and their spore germination rates were determined. The germination rate was determined after culturing the samples at 25. + -. 1 ℃ for 24 hours using the Sasa medium SDAY, and the spores were considered to have germinated when the length of the germ tube was not less than the diameter of the spores. Three replicates of each sample were used and the results are shown in table 1 below.
TABLE 1 Germination ratio (%)
| Control | Process 1 | Treatment 2 | Treatment 3 | |
| Repetition of 1 | 93.4 | 92.5 | 93.3 | 92.6 |
| Repetition 2 | 94.1 | 93.1 | 92.9 | 93.5 |
| Repetition of 3 | 93.8 | 92.8 | 93.7 | 92.1 |
| Average germination rate | 93.77 | 92.80 | 93.30 | 92.73 |
The data results in table 1 show that: trehalose, glycerol and xanthan gum have no obvious effect on promoting germination of beauveria bassiana spores.
Respectively sampling 5mL of the samples from the comparison and treatment 1-7 into sterile test tubes, placing the samples in a constant-temperature water bath kettle at 50 ℃ for treatment for 30min (simultaneously shaking), immediately taking out the samples, and placing the samples in an ice-water mixture for ice-bath cooling to room temperature; the cooled mixture was transferred to a 250mL Erlenmeyer flask, and 45mL of liquid Sasa's medium SDAY was added thereto, and after mixing, the mixture was cultured at 25. + -. 1 ℃ for 24 hours, and then the germination rate was measured, and the results are shown in Table 2.
TABLE 2 germination percentage (%)
| Control | Process 1 | Treatment of2 | Treatment 3 | Treatment 4 | Treatment 5 | Treatment 6 | Treatment 7 | |
| Repetition of 1 | 3.5 | 23.5 | 24.5 | 26.8 | 36.8 | 38.8 | 39.5 | 79.5 |
| Repetition 2 | 3.7 | 22.9 | 25.2 | 27.3 | 36.2 | 38.2 | 39.1 | 79.8 |
| Repetition of 3 | 4.2 | 23.1 | 24.8 | 27.1 | 37.5 | 37.9 | 38.8 | 80.3 |
| Average germination rate | 3.8 | 23.17 | 24.83 | 27.07 | 36.83 | 38.30 | 39.13 | 79.87 |
The data results in table 2 show that: the germination rate of the treated spores 7 is 21.02 times of that of the control, when trehalose, glycerol and xanthan gum coexist, the germination rate of the thermally treated spores is obviously improved, and the thermal protection effect is obvious.
(2) Effect of trehalose, Glycerol and Xanthan Gum concentration on Heat resistance of Beauveria bassiana spores
The beauveria bassiana spore powder prepared in the example 1 is prepared by adopting normal saline with the concentration of 5 multiplied by 108Spore suspension per mL.
Comparison: 5X 108Spore suspension per mL.
And (7) treatment: 5X 108Trehalose, glycerol and xanthan gum are added into each/mL spore suspension to ensure that the final concentration of the trehalose is 8g/L, the final concentration of the glycerol is 5g/L and the final concentration of the xanthan gum is 0.5 g/L.
And (4) treatment 8: 5X 108Trehalose, glycerol and xanthan gum are added into each/mL spore suspension to ensure that the final concentration of the trehalose is 5g/L, the final concentration of the glycerol is 2g/L and the final concentration of the xanthan gum is 0.2 g/L.
And (4) treatment: 5X 108Trehalose, glycerol and xanthan gum are added into each/mL spore suspension to ensure that the final concentration of the trehalose is 10g/L, the final concentration of the glycerol is 8g/L and the final concentration of the xanthan gum is 1 g/L.
Sampling 5mL of the control and treatment 7-9 samples in sterile test tubes respectively, placing the samples in a constant-temperature water bath kettle at 50 ℃, taking out the samples immediately after water bath for 30min (simultaneous oscillation), and placing the samples in an ice-water mixture for ice bath cooling to room temperature; the cooled mixture was transferred to a 250mL Erlenmeyer flask, and 45mL of liquid Sasa's medium SDAY was added thereto, and after mixing, the mixture was cultured at 25. + -. 1 ℃ for 24 hours, and then the germination rate was measured, and the results are shown in Table 3.
TABLE 3 determination of germination percentage (%)
| Control | Treatment 7 | Treatment 8 | Process 9 | |
| Repetition of 1 | 3.6 | 78.3 | 70.4 | 72.4 |
| Repetition 2 | 3.5 | 78.2 | 69.8 | 71.9 |
| Repetition of 3 | 4.0 | 77.8 | 69.1 | 72.2 |
| Average germination rate | 3.7 | 78.1 | 69.8 | 72.2 |
The data results in table 3 show that: the trehalose concentration is 5-10g/L, the glycerol concentration is 2-8g/L, and the xanthan gum concentration is 0.2-1g/L, so that the germination rate of the spores after heat treatment is obviously improved. Treatment 7 gave the best protection, with a spore germination rate of 21.11 times that of the control.
Example 3 determination of the anti-ultraviolet Capacity of Beauveria bassiana spores
(1) Influence of various substances on anti-ultraviolet capability of beauveria bassiana spores
The beauveria bassiana spore powder prepared in the example 1 is prepared into 5 multiplied by 10 concentration by normal saline8Spore suspension per mL.
Comparison: 5X 108Spore suspension per mL.
And (4) treatment 10: at 5X 108To each mL of spore suspension was added sodium diphenylethylene biphenyl disulfonate (fluorescent whitening agent) to give a final fluorescent whitening agent concentration of 1 g/L.
And (3) treatment 11: at 5X 1082-hydroxy-4-methoxybenzophenone was added to the spore suspension per mL to give a final concentration of 1g/L of 2-hydroxy-4-methoxybenzophenone.
And (4) treatment 12: at 5X 108The spore suspension of each mL is added with sodium diphenylethylene biphenyl disulfonate (fluorescent whitening agent) and 2-hydroxy-4-methoxybenzophenone, so that the final concentration of the sodium diphenylethylene biphenyl disulfonate and the final concentration of the 2-hydroxy-4-methoxybenzophenone are both 1 g/L.
Samples were taken from control and treatment 10-12 and germination was determined after 24h incubation at 25 + -1 deg.C using Sasa medium SDAY, and the results are shown in Table 4 for three replicates per sample.
TABLE 4 germination Rate (%) of each sample
| Control | Process 10 | Process 11 | Treatment 12 | |
| Repetition of 1 | 93.5 | 92.9 | 94.2 | 93.6 |
| Repetition 2 | 93.8 | 93 | 93.7 | 93.1 |
| Repetition of 3 | 93.1 | 92.1 | 94.5 | 92.8 |
| Average germination rate | 93.5 | 92.7 | 94.1 | 93.2 |
The data in table 4 show that: the diphenyl ethylene biphenyl sodium disulfonate and the 2-hydroxy-4-methoxy benzophenone have no obvious promotion effect on the germination of beauveria bassiana spores.
10mL of the sample from the control and treatment 10-12 was placed in a sterile petri dish with a diameter of 9cm and mixed thoroughly for use. An ultraviolet lamp (integrated TB fluorescent lamp with excessive light, 15W, 0.65 power factor and 50-60 Hz) is respectively arranged above two ends in the super clean bench. Opening the ultraviolet lamp tube for 5min, opening the culture dish containing the sample into a super clean bench after the ultraviolet lamp tube is stabilized, irradiating the culture dish at a position 35cm below the ultraviolet lamp for 5min, and turning off the ultraviolet lamp. The treated liquid was mixed well, 5mL was taken in a 250mL triangular flask, and 45mL of liquid Sasa's medium SDAY was added thereto, and after mixing well, the germination rate was measured after culturing at 25. + -. 1 ℃ for 24 hours, and the results are shown in Table 5.
TABLE 5 Germination ratio (%)
| Control | Process 10 | Process 11 | Treatment 12 | |
| Repetition of 1 | 2.1 | 45.6 | 46.4 | 65.4 |
| Repetition 2 | 2.7 | 44.8 | 46.1 | 64.9 |
| Repetition of 3 | 2.3 | 45.1 | 45.9 | 65.1 |
| Average germination rate | 2.4 | 45.2 | 46.1 | 65.1 |
The data results in table 5 show that: the diphenyl ethylene biphenyl sodium disulfonate, the 2-hydroxy-4-methoxy benzophenone and the mixture thereof have the ultraviolet protection effect on the beauveria bassiana spores, the germination rate of the treated 12 is 27.13 times of that of the contrast, and the ultraviolet protection effect is obvious.
(2) Influence of concentration of sodium diphenylethene biphenyl disulfonate (fluorescent whitening agent) and 2-hydroxy-4-methoxybenzophenone on anti-ultraviolet capability of beauveria bassiana spores
The beauveria bassiana spore powder prepared in the example 1 is prepared into a concentration of 5X 10 by using normal saline8Spore suspension per mL.
Comparison: 5X 108Spore suspension per mL.
And (4) treatment 12: at 5X 108In each mL spore suspension, sodium diphenylethylene biphenyl disulfonate (fluorescent whitening agent) and 2-hydroxy-4-methoxybenzophenone were added to give final concentrations of 1 g/L.
And (4) treatment: 5X 108To each mL of spore suspension, sodium diphenylethylene biphenyl disulfonate (fluorescent whitening agent) and 2-hydroxy-4-methoxybenzophenone were added to give final concentrations of 0.5 g/L.
And (4) processing: 5X 108In each mL spore suspension, sodium diphenylethylene biphenyl disulfonate (fluorescent whitening agent) and 2-hydroxy-4-methoxybenzophenone were added to give final concentrations of 2 g/L.
10mL of the sample from control and treatment 12-14, respectively, was placed in a sterile petri dish with a diameter of 9cm and mixed well. An ultraviolet lamp (integrated TB fluorescent lamp with excessive light, 15W, 0.65 power factor and 50-60 Hz) is respectively arranged above two ends in the super clean bench. Opening the ultraviolet lamp tube for 5min, opening the culture dish containing the sample into a super clean bench after the ultraviolet lamp tube is stabilized, irradiating the culture dish 35cm below the ultraviolet lamp for 5min, and then closing the ultraviolet lamp.
The treated liquid was mixed well, 5mL was taken in a 250mL triangular flask, and 45mL of liquid Sasa's medium SDAY was added thereto, and after mixing well, the germination rate was measured after culturing at 25. + -. 1 ℃ for 24 hours, and the results are shown in Table 6.
TABLE 6 Germination ratio (%)
| Control | Treatment 12 | Treatment 13 | Process 14 | |
| Repetition of 1 | 3.1 | 65.9 | 58.7 | 64.8 |
| Repetition 2 | 2.7 | 65.2 | 58.1 | 64.2 |
| Repetition of 3 | 2.5 | 66 | 57.9 | 63.8 |
| Average germination rate | 2.8 | 65.7 | 58.2 | 64.3 |
The data results in table 6 show that: the concentration of the diphenyl ethylene sodium biphenyl disulfonate (fluorescent whitening agent) and the concentration of the 2-hydroxy-4-methoxy benzophenone are changed, and the ultraviolet protection effect on the beauveria bassiana spores is achieved. The UV protection effect of treatment 12 was more pronounced, with a spore germination rate 23.46 times that of the control.
Example 4 determination of Heat and UV resistance of Beauveria bassiana oil suspending agent
(1) Determination of heat resistance and ultraviolet resistance under constant temperature water bath and ultraviolet lamp irradiation
The following samples and treatments were set up:
control agents: the beauveria bassiana spore powder prepared in the example 1 is directly added into a mixture of polyglycerol polyricinoleate (surfactant) and polymerized linseed oil (solvent oil), and the mixture is uniformly mixed to obtain the beauveria bassiana oil suspending agent, wherein the concentration of spores is 100 hundred million/mL. Wherein the volume ratio of the polyglycerol polyricinoleate to the polymerized linseed oil is 6: 94.
treating the medicament: mixing polyglycerol polyricinoleate and polymerized linseed oil according to the volume ratio of 6: 94, mixing uniformly, adding spores with a final concentration of 100 hundred million/mL, and simultaneously adding the following protective agents: 8g/L of trehalose, 5g/L of glycerol, 0.5g/L of xanthan gum, 1g/L of sodium diphenylethylene biphenyl disulfonate (fluorescent whitening agent) and 1g/L of 2-hydroxy-4-methoxybenzophenone, wherein the concentrations are final concentrations.
And respectively carrying out heat treatment, ultraviolet treatment and ultraviolet treatment after heat treatment on the control medicament and the treatment medicament, and detecting the germination rate of the treated sample.
Heat treatment and germination rate determination: respectively sampling 5mL of each sample, placing the samples in sterile test tubes, placing the samples in a constant-temperature water bath kettle at 50 ℃, taking out the samples immediately after water bath for 30min (simultaneous oscillation), and placing the samples in an ice-water mixture for ice bath cooling to room temperature; transferring the cooled mixed solution into a 250mL triangular flask, adding 45mL liquid Sasa's medium SDAY, uniformly mixing, culturing at 25 +/-1 ℃ for 24h, and determining the germination rate.
Ultraviolet treatment and germination rate determination: 10mL of each sample was placed in a sterile petri dish with a diameter of 9cm, and mixed well. And turning off the ultraviolet lamp after the ultraviolet lamp irradiates for 5 min. And (3) uniformly mixing the treated liquid, putting 5mL of the mixed liquid into a 250mL triangular flask, adding 45mL of liquid Sasa culture medium SDAY, uniformly mixing, culturing at 25 +/-1 ℃ for 24 hours, and determining the germination rate.
Carrying out ultraviolet treatment and germination rate determination after heat treatment: respectively sampling 10mL of each sample, placing the samples in a sterile test tube, placing the samples in a constant-temperature water bath kettle at 50 ℃, taking out the samples immediately after water bath for 30min (simultaneous oscillation), and placing the samples in an ice-water mixture for ice bath cooling to room temperature; and transferring the cooled mixed solution into a sterile culture dish with the diameter of 9cm, and fully and uniformly mixing. And turning off the ultraviolet lamp after the ultraviolet lamp irradiates for 5 min. And (3) uniformly mixing the treated liquid, putting 5mL of the mixed liquid into a 250mL triangular flask, adding 45mL of liquid Sasa culture medium SDAY, uniformly mixing, culturing at 25 +/-1 ℃ for 24 hours, and determining the germination rate.
The specific results are shown in Table 7.
TABLE 7 germination rates for different treatments with different agents
As can be seen from the data in Table 7, the spore germination rate of the treatment agent added with the protective agent is 24.19 times that of the control agent when the treatment agent is subjected to single heat treatment; during single ultraviolet treatment, the spore germination rate of the treatment agent added with the protective agent is 23.54 times that of the control agent; after heat treatment, ultraviolet treatment is carried out, and the spore germination rate of the treatment medicament added with the protective agent is 32.84 times that of the control medicament. Therefore, the addition of trehalose, glycerol, xanthan gum, sodium diphenylethylene biphenyl disulfonate and 2-hydroxy-4-methoxybenzophenone obviously improves the heat resistance and ultraviolet resistance of the spores.
(2) Determination of heat and UV resistance of spore suspension preserved at room temperature for one year
The following 2 samples and treatments were set up:
control agents: mixing polyglycerol polyricinoleate and polymerized linseed oil according to the volume ratio of 6: 94, mixing evenly, and adding beauveria bassiana spores with the final concentration of 100 hundred million/mL.
Treating the medicament: mixing polyglycerol polyricinoleate and polymerized linseed oil according to the volume ratio of 6: 94, mixing uniformly, adding beauveria bassiana spores with the final concentration of 100 hundred million/mL, and simultaneously adding the following protective agents: 8g/L of trehalose, 5g/L of glycerol, 0.5g/L of xanthan gum, 1g/L of sodium diphenylethylene biphenyl disulfonate and 1g/L of 2-hydroxy-4-methoxybenzophenone, wherein the concentrations are final concentrations.
The control and treatment agents were left in a dry environment at room temperature (window south facing, no shade) for 1 year, and at the beginning of the placement (initial sample), samples were taken for 3 months, 6 months, and 12 months to determine the spore germination rate. The results are shown in Table 8.
TABLE 8 Germination Rate of samples during one year of storage at room temperature
Note: in table 8, "control" is the control agent and "treatment" is the treatment agent.
As can be seen from the data in Table 8, when 100 hundred million/mL beauveria bassiana oil suspending agent is placed at room temperature for one year, the spore germination rate of the treatment agent added with the protective agent is 2.03 times that of the control agent, so that the addition of trehalose, glycerol, xanthan gum, sodium diphenylethylene biphenyl disulfonate and 2-hydroxy-4-methoxybenzophenone obviously improves the heat resistance and the ultraviolet resistance of the beauveria bassiana spores.
Example 5 prevention and control of tea leafhoppers by Beauveria bassiana oil suspending agent in the field
The prevention and control effect of the beauveria bassiana oil suspending agent on tea leafhoppers is investigated in the field. Test site: tea garden test field of tea research institute of agricultural institute of Yunnan province. The garden is planted in single row, and the planting density is 3500 plants/mu.
And (3) experimental treatment design: blank control; control 1 is the control agent prepared in example 4 of the present invention (no heat and uv resistant protectant added); control 2 is the treatment agent prepared in example 4 of the present invention (with the addition of heat and uv resistant protective agents).
The use amount is as follows: 3 treatments (blank, control 1 and control 2) were set, each treatment was repeated 3 times for a total of 9 plots, each about 20 square meters, and randomly grouped, with the tea plants around each plot being the protection row. The medicine is applied once in 2018, 6 and 5 days, the application rates of the control 1 and the control 2 are both 100 mL/mu, and clear water is used for replacing the medicine in the blank control.
The application method comprises the following steps: the hand sprayer sprays uniformly, the main medicine application parts are tea tree fluffy surface and front and back surfaces of two side leaves, and the liquid medicine is in full contact with the lesser leafhopper bodies.
The test was conducted 3 times of investigation, 1 day, 7 days, 14 days after the drug administration, and 100 bud leaves per plot were investigated, and the survival number of tea leafhoppers was recorded. The results of the investigation are shown in Table 9 below.
TABLE 9 field test results of the efficacy of tea leafhoppers
As can be seen from Table 9, the Beauveria bassiana oil suspending agent has obvious control effect on tea leafhoppers in the field whether a protective agent is added or not. The control effect of the treatment medicament added with the protective agent is obviously higher than that of a control medicament without the protective agent.
The beauveria bassiana oil suspending agent with 100 hundred million spores/mL is a novel microbial pesticide which is safe, nontoxic and free of residue. The heat-resistant and ultraviolet-resistant protective agent is added, so that the heat resistance and ultraviolet resistance of the beauveria bassiana can be obviously improved, the adverse effects of high temperature and ultraviolet rays on the beauveria bassiana in the processes of preparation, preservation and field use of spores are reduced, the survival rate of the beauveria bassiana spores is improved, the shelf life is prolonged, and the field use effect of products is improved.
The above is only a preferred embodiment of the present invention, and it should be noted that the above preferred embodiment should not be considered as limiting the present invention, and the protection scope of the present invention should be subject to the scope defined by the claims. It will be apparent to those skilled in the art that various modifications and adaptations can be made without departing from the spirit and scope of the invention, and these modifications and adaptations should be considered within the scope of the invention.
Sequence listing
<110> Yaozha biological products of Arisaema cum bile Ltd
<120> Beauveria bassiana oil suspending agent
<130>20190507
<141>2019-05-15
<160>1
<170>SIPOSequenceListing 1.0
<210>1
<211>457
<212>DNA
<213> Beauveria bassiana (Beauveria bassiana ZD-1 strain)
<400>1
gaggtcaacg ttcagaagtt gggtgtttta cggcgtggcc gcgtcggggt tccggtgcga 60
gctgtattac tgcgcagagg tcgccgcgga cgggccgcca ctccatttca gggccggcgg 120
tgtgctgccg gtccccaacg ccgacctccc caaggggagg tcgagggttg aaatgacgct 180
cgaacaggca tgcccgccag aatgctggcg ggcgcaatgt gcgttcaaag attcgatgat 240
tcactggatt ctgcaattca cattacttat cgcgtttcgc tgcgttcttc atcgatgcca 300
gagccaagag atccgttgtt gaaagttttg attcatttgt tttgccttgc ggcgtattca 360
gaagatgctg gaatacaaga gtttgaggtc cccggcgggc cgctggtcca gtccgcgtcc 420
gggatccctc cgctggttca ccaacggaga ccttgtt 457
Claims (7)
1. The beauveria bassiana oil suspending agent is characterized in that the beauveria bassiana oil suspending agent is obtained by adding beauveria bassiana spores into a mixture of a surfactant and solvent oil and uniformly mixing, the concentration of the spores is 90-110 hundred million/mL, and the beauveria bassiana oil suspending agent also contains 5-10g/L trehalose, 2-8g/L glycerol and 0.2-1g/L xanthan gum; the beauveria bassiana is beauveria bassiana (Beauveria bassiana) ZD-1 strain, the preservation number is CGMCC NO: 16500.
2. the beauveria bassiana oil suspending agent of claim 1, wherein the beauveria bassiana oil suspending agent further comprises 0.5-2g/L of sodium diphenylethylene biphenyl disulfonate and 0.5-2g/L of 2-hydroxy-4-methoxybenzophenone.
3. The beauveria bassiana oil suspending agent as claimed in claim 2, wherein the surfactant is one or a mixture of more than two of polyglycerol ricinoleate, fatty alcohol-polyoxyethylene ether, alkylphenol polyoxyethylene ether and tween.
4. The beauveria bassiana oil suspension concentrate of claim 3, wherein the surfactant is polyglycerol polyricinoleate.
5. The beauveria bassiana oil suspension as set forth in claim 4, wherein the solvent oil is one or a mixture of two or more of soybean oil, rapeseed oil, cottonseed oil, palm oil, sesame oil, corn oil and polymerized linseed oil.
6. The beauveria bassiana oil suspension concentrate of claim 5, wherein the mineral spirits are polymerized linseed oil.
7. The beauveria bassiana oil suspending agent according to claim 6, wherein the volume ratio of the surfactant to the solvent oil is 6: 94.
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| CN102172250A (en) * | 2011-03-24 | 2011-09-07 | 中国农业科学院农业环境与可持续发展研究所 | Preparation method and application of eauveria-spinosad suspending agent |
| CN102321543A (en) * | 2011-08-30 | 2012-01-18 | 江南大学 | Carbohydrate protective agent for increasing heat resistance of beauveria bassiana spore |
| CN104642393A (en) * | 2015-01-29 | 2015-05-27 | 江西省新龙生物科技有限公司 | Insect virus and beauveria bassiana mixing insecticide suspending agent and preparation method thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102172250A (en) * | 2011-03-24 | 2011-09-07 | 中国农业科学院农业环境与可持续发展研究所 | Preparation method and application of eauveria-spinosad suspending agent |
| CN102321543A (en) * | 2011-08-30 | 2012-01-18 | 江南大学 | Carbohydrate protective agent for increasing heat resistance of beauveria bassiana spore |
| CN104642393A (en) * | 2015-01-29 | 2015-05-27 | 江西省新龙生物科技有限公司 | Insect virus and beauveria bassiana mixing insecticide suspending agent and preparation method thereof |
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| Title |
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| 假眼小绿叶蝉白僵菌优良菌株筛选及其应用研究;蔡国贵;《江西农业大学学报》;20050831;第27卷(第04期);摘要 * |
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