CN110184197A - A kind of beauveria bassiana oil-suspending agent - Google Patents
A kind of beauveria bassiana oil-suspending agent Download PDFInfo
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- CN110184197A CN110184197A CN201910404380.8A CN201910404380A CN110184197A CN 110184197 A CN110184197 A CN 110184197A CN 201910404380 A CN201910404380 A CN 201910404380A CN 110184197 A CN110184197 A CN 110184197A
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Abstract
The present invention provides a kind of beauveria bassiana oil-suspending agent, is related to microbial pesticide field.The beauveria bassiana oil-suspending agent, the beauveria bassiana oil-suspending agent is that beauveria bassiana spore is added in the mixture of surfactant and solvent naphtha to obtain after mixing, spore concentration is hundred million/mL of 90-110, the beauveria bassiana oil-suspending agent also xanthan gum containing 5-10g/L trehalose, the glycerol of 2-8g/L and 0.2-1g/L.Beauveria bassiana oil-suspending agent of the present invention, significantly improves the heat resistance of beauveria bassiana spore, can be used for the preparation, preservation and Field information of beauveria bassiana preparation.
Description
Technical field
The present invention relates to microbial pesticide fields, more particularly to a kind of beauveria bassiana oil-suspending agent.
Background technique
Beauveria bassiana belongs to Ascomycota, Hypocreales, cordyceps sinensis section, Beauveria.Beauveria bassiana is domestic and international
It is widely used in one of the disinsection fungal of biological control of insect pests, it is considered to be a kind of most insect pathogenic fungus of potentiality to be exploited.
Beauveria bassiana is broad spectrum insecticide fungi, and researchers at home and abroad are small green using beauveria bassiana prevention and treatment tealeaves
The agriculture and forestry injurious insects such as leafhopper, corn borer, pine moth, small sugarcane borer, blind Chinese toon, grain weevil, citrus red mite and aphid.Especially to tealeaves
The biological control of smaller green leaf hopper, corn borer, pine moth, has been used as conventional means to use year after year at home.Due to beauveria bassiana
Population number can be efficiently controlled, while not injuring other natural enemy insects and beneficial organism, complies fully with harmful organism Synthetic
The objective of reason, simultaneously because it is easy mass production, control cost is more competitive, thus it is with a wide range of applications.
Conidium (conidiospore) is a kind of asexual spore common in fungi, is to be born in extracellular spore,
So referred to as exogenous spore.Conidium is born in differentiated conidiophore (conidiophore) or has a setting
On the stigma of shape, also the conidium of some fungies is with raw on the top of mycelia.
Beauveria bassiana spore preparation, all can be thermally damaged in preparation, preservation and use process, this can accelerate agriculture
The degradation of effective component in medicine slows down drug effect and plays, reduces drug effect or cause control efficiency unstable.Heat comes from preparation process
In drying, the environment in preserving process, use (Field information) during sunlight irradiation or parasitic behavior.Therefore, it improves
The heat resistance of beauveria bassiana spore is most important to its killing rate of raising and biological control effect.
Summary of the invention
The object of the present invention is to provide a kind of beauveria bassiana oil-suspending agents, significantly improve beauveria bassiana spore
Heat resistance can be used for the preparation, preservation and Field information of beauveria bassiana preparation.
The purpose of the present invention adopts the following technical scheme that realization.
A kind of beauveria bassiana oil-suspending agent, the beauveria bassiana oil-suspending agent are that beauveria bassiana spore is added
To what is obtained after mixing in the mixture of surfactant and solvent naphtha, spore concentration is hundred million/mL of 90-110, described
The beauveria bassiana oil-suspending agent also xanthan gum containing 5-10g/L trehalose, the glycerol of 2-8g/L and 0.2-1g/L.
In the present invention, the beauveria bassiana oil-suspending agent also stilbene biphenyl disulfonic acid containing 0.5-2g/L
The ESCALOL 567 of sodium and 0.5-2g/L.
In the present invention, the beauveria bassiana is ZD-1 plants of beauveria bassiana (Beauveria bassiana), preservation
Number be CGMCC NO:16500.
In the present invention, the surfactant is polyglycerol polyricinoleate, fatty alcohol polyoxyethylene ether, alkyl phenol gather
One or more of ethylene oxide ether, tween.
In preferred technical solution, the surfactant is polyglycerol polyricinoleate.
In the present invention, the solvent naphtha is soybean oil, rapeseed oil, cotton seed oil, palm oil, sesame oil, corn oil and gathers
Close the mixture of one or more of linseed oil.
In preferred technical solution, the solvent naphtha is polymerization linseed oil.
In the present invention, the volume ratio of surfactant and solvent naphtha is 6:94.
Beauveria bassiana oil-suspending agent provided by the invention is significantly mentioned due to being added to trehalose, glycerol and xanthan gum
The high heat resistance of beauveria bassiana spore.Stilbene biphenyl sodium disulfonate is added in beauveria bassiana oil-suspending agent
After ESCALOL 567, the anti-uv-ray of beauveria bassiana spore is significantly improved, is conducive to
Field using when prevent damage of the ultraviolet light to beauveria bassiana.Oil-suspending agent of the present invention reduces spore in preparation preparation, guarantor
Hiding, field use process high temperature and ultraviolet light are conducive to improve beauveria bassiana spore to the adverse effect of beauveria bassiana
Survival rate, Shelf-life, improve product field using effect.Beauveria bassiana oil-suspending agent can be used for preventing and treating tealeaves
Smaller green leaf hopper.
Specific embodiment
The solvent that culture medium uses in the present invention is water.Gas liquid ratio refers to minute ventilation volume (m3) and fermentating liquid volume
(m3The ratio between).Ventilation volume refers to minute ventilation volume (m3) and solid fermentation culture volume (m3The ratio between).
The acquisition of 1 beauveria bassiana ZD-1 conidia powder of embodiment
This embodiment describes the acquisitions of beauveria bassiana ZD-1 conidia powder.
1. the screening and identification of Strain of Beauveria bassiana
(1) screening of Strain of Beauveria bassiana
Using tealeaves smaller green leaf hopper as subjects, this embodiment describes ZD-1 plants of screenings of beauveria bassiana.
Strains tested: XY-201805 plants, YN-201803 plants, ZD-1 plants, KM-4 plants, ZA-7-3 plants, B3 plants, B822 plants,
PE-55 plants, PE-23 plants.
Selected insect source: in early April, 2018 to late April acquires small in Yunnan Province's academy of agricultural science tea base
Greenery cicada.The smaller green leaf hopper adopted back is placed in wide-mouth bottle, with the fresh tea branch item interior raising with tender leaf and young shoot.
Each strains tested is inoculated in respectively on sabouraud medium SDAY (sabouraud medium SDAY formula: 4% glucose,
1% peptone, 1% yeast powder, 2% agar), under the conditions of 25 ± 1 DEG C, cultivates 8 days, obtain the spore of each bacterial strain.With containing
Spore under the sterile washing of 0.05%Tween 80, is configured to 1.0 × 107The spore suspension of a/mL, it is spare.
The consistent smaller green leaf hopper of Individual Size is chosen, cause of the spore suspension of above-mentioned each bacterial strain to smaller green leaf hopper is investigated
Sick power.The specific method is as follows: taking out after smaller green leaf hopper is soaked 10s in spore suspension, is placed on filter paper and sucks superfluous water
Point, it is transferred in artificial feeding box.Every kind of spore suspension handles 50 test worms, repeats 3 times.Contain 0.05%Tween with leaching
80 sterile water is normally raised under similarity condition as compareing.In breeding process, dead worm is transferred in sterile petri dish in time
Culture.The 6th day, the 9th day, the 12nd day statistics death toll after smaller green leaf hopper processing respectively, to grow visible mycelia on worm corpse
Or conidia powder is effectively lethal.Concrete outcome is shown in Table 1.
The cumulative mortality of smaller green leaf hopper after the different Strain of Beauveria bassiana processing of table 1
From the data in table 1, it can be seen that each beauveria bassiana spore has different degrees of pathogenicity to smaller green leaf hopper.But ZD-1
The insecticidal effect of strain is best, and at the 12nd day, cumulative mortality was up to 95.33%.
(2) it identifies
ZD-1 plants grow relatively slowly on potato dextrose agar (PDA), cultivate 14 under 28 DEG C of dark conditions
It, colony diameter is 58~61mm, and initial stage is white, after gradually become light yellow, it is velvet-like to cotton-shaped, have concentricity and put
Penetrate shape lines；The bacterium colony back side is cream-coloured to arrive fawn, no water colo(u)r.
ZD-1 plants of vegetative hyphae thin-walleds, transparent, smooth, common branch is 0.7~2.0 μm wide, conidiophore directly
On vegetative hyphae, conidium is subsphaeroidal to ellipse, monospore, thin-walled, transparent, light on conidiophore for life
It is sliding, 23.5~43.2 × 2.2~4.3 μm.
ZD-1 plants of rRNA gene orders are as shown in SEQ ID NO:1, and by sequence alignment, ZD-1 plants of discovery white for ball spore
Stiff bacterium is named as ZD-1 plants of beauveria bassiana (Beauveria bassiana), is abbreviated as ZD-1 plants of beauveria bassiana.
ZD-1 plants of beauveria bassiana (Beauveria bassiana), preservation.
Classification naming are as follows: beauveria bassiana
Beauveria bassiana。
Join the biomaterial (strain) of Ju: ZD-1.
The deposit date is on November 19th, 2018.
Depositary institution's full name is China Committee for Culture Collection of Microorganisms's common micro-organisms center, abbreviation CGMCC.
Depositary institution address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica.
Deposit number are as follows: CGMCC NO.16500.
2. the culture that ZD-1 plants of beauveria bassiana
(1) beauveria bassiana ZD-1 plants of spore activation
By ZD-1 plants of inclined-plane seeds of beauveria bassiana be inoculated in sabouraud medium SDAY (4% glucose, 1% peptone,
1% yeast powder, 2% agar) on, it is cultivated 8 days at 25 ± 1 DEG C, ZD-1 plants of spores of beauveria bassiana after being activated.
(2) ZD-1 plants of primary seed solutions of beauveria bassiana are prepared
In 250mL conical flask, the liquid amount 50mL of liquid sabouraud medium SDAY, the ball after accessing above-mentioned activation
ZD-1 plants of spores of beauveria bassiana, make spore concentration 3 × 106A/mL.Under the conditions of 25 ± 1 DEG C, 150rpm, shaken cultivation
After 36h, obtaining spore content is 30 × 108The primary seed solution of a/mL.
Wherein liquid sabouraud medium SDAY contains 40g/L glucose, 10g/L peptone and 10g/L yeast powder.
(3) ZD-1 plants of secondary seed solutions of beauveria bassiana are prepared
In 100L fermentor, the culture medium for containing following ingredient: starch 10g/L, glucose 15g/L, peptone is prepared
5g/L, yeast powder 10g/L, potassium dihydrogen phosphate 0.2g/L, magnesium sulfate 0.25g/L.Solvent is water.Sterilize 30min at 121 DEG C,
After being cooled to room temperature, be inoculated in the present embodiment title 2 gained primary seed solution according to 2.5% inoculum concentration, 25 ± 1 DEG C,
36h is cultivated under conditions of 200rpm, gas liquid ratio 1:1, obtains secondary seed solution, spore content is 1.3 × 109A/mL.
(4) beauveria bassiana ZD-1 plants of tertiary liquid fermentation cultures
It is that 1% (V/V) is respectively connected to 3000L fermentation according to inoculum concentration by ZD-1 plants of secondary seed solution of beauveria bassiana
It is cultivated in tank, obtains three-level seed liquor.Culture medium and the condition of culture difference that fermentor uses are as follows.
Culture medium contains following ingredient: rice meal 20g/L, glucose 10g/L, sucrose 5g/L, soybean cake powder 5g/L, silkworm
Pupa powder 5g/L, yeast powder 7.5g/L, potassium dihydrogen phosphate 0.25g/L, magnesium sulfate 0.2g/L, calcium chloride 0.3g/L, pH7.0.Solvent
For water.
Condition of culture: fermentor speed of agitator 150rpm, ventilatory capacity and temperature control are as follows in incubation: 0h~8h:
Gas liquid ratio is 0.5:1,28 DEG C of temperature；8h~and for 24 hours: 25 DEG C of gas liquid ratio 0.8:1, temperature；For 24 hours~48h: gas liquid ratio 1:1,
25 DEG C of temperature.44h is cultivated, the spore content of three-level seed liquor reaches 885 × 108A/mL.
(5) solid fermentation culture.
After solid medium is mixed with water, autoclave sterilization, the tiling of toilet's tray, solid medium thickness
7cm is cooled to room temperature.By ZD-1 plants of beauveria bassiana of three-level seed liquor, uniformly it is inoculated into tray and is further sent out
Ferment culture.
Culture medium: 30 parts of rice, 10 parts of corn flour, 1 part of glucose, 27 parts of husk, wheat bran 18 are weighed according to mass parts
Part, 8.5 parts of dried silkworm chrysalis meal, 5.5 parts of beancake powder, 150 parts of water are uniformly mixed.
Condition of culture: inoculum concentration 15%, condition of culture control is as follows after inoculation: 0~2 day: ventilation volume 0.3:1, temperature
Spend 27.5 DEG C~28.5 DEG C, humidity 65%；3~7 days: ventilation volume 0.5:1,24.5 DEG C~25.5 DEG C of temperature, humidity are
60%；8~9 days: ventilation volume 0.5:1,28 DEG C~29 DEG C of temperature, humidity 45%.
When fermentation time is 216h, spore content reaches 1150 × 108A/g, spore germination rate are not less than 90%.It will hair
After ferment culture is dried, spore extracting is carried out using cyclonic separation collection spore device, obtains beauveria bassiana conidia powder.
The thermal protecting agent of 2 beauveria bassiana spore of embodiment
1, heat resistance measures
Beauveria bassiana conidia powder prepared by embodiment 1 uses normal saline at content for 5 × 108A/mL
Spore suspension, as control.
Control is respectively processed as follows, investigates each substance to beauveria bassiana spore heat resistanceheat resistant performance
It influences.
Processing 1: 5 × 108Trehalose is added in the spore suspension of a/mL, makes the final concentration of 8g/L of trehalose.
Processing 2: 5 × 108Glycerol is added in the spore suspension of a/mL, makes the final concentration of 5g/L of glycerol.
Processing 3: 5 × 108Xanthan gum is added in the spore suspension of a/mL, makes the final concentration of 0.5g/ of xanthan gum
L。
Processing 4: 5 × 108Trehalose, glycerol are added in the spore suspension of a/mL, make the final concentration of of trehalose
The final concentration of 5g/L of 8g/L, glycerol.
Processing 5: 5 × 108Xanthan gum, glycerol are added in the spore suspension of a/mL, make the final concentration of of xanthan gum
The final concentration of 5g/L of 0.5g/L, glycerol.
Processing 6: 5 × 108Xanthan gum, trehalose are added in the spore suspension of a/mL, make the final concentration of xanthan gum
For 0.5g/L, the final concentration of 8g/L of trehalose.
Processing 7: 5 × 108Trehalose, glycerol, xanthan gum are added in the spore suspension of a/mL, make the end of trehalose
Concentration is the final concentration of 0.5g/L of 8g/L, the final concentration of 5g/L of glycerol, xanthan gum.
It is separately sampled from control, processing 1, processing 2 and processing 3, measure its spore germination rate.Each sample is used into Sa
Family name culture medium SDAY measures germination rate in 25 ± 1 DEG C of cultures afterwards for 24 hours, when germ tube length is not less than spore diameter, the spore
It is considered as and has sprouted.Each sample repeats three times, and the result is shown in such as the following table 1.
The germination rate (%) of 1 each sample of table
| Control | Processing 1 | Processing 2 | Processing 3 | |
| Repeat 1 | 93.4 | 92.5 | 93.3 | 92.6 |
| Repeat 2 | 94.1 | 93.1 | 92.9 | 93.5 |
| Repeat 3 | 93.8 | 92.8 | 93.7 | 92.1 |
| Average germination rate | 93.77 | 92.80 | 93.30 | 92.73 |
The data result of table 1 shows: there is no obvious for the sprouting of trehalose, glycerol, xanthan gum to beauveria bassiana spore
Promotion effect.
From control, processing 1-7, separately sampled 5mL is placed in 50 DEG C of thermostat water baths in sterile test tube, by sample
It after middle processing 30min (shaking simultaneously), takes out immediately, is placed in ice bath in mixture of ice and water and is cooled to room temperature；It will be after cooling mixed
It closes liquid to be transferred in 250mL triangular flask, adds 45mL liquid sabouraud medium SDAY, after mixing, cultivated for 24 hours at 25 ± 1 DEG C
After measure germination rate, the results are shown in Table 2.
The germination rate (%) of sample after 2 water bath processing of table
| Control | Processing 1 | Processing 2 | Processing 3 | Processing 4 | Processing 5 | Processing 6 | Processing 7 | |
| Repeat 1 | 3.5 | 23.5 | 24.5 | 26.8 | 36.8 | 38.8 | 39.5 | 79.5 |
| Repeat 2 | 3.7 | 22.9 | 25.2 | 27.3 | 36.2 | 38.2 | 39.1 | 79.8 |
| Repeat 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 result of table 2 shows: the germination rate for handling 7 is 21.02 times compareed, and trehalose, glycerol, xanthan gum are total
With in the presence of, the germination rate of spore after heat treatment is significantly improved, heat protective effect is obvious.
(2) influence of trehalose, glycerol and xanthan gum concentration to beauveria bassiana spore heat resistance
The beauveria bassiana conidia powder prepared using embodiment 1, uses normal saline concentration for 5 × 108A/mL
Spore suspension.
Control: 5 × 108The spore suspension of a/mL.
Handle 7:5 × 108Trehalose, glycerol, xanthan gum are added in the spore suspension of a/mL, make trehalose final concentration
For 8g/L, final glycerol concentration 5g/L, the final concentration of 0.5g/L of xanthan gum.
Handle 8:5 × 108Trehalose, glycerol, xanthan gum are added in the spore suspension of a/mL, make trehalose final concentration
For 5g/L, final glycerol concentration 2g/L, the final concentration of 0.2g/L of xanthan gum.
Handle 9:5 × 108Trehalose, glycerol, xanthan gum are added in the spore suspension of a/mL, make trehalose final concentration
For 10g/L, final glycerol concentration 8g/L, the final concentration of 1g/L of xanthan gum.
By control, the processing separately sampled 5mL of 7-9 in sterile test tube, sample is placed in 50 DEG C of thermostat water baths,
It after water-bath 30min (shaking simultaneously), takes out immediately, is placed in ice bath in mixture of ice and water and is cooled to room temperature；By mixing after cooling
Liquid is transferred in 250mL triangular flask, adds 45mL liquid sabouraud medium SDAY, after mixing, after 25 ± 1 DEG C of cultures for 24 hours
Germination rate is measured, the results are shown in Table 3.
Sample germination rate after 3 water bath processing of table measures (%)
| Control | Processing 7 | Processing 8 | Processing 9 | |
| Repeat 1 | 3.6 | 78.3 | 70.4 | 72.4 |
| Repeat 2 | 3.5 | 78.2 | 69.8 | 71.9 |
| Repeat 3 | 4.0 | 77.8 | 69.1 | 72.2 |
| Average germination rate | 3.7 | 78.1 | 69.8 | 72.2 |
The data result of table 3 shows: trehalose concentration 5-10g/L, glycerol concentration 2-8g/L, xanthan gum concentration are
0.2-1g/L significantly improves the germination rate of spore after heat treatment.The protecting effect of processing 7 is best, and spore germination rate is control
21.11 times.
The uvioresistant ability of 3 beauveria bassiana spore of embodiment measures
(1) influence of each substance to beauveria bassiana spore uvioresistant ability
Beauveria bassiana conidia powder prepared by embodiment 1 uses normal saline at concentration for 5 × 108A/mL's
Spore suspension.
Control: 5 × 108The spore suspension of a/mL.
Processing 10: 5 × 108(the fluorescent brightening of stilbene biphenyl sodium disulfonate is added in the spore suspension of a/mL
Agent), make the final concentration of 1g/L of fluorescent whitening agent.
Processing 11: 5 × 108ESCALOL 567 is added in the spore suspension of a/mL, makes 2- hydroxyl
The final concentration of 1g/L of base -4- methoxy benzophenone.
Processing 12: 5 × 108(the fluorescent brightening of stilbene biphenyl sodium disulfonate is added in the spore suspension of a/mL
Agent) and ESCALOL 567, make stilbene biphenyl sodium disulfonate and ESCALOL 567
Final concentration be all 1g/L.
It is sampled from compareing and handling in 10-12, using sabouraud medium SDAY, in 25 ± 1 DEG C of cultures, measurement is sprouted afterwards for 24 hours
Hair rate, each sample in triplicate, result such as table 4.
The germination rate (%) of 4 each sample of table
| Control | Processing 10 | Processing 11 | Processing 12 | |
| Repeat 1 | 93.5 | 92.9 | 94.2 | 93.6 |
| Repeat 2 | 93.8 | 93 | 93.7 | 93.1 |
| Repeat 3 | 93.1 | 92.1 | 94.5 | 92.8 |
| Average germination rate | 93.5 | 92.7 | 94.1 | 93.2 |
Table 4 statistics indicate that: stilbene biphenyl sodium disulfonate and ESCALOL 567 are white to ball spore
There is no the effects significantly promoted for the sprouting of stiff bacterium spore.
It takes 10mL sample to be placed in the sterile petri dish that diameter is 9cm from control, processing 10-12 respectively, mixes well
It is spare.Respectively there are a ultraviolet radiator (integrated TB excess light fluorescent lamp, 15W, power factor (PF) 0.65, frequency in super-clean bench above both ends
50~60Hz of rate).Quartz burner 5min is opened, opens wide the culture dish equipped with above-mentioned sample as ultra-clean after it is stablized
In platform, after irradiating 5min at 35cm under ultraviolet lamp, ultraviolet lamp is closed.Will treated liquid blending, take 5mL in 250mL triangular flask
In, 45mL liquid sabouraud medium SDAY is added, after mixing, measures germination rate afterwards for 24 hours in 25 ± 1 DEG C of cultures, as a result such as table
Shown in 5.
The germination rate (%) of each sample after 5 treatment with ultraviolet light of table
| Control | Processing 10 | Processing 11 | Processing 12 | |
| Repeat 1 | 2.1 | 45.6 | 46.4 | 65.4 |
| Repeat 2 | 2.7 | 44.8 | 46.1 | 64.9 |
| Repeat 3 | 2.3 | 45.1 | 45.9 | 65.1 |
| Average germination rate | 2.4 | 45.2 | 46.1 | 65.1 |
Data result in table 5 shows: stilbene biphenyl sodium disulfonate, ESCALOL 567 and its
Mixture has ultraviolet protection effect to beauveria bassiana spore, and the germination rate for handling 12 is 27.13 times compareed, ultraviolet protection
Effect is obvious.
(2) stilbene biphenyl sodium disulfonate (fluorescent whitening agent) and ESCALOL 567 concentration are to ball
The influence of beauveria bassiana spore uvioresistant ability
Beauveria bassiana conidia powder prepared by embodiment 1, uses physiological saline to be prepared into concentration as 5 × 108A/mL
Spore suspension.
Control: 5 × 108The spore suspension of a/mL.
Processing 12: 5 × 108(the fluorescent brightening of stilbene biphenyl sodium disulfonate is added in the spore suspension of a/mL
Agent) and ESCALOL 567, making its final concentration is all 1g/L.
Handle 13:5 × 108Stilbene biphenyl sodium disulfonate (fluorescent whitening agent) is added in the spore suspension of a/mL
And ESCALOL 567, making its final concentration is all 0.5g/L.
Handle 14:5 × 108Stilbene biphenyl sodium disulfonate (fluorescent whitening agent) is added in the spore suspension of a/mL
And ESCALOL 567, making its final concentration is all 2g/L.
10mL sample is taken to be placed in the sterile petri dish that diameter is 9cm from control, processing 12-14 respectively, it is sufficiently mixed
It is even.Respectively there are a ultraviolet radiator (integrated TB excess light fluorescent lamp, 15W, power factor (PF) 0.65, frequency above both ends in super-clean bench
50~60Hz).Quartz burner 5min is opened, opens wide the culture dish equipped with above-mentioned sample as super-clean bench after it is stablized
It is interior, after irradiating 5min at 35cm under ultraviolet lamp, close ultraviolet lamp.
Will treated liquid blending, take 5mL in 250mL triangular flask, add 45mL liquid sabouraud medium
SDAY after mixing, measures germination rate in 25 ± 1 DEG C of cultures, the results are shown in Table 6 afterwards for 24 hours.
The germination rate (%) of each sample after 6 treatment with ultraviolet light of table
| Control | Processing 12 | Processing 13 | Processing 14 | |
| Repeat 1 | 3.1 | 65.9 | 58.7 | 64.8 |
| Repeat 2 | 2.7 | 65.2 | 58.1 | 64.2 |
| Repeat 3 | 2.5 | 66 | 57.9 | 63.8 |
| Average germination rate | 2.8 | 65.7 | 58.2 | 64.3 |
The data result of table 6 shows: changing stilbene biphenyl sodium disulfonate (fluorescent whitening agent) and 2- hydroxyl -4- first
The concentration of oxygroup benzophenone has ultraviolet protection effect to beauveria bassiana spore.The ultraviolet protection effect of processing 12 is more
To be obvious, spore germination rate is 23.46 times of control.
4 beauveria bassiana oil-suspending agent heat resistanceheat resistant of embodiment, the measurement of uvioresistant ability
(1) water bath with thermostatic control and heat resistanceheat resistant and the measurement of uvioresistant ability under ultraviolet light irradiation
Following several samples and processing are set:
Comparison medicament: beauveria bassiana conidia powder prepared by embodiment 1 is directly added into polyglycerol polyricinoleate (surface
Activating agent) and polymerize in the mixture of linseed oil (solvent naphtha), beauveria bassiana oil-suspending agent, spore are obtained after mixing
Concentration is 10,000,000,000/mL.Wherein the volume ratio of polyglycerol polyricinoleate and polymerization linseed oil is 6:94.
Treatment agent: polyglycerol polyricinoleate and polymerization linseed oil are uniformly mixed according to volume ratio for 6:94, addition is eventually
Concentration is the spore of 10,000,000,000/mL, while adding following protective agent: trehalose 8g/L, glycerol 5g/L, xanthan gum 0.5g/L,
Stilbene biphenyl sodium disulfonate (fluorescent whitening agent) 1g/L, ESCALOL 567 1g/L, concentration herein
It is final concentration.
Comparison medicament and treatment agent are heat-treated respectively, after UV treatment and heat treatment again at ultraviolet light
It manages, the germination rate of sample after detection processing.
Heat treatment and germination rate measurement: it is placed in sterile test tube from 5mL separately sampled in each sample, sample is placed in
It in 50 DEG C of thermostat water baths, after water-bath 30min (shaking simultaneously), takes out immediately, is placed in ice bath in mixture of ice and water and is cooled to room
Temperature；Mixed liquor after cooling is transferred in 250mL triangular flask, 45mL liquid sabouraud medium SDAY is added, after mixing,
Germination rate is measured afterwards for 24 hours in 25 ± 1 DEG C of cultures.
UV treatment and germination rate measurement: separately sampled product 10mL is placed in the sterile training that diameter is 9cm from each sample
It supports in ware, mixes well.Ultraviolet lamp is closed after ultraviolet light irradiation 5min.Will treated liquid blending, take 5mL in 250mL triangle
In bottle, 45mL liquid sabouraud medium SDAY is added, after mixing, measures germination rate afterwards for 24 hours in 25 ± 1 DEG C of cultures.
Ultraviolet processing and germination rate measurement again after heat treatment: separately sampled 10mL is placed in sterile test tube from each sample
In, sample is placed in 50 DEG C of thermostat water baths, after water-bath 30min (shaking simultaneously), takes out immediately, is placed in mixture of ice and water
Middle ice bath is cooled to room temperature；Mixed liquor after cooling transfer is placed in the sterile petri dish that diameter is 9cm, is mixed well.It is purple
Ultraviolet lamp is closed after outer light irradiation 5min.Will treated liquid blending, take 5mL in 250mL triangular flask, add 45mL liquid
Body sabouraud medium SDAY after mixing, measures germination rate in 25 ± 1 DEG C of cultures afterwards for 24 hours.
Concrete outcome is shown in Table 7.
Germination rate under 7 different agents different disposal of table
From the data in table 7, it can be seen that being added to protectant treatment agent spore germination rate is comparison medicine when being individually heat-treated
24.19 times of agent；When single UV treatment, it is added to 23.54 that protectant treatment agent spore germination rate is comparison medicament
Times；UV treatment again after heat treatment is added to 32.84 times that protectant treatment agent spore germination rate is comparison medicament.
It follows that being added to trehalose, glycerol, xanthan gum, stilbene biphenyl sodium disulfonate and 2- hydroxyl -4- methoxyl group hexichol
Ketone significantly improves the heat resistanceheat resistant and uvioresistant ability of spore.
(2) spore suspension heat resistanceheat resistant in preservation 1 year and the measurement of uvioresistant ability at room temperature
Following 2 samples and processing are set:
Comparison medicament: polyglycerol polyricinoleate and polymerization linseed oil are uniformly mixed according to volume ratio for 6:94, addition is eventually
Concentration is the beauveria bassiana spore of 10,000,000,000/mL.
Treatment agent: polyglycerol polyricinoleate and polymerization linseed oil are uniformly mixed according to volume ratio for 6:94, addition is eventually
Concentration is the beauveria bassiana spore of 10,000,000,000/mL, while adding following protective agent: trehalose 8g/L, glycerol 5g/L, xanthan
Glue 0.5g/L, stilbene biphenyl sodium disulfonate 1g/L, ESCALOL 567 1g/L, concentration herein are
Final concentration.
Comparison medicament and treatment agent are placed at room temperature for (window towards south, no curtain sunshade) 1 year, respectively in dry environment
When placing beginning (initial sample), places 3 months, 6 months, 12 months and its spore germination rate is measured by sampling.It the results are shown in Table 8.
The germination rate of each sample during 8 room temperature preservation of table 1 year
Note: " control " is comparison medicament in table 8, and " processing " is treatment agent.
From the data in table 8, it can be seen that the beauveria bassiana oil-suspending agent of 10,000,000,000/mL, is placed at room temperature for one-year age, is added to
Protectant treatment agent spore germination rate is 2.03 times of comparison medicament, it can be seen that, it is added to trehalose, glycerol, xanthan
Glue, stilbene biphenyl sodium disulfonate and ESCALOL 567 significantly improve the anti-of beauveria bassiana spore
Heat and uvioresistant ability.
Prevention and treatment of the 5 beauveria bassiana oil-suspending agent of embodiment in field to tealeaves smaller green leaf hopper
Beauveria bassiana oil-suspending agent has been investigated to the control efficiency of tealeaves smaller green leaf hopper in field.Test site: Yunnan
Tealeaves research institute, academy of agricultural sciences, province tea place is experimental field.Garden uniline plantation, 3500 plants/acre of planting density.
Test process design: blank control；Control 1 be prepared in the embodiment of the present invention 4 comparison medicament (no heat resistanceheat resistant and
Ultraviolet resistance protectant addition)；Control 2 is that the treatment agent prepared in the embodiment of the present invention 4 (is added with heat resistanceheat resistant and uvioresistant
Line protective agent).
Usage amount: setting 3 are handled (blank control, control 1 and control 2), 3 repetitions of each processing, totally 9 cells,
20 square metres or so of every cell, random district's groups arrangement, the tea tree of each cell surrounding is protection row.It is administered on June 5th, 2018
Once, the amount of application of control 1 and control 2 is 100mL/ mus, with clear water surrogate-data technique in blank control.
Insecticide-applying way: hand sprayer even spraying, medication main portions are the front and back sides of the fluffy face of tea tree and two lateral lobes,
Medical fluid is set to come into full contact with smaller green leaf hopper polypide.
Test carries out 3 investigation, and 1 day respectively after medicine, 7 days, 14 days, every cell investigates 100 bud-leafs, records tealeaves
The survival number of smaller green leaf hopper.Investigation result see the table below 9.
9 tealeaves smaller green leaf hopper field control effectiveness test result of table
As shown in Table 9, regardless of whether addition protective agent, beauveria bassiana oil-suspending agent is in field to tealeaves smaller green leaf hopper
With apparent control efficiency.It is added to the control efficiency of protectant treatment agent, hence it is evident that be higher than protectant without adding
Comparison medicament.
10000000000 spore/mL beauveria bassiana oil-suspending agents are a kind of safe and nontoxic, noresidue novel microbials
Pesticide.Heat resistanceheat resistant and the protectant addition of uvioresistant can significantly improve the heat resistance and ultraviolet-resistent property of beauveria bassiana, reduce
Spore to the adverse effect of beauveria bassiana, is conducive in preparation preparation, preservation, field use process high temperature, ultraviolet light
Improve survival rate, the Shelf-life, the field using effect for improving product of beauveria bassiana spore.
The above is only the preferred embodiment of the present invention, it is noted that above-mentioned preferred embodiment is not construed as pair
Limitation of the invention, protection scope of the present invention should be defined by the scope defined by the claims..For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, several improvements and modifications can also be made, these
Improvements and modifications also should be regarded as protection scope of the present invention.
Sequence table
<110>Yunnan Xing Yao biological products Co., Ltd
<120>a kind of 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 (bassianaZD-1 plants of Beauveria)
<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 (6)
1. one plant of beauveria bassiana, classification naming be beauveria bassiana (Beauveria bassiana) ZD-1 plants, deposit number is
CGMCC NO:16500.
2. the cultural method of beauveria bassiana described in claim 1, being characterized in that including the following steps: will be described in claim 1
Beauveria bassiana obtains three-level seed liquor by first order seed culture, secondary seed culture and three-level seed culture, by described three
Grade seed liquor is seeded to solid medium, carries out solid fermentation, obtains beauveria bassiana conidia powder.
3. the cultural method of beauveria bassiana according to claim 2, it is characterised in that: in three-level seed culture, fermentation training
Feeding base contain 10 ~ 30g/L of rice meal, 10 ~ 30g/L of glucose, 5 ~ 20g/L of sucrose, 5 ~ 20g/L of soybean cake powder, dried silkworm chrysalis meal 5 ~
10g/L, 5 ~ 10g/L of yeast powder, 0.1 ~ 0.5g/L of potassium dihydrogen phosphate, 0.1 ~ 0.5g/L of magnesium sulfate, 0.1 ~ 0.5g/L of calcium chloride,
pH7.0；In three-level seed culture, speed of agitator is 120 ~ 200rpm, and ventilatory capacity and temperature control are as follows: 0h~8h: gas liquid ratio
It is 26 DEG C~28 DEG C for 0.4~0.6:1, temperature；8h~and for 24 hours: gas liquid ratio is 0.8~1.0:1, temperature is 24 DEG C~26 DEG C；24h
~48h: gas liquid ratio is 0.8~1.2:1, temperature is 24 DEG C~26 DEG C.
4. the cultural method of the beauveria bassiana according to Claims 2 or 3, it is characterised in that: the solid training in solid fermentation
Feeding base is prepared with the following method: by 30 ~ 50 mass parts of rice, 10 ~ 20 mass parts of corn flour, 1 ~ 3 mass parts of glucose, husk
10 ~ 30 mass parts, 5 ~ 20 mass parts of wheat bran, 100 ~ 200 mass parts of 5 ~ 10 mass parts of dried silkworm chrysalis meal, 2 ~ 10 mass parts of beancake powder and water
It is uniformly mixed.
5. the cultural method of beauveria bassiana according to claim 4, it is characterised in that: in solid fermentation, three-level seed liquor
Inoculum concentration be 10 ~ 15%, ventilation volume, temperature and humidity control are as follows in solid state fermentation: 0~48h: ventilation volume is 0.2~0.4:
1, temperature is 27 DEG C~29 DEG C, humidity is 60~70%；48~168h: ventilation volume is 0.4~0.6:1, temperature is 24 DEG C~26
DEG C, humidity be 50~65%；168~216h: ventilation volume is 0.4~0.6:1, temperature is 27 DEG C~29 DEG C, humidity is 40~50%.
6. the cultural method of beauveria bassiana according to claim 5, it is characterised in that solid medium in solid fermentation
With a thickness of 5-8 centimetres.
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| CN114438011A (en) * | 2022-03-07 | 2022-05-06 | 广西大学 | Fermentation production method and application of beauveria bassiana PfBb spore powder |
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