CN106497805A - A kind of cultural method of beauveria bassiana - Google Patents
A kind of cultural method of beauveria bassiana Download PDFInfo
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Abstract
The present invention discloses a kind of cultural method of beauveria bassiana, including (1) primary inclined plane culture：Beauveria bassiana thalline is inoculated in primary inclined plane culture medium, is cultivated 56 days at 24 25 DEG C；(2) secondary liquid culture：The strain obtained after step (1) is cultivated is seeded in seed tank, cultivates 24 28h, (3) three-level fluid enlargement culture at 24 25 DEG C：The strain obtained after step (2) is cultivated is seeded in fermentation tank, cultivates 28 32h at 25 28 DEG C；(4) level Four solid culture：Strain after step (3) amplification culture is inoculated in solid medium, and 5 6d are cultivated at 22 25 DEG C.By screening and the proportioning of nutrient media components in each stage, while coordinating the temperature and humidity condition of culture, cultivation cycle can be shortened, improve the conidial quality of beauveria bassiana and yield, improve conidial activity.
Description
Technical field
The present invention relates to a kind of cultural method of beauveria bassiana.
Background technology
Muscardine is a kind of ascomycetous entomogenous fungi, and main species include beauveria bassiana and muscardine etc.,
Conidium is generated usually through asexual propagation, mycelia has tabula to have branch.Muscardine can invade more than 200 kinds of elder brother of 6 Ge Mu, 15 sections
Amount reproduction in worm, the polypide of mite class, while produce Bombyx Batryticatus element (Non-ribosomal peptide toxoid), oosporein (benzoquinones poison
Element) and calcium oxalate crystal, these materials can cause insecticide to be poisoned, upset metabolism so that dead.Wherein, beauveria bassiana is
Ascomycetous funguses, can industrialization culture production, people can be processed into microbial insecticide using its conidium for producing；
Chinese medicine Bombyx Batryticatus can also be obtained by Infected by Beauveria bassiana silkworm.
The comparison of ingredients of existing beauveria bassiana culture medium is single, not good to the growth result for promoting beauveria bassiana,
The design carried out for the growth course of beauveria bassiana is less, is easily bacterial contamination in incubation, therefore, the ball that obtains
The conidial quality of beauveria bassiana is unstable, and virulence is weaker, and the breeding cycle is longer.
Content of the invention
In view of this, the application provides a kind of cultural method of beauveria bassiana, by nutrient media components in each stage
Screening and proportioning, while coordinating the temperature and humidity condition of culture, cultivation cycle can be shortened, beauveria bassiana is improved mitogenetic
The quality and yield of spore, improves conidial activity.
For solving above technical problem, the technical scheme that the present invention is provided is a kind of cultural method of beauveria bassiana, institute
State cultural method to comprise the following steps：
(1) primary inclined plane culture：Beauveria bassiana thalline is inoculated in primary inclined plane culture medium, is cultivated at 24-25 DEG C
5-6 days；
(2) secondary liquid culture：The strain obtained after step (1) is cultivated is seeded in seed tank, is cultivated at 24-25 DEG C
24-28h, the component of the fluid medium in the seed tank include by weight percentage：PDA culture medium, supplements 10-20%
Peptone, 5-10% carbamide, 5-20% sucrose, 10-15% Testa Tritici, 0.1-0.5% potassium dihydrogen phosphates, 0.01-0.05% sulphuric acid
Magnesium, 0.01-0.05% vitamin Bs, 0.01-0.05% vitamin Cs, 0.01-0.02% zinc sulfate, 0.01-0.02% copper sulfate,
0.05-0.1% iron chloride, 0.05-0.1% calcium chloride；
(3) three-level fluid enlargement culture：The strain obtained after step (2) is cultivated is seeded in fermentation tank, at 25-28 DEG C
Culture 28-32h, the component of the fluid enlargement culture base in the fermentation tank include by weight percentage：PDA culture medium, mends
Fill 10-20% peptones, 10-20% sucrose, 10-20% analysis for soybean powder, 0.1-0.5% potassium dihydrogen phosphates, 0.05-0.1% sulphuric acid
Magnesium, 0.02-0.05% vitamin Bs, 0.01-0.02% vitamin Cs, 0.05-0.15% calcium chloride, 0.2-1.0% sodium alginates；
(4) level Four solid culture：Strain after step (3) amplification culture is inoculated in solid medium, at 22-25 DEG C
Lower culture 5-6d, the component of the solid medium count by weight including：Testa Tritici 20-30 parts, dried silkworm chrysalis meal 10-20 parts, Huang
Semen Glycines powder 10-20 parts, vitamin B 1-3 parts, vitamin C 1-2 parts, sucrose 2-10 parts, Testa oryzae 20-40 parts.
Preferably, the component of step (1) slant medium count by weight including：Potato starch 20-30 parts,
Peptone 1-2 parts, Lactose 5-10 part, sucrose 1-5 parts, potassium dihydrogen phosphate 0.05-0.1 parts, magnesium sulfate 0.05-0.1 parts, agar 1-
2 parts.
Be more highly preferred to, the component of step (1) slant medium count by weight including：Potato starch 25
Part, 1 part of peptone, 8 parts of Lactose, 3 parts of sucrose, 0.08 part of potassium dihydrogen phosphate, 0.06 part of magnesium sulfate, 2 parts of agar.
Preferably, the component of the fluid medium in step (2) seed tank includes by weight percentage：PDA is trained
Foster base, supplement 18% peptone, 7% carbamide, 15% sucrose, 13% Testa Tritici, 0.3% potassium dihydrogen phosphate, 0.02% magnesium sulfate,
0.04% vitamin B, 0.02% vitamin C, 0.015% zinc sulfate, 0.018% copper sulfate, 0.08% iron chloride, 0.07% chlorine
Change calcium.
Preferably, the component of the fluid enlargement culture base in step (3) fermentation tank includes by weight percentage：
PDA culture medium, supplement 15% peptone, 18% sucrose, 17% analysis for soybean powder, 0.3% potassium dihydrogen phosphate, 0.07% magnesium sulfate,
0.04% vitamin B, 0.03% vitamin C, 0.10% calcium chloride, 0.8% sodium alginate.
Preferably, in step (2) and step (3), the vitamin B is vitamin B1, vitamin B2, vitamin B3, dimension
At least one in raw element B6.
It is more highly preferred to, in step (2) and step (3), the vitamin B is vitamin B2, vitamin B3, vitamin B6
Compositionss, the vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:(1-2):(1-2).
It is more highly preferred to, in step (2) and step (3), the vitamin B is vitamin B2, vitamin B3, vitamin B6
Compositionss, the vitamin B2, vitamin B3, vitamin B6 weight ratio be 1:1.5:1.8.
Preferably, step (1) slant medium first adjusts pH value before inoculation and to 6.5-6.8, then in pressure is
Pressurize sterilizing 40-60min under 100-110kPa.
Preferably, the component of step (4) solid medium count by weight including：25 parts of Testa Tritici, dried silkworm chrysalis meal 15
Part, 15 parts of analysis for soybean powder, 2 parts of vitamin B, Catergen part, 7 parts of sucrose, 30 parts of Testa oryzae.
Preferably, in step (4), the humidity of level Four solid culture is 95-98%.
Preferably, in step (4), the thickness of solid medium is 5-6cm, and the ratio of the affixed kind of liquid is 1:(1.2-1.3).
Preferably, step (4) level Four solid culture is covered with after conidium to solid medium, you can be dried place
Reason.
Abbreviation of the described PDA culture medium for potato dextrose agar, i.e. Potato Dextrose Agar
Medium.Its way is：Weigh 200g and clean the Rhizoma Solani tuber osi of peeling and be cut into small pieces, add water well-done (boil 20 30min, can quilt
Glass rod is poked), with eight layers of filtered through gauze, heat, need further according to experiment plus 1 10g agar, continue heated and stirred and mix
Even, after agar has dissolved, add glucose, stir, supply moisture after slightly cooling down again to 1000 milliliters, subpackage test tube or
Person's conical flask, jumps a queue, wraps up, and 115 DEG C of sterilizings are taken out test tube pendulum after 20 minutes and inclined-plane or shaken up, and obtain final product after cooling.
In technical scheme, the culture of described beauveria bassiana includes primary inclined plane culture, secondary liquid
The step of culture, three-level fluid enlargement culture, level Four solid culture, by primary inclined plane culture, beauveria bassiana strain is obtained,
The component and proportioning of control slant medium, and add Element Potassium needed for growth and magnesium, while the temperature for controlling culture exists
Between 24-25 DEG C, accelerate the growth of strain；By secondary liquid culture, strain quantity is made to improve rapidly, its fluid medium
Component includes carbon source necessary to growth, nitrogen source, also add potassium dihydrogen phosphate, magnesium sulfate, zinc sulfate, copper sulfate,
Iron chloride, calcium chloride, there is provided element needed for thalli growth, while also add vitamin B and vitamin C, the two can improve
Thalli growth speed, improves thalline quantity, and above compounding can greatly improve the speed of growth of strain, improve strain quantity, this
Outward, vitamin B is vitamin B2, vitamin B3, the compounding of vitamin B6, it is possible to increase ensures bacterial activity, and improves strain
The speed of growth；Through three-level fluid enlargement culture, strain continued growth, quantity increase, while strain generates mycelium, its liquid
The component of body amplification culture base includes carbon source necessary to growth, nitrogen source, also add promotion mycelial growth phosphoric acid
Potassium dihydrogen, magnesium sulfate, calcium chloride, while also add vitamin B and vitamin C, the two can improve thalline and mycelium
The speed of growth, vitamin B are vitamin B2, vitamin B3, the compounding of vitamin B6, it is possible to increase ensure mycelia active, this
Outward, sodium alginate is also added in described fluid enlargement culture base, it is possible to increase the viscosity of liquid, be conducive to nutrient media components
Suspension, improve the utilization rate of component in culture medium, while by the adjustment of its proportioning, make broth viscosity control adapt to model
Enclose, it is to avoid during liquid sloshing, mycelium rocks generation therewith and pulls, destroy mycelium；By level Four solid culture, conidium
A large amount of generations, can be now dried, the beauveria bassiana spore powder needed for obtaining, and in solid medium, add
Testa Tritici, dried silkworm chrysalis meal, analysis for soybean powder, Testa oryzae, sucrose, are that mycelium and conidium provide carbon source and nitrogen source, while Testa Tritici, Huang
The lax quality of Semen Glycines powder, Testa oryzae, with preferable moisture retention and breathability, there is provided moistening and ventilative environment, is conducive to mycelia
The quick breeding of body and product spore, additionally, being also added into vitamin B and vitamin C, can accelerate the speed that mycelium produces spore, with
When can improve conidial activity with the compounding of dried silkworm chrysalis meal.
Compared with prior art, the cultural method of the beauveria bassiana described in technical scheme, including primary inclined plane
Culture, secondary liquid culture, three-level fluid enlargement culture, level Four solid culture, by the sieve of nutrient media components in each stage
Choosing and proportioning, while coordinating the temperature and humidity condition of culture, can shorten cultivation cycle, improve beauveria bassiana conidium
Quality and yield, improve conidial activity.
Specific embodiment
In order that those skilled in the art more fully understands technical scheme, with reference to specific embodiment pair
The present invention is described in further detail.
Embodiment 1
A kind of cultural method of the beauveria bassiana described in the present embodiment, the cultural method are comprised the following steps：
(1) primary inclined plane culture：Slant medium is adjusted pH value to 6.8, then is pressurize sterilizing under 105kPa in pressure
50min, after cooling, beauveria bassiana thalline is inoculated in primary inclined plane culture medium, is cultivated 5 days at 25 DEG C, the inclined-plane training
The component of foster base count by weight including：25 parts of potato starch, 1 part of peptone, 8 parts of Lactose, 3 parts of sucrose, biphosphate
0.08 part of potassium, 0.06 part of magnesium sulfate, 2 parts of agar；
(2) secondary liquid culture：The strain obtained after step (1) is cultivated is seeded in seed tank, is cultivated at 25 DEG C
26h, the component of the fluid medium in the seed tank include by weight percentage：PDA culture medium, supplements 18% albumen
Peptone, 7% carbamide, 15% sucrose, 13% Testa Tritici, 0.3% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.04% vitamin B,
0.02% vitamin C, 0.015% zinc sulfate, 0.018% copper sulfate, 0.08% iron chloride, 0.07% calcium chloride；The dimension life
Plain B be vitamin B2, vitamin B3, the compositionss of vitamin B6, the vitamin B2, vitamin B3, the weight of vitamin B6
Than for 1:1.5:1.8；
(3) three-level fluid enlargement culture：The strain obtained after step (2) is cultivated is seeded in fermentation tank, is trained at 28 DEG C
Foster 30h, the component of the fluid enlargement culture base in the fermentation tank include by weight percentage：PDA culture medium, supplements 15%
Peptone, 18% sucrose, 17% analysis for soybean powder, 0.3% potassium dihydrogen phosphate, 0.07% magnesium sulfate, 0.04% vitamin B, 0.03%
Vitamin C, 0.10% calcium chloride, 0.8% sodium alginate；The vitamin B is vitamin B2, vitamin B3, vitamin B6
Compositionss, the vitamin B2, vitamin B3, the weight ratio of vitamin B6 are 1:1.5:1.8；
(4) level Four solid culture：Strain after step (3) amplification culture is inoculated in solid medium, at 24 DEG C
Culture 5d, the humidity for control level Four solid culture are 96%, the component of the solid medium count by weight including：Testa Tritici
25 parts, 15 parts of dried silkworm chrysalis meal, 15 parts of analysis for soybean powder, 2 parts of vitamin B, Catergen part, 7 parts of sucrose, 30 parts of Testa oryzae；Wherein, solid
The thickness of culture medium is 5.5cm, and the ratio of the affixed kind of liquid is 1:1.2, cover with level Four solid culture to solid medium mitogenetic
After spore, you can be dried process, after dried, spore extracting is carried out using cyclonic separation collection spore device, ball spore is obtained white
Stiff bacterium spore powder.
In the present embodiment, the beauveria bassiana spore powder that obtained using above-mentioned cultural method, its spore content are 8.8 × 1010/
G, spore germination rate are 96%, and spore rate living is 98%；The bioassay that spore virulence is carried out with 3 instar larvae of pine moth, after spray bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, humidity more than 90%) 4 days, there is peak mortality in pine moth, 7 days
Pine moth mortality rate is up to 95%.
Embodiment 2
A kind of cultural method of the beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
With.
In the cultural method：
(1) primary inclined plane culture：The component of the slant medium count by weight including：20 parts of potato starch,
1 part of peptone, 5 parts of Lactose, 1 part of sucrose, 0.05 part of potassium dihydrogen phosphate, 0.05 part of magnesium sulfate, 1 part of agar；
(2) secondary liquid culture：The component of the fluid medium in the seed tank includes by weight percentage：PDA
Culture medium, supplement 10% peptone, 5% carbamide, 5% sucrose, 10% Testa Tritici, 0.1% potassium dihydrogen phosphate, 0.01% magnesium sulfate,
0.01% vitamin B, 0.01% vitamin C, 0.01% zinc sulfate, 0.01% copper sulfate, 0.05% iron chloride, 0.05% chlorination
Calcium；The vitamin B is vitamin B2, vitamin B3, the compositionss of vitamin B6, and the vitamin B2, vitamin B3, dimension are given birth to
The weight ratio of plain B6 is 1:1:1；
(3) three-level fluid enlargement culture：The component of the fluid enlargement culture base in the fermentation tank is by weight percentage
Including：PDA culture medium, supplements 10% peptone, 10% sucrose, 10% analysis for soybean powder, 0.1% potassium dihydrogen phosphate, 0.05% sulphuric acid
Magnesium, 0.02% vitamin B, 0.01% vitamin C, 0.05% calcium chloride, 0.2% sodium alginate；The vitamin B is vitamin
B2, vitamin B3, the compositionss of vitamin B6, the vitamin B2, vitamin B3, the weight ratio of vitamin B6 are 1:1:1；
(4) level Four solid culture：The component of the solid medium count by weight including：Testa Tritici 20-30 parts, Pupa bombycises
10 parts of powder, 10 parts of analysis for soybean powder, 1 part of vitamin B, 1 part of vitamin C, 2 parts of sucrose, 20 parts of Testa oryzae；Wherein, the thickness of solid medium
Spend for 5.5cm, the ratio of the affixed kind of liquid is 1:1.2.
In the present embodiment, the beauveria bassiana spore powder that obtained using above-mentioned cultural method, its spore content are 8.2 × 1010/
G, spore germination rate are 95%, and spore rate living is 98%；The bioassay that spore virulence is carried out with 3 instar larvae of pine moth, after spray bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, humidity more than 90%) 6 days, there is peak mortality in pine moth, 8 days
Pine moth mortality rate is up to 95%.
Embodiment 3
A kind of cultural method of the beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
With.
In the cultural method：
(1) primary inclined plane culture：The component of the slant medium count by weight including：30 parts of potato starch,
2 parts of peptone, 10 parts of Lactose, 5 parts of sucrose, 0.1 part of potassium dihydrogen phosphate, 0.1 part of magnesium sulfate, 2 parts of agar；
(2) secondary liquid culture：The component of the fluid medium in the seed tank includes by weight percentage：PDA
Culture medium, supplements 20% peptone, 10% carbamide, 20% sucrose, 15% Testa Tritici, 0.5% potassium dihydrogen phosphate, 0.05% sulphuric acid
Magnesium, 0.05% vitamin B, 0.05% vitamin C, 0.02% zinc sulfate, 0.02% copper sulfate, 0.1% iron chloride, 0.1% chlorine
Change calcium；The vitamin B be vitamin B2, vitamin B3, the compositionss of vitamin B6, the vitamin B2, vitamin B3, dimension
The weight ratio of raw element B6 is 1:2:2；
(3) three-level fluid enlargement culture：The component of the fluid enlargement culture base in the fermentation tank is by weight percentage
Including：PDA culture medium, supplements 20% peptone, 20% sucrose, 20% analysis for soybean powder, 0.5% potassium dihydrogen phosphate, 0.1% sulphuric acid
Magnesium, 0.05% vitamin B, 0.02% vitamin C, 0.15% calcium chloride, 1.0% sodium alginate；The vitamin B is vitamin
B2, vitamin B3, the compositionss of vitamin B6, the vitamin B2, vitamin B3, the weight ratio of vitamin B6 are 1:2:2；
(4) level Four solid culture：The component of the solid medium count by weight including：30 parts of Testa Tritici, dried silkworm chrysalis meal
20 parts, 20 parts of analysis for soybean powder, vitamin B3 part, Catergen part, 10 parts of sucrose, 40 parts of Testa oryzae；Wherein, the thickness of solid medium
For 5.5cm, the ratio of the affixed kind of liquid is 1:1.3.
In the present embodiment, the beauveria bassiana spore powder that obtained using above-mentioned cultural method, its spore content are 8.8 × 1010/
G, spore germination rate are 97%, and spore rate living is 98%；The bioassay that spore virulence is carried out with 3 instar larvae of pine moth, after spray bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, humidity more than 90%) 4 days, there is peak mortality in pine moth, 8 days
Pine moth mortality rate is up to 95%.
Embodiment 4
A kind of cultural method of the beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
With.
In the cultural method：
(1) primary inclined plane culture：The component of the slant medium count by weight including：20 parts of potato starch,
2 parts of peptone, 5 parts of Lactose, 5 parts of sucrose, 0.05 part of potassium dihydrogen phosphate, 0.1 part of magnesium sulfate, 1 part of agar；
(2) secondary liquid culture：The component of the fluid medium in the seed tank includes by weight percentage：PDA
Culture medium, supplement 20% peptone, 5% carbamide, 20% sucrose, 10% Testa Tritici, 0.5% potassium dihydrogen phosphate, 0.01% magnesium sulfate,
0.05% vitamin B, 0.01% vitamin C, 0.02% zinc sulfate, 0.01% copper sulfate, 0.1% iron chloride, 0.05% chlorination
Calcium；The vitamin B is vitamin B2, vitamin B3, the compositionss of vitamin B6, and the vitamin B2, vitamin B3, dimension are given birth to
The weight ratio of plain B6 is 1:1:2；
(3) three-level fluid enlargement culture：The component of the fluid enlargement culture base in the fermentation tank is by weight percentage
Including：PDA culture medium, supplements 20% peptone, 10% sucrose, 20% analysis for soybean powder, 0.1% potassium dihydrogen phosphate, 0.1% sulphuric acid
Magnesium, 0.02% vitamin B, 0.02% vitamin C, 0.05% calcium chloride, 1.0% sodium alginate；The vitamin B is vitamin
B2, vitamin B3, the compositionss of vitamin B6, the vitamin B2, vitamin B3, the weight ratio of vitamin B6 are 1:2:1；
(4) level Four solid culture：The component of the solid medium count by weight including：20 parts of Testa Tritici, dried silkworm chrysalis meal
20 parts, 10 parts of analysis for soybean powder, 3 parts of vitamin B, 1 part of vitamin C, 10 parts of sucrose, 20 parts of Testa oryzae；Wherein, the thickness of solid medium
Spend for 5.5cm, the ratio of the affixed kind of liquid is 1:1.3.
In the present embodiment, the beauveria bassiana spore powder that obtained using above-mentioned cultural method, its spore content are 8.6 × 1010/
G, spore germination rate are 95%, and spore rate living is 98%；The bioassay that spore virulence is carried out with 3 instar larvae of pine moth, after spray bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, humidity more than 90%) 5 days, there is peak mortality in pine moth, 7 days
Pine moth mortality rate is up to 95%.
Embodiment 5
A kind of cultural method of the beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
With.
In the cultural method：
(1) primary inclined plane culture：The component of the slant medium count by weight including：30 parts of potato starch,
1 part of peptone, 10 parts of Lactose, 1 part of sucrose, 0.1 part of potassium dihydrogen phosphate, 0.05 part of magnesium sulfate, 2 parts of agar；
(2) secondary liquid culture：The component of the fluid medium in the seed tank includes by weight percentage：PDA
Culture medium, supplement 10% peptone, 10% carbamide, 5% sucrose, 15% Testa Tritici, 0.1% potassium dihydrogen phosphate, 0.05% magnesium sulfate,
0.01% vitamin B, 0.05% vitamin C, 0.01% zinc sulfate, 0.02% copper sulfate, 0.05% iron chloride, 0.1% chlorination
Calcium；The vitamin B is vitamin B2, vitamin B3, the compositionss of vitamin B6, and the vitamin B2, vitamin B3, dimension are given birth to
The weight ratio of plain B6 is 1:2:1；
(3) three-level fluid enlargement culture：The component of the fluid enlargement culture base in the fermentation tank is by weight percentage
Including：PDA culture medium, supplements 10% peptone, 20% sucrose, 10% analysis for soybean powder, 0.5% potassium dihydrogen phosphate, 0.05% sulphuric acid
Magnesium, 0.05% vitamin B, 0.01% vitamin C, 0.15% calcium chloride, 0.2% sodium alginate；The vitamin B is vitamin
B2, vitamin B3, the compositionss of vitamin B6, the vitamin B2, vitamin B3, the weight ratio of vitamin B6 are 1:1:2；
(4) level Four solid culture：The component of the solid medium count by weight including：30 parts of Testa Tritici, dried silkworm chrysalis meal
10 parts, 20 parts of analysis for soybean powder, 1 part of vitamin B, Catergen part, 2 parts of sucrose, 40 parts of Testa oryzae；Wherein, the thickness of solid medium
For 5.5cm, the ratio of the affixed kind of liquid is 1:1.2.
In the present embodiment, the beauveria bassiana spore powder that obtained using above-mentioned cultural method, its spore content are 8.7 × 1010/
G, spore germination rate are 95%, and spore rate living is 98%；The bioassay that spore virulence is carried out with 3 instar larvae of pine moth, after spray bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, humidity more than 90%) 5 days, there is peak mortality in pine moth, 6 days
Pine moth mortality rate is up to 95%.
Embodiment 6
A kind of cultural method of the beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
With.
In the cultural method：
(1) primary inclined plane culture：Slant medium is adjusted pH value to 6.5, then is pressurize sterilizing under 100kPa in pressure
60min, after cooling, beauveria bassiana thalline is inoculated in primary inclined plane culture medium, is cultivated 5 days at 24 DEG C；
(2) secondary liquid culture：The strain obtained after step (1) is cultivated is seeded in seed tank, is cultivated at 24 DEG C
24h；
(3) strain obtained after step (2) culture is seeded in fermentation tank, 28h is cultivated at 25 DEG C；
(4) level Four solid culture：Strain after step (3) amplification culture is inoculated in solid medium, at 22 DEG C
Culture 5d, the humidity for controlling level Four solid culture are 95%.
In the present embodiment, the beauveria bassiana spore powder that obtained using above-mentioned cultural method, its spore content are 8.6 × 1010/
G, spore germination rate are 95%, and spore rate living is 98%；The bioassay that spore virulence is carried out with 3 instar larvae of pine moth, after spray bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, humidity more than 90%) 5 days, there is peak mortality in pine moth, 7 days
Pine moth mortality rate is up to 95%.
Embodiment 7
A kind of cultural method of the beauveria bassiana described in the present embodiment, in addition to following characteristics, remaining with 1 phase of embodiment
With.
In the cultural method：
(1) primary inclined plane culture：Slant medium is adjusted pH value to 6.8, then is pressurize sterilizing under 110kPa in pressure
40min, after cooling, beauveria bassiana thalline is inoculated in primary inclined plane culture medium, is cultivated 5-6 days at 25 DEG C；
(2) secondary liquid culture：The strain obtained after step (1) is cultivated is seeded in seed tank, is cultivated at 25 DEG C
28h；
(3) strain obtained after step (2) culture is seeded in fermentation tank, 32h is cultivated at 28 DEG C；
(4) level Four solid culture：Strain after step (3) amplification culture is inoculated in solid medium, at 25 DEG C
Culture 6d, the humidity for controlling level Four solid culture are 98%.
In the present embodiment, the beauveria bassiana spore powder that obtained using above-mentioned cultural method, its spore content are 8.7 × 1010/
G, spore germination rate are 96%, and spore rate living is 98%；The bioassay that spore virulence is carried out with 3 instar larvae of pine moth, after spray bacterium
(spore liquid 0.1 × 108/ mL concentration, temperature are 20-28 DEG C, humidity more than 90%) 4 days, there is peak mortality in pine moth, 6 days
Pine moth mortality rate is up to 95%.
The above is only the preferred embodiment of the present invention, it is noted that it is right that above-mentioned preferred implementation is not construed as
The restriction of the present invention, protection scope of the present invention should be defined by claim limited range.For the art
For those of ordinary skill, without departing from the spirit and scope of the present invention, some improvements and modifications can also be made, these change
Enter and retouch also to should be regarded as protection scope of the present invention.
Claims (10)
1. a kind of cultural method of beauveria bassiana, it is characterised in that：The cultural method is comprised the following steps：
(1) primary inclined plane culture：Beauveria bassiana thalline is inoculated in primary inclined plane culture medium, 5-6 is cultivated at 24-25 DEG C
My god；
(2) secondary liquid culture：The strain obtained after step (1) is cultivated is seeded in seed tank, cultivates 24- at 24-25 DEG C
28h, the component of the fluid medium in the seed tank include by weight percentage：PDA culture medium, supplements 10-20% eggs
White peptone, 5-10% carbamide, 5-20% sucrose, 10-15% Testa Tritici, 0.1-0.5% potassium dihydrogen phosphates, 0.01-0.05% magnesium sulfate,
0.01-0.05% vitamin Bs, 0.01-0.05% vitamin Cs, 0.01-0.02% zinc sulfate, 0.01-0.02% copper sulfate,
0.05-0.1% iron chloride, 0.05-0.1% calcium chloride；
(3) three-level fluid enlargement culture：The strain obtained after step (2) is cultivated is seeded in fermentation tank, is cultivated at 25-28 DEG C
28-32h, the component of the fluid enlargement culture base in the fermentation tank include by weight percentage：PDA culture medium, supplements 10-
20% peptone, 10-20% sucrose, 10-20% analysis for soybean powder, 0.1-0.5% potassium dihydrogen phosphates, 0.05-0.1% magnesium sulfate,
0.02-0.05% vitamin Bs, 0.01-0.02% vitamin Cs, 0.05-0.15% calcium chloride, 0.2-1.0% sodium alginates；
(4) level Four solid culture：Strain after step (3) amplification culture is inoculated in solid medium, is trained at 22-25 DEG C
Foster 5-6d, the component of the solid medium count by weight including：Testa Tritici 20-30 parts, dried silkworm chrysalis meal 10-20 parts, analysis for soybean powder
10-20 parts, vitamin B 1-3 parts, vitamin C 1-2 parts, sucrose 2-10 parts, Testa oryzae 20-40 parts.
2. the cultural method of a kind of beauveria bassiana according to claim 1, it is characterised in that：Step (1) inclined-plane
The component of culture medium count by weight including：Potato starch 20-30 parts, peptone 1-2 parts, Lactose 5-10 part, sucrose 1-5
Part, potassium dihydrogen phosphate 0.05-0.1 parts, magnesium sulfate 0.05-0.1 parts, agar 1-2 parts.
3. the cultural method of a kind of beauveria bassiana according to claim 2, it is characterised in that：Step (1) inclined-plane
The component of culture medium count by weight including：25 parts of potato starch, 1 part of peptone, 8 parts of Lactose, 3 parts of sucrose, di(2-ethylhexyl)phosphate
0.08 part of hydrogen potassium, 0.06 part of magnesium sulfate, 2 parts of agar.
4. the cultural method of a kind of beauveria bassiana according to claim 1, it is characterised in that：Step (2) seed
The component of the fluid medium in tank includes by weight percentage：PDA culture medium, supplement 18% peptone, 7% carbamide,
15% sucrose, 13% Testa Tritici, 0.3% potassium dihydrogen phosphate, 0.02% magnesium sulfate, 0.04% vitamin B, 0.02% vitamin C,
0.015% zinc sulfate, 0.018% copper sulfate, 0.08% iron chloride, 0.07% calcium chloride.
5. the cultural method of a kind of beauveria bassiana according to claim 1, it is characterised in that：Step (3) fermentation
The component of the fluid enlargement culture base in tank includes by weight percentage：PDA culture medium, supplements 15% peptone, 18% sugarcane
Sugar, 17% analysis for soybean powder, 0.3% potassium dihydrogen phosphate, 0.07% magnesium sulfate, 0.04% vitamin B, 0.03% vitamin C, 0.10%
Calcium chloride, 0.8% sodium alginate.
6. the cultural method of a kind of beauveria bassiana according to claim 1, it is characterised in that：Step (2) and step (3)
In, the vitamin B is vitamin B1, vitamin B2, vitamin B3, at least one in vitamin B6.
7. the cultural method of a kind of beauveria bassiana according to claim 6, it is characterised in that：Step (2) and step (3)
In, the vitamin B is vitamin B2, vitamin B3, the compositionss of vitamin B6, and the vitamin B2, vitamin B3, dimension are given birth to
The weight ratio of plain B6 is 1:(1-2):(1-2).
8. the cultural method of a kind of beauveria bassiana according to claim 1, it is characterised in that：Step (1) inclined-plane
Culture medium first adjusts pH value before inoculation to 6.5-6.8, then is pressurize sterilizing 40-60min under 100-110kPa in pressure.
9. the cultural method of a kind of beauveria bassiana according to claim 1, it is characterised in that：In step (4), level Four is solid
The humidity of body culture is 95-98%.
10. the cultural method of a kind of beauveria bassiana according to claim 1, it is characterised in that：Solid training in step (4)
The thickness of foster base is 5-6cm, and the ratio of the affixed kind of liquid is 1:(1.2-1.3).
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| CN106906148A (en) * | 2017-04-25 | 2017-06-30 | 四川省农业科学院蚕业研究所 | A kind of culture medium of beauveria bassiana, the cultural method of beauveria bassiana |
| CN108719292A (en) * | 2018-07-07 | 2018-11-02 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of special synergistic agent of beauveria bassiana class biological pesticide and the preparation method and application thereof |
| CN109628333A (en) * | 2019-02-28 | 2019-04-16 | 广西地源之本肥业有限公司 | A kind of preparation method for the microorganism biological and ecological methods to prevent plant disease, pests, and erosion agent inhibiting insect pest |
| CN110093283A (en) * | 2019-05-15 | 2019-08-06 | 云南星耀生物制品有限公司 | Strain of Beauveria bassiana and its cultural method |
| CN110305801A (en) * | 2019-07-31 | 2019-10-08 | 四川省农业科学院经济作物育种栽培研究所 | A kind of method of cultivating in a fungus bag beauveria bassiana conidia powder |
| CN110352920A (en) * | 2019-08-30 | 2019-10-22 | 江苏海洋大学 | A method of the larva of a silkworm with batrytis is manually cultivated based on muscardine |
| CN111676142A (en) * | 2020-04-14 | 2020-09-18 | 中国烟草总公司陕西省公司 | Preparation and application method of beauveria bassiana powder |
| CN114058518A (en) * | 2021-11-26 | 2022-02-18 | 盐城市神微微生物菌种科技有限公司 | Culture method of beauveria bassiana |
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| CN106906148A (en) * | 2017-04-25 | 2017-06-30 | 四川省农业科学院蚕业研究所 | A kind of culture medium of beauveria bassiana, the cultural method of beauveria bassiana |
| CN108719292A (en) * | 2018-07-07 | 2018-11-02 | 安徽省农业科学院植物保护与农产品质量安全研究所 | A kind of special synergistic agent of beauveria bassiana class biological pesticide and the preparation method and application thereof |
| CN108719292B (en) * | 2018-07-07 | 2021-06-08 | 安徽省农业科学院植物保护与农产品质量安全研究所 | Special synergist for beauveria bassiana biological pesticide and preparation method and application thereof |
| CN109628333A (en) * | 2019-02-28 | 2019-04-16 | 广西地源之本肥业有限公司 | A kind of preparation method for the microorganism biological and ecological methods to prevent plant disease, pests, and erosion agent inhibiting insect pest |
| CN110093283A (en) * | 2019-05-15 | 2019-08-06 | 云南星耀生物制品有限公司 | Strain of Beauveria bassiana and its cultural method |
| CN110305801A (en) * | 2019-07-31 | 2019-10-08 | 四川省农业科学院经济作物育种栽培研究所 | A kind of method of cultivating in a fungus bag beauveria bassiana conidia powder |
| CN110352920A (en) * | 2019-08-30 | 2019-10-22 | 江苏海洋大学 | A method of the larva of a silkworm with batrytis is manually cultivated based on muscardine |
| CN110352920B (en) * | 2019-08-30 | 2021-09-14 | 江苏海洋大学 | Method for artificially culturing white muscardine silkworms based on beauveria bassiana |
| CN111676142A (en) * | 2020-04-14 | 2020-09-18 | 中国烟草总公司陕西省公司 | Preparation and application method of beauveria bassiana powder |
| CN114058518A (en) * | 2021-11-26 | 2022-02-18 | 盐城市神微微生物菌种科技有限公司 | Culture method of beauveria bassiana |
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