CN109722397A - Bacillus cercus YUPP-10 microbial inoculum and its preparation method and application - Google Patents
Bacillus cercus YUPP-10 microbial inoculum and its preparation method and application Download PDFInfo
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Abstract
The invention belongs to agricultural biological technical fields, and in particular to Bacillus cercus YUPP-10 microbial inoculum and its preparation method and application.The microbial bacterial agent is fermented by Bacillus cercus (Bacillus cereus) YUPP-10, and deposit number is CCTCC M 2017141.The microbial-bacterial fertilizer shows preferable control efficiency to cotton verticillium wilt；It can be directly used for seed pelleting, also can be used for trickle irrigation.YUPP-10 bacterial strain is to separate to obtain from healthy cotton plants, nontoxic, no pathogenicity compatible with soil ecology harmony, therefore has biggish application potential in the biological control of cotton verticillium wilt.
Description
Technical field
The invention belongs to agricultural biological technical fields, and in particular to Bacillus cercus YUPP-10 microbial inoculum and its preparation side
Method and application.
Background technique
Cotton verticillium wilt is the silborne fungal diseases as caused by verticillium dahliae, seriously affects output of cotton and fabric
Matter.Due to long-term continuous cropping, verticillium wilt pathogen is increasingly accumulated in soil, lacks disease-resistant variety and the agriculture with preferable control efficiency in addition
Medicine, Cotton in China verticillium wilt are on the rise, and become one of the main limiting factor that cotton is developed production.Currently, economical and effective control
For the approach of verticillium wilt processed other than planting disease-resistant variety, biological control is that one kind is suitble to current China's agricultural production " weight-reducing subtracts medicine "
The important channel of target.
The biological control of plant disease is to utilize beneficial microbe and microbial metabolic products prevention and control crop disease
Technology and methods.Biological control has the characteristics of pollution-free, sustainable development.It is developed as the bacterium of biological pesticide at present
Bacterial strain focuses primarily upon agrobacterium (Agrobacterium sp.), bacillus (Bacillus sp.) and pseudomonad
(Pseudomonas sp.), wherein bacillus accounts for larger proportion.
Although the biocontrol microorganisms for having some pairs of plant diseases that there are better effects in the prior art, by biocontrol microorganisms application
When into control of plant disease work, often due to biocontrol microorganisms product does not have higher activity or stability, is unable to satisfy storage
It is required that or can not reform of nature environment, so as to cause preferable control in field effect can not be obtained.
Summary of the invention
The purpose of the present invention is to provide a kind of Bacillus cercus YUPP-10 microbial inoculums.
A further object of the present invention is to provide the preparation methods of above-mentioned microbial inoculum.
A further object of the present invention is to provide the applications of above-mentioned Bacillus cercus YUPP-10 microbial inoculum.
The Bacillus cercus YUPP-10 microbial inoculum of specific embodiment according to the present invention, by what is included the following steps
Method preparation:
(1) fermentation liquid of bacillus YUPP-10 is prepared, Bacillus cercus YUPP-10 (Bacillus cereus) is protected
It is hidden in China typical culture collection center (CCTCC), deposit number is CCTCC No:M2017141；
(2) the resulting fermentation liquid of step (1) is added to the CaCl of mass percent 1%~2%2Solution is stood, centrifugation,
Collect bacterium mud；
(3) it is added calcium carbonate into step (2) resulting bacterium mud, the mass ratio of bacterium mud and calcium carbonate is 1:0.5~1,38
~42 DEG C of crushed after being dried obtain said waxy bacillus YUPP-10 microbial inoculum.
CaCl2Solution plays the role of accelerating precipitating in fermentation liquid, can achieve the goal greater than 1%, general control
CaCl21%~2% that volume is fermentating liquid volume is added in solution.
The Bacillus cercus YUPP-10 microbial inoculum of specific embodiment according to the present invention, in step (1), bacillus
YUPP-10 successively passes through actication of culture, seed liquor preparation, seeding tank fermented and cultured and fermented and cultured step, obtains the gemma
The fermentation liquid of bacillus YUPP-10.
The Bacillus cercus YUPP-10 microbial inoculum of specific embodiment according to the present invention, in actication of culture step, by wax
Shape bacillus YUPP-10 streak inoculation is in LB solid medium tablets, for 24 hours in 37 DEG C of dark cultures.
The Bacillus cercus YUPP-10 microbial inoculum of specific embodiment according to the present invention will in seed liquor preparation step
Bacillus cercus YUPP-10 after activation is inoculated in the triangular flask equipped with LB liquid medium, 37 in constant temperature oscillator
DEG C, 200rpm shaken cultivation for 24 hours, obtains seed liquor.
The Bacillus cercus YUPP-10 microbial inoculum of specific embodiment according to the present invention, seeding tank fermented and cultured step
In, seed liquor is inoculated into 10L seeding tank with the inoculum concentration of percentage by volume 4%~6%, fluid nutrient medium fills in seeding tank
Liquid measure is 6L, in 37 DEG C of culture 12h, obtains fermentation culture medium.
The Bacillus cercus YUPP-10 microbial inoculum of specific embodiment according to the present invention, in fermented and cultured step, by institute
It states fermentation culture medium to be inoculated into 100L fermentor with the inoculum concentration of percentage by volume 4%~6%, fluid nutrient medium in fermentor
Liquid amount is 70L, and 37 DEG C of culture 48h obtain the fermentation liquid of said waxy bacillus YUPP-10.
The method of Bacillus cercus YUPP-10 microbial inoculum provided by the invention comprising following steps:
(1) fermentation liquid of Bacillus cercus YUPP-10 is prepared；
(2) the resulting fermentation liquid of step (1) is added to the CaCl of percentage by volume 1%~2%2In solution, stand, from
The heart collects bacterium mud；
(3) it is added calcium carbonate into step (2) resulting bacterium mud, the mass ratio of bacterium mud and calcium carbonate is 1:0.5~1, is done
After dry, said waxy bacillus YUPP-10 microbial inoculum is obtained.
The present invention also provides application of the Bacillus cercus YUPP-10 microbial inoculum in prevention and treatment cotton verticillium wilt.
The application mode of the Bacillus cercus YUPP-10 microbial inoculum of specific embodiment according to the present invention are as follows:
(1) cotton seeds are coated with the microbial inoculum, the usage amount of the microbial inoculum is 0.5Kg/10Kg seed~1Kg/
10Kg seed.
(2) at the end of May to the initial stage that cotton verticillium wilt at the beginning of 6 months is fallen ill, with the microbial inoculum with water Stage of Cotton Under-Mulch-Drip Irrigation, the microbial inoculum
Usage amount be 1kg/ mus~2kg/ mus.
Bacillus cercus YUPP-10 used in the present invention is isolated from cotton stalk, compatible with soil ecology harmony, has
Conducive to the advantage for giving full play to bacterial strain.
Microbial inoculum living bacteria count of the invention is 1.0 × 1010~1.5 × 1010CFU/g can significantly mitigate cotton yellow and wither
The generation of disease, can reduce farmland pollution, and can improve soil texture.
Biocontrol Strain condition of culture used in the present invention is simple, easy to industrialized production, and there is good exploitation to answer
Use prospect.
Detailed description of the invention
Fig. 1 is Bacillus cercus YUPP-10 microbial inoculum to cotton seeds coating effect figure, and wherein A is capsuled seed, and B is
Uncoated seed.
Bacillus cercus (Bacillus cereus) YUPP-10 of the invention, was preserved on March 24th, 2017
State's Type Tissue Collection (Wuhan, China Wuhan University, postcode 430072), deposit number are CCTCC No:
M2017141。
Specific embodiment
Embodiment 1
The preparation process of Bacillus cercus YUPP-10 microbial inoculum of the invention:
(1) actication of culture: by Bacillus cercus YUPP-10 streak inoculation in LB solid medium tablets, in 37 DEG C
Dark culture is for 24 hours；
(2) prepared by seed liquor: the Bacillus cercus YUPP-10 after activation is inoculated in three equipped with LB liquid medium
In the bottle of angle, 37 DEG C in constant temperature oscillator, 200rpm shaken cultivation obtains seed liquor for 24 hours；
(3) seed liquor in step (2) seeding tank fermented and cultured: is inoculated into 10L with the inoculum concentration of percentage by volume 4%
In seeding tank, fluid nutrient medium liquid amount is 6L in seeding tank, obtains fermentation culture medium in 37 DEG C of culture 12h；
(4) fermentation culture medium in step (3) fermented and cultured: is inoculated into 100L with the inoculum concentration of percentage by volume 4%
In fermentor, fluid nutrient medium liquid amount is 70L in fermentor, and 37 DEG C of culture 48h obtain Bacillus cercus YUPP-10's
Fermentation liquid；
(5) CaCl of mass percent 1% is added into the fermentation liquid of step (4)2Solution, CaCl2Volume is added in solution
The 1% of fermentating liquid volume, after standing 30min, bacterium mud is collected in centrifugation；
(6) it is added calcium carbonate powder into bacterium mud, the mass ratio of bacterium mud and calcium carbonate is 1:0.5, is subsequently placed in 38 DEG C of perseverances
80 meshes are crossed after dry, pulverize in warm drying box obtains microbial inoculum.
Embodiment 2
The preparation process of Bacillus cercus YUPP-10 microbial inoculum of the invention:
(1) actication of culture: by Bacillus cercus YUPP-10 streak inoculation in LB solid medium tablets, in 37 DEG C
Dark culture is for 24 hours；
(2) prepared by seed liquor: the Bacillus cercus YUPP-10 after activation is inoculated in three equipped with LB liquid medium
In the bottle of angle, 37 DEG C in constant temperature oscillator, 200rpm shaken cultivation obtains seed liquor for 24 hours；
(3) seed liquor in step (2) seeding tank fermented and cultured: is inoculated into 10L with the inoculum concentration of percentage by volume 5%
In seeding tank, fluid nutrient medium liquid amount is 6L in seeding tank, obtains fermentation culture medium in 37 DEG C of culture 12h；
(4) fermentation culture medium in step (3) fermented and cultured: is inoculated into 100L with the inoculum concentration of percentage by volume 5%
In fermentor, fluid nutrient medium liquid amount is 70L in fermentor, and 37 DEG C of culture 48h obtain Bacillus cercus YUPP-10's
Fermentation liquid；
(5) CaCl of mass percent 2% is added into the fermentation liquid of step (4)2Solution, CaCl2Volume is added in solution
The 2% of fermentating liquid volume, after standing 30min, bacterium mud is collected in centrifugation；
(6) it is added calcium carbonate powder into bacterium mud, the mass ratio of bacterium mud and calcium carbonate is 1:0.6, is subsequently placed in 40 DEG C of perseverances
80 meshes are crossed after dry, pulverize in warm drying box obtains microbial inoculum.
Embodiment 3
The preparation process of Bacillus cercus YUPP-10 microbial inoculum of the invention:
(1) actication of culture: by Bacillus cercus YUPP-10 streak inoculation in LB solid medium tablets, in 37 DEG C
Dark culture is for 24 hours；
(2) prepared by seed liquor: the Bacillus cercus YUPP-10 after activation is inoculated in three equipped with LB liquid medium
In the bottle of angle, 37 DEG C in constant temperature oscillator, 200rpm shaken cultivation obtains seed liquor for 24 hours；
(3) seed liquor in step (2) seeding tank fermented and cultured: is inoculated into 10L with the inoculum concentration of percentage by volume 6%
In seeding tank, fluid nutrient medium liquid amount is 6L in seeding tank, obtains fermentation culture medium in 37 DEG C of culture 12h；
(4) fermentation culture medium in step (3) fermented and cultured: is inoculated into 100L with the inoculum concentration of percentage by volume 6%
In fermentor, fluid nutrient medium liquid amount is 70L in fermentor, and 37 DEG C of culture 48h obtain Bacillus cercus YUPP-10's
Fermentation liquid；
(5) CaCl of mass percent 1% is added into the fermentation liquid of step (4)2Solution, CaCl2Volume is added in solution
The 1% of fermentating liquid volume, after standing 30min, bacterium mud is collected in centrifugation；
(6) it is added calcium carbonate powder into bacterium mud, the mass ratio of bacterium mud and calcium carbonate is 1:1, is subsequently placed in 42 DEG C of constant temperature
80 meshes are crossed after dry, pulverize in drying box obtains microbial inoculum.
The detection of 4 Bacillus cercus YUPP-10 microbial inoculum viable count of embodiment
(1) bacterium mud that step (5) obtains in bacterial preparation process of the present invention is divided into 4 parts, every part of 1Kg, then respectively with
The calcium carbonate powder of 0.3Kg, 0.6Kg, 0.9Kg, 1.2Kg mix, number consecutively A, B, C, D, then A, B, C, D are each separated into
3 parts, number A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, D3, A1, B1, C1 and D1 are then placed in 35 DEG C of constant temperature
Dry in drying box, A2, B2, C2 and D2 are placed in drying in 40 DEG C of thermostatic drying chambers, and it is dry that A3, B3, C3 and D3 are placed in 45 DEG C of constant temperature
It is dry in dry case, 80 meshes are smashed it through respectively after dry.
(2) it after mixing well above-mentioned sample A1, A2, A3, B1, B2, B3, C1, C2, C3, D1, D2, D3, respectively weighs
0.1g is added in sterile water containing 10mL, is placed in 25 DEG C of shaking tables and shakes 10min, then carry out 10 times of gradient dilutions, until 10-8g/
Then mL takes 0.5mL to be spread evenly across on LB plate, dries up in superclean bench, be placed in 37 DEG C of constant incubators and cultivate,
The bacterium colony of planar surface is counted after 1 day.Each processing is repeated 5 times.
The quantity of viable bacteria in the different samples of table 1
As shown in Table 1, the content highest that viable bacteria in B2 and C2 is handled in the present invention, respectively up to 1.576 × 1010/ g and
1.692×1010/ g, remaining processing are substantially less than processing B2 and C2.The result illustrates the bacterium mud and carbon of YUPP-10 in the present invention
The optimal proportion of sour calcium powder is 1:0.6~0.9；The temperature that YUPP-10 bacterium mud and calcium carbonate powder are most preferably dried after mixing is
40℃。
Application of the 5 Bacillus cercus YUPP-10 microbial inoculum of embodiment in a manner of seed coat agent in verticillium wilt prevention and treatment
4 parts of cotton seeds that 2000g kind is Lu Mianyan 21 are weighed, 50mL clear water is separately added into, the surface of the seed is moistened,
It is then respectively adding Bacillus cercus YUPP-10 microbial inoculum 100g, 150g, 200g and bacillus subtilis (commercially available) of the invention
150g is mixed well, and is placed in porcelain dish and is dried, as shown in Figure 1, spare.
It chooses one piece of cotton verticillium wilt and lays particular stress on and plot occurs, carry out prevention and treatment verticillium wilt test.5 processing of test setting, point
It Wei not Bacillus cercus YUPP-10 microbial inoculum 0.5Kg/10Kg seed (processing 1), Bacillus cercus YUPP-10 microbial inoculum
0.75Kg/10Kg seed (processing 2), Bacillus cercus YUPP-10 microbial inoculum 1.0Kg/10Kg seed (processing 3), withered grass gemma
Bacillus 0.75Kg/10Kg seed and uncoated blank control, 3 repetitions of each processing.It is broadcast in cotton normal sowing time
Kind, it after planting routinely requires to be managed.A situation arises for 45 days and 60 days investigation diseases after sowing cotton seed, and investigation method is pressed
It is carried out according to national standard, calculates disease index and protection effect.The calculation formula of control efficiency is as follows:
Control efficiency (%)=(control disease index-processing disease index)/control disease index × 100
Different disposal the results are shown in Table 2 to the control efficiency of cotton verticillium wilt:
Control efficiency of the 2 Bacillus cercus YUPP-10 microbial inoculum seed pelleting of table to cotton verticillium wilt disease
As shown in Table 2, the Bacillus cercus YUPP-10 microbial inoculum in the present invention is by seed coat agent mode to cotton verticillium wilt
With preferable control efficiency.When Bacillus cercus YUPP-10 microbial inoculum dosage is 0.5Kg/10Kg seed~1Kg/10Kg kind
The period of the day from 11 p.m. to 1 a.m, to cotton verticillium wilt control efficiency be 33.3%~44.9% (after planting 45 days) and 29.2%~39.2% (after planting
60 days), better than the control efficiency of commercially available bacillus subtilis.
Application of the 6 Bacillus cercus YUPP-10 microbial inoculum of embodiment with drip irrigation in verticillium wilt prevention and treatment
It chooses one piece of cotton verticillium wilt and lays particular stress on and plot occurs, carry out prevention and treatment verticillium wilt test, be new land for examination cotton variety
In 37.
It is sowed in cotton normal sowing time, after planting routinely requires to be managed.On May 25 or so started to carry out
Disease control test.It is 6 processing of test setting, respectively 1Kg/ mu of Bacillus cercus YUPP-10 microbial inoculum (handle 1), wax-like
It is 1.5Kg/ mus of bacillus YUPP-10 microbial inoculum (processing 2), 2.0Kg/ mu of Bacillus cercus YUPP-10 microbial inoculum (handling 3), withered
The YUPP-10 fermentation liquid (processing 5) of the bacterium amounts such as careless bacillus 1.5Kg/ mus of seeds (processing 4) and processing 2 and blank control,
3 repetitions of each processing.Fill with water droplet according to the dosage in each processing and test.When trickle irrigation, 2 hours clear water of first trickle irrigation, then
2 hours microbial inoculums of trickle irrigation, then 1 hour clear water of trickle irrigation, total water consumption are 10 tons/mu.Trickle irrigation is carried out after 10 days.After second of trickle irrigation
A situation arises for 15 days and 30 days investigation diseases, and investigation method carries out according to national standards, calculates disease index and protection effect.
The calculation formula of control efficiency is as follows:
Control efficiency (%)=(control disease index-processing disease index)/control disease index × 100
Different disposal the results are shown in Table 3 to the control efficiency of cotton verticillium wilt:
Control efficiency of the 3 Bacillus cercus YUPP-10 microbial inoculum trickle irrigation of table to cotton verticillium wilt disease
As shown in Table 3, the Bacillus cercus YUPP-10 microbial inoculum in the present invention has cotton verticillium wilt by drip irrigation
There is preferable control efficiency.When Bacillus cercus YUPP-10 microbial inoculum dosage is 1.0Kg/ mus~2.0Kg/ mus, to cotton yellow
Disease of withering control efficiency is 39.2%~51.3% (after trickle irrigation 15 days) and 33.2%~45.6% (after trickle irrigation 30 days), better than commercially available
The control efficiency of bacillus subtilis.
Claims (10)
1. Bacillus cercus YUPP-10 microbial inoculum, which is characterized in that said waxy bacillus YUPP-10 microbial inoculum by include with
It is prepared by the method for lower step:
(1) fermentation liquid of Bacillus cercus YUPP-10 is prepared, the deposit number of Bacillus cercus YUPP-10 is CCTCC
No:M2017141；
(2) the resulting fermentation liquid of step (1) is added to the CaCl of mass percent 1%~2%2It in solution, stands, is centrifuged, collect
Bacterium mud；
(3) it is added calcium carbonate into step (2) resulting bacterium mud, the mass ratio of bacterium mud and calcium carbonate is 1:0.5~1,38~42
After DEG C dry, said waxy bacillus YUPP-10 microbial inoculum is obtained.
2. Bacillus cercus YUPP-10 microbial inoculum according to claim 1, which is characterized in that in step (1), gemma bar
Bacterium YUPP-10 successively passes through actication of culture, seed liquor preparation, seeding tank fermented and cultured and fermented and cultured step, obtains the bud
The fermentation liquid of spore bacillus YUPP-10.
3. Bacillus cercus YUPP-10 microbial inoculum according to claim 2, which is characterized in that, will in actication of culture step
Bacillus cercus YUPP-10 streak inoculation is in LB solid medium tablets, for 24 hours in 37 DEG C of dark cultures.
4. Bacillus cercus YUPP-10 microbial inoculum according to claim 2, which is characterized in that in seed liquor preparation step,
Bacillus cercus YUPP-10 after activation is inoculated in the triangular flask equipped with LB liquid medium, in constant temperature oscillator
37 DEG C, 200rpm shaken cultivation for 24 hours, obtains seed liquor.
5. Bacillus cercus YUPP-10 microbial inoculum according to claim 4, which is characterized in that seeding tank fermented and cultured step
In rapid, seed liquor is inoculated into 10L seeding tank with the inoculum concentration of percentage by volume 4%~6%, fluid nutrient medium in seeding tank
Liquid amount is 6L, in 37 DEG C of culture 12h, obtains fermentation culture medium.
6. Bacillus cercus YUPP-10 microbial inoculum according to claim 5, which is characterized in that, will in fermented and cultured step
The fermentation culture medium is inoculated into 100L fermentor with the inoculum concentration of percentage by volume 4%~6%, Liquid Culture in fermentor
The liquid amount of base is 70L, and 37 DEG C of culture 48h obtain the fermentation liquid of said waxy bacillus YUPP-10.
7. the method for preparing Bacillus cercus YUPP-10 microbial inoculum described in claim 1, which is characterized in that the method packet
Include following steps:
(1) fermentation liquid of Bacillus cercus YUPP-10 is prepared, the deposit number of Bacillus cercus YUPP-10 is CCTCC
No:M2017141；
(2) the resulting fermentation liquid of step (1) is added to the CaCl of percentage by volume 1%~2%2It in solution, stands, is centrifuged, collect
Bacterium mud；
(3) it is added calcium carbonate into step (2) resulting bacterium mud, the mass ratio of bacterium mud and calcium carbonate is 1:0.5~1, after dry,
Obtain said waxy bacillus YUPP-10 microbial inoculum.
8. application of the Bacillus cercus YUPP-10 microbial inoculum according to claim 1 in prevention and treatment cotton verticillium wilt.
9. application according to claim 8, which is characterized in that use said waxy bacillus YUPP-10 microbial inoculum by cotton
Seed pelleting, the usage amount of said waxy bacillus YUPP-10 microbial inoculum are 0.5Kg/10Kg seed~1Kg/10Kg seed.
10. application according to claim 8, which is characterized in that in cotton verticillium wilt early stage, with said waxy gemma
Bacillus YUPP-10 microbial inoculum is 1kg/ mus~2kg/ with water Stage of Cotton Under-Mulch-Drip Irrigation, the usage amount of said waxy bacillus YUPP-10 microbial inoculum
Mu.
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| CN115948279A (en) * | 2022-09-30 | 2023-04-11 | 精河县农业技术推广中心 | Compound microbial agent for preventing and treating cotton verticillium wilt and preparation method thereof |
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Application publication date: 20190507 |