CN103667158A - Bacillus cereus and application of bacillus cereus - Google Patents
Bacillus cereus and application of bacillus cereus Download PDFInfo
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- CN103667158A CN103667158A CN201310737546.0A CN201310737546A CN103667158A CN 103667158 A CN103667158 A CN 103667158A CN 201310737546 A CN201310737546 A CN 201310737546A CN 103667158 A CN103667158 A CN 103667158A
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02W—CLIMATE CHANGE MITIGATION TECHNOLOGIES RELATED TO WASTEWATER TREATMENT OR WASTE MANAGEMENT
- Y02W30/00—Technologies for solid waste management
- Y02W30/40—Bio-organic fraction processing; Production of fertilisers from the organic fraction of waste or refuse
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Fertilizers (AREA)
Abstract
The invention relates to the field of microorganisms, and particularly discloses bacillus cereus. The bacillus cereus is collected in CGMCC (China general microbiological culture collection center), and has the collection number being CGMCC No.7979. The bacillus cereus can be stably colonized in the soil, the soil microbial environment is improved, the biological continuous cropping obstacle is eliminated, in addition, an obvious inhibition effect is realized on fusarium oxysporum, in addition, the growth of vanilla can be prompted, and the bacillus cereus can be applied to prevention and treatment of fusarium oxysporum, vanilla growth promotion and preparation of relevant products.
Description
Technical field
The present invention relates to microorganism field, relate in particular a kind of bacillus cereus and application thereof.
Background technology
Herba vanillae Planifoliae (Vanilla planifolia Andrews) is the orchid family torrid zone liana spice berry, has the good reputation of " king of natural flavor ".Its fresh beanpod contains more than 250 kind of fragrance ingredient after raw fragrant processing, and fragrance is unique, is widely used in modulating various high-grade cigarettes, famous brand of wine, tealeaves, is the perfume material of joining of all kinds of luxury foods and beverage, and supply falls short of demand in the international market.
Herba vanillae Planifoliae is perennial crop, increase along with Planting Years, soil microflora imbalance, the quantity of soil-borne pathogen rhizome maize ear rot pathogenic bacterium-Fusarium oxysporum obviously increases, it is simple that diversity of soil microorganism is tending towards, a little less than Herba vanillae Planifoliae growing way, there is continuous cropping biological barrier problem, seriously restricted the Sustainable development of Herba vanillae Planifoliae plant husbandry.For reducing pathogenic bacteria quantity, a large amount of agricultural chemicals that use in production, cause production cost increase, peasant burden to increase the weight of, and beanpod quality declines, ecotope severe contamination.At present, Herba vanillae Planifoliae plantation operation period garden mould passes pathogenic bacterium to be increased, and beneficial bacteria reduces, and soil microflora is unbalance has become the principal element of restriction Herba vanillae Planifoliae large-scale planting, is also key issue urgently to be resolved hurrily.
Summary of the invention
In view of this, the object of the present invention is to provide a kind of bacillus cereus, make this bacterial strain there is obvious restraining effect to Fusarium oxysporum, and can in soil, stablize and surely grow.
In addition, another goal of the invention of the present invention is the growth that described bacillus cereus microbial fertilizer can promote Herba vanillae Planifoliae.
For achieving the above object, the invention provides the bacterial strain that in Herba vanillae Planifoliae in age plantation, a kind of Cong20Nian garden, in the good rhizosphere soil of growing way, separation obtains, called after bacterial strain F-6, method for screening and separating is as follows:
Select the good rhizosphere soil of growing way in Herba vanillae Planifoliae in age plantation, 20Nian garden, sample is taken back laboratory dilution spread, cultivation.Select and on blood agar culture-medium, have the bacterial strain of obvious haemolysis circle (showing that it can produce antifungal lipopeptid class active substance), then sift out again Herba vanillae Planifoliae rhizome maize ear rot pathogenic bacterium-Fusarium oxysporum is had to remarkable inhibiting bacterial strain F-6 by dull and stereotyped dual test.By 16S rDNA method (its 16S rDNA is as shown in SEQ ID NO:1), identify that bacterial classification is bacillus cereus.Bacterial strain F-6 can produce antifungal lipopeptid class material, take well-grown under the condition that maltose, peptone is unique carbon and nitrogen sources.
Bacillus cereus of the present invention has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 3rd, 2013, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.7979.
Bacillus cereus of the present invention can significantly suppress Fusarium oxysporum, meanwhile, and with 10
7cfu/g quantity was used bacillus cereus of the present invention after 4 months, and its quantity in soil still remains on 10
5more than cfu/g, fully show that bacillus cereus of the present invention can be stablized surely to grow in soil, improve soil microorganisms environment, eliminate continuous cropping biological barrier.
Therefore, the present invention also provides application and the application in the product of preparation control Fusarium oxysporum of described bacillus cereus in control Fusarium oxysporum, and wherein, described product is preferably biological prevention and control agent or microbial fertilizer.
In addition, the present invention also provides a kind of biological prevention and control agent and preparation method thereof, and a kind of microbial fertilizer and preparation method thereof.
When bacillus cereus of the present invention is applied in the preparation of biological prevention and control agent, described biological prevention and control agent comprises that deposit number is acceptable auxiliary agent on the bacillus cereus of CGMCC No.7979 and biological prevention and control agent.On described biological prevention and control agent, acceptable auxiliary agent can be for the conventional material in this area, as carrier oil, activity increase agent and uv-protector etc.
The preparation method of described biological prevention and control agent is, the bacillus cereus that is CGMCC No.7979 by deposit number is inoculated in shaking culture in the NA liquid nutrient medium that pH value is 6-8, centrifugal thalline, resuspended with sterilized water, centrifugal again, after suspending with isopyknic sterilized water, add the acceptable auxiliary agent of biological prevention and control agent and get final product.Wherein, as preferably, described culture condition is 28 ℃-35 ℃, and hunting speed is turn/min of 160-220, and incubation time is 24h-48h, most preferably is 36h.As preferably, described NA liquid culture based formulas is: extractum carnis 5g/L, and peptone 10g/L, sodium-chlor 5g/L, surplus is water.On described biological prevention and control agent, acceptable auxiliary agent can be for the conventional material in this area, as carrier oil, activity increase agent and uv-protector etc.
When bacillus cereus of the present invention is applied in the preparation of microbial fertilizer, described microbial fertilizer, the bacillus cereus and the livestock poultry manure that by deposit number, are CGMCC No.7979 obtain through solid fermentation.As preferably, described livestock poultry manure is cow dung, pig manure, sheep excrement, chicken manure, duck excrement, goose excrement or their any number of mixture, preferred, described livestock poultry manure is cow dung, pig manure, sheep excrement, chicken manure, duck excrement, goose excrement or their any number of mixture after becoming thoroughly decomposed.Most preferred, described in each livestock poultry manure of becoming thoroughly decomposed, quality of organic matter ratio content >=35%, organonitrogen quality is 1.0%-2.0% than content, water content mass ratio is 25%-30%.
Described microorganism fertilizer preparation method for material comprises the following steps:
Step 1, the bacillus cereus that is CGMCC No.7979 by deposit number are inoculated in shaking culture in the NA liquid nutrient medium that pH value is 6-8, and centrifugal thalline is resuspended with sterilized water, more centrifugal, after suspending, obtain bacteria suspension with isopyknic sterilized water;
Wherein, as preferably, described culture condition is 28 ℃-35 ℃, and hunting speed is turn/min of 160-220, and incubation time is 24h-48h, most preferably is 36h.As preferably, described NA liquid culture based formulas is: extractum carnis 5g/L, and peptone 10g/L, sodium-chlor 5g/L, surplus is water.
As preferably, described livestock poultry manure is cow dung, pig manure, sheep excrement, chicken manure, duck excrement, goose excrement or their any number of mixture, preferred, described livestock poultry manure is cow dung, pig manure, sheep excrement, chicken manure, duck excrement, goose excrement or their any number of mixture after becoming thoroughly decomposed.Most preferred, described in each livestock poultry manure of becoming thoroughly decomposed, quality of organic matter ratio content >=35%, organonitrogen quality is 1.0%-2.0% than content, water content mass ratio is 25%-30%.
As preferably, the volume mass of described bacteria suspension and livestock poultry manure is than being 1mL-2mL:10g.
The preferred version of preparing as described microbial fertilizer, also comprises step 3:
Described microbial-bacterial fertilizer is carried out to after-ripening 3-5 days, finally in temperature 45 C-60, ℃ its water content is evaporated to below 30%.
Microbial fertilizer of the present invention, except can obviously suppressing Fusarium oxysporum, can also promote the growth of Herba vanillae Planifoliae, as improved Herba vanillae Planifoliae weight of root system, stem weight, leaf dry weight, root system diameter, root system cumulative volume etc.Use and contain 10 simultaneously
7the microbial fertilizer of cfu/g quantity bacillus cereus is after 4 months, and its quantity in soil still remains on 10
7more than cfu/g, fully show that microbial fertilizer of the present invention can be stablized surely to grow in soil, improve soil microorganisms environment, eliminate continuous cropping biological barrier.
From above technical scheme, bacillus cereus of the present invention can be stablized and surely grows in soil, improve soil microorganisms environment, eliminate continuous cropping biological barrier, and Fusarium oxysporum is had to obvious restraining effect, and can promote to can be applicable to the growth of Herba vanillae Planifoliae prevent and treat in the preparation of Fusarium oxysporum and related products.
Biological preservation explanation
Classification And Nomenclature: bacillus cereus, Bacillus cereus. is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 3rd, 2013, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.7979.
Accompanying drawing explanation
Figure 1 shows that the column diagram of different treatment on the impact of Herba vanillae Planifoliae plant overground part and weight of root system, the implication of each column diagram representative is referring to Fig. 1 legend, a, b, c, ab, bc, cd letter representation significant difference degree on column diagram;
Figure 2 shows that the column diagram of different treatment on the impact of Herba vanillae Planifoliae root system diameter and root system cumulative volume, the implication of each column diagram representative indicates referring to Fig. 2 transverse and longitudinal coordinate, a, b, ab letter representation significant difference degree on column diagram;
Figure 3 shows that the column diagram of different treatment on the impact of Herba vanillae Planifoliae rhizosphere soil Fusarium oxysporum quantity, the implication of each column diagram representative indicates referring to Fig. 3 transverse and longitudinal coordinate, a, b, c letter representation significant difference degree on column diagram;
Figure 4 shows that bacterial strain F-6 grows the column diagram of situation surely at Herba vanillae Planifoliae rhizosphere soil, the implication of each column diagram representative indicates referring to Fig. 4 transverse and longitudinal coordinate, a, b, c letter representation significant difference degree on column diagram.
Embodiment
The invention discloses a kind of bacillus cereus and application thereof, those skilled in the art can use for reference content herein, suitably improve processing parameter and realize.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the artly, they are all deemed to be included in the present invention.Bacillus cereus of the present invention and application thereof are described by preferred embodiment, related personnel obviously can change methods and applications as herein described or suitably change and combination within not departing from content of the present invention, spirit and scope, realizes and apply the technology of the present invention.
Below in conjunction with embodiment, further set forth the present invention.
Embodiment 1: the separated and evaluation of bacillus cereus of the present invention
Select the good rhizosphere soil of growing way in Herba vanillae Planifoliae in age plantation, 20Nian garden, sample is taken back laboratory dilution spread, cultivation.Select and on blood agar culture-medium, have the bacterial strain of obvious haemolysis circle (showing that it can produce antifungal lipopeptid class active substance), then sift out again Herba vanillae Planifoliae rhizome maize ear rot pathogenic bacterium-Fusarium oxysporum is had to remarkable inhibiting bacterial strain F-6 by dull and stereotyped dual test.By 16S rDNA method, identify bacterial classification, this bacterial strain F-6 belongs to bacillus cereus (Bacillus cereus), can produce antifungal lipopeptid class material, take well-grown under the condition that maltose, peptone is unique carbon and nitrogen sources.Bacterial classification is-80 ℃ of preservations in 30% glycerine.
Described bacillus cereus has been deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 3rd, 2013, address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.7979.
Embodiment 2: the preparation of biological prevention and control agent of the present invention
The bacillus cereus that is CGMCC No.7979 by deposit number is inoculated in the NA liquid nutrient medium that pH value is 6-8; shaking culture 36h under 28 ℃-35 ℃, turn/min of 160-220 rotating speed; centrifugal thalline; resuspended with sterilized water; centrifugal again, after suspending with isopyknic sterilized water, add carrier oil, activity increase agent and uv-protector and get final product.
Wherein, NA liquid culture based formulas is: extractum carnis 5g/L, and peptone 10g/L, sodium-chlor 5g/L, surplus is water.
Embodiment 3: the preparation of microbial fertilizer of the present invention
The bacillus cereus that is CGMCC No.7979 by deposit number is inoculated in the NA liquid nutrient medium that pH value is 6-8, shaking culture 36h under 28 ℃-35 ℃, turn/min of 160-220 rotating speed, centrifugal thalline, resuspended with sterilized water, centrifugal again, after suspending with isopyknic sterilized water, obtain bacteria suspension; Wherein, NA liquid culture based formulas is: extractum carnis 5g/L, and peptone 10g/L, sodium-chlor 5g/L, surplus is water.
By bacterial suspension inoculation, in the cow dung after becoming thoroughly decomposed, (volume mass of bacteria suspension and decomposed cow dung is than being 2mL:10g) carried out solid fermentation to described bacillus cereus quantity and reached 10
8more than cfu/g, during solid fermentation temperature not higher than 50 ℃, stir every day 1 time, obtain microbial-bacterial fertilizer.Wherein, quality of organic matter ratio content >=35% in the cow dung of becoming thoroughly decomposed, organonitrogen quality is 1.0%-2.0% than content, water content mass ratio is 25%-30%.
Resulting microbial-bacterial fertilizer is carried out to after-ripening 4 days, finally in temperature 45 C-60, ℃ its water content is evaporated to below 30%.
Microbial fertilizer through after-ripening contains 10
8bacillus cereus more than cfu/g, total npk nutrient quality is 5%-11% than content, quality of organic matter ratio content is 30%-40%.
Embodiment 4: pot experiment
The Herba vanillae Planifoliae plantation soil that gather 20Nian garden age, has an obvious continuous cropping biological barrier is as for examination soil, and test basin alms bowl adopts Turnover Box (inside dimension 600 * 420 * 165mm), chooses healthy Herba vanillae Planifoliae that growing way is consistent as for examination seedling.
Bacterial strain F-6 after green fluorescent protein molecule marker is seeded to 2000ml containing in the NA liquid nutrient medium of 0.3mg/ml kantlex, and shaking table is cultivated 36h, and centrifugal thalline is resuspended with sterilized water, more centrifugal, the bacteria suspension obtaining after suspending with isopyknic sterilized water; Wherein, NA liquid culture based formulas is: extractum carnis 5g/L, and peptone 10g/L, sodium-chlor 5g/L, surplus is water.
By bacterial suspension inoculation, in the cow dung after becoming thoroughly decomposed, (volume mass of bacteria suspension and decomposed cow dung is than being 2mL:10g) carried out solid fermentation to described bacillus cereus quantity and reached 10
8cfu/g, during solid fermentation temperature not higher than 50 ℃, stir every day 1 time, obtain microbial-bacterial fertilizer.Wherein, quality of organic matter ratio content >=35% in the cow dung of becoming thoroughly decomposed, organonitrogen quality is 1.0%-2.0% than content, water content mass ratio is 25%-30%.
4 processing are established in test: 1) contrast (CK uses equivalent chemical fertilizer); 2) use cow dung compost (OM, rate of fertilizer application 3%); 3) (F-6, inoculum density is 10 to use mark bacterial strain F-6 bacteria suspension
7cfu/g soil); 4) (F-6 inoculum density is 10 for BIO, rate of fertilizer application 3% to use microbial fertilizer
7cfu/g soil).The every potted plant kind 1 strain Herba vanillae Planifoliae of this pot experiment, every processing repeats 4 times, three independent experiments, totally 48 basins.
Testing index: crushing sampling after Herba vanillae Planifoliae is transplanted 4 months, adopts root scanner to measure Root morphology; Plant overground part and weight of root system; Rhizosphere soil mark bacterial strain F-6 and Fusarium oxysporum quantity.Result is shown in respectively Fig. 1-Fig. 4.
As shown in Figure 1, processing Herba vanillae Planifoliae weight of root system, leaf dry weight and the stem of using microbial fertilizer is heavily except stem is heavy and use cow dung compost processes significantly, and all the other are all significantly higher than other processing.
As shown in Figure 2, use processing root system diameter and the root system cumulative volume of microbial fertilizer and all higher than other, process, microbial fertilizer is used in this explanation can promote Herba vanillae Planifoliae root growth.In addition, use the processing root system diameter of cow dung and cumulative volume no significant difference compared with the control, show to use cow dung fertilizer to improving Herba vanillae Planifoliae root growth situation without unusual effect.
As shown in Figure 3, use the processing Herba vanillae Planifoliae rhizosphere soil Fusarium oxysporum quantity of microbial fertilizer and bacterium liquid F-6 significantly lower than the processing that contrasts and use cow dung, use the processing Fusarium oxysporum quantity of microbial fertilizer significantly lower than the processing of using F-6, in addition, contrast and use between the processing of cow dung difference not remarkable.Show to use microbial fertilizer or F-6 bacterium liquid all can significantly suppress the growth of Herba vanillae Planifoliae rhizosphere soil harmful fungoid Fusarium oxysporum, reduce it in rhizosphere soil quantity.
As shown in Figure 4, use F-6 bacterium liquid or its microbial fertilizer after 4 months, the concentration of F-6 in soil is respectively 10
5cfu/g and 10
7more than cfu/g, and this bacterial strain do not detected in contrast and in using the processing of cow dung compost, this explanation F-6 can stablize and surely grow in soil, is beneficial to Herba vanillae Planifoliae growth, improves soil microorganisms environment, elimination continuous cropping biological barrier.And the processing F-6 that uses microbial fertilizer is significantly higher than the processing of using separately bacterium liquid in the quantity of rhizosphere soil, this shows that microbial organic fertilizer can be F-6 abundant carbon and nitrogen sources is provided, and is more conducive to it and survives in soil.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention; can also make some improvements and modifications, these improvements and modifications also should be considered as protection scope of the present invention.
Claims (10)
1. a bacillus cereus, deposit number is CGMCC No.7979.
2. bacillus cereus according to claim 1, is characterized in that, 16S rDNA is as shown in SEQ ID NO:1.
3. the application of bacillus cereus in control Fusarium oxysporum described in claim 1.
4. the application of bacillus cereus in the product of preparation control Fusarium oxysporum described in claim 1.
5. application according to claim 4, is characterized in that, described product is biological prevention and control agent or microbial fertilizer.
6. a biological prevention and control agent, is characterized in that, comprises that deposit number is acceptable auxiliary agent on the bacillus cereus of CGMCC No.7979 and biological prevention and control agent.
7. the preparation method of a biological prevention and control agent, it is characterized in that, the bacillus cereus that is CGMCC No.7979 by deposit number is inoculated in shaking culture in the NA liquid nutrient medium that pH value is 6-8, centrifugal thalline, resuspended with sterilized water, centrifugal again, after suspending with isopyknic sterilized water, add the acceptable auxiliary agent of biological prevention and control agent and get final product.
8. a microbial fertilizer, is characterized in that, the bacillus cereus and the livestock poultry manure that by deposit number, are CGMCC No.7979 obtain through solid fermentation.
9. a preparation method for microbial fertilizer, is characterized in that, comprises the following steps:
Step 1, the bacillus cereus that is CGMCC No.7979 by deposit number are inoculated in shaking culture in the NA liquid nutrient medium that pH value is 6-8, and centrifugal thalline is resuspended with sterilized water, more centrifugal, after suspending, obtain bacteria suspension with isopyknic sterilized water;
Step 2, bacterial suspension inoculation is reached to 10 to carrying out solid fermentation to described bacillus cereus quantity in livestock poultry manure
8more than cfu/g, during solid fermentation temperature not higher than 50 ℃, obtain microbial-bacterial fertilizer.
10. preparation method according to claim 9, is characterized in that, also comprises step 3:
Described microbial-bacterial fertilizer is carried out to after-ripening 3-5 days, finally in temperature 45 C-60, ℃ its water content is evaporated to below 30%.
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