CN103103149A - Bacillus subtilis S001, application of bacillus subtilis S001, microbial preparation and preparation method of microbial preparation - Google Patents
Bacillus subtilis S001, application of bacillus subtilis S001, microbial preparation and preparation method of microbial preparation Download PDFInfo
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Abstract
The invention discloses bacillus subtilis S001 capable of inhibiting a variety of pathogens and further discloses a microbial preparation prepared from the bacillus subtilis S001 and a preparation method the microbial preparation. The bacillus subtilis S001 with the collection number of CGMCC No.6425 has the function of promoting growth and inhibiting pathogens including the fusarium, pepper rhizoctonia solani, tomato leaf mold, tomato early blight pathogen, tomato grey mold, eggplant sclerotia pathogen, wheat full-rotting pathogen, wheat root rotting pathogen and wheat sheath blight pathogen. The microbial pesticides and microbial fertilizers prepared from the bacillus subtilis S001 are environmentally-friendly and have good safety, and pathogens can not resist the microbial pesticides and the microbial fertilizers easily.
Description
Technical field
The present invention relates to the biological and ecological methods to prevent plant disease, pests, and erosion field, the microbiobacterial agent and the preparation method that are specifically related to subtilis S001 and its application and prepare with it.
Background technology
Along with the raising of people's living standard and the enhancing of environmental consciousness, in International or National market, vegetable products is carried out to access system, the vegetables that come into the market must be pollution-free vegetables.Chemical bactericide can be residual in environment and agricultural-food, and it is having a strong impact on the whole people's physical and mental health, influence ecological environment and agricultural trade simultaneously, the also murder by poisoning as people, domestic animal, beneficial insect and wildlife etc. to nontarget organism.The vegetable pesticide residue amount exceeds standard, and faces " Green Trade Barrier ", under the overall situation of WTO, can't enter world market.This will ask for help pesticide species and performance are adjusted, to the public nuisance-free agricultural chemicals future development.Biological pesticide is to produce the good option of preventing and treating disease in the non-polluted farm product process.
Microbial pesticide is " public nuisance-free agricultural chemicals " of generally acknowledging, can prevent and treat disease by the direct or indirect mode of action, and its prevention effect is better, nontoxic to people and animals, free from environmental pollution, noresidue; The selectivity that tool is stronger, do not injure beneficial organism and natural enemy, do not destroy the eubiosis, is difficult for developing immunity to drugs; Raw materials for production and effective constituent are natural product, and easily degraded, can go back to nature, and guarantee Sustainable development; Along with carrying out of Law on Environmental Protection, and participate in competition in the international market, microbial pesticide will, as a kind of new and high technology, in protection of the environment, assurance human health, in agricultural sustainable development, be brought into play more and more important effect.
The chemical nitrogen fertilizer be manured into soil in a large number, phosphate fertilizer and potash fertilizer can cause heavy metal to be accumulated in the soil body, natural resources and energy resources consumes in a large number, environmental pollution, and its utilization ratio is low, the nitrogenous fertilizer be not absorbed and used runs off or volatilizees in farmland, underground water resources, soil and air have been caused to severe contamination, the toxic substance of agricultural and animal products is exceeded standard, to human health, brought great harm, simultaneously chemical fertilizer uses the spoiled soil crumb structure for a long time in a large number, cause soil compaction, fertility descends.
Microbial fertilizer is as a kind of new-type fertilizer, after being manured into soil, Fast-propagation by its specific bacterial strain, can be fixedly phosphorus, the potassium element of stationary state in nitrogen in atmosphere, release soil, make the nutrient potentiality of environment be given full play to, and build a good soil microorganisms environment for plant growth, reducing, the aspects such as fertilizer amount, reduction environmental pollution, raising crops quality are significant.Especially integrate the research and development of the composite microbiological fertilizer of fixed nitrogen, phosphorus decomposing, potassium decomposing and plant growth element, very important effect is arranged in agricultural sustainable development.
At present, although microbial pesticide and microbial fertilizer aspect have all been dropped into to huge manpower and material resources both at home and abroad, with regard to quantity, chemical pesticide and fertilizer are far away more than biological pesticide and fertilizer; In addition, biological pesticide and fertilizer having relatively high expectations to environment.Therefore, develop the microbial pesticide and the fertilizer that meet the local environment condition particularly important, can promote the development of local green agriculture, the competition that participates in international market for farm products, protection of the environment and assurance human health just to seem particularly important.
Summary of the invention
Purpose of the present invention is exactly for above-mentioned defect of the prior art, and the subtilis S001 that can have broad-spectrum sterilization activity and growth-promoting ability is provided.
To achieve these goals, technical scheme provided by the invention is: subtilis
(Bacillus subtilis), be bacillus
(Bacillus Cohn), being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on August 13rd, 2012, address is No. 3, No. 1, North Star West Road, Chaoyang District, BeiJing, China city institute, culture presevation is numbered CGMCC NO.6425.
Second purpose of the present invention has been to provide the application of above-mentioned subtilis S001 in suppressing pathogenic bacteria, and described pathogenic bacteria is tomato gray mould bacterium, eggplant sclerotium bacteria, wheat total eclipse bacterium, Helminthosporium or Rhizoctonia cereali, and bacteriostasis rate is more than or equal to 90%.
The application of above-mentioned subtilis S001 in suppressing pathogenic bacteria, described pathogenic bacteria is sharp Fusariumsp, capsicum dry thread Pyrenomycetes, Cladosporium fulvum or Alternaria solani, bacteriostasis rate is more than or equal to 71.1%.
The application of above-mentioned subtilis S001 in suppressing pathogenic bacteria, described subtilis S001 is containing 100 mg/L tryptophanes or containing the concentration of secreting indolylacetic acid in the King nutrient solution of tryptophane, be not respectively 33.50 mg/L and 10.88mg/L.
The 3rd purpose of the present invention has been to provide microbiobacterial agent prepared by subtilis S001, and the composition of described microbiobacterial agent is counted subtilis S001 5-10% according to weight percent, subtilis S001 substratum 90-95%.
Further, the mentioned microorganism microbial inoculum, described subtilis S001 substratum comprises following component by weight percentage: Semen Maydis powder 1.5-2.5%, analysis for soybean powder 0.5-1.2%, beef extract 0.25-0.75%, distilled water or deionized water 1000ml; The pH value of described subtilis S001 substratum is 7-8.
Further, the mentioned microorganism microbial inoculum, described subtilis S001 substratum comprises following component by weight percentage: Semen Maydis powder 2%, analysis for soybean powder 0.85%, beef extract 0.5%, distilled water or deionized water 1000ml; The pH value of described subtilis S001 substratum is 7.5.
The 4th purpose of the present invention has been to provide the preparation method of microbiobacterial agent prepared by subtilis S001, and subtilis S001 liquid fermenting 24-36h on subtilis S001 substratum obtained; Described liquid fermentation condition is temperature 26-30 ℃, rotating speed 150-250r/min, air flow 800-1000L/h; The viable count of described subtilis S001 is 5.78 * 10
11cfu/mL-8.13 * 10
12cfu/mL.
Further, the preparation method of microbiobacterial agent prepared by above-mentioned subtilis S001, described liquid fermentation condition is 28 ℃ of temperature, rotating speed 200r/min, air flow 900L/h, fermentation time is 30 hours.
The 5th purpose of the present invention has been to provide the application of microbiobacterial agent in preparing microbial pesticide or microbial fertilizer prepared by subtilis S001.
specific operation process of the present invention is as follows:
One, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) identification of morphology.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425) be inoculated on beef extract peptone plate culture medium, 28 ℃ of cultivation proterties of cultivating 3d are: the soil subtilis
bacillus subtiliss001(CGMCC No.6425) Gram-positive, the blunt circle in shaft-like, two ends, minority is catenation in various degree.Size (0.62～2.54) um * (1.59～4.30) um, produce gemma after cultivation 48h, the gemma ellipse, and most of proximal ends are given birth to, and in minority, give birth to, and sporocyst expands not obvious or does not expand.On beef extract peptone flat board, bacterium colony oyster white, tarnish, surface are convex rugose, edge is irregular, out-of-shape shape.Lawn oyster white, edge waviness shape, opaque, surface dead color on inclined-plane.
Two, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) 16S rDNA gene order is identified.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425) 16S rDNA gene order in Genebank with
bacillus subtilis(AB017591.1) 16S rDNA gene order is carried out homology relatively.Comparative result shows, the soil subtilis
bacillus subtiliss001(CGMCC No.6425) with
bacillus subtilis(AB017591.1) homology can reach more than 99%.By 16S rDNA gene order result, soil subtilis
bacillus subtiliss001(CGMCC No.6425) with
bacillus subtilis(AB017591.1) homology can prove the soil subtilis
bacillus subtiliss001(CGMCC No.6425) really belong to subtilis
bacillus subtilis.
Three, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) indoor bacteriostatic activity is measured.
Filter out first the soil subtilis
bacillus subtiliss001(CGMCC No.6425) antagonistic strain, be inoculated in S001(CGMCC No.6425 by this antagonistic strain) cultivated in substratum; Simultaneously, indicator strain is inoculated in potato dextrose agar (being PDA) substratum and is cultivated.After antagonistic strain and indicator strain are cultivated activation in substratum separately, break into punch tool the bacterium cake that diameter is 7mm respectively.Choose one of the bacterium cake of indicator strain, be placed in the PDA substratum; Concrete, can be by this pure culture biscuits involvng inoculation the middle position (plate diameter 9cm) in the plate of splendid attire PDA substratum; Choose again four of the bacterium cakes that obtained by antagonistic strain, according to the face-off method, by this four ferfas cake circumference that equidistantly to be inoculated in apart from this plate middle position be 2.5cm.After this culture dish is cultivated to 5 days under the envrionment temperature of 26 ℃, with the antibacterial circle diameter of above-mentioned bacterial strains in cross fado time this PDA substratum of replication, in this test, replication 3 times.
The soil subtilis
bacillus subtiliss001(CGMCC No.6425) sterile liquid can be referring to table 1 to the measurement result of part pathogenic bacteria bacteriostasis.
As shown in Table 1, soil subtilis
bacillus subtiliss001(CGMCC No.6425) in his-and-hers watches one, the inhibiting rate of 10 kinds of frequently seen plants germs at least can reach 71.1%, and DEVELOPMENT PROSPECT is better.
Four, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) generation indolylacetic acid ability and the growth-promoting performance of microbiobacterial agent thereof are detected.
1, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) generation indolylacetic acid (IAA) ability is measured.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425) being inoculated in Jin Shi (King) nutrient solution that contains 100mg/L tryptophane (or not containing tryptophane) cultivates after 12 days bacterium suspension and centrifugal 10 min of blank, rotating speed is 10000 r/min, get supernatant liquor 4 mL and add respectively the equivalent color solution, use immediately spectrophotometric determination OD in dark after standing 30min
530value, the soil subtilis
bacillus subtiliss001(CGMCC No.6425) in containing tryptophane (100 mg/L) and not containing the Jin Shi substratum of tryptophane, the amount of producing IAA is respectively 33.50 mg/L and 10.88mg/L.
2, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) the growth-promoting ability is measured.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425) inoculation is in inoculum, after putting under 28 ℃ and cultivating 48h, be diluted to concentration ratio 1:30 with sterilized water, 1:40, the bacterium liquid of 1:50 soaks respectively wheat, corn, tomato, after muskmelon and watermelon seed 24h, take the same time Seed soaking as contrast, be placed on culture dish filter paper, 30, every kind of every ware of crop, repeat 3 times, observe record seed germination situation every day, Continuous Observation 5d, the radicle of usining stretches out kind of skin 2mm as the germination standard, take 3d as specified time survey germinating energy, after having surveyed percentage of germination and germinating energy, from 10 of every ware chitting piece random chooses, plant respectively small flower again, growth 2-3 week is measured underground part and over-ground part length.Result shows, the bacteria suspension of 1:50 is the most obvious to the effect of wheat, corn, tomato, muskmelon and Germination of Watermelon Seed rate, contrast has improved respectively 14,17,40,20 and 31 percentage points, and the bacteria suspension of 1:30 is the most obvious to the effect of muskmelon seeds percentage of germination, contrast has improved 27 percentage points.This result can be found out the soil subtilis
bacillus subtiliss001(CGMCC No.6425) especially more obvious to the growth-promoting functions of the crop seeds such as tomato, muskmelon, watermelon.
The soil subtilis
bacillus subtiliss001(CGMCC No.6425) the measurement result to the part rate of emergence can be referring to table 2.
Five, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) optimum medium and the performance of microbiobacterial agent thereof are detected.
1, determine the soil subtilis
bacillus subtiliss001(CGMCC No.6425) optimum medium.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425) be inoculated in respectively in the substratum that label is followed successively by A, B, C, D, E, F and G observation soil subtilis
bacillus subtiliss001(CGMCC No.6425) energy for growth in these 7 kinds of substratum.
Through observation, find the soil subtilis
bacillus subtiliss001(CGMCC No.6425) in these 7 kinds of substratum, all can grow, but viable count is variant.Wherein, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) in these 7 kinds of substratum, the measurement result of viable count can be referring to table 3.
In table 3, " A " means nutrient agar medium (Nutrient Agar is called for short NA) substratum, in the NA substratum, comprises beef extract 5.0g, peptone 10g, sucrose 20g and distilled water/deionized water 1000mL; " B " means the PDA substratum, in the PDA substratum, comprises potato 200g, peptone 10g, sucrose 20g and distilled water/deionized water 1000mL; " C " means nutrient broth (Nutrient Broth is called for short NB) substratum, in the NB substratum, comprises beef extract 3g, albumen 5.0g, glucose 2.5g and distilled water/deionized water 1000mL; " D " means yeast glucose solids (being NYDA) substratum, in the NYDA substratum, comprises beef extract 8g, yeast extract 5.0g, glucose 10g and distilled water/deionized water 1000mL; " E " means the first W-Gum substratum, in the first W-Gum substratum, comprises W-Gum 2.5g, (NH
4)
2sO
410g, sucrose 10g and distilled water/deionized water 1000mL; " F " means the second W-Gum substratum, in the second W-Gum substratum, comprises W-Gum 3.0g, (NH
4)
2sO
410g, KH
2pO
415g, sucrose 10g and distilled water/deionized water 1000mL; " G " means protein culture medium, in protein culture medium, comprises peptone 10g, yeast extract 5.0g, NaCl 10g and distilled water/deionized water 1000mL.
As shown in Table 3, under identical condition, select 7 kinds of different nutrient solutions to cultivate S001(CGMCC No.6425), its viable bacteria amount has notable difference.In nutrient solution E and F, viable count is 0, and the viable count of nutrient solution A reaches 2.84 * 10
12cfu/mL, be significantly higher than other substratum.Visible, nutrient solution A is comparatively desirable, and therefore, beef extract 5.0g/L, peptone 10g/L, sucrose 20g/L are S001(CGMCC No.6425) basic medium.
At definite NA substratum, it is the most applicable soil subtilis
bacillus subtiliss001(CGMCC No.6425) on the basis of the substratum of growth, further utilize Orthogonal Method to be optimized carbon nitrogen source content in the NA substratum and pH value.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425) with 5% inoculum size, with the rotating speed of 150r/min, after the temperature condition bottom fermentation 24h of 28 ℃, take Semen Maydis powder, yam starch, W-Gum, white sugar and do not add carbon source as processing, measure the viable count in fermented liquid.
Through synthesis measuring, cultivate the soil subtilis by 4 kinds of different carbon sources
bacillus subtiliss001(CGMCC No.6425) the viable bacteria amount has notable difference, and take the viable bacteria amount of W-Gum in processing, is 4.18 * 10
12cfu/mL, be 15.03 times that white sugar is processed, and is that cultivate on the CK(basis) 6.92 times.Therefore, W-Gum is the preferably carbon source filtered out.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425) with 5% inoculum size, with the rotating speed of 150r/min, after the temperature condition bottom fermentation 24h of 28 ℃, with analysis for soybean powder, NH
4cl, (NH
4)
2sO
4, KNO
3do not add nitrogenous source for processing, measure the viable count in fermented liquid.
Through synthesis measuring, cultivate the soil subtilis with 4 kinds of different nitrogenous sources
bacillus subtiliss001(CGMCC No.6425) the viable bacteria amount has notable difference, with KNO
3during for nitrogenous source, the viable bacteria amount is 2.03 * 10
11cfu/mL, and take analysis for soybean powder during as nitrogenous source the viable bacteria amount reach 4.77 * 10
12cfu/mL is KNO
323.50 times, be 5.02 times that cultivate on basis, analysis for soybean powder is the preferably nitrogenous source filtered out.
Carry out orthogonal test with Semen Maydis powder, analysis for soybean powder, beef extract and pH, through synthesis measuring, the substratum after finding to optimize is more suitable for the soil subtilis than above-mentioned NA substratum
bacillus subtiliss001(CGMCC No.6425) growth and antibacterial.Wherein, the component of the substratum after above-mentioned optimization, content and pH value are respectively: Semen Maydis powder 2%, analysis for soybean powder 0.5%, beef extract 0.5%, pH value 8.0, distilled water/deionized water 1000mL.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425) in the substratum after above-mentioned optimization, ferment, in fermented liquid, viable count can reach 4.00 * 10
11cfu/mL～1.60 * 10
12cfu/mL.In addition, by the soil subtilis
bacillus subtiliss001(CGMCC No.6425) comprising Semen Maydis powder 2%, analysis for soybean powder 0.5%, beef extract 0.5%, pH value 7.5, in the substratum of distilled water/deionized water 1000mL, ferment, in fermented liquid, viable count can reach 6.13 * 10
12cfu/mL.
2, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) activity of microbiobacterial agent is detected.
Determined the soil subtilis in above-mentioned test one
bacillus subtilison the basis of optimum medium S001(CGMCC No.6425), by the soil subtilis be kept in slant medium
bacillus subtiliss001(CGMCC No.6425), cultivate activation 24h on the plate of nutrient agar medium (Nutrient Agar is called for short NA) substratum after, be inoculated in nutrient broth (Nutrient Broth is called for short NB) substratum; After cultivating 24h, after in 5% inoculum size access seed fermentation tank, cultivating 20h, turn in the fermentor tank that is inoculated in 10L and fermented, the viable count in fermented liquid of take is index, respectively air flow, rotating speed, inoculum size and liquid amount are optimized final soil subtilis
bacillus subtiliss001(CGMCC No.6425) the most adaptable method in the 10L fermentor tank is: temperature 26-30 ℃, and inoculum size 5%-10%, pH7.0-8.0, air flow 800-1000L/h, rotating speed 150-250 r/min, incubation time is 24-36h, viable count is 5.78 * 10
11cfu/mL-8.13 * 10
12cfu/mL.
By the soil subtilis
bacillus subtiliss001(CGMCC No.6425), in the fermentation cylinder for fermentation of above-mentioned fermentation condition, can make the microbiobacterial agent with good growth-promoting ability and bacteriostasis.Wherein, in temperature, be 28 ℃, inoculum size is that 10%, pH value is 7.5, and air flow is 1000L/h, and rotating speed is that under 200r/min and the incubation time fermentation condition that is 24h, in fermented liquid, viable count can reach 8.13 * 10
12cfu/mL.
3, to the soil subtilis
bacillus subtiliss001(CGMCC No.6425) quality determining method of microbiobacterial agent is verified.
The method of plate culture count is for the density of assay plate bacterium colony viable bacteria body, and turbidimetry is for the number of assay plate bacterium colony viable bacteria body.Here, with density and the number of viable bacteria body in the method for plate culture count and turbidimetry for Determination flat-plate bacterial colony, measurement result is basically identical respectively, and on flat-plate bacterial colony, the density of viable bacteria body is larger, number is more, and ultraviolet spectrophotometer is measured OD
600the absorption value at nm place is higher.
Visible, the soil subtilis
bacillus subtiliss001(CGMCC No.6425) quality of microbiobacterial agent can adopt the method for plate culture count to detect, and also can adopt turbidimetry to detect.
Beneficial effect of the present invention is: deposit number provided by the invention is S001(CGMCC No.6425) the soil subtilis
bacillus subtiliss001 has antibacterial and growth-promoting ability, to sharp Fusariumsp
fusarium oxysporum, the capsicum dry thread Pyrenomycetes
rhizoctonia solani, Cladosporium fulvum
fulvia fulva, Alternaria solani
alternaria solani, the tomato gray mould bacterium
botrytis cinerea, the eggplant sclerotium bacteria
sclerotinia sclerotiorum, wheat total eclipse bacterium
gaeumannomyces graminisvar
.tritici, Helminthosporium
bipolaris sativum, Rhizoctonia cereali
rhizoctonia celeresdeng pathogenic bacteria, restraining effect is arranged; The microbial pesticide prepared with it and the microbial fertilizer feature of environmental protection are good, are difficult for developing immunity to drugs, and security is good.
The accompanying drawing explanation
The colonial morphology that Fig. 1 is subtilis S001 provided by the invention (left side) and thalli morphology (right side).
Embodiment
embodiment 1:
One bacillus subtilis S001, bacterial strain S001 is bacillus
(Bacillus Cohn), being preserved in Chinese common micro-organisms culture presevation administrative center on August 13rd, 2012, culture presevation is numbered CGMCC NO.6425.
The optical microscope photograph figure of subtilis S001 is provided as shown in Figure 1.
embodiment 2:
According to the embodiment of the present invention, provide a kind of soil subtilis
bacillus subtiliss001(CGMCC No.6425) application, comprise sharp Fusariumsp
fusarium oxysporum, the capsicum dry thread Pyrenomycetes
rhizoctonia solani, Cladosporium fulvum
fulvia fulva, Alternaria solani
alternaria solani, the tomato gray mould bacterium
botrytis cinerea, the eggplant sclerotium bacteria
sclerotinia sclerotiorum, wheat total eclipse bacterium
gaeumannomyces graminisvar.
tritici, Helminthosporium
bipolaris sativum, Rhizoctonia cereali
rhizoctonia celeresinhibition Deng pathogenic bacteria.
In the present embodiment, soil subtilis
bacillus subtiliss001(CGMCC No.6425) to the eggplant sclerotium bacteria
sclerotinia sclerotiorumbacteriostasis rate be 98.8%.
embodiment 3:
In the present embodiment, soil subtilis
bacillus subtiliss001(CGMCC No.6425) to sharp Fusariumsp
fusarium oxysporum, the capsicum dry thread Pyrenomycetes
rhizoctonia solani, Cladosporium fulvum
fulvia fulvaand Alternaria solani
alternaria solani, bacteriostasis rate is at least 71.1%.
embodiment 4:
In the present embodiment, soil subtilis
bacillus subtiliss001(CGMCC No.6425) to the tomato gray mould bacterium
botrytis cinereainhibited, bacteriostasis rate is 96.1%.
embodiment 5:
In the present embodiment, soil subtilis
bacillus subtiliss001(CGMCC No.6425) fermented liquid can make the tomato seeds percentage of germination improve 40 percentage points, makes over-ground part and underground part increase respectively 4.60cm and 3.76cmm.
embodiment 6:
According to the embodiment of the present invention, provide a kind of soil subtilis
bacillus subtiliss001(CGMCC No.6425) microbiobacterial agent, biological activity component and the weight percent content of this microbiobacterial agent comprise: the soil subtilis
bacillus subtiliss001(CGMCC No.6425) 5% and S001(CGMCC No.6425) the substratum surplus.
In the present embodiment, S001(CGMCC No.6425) component and the content of substratum comprise: Semen Maydis powder 1.5%, analysis for soybean powder 0.5%, beef extract 0.25%, pH value 7.0, distilled water/deionized water 1000mL.
embodiment 7:
Different from above-described embodiment, in the present embodiment, the soil subtilis
bacillus subtiliss001(CGMCC No.6425) component of microbiobacterial agent and weight percent content comprise: the soil subtilis
bacillus subtiliss001(CGMCC No.6425) 8% and S001(CGMCC No.6425) the substratum surplus.
In the present embodiment, S001(CGMCC No.6425) component and the content of substratum comprise: Semen Maydis powder 2%, analysis for soybean powder 0.85%, beef extract 0.5%, pH value 7.5, distilled water/deionized water 1000mL.
embodiment 8:
Different from above-described embodiment, in the present embodiment, the soil subtilis
bacillus subtiliss001(CGMCC No.6425) component of microbiobacterial agent and weight percent content comprise: the soil subtilis
bacillus subtiliss001(CGMCC No.6425) 10% and S001(CGMCC No.6425) the substratum surplus.
In the present embodiment, S001(CGMCC No.6425) component and the content of substratum comprise: Semen Maydis powder 2.5%, analysis for soybean powder 1.2%, beef extract 0.75%, pH value 8.0, distilled water/deionized water 1000mL.
embodiment 9:
According to the embodiment of the present invention, provide a kind of soil subtilis
bacillus subtiliss001(CGMCC No.6425) preparation method of microbiobacterial agent, this preparation method comprises: by the soil subtilis
bacillus subtiliss001(CGMCC No.6425) 5% be inoculated in S001(CGMCC No.6425) in substratum, in temperature, be that 26 ℃, rotating speed are under 150r/min and the air flow condition that is 800L/h, carry out liquid fermenting 24h, the fermented liquid obtained can be directly as the soil subtilis
bacillus subtiliss001(CGMCC No.6425) microbiobacterial agent is used.
In the present embodiment, S001(CGMCC No.6425) the pH value of substratum is 7.0, and component and content can comprise: Semen Maydis powder 1.5%, analysis for soybean powder 0.5%, beef extract 0.25%, pH value 7.0, distilled water/deionized water 1000mL.
After measured, soil subtilis in above-mentioned fermented liquid
bacillus subtiliss001(CGMCC No.6425) viable count can reach 8.83 * 10
11cfu/mL.
embodiment 10:
Different from above-described embodiment, in the present embodiment, the soil subtilis
bacillus subtiliss001(CGMCC No.6425) preparation method of microbiobacterial agent comprises: the soil subtilis
bacillus subtiliss001(CGMCC No.6425) 8% be inoculated in S001(CGMCC No.6425) in substratum, in temperature, be that 28 ℃, rotating speed are under 200r/min and the air flow condition that is 900L/h, carry out liquid fermenting 30h, the fermented liquid obtained can be directly as the soil subtilis
bacillus subtiliss001(CGMCC No.6425) microbiobacterial agent is used.
In the present embodiment, S001(CGMCC No.6425) the pH value of substratum is 7.5, and component and content can comprise: Semen Maydis powder 2%, analysis for soybean powder 0.85%, beef extract 0.5%, pH value 7.5, distilled water/deionized water 1000mL.
After measured, soil subtilis in above-mentioned fermented liquid
bacillus subtiliss001(CGMCC No.6425) viable count can reach 7.53 * 10
12cfu/mL.
embodiment 11:
Different from above-described embodiment, in the present embodiment, the soil subtilis
bacillus subtiliss001(CGMCC No.6425) preparation method of microbiobacterial agent comprises: by the soil subtilis
bacillus subtiliss001(CGMCC No.6425) 10% be inoculated in S001(CGMCC No.6425) in substratum, in temperature, be that 30 ℃, rotating speed are under 250r/min and the air flow condition that is 1000L/h, carry out liquid fermenting 36h, the fermented liquid obtained can be directly as the soil subtilis
bacillus subtiliss001(CGMCC No.6425) microbiobacterial agent is used.
In the present embodiment, S001(CGMCC No.6425) the pH value of substratum is 8.0, and component and content can comprise: Semen Maydis powder 2.5%, analysis for soybean powder 1.2%, beef extract 0.75%, distilled water/deionized water 1000mL.
After measured, soil subtilis in above-mentioned fermented liquid
bacillus subtiliss001(CGMCC No.6425) viable count can reach 6.859 * 10
12cfu/mL.
Through verification experimental verification, above-mentioned soil subtilis
bacillus subtiliss001(CGMCC No.6425) the soil subtilis of microbiobacterial agent and preparation method thereof embodiment
bacillus subtiliss001(CGMCC No.6425) microbiobacterial agent, suitable to arid, the semiarid and area use that cools.
Finally it should be noted that: the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, although with reference to previous embodiment, the present invention is had been described in detail, for a person skilled in the art, its technical scheme that still can put down in writing aforementioned each embodiment is modified, or part technical characterictic wherein is equal to replacement.Within the spirit and principles in the present invention all, any modification of doing, be equal to replacement, improvement etc., within all should being included in protection scope of the present invention.
Claims (10)
1. subtilis S001, bacterial strain S001 is bacillus
(Bacillus Cohn), being preserved in Chinese common micro-organisms culture presevation administrative center on August 13rd, 2012, culture presevation is numbered CGMCC NO.6425.
2. the application of subtilis S001 according to claim 1 in suppressing pathogenic bacteria is characterized in that described pathogenic bacteria is tomato gray mould bacterium, eggplant sclerotium bacteria, wheat total eclipse bacterium, Helminthosporium or Rhizoctonia cereali, and bacteriostasis rate is more than or equal to 90%.
3. the application of subtilis S001 according to claim 1 in suppressing pathogenic bacteria is characterized in that described pathogenic bacteria is sharp Fusariumsp, capsicum dry thread Pyrenomycetes, Cladosporium fulvum or Alternaria solani, and bacteriostasis rate is more than or equal to 71.1%.
4. the application of subtilis S001 according to claim 1 in suppressing pathogenic bacteria, it is characterized in that, described subtilis S001 is containing 100 mg/L tryptophanes or containing the concentration of secreting indolylacetic acid in the King nutrient solution of tryptophane, be not respectively 33.50 mg/L and 10.88mg/L.
5. the microbiobacterial agent that prepared by subtilis S001, is characterized in that, the composition of described microbiobacterial agent is counted subtilis S001 5-10% according to weight percent, subtilis S001 substratum 90-95%.
6. microbiobacterial agent according to claim 5, it is characterized in that, described subtilis S001 substratum comprises following component by weight percentage: Semen Maydis powder 1.5-2.5%, analysis for soybean powder 0.5-1.2%, beef extract 0.25-0.75%, distilled water or deionized water 1000ml; The pH value of described subtilis S001 substratum is 7-8.
7. microbiobacterial agent according to claim 6, is characterized in that, described subtilis S001 substratum comprises following component by weight percentage: Semen Maydis powder 2%, analysis for soybean powder 0.85%, beef extract 0.5%, distilled water or deionized water 1000ml; The pH value of described subtilis S001 substratum is 7.5.
8. the preparation method of the microbiobacterial agent that prepared by subtilis S001, is characterized in that, subtilis S001 liquid fermenting 24-36h on subtilis S001 substratum is obtained; Described liquid fermentation condition is temperature 26-30 ℃, rotating speed 150-250r/min, air flow 800-1000L/h; The viable count of described subtilis S001 is 5.78 * 10
11cfu/mL-8.13 * 10
12cfu/mL.
9. the preparation method of the microbiobacterial agent that prepared by subtilis S001 according to claim 8, is characterized in that, described liquid fermentation condition is 28 ℃ of temperature, rotating speed 200r/min, and air flow 900L/h, fermentation time is 30 hours.
10. the application of microbiobacterial agent in preparing microbial pesticide or microbial fertilizer that prepared by subtilis S001.
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