CN104152385A - Bacillus mojavensis QLY002 strain, microbial inoculum, preparation method of microbial inoculum and use of Bacillus mojavensis QLY002 strain and microbial inoculum - Google Patents
Bacillus mojavensis QLY002 strain, microbial inoculum, preparation method of microbial inoculum and use of Bacillus mojavensis QLY002 strain and microbial inoculum Download PDFInfo
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Abstract
The invention provides a Bacillus mojavensis QLY002 strain. The Bacillus mojavensis QLY002 strain has a preservation number of CGMCC No. 8905. The invention also provides a microbial inoculum of the Bacillus mojavensis QLY002 strain and its preparation method and use in potato pathogen inhibition and potato growth promotion. The Bacillus mojavensis QLY002 strain and the microbial inoculum can be used as microbe pesticides and fertilizer, can inhibit Phoma foveata, Alternaria solani, Fusarium oxysporum and Colletotrichum coccodes, have an antibacterial rate more than 61%, have potato growth promotion effects, can be used as potato special-purpose microbial pesticide and microbial fertilizer, solve the problem that the existing chemical pesticide has poor environmental friendliness, easily causes pesticide resistance and has poor safety, and have the advantages of good environmental friendliness, no pesticide resistance and good safety.
Description
Technical field
The invention belongs to microbial pesticide and microbial fertilizer technical field, be specifically related to strain Mo Hawei bacillus mojavensis QLY002 bacterial strain and microbiobacterial agent and an application.
Background technology
Along with in a large number, frequently using of chemical pesticide, the problems such as environmental pollution, pesticide residue and increase resistance are also more and more serious, the biological control of Plant diseases becomes study hotspot, and the endophyte of plant of wherein especially usining obtains numerous scholars' concern as the research of Antagonistic Fungi.Endophytic bacterium refers in root, stem, leaf, flower, fruit and the seed of living in plant, but the bacterium that plant is injured without any substance, it has the powerful support that various biological function, particularly bacteria resistance function become biocontrol microorganisms resource.Research finds that endophyte of plant has the effect that promotes growth to plant, these growth-promoting functions are mainly reflected in endogenetic bacteria by producing IAA, nitrogen fixation and making plant can obtain as much as possible supplementing of nutrient by phosphorus decomposing or molten phosphorus effect, thereby plant is accumulated biomass; Endophytic bacterium also can stop or reduce the harm of infecting or causing of pathogenic bacteria to plant by secretion antibacterial substance, with pathogenic bacteria competition nutrition space and inducing plant acquisition disease resistance.The microbial pesticide that the endogenetic bacteria of take is microorganism resource development is difficult for developing immunity to drugs, and does not injure natural enemy, and can utilize even trade effluent widespread production of agricultural byproducts, and being applied to provides a new approach for producing green or non-polluted farm product after agriculture production.
A large amount of use chemical nitrogen fertilizer, phosphate fertilizer and potash fertilizer can cause heavy metal to be accumulated, natural resources and energy resources consumes in a large number, environmental pollution in the soil body, and its utilization ratio is low, the nitrogenous fertilizer not being absorbed and used runs off or volatilizees in farmland, groundwater resource, 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 spoiled soil crumb structure for a long time in a large number, cause soil compaction, fertility declines.
Microbial fertilizer is as a kind of new-type fertilizer, after being manured into soil, by the Fast-propagation of its specific bacterial strain, can be fixedly phosphorus, the potassium element of stationary state in nitrogen in atmosphere, release soil, the nutrient potentiality of environment are 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 the research and development that integrate the composite microbiological fertilizer of antibacterial, fixed nitrogen, phosphorus decomposing, potassium decomposing and plant growth element have very important effect in agricultural sustainable development.
Summary of the invention
The object of the invention is poor for chemical pesticide in prior art and the chemical fertilizer feature of environmental protection, easily develop immunity to drugs and the defect of poor stability, one strain Mo Hawei bacillus mojavensis QLY002 bacterial strain is provided, utilize the active and growth-promoting ability of the broad-spectrum sterilization of described endogenetic bacteria Mo Hawei genus bacillus, prepare the feature of environmental protection good, be difficult for the endogenetic bacteria Mo Hawei micro-organisms bacillus microbial inoculum that develops immunity to drugs and security is good.
The present invention is from Alpine Grasslands serpentgrass (Polygonum viviparum), Stipa purpurea (Stipa purpurca Griseb), line leaf high (Kobreasia Capillifolia), in the endogenetic bacteria of bark of ash (Gentiana macrophy pall) and Herba Anaphalidis Lacteae (Anaphalis Lactea Maxin) etc., screening acquisition one strain has endogenetic bacteria antibacterial and to potato tool growth-promoting functions to potato cause of disease bacterium: Mo Hawei Bacillus strain, the Latin name of this bacterial strain is called Bacillus mojavensis QLY002, and its biological function is studied, its culture condition and fermentation condition have been optimized, and carried out control and tested, one strain biological control and the microorganism resource of bio-bacterial manure and the preparation method of its microbial inoculum are provided.The applicant is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center by Mo Hawei bacillus mojavensis QLY002 bacterial strain, deposit number is CGMCC No.8905, preservation address is No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, depositary institution is called for short: CGMCC, preservation date is on 03 10th, 2014.
Second object of the present invention is to provide a kind of microbiobacterial agent, and this microbiobacterial agent contains thalline and substratum, and described thalline is Mo Hawei Bacillus strain CGMCC No.8905.
Preferably, in microbiobacterial agent every milliliter described, the viable count of Mo Hawei Bacillus strain CGMCC No.8905 is 4.12 * 10
10-1.59 * 10
11cFU.
Preferably, component and the content of described substratum comprise: ammonium chloride 10-18.5g/L, Semen Maydis powder 14-24g/L, potato 180-280g/L; PH value is 7.2-8.2, and solvent is distilled water or deionized water.
Most preferably, component and the content of described substratum comprise: ammonium chloride 14.25g/L, Semen Maydis powder 19g/L, potato 230g/L; PH value is 7.7, and solvent is distilled water or deionized water.
Preferably, in microbiobacterial agent, the mass percentage content of described thalline is 5-11%, and all the other are substratum.
The present invention determines the optimum medium of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) by following test.
Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is inoculated in respectively in the substratum that label is followed successively by A, B, C, D, E, F and G to the energy for growth of observation Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) in these 7 kinds of substratum.
Through observation, find that Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) all can grow in these 7 kinds of substratum, but viable count is variant.Wherein, to the measurement result of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) viable count in these 7 kinds of substratum referring to Fig. 1.
In Fig. 1, " A " represents nutrient agar medium (Nutrient Agar is called for short NA) substratum, in NA substratum, comprises beef peptone 5.0g, peptone 10.0g, sucrose 20.0g and distilled water/deionized water 1000mL; " B " represents PDA substratum, in PDA substratum, comprises potato 200.0g, peptone 10.0g, sucrose 20.0g and distilled water/deionized water 1000mL; " C " represents nutrient broth (Nutrient Broth is called for short NB) substratum, in NB substratum, comprises beef peptone 3.0g, peptone 5.0g, glucose 2.5g and distilled water/deionized water 1000mL; " D " represents yeast glucose solids (being NYDA) substratum, in NYDA substratum, comprises beef peptone 8.0g, yeast extract 5.0g, glucose 10.0g and distilled water/deionized water 1000mL; " E " represents the first W-Gum substratum, in the first W-Gum substratum, comprises W-Gum 2.5g, (NH
4)
2sO
410.0g, sucrose 10.0g and distilled water/deionized water 1000mL; " F " represents the second W-Gum substratum, in the second W-Gum substratum, comprises W-Gum 3.0g, (NH
4)
2sO
410.0g, KH
2pO
415.0g, sucrose 10g and distilled water/deionized water 1000mL; " G " represents protein culture medium, in protein culture medium, comprises peptone 10.0g, yeast extract 5.0g, NaCl 10.0g and distilled water/deionized water 1000mL.
As shown in Figure 1, under identical condition, select 7 kinds of different nutrient solutions to cultivate Mo Hawei genus bacillus CGMCC No.8905, its viable bacteria amount has notable difference.In B nutrient solution, viable count 1.045 * 10
9cFU/mL, in nutrient solution E and F, viable count is all lower than 3 * 10
3cFU/mL, nutrient solution A, C, D and G viable count are significantly lower than B nutrient solution.Visible, nutrient solution B is comparatively desirable, and therefore, potato 200.0g, peptone 10.0g, sucrose 20.0g, water 1000mL are the basic medium of Mo Hawei genus bacillus CGMCC No.8905.
Determining that PDA substratum be on the basis of the most applicable Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) substratum of growing, and further utilizes Orthogonal Method to be optimized carbon nitrogen source content in PDA substratum and pH value.
1, the optimization of carbon source:
Inoculum size by Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) with 5%, take Semen Maydis powder, potato, W-Gum, white sugar and do not add carbon source as processing, rotating speed with 150r/min, after the temperature condition bottom fermentation 24h of 28 ℃, measure the viable count in fermented liquid.
Through synthesis measuring, by 4 kinds of different carbon sources, to cultivate, Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) viable bacteria amount has notable difference, and the viable bacteria amount of take in Semen Maydis powder processing is 5.5 * 10
9cFU/mL is significantly higher than other carbon sources.Therefore, Semen Maydis powder is the more excellent carbon source filtering out.
2, the optimization of nitrogenous source:
Inoculum size by Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) with 5%, take saltpetre, ammonium chloride, ammonium sulfate, analysis for soybean powder and do not add nitrogenous source as processing, rotating speed with 150r/min, after the temperature condition bottom fermentation 24h of 28 ℃, measure the viable count in fermented liquid.
Through synthesis measuring, with 4 kinds of different nitrogenous sources, to cultivate, Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) viable bacteria amount has notable difference, and when nitrogenous source is ammonium chloride, viable count is 1.41 * 10
10cFU/mL, is significantly higher than the corresponding viable count of other nitrogenous sources, therefore, chooses ammonium chloride as the nitrogenous source of best medium.
3, the optimization of pH value:
By substratum and carbon nitrogen source, screen, the main component of determining basic culture solution is ammonium chloride, Semen Maydis powder and potato, and the pH of nutrient solution is 7, each factor is got to the value (referring to table 1) of different levels.According to Response Surface Method (Box-Behnken), design, test design with the results are shown in Table 2, utilize Design-Expert 7.0 softwares to carry out the multinomial regression fit of secondary, the dynamic response model of the viable count that draws bacterial strain QLY002 (CGMCC No.8905) to ammonium chloride, Semen Maydis powder, potato and pH:
Y＝
2.503×10
10+5.939×10
9X
1+5.569×10
9X
2+6.165×10
7X
3+3.905×10
8X
4-1.151×10
9X
1X
2+2.918×10
9X
1X
3+6.576×10
8X
1X
4-7.193×10
9X
2X
4+2.507×10
9X
3X
4-7.751×10
9X
1 2-4.669×10
9X
2 2-1.689×10
9X
3 2-2.059×10
9X
4 2-6.144×10
9X
1 2X
2+3.103×10
9X
1 2X
3+2.774×10
9X
1X
2 2+1.541×10
9X
2 2X
4
R
2=0.9714, correct coefficient of determination Adj-R
2=0.9273, in formula, Y is the predictor of Mo Hawei genus bacillus CGMCC No.8905 viable count.By correcting the coefficient of determination, can think that Regression Model Simulator degree is better; Ammonium chloride, Semen Maydis powder, potato and pH are remarkable on the impact of viable count, this model (P>F) P<0.0001, the degree of fitting that shows regression equation is extremely remarkable, vow to intend (P>F) P=0.196, show that this model shared Non-normal errors in actual matching is less; Therefore, can pass through the optimal culture condition of forecast of regression model bacterial strain Mo Hawei genus bacillus CGMCC No.8905.The addition that ammonium chloride, Semen Maydis powder and potato are worked as in this model prediction is respectively 14.25g/L, 19g/L, 230g/L; Nutrient solution pH is 7.7 o'clock, and the viable count of Mo Hawei genus bacillus CGMCC No.8905 bacterial strain has maximum value 3.27 * 10
10cFU/mL; Through verifying under this culture condition, the viable count of Mo Hawei genus bacillus CGMCC No.8905 bacterial strain can reach 3.48 * 10
10cFU/mL is not remarkable with predictor difference.The best nutrient solution formula that Mo Hawei genus bacillus CGMCC No.8905 bacterial strain is described is ammonium chloride 14.25g/L, Semen Maydis powder 19g/L, potato 230g/L, pH7.7.
Table 1: response curved design is tested each factor span
Table 2: response curved design and test-results
Substratum (ammonium chloride 10-18.5g/L, Semen Maydis powder 14-24g/L, potato 180-280g/L by Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) after above-mentioned optimization; PH value is 7.2-8.2, and solvent is distilled water/deionized water) middle fermentation, in fermented liquid, viable count can reach 3.03 * 10
9cFU/mL～1.59 * 10
11cFU/mL.In addition, Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is being comprised to ammonium chloride 14.25g/L, Semen Maydis powder 19g/L, potato 230g/L, pH7.7, in the substratum of distilled water/deionized water 1000mL, ferment, in fermented liquid, viable count can reach 1.59 * 10
11cFU/mL.
The 3rd object of the present invention is to provide the preparation method of mentioned microorganism microbial inoculum, step is as follows: Mo Hawei Bacillus strain CGMCC No.8905 is seeded in substratum, in temperature, be that 26-30 ℃, rotating speed are under 150-210r/min and the dissolved oxygen amount condition that is 50%-70%, carry out liquid fermenting 32-40h, make fermented liquid, obtain microbiobacterial agent.
Preferably, be in inoculum size, be 8%, temperature is that 28 ℃, rotating speed are under 180r/min and the dissolved oxygen amount condition that is 60%, carries out liquid fermenting 36h, makes fermented liquid, obtains microbiobacterial agent.
On the basis of above-mentioned fixed Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) optimum medium, by the Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) being kept in slant medium, at nutrient agar medium (Nutrient Agar, abbreviation NA) on the plate of substratum, cultivate after activation 24h, be inoculated in nutrient broth (Nutrient Broth is called for short NB) substratum; After cultivating 24h, to cultivate 24h in 5% inoculum size access seed fermentation tank; Afterwards, turn in the fermentor tank that is inoculated in 10L and ferment, the viable count in fermented liquid of take is index, respectively dissolved oxygen amount, rotating speed, pH value and inoculum size is optimized, and the most adaptable method of final Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) in 10L fermentor tank is: temperature 26-30 ℃, inoculum size 5%-11%, pH7.2-8.2, dissolved oxygen amount 50%-70%, rotating speed 150-210r/min, incubation time is 32-40h, and viable count is 4.12 * 10
10cFU/mL-1.59 * 10
11cFU/mL.
Fermentation cylinder for fermentation by Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) at 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 8%, pH value is 7.7, dissolved oxygen amount 60%, rotating speed is that under 180r/min and the incubation time fermentation condition that is 36h, in fermented liquid, viable count can reach 1.59 * 10
11cFU/mL, it is best that growth-promoting ability now and bacteriostasis reach.
The quality determining method of Xia Mian Dui Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) 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 quality of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) microbiobacterial agent can adopt the method for plate culture count to detect, and also can adopt turbidimetry to detect.
The 4th object of the present invention is to provide above-mentioned bacterial strains and the application of microbiobacterial agent in preparing potato special microorganism agricultural chemicals.
Wherein, described potato cause of disease bacterium is potato gangrene germ, the former bacterium of target, dry rot of potato bacterium and potato anthrax bacteria.Bacteriostasis rate is respectively: the bacteriostasis rate to potato gangrene germ (Phoma foveata) is 71.83%; To potato anthrax bacteria (Colletotrichum coccodes) bacteriostasis rate, be 74.92%; Inhibited to dry rot of potato bacterium (Fusarium oxysporum), bacteriostasis rate is 61.42%; Inhibited to the former bacterium of target (Alternaria solani), bacteriostasis rate is 64.30%.
The 5th object of the present invention is to provide above-mentioned bacterial strains and microbiobacterial agent is being prepared potato microbial fertilizer special and the application in potato growth-promoting functions.
The 6th object of the present invention is to provide a kind of microbial pesticide, and it contains above-mentioned Mo Hawei Bacillus strain or above-mentioned microbiobacterial agent.
The 7th object of the present invention is to provide a kind of microbial fertilizer, and it contains above-mentioned Mo Hawei Bacillus strain or above-mentioned microbiobacterial agent.
In sum, the invention provides a kind of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) bacterial strain, microbiobacterial agent, preparation method and application, being about to have bioactive Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is 5-11% according to inoculum size, be inoculated in and in optimum medium, carry out liquid fermenting, the fermented liquid obtaining can directly be used as Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) microbiobacterial agent, this Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) microbiobacterial agent has bacteriostatic action and growth-promoting functions, can be as microbial pesticide and Fertilizer application, can inhibition of potato gangrene germ (Phoma foveata), the former bacterium of target (Alternaria solani), dry rot of potato bacterium (Fusarium oxysporum) and potato anthrax bacteria (Colletotrichum coccodes), its bacteriostasis rate is more than 61%, potato is had to growth-promoting functions, can be used as microbial pesticide and microbial fertilizer uses, thereby can overcome the chemical pesticide feature of environmental protection in prior art poor, easily develop immunity to drugs and the defect of poor stability, with realize the feature of environmental protection good, be difficult for the advantage that develops immunity to drugs and security is good.
Accompanying drawing explanation
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for specification sheets, for explaining the present invention, is not construed as limiting the invention together with embodiments of the present invention.In the accompanying drawings:
Fig. 1 is the impact of the different nutrient solutions cultivations of application on Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) viable count; Letter representation level difference is remarkable, small letter: P<0.05; Capitalization: P<0.01;
Fig. 2 is the optical microscope photograph of endogenetic bacteria Mo Hawei bacillus mojavensis (CGMCC No.8905); Wherein A is thalli morphology; B is colonial morphology.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.
Acquisition and the evaluation of embodiment 1 Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905)
One, the acquisition of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905)
The present invention is from Alpine Grasslands serpentgrass (Polygonum viviparum), Stipa purpurea (Stipa purpurca Griseb), line leaf high (Kobreasia Capillifolia), in the endogenetic bacteria of bark of ash (Gentiana macrophy pall) and Herba Anaphalidis Lacteae (Anaphalis Lactea Maxin) etc., utilizing face-off culture method is culture dish central authorities access pathogenic bacterias, the equidistant inoculation endogenetic bacteria of surrounding 2.5 centimeters, the right-angled intersection method after 5-7 days of cultivating is measured colony diameter, calculate bacteriostasis rate, the bacteriostasis rate of take has endogenetic bacteria antibacterial and to potato tool growth-promoting functions as obtaining a strain according to screening to potato cause of disease bacterium: Mo Hawei Bacillus strain, the Latin name of this bacterial strain is called Bacillus mojavensis QLY002, the applicant is deposited in Chinese common micro-organisms culture presevation administrative center, deposit number is CGMCC No.8905, the preservation time is on 03 10th, 2014.
Two, the evaluation of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905).
(1) identification of morphology to Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905).
Bacillus mojavensis QLY002 is inoculated on beef extract-peptone plate culture medium, and 28 ℃ of cultivation proterties of cultivating 3d are: bacterium colony size is 4-6 mm, and there is ridge shape protuberance centre, and neat in edge is opaque, beige; Bacillus mojavensis QLY002 is Gram-positive, and raw raw to time end in gemma, thalline size is 0.45～0.75 μ m * 1.55～3.20 μ m.
(2) the 16S rDNA gene order of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is identified.
By the 16S rDNA gene order of Bacillus mojavensis QLY002 in Genebank with Bacillus amylolique-faciens (KC250199.1), Bacillus subtilis (JN584217.1) comparison, with the kind homology of many bacillus genus such as Bacillus methylotrophicus (JN661700.1) and Bacillus mojavensis (JX126863.1) more than 99%; Bacillus mojavensis QLY002 is accredited as to the bacterial strain of bacillus.
(3) the gyrB gene order of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is identified.
The gyrB gene order of Bacillus mojavensis QLY002 in Genebank with the homology of the bacterial strains such as Bacillus mojavensis (DQ309297.1) more than 99%, with Mega (4.0) software adjacent method phylogenetic tree construction, Bacillus mojavensis QLY002 and B.mojavensis consistence are the highest, in conjunction with cultivating proterties, morphological specificity and 16S rDNA Gene sequence comparison result, be accredited as the Mo Hawei bacillus mojavensis in bacillus again.
The preparation of embodiment 2 Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) microbiobacterial agents
Biological activity component and the weight percent content of microbiobacterial agent of the present invention comprise: Mo Hawei genus bacillus CGMCC No.89055% and substratum surplus.
Component and the content of substratum comprise: ammonium chloride 10g, Semen Maydis powder 14g, potato 180g, pH value 7.2, distilled water/deionized water 1000mL.
The preparation method of microbiobacterial agent is: Mo Hawei genus bacillus CGMCC No.8905 is inoculated in substratum in proportion, in temperature, be that 26 ℃, rotating speed are under the condition of 150r/min and dissolved oxygen amount 50%, carry out liquid fermenting 32h, the fermented liquid obtaining is microbiobacterial agent.
After measured, in above-mentioned fermented liquid, the viable count of Mo Hawei genus bacillus CGMCC No.8905 can reach 4.12 * 10
10cFU/mL.
The preparation of embodiment 3 Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) microbiobacterial agents
Biological activity component and the weight percent content of microbiobacterial agent of the present invention comprise: Mo Hawei genus bacillus CGMCC No.89058% and substratum surplus.
Component and the content of substratum comprise: ammonium chloride 14.25g, Semen Maydis powder 19g, potato 230g, pH value 7.7, distilled water/deionized water 1000mL.
The preparation method of microbiobacterial agent is: Mo Hawei genus bacillus CGMCC No.8905 is inoculated in substratum in proportion, in temperature, be that 28 ℃, rotating speed are under the condition of 180r/min and dissolved oxygen amount 60%, carry out liquid fermenting 36h, the fermented liquid obtaining is microbiobacterial agent.
After measured, in above-mentioned fermented liquid, the viable count of Mo Hawei genus bacillus CGMCC No.8905 can reach 1.59 * 10
11cFU/mL.
The preparation of embodiment 4 Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) microbiobacterial agents
Biological activity component and the weight percent content of microbiobacterial agent of the present invention comprise: Mo Hawei genus bacillus CGMCC No.890511% and substratum surplus.
Component and the content of substratum comprise: ammonium chloride 18.5g, Semen Maydis powder 24g, potato 280g, pH value 8.2, distilled water/deionized water 1000mL.
The preparation method of microbiobacterial agent is: Mo Hawei genus bacillus CGMCC No.8905 is inoculated in substratum in proportion, in temperature, be that 30 ℃, rotating speed are under the condition of 210r/min and dissolved oxygen amount 70%, carry out liquid fermenting 40h, the fermented liquid obtaining is microbiobacterial agent.
After measured, in above-mentioned fermented liquid, the viable count of Mo Hawei genus bacillus CGMCC No.8905 can reach 1.51 * 10
11cFU/mL.
Indoor bacteriostatic activity and the field control effect of embodiment 5 Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905)
Test one: the indoor Antibacterial Activity of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905).
Filter out first Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) antagonistic strain, this antagonistic strain is inoculated in the substratum described in embodiment 3 and is cultivated; Meanwhile, indicator strain (potato gangrene germ (Phoma foveata), the former bacterium of target (Alternaria solani), dry rot of potato bacterium (Fusarium oxysporum) and potato anthrax bacteria (Colletotrichum coccodes)) is inoculated in potato dextrose agar (being PDA) substratum and is cultivated.When antagonistic strain and indicator strain, in substratum separately, cultivate after activation, with punch tool, break into the bacterium cake that diameter is 6mm respectively.One of bacterium cake choosing indicator strain, is placed in PDA substratum; Concrete, can be by this pure culture biscuits involvng inoculation in the middle position (plate diameter 9cm) of the plate of splendid attire PDA substratum; Choose again four of the bacterium cakes that obtained by antagonistic strain, according to face-off method, by this four ferfas cake circumference that to be equidistantly inoculated in apart from this plate middle position be 2.5cm.This culture dish is cultivated under the envrionment temperature of 28 ℃ after 5 days, 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.
Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) can be referring to table 3 to the measurement result of part pathogenic bacteria bacteriostasis.
Table 3: the measurement result of Mo Hawei genus bacillus CGMCC No.8905 to part pathogenic bacteria bacteriostasis
As shown in Table 3, in Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) his-and-hers watches 1, the inhibiting rate of 4 kinds of common potato germs at least can reach 61.42%, and DEVELOPMENT PROSPECT is better.
Test two: Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) microbial inoculum is measured the prevention effect of storage of potato phase gangrenosis.
When the storage of Dingxi City, Gansu Province potato warehouse-in, selection comes from the potato piece of 200 uniformities in grave illness field, the Mo Hawei genus bacillus CGMCC No.8905 microbial inoculum of embodiment 4 preparations is mixed with to 1:10 times of liquid and is sprayed on potato piece surface, with 5000 times of liquid of 10% fluzilazol and 3000 times of liquid of 50% kresoxim-methyl, do medicament contrast respectively, take clear water as blank, in spray medicine November 15 in 2011, on March 23rd, 2012, Investigate incidence, and calculated preventive effect.Result shows, the sickness rate of clear water contrast potato gangrenosis is 38.60%, it is 13.77% that Mo Hawei genus bacillus CGMCC No.8905 microbial inoculum is processed sequela rate, its preventive effect is 64.31%, preventive effect than 5000 times of liquid 10% fluzilazols is low 22.62 percentage points, but than 3000 times of high 11.83 percentage points (tables 4) of liquid 50% kresoxim-methyl, illustrate that Mo Hawei genus bacillus CGMCC No.8905 microbial inoculum of the present invention has good prevention effect to potato gangrenosis (P.foveata) in storage.
Table 4: the preventive effect of Mo Hawei genus bacillus CGMCC No.8905 microbial inoculum to potato gangrenosis
Note: it is remarkable that after numerical value, lowercase is illustrated in 0.05 level difference, " ± " represents mean error.
6 couples of Mo Hawei bacillus mojavensis QLY002 of embodiment (CGMCC No.8905) produce the detection of the growth-promoting performance of indolylacetic acid ability and microbiobacterial agent thereof.
Test one: the mensuration that Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is produced to indolylacetic acid (IAA) ability.
Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is inoculated in to Jin Shi (King) nutrient solution that contains 100mg/L tryptophane (or containing tryptophane) to be cultivated after 12 days bacterium suspension and the centrifugal 10min of blank, rotating speed is 10000r/min, get supernatant liquor 4mL and add respectively equivalent color solution, in dark, after standing 30min, use immediately spectrophotometric determination OD
530value, Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) amount of producing IAA in containing tryptophane (100mg/L) and not containing the Jin Shi substratum of tryptophane is respectively 12.17mg/L and 9.75mg/L.
Test two: the indoor growth-promoting ability of Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is measured.
By Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) inoculation in the nutrient solution described in the embodiment of the present invention 3, put at 28 ℃ and cultivate after 36h, with the bacterium liquid that sterilized water is diluted to concentration ratio 1:10,1:20 and the 1:30 potato seed of dressing seed respectively, the same time of take is contrast with clear water seed dressing, plant in flowerpot, and be that 35:1 fills with root by soil quality and microbial inoculum volume ratio.Under greenhouse, manage after 55 days, measure the length of potato rhizome, dry weight in wet base and diameter stem, and calculate dry weight in wet base root/shoot ratio.Result shows, it is potted plant after potato is dressed seed, root, stem and chlorophyll content etc. are all high than contrast, wherein after 10 times of liquid are processed, root is long increases respectively 8cm, 0.75g and 5.07g with dry, weight in wet base, thick and the stem of plant height, stem, weight in wet base increase respectively 2.74cm, 0.27cm, 0.52g and 5.73g, and dry wet root/shoot ratio increases respectively by 0.214 and 0.094 (table 5), and chlorophyll content increases 0.54mg/g (table 6).This result can find out that Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is obvious to the growth-promoting functions of potato.
Its biomass estimation after table 5: Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) processing potato
Note: after numerical value, " ± " represents mean error.
After table 6: Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) processes potato, its chlorophyll content in leaf blades is measured
Note: letter representation level difference is remarkable, small letter: P<0.05; Capitalization: P<0.01
Test three: Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) land for growing field crops growth-promoting ability is measured.
By Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) inoculation in the nutrient solution described in the embodiment of the present invention 3, put at 28 ℃ and cultivate after 36h, with the bacterium liquid that sterilized water the is diluted to concentration ratio 1:20 potato seed of dressing seed respectively, take same time Seed soaking as contrast, 6 communities, every community 20m
2, repeat for 3 times, during results, survey and produce and calculate stimulation ratio.Result shows, after the bacteria suspension of 1:20 is processed, potato commodity potato rate improves 36.29%.This result can find out that Mo Hawei bacillus mojavensis QLY002 (CGMCC No.8905) is obvious to the growth-promoting functions of potato.
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 the present invention is had been described in detail with reference to previous embodiment, for a person skilled in the art, its technical scheme that still can record aforementioned each embodiment is modified, or part technical characterictic is wherein 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. a strain Mo Hawei Bacillus strain, is characterized in that: described Mo Hawei Bacillus strain
bacillus mojavensisthe deposit number of QLY002 is: CGMCC No.8905.
2. a microbiobacterial agent, this microbiobacterial agent contains thalline and substratum, it is characterized in that: described thalline is Mo Hawei Bacillus strain CGMCC No.8905.
3. microbiobacterial agent according to claim 2, is characterized in that: in microbiobacterial agent every milliliter described, the viable count of Mo Hawei Bacillus strain CGMCC No.8905 is 4.12 * 10
10-1.59 * 10
11cFU.
4. according to the microbiobacterial agent described in claim 2 or 3, it is characterized in that: component and the content of described substratum comprise: ammonium chloride 10-18.5g/L, Semen Maydis powder 14-24g/L, potato 180-280g/L; PH value is 7.2-8.2, and solvent is distilled water or deionized water; As preferably, component and the content of described substratum comprise: ammonium chloride 14.25g/L, Semen Maydis powder 19g/L, potato 230g/L; PH value is 7.7, and solvent is distilled water or deionized water.
5. according to the arbitrary described microbiobacterial agent of claim 2-4, it is characterized in that: in microbiobacterial agent, the mass percentage content of described thalline is 5-11%, and all the other are substratum.
6. the preparation method of the arbitrary described microbiobacterial agent of claim 2-5, it is characterized in that: step is as follows: Mo Hawei Bacillus strain CGMCC No.8905 is seeded in substratum, in temperature, be that 26-30 ℃, rotating speed are under 150-210r/min and the dissolved oxygen amount condition that is 50%-70%, carry out liquid fermenting 32-40h, make fermented liquid, obtain microbiobacterial agent; Preferably, be in inoculum size, be 8%, temperature is that 28 ℃, rotating speed are under 180r/min and the dissolved oxygen amount condition that is 60%, carries out liquid fermenting 36h, makes fermented liquid, obtains microbiobacterial agent.
7. the application of the arbitrary described microbiobacterial agent of Mo Hawei Bacillus strain claimed in claim 1 and claim 2-5 in preparation potato special microorganism agricultural chemicals.
8. the application of the arbitrary described microbiobacterial agent of Mo Hawei Bacillus strain claimed in claim 1 and claim 2-5 in preparation potato microbial fertilizer special.
9. a microbial pesticide, it contains Mo Hawei Bacillus strain claimed in claim 1 or the arbitrary described microbiobacterial agent of claim 2-5.
10. a microbial fertilizer, it contains Mo Hawei Bacillus strain claimed in claim 1 or the arbitrary described microbiobacterial agent of claim 2-5.
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