CN103289931A - Bacillus vallismortis strain SJ and application thereof in preparation of tobacco antiviral preparation and promoter - Google Patents
Bacillus vallismortis strain SJ and application thereof in preparation of tobacco antiviral preparation and promoter Download PDFInfo
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Abstract
The invention discloses a bacillus vallismortis strain SJ and an application of the strain in preparation of a tobacco antiviral preparation and a promoter. The preservation number of the bacillus vallismortis strain SJ is CGMCC No.7571 (China General Microbiological Culture Collection Center). The invention also provides an SJ strain preparation with certain viable count, and the SJ strain preparation can be used for preparing a biological preparation capable of resisting a tobacco viral disease and a promoter for promoting the growth of the tobacco. By adopting the technical scheme, not only the utilization rates of a fertilizer and a pesticide capable of resisting the tobacco virus can be reduced, but also the environment pollution caused by the pesticide is reduced; the application of the SJ strain preparation disclosed by the invention not only can reach good antiviral effect, but also can effectively promote the growth of the tobacco; and the SJ strain preparation is low in production cost, simple and convenient to operate, long in storage time, easy to store and transport, conductive to factory expanded production, capable of achieving industrialization, and good in technical effect and market application prospect.
Description
Technical field
The invention belongs to microbial technology field, relate in particular to a kind of spend territory Bacillus strain SJ and the application in preparation tobacco disease resistance toxin preparation and growth-promoting agent thereof.
Background technology
Tobacco is China's main economic crops, and its output and relationship between quality national economy.At present, the general method of control tobacco diseases is to use chemical pesticide, yet, the life-time service chemical pesticide can cause pathogenic bacteria to develop immunity to drugs and reduce the protection effect of chemical agent, the life-time service chemical agent can cause pesticide residue to exceed standard and environmental pollution simultaneously, cause the tobacco leaf interior quality to descend, the vega disruption of ecological balance, even had a strong impact on the Sustainable development of leaf tobacco production.And biological control is a kind of new way of control Plant diseases, and it mainly is the antagonistic action of utilizing between biocontrol microorganisms and the plant pathogenic microorganisms, suppresses the growth of phytopathogen.The kind of biocontrol microorganisms and the antagonistic action size between the pathogenic micro-organism are the keys that determines its anti-virus ability in the microbial preparation, and therefore will seeking the high-activity microorganism of antagonism pathogenic bacterium or pathogenic insect and meta-bolites thereof now, to prevent and solve the viroses of plant are present research focus and breaches.
Summary of the invention
The invention provides a kind of spend territory Bacillus strain SJ and the application in preparation tobacco disease resistance toxin preparation and growth-promoting agent thereof.Use SJ bacteria preparation of the present invention can prepare biotechnological formulation and the tobacco growth-promoting agent of anti-tobacco virus disease, have antiviral effect good, be conducive to environment protection, be easy to advantages such as industrialization production.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
A kind of colored territory Bacillus strain SJ, its classification called after flower territory bacillus vallismortis is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its deposit number is: CGMCC No.7571.
The bacterium colony of described colored territory Bacillus strain SJ is circular, neat in edge, and diameter 3.0mm-8.0mm is white in color, and projection is arranged, and the surface is film, and the inside is mucus, has provoked wire drawing, and thalline is rod-short, and the gemma end is given birth to, circle.
The invention provides the SJ bacteria preparation that contains described colored territory Bacillus strain SJ.
Wherein, described SJ bacteria preparation is solid fungicide or liquid bacterial agent, and described solid fungicide living bacteria count is more than 100,000,000/g, and the liquid bacterial agent living bacteria count is more than 100,000,000/mL.
Wherein, the optimization culture medium prescription of described colored territory Bacillus strain SJ is: Semen Maydis powder 6.0-8.0g/L, starch 0.5-2.0g/L, glucose 3.0-5.0g/L, peptone 1.0-3.0g/L, soybean cake powder 8.0-10.0g/L, glucose 2.0-3.0g/L, dipotassium hydrogen phosphate 0.5-1.0g/L, calcium carbonate 3.0-5.0g/L, magnesium sulfate heptahydrate 0.8-1.0g/L, manganous sulfate 0.1-0.5g/L, defoamer 1.5-3.0g/L, pH8.0-8.5.
The present invention also provides the described application of SJ bacteria preparation in preparation tobacco disease resistance toxin preparation that contains colored territory Bacillus strain SJ.
Wherein, described virus is tobacco mosaic virus (TMV).
The present invention also provides the described application of SJ bacteria preparation in preparation tobacco growth-promoting agent that contains colored territory Bacillus strain SJ.
Compared with prior art, advantage of the present invention with positively effect is: the present invention separates the antagonistic strain SJ that obtains preventing and treating TMV from tobacco-growing soil, and by the technical scheme to its medium optimization, be made into the SJ bacteria preparation that reaches certain viable count, the SJ bacteria preparation of gained can be used for preparing the biotechnological formulation of anti-tobacco virus disease and promote the growth-promoting agent of tobacco growing.By adopting technical scheme of the present invention, not only can reduce the rate of utilization of the fertilizer and pesticide of anti-tobacco virus, alleviated the agricultural chemicals pollution on the environment; And use SJ bacteria preparation of the present invention can reach good antiviral effect, can also play effective growth-promoting functions to tobacco; Described SJ bacteria preparation production cost is low, simple to operation, the shelf time long, be easy to storage and transportation, is convenient to factory's extension and produces, and realizes industrialization, has good technical effect and market application foreground.
After reading the specific embodiment of the present invention by reference to the accompanying drawings, other characteristics of the present invention and advantage will become clearer.
Description of drawings
Fig. 1 is the plate culture medium photo of colored territory genus bacillus SJ of the present invention.
Fig. 2 is the electromicroscopic photograph figure that colored territory of the present invention genus bacillus SJ bacteria preparation influences the TMV virion, and wherein a shows undressed TMV virion form, and b shows and the mixed TMV virion of SJ preparation form.
Embodiment
Below in conjunction with the drawings and specific embodiments technical scheme of the present invention is described in further detail.
Embodiment 1
One, the separation and purification of active bacterial strain
Part rhizosphere soil surplus in the of 50 of taking a sample altogether from Yishui, Shandong cigarette district tobacco-growing soil on July 4th, 2012 is got the 2g sample respectively and is suspended in the 20ml physiological saline, 180rpm, concussion 30min.Getting 100 μ L sample suspension respectively evenly is coated with at nutrient agar plate, be inverted in 35 ℃ of incubators and cultivate 16h, single bacterium colony that picking comes in every shape is inoculated on the nutrient agar plate with method of scoring, obtain pure bacterium by the repeated multiple times line, filter out the pure bacterium of 126 strains altogether, and all bacterial strains are preserved by the inclined-plane.
Two, the screening of the SJ bacterial strain of control TMV antagonistic bacterium
The above-mentioned purifying inoculation of picking is in the LB liquid nutrient medium, 28 ℃ of constant temperature, 140rpm shaking culture 48h, after getting 20 times of TMV juice of 20ml and equal-volume mixing 30min, frictional inoculation TMV(tobacco mosaic virus (TMV), tobacco mosaic virus) withered spot host three lives-NN cigarette, 4 leaves in every strain inoculation top, be mixed into contrast with sterilized water and TMV, 3～5d investigates withered spot inhibiting rate.There is the bacterial strain of antagonistic activity to adopt the same procedure repeated test through preliminary test, every processing 3 strains, three repetitions.Withered spot inhibiting rate calculation formula:
Withered spot inhibiting rate experimental result shows that the inhibition of the TMV of bacterial strain SJ is best, and the withered spot inhibiting rate of TMV is reached 94.5%, and biocontrol bacteria has produced in the growth metabolism process has the antibiotics (seeing Table 1) that suppresses virus activity.
The restraining effect of the tobacco mosaic viruses of table 1 bacterial strain SJ
Three, the bacteria molecule genetic classification of flower territory genus bacillus is identified
The SJ bacterial strain is extracted DNA, is template with this DNA, and 16S rDNA universal primer is primer, and 16S rDNA fragment is increased.The fragment of amplification is directly carried out sequencing.The blast program that the result who checks order imports on the NCBI website is compared, the result shows that the 16s rDNA sequence homology degree of the Bacillus vallismortis in the bacillus in this bacterial strain 16S rDNA sequence and the GenBank gene pool is the highest, and homology reaches 99%.16S rDNA sequence by existing bacillus among the Genbank of DNAMAN6.0 is carried out the genetic evolution analysis, the result shows, bacterial strain SJ16S rDNA and Bacillus vallismortis homology are the highest, therefore can judge that this bacterial strain is the colored territory genus bacillus of bacillus, called after flower territory Bacillus strain SJ.
The colored territory Bacillus strain SJ that screens is carried out culture presevation, depositary institution: China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC); Address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica; Preservation date: on May 8th, 2013; Bacillus vallismortis is numbered CGMCC No.7571.
As shown in Figure 1, flower territory Bacillus strain SJ 37 ℃ of bacterium colonies of cultivating 18h formation on nutrient agar plate are circular, neat in edge, and diameter 3.0mm-8.0mm, white has projection, and the surface is film, and the inside is mucus, has provoked wire drawing.Flower territory genus bacillus thalline is rod-short, and the gemma end is given birth to, circle.
Four, spend the medium optimization of territory Bacillus strain SJ
The SJ bacterial classification streak inoculation that the test tube slant is preserved is on the NA culture medium flat plate, be inoculated in behind 28 ℃ of cultivation 64h and carry out liquid culture in the nutrient broth liquid nutrient medium, liquid amount is 200mL in every 500mL triangular flask, in 35 ℃, 180r/min. the constant temperature shaking table is cultivated 16h, is used for following test as seed liquor.
Fixedly nitrogen source urea is added single carbon sources such as Semen Maydis powder, sucrose, glucose, starch, carries out the carbon source screening; Fixedly the carbon source Semen Maydis powder adds single nitrogenous sources such as soybean cake powder, yeast powder, peptone, filters out optimum nitrogenous source, adjusts optimization best medium such as medium pH, leavening temperature, rotating speed, inoculative proportion.
The optimization culture medium prescription of selecting colored territory Bacillus strain SJ through testing sieve is: Semen Maydis powder 6.0-8.0g/L, starch 0.5-2.0g/L, glucose 3.0-5.0g/L, peptone 1.0-3.0g/L, soybean cake powder 8.0-10.0g/L, glucose 2.0-3.0g/L, dipotassium hydrogen phosphate 0.5-1.0g/L, calcium carbonate 3.0-5.0g/L, magnesium sulfate heptahydrate 0.8-1.0g/L, manganous sulfate 0.1-0.5g/L, defoamer 1.5-3.0g/L, pH8.0-8.5.
The fermentation result: about pH7.0, living bacteria count is 25,000,000,000/mL.
Five, the preparation of the SJ bacteria preparation of anti-tobacco virus disease
The SJ bacterial classification streak inoculation that the test tube slant is preserved is on the nutrient broth medium flat board, be inoculated in behind 35 ℃ of cultivation 16h and carry out liquid culture in the nutrient broth liquid nutrient medium, liquid amount is 200mL in every 500mL triangular flask, in 35 ℃, 180r/min, the constant temperature shaking table is cultivated 16h, as primary seed solution.
Primary seed solution is inoculated in the substratum of having optimized, liquid amount 240L in the 350L fermentor tank, 35 ℃, mixing speed 180r/min is inoculated into 10 tons of fermentor tanks, 35 ℃ as secondary seed solution behind the fermentation 16h, mixing speed 180r/min, approximately about 14h, gemma rate 80% is put jar, is the fermented liquid of SJ bacterial strain.With gained bacterium liquid elimination dregs, press the 1:1 mixed with carrier calcium carbonate, stir, by spray-drying tower, set 170 ℃ of inlet temperature, 60 ℃ of air outlet temperatures are removed moisture, and thalline is attracted on the carrier fully, and gained is the SJ bacteria preparation.The mensuration of living bacteria count records living bacteria count 50,000,000,000/g according to GB20287 agricultural microorganism microbial inoculum standard.
Described SJ bacteria preparation can adopt the form of solid fungicide or liquid bacterial agent, and described solid fungicide living bacteria count is more than 100,000,000/g, and the liquid bacterial agent living bacteria count is can reach good antibacterial effect more than 100,000,000/mL.
Six, the SJ bacteria preparation of Antiphytoviral is to the antagonistic action of TMV
With 1000 times of bacterial strain SJ preparation dilutions, behind 80 times of TMV juice equal-volume mixings 15min, in frictional inoculation three lives-NN cigarette, 2 leaves in every strain inoculation top are mixed into CK with sterilized water and TMV, and the while is comparison with SJ nutrient solution, NB substratum replacement SJ preparation liquid.Calculate withered spot inhibiting rate.
Table 2 SJ preparation is to the restraining effect of TMV
| ? | The SJ fermented liquid | The SJ preparation | Nutrient broth medium |
| Average withered spot number | 8.4 | 9.5 | 296 |
| CK | 324 | 351 | 349 |
| Inhibiting rate % | 97.41 | 97.29 | 15.19 |
Find out that by table 2 the SJ bacteria preparation neither influences the activity of fermented liquid, can well adsorb bacterium liquid again, the preventive effect of TMV is reached 97.29%.Illustrate and select calcium carbonate as carrier, do not influence the SJ viable bacteria to the restraining effect of TMV, calcium carbonate has advantages such as excellent adsorption, low price as filler, can produce use.
Seven, observe the SJ preparation to the influence of TMV virion under the perspective Electronic Speculum
After negative staining was handled the TMV that purifies, through the JEM-100CX transmission electron microscope observing, the TMV plastochondria in the visual field was typically shaft-like, the about 500nm of length, and upright and outspoken and marshalling, integrity is good; Behind 1000 times of SJ preparation dilutions and the pure TMV plastochondria of the equal-volume combination treatment 2min, as shown in Figure 2, through electron microscopic observation, in the visual field TMV plastochondria generation noticeable change almost complete rupture become little shaft-like, arrange confused and disordered.This shows that the SJ preparation has strong destruction to the TMV plastochondria, prove that the SJ preparation can produce passivation to virus to the antagonistic substance of TMV, suppress the polymerization of TMV coat protein, and destroy the integrity of plastochondria.
Eight, growth-promoting functions
Common cigarette NC89 of 2-3 leaf phase transplants to the small flower of diameter 10cm, uses the SJ preparation of 1000 times of dilutions to irritate root during transplanting, every strain 10ml, and interval 3d, the continuous irrigation root is 2 times again, and irritating root with nutrient broth, sterilized water is contrast, every processing 6 strains, 3 repetitions.After last is irritated root 30d, investigation fresh weight, maximum leaf length and width.
Found out by table 3, common cigarette NC89, continuous three filling roots, the SJ bacteria preparation is handled its fresh weight, and maximum leaf length and width all is higher than nutrient broth and clear water is handled, and is extremely remarkable with clear water processing difference.
Table 3 4A1 fermentation liquid irrigating root is to the growth-promoting functions of NC89
Nine, thalline stability of formulation test
The SJ bacteria preparation is at room temperature placed, at set intervals preparation is counted.1000 times of preparation dilutions are mixed with the TMV equal-volume, greenhouse inoculation three lives NN cigarette, detection is to the withered spot inhibiting rate of virus, and the result is as shown in table 4.
Active degradation rule under the table 4 SJ bacteria preparation room temperature
Find out by table 4, the SJ bacteria preparation is at room temperature preserved, and along with the prolongation of time, activity is degraded gradually, preserved 12 months, activity to TMV drops to 79.04%, and the bacterium number is 25,000,000,000/g, the SJ bacteria preparation is described at room temperature, degraded slowly, analyzing its reason mainly is to have formed gemma, is easy to store and transportation, has the potential quality that is developed as biological prevention and control agent.
Above embodiment is only in order to illustrating technical scheme of the present invention, but not limits it; Although with reference to previous embodiment the present invention is had been described in detail, for the person of ordinary skill of the art, still can make amendment to the technical scheme that previous embodiment is put down in writing, perhaps part technical characterictic wherein is equal to replacement; And these modifications or replacement do not make the essence of appropriate technical solution break away from the spirit and scope of the present invention's technical scheme required for protection.
Claims (8)
1. colored territory Bacillus strain SJ, it is characterized in that: its classification called after flower territory bacillus vallismortis, be preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, its deposit number is: CGMCC No.7571.
2. a kind of colored territory Bacillus strain SJ according to claim 1, it is characterized in that: the bacterium colony of flower territory Bacillus strain SJ is circle, neat in edge, diameter 3.0mm-8.0mm, be white in color, projection is arranged, and the surface is film, and the inside is mucus, provoked wire drawing, thalline is rod-short, and the gemma end is given birth to, circle.
3. the SJ bacteria preparation that contains the described colored territory Bacillus strain SJ of claim 1.
4. the SJ bacteria preparation that contains colored territory Bacillus strain SJ according to claim 3, it is characterized in that: described SJ bacteria preparation is solid fungicide or liquid bacterial agent, described solid fungicide living bacteria count is more than 100,000,000/g, and the liquid bacterial agent living bacteria count is more than 100,000,000/mL.
5. the SJ bacteria preparation that contains colored territory Bacillus strain SJ according to claim 3, it is characterized in that: the optimization culture medium prescription of described colored territory Bacillus strain SJ is: Semen Maydis powder 6.0-8.0 g/L, starch 0.5-2.0 g/L, glucose 3.0-5.0 g/L, peptone 1.0-3.0 g/L, soybean cake powder 8.0-10.0 g/L, glucose 2.0-3.0 g/L, dipotassium hydrogen phosphate 0.5-1.0 g/L, calcium carbonate 3.0-5.0 g/L, magnesium sulfate heptahydrate 0.8-1.0 g/L, manganous sulfate 0.1-0.5 g/L, defoamer 1.5-3.0 g/L, pH 8.0-8.5.
6. the application of SJ bacteria preparation in preparation tobacco disease resistance toxin preparation that contains colored territory Bacillus strain SJ according to claim 3.
7. the application of SJ bacteria preparation in preparation tobacco disease resistance toxin preparation that contains colored territory Bacillus strain SJ according to claim 6, it is characterized in that: described virus is tobacco mosaic virus (TMV).
8. the application of SJ bacteria preparation in preparation tobacco growth-promoting agent that contains colored territory Bacillus strain SJ according to claim 3.
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| CN110157641A (en) * | 2019-05-20 | 2019-08-23 | 黑龙江省农业科学院土壤肥料与环境资源研究所 | One plant of biocontrol bacteria BV23 for preventing and treating Causal Organism of Maize Basal Stalk and its application |
| CN112868671A (en) * | 2021-01-15 | 2021-06-01 | 湖北省生物农药工程研究中心 | Preparation method and application of killed bacillus vallismortis suspending agent for killing citrus mites |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104725136A (en) * | 2015-03-19 | 2015-06-24 | 青岛海大生物集团有限公司 | Special marine biological fertilizer for tobacco as well as preparation method and application of fertilizer |
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| CN110157641A (en) * | 2019-05-20 | 2019-08-23 | 黑龙江省农业科学院土壤肥料与环境资源研究所 | One plant of biocontrol bacteria BV23 for preventing and treating Causal Organism of Maize Basal Stalk and its application |
| CN110157641B (en) * | 2019-05-20 | 2023-05-05 | 黑龙江省农业科学院土壤肥料与环境资源研究所 | Biocontrol bacterium BV23 for preventing and treating corn stem-based rot and application thereof |
| CN112868671A (en) * | 2021-01-15 | 2021-06-01 | 湖北省生物农药工程研究中心 | Preparation method and application of killed bacillus vallismortis suspending agent for killing citrus mites |
| CN112868671B (en) * | 2021-01-15 | 2021-08-03 | 湖北省生物农药工程研究中心 | Preparation method and application of killed bacillus vallismortis suspending agent for killing citrus mites |
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