CN103289931B - 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 and a kind ofly spend territory Bacillus strain SJ and in the application of preparing in tobacco disease resistance toxin preparation and growth-promoting agent.
Background technology
Tobacco is the main cash crop of China, and its output and relationship between quality national economy.At present, the general method of controlling tobacco diseases is to use chemical pesticide, but, life-time service chemical pesticide can cause pathogenic bacteria to develop immunity to drugs and reduce the protection effect of chemical agent, life-time service chemical agent can cause pesticide residue to exceed standard and environmental pollution simultaneously, cause tobacco leaf interior quality to decline, vega disruption of ecological balance, has even had a strong impact on the Sustainable development of leaf tobacco production.And biological control is a kind of new way of controlling Plant diseases, it is mainly the antagonistic action of utilizing between biocontrol microorganisms and plant pathogenic microorganisms, the growth of Suppressing phytopathogens.Antagonistic action size in microbial preparation between kind and the pathogenic micro-organism of biocontrol microorganisms is to determine the key of its anti-virus ability, and therefore by finding the high-activity microorganism of antagonism pathogenic bacterium or pathogenic insect and meta-bolites thereof, to prevent and solve the viroses of plant are current study hotspot and breaches now.
Summary of the invention
The invention provides and a kind ofly spend territory Bacillus strain SJ and in the application of preparing in tobacco disease resistance toxin preparation and growth-promoting agent.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 the advantages such as industrialization production.
For achieving the above object, the present invention adopts following technical proposals to be achieved:
A kind of flower territory Bacillus strain SJ, its Classification And Nomenclature, for flower territory bacillus vallismortis, is preserved in China Committee for Culture Collection of Microorganisms's 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 has projection, and surface is film, and the inside is mucus, has provoked wire drawing, and thalline is rod-short, and gemma end is raw, 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 liquid bacterial agent living bacteria count is more than 100,000,000/mL.
Wherein, the Optimal Medium formula 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 SJ bacteria preparation that contains colored territory Bacillus strain SJ described in the present invention also provides is in the application of preparing in tobacco disease resistance toxin preparation.
Wherein, described virus is tobacco mosaic virus (TMV).
The application of the SJ bacteria preparation that contains colored territory Bacillus strain SJ described in the present invention also provides in preparation tobacco growth-promoting agent.
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, alleviate 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 transport, is convenient to factory's extension and produces, and realizes industrialization, has good technique effect and market application foreground.
Read by reference to the accompanying drawings after the specific embodiment of the present invention, it is clearer that the other features and advantages of the invention will become.
Brief description of the 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 of colored territory of the present invention genus bacillus SJ bacteria preparation on the impact of 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
On July 4th, 2012 samples altogether more than 50 parts of rhizosphere soils from Yishui, Shandong cigarette district tobacco-growing soil, gets respectively 2g sample and is suspended in 20ml physiological saline, 180rpm, concussion 30min.Getting respectively 100 μ L sample suspension is evenly coated with at nutrient agar plate, in 35 DEG C of incubators, be inverted and cultivate 16h, single bacterium colony method of scoring that picking comes in every shape is inoculated on nutrient agar plate, obtain pure bacterium by repeated multiple times line, filter out altogether the pure bacterium of 126 strain, and all bacterial strains are preserved by inclined-plane.
Two, the screening of the SJ bacterial strain of control TMV antagonistic bacterium
The above-mentioned purifying inoculation of picking is in LB liquid nutrient medium, 28 DEG C of constant temperature, 140rpm shaking culture 48h, get after 20 times of TMV juice mixing 30min of 20ml and equal-volume, frictional inoculation TMV(tobacco mosaic virus (TMV), tobacco mosaic virus) the withered spot host three lives-NN cigarette, 4, top of every strain inoculation leaf, be mixed into and contrast with TMV with sterilized water, 3～5d investigates withered spot inhibiting rate.Have the bacterial strain of antagonistic activity to adopt same procedure repeated test through preliminary test, every processing 3 strains, repeat for three times.Withered spot inhibiting rate calculation formula:
Withered spot inhibiting rate experimental result shows that bacterial strain SJ is best to the inhibition of TMV, and the withered spot inhibiting rate of TMV is reached to 94.5%, and biocontrol bacteria has produced in growth metabolism process has the antibiotics (in table 1) that suppresses virus activity.
The restraining effect of table 1 bacterial strain SJ to tobacco mosaic viruses
Three, the bacteria molecule genetic classification qualification to flower territory genus bacillus
SJ bacterial strain is extracted to DNA, and taking this DNA as template, 16S rDNA universal primer is primer, and 16S rDNA fragment is increased.The fragment of amplification is directly carried out to sequencing.Blast program on the result input NCBI website of order-checking is compared, result shows that the 16s rDNA sequence homology degree of the Bacillus vallismortis in bacillus in this bacterial strain 16S rDNA sequence and GenBank gene pool is the highest, and homology reaches 99%.By DNAMAN6.0, the 16S rDNA sequence of existing bacillus in Genbank is carried out to phylogenetic analysis, result shows, bacterial strain SJ16S rDNA and Bacillus vallismortis homology are the highest, therefore can judge the colored territory genus bacillus that this bacterial strain is bacillus, called after flower territory Bacillus strain SJ.
The colored territory Bacillus strain SJ screening is carried out to culture presevation, depositary institution: China Committee for Culture Collection of Microorganisms's 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 DEG C 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 surface is film, and the inside is mucus, has provoked wire drawing.Flower territory genus bacillus thalline is rod-short, and gemma end is raw, circle.
Four, spend the medium optimization of territory Bacillus strain SJ
The SJ bacterial classification streak inoculation that test tube slant is preserved is on NA culture medium flat plate, cultivate to be inoculated in after 64h for 28 DEG C and in nutrient broth liquid nutrient medium, carry out liquid culture, in every 500mL triangular flask, liquid amount is 200mL, in 35 DEG C, 180r/min. constant-temperature table is cultivated 16h, is used for test below as seed liquor.
Fixing nitrogen source urea, adds the single carbon sources such as Semen Maydis powder, sucrose, glucose, starch, carries out carbon source screening; Fixing carbon source Semen Maydis powder, adds the single nitrogenous sources such as soybean cake powder, yeast powder, peptone, filters out optimum nitrogenous source, adjusts the optimization best medium such as medium pH, leavening temperature, rotating speed, inoculative proportion.
The Optimal Medium formula 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.
Fermentation results: pH7.0 left and right, 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 test tube slant is preserved is on nutrient broth medium flat board, cultivate to be inoculated in after 16h for 35 DEG C and in nutrient broth liquid nutrient medium, carry out liquid culture, in every 500mL triangular flask, liquid amount is 200mL, in 35 DEG C, 180r/min, constant-temperature table is cultivated 16h, as primary seed solution.
Primary seed solution is inoculated in the substratum of having optimized, liquid amount 240L in 350L fermentor tank, 35 DEG C, mixing speed 180r/min, is inoculated into 10 tons of fermentor tanks, 35 DEG C as secondary seed solution after fermentation 16h, mixing speed 180r/min, approximately 14h left and right, gemma rate 80% is put tank, is the fermented liquid of SJ bacterial strain.By gained bacterium liquid elimination dregs, mix in 1:1 ratio with carrier calcium carbonate, stir, by spray-drying tower, set 170 DEG C of inlet temperature, 60 DEG C of air outlet temperatures, remove moisture, and thalline is attracted on carrier completely, and gained is SJ bacteria preparation.The mensuration of living bacteria count, according to GB20287 agricultural microorganism microbial inoculum standard, records living bacteria count 50,000,000,000/g.
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 liquid bacterial agent living bacteria count is that 100,000,000/mL can reach good antibacterial effect above.
Six, the antagonistic action of the SJ bacteria preparation of Antiphytoviral to TMV
By 1000 times of bacterial strain SJ preparation dilutions, after 80 times of TMV juice equal-volume mixing 15min, the frictional inoculation three lives-NN cigarette, 2, top of every strain inoculation leaf, is mixed into CK with sterilized water and TMV, replaces SJ preparation liquid as comparison taking SJ nutrient solution, NB substratum simultaneously.Calculate withered spot inhibiting rate.
The restraining effect of table 2 SJ preparation to TMV
| ? | SJ fermented liquid | 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 by table 2, SJ bacteria preparation neither affects the activity of fermented liquid, can well adsorb again bacterium liquid, and the preventive effect of TMV is reached to 97.29%.Illustrate and select calcium carbonate as carrier, do not affect the restraining effect of SJ viable bacteria to TMV, calcium carbonate, as filler, has the advantage such as excellent adsorption, low price, can on producing, use.
Seven, have an X-rayed the impact of electric Microscopic observation SJ preparation on TMV virion
Negative staining is processed after the TMV purifying, and through JEM-100CX transmission electron microscope observing, the TMV plastochondria in the visual field is typically shaft-like, the about 500nm of length, and upright and outspoken and marshalling, integrity is good; After 1000 times of SJ preparation dilutions and the pure TMV plastochondria of 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.As can be seen here, SJ preparation has strong destruction to TMV plastochondria, proves that SJ preparation can produce passivation to virus to the antagonistic substance of TMV, suppresses the polymerization of TMV coat protein, and destroys the integrity of plastochondria.
Eight, growth-promoting functions
Common cigarette NC89 of 2-3 leaf phase, transplants to the small flower of diameter 10cm, and the SJ preparation with 1000 times of dilutions when transplanting is filled with root, every strain 10ml, and interval 3d, then continuous irrigation root 2 times, fill with root as contrast taking nutrient broth, sterilized water, and every processing 6 strains repeat for 3 times.Last is filled with after root 30d, investigation fresh weight, maximum leaf length and width.
Found out by table 3, common cigarette NC89, fills with root continuous three times, and SJ bacteria preparation is processed its fresh weight, and maximum leaf length and width is all higher than nutrient broth and clear water processing, extremely remarkable with clear water processing difference.
The growth-promoting functions of table 3 4A1 fermentation liquid irrigating root to NC89
Nine, the stability test of fungi medicament
SJ bacteria preparation is at room temperature placed, at set intervals preparation is counted.1000 times of preparation dilutions are mixed with TMV equal-volume, and greenhouse inoculation three lives NN cigarette, detects the withered spot inhibiting rate to virus, and result is as shown in table 4.
Active degradation rule under table 4 SJ bacteria preparation room temperature
Find out by table 4, SJ bacteria preparation is at room temperature preserved, and along with the prolongation of time, activity is degraded gradually, preserve 12 months, activity to TMV drops to 79.04%, and bacterium number is 25,000,000,000/g, SJ bacteria preparation is described at room temperature, degraded slowly, analyzing its reason is mainly to have formed gemma, is easy to storage and transport, has the potential quality that is developed as biological prevention and control agent.
Above embodiment is only in order to technical scheme of the present invention to be described, but not is limited; Although the present invention is had been described in detail with reference to previous embodiment, for the person of ordinary skill of the art, the technical scheme that still can record previous embodiment is modified, or part technical characterictic is wherein equal to replacement; And these amendments or replacement do not make the essence of appropriate technical solution depart from the spirit and scope of the present invention's technical scheme required for protection.
Claims (7)
1. spend a territory Bacillus strain SJ, it is characterized in that: its Classification And Nomenclature is flower territory genus bacillus
bacillus vallismortis, being preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, its deposit number is: CGMCC No.7571.
2. a kind of flower 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, have projection, surface is film, and the inside is mucus, provoke wire drawing, thalline is rod-short, and gemma end is raw, circle.
3. contain the SJ bacteria preparation of colored territory Bacillus strain SJ claimed in 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 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 Optimal Medium formula 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 SJ bacteria preparation that contains colored territory Bacillus strain SJ according to claim 3, in the application of preparing in tobacco disease resistance toxin preparation, is characterized in that, described virus is tobacco mosaic virus (TMV).
7. the application of the SJ bacteria preparation that contains colored territory Bacillus strain SJ according to claim 3 in preparation tobacco growth-promoting agent.
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| 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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