CN102978130B - Bacillus pumilus GBSC66 and its application - Google Patents
Bacillus pumilus GBSC66 and its application Download PDFInfo
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- CN102978130B CN102978130B CN201210433938.3A CN201210433938A CN102978130B CN 102978130 B CN102978130 B CN 102978130B CN 201210433938 A CN201210433938 A CN 201210433938A CN 102978130 B CN102978130 B CN 102978130B
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- 229910002651 NO3 Inorganic materials 0.000 claims description 3
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- 201000010099 disease Diseases 0.000 abstract description 12
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 12
- 230000000694 effects Effects 0.000 abstract description 11
- 241001558929 Sclerotium <basidiomycota> Species 0.000 abstract description 5
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 1
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Classifications
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention relates to a Bacillus pumilus having a preservation number of GBSC66 CGMCC No.3379, separated in Tibet, having growth promoting and disease preventing effects and growing at a low temperature, and its application in the control of rape sclerotium diseases. The Bacillus pumilus GBSC66 is Biocontrol bacillus obtained through separating from grass rhizosphere soil in Songduo, Gongbujiada Town, Tibet, can well grown at a low temperature, is benefit for the performance of the growth promoting and biocontrol effects, has a rape sclerotium disease biocontrol effect of 69%, enables the output to be increased by 17%, and has a very good application prospect.
Description
Technical field
The invention belongs to microorganism and agricultural disease Prevention Technique field, relate to bacillus pumilus, be specifically related to suitable raw bacillus pumilus (Bacilluspumilus) GBSC66 of a kind of low temperature and the application in control sclerotinia rot of colza evil thereof.
Background technology
Rape is the main oil crops of China, and sclerotinia rot of colza is one of important disease in the Rape-seed production caused by Ascomycotina fungi Sclerotinia sclerotiorum (Sclerotinia sclerotiorum).Sclerotinia rot of colza all can be fallen ill in the whole breeding time of rape, and productive phase occurs the heaviest.Stem, leaf, flower, angle fruit all can be injured, and stem is injured the heaviest.Stem catches an illness and begins to show light brown water stain shape scab, after develop into the rectangular spot of tool wheel line shape, edge brown, table raw flocculence white hypha during high humidity, accidental black sclerotium, the rotten one-tenth cavity of marrow in sick stem, interior life a lot of black mouse excrement shape sclerotium.Sclerotinia rot of colza is first of the large disease of rape three in China, and winter, spring cultural area all have generation, bring heavy losses to production.Long-term strain sickness rate is up to 10%-30%, and serious reaches more than 80%; The general underproduction 70%-100% of diseased plant.
The Main Means of current control sclerotinia rot of colza is for spraying chemical pesticide, the extensive application of the latter not only causes the pollution of air, water source, soil and food, the prior orthoselection being through chemical pesticide makes many pathogenic bacterias create resistance, control for this disease brings larger difficulty, serious threat China Rape-seed production safety.In recent years, increasing area adopts the method for integrated control to prevent and treat sclerotinia rot of colza, wherein especially plays an important role with biological pesticide.Rhizospheric microorganism is of a great variety, wherein have many types can Promoting plant growth, controlling plant diseases, increase crop yield, this quasi-microorganism is commonly referred to as plant growth-promoting rhizobacteria (Plant Growth-Promoting Rhizobacteria is called for short PGPR).There is good mutualism relation between PGPR and plant, plant can not only be made to produce induced resistance, and the yield and quality of crop can be improved.So, be separated from PGPR and utilize the probiotic bacterium the control and the increase yield that are applied to Plant diseases with growth-promoting and prophylaxis effect, there is very important theory significance and using value.
CN 101270344A discloses a strain has inhibiting Antagonistic Fungi X-01 bacterial strain separation screening to sclerotinia rot of colza, the separation of antibacterial substance and application.The X-01 bacterial strain of this invention is Bacillus subtillis (Bacillus subtilis), can secrete the lipopeptide antibiotic ITURIN A that a kind of molecular weight is 1042.9DA.This invention also discloses the method utilizing X-01 bacterial strain to prepare lipopeptide antibiotic ITURIN A.In vitro biological and ecological methods to prevent plant disease, pests, and erosion test shows, lipopeptide antibiotic ITURINA prepared by this invention has obvious inhibition to Sclerotinia sclerotiorum, the effect of preventing and treating sclerotinia rot of colza when lipopeptide antibiotic ITURIN A concentration is 100 Μ G/ML reaches 100%, and prevention effect when concentration is 10 Μ G/ML reaches 40.7%.
CN 101603009B discloses a strain and has strong parasitism to sclerotinite sclerotium, cause point screening of rotten active shield shell trichoderma strain, qualification and the application in microbiobacterial agent, also discloses the semi-open-type solid fermentation cultural method of ZS-1SB microbial inoculum and the application in control sclerotinia rot of colza.The microbiobacterial agent of this invention has good biocontrol effect to fungal diseases of plants such as sclerotinia rot of colzas.
CN 101935620A discloses a kind of green color-producing streptomycete JZG117 (Streptomyces viridochromogenes) with suppression Sclerotinia sclerotiorum, additionally provide and utilize this bacterial strain effectively to suppress the diffusion of dish hyphal cluster germ in rape body, reduce sickness rate, improve the yield and quality, and contribute to biological control product development, and then promote the safety control of this disease.
Summary of the invention
One of the object of the invention is that provide a kind of bacillus pumilus (Bacillus pumilus), it has good fungistatic effect to pathogenic bacterias such as Sclerotinia sclerotiorums, has potential biological and ecological methods to prevent plant disease, pests, and erosion to be worth for Problems existing in current sclerotinia rot of colza control.Two of the object of the invention is to provide the application of this bacillus pumilus in control sclerotinia rot of colza.
For one of achieving the above object, the present invention adopts following technical scheme:
A kind of bacillus pumilus, Classification And Nomenclature is bacillus pumilus (Bacillus pumilus) GBSC66, and on November 2nd, 2009 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCC No.3379.
Described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 encloses soil to be separated from the many grass roots of pine in work Bu Jiada town, Tibet to obtain.
The morphological specificity of described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 and physiological and biochemical property: Gram-positive, shaft-like, can 10 DEG C of growths, it is the suitable raw bacterium of a kind of low temperature, but can not 50 DEG C of growths, the cultivation of pH5.7 and 7%NaCl can be tolerated, catalase test, Citrate trianion utilize test, glucose fermentation test and casein test to be the positive, and Starch Hydrolysis test, V-P test, nitrate reduction test, experiment of growth factor and propionic salt utilize test to be feminine gender.
The Molecular Identification feature of described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379: the part 16S rRNA sequence utilizing round pcr amplification bacillus pumilus (Bacillus pumilus) GBSC66CGMCCNo.3379, obtaining length is the fragment of 487bp, its sequencing result is as shown in sequence table, through carrying out tetraploid rice with known 16S rRNA sequence in GenBank, find that with the similarity of Bacillus pumilus GQ911554.1 be 99%.
According to the result of the gramstaining of bacterial strain, morphological specificity, physiological and biochemical property and 16S rRNA sequential analysis, with reference to " bacillus ", (Cai Miaoying etc. translate, agricultural press, 1988) and " microbial taxonomy " (Zhang Jizhong, Fudan University in Shanghai press, 1995) key is retrieved, and bacterial strain of the present invention is bacillus pumilus (Bacillus pumilus), called after GBSC66 through Bacillus subtilis.
Microbial inoculum containing bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 also belongs to protection scope of the present invention.
For achieve the above object two, the present invention adopts following technical scheme:
The application of described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 in control sclerotinia rot of colza.Concrete technical scheme is as follows:
Described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 has significant fungistatic effect for Sclerotinia sclerotiorum.
Described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 has significant fungistatic effect for yeast.
Described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 has significant growth promoting effects effect for rape.
The application method of described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 bacterial strain is: fermentation culture → dilution → sprinkling → effect measuring.
The fermentation culture of described bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 bacterial strain, its substratum consists of: glucose 20g/L, Pidolidone 5g/L, MgSO
40.5g/L, KCl0.5g/L, KH
2pO
41g/L, Fe
2sO
46H
2o 0.15mg/L, MnSO
4h
2o 5.0mg/L, CuSO
45H
2o0.16mg/L, all the other are filtered water, are adjusted to pH7.0.
Bacillus pumilus provided by the invention (Bacillus pumilus) GBSC66CGMCC No.3379 is a kind of genus bacillus sclerotinia rot of colza being had to good prevention effect, and be the suitable raw bacterium of a kind of low temperature, can grow at the temperature of 10 DEG C, environmentally safe, can significantly suppress Sclerotinia sclerotiorum and yeast, significantly can also promote growth of rape, there is potential biological and ecological methods to prevent plant disease, pests, and erosion and be worth.
Accompanying drawing explanation
Fig. 1 is the gemma photo of bacillus pumilus (Bacillus pumilus) the GBSC66CGMCC No.3379 of embodiment 2.
Fig. 2 is that bacillus pumilus (Bacillus pumilus) the GBSC66CGMCC No.3379 of embodiment 6 is to the bacteriostatic action photo of Sclerotinia sclerotiorum.
Fig. 3 is that bacillus pumilus (Bacillus pumilus) the GBSC66CGMCC No.3379 of embodiment 7 is to saccharomycetic bacteriostatic action photo.
Technical scheme of the present invention is further illustrated by embodiment below in conjunction with accompanying drawing; but following example is only simple and easy example of the present invention; do not represent or limit the scope of the present invention, interest field of the present invention is as the criterion with claims.
Embodiment
For better the present invention being described, be convenient to understand technical scheme of the present invention, typical but non-limiting embodiment of the present invention is as follows:
Embodiment 1:
Adopt five point samplings, the many grass roots of pine in work Bu Jiada town, Tibet enclose dark 5-25mm place in soil and take soil sample to load in sampler bag, have marked sampling time, place, vegetation.Take soil sample 5g, be dissolved in 45ml physiological saline, on shaking table, concussion mixing 30min, leaves standstill and makes clarification.Aspirate supernatant is respectively with sterilized water dilution 10
-1, 10
-2, 10
-3, 10
-4, 10
-5five concentration gradients, 80 DEG C of water-bath 10min.Draw 200 μ l and be coated with LB flat board, 37 DEG C of incubated overnight.After flat board grows bacterium colony, picking list bacterium colony, after line purifying, numbering is preserved, and screening obtains bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379.
Embodiment 2:
With reference to " bacillus ", (Cai Miaoying etc. translate, agricultural press, 1988) and " microbial taxonomy " (Zhang Jizhong, Fudan University in Shanghai press, 1995) test method, the morphological specificity of bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 obtain embodiment 1 and physiological and biochemical property detect.
Result shows, this bacterium Gram-positive, shaft-like, produces gemma as shown in Figure 1.Can 10 DEG C of growths, but can not 50 DEG C of growths, the cultivation of pH5.7 and 7%NaCl can be tolerated, catalase test, Citrate trianion utilize test, glucose fermentation test and casein test to be the positive, and Starch Hydrolysis test, V-P test, nitrate reduction test, experiment of growth factor and propionic salt utilize test to be feminine gender.
Embodiment 3:
Bacillus pumilus (Bacillus pumilus) GBSC66CGMCCNo.3379 embodiment 1 obtained is inoculated in LB liquid medium, 180rpm, 37 DEG C of concussions cultivate 10h, collected by centrifugation thalline, add N,O-Diacetylmuramidase and SDS broken wall after suspension, adopt phenol-chloroform method to extract genomic dna.
Be template with genomic dna, use forward primer PRB1601(5 '-GGATCCTAATACATGCAAGTCGAGCGG-3 ') (SEQ ID NO:1) and reverse primer PRB 1602(5 '-GGATCCACGTATTACCGCGGCTGCTGGC-3 ') (SEQ ID NO:2) carry out pcr amplification to 16S rRNA.PCR condition is: 95 DEG C, 4min; 94 DEG C, 1min; 50 DEG C, 1min; 72 DEG C, 2min; 34cycle; 72 DEG C, 10min, 4 DEG C of preservations.Reclaim after PCR primer and send order-checking, through confirming that 16S rRNA partial gene sequence length is 487bp, its result, as shown in sequence table, is 99% with Bacillus pumilus GQ911554.1 similarity in GenBank.
Embodiment 4:
According to table 1 formulated 1C substratum:
Table 1
| Moiety | Concentration | 1×1C | 5×1C |
| Peptone | 0.7 | 4.2g | 21g |
| Sodium-chlor | 0.5 | 3g | 15g |
| Soyflour | 0.3 | 1.8g | 9g |
| Final volume | 600ml | 600ml |
Prepare soil extract as follows:
Joined by 500g soil in 1L distilled water, 98 DEG C of heating 45min, filter paper filtering, in 4 DEG C of preservations after autoclaving.
According to table 2 formulated main medium:
Table 2
| Moiety | Final concentration | Volume (ml) |
| 5×1C | 1× | 50 |
| 20% glucose | 0.1% | 1.25 |
| Soil extract | 10% | 25 |
| Water | 173.75 |
Bacillus pumilus (Bacillus pumilus) GBSC66CGMCCNo.3379 embodiment 1 obtained is inoculated in above-mentioned main medium, 37 DEG C of overnight incubation, get the right-angled intersection point of 5 μ l bacterium liquid points to distance solid medium plate center 2.5cm, each bacterial strain 5 repetition, be placed on respectively in 4 DEG C, 10 DEG C, 14 DEG C, 18 DEG C, 30 DEG C incubators and cultivate, record strain growth situation every day, record 15 days.
Result shows, bacillus pumilus (Bacillus pumilus) GBSC66 CGMCCNo.3379 of the present invention can grow in the environment of temperature≤10 DEG C.The bacterial strain that can manifest bacterium colony at the temperature of 10 DEG C in one week is designated as suitable raw bacillus pumilus (Bacillus pumilus) GBSC66CGMCCNo.3379 of low temperature, the result of test further shows that it can grow under the condition of 4 DEG C.
Embodiment 5:
Bacillus pumilus (Bacillus pumilus) GBSC66CGMCCNo.3379 embodiment 1 obtained is inoculated into and is seeded on LB substratum, incubated overnight, then bacillus pumilus (Bacillus pumilus) the GBSC66CGMCC No.3379 of activation is transferred in Landy substratum and carries out shake flask fermentation, temperature is 30 DEG C, shaking speed is 200rpm, fermentation time is 38 hours, obtains the fermented liquid of bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379.
Wherein fermention medium consists of: glucose 20g/L, Pidolidone 5g/L, MgSO
40.5g/L, KCl0.5g/L, KH
2pO
41g/L, Fe
2sO
46H
2o 0.15mg/L, MnSO
4h
2o 5.0mg/L, CuSO
45H
2o0.16mg/L, all the other are filtered water, are adjusted to pH7.0.
Embodiment 6:
Adopt dull and stereotyped face-off method, good PDA is dull and stereotyped, the Sclerotinia sclerotiorum bacterium block of diameter 1cm is placed in dull and stereotyped central authorities, at its surrounding distance 2cm place, symmetry places four filter papers, filter paper is made a call to the fermented liquid of bacillus pumilus (Bacillus pumilus) the GBSC66CGMCC No.3379 of the embodiment 5 of 5 μ l, in triplicate, cultivate 3 days in 25 DEG C of illumination boxs, observe bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 to the fungistatic effect of Sclerotinia sclerotiorum.
As shown in Figure 2, visible average diameter of inhibition zone is 22mm to result, and namely bacillus pumilus provided by the invention (Bacillus pumilus) GBSC66CGMCC No.3379 has significant bacteriostatic action to Sclerotinia sclerotiorum.
Embodiment 7:
Suitable for the low temperature of embodiment 4 raw bacillus pumilus (Bacillus pumilus) GBSC66CGMCCNo.3379 is inoculated in 1C substratum and cultivates 8h, then be transferred in the Landy substratum of 10ml with the ratio of 1:100, ferment about 40 hours, collected by centrifugation supernatant liquor, for subsequent use in 4 DEG C of storages.
Fungal bacterial strain yeast to be measured is inoculated into overnight incubation in appropriate media, after measuring OD value, bacterium liquid is diluted to 10
7cfu/ml, mixed culture medium is made containing bacterium dull and stereotyped.To be cooled completely after, right-angled intersection punching (apart from plate center 2.5cm), suitable raw bacillus pumilus (Bacillus pumilus) the GBSC66CGMCC No.3379 of inoculation low temperature is in Landy substratum fermented supernatant fluid.Cultivate 2d under placing room temperature, observe for saccharomycetic fungistatic effect.
As shown in Figure 3, visible average diameter of inhibition zone is 29mm to result, and namely bacillus pumilus provided by the invention (Bacillus pumilus) GBSC66CGMCC No.3379 has significant bacteriostatic action to yeast.
Embodiment 8:
Semen Brassicae campestris clear water soaks 2h, vernalization under 28 DEG C of wet conditions, be seeded in the Turnover Box that aseptic soil is housed, fill with root when treating that Brassica campestris L seedling grows to 5 true leaves, average every young plant waters the fermented liquid of bacillus pumilus (Bacillus pumilus) the GBSC66CGMCC No.3379 of 20ml embodiment 5, and (concentration is 10
8cfu/ml), contrast with clear water.Extract 10 young plants after two weeks, measure plant height, root length and plant fresh weight.
Result shows, the Brassica campestris L seedling through the process of bacterium liquid is obviously good than the growing way of control group.After measured, plant height increment rate reaches 58%, and the long increment rate of root reaches 8.2%, and fresh weight increment rate reaches 121.3%.Namely bacillus pumilus provided by the invention (Bacillus pumilus) GBSC66CGMCC No.3379 significantly can promote growth of rape.
Embodiment 9:
The fermented liquid of bacillus pumilus (Bacillus pumilus) the GBSC66CGMCC No.3379 of embodiment 5 is diluted 100 times, within 10 days, starts before flowering to execute fermented liquid to rape spraying, afterwards at interval of spray in 7 days 1 time, spray 3 times altogether.
Result shows, the fermented liquid of bacillus pumilus of the present invention (Bacillus pumilus) GBSC66CGMCCNo.3379 has significant control sclerotinia rot of colza and improves the effect of yield of rape, its biocontrol effect can reach 69.3%, increases yield of rape and reaches 17.2%.
It should be noted that and understand, when not departing from the spirit and scope of the present invention required by accompanying claim, various amendment and improvement can be made to the present invention of foregoing detailed description.Therefore, the scope of claimed technical scheme is not by the restriction of given any specific exemplary teachings.
Applicant states, the present invention illustrates proportioning raw materials of the present invention and preparation process by above-described embodiment, but the present invention is not limited to said ratio and preparation process, does not namely mean that the present invention must rely on above-mentioned preparation process and could implement.Person of ordinary skill in the field should understand, any improvement in the present invention, to equivalence replacement and the interpolation of ancillary component, the concrete way choice etc. of raw material selected by the present invention, all drops within protection scope of the present invention and open scope.
Sequence table: (SEQ ID NO:3)
TTGGCGTAGTTGCCGTGGCTTTCTGGTTAGGTACCGTCAAGGTGCGAGCAGTTACTCTCGCACTTGTTCTTCCCTAACAACAGTAGCTTTACGATCCGAAGACCTTCATCACTCACGCGGCGTTGCTCCGTCAGGACTTTCGTCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTGAGCCATTACCCCACCAACTAGCTAATGCGCCGCGGGTCCATCTGTAAGTGACAGCCGAAACCGTCTTTCATCCTTGAACCATGCGGTTCAAGGAACTATCCGGTATTAGCTCCGGTTTCCCGGAGTTATCCCAGTCTTACAGGCAGGTTACCCACGTGTTACTCACCCGTCCGCCGCTAACATCCGGGAGCAAGCTCCCTTCTGTCCGCTCGACTTGCATGTATTAAAGATCCAA
Claims (1)
1. bacillus pumilus (Bacillus pumilus) GBSC66CGMCC No.3379 is suppressing the application in yeast;
Described bacillus pumilus (Bacillus pumilus) GBSC66 on November 2nd, 2009 in the center preservation of China Committee for Culture Collection of Microorganisms's common micro-organisms, preserving number is CGMCC No.3379, encloses soil to be separated to obtain from the many grass roots of pine in work Bu Jiada town, Tibet; Its morphological specificity and physiological and biochemical property are: Gram-positive, shaft-like, can 10 DEG C of growths, but can not 50 DEG C of growths, the cultivation of pH5.7 and 7%NaCl can be tolerated, catalase test, Citrate trianion utilize test, glucose fermentation test and casein test to be the positive, and Starch Hydrolysis test, V-P test, nitrate reduction test, experiment of growth factor and propionic salt utilize test to be feminine gender.
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