CN107502565B - One plant of fritillary bulb rhizosphere Paenibacillus polymyxa and its application - Google Patents
One plant of fritillary bulb rhizosphere Paenibacillus polymyxa and its application Download PDFInfo
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- 241000194105 Paenibacillus polymyxa Species 0.000 title claims abstract description 56
- 241000935235 Fritillaria meleagris Species 0.000 title claims abstract description 30
- 241000196324 Embryophyta Species 0.000 title claims abstract description 17
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 claims abstract description 19
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/12—Bacillus polymyxa ; Paenibacillus polymyxa
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Abstract
One plant of fritillary bulb rhizosphere Paenibacillus polymyxa and its application are related to one plant of Paenibacillus polymyxa and its application.The present invention provides a kind of fritillary bulb rhizosphere Paenibacillus polymyxa.Paenibacillus polymyxa SBS-SF0105 of the present invention, is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and the deposit date is on November 5th, 2015, deposit number was CGMCC No.11586.The bacterial strain antimicrobial spectrum is wide, and bacteriostasis is strong, can colonize in plant rhizospheres such as fritillary bulbs, for preventing and treating the plant soil-borne diseases such as fritillary bulb, Schisandra chinensis, wilsonii, using the bacteria agent that the bacterial strain is prepared as main component, can effectively solve fritillary bulb Soil-sickness Problem.
Description
Technical field
The present invention relates to one plant of Paenibacillus polymyxa and its applications.
Background technique
Fritillary bulb (Fritillaria ussuriensis Maxim it is) Liliaceae Fritillaria herbaceos perennial, is
One of important medicinal material in the Northeast, flat shellfish bulb effective component are alkaloid.With clearing heat and moistening lung, preventing phlegm from forming and stopping coughing, mass dissipating and swelling eliminating
And other effects, for treating a variety of diseases such as bronchitis, pneumonia, pertussis.Fritillary bulb not be only bulk medicinal materials, and medicine,
The raw material of the industries such as health care.Heilongjiang Province is fritillary bulb Main Cultivation producing region, as Forest Industry System of Heilongjiang Province stops commerciality comprehensively
It fells and management mode transition, forest economy has become the important growth point in lumber's economy of forestry.Northern medicine cultivation is lumber
The important plate of forest economy, wherein the cultivation of flat shellfish is one of the pillar industry of part forestry bureau, wherein building in Hailin forestry bureau
Vertical fritillary bulb cultivation base, cultivated area is just up to 3000 mu or more.
Black rot is the most important disease of flat shellfish also known as sclerotiniose, belongs to soil-borne disease, is passed by medium of soil
It broadcasts, seriously can lead to flat shellfish total crop failure, black rot is mainly controlled by chemical pesticides at present, but effect is undesirable, and the chemistry of many years is anti-
Controlling causes pathogen drug resistance to increase, soil degradation, and serious, flat shellfish occurs for the problems such as continuous cropping obstacle, pesticide excess residual
Female yield and quality is affected.Therefore, Many researchers are devoted to find a kind of effectively preventing side for a long time
Method, to reduce the generation of black rot.Pathogen due to endangering fritillary bulb underground bulb is soil-borne pathogen, existing in the soil
Good facultative parasitism ability, while there is stronger battalion corruption survival ability again, these pathogens diffusion in the soil and numerous
Upgrowth situation close phase light of the speed with soil environment and fritillary bulb is grown, research has shown that soil microenvironment disorder and soil reason
Change property destruction and be the main reason for causing black rot persistently to occur, therefore take agricultural measures etc. and is unable to reach ideal control
Effect, it is difficult to tackle the problem at its root, the chemical pesticides such as soil sterilants are excessively used for a long time can also destroy soil micro-ecosystem
Environment, while it is excessively high to cause harmful substance to remain in fritillary bulb bulb, therefore needs set of environmental concordance method to continue
Control the generation of fritillary bulb black rot.
The it is proposed of " Good Agricultural Practices " (GAP) and the development of relevant authentication, it is raw to medicinal plant cultivation
The influence of production is of far-reaching significance, and GAP advocates and encourage to use the medicament of biogenic pesticide and low toxicity and low residue, especially biology system
Agent, and the integrated application for the non-pollutings control method such as cooperate cultural control method, physical control, are reduced as far as possible to reach
With or without the use of the purpose of chemical synthetic pesticide.There is largely have the beneficial of antagonism to black rot in fritillary bulb rhizosphere
Microorganism carries out biological control to flat shellfish black rot using the probiotics of plant rhizosphere, can fundamentally improve plant rhizosphere
Micro-ecological environment, and have the characteristics that nontoxic, pollution-free, and adventive will not be introduced, be prevention and treatment fritillary bulb soil-borne disease
One effective way.
Summary of the invention
The present invention provides a kind of fritillary bulb rhizosphere Paenibacillus polymyxa, and the bacterial strain antimicrobial spectrum is wide, and bacteriostasis is strong, can be
The plant rhizospheres such as fritillary bulb colonize, for preventing and treating the plant soil-borne diseases such as fritillary bulb, Schisandra chinensis, wilsonii, based on the bacterial strain
The bacteria agent for wanting ingredient to prepare, can effectively solve fritillary bulb Soil-sickness Problem.
Fritillary bulb rhizosphere Paenibacillus polymyxa of the present invention be Paenibacillus polymyxa (Paenibacillus polymyxa) SBS-SF0105, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC),
Preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Institute of Microorganism, Academia Sinica, the deposit date is 2015
On November 5, in, deposit number are CGMCC No.11586.
Paenibacillus polymyxa SBS-SF0105 of the present invention is in PDA culture medium, and after 25 DEG C of culture 48h, bacterium colony is at nothing
Color transparence, non-pigment generate, and colony diameter 1.5-2.0mm, bacterium colony moistens tacky, neat in edge.The bacterium thallus is rod-shaped, bud
Spore is ellipse, and sporangium is expanded, and Gram's staining is positive.
The contact enzymatic activity of Paenibacillus polymyxa SBS-SF0105 of the present invention is the positive, and oxidase active is negative, and V-P is real
The positive is tested, degradable starch, pectin and xylan are fermented type bacterial strain, the sugar such as oxidable glucose, arabinose, mannitol
Class.
Paenibacillus polymyxa SBS-SF0105 of the present invention is by 16S rDNA sequence alignment analysis, with more viscous class gemma bars
Bacterium (Paenibacillus polymyxa) between have high homology, homology 99%.By combining thalli morphology special
Sign, growth conditions, Physiology and biochemistry qualification result determine Paenibacillus polymyxa belong to bacillus (Paenibacillus),
For Paenibacillus polymyxa (Paenibacillus polymyxa).
Root irrigation is carried out to fritillary bulb plant using Paenibacillus polymyxa SBS-SF0105 fermentation liquid of the invention, is led to
Opposite culture experiment and field control experiment are crossed, Paenibacillus polymyxa, which has, preferably prevents and treats flat shellfish soil-borne disease black rot
Effect, control efficiency can reach 55% or more, while the use of Paenibacillus polymyxa microbial inoculum has improvement soil microenvironment
Effect.
Paenibacillus polymyxa of the present invention (Paenibacillus polymyxa) SBS-SF0105, belong to bacillus
(Paenibacillus), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), preservation
Address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, and Institute of Microorganism, Academia Sinica, the deposit date is 2015 11
The moon 5, deposit number are CGMCC No.11586.
Detailed description of the invention
Fig. 1 is the 16S for the close bacterial strain included in Paenibacillus polymyxa SBS-SF0105 of the present invention and GenBank
RDNA sequence carries out phylogenetic tree constructed by sequence analysis.
Specific embodiment
The technical solution of the present invention is not limited to the following list, further includes between each specific embodiment
Any combination.
Specific embodiment 1: present embodiment fritillary bulb rhizosphere Paenibacillus polymyxa is Paenibacillus polymyxa
(Paenibacillus polymyxa) SBS-SF0105, it is deposited in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center (CGMCC), preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica,
The deposit date is on November 5th, 2015, deposit number was CGMCC No.11586.
Present embodiment Paenibacillus polymyxa SBS-SF0105 is in PDA culture medium, after 25 DEG C of culture 48h, bacterium colony
At colorless and transparent, non-pigment is generated, and colony diameter 1.5-2.0mm, bacterium colony moistens tacky, neat in edge.The bacterium thallus is bar
Shape, gemma are ellipse, and sporangium is expanded, and Gram's staining is positive.
Referring to " Berger bacterial identification manual " the 8th edition and " common bacteria system identification handbook " to Paenibacillus polymyxa
(Paenibacillus polymyxa) SBS-SF0105 progress Physiology and biochemistry identification: present embodiment Paenibacillus polymyxa
The contact enzymatic activity of SBS-SF0105 is the positive, and oxidase active is negative, and V-P experiment is positive, and degradable starch, pectin and wood are poly-
Sugar is fermented type bacterial strain, the carbohydrates such as oxidable glucose, arabinose, mannitol.
Present embodiment Paenibacillus polymyxa SBS-SF0105 can be grown in PDA culture medium, optimum growth temperature
It is 25 DEG C.
Specific embodiment 2: present embodiment Paenibacillus polymyxa SBS-SF0105 is isolated by flat shellfish rhizosphere.
Separation method sequentially includes the following steps:
1) separation of fritillary bulb rhizosphere bacteria saves: cultivating plot in flat shellfish, takes the flat shellfish plant of health, is received using method is shaken off
Collecting rhizosphere soil sample will be under the rhizosphere soil brush on root system and bulb with hairbrush after first gently shaking off the soil without root system
It collects.It takes Rhizosphere Soil 1g to be put into 250ml sterilizing triangular flask, 100ml sterile water is added, sterilizing bead 30-40 is a, on shaking table
160rpm vibrates 30min, stands 30min, supernatant is taken, using gradient dilution method diluted concentration to 10-4、10-5、10-6Three ladders
Degree, draws on 0.1ml liquid inoculation to beef extract-peptone solid medium, 30 DEG C of constant temperature incubations respectively from three gradients
48h, the different single bacterium colony of picking cultural characteristic are used as strains tested after culture purified.
2) screening of flat shellfish black rot biocontrol bacteria: antagonistic bacterium is screened using opposite culture method, in PDA culture medium
Plate (90mm) centre is inoculated with the isolated flat shellfish Pathogen of Black Rot bacterium PB-011(obtainedSclerotinia sclerotiorum)
(Song little Shuan meets Wen Jing, Yin great Chuan, Zhou Qi, the flat shellfish Pathogen of Black Rot bacterium separation identification of Deng Xun and biological characteristics
Primary Study [J] Chinese forest disease pest, 2016, (03): 7-11.)
The bacterial strain of the separated purifying of step 1) is put respectively and is connect on the point at pathogen 2.5cm, it is short of money not connect
Antibacterial strain is control, is placed in 28 DEG C of constant incubators and is cultivated.Entire culture dish is covered with wait compare, measures each processing
Bacteriostasis rate, choose to pathogen have compared with high inhibition effect bacterial strain carry out secondary screening, compare the size of bacteriostasis rate, filter out antibacterial
The good bacterial strain of effect.Antagonism fermented liquid is measured to the inhibiting effect of pathogen, takes fermentation liquid through 0.22 μm of membrane filtration, nothing
The bacteriostatic activity of bacterium filtrate Odontothrips loti measurement fermentation liquid.100 μ L bacteria-free filtrates are added in each Oxford cup, after 28 DEG C of cultures
Measure bacteriostasis rate, using non-inoculation medium supernatant as blank control, 3 repetitions of every processing.Effect is selected according to result
Optimal bacterium (such as table 1), as the Paenibacillus polymyxa SBS-SF0105 of present embodiment.
Table 1
| Number | Opposite culture bacteriostasis rate (%) | Fermentation liquid bacteriostasis rate (%) |
| Paenibacillussp SBS-SF0105 | 79.11±5.05 | 85.19±6.23 |
Molecular Identification is carried out to the Paenibacillus polymyxa SBS-SF0105 that separation screening obtains, is sequentially included the following steps:
The total DNA for extracting bacterial strain carries out PCR amplification by template of genomic DNA using the 16S rDNA universal primer of bacterium.Then
Utilize plastic recovery kit recovery purifying PCR product；Later, it cloned, converted, screening positive clone daughter colony, through expanding
It is sequenced after culture.
Paenibacillus polymyxa (Paenibacillus polymyxa) the 16SrDNA sequence length of SBS-SF0105 is
1440bp, as shown in SEQ ID NO:1 in sequence table.16S rDNA sequence in sequencing result and GenBank carries out homology
It compares, the phylogenetic tree of building, to determine the race relation of bacterial strain.Homology analysis the result shows that, the sequence and mostly viscous classes
Bacillus (Paenibacillus polymyxa) between have high homology, homology 99%.By combining thallus shape
State feature, growth conditions, Physiology and biochemistry qualification result determine that Paenibacillus polymyxa belongs to bacillus
(Paenibacillus), be Paenibacillus polymyxa (Paenibacillus polymyxa).
The indoor and outdoor fungistatic effect test of present embodiment Paenibacillus polymyxa SBS-SF0105 is specific as follows:
Test 1 flat shellfish rhizosphere Paenibacillus polymyxa SBS-SF0105 Indoor antibacterial spectrum measurement
1) cause of disease bacteria strain include flat shellfish black rot (Sclerotinia sclerotiorum), Rhizoctonia solani Kuhn (Rhizoctonia solani), fusarium solani ( Fusarium solani), Shoot-blight for Pinus sylvestris var. mongolica (Sphaeropsis sapinea), Cytospora chrysosperma (Valsa sordida), the above pathogen is purchased from Heilongjiang Province's forest conservation research
Institute.
2) antimicrobial spectrum measurement uses opposite culture method and fermentation liquid bacteriostatic method.Opposite culture, in PDA culture medium plate
The center (90mm) access 5mm pathogen bacteria cake, meets Paenibacillus polymyxa SBS-SF0105 in distance center 2.5cm location point,
2 points of every ware inoculated bacteria repeats 3 wares, is control with not inoculated bacteria.Fermentation liquid is antibacterial, takes fermentation liquid through 0.22 μm of filter membrane mistake
Filter, bacteria-free filtrate Odontothrips loti measure the bacteriostatic activity of fermentation liquid.100 μ L bacteria-free filtrates, 28 DEG C of trainings are added in each Oxford cup
Bacteriostasis rate is measured after supporting 72h, using non-inoculation medium supernatant as blank control.
3) measurement result such as table 2:
Table 2
| Pathogen | Opposite culture bacteriostasis rate (%) | Fermentation liquid bacteriostasis rate (%) |
| S.sclerotiorum | 60.54±1.15 | 62.33±1.87 |
| R.solani | 45.98±2.25 | 67.12±2.32 |
| F.solani | 49.78±2.22 | 55.25±1.99 |
| S.sapinea | 67.85±3.21 | 69.56±3.55 |
| V.sordida | 50.22±1.15 | 59.07±2.19 |
By testing result it is found that Paenibacillus polymyxa SBS-SF0105 of the invention is for flat shellfish black rot
(Sclerotinia sclerotiorum), Rhizoctonia solani Kuhn ( Rhizoctonia solani), fusarium solani (Fusarium solani), Shoot-blight for Pinus sylvestris var. mongolica (Sphaeropsis sapinea), Cytospora chrysosperma (Valsa sordida) all have inhibitory effect.
2 fritillary bulb rhizosphere Paenibacillus polymyxa SBS-SF0105 are tested to the field control effect of flat shellfish black rot
It prevents and treats place and overview: being located at the flat shellfish cultivation base in 35 forest farm of Mudanjiang City Hailin forestry bureau, flat shellfish plantation
1850 mu of area, flat shellfish cultivation starts from 1980, is many years sequential cropping cultivation mode, 2400 DEG C of effective accumulated temperature of forest farm year.Annual day
According to when number 2540 hours.550 millimeters of mean annual precipitation.Frost-free period 125 days or so, flat shellfish black rot, gray mold occurred in recent years
Seriously, 50% or more disease incidence.
Microbial inoculum preparation: the Paenibacillus polymyxa SBS-SF0105 frozen is accessed into PDA slant medium, 25 DEG C of cultures
48h carries out bacterial strain activation, and the flat shellfish rhizosphere Paenibacillus polymyxa bacterial strain access PD culture medium of picking activation (go by PDA culture medium
Fall agar) in, OD is arrived in 120rpm/min culture, culture600Value is 0.5 or so, obtains seed liquor.By seed liquor according to 5% volume
In PD culture medium than access sterilizing, 28 DEG C of culture 48h obtain fermentation liquid, are 10 by counting adjustment bacterial concentration9CFU/
Ml is 4.0 using HCl adjustment pH value, poly- three oxosilane 1%, OP-10 1%, antioxidant BHT 0.2%, anti ultraviolet agent is added
UV531 0.3%, 3 ‰ sodium benzoates are prepared into Paenibacillus polymyxa fermentation liquid microbial inoculum dosage form.
It prevents and treats time and method: in late April, 2014, emerging initial stage in flat shellfish, it is rotten to carry out Ping Beihei using root-pouring method
Disease prevention and treatment, dosage are that microbial inoculum dilutes after certain multiple (stoste, 10 times, 100 times, 1000 times), according to 500ml/m2, filled
Root processing, pouring root is primary within 7 days, continuous prevention and treatment 3 times.Investigating flat shellfish black rot in late May, a situation arises, calculates control efficiency,
It is control with clear water pouring root.
Disease incidence (%)=disease plant strain number/investigation total strain number × 100%
Control efficiency (%)=(control disease incidence-processing disease incidence)/control disease incidence × 100%
Control efficiency investigation such as table 3:
Table 3
| Number | Different disposal | Investigation number/strain | Morbidity number/strain | Disease incidence/% | Opposite control efficiency/% |
| 1 | Stoste | 500 | 119 | 23.8 | 70.39 |
| 2 | 100 times of liquid | 500 | 159 | 31.8 | 60.44 |
| 3 | 500 times of liquid | 500 | 226 | 45.2 | 43.78 |
| 4 | 1000 times of liquid | 500 | 298 | 59.6 | 25.87 |
| 5 | CK | 500 | 402 | 67.20 | - |
It is found that Paenibacillus polymyxa SBS-SF0105 of the present invention, which has, preferably prevents and treats flat shellfish soil-borne disease black rot
Effect, control efficiency can reach 55% or more.
Sequence table
<110>Song little Shuan
<120>one plants of fritillary bulb rhizosphere Paenibacillus polymyxas and its applications
<160> 1
<210> 1
<211> 555
<212>16SrDNA
<213>bacillus (Paenibacillus)
<220>
<223>the 16SrDNA sequence of Paenibacillus polymyxa SBS-SF0105
<400> 1
ctggctcctt gcggttacct caccgacttc gggtgttgta aactctcgtg gtgtgacggg 60
cggtgtgtac aagacccggg aacgtattca ccgcggcatg ctgatccgcg attactagca 120
attccgagtt catgcaggcg agttgcagcc tgcaatccga actgagaccg gcttttctag 180
gattggctcc agatcgctcc ttcgcttccc gttgtaccgg ccattgtagt acgtgtgtag 240
cccaggtcat aaggggcatg atgatttgac gtcatccccg ccttcctccg gtttgtcacc 300
ggcagtctac ttagagtgcc cagcttmacc tggtggcaac taagttcaag ggttgcgctc 360
gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccatg gcacgacctg 420
tcacctctgt cccgaaggaa aggcttatct ctaagccggt cagagggatg tcaagacctg 480
gtaaggttct tcgcgttgct tcgaattaaa ccacatactc cactgcttgt gcgggtcccc 540
gtcaattcct tagagtttca ctcttgcgag cgtactcccc aggcggcatg cttactgtgt 600
tatcttcggc accaagggta tcgaaacccc taacacctag catgcatcgt ttacggcgtg 660
gactaccagg gtatctaatc ctgtttgctc cccacgcttt cgcgcctcag cgtcagttac 720
aggccagaaa gtcgccttcg ccactggtgt tcctccacat ctctacgcat ttcaccgcta 780
cacgtggaat tccactttcc tctcctgcac tccagtctcc cagtttccgg tgcgaaccga 840
ggttgagcct cgggattaaa caccagactt aaaaaaccgc ctgcgcgcgc tttacgccca 900
ataattccgg acaacgcttg ccccctacgt attagcgcgg ctgctggcac gtagttagcc 960
ggggctttct tctcaggtac cgtcacccaa agagcagtta ctctccttgg cgttcttccc 1020
tggcaacaga gctttacgat ccgaaaacct tcatcactca cgcggcgttg ctccgtcaga 1080
cttgcgtcca ttgcggaaga ttccctactg ctgcctcccg taggagtctg ggccgtctct 1140
cagtcccagt gtggccgttc accctctcag gtcggctagg catcgtcgcc ttggtgagcc 1200
gttacctcac caactagcta atgcgccgca ggcccatccg taagccacag attgctccgt 1260
gtttcacagt ctcttcatgc gaagaaaccg gttatccggt cttagctacc gtttccggta 1320
gttatcccga tcttacaggc aggttgccta cgtgttactc acccgtccgc cgctaaccat 1380
cagagaagca agctcctctt caagtccgct cgacttgcat gtattaggca cgccgccagc 1440
Claims (2)
1. one plant of fritillary bulb rhizosphere Paenibacillus polymyxa, it is characterised in that the bacterial strain is Paenibacillus polymyxa
(Paenibacillus polymyxa) SBS-SF0105, it is deposited in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center (CGMCC), preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica,
The deposit date is on November 5th, 2015, deposit number was CGMCC No.11586.
2. fritillary bulb rhizosphere Paenibacillus polymyxa described in claim 1 answering in prevention and treatment fritillary bulb soil-borne disease black rot
With.
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