CN109504640A - Bacillus subtilis GU MT323 and its application - Google Patents
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Abstract
The invention discloses a kind of bacillus subtilis GU MT323, are deposited in China typical culture collection center, and deposit number is CCTCC NO:M 2018873.The invention also discloses a kind of application of bacillus subtilis GU MT323 in prevention and treatment tobacco black shank.Bacillus subtilis GU MT323 of the present invention has the ability of apparent antagonism tobacco black shank bacterium and the ability of antagonism pepper anthracnose.
Description
Technical field
The present invention relates to a kind of bacillus subtilis GU MT323 and its applications, belong to technical field of biological control.
Background technique
Tobacco black shank (Phytophthora nicotianae) is most destructive soil-borne disease in Guizhou Tobacco production
One of evil, nearly ten million yuan of the economic loss as caused by the disease, seriously affects the income of tobacco grower every year.At present in production
Chemical control is mostly used greatly, but primary medicament service life is longer, pathogen drug resistance increases year by year, and pesticide residue and environment are dirty
The negative effects such as dye also gradually increase.Therefore, carrying out tobacco diseases prevention and treatment using biological control is the hot fields studied at present.
Bacillus (Bacillus spp.) is most common microbe species in current biological control research, gemma bar
Bacterium has the prospect of industrialization, and mechanism of action multiplicity, production cost is low, and easy storage and transportation is not likely to produce drug resistance, to safety of human and livestock etc.
Advantage is the important directions of biological prevention and control agent exploitation.Native Bacillus separation strains excellent there are many character domestic and international at present
As living microorganism preparation viable commercial product application, development potentiality is huge.The BioYield of Gustafson company research and development is by solving
Bacillus amyloliquefaciens (B.amyloliquefaciens) FZB42 is mixed with bacillus subtilis (B.subtilis) GB122 to be made
Fungicide, be applied to the prevention and treatment of plurality of plant diseases.China is earliest by China Agricultural University's development by waxy gemma
Yield increasing fungus series of products made of the bacillus such as bacillus (B.cereus) and bacillus subtilis (B.subtilis) rice,
There is good volume increase, protection effect on wheat, vegetables and more industrial crops.
It is suitable for the biological prevention and control agent exploitation of Guizhou Tobacco balck shank disease control at present and good preparation can be used
Relatively fewer, many reports prove that the drug resistance of Guizhou Province tobacco producing region balck shank pathogen increases year by year, it is therefore necessary to grind
Better biological inoculum is made to control the generation of tobacco black shank.
In addition, pepper anthracnose is one of the Major Diseases in capsicum production, it can cause harm in capsicum entire breeding time, make
At very big loss.Guizhou Province be the Main Cultivation of China capsicum one of, the long-term continuous cropping of capsicum causes the morbidity face of anthracnose
Product constantly expands, and seriously affects farmers' income.Production at present is upper mainly to use chemical pesticide control disease, the drug resistance of pathogen
Increase year by year, field dosage is also increase accordingly, and easily causes pesticide residue and environmental pollution.Therefore, using biological control into
The prevention and treatment of row pepper diseases is the hot fields studied at present.
Summary of the invention
The technical problem to be solved by the present invention is provide a kind of bacillus subtilis GU MT323, to tobacco black shank, with
And pepper anthracnose has preferable control efficiency, with overcome the deficiencies in the prior art.
The technical scheme is that a bacillus subtilis GUMT323, which protects on December 22nd, 2018
It ensconces China typical culture collection center (address: Wuhan, China Wuhan University), deposit number is CCTCC NO:M
2018873, title are as follows: bacillus subtilis GU MT323 (Bacillus subtilis GUMT323).
The present invention also provides a kind of application of bacillus subtilis GU MT323 in prevention and treatment tobacco black shank.
The beneficial effects of the present invention are: the present invention acquires rhizosphere soil from Zunyi City, Guizhou Province tobacco planting plot, therefrom
Isolated bacillus subtilis GU MT323 is screened, there is apparent antagonism tobacco black shank bacterium (Phytophthora
Nicotianae the ability of ability and antagonism pepper anthracnose).Pass through morphology, physio-biochemical characteristics and 16S rDNA
Identified for genes GUMT323 is bacillus subtilis (B.subtilis).
Detailed description of the invention
Fig. 1 bacillus screens the antagonistic effect of tobacco black shank bacterium, and CK: tobacco black shank bacterium (Phytophthora
nicotianae)；GUMT323 indicates the Bacillus strain with antagonistic effect screened；
Fig. 2 bacterial strain GUMT323 16S rDNA phylogenetic trees；
The identification of Fig. 3 bacterial strain GUMT323 antimicrobial spectrum；
Fig. 4 bacterial strain GUMT323, GUMT323 and GUMT332 biomembrane, protease, cellulase, thermophilic iron element and phosphate
Detection.
Specific embodiment
With reference to the accompanying drawing and invention is described further in specific embodiment:
One, the separation and screening of bacillus subtilis GU MT323
1. materials and methods
1.1 material
2017-2018 chooses the serious plot of tobacco black shank morbidity from Zunyi City, Guizhou Province Meitan County, acquires health-care cigarette
Same plot soil sample is mixed and calculates 1 part of soil sample by strain rhizosphere soil, 5 point samplings.Soil sample, soil are collected using sterile valve bag
Sampling instrument is carried out disinfection with 75% alcohol, and edaphon between different fields is avoided to mix.
1.2 culture medium
LB culture medium is used for the separation and preservation of bacillus；PDA culture medium is tested for plate opposite culture；Protease
Detection culture medium, cellulase detection culture medium, thermophilic iron element detection culture medium, phosphate detection culture medium are for detecting gemma
Whether bacillus generates protease, cellulase, thermophilic iron element and phosphate etc..
1.3 strains tested
Strains tested tobacco black shank bacterium (Phytophthora nicotianae) is by agricultural college, Guizhou University plant pathology
Teaching and research room is learned to provide.
2. the separation and preservation of rhizosphere bacillus
Using dilution-plate method, by soil dilution liquid, water-bath 30min is non-to kill the overwhelming majority in 85 DEG C of water-bath
Spore bacteria.The different single colonie of picking purifies bacterial strain and is stored in -80 DEG C of refrigerators.
3. tobacco black shank bacterium and the screening of ralstonia solanacearum antagonistic Bacillus
Using tobacco black shank bacterium as test plant pathogen, prevention and treatment tobacco black shank effect is screened by tablet face-off method
Apparent biocontrol bacterial strain sees whether to generate inhibition zone by primary dcreening operation and secondary screening, and it is antibacterial straight using crossing method to measure its
Diameter.The generation of biomembrane, protease and cellulase is detected whether by specific plate again, wherein 3 plants of effects are best for selection
Bacterial strain be used for subsequent field experiment, number is GUMT323 (see Fig. 1).
Bacteriostasis rate (%)=[(control group colony diameter-processing group colony diameter)/processing group colony diameter] × 100 2,
The identification of bacillus subtilis GU MT323
1. biocontrol bacterial strain is identified
It is identified by the bacterial strain that morphology, physiological and biochemical property and 16S rDNA gene pairs screen.
1.1 colony morphological observation
In inoculating strain to the test tube containing 5mL LB culture solution, 37 DEG C are shaken training overnight, and thalline were collected by centrifugation, thallus nothing
Bacterium water cleans 3 times, is finally resuspended with sterile water and is diluted to concentration 1 × 10810 μ L bacterium solutions are added dropwise in CFU/mL on LB plate,
37 DEG C be incubated overnight after observe colonial morphology.
1.2 physiological and biochemical property
According to " primary Jie Shi systematic bacteriology handbook ", the physio-biochemical characteristics of bacterial strain are measured.
1.3 molecular biology identification
37 DEG C of water-bath 1h of 50mg/mL lysozyme handle thallus, are mentioned using Biomiga bacterial genomes DNA extraction kit
Take genomic DNA.Using genomic DNA as template, PCR amplification is carried out using 16S rDNA universal primer 27F/1492R, PCR is produced
The raw work in sea is served after object verifying is correct carries out PCR product sequencing.Sequencing result carries out BLAST in ncbi database and compares analysis,
And the Neighbor-Joining in Multiple Sequence Alignment and phylogenetic analysis software MEGA6 is carried out using CLUSTAL X software
Algorithm phylogenetic tree construction.
Through analyzing: GUMT323 can form more complicated colony structure on LB culture medium.The physio-biochemical characteristics of bacterial strain
It is shown in Table 1, bacterium G+, can decompose glucose, rhamnose and mannitol, generates catalase and nitrate reductase, methyl red dye
Color is positive, the features such as liquefiable gelatin.By 16S rDNA gene sequencing, the 16S rDNA of bacterial strain GUMT323 is constructed
Phylogenetic trees, the results show that bacterial strain GUMT323 with bacillus subtilis (B.subtilis) gather a branch (Fig. 2).
It is bacillus subtilis (B.subtilis) by morphology, physio-biochemical characteristics and 16S rDNA identified for genes GUMT323.
1 bacterial strain GUMT323 physiological and biochemical property of table
+: positive reaction；: negative reaction
2. biocontrol bacterial strain antimicrobial spectrum is identified
Bacterial strain GUMT323 has spectrum antagonistic activity, to tobacco black shank bacterium, tobacco brown spot pathogen, Colletotrichum capsici
Preferably (see Fig. 3) with a variety of disease fungus fungistatic effects such as sorghum A. mali.It is potential to show that this plant of bacterium has the above disease
Biocontrol effect.
3. bacterial strain biomembrane, protease and cellulase measurement
GUMT323 bacillus can produce more complicated biofilm structure, can produce protease, cellulase, phosphoric acid
Esterase and thermophilic iron are plain (see Fig. 4), promote plant growth related to bacillus.Bacillus colonizes and generates multiple in root system of plant
Miscellaneous biofilm structure can effectively occupy root table Advantageous sites, limit the intrusion of pathogen mycelia, and can induce plant to disease
Opportunistic pathogen generates resistance, achievees the effect that disease-preventing and yield-increasing.
Three, application of the bacillus subtilis GU MT323 in terms of controlling plant diseases
1. tobacco black shank prevents and treats field trial
Experimental cultivar: cloud and mist 87.Test site: the Meitan County town Chao Le.Experimental field: fertility is uniform, plantation flue-cured tobacco in 2017
And balck shank morbidity plot.
Experimental design: being handled with bacillus subtilis GU MT323, and with 30% first frost hymexazol aqua of medicament, (middle peasant is vertical
Magnificent (Tianjin) agricultural chemicals Co., Ltd) it is positive control, with greenery patches health, (greenery patches health is the green health of middle peasant (Beijing) Limited Liability
The probiotics of company's research and development, to improve the effect of soil micro-ecosystem controlling disease and growth-promoting volume increase) it is positive control,
Not carrying out any chemicals treatment is blank control, totally 3 processing, 3 repetitions, 30 plants of cigarettes of every cell, all random areas of processing
Group arrangement.Chemicals treatment concentration and dosage illustrate that pouring root is applied when tobacco seedlings are transplanted with reference to medicament, apply 2 times；Bacillus concentration
For 108CFU/mL, the application of 50mL bacterium solution pouring root is co-administered 3 times, pouring root 1 time when being transplanted from tobacco seedlings, after every 7-10d pouring root 1
It is secondary.Transplanting on April 23rd, 2018, field planting management method is consistent with locality, does not apply other fungicide.
Field investigation: the field investigation time is chosen and carries out 3 times altogether after the onset of blank control starts to harvesting is preceding, and investigation is all
Cell cigarette strain balck shank disease incidence is handled, and is classified investigation disease severity.Tobacco black shank disease scale standard is according to GB/T
23222-2008 is carried out, and calculates the disease incidence and disease index of disease, and data use the new multipole of DPS data processing system Duncan
Poor method carries out variance analysis.The results are shown in Table 2.
The field plot preventive effect of 2 Meitan area tobacco black shank of table
Table 2 is it is found that bacillus has certain preventive effect to tobacco black shank, but preventive effect is not as good as first frost hymexazol.First frost
Hymexazol is for preventing and treating the primary medicament of tobacco black shank in current production, to tobacco black shank control efficiency in this test
It is good.It has been reported that and shows that tobacco black shank bacterium develops drug resistance to metalaxyl-mn-zn in production.Therefore, in conjunction with chemical agent
With the advantage and disadvantage of biological bactericide, biocontrol bacterial strain can be attempted and compound application with chemical pesticide, reach the dosage for reducing chemical pesticide.
2. tobacco bacterial wilt prevents and treats field trial
Experimental cultivar: cloud and mist 87.Test site: the Meitan County town Chao Le.Experimental field: fertility is uniform, plantation flue-cured tobacco in 2017
And bacterial wilt morbidity plot.
Experimental design: being handled with bacillus subtilis GU MT323, and with Zhongshengmycin, (Zhongshengmycin is that the Chinese Academy of Agricultural Sciences is raw
A kind of anti-succeeded in developing novel agricultural antibiotic is the antibiotic generated by the mutation of Strepiomyces lavendulae Hainan, belongs to N- sugar
Glycoside alkali water-soluble substance.The processing dosage form of the bacterium is a kind of protective fungicide that fungicidal spectrum is wider, has and tags, permeates
Effect.Zhongshengmycin has very high activity to bacterial disease and some fungi the venereal disease evil of crops, while having certain
Production-increasing function.It is biological bactericide more commonly used in current production.It is obvious to prevent and treat Solanaceae bacterial wilt effect) it is positive right
According to using greenery patches health as positive control, not carrying out any chemicals treatment is blank control, totally 3 processing, 3 repetitions, every cell
30 plants of cigarettes, all random district's groups arrangements of processing.Chemicals treatment concentration and dosage illustrate that pouring root is applied when tobacco seedlings are transplanted with reference to medicament
With application 2 times；Bacillus concentration is 108The application of CFU/mL, 50mL bacterium solution pouring root, co-administers 3 times, fills when transplanting from tobacco seedlings
Root 1 time, after every 7-10d pouring root 1 time.Transplanting on April 23rd, 2018, field planting management method is consistent with locality, does not apply
Other fungicide.
Field investigation: the field investigation time is chosen and carries out 3 times altogether after the onset of blank control starts to harvesting is preceding, and investigation is all
Cell cigarette strain bacterial wilt disease incidence is handled, and is classified investigation disease severity.Tobacco bacterial wilt disease scale standard is according to GB/T
23222-2008 is carried out, and calculates the disease incidence and disease index of disease, and data use the new multipole of DPS data processing system Duncan
Poor method carries out variance analysis.The results are shown in Table 3.
The field plot preventive effect of 3 Meitan area tobacco bacterial wilt of table
The experimental results showed that this bacillus is preferable to the preventive effect of tobacco bacterial wilt, although being lower than Zhongshengmycin, it is better than
Greenery patches health.
3. pepper anthracnose prevents and treats field trial
Experimental cultivar: Dang Wu.Test site: Guiyang Huaxi District.
Experimental design: being handled with bacillus subtilis GU MT323, (the green health of the middle peasant (north the green Kang Wei of biological prevention and control agent is commercialized
Capital) Bioisystech Co., Ltd) it is used as positive control, not carrying out any chemicals treatment is blank control, totally 4 processing, 3 times
It repeats, 30 plants of capsicums of every cell, all random district's groups arrangements of processing.Chemicals treatment concentration and dosage are with reference to medicament explanation, capsicum
Pouring root is applied when transplantation of seedlings, is applied 3 times；Bacillus concentration is 108The application of CFU/mL, 50mL bacterium solution pouring root, co-administers 3 times,
From pouring root 1 time when capsicum transplantation of seedlings, after every 7-10d pouring root 1 time.Field planting management method is consistent with locality, does not apply it
Its fungicide.
The field investigation time is chosen after the onset of blank control starts and carries out 2 times altogether, and all processing cell capsicum anthraxs are investigated
Sick disease incidence, and it is classified investigation disease severity.Pepper anthracnose disease scale standard according to GB/T17980.33-2000 into
Row, calculates the disease incidence and disease index of disease, and data carry out variance using DPS data processing system Duncan duncan's new multiple range method
Analysis.
Start to carry out disease survey when blank control cell pepper anthracnose takes place.Investigation on May 22nd, 2018
It 1st time, investigates 1 time every 7-10d, investigates 3 times altogether.The results are shown in Table 4.
Field plot preventive effect of 4 bacillus to pepper anthracnose
The above content is a further detailed description of the present invention in conjunction with specific preferred embodiments, and it cannot be said that
Specific implementation of the invention is only limited to these instructions.For those of ordinary skill in the art to which the present invention belongs, exist
Under the premise of not departing from present inventive concept, a number of simple deductions or replacements can also be made, all shall be regarded as belonging to of the invention
Protection scope.
Claims (4)
1. a bacillus subtilis GUMT323 is deposited in China typical culture collection center, deposit number CCTCC
NO:M 2018873.
2. application of the bacillus subtilis GU MT323 described in claim 1 in prevention and treatment tobacco black shank.
3. application of the bacillus subtilis GU MT323 described in claim 1 in prevention and treatment tobacco bacterial wilt.
4. application of the bacillus subtilis GU MT323 described in claim 1 in prevention and treatment pepper anthracnose.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112358997A (en) * | 2020-11-20 | 2021-02-12 | 云南省烟草公司曲靖市公司 | Bacterial strain with biocontrol function on tobacco brown spot, fermentation method of bacterial strain and tobacco brown spot prevention and control compound agent |
| CN116004451A (en) * | 2022-12-08 | 2023-04-25 | 贵州大学 | Composite particle type microbial agent and application thereof |
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| 邹芳芸等: "烟草主要病原菌拮抗菌的筛选", 《食品工业科技》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112358997A (en) * | 2020-11-20 | 2021-02-12 | 云南省烟草公司曲靖市公司 | Bacterial strain with biocontrol function on tobacco brown spot, fermentation method of bacterial strain and tobacco brown spot prevention and control compound agent |
| CN112358997B (en) * | 2020-11-20 | 2022-09-27 | 云南省烟草公司曲靖市公司 | Bacterial strain with biocontrol function on tobacco brown spot, fermentation method of bacterial strain and tobacco brown spot prevention and control compound agent |
| CN116004451A (en) * | 2022-12-08 | 2023-04-25 | 贵州大学 | Composite particle type microbial agent and application thereof |
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