CN107287142B - One plant inhibits sickle-like bacteria growth and produces serratia marcescens SerEW01 and its application of poison - Google Patents
One plant inhibits sickle-like bacteria growth and produces serratia marcescens SerEW01 and its application of poison Download PDFInfo
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- CN107287142B CN107287142B CN201710713397.2A CN201710713397A CN107287142B CN 107287142 B CN107287142 B CN 107287142B CN 201710713397 A CN201710713397 A CN 201710713397A CN 107287142 B CN107287142 B CN 107287142B
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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
- C12R2001/425—Serratia
- C12R2001/43—Serratia marcescens
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- C—CHEMISTRY; METALLURGY
- 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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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
- A01N63/00—Biocides, pest repellants or attractants, or plant growth regulators containing microorganisms, viruses, microbial fungi, animals or substances produced by, or obtained from, microorganisms, viruses, microbial fungi or animals, e.g. enzymes or fermentates
- A01N63/10—Animals; Substances produced thereby or obtained therefrom
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- C—CHEMISTRY; METALLURGY
- 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
Abstract
The present invention, which isolates one plant from earthworm body surface for the first time, has serratia marcescens (Serratia marcescens) SerEW01 for inhibiting sickle-like bacteria growth and producing toxic action, can effectively inhibit fusarium moniliforme (Fusarium verticillioides), (F.proliferatum) spore germination of the raw sickle-like bacteria of layer and mycelia growth；The bacterial strain has chitinase gene (chiA, chiB), can synthesize chitinase, has the activity of degradation dimorphic fungal cell wall；The bacterium has apparent inhibiting effect to fumonisin B1's, in terms of crops sickle-like bacteria prevention and treatment, with very high application value, new thinking is provided effectively to control and reducing the pollution of agricultural product, fumonisins in fodder B1, it is significant for improving agricultural product quality and safety, there is very high application value.
Description
Technical field
The invention belongs to microorganism and field of biotechnology, and in particular to one plant inhibits sickle-like bacteria growth and produces the cement of poison
Serratieae SerEW01.
Background technique
Fumonisin is a kind of mycotoxin, by the different polyhydric alcohols double esterification similar with the structure that tricarballylic acid forms
Object is closed, molecular polarity is stronger, can be dissolved in water and other polar solvents.Fumonisin is mainly by fusarium moniliforme (Fusarium
Verticillioides it) is generated with the raw sickle-like bacteria of layer (F.proliferatum) nematophyte disease fungus.Fumonisin is frequent
Corn and corn product are polluted, people, which ingests, can cause disease by the corn food that fumonisin pollutes.In addition, animal feeding is lied prostrate
The corn feed of horse endotoxin contamination can also cause disease.Fumonisin B1 (FB1), chemical structural formula is seen below, and is most common
Fumonisin is simultaneously dangerous to the nutrition and health of humans and animals.In addition, fumonisin B1 is proved to be able to carcinogenic teratogenesis, have
Research confirms to be positively correlated with cancer of the esophagus, also results in children's neural tube defect.
To the soil-borne pathogen sickle-like bacteria (Fusarium spp.) that fumonisin can be generated existing for nature pollute into
Row effectively inhibits to reduce the generation of mycotoxin, and carries out effectively being degraded into scientific circles to already present mycotoxin
Problem in the urgent need to address.In recent years, inhibit the toxigenic research of sickle-like bacteria using biocontrol microorganisms or biological source active material
The attention of researcher is caused.(Ye Huochun, Lai Xianwen, Wang Yanli wait .1 plants of inhibition sickle-like bacteria growths and produce such as Ye Huochun
The separation of raw fumonisins streptomycete and identification [J] Journal of Northwest Sci Tech University of Agriculture and Forestry: natural science edition, 2013,41 (7):
150-156.) etc. from separation screening is picked up from the mangrove soil of Shenzhen coastal waters to inhibiting the growth of proliferation sickle-like bacteria and generate volt horse bacterium
The bacterial strain of plain B1 (FB1)；(Yang Pengfei, Chang Xiaojiao, Wu Songling wait the screening and mirror of mono- plant of fumonisin degradation bacteria of to Yang Pengfei etc.
Determine [J] China grain and oil journal, 2017,32 (4): 110-115.) one plant of sphingol box bacterium ASAG22 is separated with enrichment culture method,
The bacterial strain can carry out degradation utilization to fumonisin using fumonisin FB1 as sole carbon source.However cement is not yet found so far
Serratieae has the report for inhibiting sickle-like bacteria growth and producing toxic action.
Summary of the invention
In view of the above shortcomings of the prior art, inventor filters out through long-term technology and practical exploration from earthworm body surface
One plant is able to suppress sickle-like bacteria growth and produces serratia marcescens (Serratia marcescens) SerEW01 of poison, through testing
Verifying can effectively inhibit fusarium moniliforme and the raw sickle-like bacteria spore germination of layer and mycelia to grow, to sickle-like bacteria hyphal cell
Wall has apparent degradation, has obvious inhibiting effect to fumonisin B1.The present invention can enrich antimicrobial source
Library provides fundamental basis to the pollution of energy product for the biological control and effective control fumonisin of sickle-like bacteria.
Specifically, the present invention relates to following technical schemes:
The first aspect of the invention, providing one plant has the Serratia for inhibiting sickle-like bacteria growth and producing toxic action
Bacterium (Serratia marcescens) SerEW01, the bacterial strain have been preserved in Chinese microorganism strain guarantor on March 28th, 2017
It hides administration committee's common micro-organisms center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3),
Biological deposits number are as follows: CGMCC NO.13952.
Serratia marcescens (Serratia marcescens) SerEW01 of the present invention, is isolated from earthworm body surface
Come, bacterium colony surface bulge of the bacterial strain on NA culture medium, center is opaque, and edge is irregular, produces red pigments.
The second aspect of the invention provides above-mentioned serratia marcescens (Serratia marcescens) SerEW01's
Cultural method, including serratia marcescens (Serratia marcescens) SerEW01 is placed in NB fluid nutrient medium and is trained
It supports, condition of culture is aerobic culture at 28 DEG C, such as shaking table (28 DEG C, 120rpm) shaken cultivation.
Wherein, the NB culture medium prescription (g/L) are as follows: beef extract 3.0；Peptone 5.0；Glucose 2.5；pH7.0±
0.2；
The third aspect of the invention, provides a kind of microbial bacterial agent, and the microbial bacterial agent includes above-mentioned cement sand thunder
Salmonella (Serratia marcescens) SerEW01's or serratia marcescens (Serratia marcescens) SerEW01
Culture.The microbial bacterial agent, which has, inhibits sickle-like bacteria growth and production toxic action.
Preferably, the dosage form of microbial bacterial agent is that wettable powder, water dispersible granules, aqueous suspension agent or dispersible oil are outstanding
Floating agent；
Preferably, further include acceptable auxiliary material in Pesticide Science in microbial bacterial agent, it is acceptable in the Pesticide Science
Auxiliary material be selected from one of dispersing agent, wetting agent, disintegrating agent, binder, defoaming agent, antifreeze, thickener, filler and solvent or
It is a variety of.The present invention is not particularly limited the source etc. of acceptable auxiliary material in the Pesticide Science, is generally using commercial product
It can.
The fourth aspect of the invention provides the serratia marcescens (Serratia marcescens) SerEW01
Or microbial bacterial agent is inhibiting the application in sickle-like bacteria spore germination and/or mycelia growth；
Wherein, the sickle-like bacteria is fusarium moniliforme and/or the raw sickle-like bacteria of layer；
The fifth aspect of the invention provides the serratia marcescens (Serratia marcescens) SerEW01
Or microbial bacterial agent is inhibiting sickle-like bacteria to generate the application in fumonisin B1；
Wherein, the sickle-like bacteria is fusarium moniliforme and/or the raw sickle-like bacteria of layer.
Beneficial effects of the present invention: the present invention, which isolates one plant from earthworm body surface for the first time, has the growth of inhibition sickle-like bacteria and production
Serratia marcescens (Serratia marcescens) SerEW01 of poison, can effectively inhibit fusarium moniliforme (Fusarium
Verticillioides), (F.proliferatum) spore germination of the raw sickle-like bacteria of layer and mycelia growth；The bacterial strain has chitin
Matter enzyme gene (chiA, chiB), can synthesize chitinase, have the activity of degradation dimorphic fungal cell wall；The bacterium is to volt horse poison
Plain B1's has apparent inhibiting effect, in terms of crops sickle-like bacteria prevention and treatment, has very high application value, for effectively control
The pollution of system and reduction agricultural product, fumonisins in fodder B1 provides new thinking, has weight for improving agricultural product quality and safety
Meaning is wanted, there is very high application value.
Detailed description of the invention
Fig. 1 is bacterium colony shape of serratia marcescens (Serratia marcescens) SerEW01 on NA culture medium flat plate
State；
Fig. 2 is that bacterial strain SerEW01Biolog Automatic Analyzer for Microbes analyzes result figure；
Fig. 3 is that bacterial strain SerEW01 bacterium solution inhibits sickle-like bacteria spore germination, wherein figure A is control group, figure B is processing group；
Fig. 4 is that bacterial strain SerEW01 bacterium solution inhibits the growth of sickle-like bacteria F6S-S mycelia；
Fig. 5 is bacterial strain SerEW01 active material degradation sickle-like bacteria F6S-S hyphal cell wall, wherein figure A is control group, figure B
For processing group；
Fig. 6 is that bacterial strain SerEW01 bacterium solution inhibits to generate 88473 mycelia of fumonisin B1 reaping hook bacterial strain in rice matrix
Growth, wherein left side triangular flask is processing group, right side triangular flask is control group；
Fig. 7 is the toxin amount that rice generation is infected in toxigenic bacterium strain 88473；
Fig. 8 is SerEW01 to be added toxigenic bacterium strain 88473 after bacterium is inhibited to infect the toxin amount that rear rice generates.
Specific embodiment
It is noted that following detailed description is all illustrative, it is intended to provide further instruction to the application.Unless another
It indicates, all technical and scientific terms used herein has usual with the application person of an ordinary skill in the technical field
The identical meanings of understanding.
It should be noted that term used herein above is merely to describe specific embodiment, and be not intended to restricted root
According to the illustrative embodiments of the application.As used herein, unless the context clearly indicates otherwise, otherwise singular
Also it is intended to include plural form, additionally, it should be understood that, when in the present specification using term "comprising" and/or " packet
Include " when, indicate existing characteristics, step, operation, device, component and/or their combination.
In conjunction with specific example, the present invention is further illustrated, and following instance is not right merely to the explanation present invention
Its content is defined.If the experiment actual conditions being not specified in embodiment, usually according to normal condition, or according to reagent public affairs
Take charge of recommended condition；Reagent as used in the following examples, consumptive material etc., are commercially available unless otherwise specified.
In a kind of specific embodiment of the invention, providing one plant has the cement for inhibiting sickle-like bacteria growth and producing toxic action
Serratieae (Serratia marcescens) SerEW01, the bacterial strain are preserved in China Microbiological on March 28th, 2017
Culture presevation administration committee common micro-organisms center (abbreviation CGMCC, address are as follows: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number), biological deposits number are as follows: CGMCC NO.13952.
In still another embodiment of the invention, above-mentioned serratia marcescens (Serratia marcescens) is provided
The cultural method of SerEW01, including serratia marcescens (Serratia marcescens) SerEW01 is placed in the training of NA liquid
It supports and is cultivated in base, condition of culture is aerobic culture at 28 DEG C, such as shaking table (28 DEG C, 120rpm) shaken cultivation.
Wherein, the NB culture medium prescription (g/L) are as follows: beef extract 3.0；Peptone 5.0；Glucose 2.5；pH7.0±
0.2；
In still another embodiment of the invention, a kind of microbial bacterial agent is provided, the microbial bacterial agent includes above-mentioned
Serratia marcescens (Serratia marcescens) SerEW01 or serratia marcescens (Serratia marcescens)
The culture of SerEW01.The microbial bacterial agent, which has, inhibits sickle-like bacteria growth and production toxic action.
The dosage form of microbial bacterial agent is wettable powder, water dispersible granules, aqueous suspension agent or dispersible oil-suspending agent；
It further include acceptable auxiliary in Pesticide Science in still another embodiment of the invention, in the microbial bacterial agent
Expect, acceptable auxiliary material is selected from dispersing agent, wetting agent, disintegrating agent, binder, defoaming agent, antifreeze, thickening in the Pesticide Science
One of agent, filler and solvent are a variety of.The present invention is not special to the source of acceptable auxiliary material in the Pesticide Science etc.
Limitation generally uses commercial product.
Wherein, the dispersing agent is anionic dispersing agent and/or non-ionic dispersing agent, can be selected from lignin sulfonic acid
Sodium, naphthalenesulfonic acid-formaldehyde condensate, sodium methylene bis-naphthalene sulfonate, formaldehyde condensation products sulfate, polycarboxylate, alkyl phenol polyoxy second
One of alkenyl phosphate and polyoxyethylene carboxylate are a variety of；
The wetting agent can be selected from lauryl sodium sulfate, neopelex, spaonin powder, spaonin powder, tea seed cake
One of powder and Nekal BX are a variety of；
The disintegrating agent can be selected from one of bentonite, ammonium sulfate, aluminium chloride, urea, magnesium chloride and glucose or more
Kind；
The binder can be selected from starch, diatomite, cyclodextrin, rosin, carboxymethyl cellulose, carboxyethyl cellulose and carboxylic
One of methylcellulose salt is a variety of；
The defoaming agent can be selected from C8~C20 fatty alcohols compound, C10~C20 saturated fat acids compound, epoxy
One of soybean oil, ethyl alcohol, silicone compound and organic silicone oil are a variety of；
The antifreeze can be selected from sorbierite, ethylene glycol, polyethylene glycol, propylene glycol, glycerine, urea and sodium chloride
It is one or more；
The thickener can be selected from one of gelatin, xanthan gum, polyethylene glycol and polyvinyl alcohol or a variety of；
The filler can be selected from one of precipitated calcium carbonate, diatomite, bentonite, attapulgite and white carbon black or more
Kind；
The solvent can be selected from water (preferably deionized water) or methyl oleate；
In still another embodiment of the invention, the serratia marcescens (Serratia is provided
Marcescens) SerEW01 or microbial bacterial agent are inhibiting the application in sickle-like bacteria spore germination and/or mycelia growth；
Wherein, the sickle-like bacteria is fusarium moniliforme and/or the raw sickle-like bacteria of layer；
In still another embodiment of the invention, the serratia marcescens (Serratia is provided
Marcescens) SerEW01 or microbial bacterial agent are inhibiting sickle-like bacteria to generate the application in fumonisin B1；
Wherein, the sickle-like bacteria is fusarium moniliforme and/or the raw sickle-like bacteria of layer.
The separation screening of 1 SerEW01 bacterial strain of embodiment
Earthworms for experiment is adopted from the Laoshan District soil of Qingdao of Shandong province, 7g Eisenia Foetida (Eisenia
Foetida it) is first washed with deionized water, is then rinsed 3 times with aqua sterilisa, is put into the 50ml centrifuge tube of sterilizing after cleaning,
Enter 30ml aqua sterilisa, vortex 5min takes 500 μ l supernatants to be added in 500 μ l LB culture medium toxin pipes.28 DEG C of constant-temperature tables
After cultivating 48h, in superclean bench, culture solution is diluted to 1:100, the training after 1:1000 times, after taking 100 μ l dilution respectively
Nutrient solution is spread evenly across on NA culture medium, and 28 DEG C of constant temperature incubations observe the growing state of bacterium colony.The bacterium colony grown is pressed down
Bacterium test, select bacteriostasis it is strong carry out identification and subsequent test.
The identification of the morphology, physiological and biochemical index and 16S rDNA of 2 SerEW01 bacterial strain of embodiment
1. colony morphological observation: uniform with oese in superclean bench by a SerEW01 bacterium colony on NA plate
It is coated on NA plate, after growing 2-3d, observes the form and color of bacterium colony, bacterium colony surface of the bacterial strain on NA culture medium is convex
It rises, center is opaque, and edge is irregular, produces red pigments, experimental result is shown in Fig. 1.
2. Determination of Physiological And Biochemical Indices: the identification for carrying out physiological and biochemical index to the strain filtered out with Biolog is specific
Operating procedure is as follows: 100 μ L bacterium solutions being uniformly coated on NA culture medium with L-type glass bar, 28 DEG C of insulating box culture standby for 24 hours
With.Transmissometer is opened, in the case where not letting alone what titer, transparency T is that 60-70% indicates that transmissometer is available, then
IF-A culture solution is placed on transmissometer, it is transparent to be shown as 100%.Aseptic cotton carrier is taken to dip in inoculation liquid wet, by cotton swab in bacterium colony
Surface scrolls, it is viscous to take thallus.Then cotton swab is rotated into cotton swab along inner wall in inoculated tube ullage, thallus is made to be attached to inner wall
On, while thallus uniformly being broken up, the concentration of culture solution is measured again, makes the transparency (bacterium of culture for 24 hours between 95-98%
Fall, 2 single colonies of picking, cultivate the bacterium colony of 48h, 1 single colonie of picking, to prevent transparency reduction), the bacteria suspension that will be prepared
Mixing is poured into loading slot, and using eight electric pipettor, being inoculated in Gram-negative bacteria in 96 holes of microplate makes
With GEN III identify microplate, inoculum concentration be 100 holes μ L/, by 96 hole microplates be placed in 33 DEG C be protected from light constant incubator 48h after see
It examines, obtained analysis result such as Fig. 2.
3. the identification of 16S rDNA: the aimed strain filtered out DNA of bacteria kit is extracted DNA, concrete operations step
It is rapid as follows: the cracking of bacterium (the Gram-negative bacteria 1ml for taking 28 DEG C to be incubated overnight is placed in the 2ml centrifuge tube of sterilizing, and 12,000
× g is centrifuged 1min, as far as possible abandoning supernatant；Then 100 μ l LB11 and 10 μ l Proteinase K are added, vibrate thorough to thallus
It suspends；It is placed in 55 DEG C of incubation 15min), 10 μ l RNaseA are added in the lysate of bacterium and decompose RNA, mixes and stands 2min
Afterwards, 400 μ l BB11 and the 30s that is vortexed is added, then whole solution is added in centrifugal column, 12,000 × g is centrifuged 30s, abandons
Efflux.500 μ l CB11,12,000 × g are added and are centrifuged 30s, abandon efflux (it is primary to repeat 500 μ l CB11 of addition)；Then
500 μ l WB11 are added, 12,000 × g is centrifuged 30s, abandon efflux (repeat 500 μ l WB11 are added primary), 12,000 × g from
Heart 2min thoroughly removes remaining WB11.Centrifugal column is placed in so that it is pre- that 100 μ l are added in column center in a clean centrifuge tube
The EB of heat, is stored at room temperature 2min, and 12,000 × g is centrifuged 1min, eluted dna.The DNA of elution is saved backup in -20 DEG C.
PCR amplification: bacterial 16 S rDNA universal primer: 27F 5 ' -3 ': AGAGTTTGATCCTGGCTCAG, 1492R is used
5 ' -3 ' TACGGYTACCTTGTTACGACTT carry out PCR amplification, reaction system 50 μ l, 25 μ l 2 × Easy Taq Supper
Mix, 1 μ l DNA of bacteria template, 1.5 μ l 27F primers, 1.5 μ l 1492R primers, 21 μ l dd water.PCR amplification condition: 94 DEG C pre-
It is denaturalized 4min；94 DEG C of denaturation 1min, 50 DEG C of annealing 1min, 72 DEG C of extension 2min, totally 30 recycle；Extend 10min after 72 DEG C.Expand
Increase to sequence splice through DNAMAN software, obtain 16S rDNA sequence as shown in SEQ ID NO.1.By using American National
Biotechnology Information center (NCBI) nucleotide BLAST is compared, and finds CGMCC NO.13952 bacterium 16S rDNA's of the invention
More plants of serratia marcescens (Serratia marcescens) 16S rDNA gene order that gene order is registered with NCBI has
High homology illustrates that CGMCC NO.13952 bacterial strain is one plant of serratia marcescens (Serratia marcescens).
Inhibition of the 3 SerEW01 fermentation liquid of embodiment to sickle-like bacteria spore germination, mycelia growth
1. the inhibition of spore germination: this test carries out on concave-concave glass slide.Prepare the SerEW01 bacterial strain hair of various concentration
The mixture of zymotic fluid and sickle-like bacteria (fusarium moniliforme or the raw sickle-like bacteria of layer) spore suspension.Ferment product is for sickle-like bacteria spore
The spore count that statistics is sprouted and do not sprouted under the microscope that son is sprouted, observes 100 spores, repeats three times, calculates spore germination
The difference of rate and sprout time, control group replace SerEW01 bacterial strain with liquid PDB culture medium.SerEW01 bacterial strain fermentation liquor is to spore
Fig. 3 is shown in the inhibition that son is sprouted.
Spore germination rate=(sprouting spore count/observation spore sum) × 100%
2. the inhibition of mycelia growth: this test uses tablet face-off method.Different reaping hook bacteria strains are in superclean bench
It is inoculated on PDA plate.28 DEG C of constant temperature incubation 4d (HWS type climatic chamber, Ningbo southeast instrument plant), the sickle from PDA plate
The region of knife bacterium robust growth, beats the bacteria cake for taking diameter 6mm, furthermore beats and takes an equal amount of NA culture medium flat plate, distinguishes thereon
50 μ l fermentation liquids, supernatant are inoculated with, the fermentation liquid and aqua sterilisa of sterilizing are as control.SerEW01 bacterial strain fermentation liquor is raw to mycelia
Fig. 4 is shown in long inhibition.
Degradation of the 4 SerEW01 fermentation liquid of embodiment to sickle-like bacteria hyphal cell wall
By micro- sem observation sickle-like bacteria F6S-S hypha form, compare through various concentration microorganism treated mycelia
Form and the difference for compareing mycelia, observe the variation of hyphal cell wall, and are photographed to record.SerEW01 bacterial strain fermentation liquor pair
Fig. 5 is shown in the inhibition of spore germination.
Lot of documents proves that serratia marcescens is a kind of strain of high yield chitinase.Chitinase (Chitinase)
Belong to a kind of glycosyl hydrolase, single-minded degradation chitin.The main component of fungal cell wall is that chitin has been found.We push away
Disconnected serratia marcescens SerEW01 bacterial strain fermentation liquor can degrade the cell wall of sickle-like bacteria mycelia.For the deduction for proving us, I
To SerEW01 strain gene group DNA extract after, with chitinase specific primer (being shown in Table 1) carry out PCR amplification.To
To PCR result be sequenced, sequence results are spliced through DNAMAN, chiA sequence results as shown in SEQ ID NO.2, chiB's
Sequence results are as shown in SEQ ID NO.3.
The relevant gene of 1 biocontrol microorganisms active material of table
The generation of 5 SerEW01 bacterial strain of embodiment inhibition fumonisin B1
1. the preparation of sample: after rice (Heilungkiang, 5 constant virtues) is impregnated 18h with aqua sterilisa, draining weighs 20g and is dispensed into
In the conical flask of sterilizing, then into conical flask after the aqua sterilisa (moisture evaporated in supply autoclaving process) of addition 4ml
High-temperature sterilization (121 DEG C, 15min).Rice after subject to sterilization is cooled to room temperature, processing one: by the sickle-like bacteria spore suspension of 6ml
+ 3ml aqua sterilisa is inoculated on the rice of sterilizing；Processing two: by the sickle-like bacteria spore suspension (10 of 6ml6~107)+3ml
SerEW01(107) bacterium solution is inoculated on the rice of sterilizing；Control group rice replaces spore suspension with aqua sterilisa.
2. the collection and grinding of sample: being taken 0,3,5,7d do not infect rice respectively, by the rice and sickle of fusarium infection
The rice about 1g that knife bacterium and bacterium are inoculated with simultaneously, the rice sample of collection is frozen at -80 DEG C, is then freeze-dried at -48 DEG C
Dry 48h keeps sample sufficiently dry in instrument.The rice that will be collected into using tissue grinder instrument (Qiagen, Tissuelyser II)
Sample comminution.The revolving speed of beveller is 3000rpm/min, time 2min.It checks and situation is ground by sample, if grinding is not filled
Point, it can repeat to grind primary.SerEW01 is shown in Fig. 6 to the inhibition of mycelia growth in rice matrix.
3. the extraction of fumonisin B1: weighing the powder of the levigate sample of 100mg, be put into the centrifuge tube of 2ml, add
After the methanol/water (75:25, methanol are HPLC rank) for entering 1ml, sufficient vortex centrifuge tube makes sample be suspended in extractant completely
In and shake overnight.Next day pipettes 800 μ l supernatants with liquid-transfering gun after centrifuge tube is centrifuged 10min with the revolving speed of 12000rpm
Into new centrifuge tube, the hexamethylene degreasing of 800 μ l is then added, vortex mixes well hexamethylene and supernatant, then takes
The liquid of 600 μ l bottoms is stored in -20 DEG C into new centrifuge tube, to be detected.Sample ultrasonic is mixed before loading.
Chromatographic condition: Waters ACQUITY UPLCTM BEH C18Column (50mm × 2.1mm, 1.7 μm of partial size)；Flowing
Phase: A :+0.1% formic acid B of acetonitrile :+0.1% formic acid solution of water, gradient elution (are shown in Table 2)；Flow velocity: 0.4mL/min；Sample volume:
10μL；Column temperature: 35 DEG C.
2 eluent gradient elution program of table
Mass Spectrometry Conditions: ionization mode: electrospray ionisation positive ion mode (ESI+)；Scanning of the mass spectrum mode: more reaction prisons
It surveys (MRM)；Capillary voltage: 3.5kV；Remove solvent temperature degree: 350 DEG C；Cone hole backflow airflow speed: 50L/h；Go solvent stream
Speed: 500L/h；Ion source temperature: 110 DEG C；Collide atmospheric pressure: 3.2x103mbar；The Mass Spectrometry Conditions parameter of fumonisin B1 is shown in
Table 3.
The Mass Spectrometry Conditions parameter of 3 fumonisin B1 of table
* quota ion is indicated
According to experimental data, the rice kernel detection that does not infect to the amount of fumonisin B1 can ignore, toxigenic bacterium strain 88473
It is 18.86ppm that the amount that the rice infected generates fumonisin B1, which reaches peak in 5d, and statistical result is shown in Fig. 7；It is being added
After SerEW01 inhibits bacterium, the yield chance of fumonisin B1 is zero, and SerEW01 has as the result is shown inhibits toxin to generate well
Ability, statistical result is shown in Fig. 8.
The foregoing is merely preferred embodiment of the present application, are not intended to limit this application, for the skill of this field
For art personnel, various changes and changes are possible in this application.Within the spirit and principles of this application, made any to repair
Change, equivalent replacement, improvement etc., should be included within the scope of protection of this application.
SEQUENCE LISTING
<110>Tobacco Institute, Chinese Academy of Agricultural Science
<120>one plants inhibit sickle-like bacteria growth and produce serratia marcescens SerEW01 and its application of poison
<130> 2010
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1443
<212> DNA
<213>serratia marcescens (Serratia marcescens)
<400> 1
cggggcggca gcttacacat gcagtcgagc ggtagcacag ggggagcttg ctccctgggt 60
gacgagcggc ggacgggtga gtaatgtctg ggaaactgcc tgatggaggg ggataactac 120
tggaaacggt agctaatacc gcataacgtc gcaagaccaa agagggggac cttcgggcct 180
cttgccatca gatgtgccca gatgggatta gctagtaggt ggggtaatgg ctcacctagg 240
cgacgatccc tagctggtct gagaggatga ccagccacac tggaactgag acacggtcca 300
gactcctacg ggaggcagca gtggggaata ttgcacaatg ggcgcaagcc tgatgcagcc 360
atgccgcgtg tgtgaagaag gccttcgggt tgtaaagcac tttcagcgag gaggaaggtg 420
gtgaacttaa tacgttcatc aattgacgtt actcgcagaa gaagcaccgg ctaactccgt 480
gccagcagcc gcggtaatac ggagggtgca agcgttaatc ggaattactg ggcgtaaagc 540
gcacgcaggc ggtttgttaa gtcagatgtg aaatccccgg gctcaacctg ggaactgcat 600
ttgaaactgg caagctagag tctcgtagag gggggtagaa ttccaggtgt agcggtgaaa 660
tgcgtagaga tctggaggaa taccggtggc gaaggcggcc ccctggacga agactgacgc 720
tcaggtgcga aagcgtgggg agcaaacagg attagatacc ctggtagtcc acgctgtaaa 780
cgatgtcgat ttggaggttg tgcccttgag gcgtggcttc cggagctaac gcgttaaatc 840
gaccgcctgg ggagtacggc cgcaaggtta aaactcaaat gaattgacgg gggcccgcac 900
aagcggtgga gcatgtggtt taattcgatg caacgcgaag aaccttacct actcttgaca 960
tccagagaac tttccagaga tggattggtg ccttcgggaa ctctgagaca ggtgctgcat 1020
ggctgtcgtc agctcgtgtt gtgaaatgtt gggttaagtc ccgcaacgag cgcaaccctt 1080
atcctttgtt gccagcggtt cggccgggaa ctcaaaggag actgccagtg ataaactgga 1140
ggaaggtggg gatgacgtca agtcatcatg gcccttacga gtagggctac acacgtgcta 1200
caatggcgta tacaaagaga agcgacctcg cgagagcaag cggacctcat aaagtacgtc 1260
gtagtccgga ttggagtctg caactcgact ccatgaagtc ggaatcgcta gtaatcgtag 1320
atcagaatgc tacggtgaat acgttcccgg gccttgtaca caccgcccgt cacaccatgg 1380
gagtgggttg caaaagaagt aggtagctta accttcggga gggcgctacc acttttgatt 1440
cct 1443
<210> 2
<211> 1664
<212> DNA
<213>artificial sequence
<400> 2
agggttgtgg cgttaatcgg cagcacgctg tgttccgcgg cgcaggccgc cgcgccgggc 60
aagccgacca tcgcctgggg caacaccaag ttcgccatcg ttgaagttga ccaggcggct 120
accgcttata atagtttggt gaaggtaaaa aatgccgccg atgtttcggt ctcctggaat 180
ttatggaatg gcgacaccgg tacgacggca aaagttttat taaatggcaa agaggcgtgg 240
agcggcccgt caaccggttc ttccggtacg gcgaatttta aagtcaataa aggcggccgt 300
tatcaaatgc aggtggcatt gtgcaatgcc gacggctgca gcgccagcga tgccaccgaa 360
attgtggtgg ccgacaccga cggcagccat ttggcgccgt tgaaagagcc gctgctggaa 420
aagaataaac cgtataaaca gaactccggc aaagtggtcg gttcttattt cgtcgagtgg 480
ggcgtttacg gggcgcaatt tcaccgtcga acaagatccc ggcgcagaac ctgacccacc 540
tgctgtacgg ctttatcccg atctgcggcg gcaacggtat caacgacagc ctgaaagaag 600
atcgaagggc agcttccagg cgctgcagcg ctcctgacca gggccgcgag gacttcaaag 660
tctcgatcca cgatccgttc gccgcgctgc aaaaagcgca gaagggcgtt accgcttggg 720
atgaccccta caagggcaac ttcggccagc tgatggcgct gaaacaggcg catcctgacc 780
tgaaaattct gccgtcgatc ggcggctgga cgctgtccga cccgttcttc ttcatgggcg 840
ataaggtgaa gcgcgatcgc ttcgtcggtt cggtgaaaga gttcctgcag acctggaagt 900
tcttcgatgg cgtggatatc gactgggagt tcccgggcgg caaaagcgcc aacccgaacc 960
tgggcagccc gcaggacggg gaaacctatg tgctgctgat gaaggagctg cgggcgatgc 1020
tggatcagct gtcggcggaa accggccgca aatatgaact gacctctgcc atcagcgccg 1080
gcaaggacaa gatcgacaag gtggcttaca acgttgcgca gaactcgatg gatcacattc 1140
ttcctgatga gctacgactt tcttatgggc gcccttcgat ctgaagaacc tggggcatca 1200
gaccgcgctg aatgcgccgg cctggaagcc ggacaccgct tacaccacgg tgaacggcgt 1260
caatgcgctg ctggcgcagg gcgtcaagcc gggcaagatc gtggtcggca ccgccatgta 1320
tggccgcggc tggaccgggg tgaacggcta ccagaacaac attccgttca ctggtaccgc 1380
caccgggccg gtcaaaggca cctgggagaa cggcatcgtg gactaccgcc aaatcgccgg 1440
ccagttcatg agcggcgagt ggcagtatac ctacgacgcc acggcggaag cgccttacgt 1500
gttcaagcct tccaccggcg atctgatcac cttcgacgat acccgctcgg tgcaggccaa 1560
aggcaagtac gtgctggata agcagctggg cggcctgttc tcctgggaga tcgacgcgga 1620
taacggcgat attctcaaca gcagaacgcc aatcctgcaa cccg 1664
<210> 3
<211> 1442
<212> DNA
<213>artificial sequence
<400> 3
gtttttttca ccaaccaaat caataattac accgagaccg atacgtccgt cgtgccattc 60
ccggtttcca acattacgcc ggccaaagcc aaacagctga cgcacatcaa cttctcgttc 120
ctggatatca acagcaacct ggaatgcgcc tgggatccgg ccaccaacga cgccaaggcg 180
cgcgatgtgg tcaaccgttt gaccgcgctc aaagcgcata accccagcct gcgcatcatg 240
ttctccatcg gcggctggta ctactccaac gatctgggcg tatcgcacgc caactacgtc 300
aacgcaggtg aaaaccccgg cgtcgcggcg ccaaggtcgc ccaatcctgc gtgcgcatca 360
tgaaggatta cggtttcgac ggtgtggaca tcgactggga gtatccgcag gcggccgaag 420
tggacggctt catcgccgcg ctgcaggaga tccgcacctt gctgaatcag caaaccgtcg 480
ccgacggccg ccaggcgctg ccgtatcagt tgaccatcgc cggcgccggg cggcgccttc 540
ttcctgtcgc gctattacag caagctggcg cagatcgtcg cgccgctcga ttacatcaac 600
ctgatgacct acgatctggc cggcccctgg gagaaggtaa ccaaccacca ggcggcgctg 660
ttcggcgacg cggccgggcc gaccttctac aacgcgctgc gcgaagccaa tctgggctgg 720
agctgggaag agctgacccg cgccttcccc agcccgttca gcctgacggt cgacgccgcg 780
gtgcagcagc acctgatgat ggaaggcgtg ccgagcgcca aaatcgtcat gggcgtgccc 840
ttctatggcc gcgccttcaa gggcgtcagc ggcggcaacg gtgggcaata cagcagccac 900
agcacgccgg gcgaagatcc gtatccgagc accgactact ggctggtggg ctgcgaagag 960
tgcgtgcgcg acaaggatcc gcgcatcgcc tcctatcgcc agttggagca gatgctgcag 1020
ggcaactacg gctatcagcg gttgtggaac gacaagacca aaacccctta tctgtatcat 1080
gcgcagaacg ggctgttcgt cacctatgac gatgccgaga gcttcaaata caaagcgaag 1140
tacatcaagc agcagcagct gggcggcgtg atgttctggc atctggggca agacaaccgc 1200
aacggcgatc tgctggccgc gctggatcgc tatttcaacg ccgcggacta cgacgacagc 1260
cagctggata tgggcaccgg gctgcgctac accggcgtcg gccccggcaa cctgcctatc 1320
atgaccgcgc cggcctatgt gccgggcacc acttacgcgc agggcgcgct ggtgtcctac 1380
cagggctacg tctggcagac caagtggggt tacatcacct ctgcaccggt ccagacgccc 1440
ct 1442
Claims (9)
1. one plant has the serratia marcescens (Serratia marcescens) for inhibiting sickle-like bacteria growth and producing toxic action
SerEW01, the bacterial strain are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on March 28th, 2017
The heart, biological deposits number are as follows: CGMCC NO.13952.
The cultural method of serratia marcescens described in claim 1 2. (Serratia marcescens) SerEW01, feature
It is, serratia marcescens (Serratia marcescens) SerEW01 is placed in NA fluid nutrient medium and is cultivated, cultivates item
Part is aerobic culture at 28 DEG C；
Wherein, the NA Liquid Culture based formulas are as follows: beef extract 3.0g/L；Peptone 5.0g/L；Glucose 2.5g/L；
pH7.0±0.2。
3. a kind of microbial bacterial agent, which is characterized in that the microbial bacterial agent includes serratia marcescens described in claim 1
(Serratia marcescens)SerEW01。
4. a kind of microbial bacterial agent as claimed in claim 3, which is characterized in that the dosage form of the microbial bacterial agent is wettable
Pulvis, water dispersible granules, aqueous suspension agent or dispersible oil-suspending agent.
5. a kind of microbial bacterial agent as claimed in claim 3, which is characterized in that further include Pesticide Science in the microbial bacterial agent
Acceptable auxiliary material is gone up, acceptable auxiliary material is selected from dispersing agent, wetting agent, disintegrating agent, binder, defoaming in the Pesticide Science
One of agent, antifreeze, thickener, filler and solvent are a variety of.
6. serratia marcescens (Serratia marcescens) SerEW01 or claim 3-5 described in claim 1 appoints
Microbial bacterial agent described in one is inhibiting the application in sickle-like bacteria spore germination and/or mycelia growth.
7. application as claimed in claim 6, which is characterized in that the sickle-like bacteria is fusarium moniliforme and/or the raw sickle-like bacteria of layer.
8. serratia marcescens (Serratia marcescens) SerEW01 or claim 3-5 described in claim 1 appoints
Microbial bacterial agent described in one is inhibiting the application in sickle-like bacteria generation fumonisin B1.
9. application as claimed in claim 8, which is characterized in that the sickle-like bacteria is fusarium moniliforme and/or the raw sickle-like bacteria of layer.
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| CN109234196A (en) * | 2018-09-28 | 2019-01-18 | 上海市农业科学院 | A kind of mixed bacterial and its crude enzyme preparation of the fumonisin B1 that degrades |
| CN110800736A (en) * | 2019-11-27 | 2020-02-18 | 南京林业大学 | Oil suspending agent for preventing and treating termites and preparation method thereof |
| CN112442524A (en) * | 2020-11-26 | 2021-03-05 | 南京思农生物有机肥研究院有限公司 | Evaluation and analysis method for preventing and controlling fusarium verticillium by chitin-enhanced trichoderma |
Citations (2)
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|---|---|---|---|---|
| KR20020097085A (en) * | 2002-10-30 | 2002-12-31 | 주식회사 메가바이오텍 | Novel Serratia marscens mutants having activities for plant growth promotion and damping-off control |
| CN103333810A (en) * | 2013-07-25 | 2013-10-02 | 中国热带农业科学院热带生物技术研究所 | Serratia marcescens and application thereof |
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2017
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20020097085A (en) * | 2002-10-30 | 2002-12-31 | 주식회사 메가바이오텍 | Novel Serratia marscens mutants having activities for plant growth promotion and damping-off control |
| CN103333810A (en) * | 2013-07-25 | 2013-10-02 | 中国热带农业科学院热带生物技术研究所 | Serratia marcescens and application thereof |
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| 粘质沙雷氏菌FS14全基因组测序分析及其Ⅵ型分泌系统翻译后调控蛋白和免疫蛋白的晶体结构;李鹏鹏;《中国博士学位论文全文数据库 农业科技辑》;20170615;D046-5,参见全文 * |
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