CN110079478A - Bateriostatics of bacillus amyloliquefaciens and preparation method thereof and the application in prevention and treatment Fusarium moniliforme peanut root rot - Google Patents
Bateriostatics of bacillus amyloliquefaciens and preparation method thereof and the application in prevention and treatment Fusarium moniliforme peanut root rot Download PDFInfo
- Publication number
- CN110079478A CN110079478A CN201910354871.6A CN201910354871A CN110079478A CN 110079478 A CN110079478 A CN 110079478A CN 201910354871 A CN201910354871 A CN 201910354871A CN 110079478 A CN110079478 A CN 110079478A
- Authority
- CN
- China
- Prior art keywords
- bateriostatics
- bacillus amyloliquefaciens
- fusarium moniliforme
- peanut
- root rot
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
-
- 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
-
- 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
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Virology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Tropical Medicine & Parasitology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Environmental Sciences (AREA)
- Plant Pathology (AREA)
- Dentistry (AREA)
- Pest Control & Pesticides (AREA)
- Agronomy & Crop Science (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
The invention belongs to technical field of microbe application, provide bateriostatics of bacillus amyloliquefaciens and preparation method thereof and the application in prevention and treatment Fusarium moniliforme peanut root rot.Separated from Shanxi mature vinegar vinegar fermented grain, filter out have strong bacteriostatic activity to Fusarium moniliforme bacillus amyloliquefaciens (Bacillus amyloliquefaciens), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, is numbered are as follows: CGMCC 15732.The fermented supernatant fluid of the strain is made to the bateriostatics of 1.0mg/mL after ammonium sulfate precipitation, dialysis, freeze-drying, applies in prevention and treatment peanut root rot, Fusarium moniliforme can be reduced to the inhibitory effect of peanut seed germinating growth；And bateriostatics have no adverse effects to peanut, can prevent and treat the root rot of peanut, and control efficiency can more preferably meet the requirement that peanut root rot prevents and treats in agricultural production up to 71.79%.
Description
Technical field
The invention belongs to technical field of microbe application, and in particular to the bateriostatics of bacillus amyloliquefaciens and its preparation side
Method and the application in prevention and treatment Fusarium moniliforme peanut root rot.
Background technique
Peanut root rot can all occur in peanut each breeding time, after peanut seeding emerge before catch an illness, can cause rotten kind, it is rotten
Bud.Seedling stage is aggrieved to be led to that root-rot, seedling are withered；Adult plant is aggrieved to be led to that root-rot, brown foot rot and pod are rotten, the performance of diseased plant overground part is short and small,
Undergrowth, blade turn yellow, and cause complete stool withered eventually.Since this disease site of pathological change is mainly in root and vascular bundle, become diseased plant root
Brown rot is rotten, vascular bundle browning, main root shrinkage dry rot, is similar to mouse shape of tail, affected part surface has yellow-white to the mould layer of pale red.
Peanut root rot is as caused by the fusarium infection of Deuteromycotina, and including Fusarium moniliforme, it be can produce
Microspore, megaspore and the chlamydospore of Invisible element.It in germ habitat soil, can survive the several years in soil, it is parasitic to belong to vascular bundle
Bacterium obstruction conduit and can excrete poison and keep plant withered, so causing peanut yield loss very big.
Peanut root rot prevention and treatment at present can use the cultivation management of efficent rotation, it is possible to use chemicals prevention and treatment, such as
Triazolone, carbendazim etc., however the problems such as drug resistance and serious chemical prevention environmental pollution due to Fusarium moniliforme, it opens
The biological control research of exhibition root rot is of great significance.Biocontrol microorganisms have the characteristics that safety, sphere of action are wide, nontoxic, therefore
It is applied in production from active material is extracted in biocontrol microorganisms, reduces the link of thallus preservation, improve utilization rate.The present invention from
The Bacillus that there is bacteriostatic activity to Fusarium moniliforme is isolated in Shanxi mature vinegar vinegar fermented grain, and is applied after extracting its antibacterial substance
In prevention and treatment peanut root rot.
Summary of the invention
The present invention provides bateriostatics of a kind of bacillus amyloliquefaciens and preparation method thereof and in prevention and treatment Fusarium moniliforme
Application in peanut root rot.
It is prepared the present invention provides a kind of bacillus amyloliquefaciens CGMCC 15732 for being isolated from Shanxi mature vinegar vinegar fermented grain
Bateriostatics, and applied in prevention and treatment Fusarium moniliforme peanut root rot.Technical scheme is as follows:
Screen the Bacillus that there is high bacteriostatic activity to Fusarium moniliforme: the vinegar fermented grain sample in acquisition Shanxi mature vinegar acetic fermentation stage
Product are diluted coating using MRS culture medium, finally isolate 38 plants of Bacillus, are therefrom sieved using Oxford cup inhibition zone method
Select the Bacillus 2014 for having high bacteriostatic activity to Fusarium moniliforme CGMCC 39030.
The MRS culture medium the preparation method comprises the following steps: glucose 20g, peptone 10g, beef extract 10g, yeast extract 5g,
K2HPO42g, sodium acetate 2g, Triammonium citrate 2g, Tween 80 1g, MgSO40.2g, MnSO40.05g, distilled water 1000mL.
PH to 6.2 6.6 is adjusted, 121 DEG C of sterilizing 20min are spare.
16Sr DNA sequencing, primer are as follows: upstream: 5 '-CAGATGGGAGCTTGCTCCCT are carried out to Bacillus 2014
G-3 ', downstream: 5 '-CGACTTCACCCAATCATCTG-3 ', identified Bacillus 2014 are bacillus amyloliquefaciens
(Bacillus amyloliquefaciens), and it is deposited in China Committee for Culture Collection of Microorganisms's commonly micro- life
Object center, deposit number are CGMCC 15732.
The preparation of 15732 bateriostatics of bacillus amyloliquefaciens CGMCC: by the bacillus amyloliquefaciens CGMCC of activation
15732 press 5% inoculum concentration, are inoculated in MRS fluid nutrient medium respectively, 37 DEG C, after 160rpm/min is cultivated for 24 hours respectively, 4 DEG C,
10000 r/min are centrifuged 10 min, take supernatant；Supernatant is tuned into neutrality with the HCl solution of 6mol/L, is added while stirring
Solid ammonium sulfate, when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；4000r/min collects precipitating after being centrifuged 20 min, uses pH
=7.2 phosphate buffer dissolves, desalination of dialysing, up to bateriostatics after vacuum freeze drying.
The preparation method of phosphate buffer described above are as follows: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, chlorine
Change sodium 8.0g, potassium chloride 0.2g, 1000mL, pH=7.2.
Vacuum freeze drying described above be -40 DEG C, vacuum degree be 100 Pa under the conditions of dry 40 h.
Antifungal mechanism of 15732 bateriostatics of bacillus amyloliquefaciens CGMCC to Fusarium moniliforme: bacillus amyloliquefaciens
The growth lag phase of the Fusarium moniliforme of 15732 bateriostatics of CGMCC effect is longer, and the logarithmic growth phase time is shorter, so to string
The mycelium morphology factor and spore germination rate of pearl Fusariumsp have apparent inhibiting effect；And to the penetrating of Fusarium moniliforme cell membrane
Property causes very big influence, destroys the integrality of cell wall；Scanning electron microscopic observation finds that the atrophy of Fusarium moniliforme mycelia is abnormal
Shape and collapse rupture；The cellular morphology of transmission electron microscope observing discovery Fusarium moniliforme has occurred significant change, cell wall collapses,
It is thinning, there is plasmolysis phenomenon, plasma membrane is kept completely separate from cell wall, and plasm agglutination, cytoplasmic densitometric increases.
The bateriostatics of bacillus amyloliquefaciens CGMCC 15732 are reducing Fusarium moniliforme to the production of peanut seed germinating growth
Application in raw inhibition: Fusarium moniliforme being connected in PDA liquid medium, 30 DEG C, after 160rpm/min is cultivated 5 days, filtering
Obtain Fusorium moniliforme Sheldon bacteria culture fluid.Full peanut seed is selected, after clear water impregnates 12 h at 30 DEG C, rinses 3 times, sets vernalization at 30 DEG C
24 h；Peanut seed is placed on the culture dish equipped with filter paper, 10, every ware pours into 5mL Fusorium moniliforme Sheldon bacteria culture fluid, adds
10mL concentration is 15732 bateriostatics of 1.0mg/mL bacillus amyloliquefaciens CGMCC, and control group is added 10 mL sterile waters, is placed in
It is cultivated at 25 DEG C of constant temperature, measurement germination length after 48h.As a result, it has been found that Fusarium moniliforme plays the germinating growth of peanut seed
Inhibiting effect is germinateed with the peanut that bateriostatics prepared by bacillus amyloliquefaciens CGMCC 15732 impregnate and is given birth to compared with the control group
Long inhibiting rate is lower, illustrates that bateriostatics can reduce Fusarium moniliforme to the inhibitory effect of peanut seed germinating growth.
Application of the bateriostatics of bacillus amyloliquefaciens CGMCC 15732 in prevention and treatment Fusarium moniliforme peanut root rot:
Fusorium moniliforme Sheldon is connected in PDA liquid medium 30 DEG C, after 160rpm/min is cultivated 5 days, 20mL Fusarium moniliforme bacterium solution is taken to fill
The root for irrigating 100 plants of potting peanuts, with 50mL concentration be 15732 bateriostatics of 1.0mg/mL bacillus amyloliquefaciens CGMCC and
50mL sterile water irrigates the root of 50 plants of peanuts respectively.As a result, it has been found that there are 39 plants of peanuts to have root rot with what sterile water was irrigated, use
What the bateriostatics of bacillus amyloliquefaciens preparation were irrigated has 11 plants of peanuts to have root rot, so bacillus amyloliquefaciens CGMCC
The bateriostatics of 15732 preparations have no adverse effects to peanut, and can prevent and treat the root rot of peanut well, and control efficiency is reachable
71.79%。
The root that peanut is irrigated with bateriostatics prepared by bacillus amyloliquefaciens CGMCC 15732, can prevent and treat well
The root rot of peanut, the prevention and treatment peanut root rot of energy efficient stable preferably meet what peanut root rot in agricultural production prevented and treated
It is required that.
Detailed description of the invention
Fig. 1 is the colonial morphology and cellular morphology of bacillus amyloliquefaciens CGMCC 15732, in figure: A is solution starch gemma
The colonial morphology of bacillus CGMCC 15732；1000 × lower the cellular morphology that B is bacillus amyloliquefaciens CGMCC 15732；
Fig. 2 is the 16SrDNA phylogenetic tree of bacillus amyloliquefaciens CGMCC 15732；
Fig. 3 is influence of 15732 fermented supernatant fluid of bacillus amyloliquefaciens CGMCC to Fusarium moniliforme mycelium morphology factor；
Fig. 4 is influence of 15732 fermented supernatant fluid of bacillus amyloliquefaciens CGMCC to Fusarium moniliforme spore germination rate；
Fig. 5 is influence of 15732 fermented supernatant fluid of bacillus amyloliquefaciens CGMCC to Fusarium moniliforme physical and chemical index, in figure: A
For the influence to Fusarium moniliforme extracellular potassium ion concentration；B is the influence of AKP content extracellular to Fusarium moniliforme；C is to a beading
The influence of Fusariumsp bacterium solution conductivity；
Fig. 6 is influence of 15732 fermented supernatant fluid of bacillus amyloliquefaciens CGMCC to Fusarium moniliforme mycelia and cellular morphology,
In figure: A is the Fusarium moniliforme hypha form of control group under scanning electron microscope；B is the Fusarium moniliforme of experimental group under scanning electron microscope
Hypha form；C is the Fusarium moniliforme cellular morphology of control group under scanning electron microscope；D is the beading sickle of experimental group under transmission electron microscope
Spore bacterium cellular morphology.
Specific embodiment
Technical solution of the present invention is described further below by specific embodiment.
Embodiment 1: the Bacillus that there is high bacteriostatic activity to Fusarium moniliforme is screened
(1) Bacillus that there is high bacteriostatic activity to Fusarium moniliforme is screened: the vinegar in acquisition Shanxi mature vinegar acetic fermentation stage
Unstrained spirits sample is diluted coating using MRS culture medium, finally isolates 38 plants of Bacillus.Using Oxford cup inhibition zone method from
In filter out to Fusarium moniliforme CGMCC 39030(buy in China Committee for Culture Collection of Microorganisms's common micro-organisms
Center) there is the Bacillus 2014 of high bacteriostatic activity.
Above-mentioned MRS solid medium the preparation method comprises the following steps: peptone 10g, beef extract 10g, yeast extract 5g, K2HPO42g,
Sodium acetate 2g, Triammonium citrate 2g, glucose 20g, Tween 80 1g, MgSO40.2g, MnSO40.05g, agar 20g, distillation
Water 1000mL, adjusts pH to 6.2 6.6, and 121 DEG C of sterilizing 20min are spare.
Above-mentioned screening has high bacteriostatic activity Bacillus to Fusarium moniliforme method particularly includes: by isolate and purify 38 plants
Bacillus is connected to MRS fluid nutrient medium, and 37 DEG C, 160 r/min cultivate 24 h, and 10000 r/min are centrifuged 10 min and collect gemma
Bacterium fermented supernatant fluid, it is spare after being adjusted to neutrality with the NaOH of 6.0 mol/L respectively；Fusarium moniliforme point is connected to and is placed with
Bacillus fermented supernatant fluid is pipetted 200 μ L into Oxford cup by the PDA culture medium plate of Oxford cup, and puts 30 DEG C of incubator trainings
It supports.
(2) there is the identification and preservation of high bacteriostatic activity Bacillus 2014 to Fusarium moniliforme
Morphological Identification: with oese picking Bacillus 2014 it is a little after cross on MRS plating medium, isolate list
The bacterium colony grown on culture medium is taken pictures, is recorded by bacterium colony, then by gram stain microscopy, observes its cellular morphology
Feature.Bacillus 2014 forms 4.58 mm of colony diameter on MRS plating medium, and bacterium colony is rounded, more sticky, surface is thick
It is rough protrusion, neat in edge, milky, opaque；Its cellular morphology: Gram's staining is in G+, rod-shaped, there are gemma, such as Fig. 1 in centre
It is shown.
16Sr DNA sequencing: extracting the genome of Bacillus 2014 with kit, carries out 16Sr DNA sequencing and strain mirror
It is fixed, primer are as follows: upstream: 5 '-CAGATGGGAGCTTGCTCCCTG-3 ', downstream: 5 '-CGACTTCACCCAA
TCATCTG-3 ', using BLAST software through tetraploid rice, the results showed that Bacillus 2014 is bacillus amyloliquefaciens
(Bacillus amyloliquefaciens), and systematic evolution tree is constructed, see Fig. 2.It is deposited in Chinese microorganism strain
Preservation administration committee common micro-organisms center, deposit number are CGMCC 15732, and the preservation time is on May 7th, 2018.
Embodiment 2: the preparation of 15732 bateriostatics of bacillus amyloliquefaciens CGMCC
The inoculum concentration that the bacillus amyloliquefaciens CGMCC 15732 of activation is pressed to 5%, is inoculated in respectively in MRS fluid nutrient medium,
37 DEG C, after 160rpm/min is cultivated for 24 hours respectively, 4 DEG C, 10000 r/min are centrifuged 10 min, take supernatant；With the HCl of 6mol/L
Supernatant is tuned into neutrality by solution, and solid ammonium sulfate is added while stirring, and when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；
4000r/min collects precipitating after being centrifuged 20 min, is dissolved with the phosphate buffer of pH=7.2, desalination of dialysing, and vacuum refrigeration is dry
Up to bateriostatics after dry.
The preparation method of phosphate buffer described above are as follows: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, chlorine
Change sodium 8.0g, potassium chloride 0.2g, 1000mL, pH=7.2.
Vacuum freeze drying described above be -40 DEG C, vacuum degree be 100 Pa under the conditions of dry 40 h.
Embodiment 3: antifungal mechanism of 15732 bateriostatics of bacillus amyloliquefaciens CGMCC to Fusarium moniliforme
15732 bateriostatics of bacillus amyloliquefaciens CGMCC are to the antifungal mechanism of Fusarium moniliforme mainly from mycelium morphology factor, spore
Sub- germination rate, extracellular potassium ion concentration, extracellular AKP content, bacterium solution conductivity and hypha form and cellular morphology etc. come
Research.From the figure 3, it may be seen that 15732 bateriostatics of bacillus amyloliquefaciens CGMCC effect Fusarium moniliforme growth lag phase compared with
Long, the logarithmic growth phase time is shorter, so the mycelium morphology factor to Fusarium moniliforme has inhibiting effect；It can obviously be seen in Fig. 4
Out, 15732 bateriostatics of bacillus amyloliquefaciens CGMCC have apparent inhibiting effect to the spore germination rate of Fusarium moniliforme；And
And very big influence is caused to Fusarium moniliforme permeability of cell membranes, the integrality (see figure 5) of cell wall is destroyed, is scanned
Electronic Speculum observation discovery Fusarium moniliforme mycelia atrophy is lopsided and collapses rupture；Transmission electron microscope observing finds the thin of Fusarium moniliforme
Significant change has occurred in born of the same parents' form, and cell wall collapses, is thinning, plasmolysis phenomenon occurs, and plasma membrane is kept completely separate from cell wall,
Plasm agglutination, cytoplasmic densitometric increase (see figure 6).
The specific side that above-mentioned 15732 bateriostatics of bacillus amyloliquefaciens CGMCC influence Fusarium moniliforme mycelium morphology factor
Method are as follows: connect Fusarium moniliforme point behind PDA culture medium inclined-plane, 30 DEG C of culture 5d with normal saline flushing spore, and by spore
Suspension concentration is adjusted to 106 Then cfu/mL is accessed in 100 mL PDA liquid mediums by 5% bacterium amount that connects, experimental group is added
The 15732 bateriostatics solution of 1.0mg/mL bacillus amyloliquefaciens CGMCC (sterile water preparation) of 5 mL, remaining blank control group
Add the MRS culture medium of 5 mL, continuation is cultivated under the conditions of 30 DEG C, 140 r/min, experimental group and blank control group respectively 1d,
2d, 4d, 6d, 8d take the filtering of 1 bottle of Fusarium moniliforme bacterium solution to discard culture solution daily, and 65 DEG C claim mycelia weight after drying to constant weight
Amount.Using the time as slogan banner, mycelium weight is that ordinate draws growth curve, observes bacillus amyloliquefaciens CGMCC 15732
Influence of the fermented supernatant fluid to Fusarium moniliforme mycelium morphology factor.
The specific side that above-mentioned 15732 bateriostatics of bacillus amyloliquefaciens CGMCC influence Fusarium moniliforme spore germination rate
Method are as follows: connect Fusarium moniliforme point behind PDA culture medium inclined-plane, 30 DEG C of 5 d of culture with normal saline flushing spore, and by spore
Suspension concentration is adjusted to 106 Cfu/mL pipettes the 1.0mg/mL bacillus amyloliquefaciens of 10 μ L spore suspensions and 50 μ L
15732 bateriostatics solution of CGMCC mixes, and is mixed into blank control with 10 μ L spore suspensions and 50 μ L PDA liquid.Hanging drop
Method is placed in sterilized slide glass center, 30 DEG C of constant temperature incubations.Respectively in 6 h, 8 h, 12 h, 16 h, 20 h microscopies, observes spore and sprout
Heat condition observes spore under low power lens (400x), calculates spore germination rate.Spore germination rate=(sprouting spore count/spore sum)
×100%。
The tool that above-mentioned 15732 bateriostatics of bacillus amyloliquefaciens CGMCC influence Fusarium moniliforme extracellular potassium ion concentration
Body method are as follows: by the experimental group of above-mentioned Fusarium moniliforme culture and blank control group respectively in 0 h, 3 h, 8 h, 13 h, 23 h
Timing takes 5 mL of bacteria suspension, and 4 DEG C of centrifugations (10000 r/min, 15 min) take supernatant, according to the side provided in potassium ion agent box
Method is measured using extracellular potassium ion concentration of the spectrophotometer to Fusarium moniliforme.
Above-mentioned 15732 bateriostatics of bacillus amyloliquefaciens CGMCC AKP content extracellular on Fusarium moniliforme influences specific
Method are as follows: by the experimental group of above-mentioned Fusarium moniliforme culture and blank control group respectively in 0 h, 3 h, 8 h, 13 h, 23 h
5 mL of bacteria suspension is taken, 4 DEG C of centrifugations (10000 r/min, 15 min) take supernatant, according to providing in alkaline phosphatase (AKP) agent box
Method, be measured using extracellular AKP content of the spectrophotometer to Fusarium moniliforme.
The specific side that above-mentioned 15732 bateriostatics of bacillus amyloliquefaciens CGMCC influence Fusarium moniliforme bacterium solution conductivity
Method are as follows: take the experimental group of above-mentioned Fusarium moniliforme culture and blank control group in 0 h, 3 h, 8 h, 13 h, 23 h respectively
5 mL of bacteria suspension, 4 DEG C of centrifugations (10000 r/min, 15 min) take supernatant, using the conductance of conductivity gauge measurement Fusarium moniliforme
Rate.
The method that Fusarium moniliforme hypha form is influenced using scanning electron microscopic observation are as follows: prepare Fusarium moniliforme 106
The spore suspension of cfu/mL is connected in 100mL PDA liquid medium, 30 DEG C by 5% bacterium amount that connects, and 140r/min is cultivated 2 days
Afterwards, the 15732 bateriostatics solution of 1.0mg/mL bacillus amyloliquefaciens CGMCC (sterile water preparation) of 5mL, control group are added
Add the MRS culture medium of 5mL, 30 DEG C, after cultivating 72h under the conditions of 140r/min, mycelia is collected by centrifugation；Respectively with 0.1 moL/L pH
It rinses mycelia 3 times, and is fixed with 2.5% glutaraldehyde, 4 DEG C of refrigerator overnights for 7.2 PBS buffer solution；Fixer is outwelled in centrifugation,
Mycelia is with connecing acicula even spread on the cover slip, and with rinsing 3 times in the PBS buffer solution of 0.1 moL/L, 15 min every time.
Treated coverslip is carried out at dehydration in the ethanol solution of concentration gradient 50%, 70%, 80%, 90% and 95% respectively
Reason, every kind of concentration handle 10 min, then with 100% alcohol treatment twice, 15 min every time, finally by the lid after dehydration
Slide is placed in plate, and plate is placed dry 4 h, metal spraying in drier and is simultaneously observed under scanning electron microscope.
The specific method for using transmission electron microscope observing to influence Fusarium moniliforme cellular morphology is prepares Fusarium moniliforme 106
The spore suspension of cfu/mL is connected in 100mL PDA liquid medium, 30 DEG C by 5% bacterium amount that connects, and 140r/min is cultivated 2 days
Afterwards, the 15732 bateriostatics solution of 1.0mg/mL bacillus amyloliquefaciens CGMCC (sterile water preparation) of 5mL, control group are added
Add the MRS culture medium of 5mL, 30 DEG C, after cultivating 72h under the conditions of 140r/min, mycelia is collected by centrifugation；It is with 0.1 moL/L pH
7.2 PBS buffer solution rinses thallus 3 times, and is fixed with 2.5% glutaraldehyde of 20 times of thallus volumes, and 4 DEG C of refrigerator overnights are outwelled
Fixer is rinsed 3 times in the PBS buffer solution of 0.1 moL/L；15 min every time；Thallus 2 is fixed with 1% osmic acid solution again
H outwells fixer, with rinsing 3 times in the PBS buffer solution of 0.1 moL/L, 15 min every time；And with concentration gradient 50%, 70%,
80%, it is carried out dehydrating in 90% and 95% ethanol solution, every kind of concentration handles 10 min, then with 100% alcohol treatment two
It is secondary, 15 min every time.Sample 1h is handled with the mixed liquor (V/V=1/1) of embedding medium and acetone, treated sample is dispensed
Into the Eppendorf pipe of 0.5mL, embedding is got up, 24 h in 37 DEG C of baking ovens, 24 h in 45 DEG C of baking ovens, 48 h in 60 DEG C of baking ovens；
Final sample is sliced in ultramicrotome, obtains the slice of 70-90nm, and observe in transmission electron microscope.
Above-mentioned PDA culture medium the preparation method comprises the following steps: take potato 20g, glucose 2g, peptone 0.2g, KH2PO4
0.2g, MgSO4·2H2O 0.1g adds distilled water 100mL, 121 DEG C of sterilizing 20min.
Embodiment 4: the bateriostatics of bacillus amyloliquefaciens CGMCC 15732 are reducing Fusarium moniliforme to peanut seed hair
Application in bud growth inhibition
Fusarium moniliforme is connected in PDA liquid medium, 30 DEG C, after 160rpm/min is cultivated 5 days, filters to obtain Fusarium moniliforme
Culture solution.Full peanut seed is selected, after clear water impregnates 12 h at 30 DEG C, rinses 3 times, sets 24 h of vernalization at 30 DEG C；By peanut
Seed is placed on the culture dish equipped with filter paper, and 10, every ware pours into 5mL Fusorium moniliforme Sheldon bacteria culture fluid, is added 10mL concentration and is
15732 bateriostatics of 1.0mg/mL bacillus amyloliquefaciens CGMCC (sterile water preparation), control group are added 10 mL sterile waters, set
It is cultivated at 25 DEG C of constant temperature, measurement germination length after 48h calculates the inhibition that Fusarium moniliforme grows germination according to formula
Rate, and determine that the bateriostatics of bacillus amyloliquefaciens CGMCC 15732 are reducing Fusarium moniliforme pair according to the size of inhibiting rate
The inhibitory effect that peanut seed germinating growth generates.Germinating growth inhibiting rate (%)=(control group germination length-processing group germination length
Degree)/control group germination length.
Fusarium moniliforme plays inhibiting effect to the germinating growth of peanut seed as the result is shown, compared with the control group, uses
The inhibiting rate for the peanut germinating growth that bateriostatics prepared by bacillus amyloliquefaciens CGMCC 15732 impregnate is lower, illustrates antibacterial
Object can reduce Fusarium moniliforme to the inhibitory effect of peanut seed germinating growth.
Embodiment 5: the bateriostatics of bacillus amyloliquefaciens CGMCC 15732 are in prevention and treatment Fusarium moniliforme peanut root rot
Application
Fusarium moniliforme is connected in PDA liquid medium 30 DEG C, after 160rpm/min is cultivated 5 days, takes 20mL Fusarium moniliforme
Bacterium solution irrigates the root of 100 plants of potting peanuts, is that 1.0mg/mL bacillus amyloliquefaciens CGMCC 15732 is antibacterial with 50mL concentration
Object and 50mL sterile water irrigate the root of 50 plants of peanuts respectively.As a result, it has been found that thering are 39 plants of peanuts to have root-rot with what sterile water was irrigated
Disease, what the bateriostatics prepared with bacillus amyloliquefaciens were irrigated has 11 plants of peanuts to have root rot, so bacillus amyloliquefaciens
Bateriostatics prepared by CGMCC 15732 have no adverse effects to peanut, and can prevent and treat the root rot of peanut well, prevention and treatment effect
Fruit is up to 71.79%.
Claims (7)
1. a bacillus amyloliquefaciens, it is characterised in that: the bacterial strain separates from Shanxi mature vinegar vinegar fermented grain, screens gained, protects
Hiding number is CGMCC 15732, depositary institution: China Committee for Culture Collection of Microorganisms's common micro-organisms center, address: north
The institute 3 of the Chaoyang District Jing Shi North Star West Road 1, preservation date: on May 7th, 2018.
2. the bateriostatics prepared using bacillus amyloliquefaciens described in claim 1, it is characterised in that: specific preparation method is such as
Under: the bacillus amyloliquefaciens CGMCC 15732 of activation is pressed to 5% inoculum concentration, is inoculated in MRS fluid nutrient medium, 37 DEG C,
After 160rpm/min is cultivated for 24 hours respectively, 4 DEG C, 10000 r/min are centrifuged 10 min, take supernatant；With the HCl solution of 6mol/L
Supernatant is tuned into neutrality, solid ammonium sulfate is added while stirring, when saturation degree reaches 80%, 4 DEG C of refrigerators are stood overnight；
4000r/min collects precipitating after being centrifuged 20 min, is dissolved with the phosphate buffer of pH=7.2, desalination of dialysing, and vacuum refrigeration is dry
Up to bateriostatics after dry.
3. the bateriostatics according to claim 2 prepared using bacillus amyloliquefaciens, it is characterised in that: the phosphoric acid
The preparation method of salt buffer are as follows: potassium dihydrogen phosphate 0.24g, disodium hydrogen phosphate 1.42g, sodium chloride 8.0g, potassium chloride 0.2g,
1000mL, pH=7.2.
4. the bateriostatics according to claim 2 prepared using bacillus amyloliquefaciens, it is characterised in that: the vacuum
Freeze-drying be -40 DEG C, vacuum degree be 100 Pa under the conditions of dry 40 h.
5. the bateriostatics as claimed in claim 2 using bacillus amyloliquefaciens preparation are in prevention and treatment Fusarium moniliforme peanut root rot
In application, it is characterised in that: the bateriostatics reduce Fusarium moniliforme in peanut seed germinating growth inhibiting effect
Method are as follows: Fusarium moniliforme is connected in PDA liquid medium, 30 DEG C, 160rpm/min cultivate 5 days after, filter to obtain a beading
Fusarium bacteria culture fluid；Full peanut seed is selected, after clear water impregnates 12 h at 30 DEG C, rinses 3 times, sets 24 h of vernalization at 30 DEG C；
Peanut seed is placed on the culture dish equipped with filter paper, 10, every ware pours into 5mL Fusorium moniliforme Sheldon bacteria culture fluid, adds 10mL
Concentration is 15732 bateriostatics of 1.0mg/mL bacillus amyloliquefaciens CGMCC, and control group is added 10 mL sterile waters, is placed in constant temperature
It is cultivated at 25 DEG C, measurement germination length after 48h, and calculates Fusarium moniliforme to the inhibiting rate of peanut seed germinating growth, and press
Determine that bateriostatics are reducing the inhibitory effect that generates to peanut seed germinating growth of Fusarium moniliforme according to the size of inhibiting rate.
6. the bateriostatics as claimed in claim 2 using bacillus amyloliquefaciens preparation are in prevention and treatment Fusarium moniliforme peanut root rot
In application, it is characterised in that: bateriostatics method for applying in prevention and treatment Fusarium moniliforme peanut root rot are as follows: will go here and there
Pearl fusarium is connected in PDA liquid medium 30 DEG C, after 160rpm/min is cultivated 5 days, and 20mL Fusarium moniliforme bacterium solution is taken to irrigate 100
The root of strain potting peanut, with 50mL concentration be 15732 bateriostatics of 1.0mg/mL bacillus amyloliquefaciens CGMCC and 50mL without
Bacterium water irrigates the root of 50 plants of peanuts respectively.
7. the bateriostatics according to claim 5 or 6 using bacillus amyloliquefaciens preparation are in prevention and treatment Fusarium moniliforme flower
Application in maize ear rot of taking root, it is characterised in that: the concentration is that the bateriostatics solution of 1.0mg/mL is formulated with sterile water.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910354871.6A CN110079478B (en) | 2019-04-29 | 2019-04-29 | Bacteriostatic agent of bacillus amyloliquefaciens, preparation method thereof and application of bacteriostatic agent in prevention and treatment of rhizoctonia solani flowers |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910354871.6A CN110079478B (en) | 2019-04-29 | 2019-04-29 | Bacteriostatic agent of bacillus amyloliquefaciens, preparation method thereof and application of bacteriostatic agent in prevention and treatment of rhizoctonia solani flowers |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110079478A true CN110079478A (en) | 2019-08-02 |
| CN110079478B CN110079478B (en) | 2022-07-29 |
Family
ID=67417574
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910354871.6A Active CN110079478B (en) | 2019-04-29 | 2019-04-29 | Bacteriostatic agent of bacillus amyloliquefaciens, preparation method thereof and application of bacteriostatic agent in prevention and treatment of rhizoctonia solani flowers |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110079478B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699264A (en) * | 2019-11-08 | 2020-01-17 | 山东省花生研究所 | Rapid culture method of pathogenic fungi of peanut rot |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080152684A1 (en) * | 2006-12-20 | 2008-06-26 | Dean Der-Syh Tzeng | Method for preparing a composition containing Bacillus subtilis WG6-14 and related use |
| CN105368747A (en) * | 2015-12-02 | 2016-03-02 | 中国农业科学院麻类研究所 | Bacillus amyloliquefaciens strain and application thereof |
| US20160286803A1 (en) * | 2012-01-25 | 2016-10-06 | Bayer Intellectual Property Gmbh | Active Compounds Combination Containing Fluopyram Bacillus and Biologically Control Agent |
| CN108017447A (en) * | 2017-12-11 | 2018-05-11 | 河南邑鸿善成生物技术有限公司 | Prepare the method for bacillus amyloliquefaciens fermentation fertilizer and its application of fertilizer |
| CN109402027A (en) * | 2018-12-14 | 2019-03-01 | 齐齐哈尔大学 | One plant has the bacillus amyloliquefaciens for inhibiting the Fusarium oxysporum effect of watermelon specialized form and its application |
| CN113897317A (en) * | 2021-10-29 | 2022-01-07 | 中国农业科学院农产品加工研究所 | Bacillus amyloliquefaciens A-1 and application thereof |
-
2019
- 2019-04-29 CN CN201910354871.6A patent/CN110079478B/en active Active
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20080152684A1 (en) * | 2006-12-20 | 2008-06-26 | Dean Der-Syh Tzeng | Method for preparing a composition containing Bacillus subtilis WG6-14 and related use |
| US20160286803A1 (en) * | 2012-01-25 | 2016-10-06 | Bayer Intellectual Property Gmbh | Active Compounds Combination Containing Fluopyram Bacillus and Biologically Control Agent |
| CN105368747A (en) * | 2015-12-02 | 2016-03-02 | 中国农业科学院麻类研究所 | Bacillus amyloliquefaciens strain and application thereof |
| CN108017447A (en) * | 2017-12-11 | 2018-05-11 | 河南邑鸿善成生物技术有限公司 | Prepare the method for bacillus amyloliquefaciens fermentation fertilizer and its application of fertilizer |
| CN109402027A (en) * | 2018-12-14 | 2019-03-01 | 齐齐哈尔大学 | One plant has the bacillus amyloliquefaciens for inhibiting the Fusarium oxysporum effect of watermelon specialized form and its application |
| CN113897317A (en) * | 2021-10-29 | 2022-01-07 | 中国农业科学院农产品加工研究所 | Bacillus amyloliquefaciens A-1 and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| SARWAR, A等: ""Qualitative analysis of biosurfactants from Bacillus species exhibiting antifungal activity"", 《PLOS ONE》 * |
| 史改玲等: ""山西老陈醋源优良芽孢杆菌菌株的鉴定及筛选"", 《中国酿造》 * |
| 张玉栋: ""芽孢杆菌对山东临沂地区花生和生姜常见病害的防效研究"", 《中国优秀硕士学位论文全文数据库（电子期刊）农业科技辑》 * |
| 贾瑞娟: ""山西老陈醋源芽孢菌抑菌机理及对腐败菌群体感应抑制的研究"", 《中国优秀硕士学位论文全文数据库（电子期刊）基础科学辑》 * |
| 赵欣等: ""解淀粉芽胞杆菌菌株HRH317对感染串珠镰孢菌玉米幼苗伏马毒素B1含量及相关防御酶活性的影响"", 《植物保护学报》 * |
| 陈志谊: ""芽孢杆菌类生物杀菌剂的研发与应用"", 《中国生物防治学报》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110699264A (en) * | 2019-11-08 | 2020-01-17 | 山东省花生研究所 | Rapid culture method of pathogenic fungi of peanut rot |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110079478B (en) | 2022-07-29 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113025501B (en) | Multifunctional trichoderma asperellum and application thereof | |
| CN106754426B (en) | Trichoderma asperellum and application thereof | |
| CN104694397A (en) | Chaetomium globosum and application thereof | |
| CN104630071A (en) | Polysporus trichoderma and application thereof | |
| CN102367421B (en) | Penicilliumgriseofulvum XJ-EP-058 strain as well as microbial preparation resisting banana vascular wilt and application thereof | |
| CN105368720B (en) | Cotton endogenetic fungus CEF-082 and its application in cotton verticillium wilt prevention and treatment | |
| CN113862156B (en) | Fusarium oxysporum (Fusarium oxysporum) K2018-1418 and application thereof | |
| CN102732430A (en) | Aspergillus niger strain and application thereof | |
| CN110079478A (en) | Bateriostatics of bacillus amyloliquefaciens and preparation method thereof and the application in prevention and treatment Fusarium moniliforme peanut root rot | |
| CN103897992A (en) | Endophytic fungi CEF-193 of cotton and application thereof | |
| CN107012110A (en) | A kind of entomopathogenic nematode symbiotic bacteria and its application with inhibition of potato late blight | |
| CN114032182B (en) | Fungus with functions of antagonizing pathogenic bacteria of garlic root rot and promoting growth | |
| CN114456973B (en) | Streptomyces rochei in tobacco and application thereof in prevention and control of tobacco diseases | |
| CN115873742A (en) | Streptomyces aureus and application thereof in preventing and treating cucumber rhizoctonia rot | |
| CN103627643B (en) | Cotton endogenetic fungus CEF-818 and the application in cotton verticillium wilt control thereof | |
| CN107603904B (en) | Streptomyces and application thereof | |
| CN114854665B (en) | Production method and application of paecilomyces lilacinus microsclerotia | |
| CN114134087B (en) | Streptomyces alginolyticus and application thereof in prevention and control of wheat stem basal rot | |
| CN116769607B (en) | Application of endophytic fungus A21-1-1 in preventing and controlling rice blast | |
| CN114164126B (en) | Rhizopus oryzae XERF-1 and application thereof in relieving apple continuous cropping obstacle | |
| KR100347388B1 (en) | Microorganism having predacious ability for soil nematoda parasitic on plant from soil, and method for incubating the same | |
| CN116649374B (en) | Pseudomonas aeruginosa QSE-TM7 and application thereof in preparation of biological agent for preventing and treating tomato gray mold | |
| CN113817613B (en) | Trichoderma asperellum 6S-2 and application thereof in relieving apple continuous cropping obstacle | |
| CN107603903B (en) | Streptomycin and application thereof | |
| CN116376710A (en) | Fusarium layering (Fusarium proliferatum) APF-1 strain and application and separation method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |