CN109762778A - The preparation method of series bacillus and its biological control agent - Google Patents
The preparation method of series bacillus and its biological control agent Download PDFInfo
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- CN109762778A CN109762778A CN201910287097.1A CN201910287097A CN109762778A CN 109762778 A CN109762778 A CN 109762778A CN 201910287097 A CN201910287097 A CN 201910287097A CN 109762778 A CN109762778 A CN 109762778A
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Abstract
The present invention relates to the preparation methods of a Bacillus species and its biological control agent, belong to microorganism field.Its taxology name are as follows: series bacillus (Paenibacillus tyrfis) KXJ51 has been preserved in China General Microbiological culture presevation administrative center, deposit number are as follows: CGMCC No.17155, the deposit date is on January 10th, 2019.The method that series bacillus prepares biological control agent, including bacterial strain activation, liquid seeds preparation and liquid fermentation.Series bacillus (Paenibacillus tyrfis) KXJ51 is applied in prevention and treatment phytopathogen.The beneficial effects of the present invention are: the series bacillus bacterial strain KXJ51 speed of growth is fast, fermentation density is high, it is strong that action spectrum can extensively prevent and treat plurality of plant diseases, genetic stability, and environment compatibility is good, the diseases prevention lasting period is long.
Description
Technical field
The present invention relates to the preparation methods of a Bacillus species and its biological control agent, belong to microorganism field.
Background technique
Plant disease is the antagonistic symbiosis of host plant and pathogen, occurs and prevalence is host plant and pathogen
The result of interaction.Disease occurs for crops and forest, and national economy and people's lives is often made to sustain losses severely.In recent years
Come, as the public is to the common concern of chemical pesticide safety in food and environment and the taboo of some dangerous chemical pesticides
With promoting people to seek other safely and effectively plant disease control new ways, utilize beneficial microbe controlling plant diseases day
Become to being taken seriously.It is to introduce the external source Antagonistic Fungi beneficial to plant into the plant natural ecosystem being broken or pass through
It adjusts environment to promote natural beneficial microbe group to increase and show biological and ecological methods to prevent plant disease, pests, and erosion activity, be reached by the interaction between microorganism
To the purpose of diseases prevention.Although and biological agent made of existing some microorganisms can reach diseases prevention to a certain extent
Effect, but the perfect condition that people want far can not be reached.
Bacillus genus (Paenibacillus) is the classificatory new development of bacillus, bacillus genus
Bacterial strain has certain general character of Bacillus strain, not only has significant Biocontrol Potential, moreover it is possible to generate the inverse bud of heat-resistant
Spore is conducive to the production of biocontrol agent, formulation and survives, colonizes and breed in the environment, is the micro- life of plant disease biological and ecological methods to prevent plant disease, pests, and erosion
The important component of object.Since Paenibacilli growth is fast, nutrition is simple, is capable of forming with stronger anti-adversity ability
Gemma, more non-bacillus living bacteria count amount is high in product development, performance is stablized, thus attracts attention, be
Compare one of the strain with application potential in bacillus.
Summary of the invention
The present invention provides a Bacillus species and its biology for deficiency present in above-mentioned existing Techniques For Chemical Control
The preparation method for preventing and treating preparation.
The technical scheme to solve the above technical problems is that
One Bacillus species, taxology name are as follows: series bacillus (Paenibacillus tyrfis) KXJ51, preservation
In China General Microbiological culture presevation administrative center, deposit number are as follows: CGMCC No. 17155, the deposit date is 2019 1
The moon 10.
The method that series bacillus prepares biological control agent, comprising the following steps:
Step 1: bacterial strain activation, picking strain streak inoculation to solid slope culture medium, in being cultivated in constant incubator, to oblique
Face covers with bacterium colony for liquid seeds of transferring；
Step 2: liquid seeds preparation, picking activated strains are inoculated in the container equipped with seed culture medium, eight layers of gauze envelope
Mouthful, in 20 DEG C -37 DEG C, 24 h of 150r/min~240r/min shaking table shaken cultivation~48h, somatic cells are grown, liquid strain is obtained
Son；
Step 3: liquid fermentation, the liquid strain that 5~20% inoculum concentrations are prepared into fermentation medium access step 2 by volume
Son, 300 r/min of revolving speed~800r/min, dissolved oxygen control 20%, pH6.0~7.5, and 20~37 DEG C of temperature, 10L fermentor is ventilated
Cultivate 24~56h.
Preferably, stating the temperature in constant incubator is 20 DEG C~37 DEG C, and incubation time is 1~2 day.
Preferably, the internal composition of the solid slope culture medium are as follows: 2 g/L of glucose, 5 g/L of yeast extract,
20 min of peptone 10 g/L, NaCl 5 g/L, agar 20 g/L, pH7.0,121 DEG C high pressure steam sterilization.
Preferably, the internal composition of the seed culture medium are as follows: glucose 10g/L, 5 g/L of yeast extract, albumen
Peptone 10 g/L, KH2PO41 g/L, MgSO40.5 g/L, corn pulp 10 g/L, pH7.0,121 DEG C high pressure steam sterilization 20
min。
Preferably, the internal composition of the fermentation medium are as follows: glucose 20g/L, 5 g/L of peptone, yeast leaching
Powder 2 g/L, MgSO40.4 g/L, KH2PO41 g/L, 3 g/L of ammonium citrate, corn pulp 10 g/L, FeSO41mg/L,
Vitamin B17.0,121 DEG C of 20 min of high pressure steam sterilization of 2 mg/L, pH.
Preferably, series bacillus (Paenibacillus tyrfis) KXJ51 can be applied in prevention and treatment phytopathogen.
Preferably, the phytopathogen includes soft rot Erwinia (Erwinia carotovora), the pathogen of Botrytis cinerea
(otrytis cinerea), Fusarium oxysporum (usarium oxysporum), gibberella saubinetii (Fusarium
Graminearum Schw), rice leaf pseudomonad (Pseudomonas oryzihabitans).
Compared with prior art, the beneficial effects of the present invention are: the series bacillus bacterial strain KXJ51 speed of growth is fast, ferments
Density is high, and action spectrum can extensively prevent and treat plurality of plant diseases, and genetic stability is strong, and good with environment compatibility, the diseases prevention lasting period is long.
Detailed description of the invention
Fig. 1 is the colonial morphology figure of Paenibacillus tyrfis bacterial strain KXJ51.
Fig. 2 is the phylogenetic tree based on 16S rRNA sequence.
Specific embodiment
The principle and features of the present invention will be described below with reference to the accompanying drawings, and the given examples are served only to explain the present invention, and
It is non-to be used to limit the scope of the invention.
Embodiment 1: the method for bacterial strain KXJ51 separation screening
Separation: bacterial strain KXJ51 is separated using method of dilution butteron on plate, is weighed 10g bottom sediment sample first and is dissolved in 90mL sterile water
In, oscillation, which shakes up, is sufficiently mixed sample with water, is coated in LB culture medium flat plate after gradient dilution, plate is then placed in 30
DEG C insulating box in cultivate 5 days, the bacterium colony of different shape on LB culture medium is crossed in LB culture medium flat plate, purifying obtains
The bacterial strain obtained, and number saves respectively.Wherein LB nutrient media components are as follows: 5 g/L of yeast extract, peptone 10 g/L, NaCl
20 min of 5 g/L, agar 20 g/L, pH7.0,121 DEG C high pressure steam sterilization.
Screening: opposite culture method is used, the pathogen of Botrytis cinerea is inoculated into PDA culture medium with the punch of 5 mm of diameter
Point, fungus block from two sides symmetrical at 2 cm of center be inoculated with 10 μ L antagonism bacterium solutions, be inverted, 28 DEG C constant temperature incubation 5 days, not connect
The culture dish of kind Antagonistic Fungi compares, checkout facility result when compareing ware mycelia and covering with culture dish.Wherein PDA culture medium component
Are as follows: potato 200g/L, glucose 20g/L, agar 20g/L, pH are naturally, 121 DEG C of 20 min of high pressure steam sterilization.
Embodiment 2: the identification of bacterial strain KXJ51
Strain properties: two days later with LB culture medium culture bacterial strain KXJ51, bacterium colony is flat, smooth circular, creamy white or light yellow,
Non-pigment；Gram-positive corynebacteria.Bacterial strain KXJ51 can decomposition glucose, fructose, glycerol, cannot be using mannose, sweet
Reveal alcohol.The test results such as oxidizing ferment, nitrate reduction are the positive.Paenibacillus tyrfis KXJ51 bacterial strain colonial morphology
See Fig. 1.
Bacterial strain identification: the 16S rRNA gene order length obtained through DNA extraction, PCR amplification and sequencing is 1412 bp,
BLAST comparison is carried out on GenBank, obtains the bacterial strain and Paenibacillus tyrfis(KT216503) base similitude
Up to 99%, bacterial strain KXJ51 is accredited as Paenibacillus tyrfis by combining form and physiological and biochemical index, is based on 16S
The phylogenetic tree of rRNA sequence is shown in Fig. 2.
Genetic stability: Paenibacillus tyrfis KXJ51 is connected in solid slope culture medium using method of scoring
It resumes generation 50 times, the ability of measurement thalli morphology, the speed of growth and anti-the pathogen of Botrytis cinerea is lost with primary bacterial strain no significant difference
Biography has good stability.
Culture presevation: it is general that the bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms on January 10th, 2019
Logical microorganism center, deposit number: CGMCC No. 17155.
Embodiment 3: the fermentation process of series bacillus KXJ51
Bacterial strain activation: picking strain streak inoculation to solid slope culture medium (2 g/L of glucose, 5 g/L of yeast extract, egg
White 10 g/L of peptone, 5 g/L of NaCl, 20 min of agar 20 g/L, pH7.0,121 DEG C high pressure steam sterilization), in constant temperature incubation
30 DEG C of case are cultivated 2 days, grow a large amount of somatic cells for liquid seeds of transferring to inclined-plane；
Liquid seeds preparation: picking activated strains are inoculated in equipped with seed culture medium (glucose 10g/L, 5 g/ of yeast extract
L, peptone 10 g/L, KH2PO4 0.5 g/L of 1 g/L, MgSO4, corn pulp 10 g/L, pH7.0,121 DEG C high steam
Sterilize 20 min) in, gauze sealing, in 30 DEG C, 220r/min shaking table shaken cultivation 28h grows a large amount of thallus, obtains liquid strain
Son；
Liquid fermentation: 5% inoculum concentration is to fermentation medium (glucose 20g/L, 5 g/L of peptone, yeast extract by volume
2 g/L, MgSO4 0.4 g/L, KH2PO4 1 g/L, 3 g/L of ammonium citrate, corn pulp 10 g/L, FeSO4 1mg/L, dimension
Raw 7.0,121 DEG C of 20 min of high pressure steam sterilization of element B1 2 mg/L, pH) access liquid seeds prepared by step 2, revolving speed 200
~700r/min, dissolved oxygen control 30%, pH7.0, and 30 DEG C of temperature, 50h is cultivated in ventilation.
Embodiment 4: the disease-resistant test of the fermentation liquid of bacterial strain KXJ51
The inspection of the anti-phytopathogen ability of Paenibacillus tyrfis KXJ51 fermentation liquid is carried out by cylinder-plate method, as a result
As shown in table 1.Paenibacillus tyrfis KXJ51 fermentation liquid to soft rot Erwinia (Erwinia carotovora),
The pathogen of Botrytis cinerea (Botrytis cinerea), Fusarium oxysporum (Fusarium oxysporum), gibberella saubinetii
The phytopathies such as (Fusarium graminearum Schw.), rice leaf pseudomonad (Pseudomonas oryzihabitans)
The growth of opportunistic pathogen all has significant antagonism.It is wherein the most significant to the inhibitory effect of the pathogen of Botrytis cinerea, 10 μ L fermentation liquids
Average inhibition diameter reach 3cm or more；Good inhibiting effect is also shown to other phytopathogens, averagely inhibition diameter
All in 2.0 cm or more, there are the potentiality of development and utilization.
The antibacterial circle diameter and area of 1 bacterial strain KXJ51 fermentation liquid of table inhibition phytopathogen
Embodiment 5:Paenibacillus tyrfis KXJ51 fermentation liquid verifies the pathogen of Botrytis cinerea field efficacy
2 needles are pierced in Strawberry Leaves and fruit with No. 5 syringe needles, the pathogen of Botrytis cinerea, every processing 50 are then inoculated at needle thorn
Strain, 3 repetitions spray 500 times of dilutions of Paenibacillus tyrfis KXJ51 fermentation liquid, 50% carbendazim after 2d respectively
500 times of liquid and be not administered clear water compare totally 3 processing, 7 days investigation disease indexs after application, calculating control efficiency.Test knot
Fruit is shown in Table 2, and 7 days after application, Paenibacillus tyrfis KXJ51 500 times of dilution control efficiency of fermentation liquid are
71.7%, it is significantly higher than the effect of 50% 500 times of liquid of carbendazim.
Control efficiency of the 2 Paenibacillus tyrfis KXJ51 fermentation liquid of table to grey mould fruit rot of strawberry
The foregoing is merely presently preferred embodiments of the present invention, is not intended to limit the invention, all in the spirit and principles in the present invention
Within, any modification, equivalent replacement, improvement and so on should all be included in the protection scope of the present invention.
Sequence table
<110>Ludong University
<120>preparation method of series bacillus and its biological control agent
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1412
<212> DNA
<213> Paenibacillus tyrfis
<400> 1
ctatacatgc aagtcgagcg gacccttcgg ggttagcggc ggacgggtga gtaacacgta 60
ggcaacctgc ctgtaagact gggataacta ccggaaacgg tagctaagac cggataagtg 120
attctctcgc atgagaggat caagaaacac ggggcaacct gtggcttaca gatgggcctg 180
cggcgcatta gctagttggt ggggtaacgg ctcaccaagg cgacgatgcg tagccgacct 240
gagagggtga tcggccacac tgggactgag acacggccca gactcctacg ggaggcagca 300
gtagggaatc ttccgcaatg gacgcaagtc tgacggagca acgccgcgtg agtgatgaag 360
gttttcggat cgtaaagctc tgttgccagg gaagaacgtc gcggagagta actgctctgc 420
gaatgacggt acctgagaag aaagccccgg ctaactacgt gccagcagcc gcggtaatac 480
gtagggggca agcgttgtcc ggaattattg ggcgtaaagc gcgcgcaggc ggccgcttaa 540
gtctggtgtt taagcccgag gctcaacctc ggttcgcact ggaaactggg tggcttgagt 600
gcaggagagg aaagcggaat tccacgtgta gcggtgaaat gcgtagagat gtggaggaac 660
accagtggcg aaggcggctt tctggcctgt aactgacgct gaggcgcgaa agcgtgggga 720
gcaaacagga ttagataccc tggtagtcca cgccgtaaac gatgagtgct aggtgttagg 780
ggtttcgata cccttggtgc cgaagtaaac acaataagca ctccgcctgg ggagtacgct 840
cgcaagagtg aaactcaaag gaattgacgg ggacccgcac aagcagtgga gtatgtggtt 900
taattcgaag caacgcgaag aaccttacca ggtcttgaca tccctatgaa taccctagag 960
atagggcagg ccttcgggac agaggagaca ggtggtgcat ggttgtcgtc agctcgtgtc 1020
gtgagatgtt gggttaagtc ccgcaacgag cgcaaccctt gaacttagtt gccagcatta 1080
agttgggcac tctaagttga ctgccggtga caaaccggag gaaggtgggg atgacgtcaa 1140
atcatcatgc cccttatgac ctgggctaca cacgtactac aatggccggt acaacgggaa 1200
gcgaagtcgc gagatggagc caatcctaag aaagccggtc tcagttcgga ttgcaggctg 1260
caactcgcct gcatgaagtc ggaattgcta gtaatcgcgg atcagcatgc cgcggtgaat 1320
acgttcccgg gtcttgtaca caccgcccgt cacaccacga gagtttacaa cacccgaagt 1380
cggtggggta accgcaagga gccagccgcc ga 1412
Claims (6)
1. a Bacillus species, taxology name are as follows: series bacillus (Paenibacillus tyrfis) KXJ51 has been protected
It is hidden in China General Microbiological culture presevation administrative center, deposit number are as follows: CGMCC No. 17155, the deposit date is 2019
On January 10, in.
2. the method that series bacillus according to claim 1 prepares biological control agent, it is characterised in that: including following
Step:
Step 1: bacterial strain activation, picking strain streak inoculation to solid slope culture medium, in being cultivated in constant incubator, to oblique
Face covers with bacterium colony for liquid seeds of transferring；
Step 2: liquid seeds preparation, picking activated strains are inoculated in the container equipped with seed culture medium, eight layers of gauze envelope
Mouthful, in 20 DEG C -37 DEG C, 24 h of 150r/min~240r/min shaking table shaken cultivation~48h, somatic cells are grown, liquid strain is obtained
Son；
Step 3: liquid fermentation, the liquid strain that 5~20% inoculum concentrations are prepared into fermentation medium access step 2 by volume
Son, 300~800r/min of revolving speed, dissolved oxygen control 20%, pH6.0~7.5, and 20~37 DEG C of temperature, the ventilation of 10L fermentor cultivates 24
~56h.
3. the method that series bacillus according to claim 2 prepares biological control agent, it is characterised in that: the constant temperature
Temperature in incubator is 20 DEG C~37 DEG C, and incubation time is 1~2 day.
4. the method that series bacillus according to claim 2 prepares biological control agent, which is characterized in that described consolidates
The internal composition of body slant medium are as follows: 2 g/L of glucose, 5 g/L of yeast extract, 5 g/ of peptone 10 g/L, NaCl
20 min of L, agar 20 g/L, pH7.0,121 DEG C high pressure steam sterilization.
5. the method that series bacillus according to claim 2 prepares biological control agent, which is characterized in that the kind
The internal composition of sub- culture medium are as follows: glucose 10g/L, 5 g/L of yeast extract, peptone 10 g/L, KH2PO41 g/L,
MgSO420 min of 0.5 g/L, corn pulp 10 g/L, pH7.0,121 DEG C high pressure steam sterilization.
6. the method that series bacillus according to claim 2 prepares biological control agent, which is characterized in that the hair
The internal composition of ferment culture medium are as follows: glucose 20g/L, 5 g/L of peptone, yeast extract 2 g/L, MgSO40.4 g/L,
KH2PO41 g/L, 3 g/L of ammonium citrate, corn pulp 10 g/L, FeSO41mg/L, vitamin B12 mg/L, pH 7.0,
121 DEG C of 20 min of high pressure steam sterilization.
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Cited By (3)
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|---|---|---|---|---|
| CN110684695A (en) * | 2019-11-11 | 2020-01-14 | 乔志伟 | Paenibacillus polymyxa QZY-1 and application thereof |
| CN111454860A (en) * | 2020-04-09 | 2020-07-28 | 广西乐土生物科技有限公司 | Bacillus polymyxa microbial inoculum and preparation method thereof |
| CN115851507A (en) * | 2022-10-12 | 2023-03-28 | 山东佰渥康生物科技有限公司 | Paenibacillus ehenii, microbial inoculum and application thereof |
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| WO2014145964A1 (en) * | 2013-03-15 | 2014-09-18 | Spogen Biotech Inc. | Fusion proteins and methods for stimulating plant growth, protecting plants, and immobilizing bacillus spores on plants |
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| CN111454860A (en) * | 2020-04-09 | 2020-07-28 | 广西乐土生物科技有限公司 | Bacillus polymyxa microbial inoculum and preparation method thereof |
| CN115851507A (en) * | 2022-10-12 | 2023-03-28 | 山东佰渥康生物科技有限公司 | Paenibacillus ehenii, microbial inoculum and application thereof |
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Denomination of invention: Preparation method of Bacillus like bacteria and its biological control preparation Effective date of registration: 20211216 Granted publication date: 20190719 Pledgee: Yantai financing guarantee Group Co.,Ltd. Pledgor: LUDONG University Registration number: Y2021980015152 |
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