CN106479938B - A kind of Brevibacillus brevis bacterial strain and its application - Google Patents
A kind of Brevibacillus brevis bacterial strain and its application Download PDFInfo
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Abstract
The invention discloses a kind of Brevibacillus brevis bacterial strain and its application, the bacterial strain is Brevibacillus brevis Bb-1, is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and deposit number is CGMCC NO.12365.Application of the biocontrol agent of Brevibacillus brevis Bb-1 preparation in prevention and treatment root-knot nematode.Brevibacillus brevis in the present invention is isolated from soil, there is good preventive effect for root-knot nematode, it is easy to colonize in the soil, and it is conducive to early stage prevention and control root-knot nematode and endangers, the bacterial strain has huge application potential for the prevention and treatment for solving root-knot nematode, helps to realize high yield, safety, the No-harmful apple orchard of vegetables；The fermentation liquid of Brevibacillus brevis Bb-1 bacterial strain reaches 100% to the lethality of root-knot nematode, and the control efficiency in potted plant experiment reaches 81.6%, and control efficiency is high and stablizes, and is suitable for agricultural production demand.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of Brevibacillus brevis bacterial strain and its application.
Background technique
Root-knot nematode (Meloidogyne) is a kind of omnivorousness plant pathogeny line insect of height specialized form.It is known to endanger vegetable
The nematode of dish mainly has peanut root-knot nematode, M hapla, Meloidogyne incognita, javanese root knot nematode and beet root knot
Nematode etc..Nematode host range is extensive, often endangers more than 30 kinds of melon, solanaceous vegetables, beans and radish, carrot, lettuce, Chinese cabbage etc.
Vegetables, moreover it is possible to propagate some fungies and bacterial disease.Root-knot nematode is a kind of important crop disease original, is caused every year big
The loss of about billions members.The root of the main infection crops of root-knot nematode, the root swelling after infecting form huge root knot,
Beading can be interconnected to form, since root is destroyed, influence normal absorbing function, thus overground part growth and development by
Resistance, the plant above ground portion after being infected, which shows, to be downgraded, and leaf portion yellow is as a result small and few.It easily wilts or withers when dry weather.
In the greenhouse of long-term continuous cropping, watermelon, watermelon, tomato etc. can especially be made to have no harvest.
Since the germ plasm resource of root-knot nematode resistant is rare, so chemical prevention is still prevention and treatment root-knot nematode in production
Important measures, such as dazomet, chloropicrin and Mobam can seriously reduce vegetables quality although these medicament desinsections are fast,
Environment is polluted, the ecological balance is destroyed, it is dangerous to people, animal in use process, therefore bio-control method is increasingly subject to people's
Concern.
Summary of the invention
The purpose of the present invention is to solve existing for chemical prevention in the prior art pollute environment, unsafe problem,
A kind of Brevibacillus brevis bacterial strain and its application are provided.
To achieve the above object, the present invention provides a kind of Brevibacillus brevis, the bacterial strain is Brevibacillus brevis Bb-
1, it is proposed that classification naming be Brevibacillus brevis Brevibacillus brevis；It is preserved in Chinese microorganism strain preservation pipe
Reason committee common micro-organisms center (CGMCC), preservation address: the Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3 Chinese Academy of Sciences
Institute of microbiology, the deposit date is on April 18th, 2016, deposit number was CGMCC 12365.
The present invention also provides the biocontrol agents using Brevibacillus brevis preparation.
The present invention also provides application of the biocontrol agent of Brevibacillus brevis preparation in prevention and treatment root-knot nematode.
Further, the root-knot nematode is Meloidogyne incognita.
The present invention also provides the preparation methods of biocontrol agent, method includes the following steps:
(1) single plant bacterium colony is obtained by Brevibacillus brevis Bb-1 streak inoculation on LB culture medium, after constant temperature incubation；
(2) the single plant bacterium colony of Brevibacillus brevis Bb-1 is seeded in seed culture medium, is obtained after constant-temperature shaking culture
Seed liquor；
(3) seed liquor is inoculated into sterilized fermentation medium and carries out fermented and cultured, obtaining concentration is 106-
108The fermentation liquid of cfu/mL；Then by fermentation liquid direct packaging at liquid dosage form.
Further, in step (1), 48-72h is cultivated in constant incubator, cultivation temperature is 35-40 DEG C.
Further, in step (1), LB culture medium prescription are as follows: yeast powder 5g/L, peptone 10g/L, NaCl 10g/L,
Surplus is water.
Further, in step (2), in constant-temperature table shaken cultivation, revolving speed 160rpm, temperature is 35-40 DEG C, oscillation training
Support 24-48h.
Further, in step (3), seed liquor is inoculated into fermentation medium with inoculum concentration 1-2%, fermentation temperature is
35-40℃。
Further, the seed culture medium in step (2) and the fermentation medium in step (3) are LB culture medium.
The invention has the benefit that the Brevibacillus brevis in the present invention is isolated from soil, have for root-knot nematode
Good preventive effect is easy to colonize in the soil, and is conducive to early stage prevention and control root-knot nematode and endangers, and the bacterial strain is for solving root-knot nematode
Prevention and treatment have huge application potential, help to realize high yield, safety, the No-harmful apple orchard of vegetables；Brevibacillus brevis Bb-
The fermentation liquid of 1 bacterial strain reaches 100% to the lethality of root-knot nematode, and the control efficiency in potted plant experiment reaches 81.6%, prevents
It is high and stable to control effect, is suitable for agricultural production demand.
Detailed description of the invention
Fig. 1 is the ne ar figure of Brevibacillus brevis Bb-1 of the present invention；
Fig. 2 is the colonial morphology figure of Brevibacillus brevis Bb-1 of the present invention.
Specific embodiment
The present invention is carried out below with reference to embodiment and attached drawing explanation is explained in detail.
Embodiment 1
Separation, purifying and the identification of Brevibacillus brevis:
Pedotheque is uniformly taken from the tomato planting field of Shunyi, 10g soil carries out mixing system respectively with 100mL aqua sterilisa
At suspension, gradient dilution is then successively carried out, takes the soil of 10000 times of dilution to mix 100 μ L of liquid and is coated on LB culture medium flat plate
On, the formula of LB culture medium are as follows: yeast powder 5g/L, peptone 10g/L, NaCl 10g/L, agar powder 1.3-1.5g/100mL, it is fixed
Hold to 1L, sealing.After autoclave sterilization, plate is made in culture dish middle berth in 121 DEG C, 20min.Culture medium is placed in 37 DEG C
Culture, after tissue block grows bacterium colony, picking single colonie purifies on LB culture medium, is used for taxonomic identification.According to " primary Jie Shi is thin
Dientification of bacteria handbook " morphology measurement is carried out to bacterial strain.
It with the toothpick picking single colonie of sterilizing, is put into the EP pipe for filling 300 μ L bacterial lysate SDS, 65 DEG C of cracking 2 are small
When, DNA of bacteria then is extracted with bacterial genomes DNA extraction kit (Tiangeng Biotechnology Co., Ltd), using DNA as mould
Plate carries out 16srRNA sequence amplification with primer 2 7F and 1492R respectively.Pcr amplification reaction system is 50 μ L, containing 5 μ L 10 ×
Ex Taq Buffer, 1 μ L Ex Taq enzyme, 5 μ L dNTP (2.5mM each), 1 μ L forward primer, 1 μ L reverse primer, 3 μ
LDNA template, 34 μ L of sterile water.Amplification condition: 94 DEG C of initial denaturation 5min, 94 DEG C of denaturation 30s, 55 DEG C of annealing 30s, 72 DEG C extend
2.5min, 30 circulations；72 DEG C of extension 10min.Amplified production is separated through 1% agarose gel electrophoresis and is identified, PCR product is direct
Carry out bidirectional sequencing.
As a result:
Referring to attached drawing 1-2, Bb-1 bacterial strain is obtained on LB culture medium flat plate, and after 37 DEG C, culture 3 days, bacterium colony is rounded, table
Complexion is dark, thicken with it is opaque.Thallus is rod-shaped, seldom chaining, even dyeing.Flagellum side is raw, raw to time end life, column in spore
Spore makes sporangiocyst be expanded into fusiform or club-like, and spore shows strong tinting strength, tinting power.Spore coat equator is split when sprouting.From nutrition
After cell exposes, spore coat resolution is slow.Bb-1 strain morphology is measured according to " primary Jie Shi Bacteria Identification handbook ", is determined
The bacterial strain is Brevibacillus brevis (Brevibacillus brevis).16s rRNA is expanded using primer 2 7F and 1492R respectively
Sequence, it is similar to the 16SrRNA sequence of Brevibacillus brevis (Brevibacillus brevis) by the homologous comparison of BLAST
Property reaches 100%, therefore Bb-1 bacterial strain is determined as Brevibacillus brevis (Brevibacillus brevis).
16SrRNA and pheS sequence amplification is carried out respectively with primer 1492R, 27F and 21FA, 22RA, and PCR product is through 1%
Agarose gel electrophoresis.The a length of 1400bp of 16SrRNA sequence expanded through sequencing analysis bacterial strain.By the 16SrRNA sequence of bacterial strain
After carrying out BLAST analysis, the results show that Bb-1 bacterial strain is reached with Brevibacillus brevis (Brevibacillus brevis) similarity
To 100%.Identify that the bacterial strain is Brevibacillus brevis (Brevibacillus brevis).
Embodiment 2
1, the preparation method of Bb-1 bacterial strain fermentation liquor, comprising the following steps:
(1) by Brevibacillus brevis Bb-1 streak inoculation on LB culture medium, the plate culture 60h in constant incubator,
Cultivation temperature is set as 37 DEG C, obtains single plant bacterium colony；
(2) the single plant bacterium colony of Brevibacillus brevis Bb-1 is seeded in seed culture medium (LB culture medium), is shaken in constant temperature
Bed shaken cultivation, revolving speed 160rpm, temperature are 37 DEG C, shaken cultivation 36h；Liquid seed culture medium formula is LB culture medium, i.e.,
Yeast powder 5g/L, peptone 10g/L, NaCl10g/L, are settled to 1L；
(3) seed liquor is inoculated into sterilized fermentation medium and carries out fermented and cultured, inoculum concentration 1%, temperature is set
It is set to 37 DEG C, obtaining concentration is 106The fermentation liquid (stoste) of cfu/mL；Liquid fermentation medium formula is LB culture medium, i.e. ferment
Female powder 5g/L, peptone 10g/L, NaCl 10g/L, is settled to 1L.
Fermentation liquid and its 10 times, 100 times of dilutions are handled root-knot nematode (Meloidogyne spp.) respectively, are measured short
Kill effect of the bacillus brevis tunning to root-knot nematode.
2, nematode is used in preparation test
Pick up from Vegetable & Flower Inst., Chinese Academy of Agriculture Science greenhouse.Capsicum root system is taken out, is gently rinsed with water, carefully
Pieces of an egg are removed, is placed in 1% sodium hypochlorite and sterilizes 3min, then with aseptic water washing 3 times, are placed on the training containing a small amount of sterile water
It supports in ware, is cultivated in 25 DEG C of insulating boxs, it is every to collect the root-knot nematode J2 larva once hatched for 24 hours.
3, test method
On sterile tissue culture plate, each 1mL of fermentation liquid of various concentration is added into hole respectively, and is made with sterile water
For control, 100 μ L nematode suspension (100 root-knot nematode J2 larvas) is then added into processing and control respectively, is placed on room
Under temperature for 24 hours, the death condition of root-knot nematode is observed, corrected mortality, as nematicidal effect, 24 holes of each processing are calculated
(sample), each test are repeated 3 times.
Corrected mortality (%)=(processing nemic death rate-control nemic death rate)/(1- compares nemic death rate)
× 100%
4, test result:
By above-mentioned test, different multiples fermentation liquor treatment is measured for 24 hours to the virulence effect of root-knot nematode, the results showed that no
Different with function and effect of the fermentation liquid multiple to nematode, the corrected mortality of the effect root-knot nematode of fermentation liquid stoste is 100%,
Control efficiency is substantially reduced the nematicidal in 10 times of dilutions and crosses only 77.8% after fermentation liquid dilution, and in 100 times of dilutions
Middle nemic death rate is very low (< 10%), with the difference very little compareed.Test illustrates fermentation liquid in higher concentrations and has good anti-
Control root-knot nematode effect.
The Bb-1 bacterial strain fermentation liquor nematicidal effect of 1 various concentration of table
Embodiment 3
1, the preparation method of Bb-1 bacterial strain fermentation liquor, comprising the following steps:
(1) by Brevibacillus brevis Bb-1 streak inoculation on LB culture medium, the plate culture 60h in constant incubator,
Cultivation temperature is 37 DEG C, obtains single plant bacterium colony；
(2) the single plant bacterium colony of Brevibacillus brevis Bb-1 is seeded in seed culture medium, in constant-temperature table shaken cultivation,
Revolving speed 160rpm, temperature are 37 DEG C, shaken cultivation 36h；Liquid seed culture medium formula be LB culture medium, i.e. yeast powder 5g/L,
Peptone 10g/L, NaCl 10g/L, are settled to 1L；
(3) seed liquor is inoculated into sterilized fermentation medium and carries out fermented and cultured, inoculum concentration 1%, temperature is set
It is set to 37 DEG C, obtaining concentration is 106The fermentation liquid of cfu/mL；Liquid fermentation medium formula is LB culture medium, i.e. yeast powder 5g/
L, peptone 10g/L, NaCl 10g/L, are settled to 1L.
It is inoculated with the 10ml bacteria culture fluid on watermelon seedlings, after 2 days, is inoculated with Meloidogyne incognita J2 larva, every plant of inoculation
500, and root knot quantity is detected after 5 weeks, calculate control efficiency.30 plants of watermelon seedlings of each processing, each processing are repeated 3 times.
And using clear water processing as control.
Root knot nematode disease mutual affection grade standard are as follows:
2 watermelon root-knot disease state of an illness partition of the level of table
Root knot index calculates:
Every part of expert evidence root incidence is investigated, is described according to Disease symptoms, material is investigated portionwise, records disease
Feelings rank calculates root knot index (RKI).
Root knot index is calculated according to the following formula:
Root knot index (%)=∑ (s × n)/N × M × 100
In formula:
Each state of an illness rank of ∑-represents the summation of numerical value state of an illness rank diseased plant number product corresponding thereto；
Each state of an illness rank of S-represents numerical value；
Each state of an illness rank diseased plant number of n-；
N-investigation total strain number；
M-highest disease index.
Control efficiency calculation formula:
Control efficiency (%)=(1- handles root knot number/control root knot number) × 100
2, test result
Processing result is shown in Table 2, by test it can be seen that the root knot quantity after biocontrol microorganisms processing on watermelon plant is significant
It reduces, 44.42/plant of root knot quantity average out in control treatment, and the root knot quantity in the watermelon of Bb-1 bacterial strain processing
8.17/plant of average out to, Bb-1 bacterial strain have reached 81.6% to the control efficiency of root-knot nematode, illustrate that biocontrol agent can be effective
Prevention and treatment watermelon root-knot.Root knot nematode disease is prevented and treated in agricultural production, especially facility cultivating watermelon has important value.
2 different disposal root knot quantity of table and control efficiency
Embodiment 4
A kind of preparation method of Bb-1 Biocontrol Activity microbial inoculum, comprising the following steps:
(1) by Brevibacillus brevis Bb-1 streak inoculation on LB culture medium, the plate culture 48h in constant incubator,
Cultivation temperature is set as 35 DEG C, obtains single plant bacterium colony；
(2) the single plant bacterium colony of Brevibacillus brevis Bb-1 is seeded in seed culture medium (LB culture medium), is shaken in constant temperature
Bed shaken cultivation, revolving speed 160rpm, temperature are 35 DEG C, and shaken cultivation is for 24 hours；Liquid seed culture medium formula is LB culture medium, i.e.,
Yeast powder 5g/L, peptone 10g/L, NaCl10g/L, are settled to 1L；
(3) seed liquor is inoculated into sterilized fermentation medium and carries out fermented and cultured, inoculum concentration 1%, temperature is set
It is set to 35 DEG C, obtaining concentration is 106The fermentation liquid (stoste) of cfu/mL, liquid fermentation medium formula are LB culture medium, i.e. ferment
Female powder 5g/L, peptone 10g/L, NaCl 10g/L, is settled to 1L；Then by obtained fermentation liquid direct packaging at liquid agent
Type.
Embodiment 5
A kind of preparation method of Bb-1 Biocontrol Activity microbial inoculum, comprising the following steps:
(1) by Brevibacillus brevis Bb-1 streak inoculation on LB culture medium, the plate culture 72h in constant incubator,
Cultivation temperature is 40 DEG C, obtains single plant bacterium colony；
(2) the single plant bacterium colony of Brevibacillus brevis Bb-1 is seeded in seed culture medium, in constant-temperature table shaken cultivation,
Revolving speed 160rpm, temperature are 40 DEG C, shaken cultivation 48h；Liquid seed culture medium formula be LB culture medium, i.e. yeast powder 5g/L,
Peptone 10g/L, NaCl 10g/L, is settled to 1L；
(3) seed liquor is inoculated into sterilized fermentation medium and carries out fermented and cultured, inoculum concentration 2%, temperature is set
It is set to 40 DEG C, obtaining concentration is 108The fermentation liquid of cfu/mL；Liquid fermentation medium formula is LB culture medium, i.e. yeast powder 5g/
L, peptone 10g/L, NaCl 10g/L, are settled to 1L；Then by obtained fermentation liquid direct packaging at liquid dosage form.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modification, equivalent replacement and simple modifications etc., should all be included in the protection scope of the present invention in content.
Claims (10)
1. a kind of Brevibacillus brevis bacterial strain, which is characterized in that the bacterial strain is Brevibacillus brevis Bb-1, and it is micro- to be preserved in China
Biological inoculum preservation administration committee common micro-organisms center, deposit number are CGMCC NO.12365.
2. the biocontrol agent prepared using Brevibacillus brevis bacterial strain described in claim 1.
3. application of the biocontrol agent of Brevibacillus brevis bacterial strain preparation as claimed in claim 2 in prevention and treatment root-knot nematode.
4. biocontrol agent the answering in prevention and treatment root-knot nematode of Brevibacillus brevis bacterial strain preparation according to claim 3
With, which is characterized in that the root-knot nematode is Meloidogyne incognita.
5. the preparation method of biocontrol agent as claimed in claim 2, which is characterized in that method includes the following steps:
(1) single plant bacterium colony is obtained by Brevibacillus brevis Bb-1 streak inoculation on LB culture medium, after constant temperature incubation；
(2) the single plant bacterium colony of Brevibacillus brevis Bb-1 is seeded in seed culture medium, seed is obtained after constant-temperature shaking culture
Liquid；
(3) seed liquor is inoculated into the fermentation medium of sterilizing and carries out fermented and cultured, obtaining concentration is 106-108Cfu/mL's
Fermentation liquid；Then by fermentation liquid direct packaging at liquid dosage form.
6. the preparation method of biocontrol agent according to claim 5, which is characterized in that in step (1), in constant incubator
Middle culture 48-72h, cultivation temperature are 35-40 DEG C.
7. the preparation method of biocontrol agent according to claim 5, which is characterized in that in step (1), LB culture medium prescription
Are as follows: yeast powder 5g/L, peptone 10g/L, NaCl 10g/L, surplus is water.
8. the preparation method of biocontrol agent according to claim 5, which is characterized in that in step (2), on constant-temperature table
Shaken cultivation, revolving speed 160rpm, temperature are 35-40 DEG C, shaken cultivation 24-48h.
9. the preparation method of biocontrol agent according to claim 5, which is characterized in that in step (3), by seed liquor to connect
Kind amount 1-2% is inoculated into fermentation medium, and fermentation temperature is 35-40 DEG C.
10. the preparation method of biocontrol agent according to claim 5, which is characterized in that the seed culture medium in step (2)
It is LB culture medium with the fermentation medium in step (3).
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| CN107164438B (en) * | 2017-07-13 | 2021-03-23 | 陕西科技大学 | Fermentation method for improving production level of brevibacillus brevis bacteriocin |
| CN109868237B (en) * | 2019-02-15 | 2020-12-08 | 湖南农业大学 | Bacillus megaterium and application thereof |
| CN110172422B (en) * | 2019-05-28 | 2020-12-25 | 中国农业科学院蔬菜花卉研究所 | Bacillus proteolicus and application thereof in preventing and controlling root-knot nematodes |
| CN111778190B (en) * | 2020-07-16 | 2022-03-08 | 山东滇鲁生物科技有限公司 | Brevibacillus brevis for preventing and treating root knot nematode disease and application thereof |
| CN112725236B (en) * | 2020-12-08 | 2022-02-01 | 青岛农业大学 | Brevibacillus agri DR2-1 and application thereof |
| CN115281216A (en) * | 2022-08-10 | 2022-11-04 | 河北省科学院生物研究所 | Application of brevibacillus brevis ZLP-151 in biological prevention and control |
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